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In JoVE (2)
- Competitive Genomic Screens of Barcoded Yeast Libraries
- Compact Quantum Dots for Single-molecule Imaging
Other Publications (95)
- Alcoholism, Clinical and Experimental Research
- The Biological Bulletin
- Integrative and Comparative Biology
- Integrative and Comparative Biology
- Foot & Ankle International / American Orthopaedic Foot and Ankle Society [and] Swiss Foot and Ankle Society
- The Analyst
- Angewandte Chemie (International Ed. in English)
- Photochemistry and Photobiology
- Journal of Gastrointestinal Surgery : Official Journal of the Society for Surgery of the Alimentary Tract
- Brain Research. Developmental Brain Research
- Annals of Biomedical Engineering
- Expert Review of Molecular Diagnostics
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Life Sciences
- Journal of Molecular Biology
- Alcohol (Fayetteville, N.Y.)
- Physical Chemistry Chemical Physics : PCCP
- Nanomedicine (London, England)
- International Journal of Nanomedicine
- Hernia : the Journal of Hernias and Abdominal Wall Surgery
- Proceedings of the National Academy of Sciences of the United States of America
- The Journal of Investigative Dermatology
- Gastrointestinal Endoscopy
- Analytical Chemistry
- Chemical Society Reviews
- The Journal of Pediatrics
- Inflammatory Bowel Diseases
- Genome Research
- Advanced Drug Delivery Reviews
- Journal of Medical Case Reports
- Neurotoxicology and Teratology
- Nature Chemical Biology
- Journal of the American Chemical Society
- Journal of the American Chemical Society
- Journal of the American Chemical Society
- Clinical Allergy and Immunology
- Angewandte Chemie (International Ed. in English)
- Nature Nanotechnology
- Nature Nanotechnology
- Neurobiology of Learning and Memory
- Analytica Chimica Acta
- Nature Protocols
- The Journal of Allergy and Clinical Immunology
- Annals of the Royal College of Surgeons of England
- Science (New York, N.Y.)
- Biochemical Pharmacology
- Langmuir : the ACS Journal of Surfaces and Colloids
- HPB : the Official Journal of the International Hepato Pancreato Biliary Association
- Biochemical Society Transactions
- Genome Research
- Biochemical Pharmacology
- The Journal of Experimental Medicine
- Nature Biotechnology
- BMC Genomics
- Nature Nanotechnology
- PloS One
- Nano Research
- Langmuir : the ACS Journal of Surfaces and Colloids
- Nucleic Acids Research
- Pharmacology & Therapeutics
- Expert Review of Clinical Immunology
- Science (New York, N.Y.)
- The Biochemist
- Inflammatory Bowel Diseases
- JOP : Journal of the Pancreas
- Life Sciences
- Annals of Surgery
- Biochemical Pharmacology
- PloS One
- Accounts of Chemical Research
- Journal of the American Chemical Society
- British Journal of Pharmacology
- Allergy and Asthma Proceedings : the Official Journal of Regional and State Allergy Societies
- European Journal of Pharmacology
- The Journal of Experimental Biology
- BMC Genomics
- Nature Biotechnology
- Digestive Diseases and Sciences
- Immunology and Allergy Clinics of North America
- Musculoskeletal Care
- Journal of Gastrointestinal Surgery : Official Journal of the Society for Surgery of the Alimentary Tract
- Journal of Experimental Psychology. Animal Behavior Processes
- Inflammatory Bowel Diseases
- Hepatobiliary & Pancreatic Diseases International : HBPD INT
- Annals of Allergy, Asthma & Immunology : Official Publication of the American College of Allergy, Asthma, & Immunology
- Journal of Gastrointestinal Surgery : Official Journal of the Society for Surgery of the Alimentary Tract
- Molecular BioSystems
- Methods in Molecular Biology (Clifton, N.J.)
- Nucleic Acids Research
Articles by Andrew M. Smith in JoVE
Competitive Genomic Screens of Barcoded Yeast Libraries
Andrew M. Smith*1,2, Tanja Durbic*2,3, Julia Oh*4, Malene Urbanus1,2, Michael Proctor5, Lawrence E. Heisler2,3, Guri Giaever2,6, Corey Nislow1,2,3
1Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 3Donnelly Sequencing Centre, University of Toronto, 4Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, 5Stanford Genome Technology Center, Stanford School of Medicine, Stanford University, 6Department of Pharmaceutical Sciences, University of Toronto
We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.
Compact Quantum Dots for Single-molecule Imaging
Andrew M. Smith1, Shuming Nie1,2
1Department of Biomedical Engineering, Emory University, 2Department of Chemistry, Georgia Institute of Technology
We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.
Other articles by Andrew M. Smith on PubMed
Chronic Continuous Nicotine Exposure During Periadolescence Does Not Increase Ethanol Intake During Adulthood in Rats
Alcoholism, Clinical and Experimental Research. Jul, 2002 | Pubmed ID: 12170106
Nicotine has been considered as the gateway drug, because many teenagers experience cigarette smoking before seeking out other drugs. By using an animal model system, we assessed the effects of chronic continuous nicotine exposure during periadolescence on ethanol intake during young adulthood.
The Biological Bulletin. Dec, 2002 | Pubmed ID: 12480724
The composition of the adhesive form of marsh periwinkle mucus was compared to the trail mucus used during locomotion. The trail mucus consists primarily of large, carbohydrate-rich molecules with some relatively small proteins. In contrast, the adhesive mucus has 2.7 times as much protein with no significant difference in carbohydrate concentration. The resulting gel has roughly equal amounts of protein and carbohydrate. This substantial increase in protein content is due to the additional presence of two proteins with molecular weights of 41 and 36 kD. These two proteins are absent from the trail mucus. Both proteins are glycosylated, have similar amino acid compositions, and have isoelectric points of 4.75. This change in composition corresponds to an order of magnitude increase in tenacity with little clear change in overall concentration. The difference between adhesive and non-adhesive mucus suggests that relatively small proteins are important for controlling the mechanics of periwinkle mucus.
Integrative and Comparative Biology. Dec, 2002 | Pubmed ID: 21680399
Octopus suckers consist of a tightly packed three-dimensional array of muscle with three major muscle fiber orientations: 1) radial muscles that traverse the wall; 2) circular muscles arranged circumferentially around the sucker; and 3) meridional muscles oriented perpendicular to the circular and radial muscles. The sucker also includes inner and outer fibrous connective tissue layers and an array of crossed connective tissue fibers embedded in the musculature. Adhesion results from reducing the pressure inside the sucker cavity. This can be achieved by the three-dimensional array of muscle functioning as a muscular-hydrostat. Contraction of the radial muscles thins the wall, thereby increasing the enclosed volume of the sucker. If the sucker is sealed to a surface the cohesiveness of water resists this expansion. Thus, the pressure of the enclosed water decreases instead. The meridional and circular muscles antagonize the radial muscles. The crossed connective tissue fibers may store elastic energy, providing an economical mechanism for maintaining attachment for extended periods. Measurements using miniature flush-mounted pressure transducers show that suckers can generate hydrostatic pressures below 0 kPa on wettable surfaces but cannot do so on non-wettable surfaces. Thus, cavitation, the failure of water in tension, may limit the attachment force of suckers. As depth increases, however, cavitation will cease to be limiting because ambient pressure increases with depth while the cavitation threshold is unchanged. Structural differences between suckers will then determine the attachment force.
Integrative and Comparative Biology. Dec, 2002 | Pubmed ID: 21680401
Many marine invertebrates form strong, temporary attachments using viscoelastic gels. To better understand these adhesives, an analysis of what is known of gel structure and function was performed. There are different ways of making gels, ranging from entangling of giant glycoproteins to crosslinking of smaller proteins. The mechanics of the gel depend largely on the size of the polymer, its concentration, and whether it is crosslinked. Compared to gels such as mammalian mucus, the mechanics of adhesive mucous gels often appear to depend more heavily on relatively small proteins than on megadalton-sized glycoproteins. In addition, changes in concentration and the presence of specific proteins have been associated with the change from a non-adhesive to an adhesive form. The attachment strengths produced by different gels at different concentrations were compared with the changes in attachment strength seen in living animals. These data suggest that changes in concentration are not sufficient to account for adhesion. Thus, it is likely that the changes in protein composition may play a large role.
Foot & Ankle International / American Orthopaedic Foot and Ankle Society [and] Swiss Foot and Ankle Society. Mar, 2003 | Pubmed ID: 12793484
We reporting the perioperative complications during our early experience using the Scarf osteotomy to correct hallux valgus. A case note review was carried out for the first 100 Scarf osteotomy procedures completed by the senior author. There were six patients (6%) with perioperative complications. Four of these were intraoperative complications including a split first metatarsal in three cases, a shearing of the K-wire in one case and there were two cases of postoperative stress fracture. These complications should be considered by those beginning to master the Scarf osteotomy procedure and by surgeons teaching surgical trainees.
The Analyst. Aug, 2004 | Pubmed ID: 15344262
Quantum dots are tiny light-emitting particles on the nanometer scale. They are emerging as a new class of biological labels with properties and applications that are not available with traditional organic dyes and fluorescent proteins. Their novel properties such as improved brightness, resistance against photobleaching, and multicolor light emission, have opened new possibilities for ultrasensitive chemical analysis and cellular imaging. In this Research Highlight article , we discuss the unique optical properties of semiconductor quantum dots, surface chemistry and bioconjugation, current applications in bioanalytical chemistry and cell biology, and future research directions.
Angewandte Chemie (International Ed. in English). Dec, 2004 | Pubmed ID: 15614890
Photochemistry and Photobiology. Nov-Dec, 2004 | Pubmed ID: 15623319
Semiconductor quantum dots (QD) are nanometer-sized crystals with unique photochemical and photophysical properties that are not available from either isolated molecules or bulk solids. In comparison with organic dyes and fluorescent proteins, QD are emerging as a new class of fluorescent labels with improved brightness, resistance against photobleaching and multicolor fluorescence emission. These properties could improve the sensitivity of biological detection and imaging by at least 10- to 100-fold. Further development in high-quality near-infrared-emitting QD should allow ultrasensitive and multicolor imaging of molecular targets in deep tissue and living animals. Here, we discuss recent developments in QD synthesis and bioconjugation, applications in molecular and cellular imaging as well as promising directions for future research.
Journal of Gastrointestinal Surgery : Official Journal of the Society for Surgery of the Alimentary Tract. Jul-Aug, 2005 | Pubmed ID: 15985240
An unusual case of variceal bleeding at the site of a biliary enteric anastomosis is presented. This entity can occur when a high-to-low pressure gradient forms in a variceal field. In this case the anastomotic site was the location of the pressure gradient from the high-pressure small bowel varices to the low-pressure biliary tract. This was successfully treated by disconnection of the anastomosis. The resulting biliary defect was patched with small intestinal submucosa, which functioned successfully as a scaffold for biliary epithelial ingrowth.
Neonatal Alcohol Exposure Increases Malondialdehyde (MDA) and Glutathione (GSH) Levels in the Developing Cerebellum
Brain Research. Developmental Brain Research. Dec, 2005 | Pubmed ID: 16256207
It has been suggested that developmental alcohol-induced brain damage is mediated through increases in oxidative stress. In this study, the concentrations of malondialdehyde (MDA) and reduced glutathione (GSH) were measured to indicate alcohol-mediated oxidative stress. In addition, the ability of two known antioxidants, melatonin (MEL) and lazaroid U-83836E (U), to attenuate alcohol-induced oxidative stress was investigated. Sprague-Dawley rat pups were randomly assigned to six artificially-reared groups, ALC (alcohol), MEL, MEL/ALC, U, U/ALC, and GC (gastrostomy control), and one normal suckle control (to control for artificial-rearing effects on the dependent variables). The daily dosages for ALC, MEL, and U were 6 g/kg, 20 mg/kg, and 20 mg/kg, respectively. Alcohol was administered in 2 consecutive feedings, and antioxidant (MEL or U) was administered for a total of 4 consecutive feedings (2 feedings prior to and 2 feedings concurrently with alcohol). The animals received treatment from postnatal days (PD) 4 through 9. Cerebellar, hippocampal, and cortical samples were collected on PD 9 and analyzed for MDA and GSH content. The results indicated that MDA concentrations in the cerebellum were significantly elevated in animals receiving alcohol; however, MDA levels in the hippocampus and cortex were not affected by alcohol treatment. Additionally, GSH levels in the cerebellum were significantly elevated in groups receiving alcohol, regardless of antioxidant treatment. Neither antioxidant was able to protect against alcohol-induced alterations of MDA or GSH. These findings suggest that alcohol might increase GSH levels indirectly as a compensatory mechanism designed to protect the brain from oxidative-stress-mediated insult.
Annals of Biomedical Engineering. Jan, 2006 | Pubmed ID: 16450199
Semiconductor quantum dots are luminescent nanoparticles that are under intensive development for use as a new class of optical imaging contrast agents. Their novel properties such as optical tunability, improved photostability, and multicolor light emission have opened new opportunities for imaging living cells and in vivo animal models at unprecedented sensitivity and spatial resolution. Combined with biomolecular engineering strategies for tailoring the particle surfaces at the molecular level, bio-conjugated quantum dot probes are well suited for imaging single-molecule dynamics in living cells, for monitoring protein-protein interactions within specific intracellular locations, and for detecting diseased sites and organs in deep tissue. In this article, we describe the engineering principles for preparing high-quality quantum dots and for conjugating the dots to biomolecular ligands. We also discuss recent advances in using quantum dots for in vivo molecular and cellular imaging.
Expert Review of Molecular Diagnostics. Mar, 2006 | Pubmed ID: 16512782
In the pursuit of sensitive and quantitative methods to detect and diagnose cancer, nanotechnology has been identified as a field of great promise. Semiconductor quantum dots are nanoparticles with intense, stable fluorescence, and could enable the detection of tens to hundreds of cancer biomarkers in blood assays, on cancer tissue biopsies, or as contrast agents for medical imaging. With the emergence of gene and protein profiling and microarray technology, high-throughput screening of biomarkers has generated databases of genomic and expression data for certain cancer types, and has identified new cancer-specific markers. Quantum dots have the potential to expand this in vitro analysis, and extend it to cellular, tissue and whole-body multiplexed cancer biomarker imaging.
Behavioral and Molecular Effects of Dopamine D1 Receptor Stimulation During Naloxone-precipitated Morphine Withdrawal
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2006 | Pubmed ID: 16775132
Morphine dependence is characterized by somatic and motivational signs of withdrawal that likely contribute to the maintenance of addictive behavior. The nucleus accumbens (NAc) receives extensive dopaminergic input and is an important substrate for mediating these aversive states. In the NAc, the function of the transcription factor cAMP response element binding protein (CREB) and AMPA glutamate receptor subunit, type 1 (GluR1) can be regulated by dopamine (DA) D1 receptor-mediated phosphorylation (P-CREB, P-GluR1). However, the roles of D1 receptors, CREB, and GluR1 in morphine dependence are not well understood. Here, we show that somatic signs of naloxone-precipitated withdrawal were associated with increased P-CREB, but not P-GluR1, in the NAc of morphine-dependent rats. The D1 receptor agonist chloro-APB hydrobromide (SKF 82958) was rewarding in morphine-dependent rats and blocked naloxone-induced place aversions and somatic signs of withdrawal. Surprisingly, SKF 82958 increased P-GluR1, but not P-CREB, in the NAc, and naloxone reduced SKF 82958-mediated P-GluR1 induction specifically in morphine-dependent rats. Together, these results confirm that aversive treatments can increase CREB function in the NAc. Furthermore, they suggest a dependence-associated shift in the molecular mechanisms that regulate the consequences of D1 receptor stimulation, favoring activation of GluR1 rather than CREB. These data raise the possibility that the rewarding effects of SKF 82958 in morphine-dependent rats involve increased P-GluR1 in the NAc, although the involvement of other brain regions cannot be ruled out. Regardless, these findings suggest for the first time that D1 agonists might be useful for the treatment of withdrawal symptoms that contribute to the maintenance of opiate addiction in humans.
Effects of Nicotine Exposure During Prenatal or Perinatal Period on Cell Numbers in Adult Rat Hippocampus and Cerebellum: a Stereology Study
Life Sciences. Nov, 2006 | Pubmed ID: 16959275
Smoking during pregnancy poses a potential risk to unborn children. The present study examined the long-term effects of early nicotine exposure on the number of pyramidal and granule cells in the hippocampus, and Purkinje cells in the cerebellar vermis. The loss of neurons is the most severe form of brain injury with significant functional implications. In this study, rats were exposed to nicotine during either the prenatal (PRE) period or both the prenatal and early postnatal (PERI) period. It was hypothesized that nicotine treatment would result in long-term decreases in neuronal numbers, and that PERI treatment would be more detrimental to these cell populations than the PRE treatment. The results showed that neither PRE nor PERI nicotine exposure reduces the numbers of pyramidal, granule or Purkinje cells. Neither the regions where these cells reside, nor the cell densities were affected by nicotine. Although no significant cell loss was observed, the current nicotine exposure regimens may lead to alterations in cellular functions or cytoarchitectures. The present results in conjunction with previous reports showing significant cell loss from nicotine exposure during the brain growth spurt suggest that "patch-like" nicotine exposure during prenatal period may alter the sensitivity or the responsiveness of the developing brain to the injurious effects of nicotine during the most vulnerable stage of brain development - the brain growth spurt. Furthermore, the current stereology cell counting results are not in agreement with some reports in the literature, and this discrepancy may simply be a function of different cell counting techniques used.
Direct Observation of Oligomeric Species Formed in the Early Stages of Amyloid Fibril Formation Using Electrospray Ionisation Mass Spectrometry
Journal of Molecular Biology. Nov, 2006 | Pubmed ID: 17005201
Numerous debilitating human disorders result from protein misfolding and amyloid formation. Despite the grave nature of these maladies, our understanding of the structural mechanism of fibril assembly is limited. Of paramount importance is the need to identify and characterize oligomeric species formed early during fibril assembly, so that the nature of the initiating assembly mechanism can be revealed and species that may be toxic to cells identified. However, the transient nature of early oligomeric species, combined with their heterogeneity and instability, has precluded detailed analysis to date. Here, we have used electrospray ionisation mass spectrometry (ESI-MS), complemented by analytical ultracentrifugation (AUC) and measurements of thioflavin-T fluorescence, to monitor the early stages of assembly of amyloid-like fibrils formed from human beta-2-microglobulin (beta2m) in vitro. We show that worm-like fibrils that form with nucleation-independent kinetics assemble by a mechanism consistent with monomer addition, with species ranging from monomer to > or = 13-mer being identified directly and uniquely as transient assembly intermediates. By contrast, only monomers, dimers, trimers and tetramers are observed during nucleated growth, which leads to the formation of long straight fibrils. The results highlight the unique power of non-covalent ESI-MS to identify protein assembly intermediates in complex heterogeneous systems and demonstrate its great potential to identify and characterise individual species formed early during amyloid assembly.
Alcohol (Fayetteville, N.Y.). Jun, 2006 | Pubmed ID: 17134658
Previous studies have reported that alcohol exposure reduces the number of neuronal-like pheochromocytoma (PC12) cells in culture. In this study, the interactive effect of coexposure of alcohol and nicotine on PC12 cell numbers was examined in comparison with the effect derived from alcohol or nicotine exposure individually. Moreover, the role of apoptosis in mediating changes in PC12 cell numbers was also investigated. It was hypothesized that alcohol would result in cell loss, and the presence of nicotine would attenuate the damaging effects of alcohol. PC12 cells were exposed to alcohol (100 mM), nicotine (10 microM), or both alcohol and nicotine for 24, 48, 72, or 96 h. Caspase-3 activity and DNA fragmentation, markers for apoptotic cell death, were measured to determine the role of apoptosis in mediating decreases in PC12 cell numbers. The findings indicated that both alcohol and nicotine exposure significantly decreased PC12 cell numbers when compared with the control treatment. Furthermore, the coexposure of these two drugs caused a significantly greater decrease in cell numbers when compared with cells exposed to either alcohol or nicotine alone. This additive effect was related to the duration of exposure with a marked reduction in cell numbers following 96 h of coexposure to alcohol and nicotine. Neither alcohol nor nicotine exposure appeared to alter caspase-3 activity or DNA fragmentation levels, suggesting that the reduction in PC12 cell numbers following alcohol and/or nicotine exposure may possibly be due to factors other than apoptosis, such as interference with proliferation rates.
A Systematic Examination of Surface Coatings on the Optical and Chemical Properties of Semiconductor Quantum Dots
Physical Chemistry Chemical Physics : PCCP. Sep, 2006 | Pubmed ID: 19817050
A number of procedures are currently available to encapsulate and solubilize hydrophobic semiconductor Quantum Dots (QDs) for biological applications. Most of these procedures are based on the use of small-molecule coordinating ligands, amphiphilic polymers, or amphiphilic lipids. However, it is still not clear how these different surface coating molecules affect the optical, colloidal, and chemical properties of the solubilized QDs. Here we report a systematic study to examine the effects of surface coating chemistry on the hydrodynamic size, fluorescence quantum yield, photostability, chemical stability, and biocompatibility of water-soluble QDs. The results indicate that quantum dots with the smallest hydrodynamic sizes are best prepared by direct ligand exchange with hydrophilic molecules, but the resulting particles are less stable than those encapsulated in amphiphilic polymers. For stability against chemical oxidation, QDs should be protected with a hydrophobic bilayer. For high stability under acidic conditions, the best QDs are prepared by using hyperbranched polyethylenimine. For stability in high salt buffers, it is preferable to have uncharged, sterically-stabilized QDs, like those coated with polyethylene glycol (PEG). These insights are expected to benefit the development of quantum dots and related nanoparticle probes for molecular and cellular imaging applications.
Nanomedicine (London, England). Aug, 2006 | Pubmed ID: 17716110
Nanometer-sized particles, such as semiconductor quantum dots and iron oxide nanocrystals, have novel optical, electronic, magnetic or structural properties that are not available from either molecules or bulk solids. When linked with tumor-targeting ligands, such as monoclonal antibodies, peptide fragments of tumor-specific proteins or small molecules, these nanoparticles can be used to target tumor antigens (biomarkers) and tumor vasculatures with high affinity and specificity. In the mesoscopic size range of 5-100 nm diameter, quantum dots and related nanoparticles have large surface areas and functional groups that can be linked to multiple diagnostic (e.g., optical, radioisotopic or magnetic) and therapeutic (e.g., anticancer) agents. In this review, recent advances in the development and applications of bioconjugated quantum dots and multifunctional nanoparticles for in vivo tumor imaging and targeting are discussed.
Molecular Profiling of Single Cancer Cells and Clinical Tissue Specimens with Semiconductor Quantum Dots
International Journal of Nanomedicine. 2006 | Pubmed ID: 17722280
Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens.
Hernia : the Journal of Hernias and Abdominal Wall Surgery. Aug, 2007 | Pubmed ID: 17342385
Bochdalek hernias on the right side of the diaphragm are very rarely diagnosed in adults. We review a case of a 35-year-old female who presented acutely with intestinal obstruction. Plain and cross-sectional imaging identified a large right-sided Bochdalek hernia, containing colon, causing a mechanical obstruction and, surprisingly, concurrent appendicitis. The patient underwent an emergency laparotomy. At surgery the colon was reduced and was viable. The diaphragmatic defect was repaired using non-absorbable sutures and an appendicectomy was then performed for purulent appendicitis. She made an uneventful recovery and remains well at 9-month follow-up. We discuss what we believe to be the first reported case of an obstructed right-sided Bochdalek's hernia associated with appendicitis in an adult and review the published literature on this rare condition.
Proceedings of the National Academy of Sciences of the United States of America. Jun, 2007 | Pubmed ID: 17567757
We have established a designed system comprising two peptides that coassemble to form long, thickened protein fibers in water. This system can be rationally engineered to alter fiber assembly, stability, and morphology. Here, we show that rational mutations to our original peptide designs lead to structures with a remarkable level of order on the nanoscale that mimics certain natural fibrous assemblies. In the engineered system, the peptides assemble into two-stranded alpha-helical coiled-coil rods, which pack in axial register in a 3D hexagonal lattice of size 1.824 nm, and with a periodicity of 4.2 nm along the fiber axis. This model is supported by both electron microscopy and x-ray diffraction. Specifically, the fibers display surface striations separated by nanoscale distances that precisely match the 4.2-nm length expected for peptides configured as alpha-helices as designed. These patterns extend unbroken across the widths (>/=50 nm) and lengths (>10 microm) of the fibers. Furthermore, the spacing of the striations can be altered predictably by changing the length of the peptides. These features reflect a high level of internal order within the fibers introduced by the peptide-design process. To our knowledge, this exceptional order, and its persistence along and across the fibers, is unique in a biomimetic system. This work represents a step toward rational bottom-up assembly of nanostructured fibrous biomaterials for potential applications in synthetic biology and nanobiotechnology.
The Journal of Investigative Dermatology. Mar, 2007 | Pubmed ID: 17008874
T-cell mediated contact sensitization by small molecular weight xenobiotics results in significant morbidity and absences from work. To be recognized by T-cells, xenobiotics must act as haptens, becoming protein-bound. At present, the requirement for processing and presentation of xenobiotics, the nature of the T-cell responses to them and the mechanisms that confer individual susceptibility in humans are unclear. We have investigated the T-cell response to the hapten 2,4-dinitrochlorobenzene (DNCB) which can sensitize all immunocompetent people. Fourteen healthy adults were sensitized with DNCB; 11 demonstrated positive T-cell responses to the chemical in vitro. Responding cells were of both CD4+ and CD8+ subsets, of Th1 and Tc1 phenotypes, producing high levels of IFN-gamma and low levels of IL-10. DNCB-specific T-cell clones were raised from 2 subjects, which in the presence of fixed and unfixed autologous Epstein-Barr virus transformed B cells as antigen-presenting-cells (APC), demonstrated that the chemical requires metabolic processing by the APC in order to initiate the T-cell response. Intracellular-reduced glutathione is consumed in detoxication of DNCB, leaving residual non-detoxified DNCB free to bind to proteins. The results suggest that DNCB forms multiple haptens with intracellular and extracellular proteins leading to Th1 and Tc1 responses in individuals exposed to this compound.
Gastrointestinal Endoscopy. Mar, 2008 | Pubmed ID: 17981279
Analytical Chemistry. Apr, 2008 | Pubmed ID: 18324840
Quantum-dot (QD) nanocrystals are promising fluorescent probes for multiplexed staining assays in biological applications. However, nonspecific QD binding to cellular membranes and proteins remains a limiting factor in detection sensitivity and specificity. Here we report a new class of hydroxyl (-OH)-coated QDs for minimizing nonspecific cellular binding and for overcoming the bulky size problems encountered with previous surface coatings. The hydroxylated QDs are prepared from carboxylated (-COOH) dots via a hydroxylation and cross-linking process. With a compact hydrodynamic size of 13-14 nm (diameter), they are highly fluorescent (>60% quantum yields) and stable under both basic and acidic conditions. By using human cancer cells, we have evaluated their superior nonspecific binding properties against that of carboxylated, protein-coated, and poly(ethylene glycol) (PEG)-coated QDs. Quantitative cellular staining data indicate that the hydroxylated QDs result in a dramatic 140-fold reduction in nonspecific binding relative to that of carboxylated dots and a still significant 10-20-fold reduction relative to that of PEG- and protein-coated dots.
Chemical Society Reviews. Apr, 2008 | Pubmed ID: 18362975
This tutorial review looks at the design rules that allow peptides to be exploited as building blocks for the assembly of nanomaterials. These design rules are either derived by copying nature (alpha-helix, beta-sheet) or may exploit entirely new designs based on peptide derivatives (peptide amphiphiles, pi-stacking systems). We will examine the features that can be introduced to allow self-assembly to be controlled and directed by application of an externally applied stimulus, such as pH, light or enzyme action. Lastly the applications of designed self-assembly peptide systems in biotechnology (3D cell culture, biosensing) and technology (nanoelectronics, templating) will be examined.
Environmental Tobacco Smoke and Interleukin 4 Polymorphism (C-589T) Gene: Environment Interaction Increases Risk of Wheezing in African-American Infants
The Journal of Pediatrics. May, 2008 | Pubmed ID: 18410779
To determine whether infants exposed to environmental tobacco smoke (ETS) having the interleukin 4 (IL-4) or interleukin 13 (IL-13) gene polymorphisms were at increased risk of wheezing.
Inflammatory Bowel Diseases. Oct, 2008 | Pubmed ID: 18421761
Inflammatory bowel diseases are common chronic inflammatory disorders. The majority are idiopathic and can be broadly divided into Crohn's disease and ulcerative colitis. Their cause is unknown, but most hypotheses focus on a primary role for T-cell dysfunction. Conversely, there is a collection of congenital disorders of phagocyte function that result not only in immunodeficiency but also in noninfectious inflammatory bowel disease. In all cases, the latter is strikingly reminiscent of the clinical and pathological features of Crohn's disease. This coincides with recent work demonstrating that despite previous emphasis on adaptive immune dysfunction, patients with Crohn's disease actually possess an unusually weak acute innate inflammatory response. This review consolidates the literature on inflammatory bowel disease in congenital immunodeficiencies and considers the role of phagocyte dysfunction in Crohn's disease. Concepts about pathogenesis and treatment that can be carried across these disorders are also discussed.
Genome Research. Jul, 2008 | Pubmed ID: 18463300
Since complete redundancy between extant duplicates (paralogs) is evolutionarily unfavorable, some degree of functional congruency is eventually lost. However, in budding yeast, experimental evidence collected for duplicated metabolic enzymes and in global physical interaction surveys had suggested widespread functional overlap between paralogs. While maintained functional overlap is thought to confer robustness against genetic mutation and facilitate environmental adaptability, it has yet to be determined what properties define paralogs that can compensate for the phenotypic consequence of deleting a sister gene, how extensive this epistasis is, and how adaptable it is toward alternate environmental states. To this end, we have performed a comprehensive experimental analysis of epistasis as indicated by aggravating genetic interactions between paralogs resulting from an ancient whole-genome duplication (WGD) event occurring in the budding yeast Saccharomyces cerevisiae, and thus were able to compare properties of large numbers of epistatic and non-epistatic paralogs with identical evolutionary times since divergence. We found that more than one-third (140) of the 399 examinable WGD paralog pairs were epistatic under standard laboratory conditions and that additional cases of epistasis became obvious only under media conditions designed to induce cellular stress. Despite a significant increase in within-species sequence co-conservation, analysis of protein interactions revealed that paralogs epistatic under standard laboratory conditions were not more functionally overlapping than those non-epistatic. As experimental conditions had an impact on the functional categorization of paralogs deemed epistatic and only a fraction of potential stress conditions have been interrogated here, we hypothesize that many epistatic relationships remain unresolved.
Advanced Drug Delivery Reviews. Aug, 2008 | Pubmed ID: 18495291
Semiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometer scale, and are emerging as a new class of fluorescent labels for biology and medicine. In comparison with organic dyes and fluorescent proteins, they have unique optical and electronic properties, with size-tunable light emission, superior signal brightness, resistance to photobleaching, and broad absorption spectra for simultaneous excitation of multiple fluorescence colors. QDs also provide a versatile nanoscale scaffold for designing multifunctional nanoparticles with both imaging and therapeutic functions. When linked with targeting ligands such as antibodies, peptides or small molecules, QDs can be used to target tumor biomarkers as well as tumor vasculatures with high affinity and specificity. Here we discuss the synthesis and development of state-of-the-art QD probes and their use for molecular and cellular imaging. We also examine key issues for in vivo imaging and therapy, such as nanoparticle biodistribution, pharmacokinetics, and toxicology.
Journal of Medical Case Reports. 2008 | Pubmed ID: 18505563
This report describes a rare complication of colonoscopy and reviews the literature with regard to other rare causes of acute abdominal presentations following colonoscopy.
Estimation of Neuronal Numbers in Rat Hippocampus Following Neonatal Amphetamine Exposure: a Stereology Study
Neurotoxicology and Teratology. Nov-Dec, 2008 | Pubmed ID: 18558471
In this study, the effects of amphetamine exposure during a portion of the brain growth spurt on the total number of hippocampal pyramidal cells (CA1/CA3 subregions) and the granule cells (dentate gyrus) were examined in both neonatal and adult rats. Intragastric intubation was used to administer 5, 15 or 25 mg/kg/day of amphetamine to Sprague-Dawley rat pups from PDs 4-9. Unbiased stereology was used to estimate the total number of cells present within each hippocampal subregion at both PD 9 and PD 68. The results indicated that neonatal amphetamine exposure did not alter the cell number, the reference volume or the density in any of the hippocampal subregions assessed, regardless of age. However, amphetamine significantly altered the rate of neuronal incorporation in both the hippocampal CA3 subregion and the dentate gyrus, and this effect appeared to be dose-related with the most robust effect observed in the highest amphetamine dose. While these findings did not demonstrate significant injurious effects of neonatal amphetamine treatment on the number of hippocampal neurons, these data suggest that amphetamine may interfere with proper hippocampal development. Future studies employing more sensitive measurements or exposing amphetamine during an alternate period of development may provide more information regarding amphetamine-mediated developmental neurotoxicity.
Nature Chemical Biology. Aug, 2008 | Pubmed ID: 18622389
Bioactive compounds are widely used to modulate protein function and can serve as important leads for drug development. Identifying the in vivo targets of these compounds remains a challenge. Using yeast, we integrated three genome-wide gene-dosage assays to measure the effect of small molecules in vivo. A single TAG microarray was used to resolve the fitness of strains derived from pools of (i) homozygous deletion mutants, (ii) heterozygous deletion mutants and (iii) genomic library transformants. We demonstrated, with eight diverse reference compounds, that integration of these three chemogenomic profiles improves the sensitivity and specificity of small-molecule target identification. We further dissected the mechanism of action of two protein phosphatase inhibitors and in the process developed a framework for the rational design of multidrug combinations to sensitize cells with specific genotypes more effectively. Finally, we applied this platform to 188 novel synthetic chemical compounds and identified both potential targets and structure-activity relationships.
Oxidative Quenching and Degradation of Polymer-encapsulated Quantum Dots: New Insights into the Long-term Fate and Toxicity of Nanocrystals in Vivo
Journal of the American Chemical Society. Aug, 2008 | Pubmed ID: 18652463
We report quenching and chemical degradation of polymer-coated quantum dots by reactive oxygen species (ROS), a group of oxygen-containing molecules that are produced by cellular metabolism and are involved in both normal physiological and disease processes such as oxidative signaling, cancer, and atherosclerosis. A major new finding is that hypochlorous acid (HOCl) in its neutral form is especially potent in degrading encapsulated QDs, due to its small size, neutral charge, long half-life, and fast reaction kinetics under physiologic conditions. Thus, small and neutral molecules such as HOCl and hydrogen peroxide (H2O2) are believed to diffuse across the polymer coating layer, leading to chemical oxidation of sulfur or selenium atoms on the QD surface. This "etching" process first generates lattice structural defects (which cause fluorescence quenching) and then produces soluble metal (e.g., cadmium and zinc) and chalcogenide (e.g., sulfur and selenium) species. We also find that significant fluorescence quenching occurs before QD dissolution and that localized surface defects can be repaired or "annealed" by UV light illumination. These results have important implications regarding the long-term fate and potential toxicity of semiconductor nanocrystals in vivo.
Journal of the American Chemical Society. Aug, 2008 | Pubmed ID: 18680294
We report a new strategy to minimize the hydrodynamic size of quantum dots (QDs) and to overcome their colloidal stability and photobleaching problems based on the use of multifunctional and multidentate polymer ligands. A novel finding is that a balanced composition of thiol (-SH) and amine (-NH 2) coordinating groups grafted to a linear polymer chain leads to highly compact nanocrystals with exceptional colloidal stability, a strong resistance to photobleaching, and high fluorescence quantum yields. In contrast to the standing brushlike conformation of PEGylated dihydrolipoic acid molecules, mutlidentate polymer ligands can wrap around the QDs in a closed "loops-and-trains" conformation. This structure is highly stable thermodynamically and is responsible for the excellent colloidal and optical properties. We have optimized this process for the preparation of ultrastable CdTe nanocrystals and have found the strategy to be broadly applicable to a wide range of nanocrystalline materials and heterostructures. This work has led to a new generation of bright and stable QDs with small hydrodynamic diameters between 5.6 and 9.7 nm with tunable fluorescence emission from the visible (515 nm) to the near-infrared (720 nm). These QDs are well suited for molecular and cellular imaging applications in which the nanoparticle hydrodynamic size must be minimized.
One-pot Synthesis, Encapsulation, and Solubilization of Size-tuned Quantum Dots with Amphiphilic Multidentate Ligands
Journal of the American Chemical Society. Oct, 2008 | Pubmed ID: 18774812
We report one-pot synthesis, encapsulation, and solubilization of high-quality quantum dots (QDs) based on the use of amphiphilic and multidentate polymer ligands. In this "all-in-one" procedure, the resulting QDs are first capped by the multidentate ligand and are then spontaneously encapsulated and solubilized by a second layer of the same multidentate polymer upon exposure to water. In addition to providing better control of nanocrystal nucleation and growth kinetics (including resistance to Ostwald ripening), this procedure allows for in situ growth of an inorganic passivating shell on the nanocrystal core, enabling one-pot synthesis of both type-I and type-II core-shell QDs with tunable light emission from visible to near-infrared wavelengths.
Nanocrystal Synthesis in an Amphibious Bath: Spontaneous Generation of Hydrophilic and Hydrophobic Surface Coatings
Angewandte Chemie (International Ed. in English). 2008 | Pubmed ID: 19016290
Nature Nanotechnology. Jan, 2009 | Pubmed ID: 19119277
The production of functional molecular architectures through self-assembly is commonplace in biology, but despite advances, it is still a major challenge to achieve similar complexity in the laboratory. Self-assembled structures that are reproducible and virtually defect free are of interest for applications in three-dimensional cell culture, templating, biosensing and supramolecular electronics. Here, we report the use of reversible enzyme-catalysed reactions to drive self-assembly. In this approach, the self-assembly of aromatic short peptide derivatives provides a driving force that enables a protease enzyme to produce building blocks in a reversible and spatially confined manner. We demonstrate that this system combines three features: (i) self-correction--fully reversible self-assembly under thermodynamic control; (ii) component-selection--the ability to amplify the most stable molecular self-assembly structures in dynamic combinatorial libraries; and (iii) spatiotemporal confinement of nucleation and structure growth. Enzyme-assisted self-assembly therefore provides control in bottom-up fabrication of nanomaterials that could ultimately lead to functional nanostructures with enhanced complexities and fewer defects.
Nature Nanotechnology. Jan, 2009 | Pubmed ID: 19119284
Strain can have a large influence on the properties of materials at the nanoscale. The effect of lattice strain on semiconductor devices has been widely studied, but its influence on colloidal semiconductor nanocrystals is still poorly understood. Here we show that the epitaxial deposition of a compressive shell (ZnS, ZnSe, ZnTe, CdS or CdSe) onto a soft nanocrystalline core (CdTe) to form a lattice-mismatched quantum dot can dramatically change the conduction and valence band energies of both the core and the shell. In particular, standard type-I quantum-dot behaviour is replaced by type-II behaviour, which is characterized by spatial separation of electrons and holes, extended excited-state lifetimes and giant spectral shifts. Moreover, the strain induced by the lattice mismatch can be used to tune the light emission--which displays narrow linewidths and high quantum yields--across the visible and near-infrared part of the spectrum (500-1,050 nm). Lattice-mismatched core-shell quantum dots are expected to have applications in solar energy conversion, multicolour biomedical imaging and super-resolution optical microscopy.
Neurobiology of Learning and Memory. Mar, 2009 | Pubmed ID: 19146964
Previous studies linking amphetamine use during pregnancy to changes in the behavioral development of affected infants have greatly increased society's level of concern regarding amphetamine use by women of reproductive age. The aim of this study was to investigate whether exposure to d-amphetamine sulfate during the brain growth spurt, the most dynamic period of brain development, alters hippocampus-mediated behaviors during both pre-adolescence and young adulthood. Sprague-Dawley rat pups were intragastrically administered a milk formula containing 0, 5, 15 or 25 mg/kg/day of amphetamine from postnatal day (PD) 4-9. Following weaning, the effects of neonatal amphetamine exposure on hippocampus-mediated behaviors were assessed using the open-field, the water maze, and the conditioned taste aversion behavioral tasks. Results from these behavioral tests revealed that while amphetamine exposure during the brain growth spurt alters behaviors in open-field testing, it does not interfere with performance in either the water maze or the conditioned taste aversion paradigm. These results offer speculation that the effects of neonatal amphetamine exposure on hippocampus-mediated behaviors may be related to interactions between the "temporal" (time of drug exposure) and "regional" (different regions of the hippocampus) vulnerability issues.
An Ultra-clean Technique for Accurately Analysing Pb Isotopes and Heavy Metals at High Spatial Resolution in Ice Cores with Sub-pg G(-1) Pb Concentrations
Analytica Chimica Acta. Feb, 2009 | Pubmed ID: 19185125
Measurements of Pb isotope ratios in ice containing sub-pg g(-1) concentrations are easily compromised by contamination, particularly where limited sample is available. Improved techniques are essential if Antarctic ice cores are to be analysed with sufficient spatial resolution to reveal seasonal variations due to climate. This was achieved here by using stainless steel chisels and saws and strict protocols in an ultra-clean cold room to decontaminate and section ice cores. Artificial ice cores, prepared from high purity water were used to develop and refine the procedures and quantify blanks. Ba and In, two other important elements present at pg g(-1) and fg g(-1) concentrations in Polar ice, were also measured. The final blank amounted to 0.2+/-0.2 pg of Pb with (206)Pb/(207)Pb and (208)Pb/(207)Pb ratios of 1.16+/-0.12 and 2.35+/-0.16, respectively, 1.5+/-0.4 pg of Ba and 0.6+/-2.0 fg of In, most of which probably originates from abrasion of the steel saws by the ice. The procedure was demonstrated on a Holocene Antarctic ice core section and was shown to contribute blanks of only approximately 5%, approximately 14% and approximately 0.8% to monthly resolved samples with respective Pb, Ba and In concentrations of 0.12 pg g(-1), 0.3 pg g(-1) and 2.3 fg g(-1). Uncertainties in the Pb isotopic ratio measurements were degraded by only approximately 0.2%.
Nature Protocols. 2009 | Pubmed ID: 19197268
Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.
Biomaterials. May, 2009 | Pubmed ID: 19201459
We report here the design of a biomimetic nanofibrous hydrogel as a 3D-scaffold for anchorage-dependent cells. The peptide-based bioactive hydrogel is formed through molecular self-assembly and the building blocks are a mixture of two aromatic short peptide derivatives: Fmoc-FF (Fluorenylmethoxycarbonyl-diphenylalanine) and Fmoc-RGD (arginine-glycine-aspartate) as the simplest self-assembling moieties reported so far for the construction of small-molecule-based bioactive hydrogels. This hydrogel provides a highly hydrated, stiff and nanofibrous hydrogel network that uniquely presents bioactive ligands at the fibre surface; therefore it mimics certain essential features of the extracellular matrix. The RGD sequence as part of the Fmoc-RGD building block plays a dual role of a structural component and a biological ligand. Spectroscopic and imaging analysis using CD, FTIR, fluorescence, TEM and AFM confirmed that FF and RGD peptide sequences self-assemble into beta-sheets interlocked by pi-pi stacking of the Fmoc groups. This generates the cylindrical nanofibres interwoven within the hydrogel with the presence of RGDs in tunable densities on the fibre surfaces. This rapid gelling material was observed to promote adhesion of encapsulated dermal fibroblasts through specific RGD-integrin binding, with subsequent cell spreading and proliferation; therefore it may offer an economical model scaffold to 3D-culture other anchorage-dependent cells for in-vitro tissue regeneration.
The Journal of Allergy and Clinical Immunology. Mar, 2009 | Pubmed ID: 19281902
The physician managing work-related asthma (WRA) assumes many roles. The first is to confirm an accurate diagnosis, recognizing that WRA has multiple phenotypes, including sensitizer-induced occupational asthma (OA) caused by high-molecular-weight (HMW) proteins or low-molecular-weight (LMW) chemicals; irritant-induced asthma; and work-exacerbated asthma. Pharmacotherapy for WRA is identical to nonwork-related asthma and should be guided by current asthma guidelines emphasizing control of both asthma impairment and risk domains. It is well established that the majority of workers diagnosed with OA caused by sensitizers experience persistent asthma after leaving the workplace. However, the long-term risk of persistent unremitting asthma can be prevented in a minority of cases, particularly with OA caused by LMW sensitizers, by establishing an early diagnosis of OA and reducing or eliminating exposure. The physician consultant may advise employers on workplace interventions needed to minimize effectively an affected employee's exposure to a causative agent or condition, and what measures are required to prevent new cases of WRA (ie, primary prevention). Although allergen immunotherapy has a putative role in treating and preventing WRA caused by HMW sensitizers, further study is needed.
Annals of the Royal College of Surgeons of England. Mar, 2009 | Pubmed ID: 19317932
Science (New York, N.Y.). Apr, 2009 | Pubmed ID: 19390044
The cause of a large increase of atmospheric methane concentration during the Younger Dryas-Preboreal abrupt climatic transition (approximately 11,600 years ago) has been the subject of much debate. The carbon-14 (14C) content of methane (14CH4) should distinguish between wetland and clathrate contributions to this increase. We present measurements of 14CH4 in glacial ice, targeting this transition, performed by using ice samples obtained from an ablation site in west Greenland. Measured 14CH4 values were higher than predicted under any scenario. Sample 14CH4 appears to be elevated by direct cosmogenic 14C production in ice. 14C of CO was measured to better understand this process and correct the sample 14CH4. Corrected results suggest that wetland sources were likely responsible for the majority of the Younger Dryas-Preboreal CH4 rise.
Biochemical Pharmacology. Oct, 2009 | Pubmed ID: 19523455
Tobacco dependence is the most preventable cause of death and is a chronic, relapsing disorder in which compulsive tobacco use persists despite known negative health consequences. All currently available cessation agents (nicotine, varenicline and bupropion) have limited efficacy and are associated with high relapse rates, revealing a need for more efficacious, alternative pharmacotherapies. The major alkaloid in tobacco, nicotine, activates nicotinic receptors (nAChRs) which increase brain extracellular dopamine producing nicotine reward leading to addiction. nAChRs are located primarily presynaptically and modulate synaptic activity by regulating neurotransmitter release. Subtype-selective nAChR antagonists that block reward-relevant mesocorticolimbic and nigrostriatal dopamine release induced by nicotine may offer advantages over current therapies. An innovative approach is to provide pharmacotherapies which are antagonists at nAChR subtypes mediating nicotine evoked dopamine release. In addition, providing multiple medications with a wider array of targets and mechanisms should provide more treatment options for individuals who are not responsive to the currently available pharmacotherapies. This review summarizes the currently available smoking cessation therapies and discusses emerging potential therapeutic approaches employing pharmacological agents which act as antagonists at nicotinic receptors.
Langmuir : the ACS Journal of Surfaces and Colloids. Aug, 2009 | Pubmed ID: 19537819
We report the effect of pH on the self-assembly process of Fmoc-diphenylalanine (Fmoc-FF) into fibrils consisting of antiparallel beta-sheets, and show that it results in two apparent pKa shifts of approximately 6.4 and approximately 2.2 pH units above the theoretical pKa (3.5). Using Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), wide angle X-ray scattering (WAXS), and oscillatory rheology, these two transitions were shown to coincide with significant structural changes. An entangled network of flexible fibrils forming a weak hydrogel dominates at high pH, while nongelling flat rigid ribbons form at intermediate pH values. Overall, this study provides further understanding of the self-assembly mechanism of aromatic short peptide derivatives.
Impact of Margin Status on Survival Following Pancreatoduodenectomy for Cancer: the Leeds Pathology Protocol (LEEPP)
HPB : the Official Journal of the International Hepato Pancreato Biliary Association. Feb, 2009 | Pubmed ID: 19590619
In a previous study we reported an 85% R1 rate for pancreatic cancer following the use of the rigorous, fully standardized Leeds Pathology Protocol (LEEPP). As this significantly exceeded R1 rates observed by others, we investigated the reproducibility of margin assessment using the LEEPP in a larger, prospective, observational cohort study and correlated clinicopathological data with survival.
Biochemical Society Transactions. Aug, 2009 | Pubmed ID: 19614571
In the present paper, we report on enzyme-initiated self-assembly of Fmoc (fluoren-9-ylmethoxycarbonyl)-tyrosine hydrogels by enzymatic dephosphorylation under physiological conditions and provide evidence for the ability to control the modulus. Upon enzyme action, a self-assembling network of interconnecting fibres is formed, observed by cryo-SEM (scanning electron microscopy) and TEM (transmission electron microscopy). The concentration of alkaline phosphatase added to the Fmoc-tyrosine phosphate ester precursor solution had a direct effect on the gelation time, mechanical properties and molecular arrangements as determined through oscillatory rheology, fluorescence spectroscopy and CD spectroscopy. This highly tuneable cost-effective gel system may have applications in three-dimensional cell culture.
Genome Research. Oct, 2009 | Pubmed ID: 19622793
Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or "Bar-seq," outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that approximately 20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.
The Novel Nicotinic Receptor Antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium Dibromide (bPiDDB), Inhibits Nicotine-evoked [(3)H]norepinephrine Overflow from Rat Hippocampal Slices
Biochemical Pharmacology. Oct, 2009 | Pubmed ID: 19631612
Smoking is a significant health concern and strongly correlated with clinical depression. Depression is associated with decreased extracellular NE concentrations in brain. Smokers may be self-medicating and alleviating their depression through nicotine stimulated norepinephrine (NE) release. Several antidepressants inhibit NE transporter (NET) function, thereby augmenting extracellular NE concentrations. Antidepressants, such as bupropion, also inhibit nicotinic receptor (nAChR) function. The current study determined if a recently discovered novel nAChR antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), inhibits nicotine-evoked NE release from superfused rat hippocampal slices. Previous studies determined that bPiDDB potently (IC(50)=2 nM) inhibits nicotine-evoked striatal [(3)H]dopamine (DA) release in vitro, nicotine-evoked DA release in nucleus accumbens in vivo, and nicotine self-administration in rats. In the current study, nicotine stimulated [(3)H]NE release from rat hippocampal slices (EC(50)=50 microM). bPiDDB inhibited (IC(50)=430 nM; I(max)=90%) [(3)H]NE release evoked by 30 microM nicotine. For comparison, the nonselective nAChR antagonist, mecamylamine, and the alpha7 antagonist, methyllycaconitine, also inhibited nicotine-evoked [(3)H]NE release (IC(50)=31 and 275 nM, respectively; I(max)=91% and 72%, respectively). Inhibition by bPiDDB and mecamylamine was not overcome by increasing nicotine concentrations; Schild regression slope was different from unity, consistent with allosteric inhibition. Thus, bPiDDB was 200-fold more potent inhibiting nAChRs mediating nicotine-evoked [(3)H]DA release from striatum than those mediating nicotine-evoked [(3)H]NE release from hippocampus.
Disordered Macrophage Cytokine Secretion Underlies Impaired Acute Inflammation and Bacterial Clearance in Crohn's Disease
The Journal of Experimental Medicine. Aug, 2009 | Pubmed ID: 19652016
The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of (111)In-labeled neutrophils at these sites and clearance of (32)P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.
Immunology. Oct, 2009 | Pubmed ID: 19740382
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical for phagocyte anti-microbial activity and plays a major role in innate immunity. Defects in genes coding for components of the NADPH oxidase enzyme system are responsible for chronic granulomatous disease (CGD), a rare primary neutrophil immunodeficiency associated with recurrent, life-threatening bacterial and fungal infections. Microbial killing and digestion within the neutrophil phagosomal compartment are defective in these patients. NADPH oxidase activity is also crucial for optimal macrophage and dendritic cell function and has recently been implicated in both cross-presentation and T-cell priming. We present evidence of impaired macrophage function in CGD, with attenuated pro-inflammatory cytokine and increased interleukin-10 secretion following bacterial stimulation. These results highlight additional abnormalities in macrophage function associated with CGD and the importance of NADPH oxidase activity in immunity.
BMC Genomics. 2009 | Pubmed ID: 19825181
Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density), but this increase in resolution can compromise sensitivity.
Nature Nanotechnology. Nov, 2009 | Pubmed ID: 19898521
Diminished Macrophage Apoptosis and Reactive Oxygen Species Generation After Phorbol Ester Stimulation in Crohn's Disease
PloS One. 2009 | Pubmed ID: 19907654
Crohn's Disease (CD) is a chronic relapsing disorder characterized by granulomatous inflammation of the gastrointestinal tract. Although its pathogenesis is complex, we have recently shown that CD patients have a systemic defect in macrophage function, which results in the defective clearance of bacteria from inflammatory sites.
Proton-Resistant Quantum Dots: Stability in Gastrointestinal Fluids and Implications for Oral Delivery of Nanoparticle Agents
Nano Research. Jun, 2009 | Pubmed ID: 20379372
Semiconductor quantum dots (QDs) have shown great promise as fluorescent probes for molecular, cellular and in-vivo imaging. However, the fluorescence of traditional polymer-encapsulated QDs is often quenched by proton-induced etching in acidic environments. This is a major problem for QD applications in the gastrointestinal tract because the gastric (stomach) environment is strongly acidic (pH 1-2). Here we report the use of proton-resistant surface coatings to stabilize QD fluorescence under acidic conditions. Using both hyperbranched polyethylenimine (PEI) and its polyethylene glycol derivative (PEG grafted PEI), we show that the fluorescence of core-shell CdSe/CdS/ZnS QDs is effectively protected from quenching in simulated gastric fluids. In comparison, amphiphilic lipid or polymer coatings provide no protection under similarly acidic conditions. The proton-resistant QDs are found to cause moderate membrane damage to cultured epithelial cells, but PEGylation (PEG grafting) can be used to reduce cellular toxicity and to improved nanoparticle stability.
Langmuir : the ACS Journal of Surfaces and Colloids. Jul, 2010 | Pubmed ID: 20408518
We investigate the possibility of using the protease thermolysin to drive the synthesis and gelation of ionic-complementary peptides from nongelling precursors. In this system, short peptide fragments are continuously interconverted to form a dynamic peptide library, which eventually favors synthesis of peptides that are thermodynamically stabilized by molecular self-assembly. Thermolysin was added at a fixed concentration (0.3 mg mL(-1)) to solutions (0-300 mg mL(-1)) of the short tetrapeptide FEFK. Initially, the protease partially hydrolyzed the tetrapeptide into dipeptides in all samples. Subsequently, longer peptide sequences were found to form through reverse-hydrolysis. The stability of the different sequences was found to be dependent on their self-assembling properties. The sequences that self-assembled into antiparallel beta-sheet rich fibers became the stable products for the reverse hydrolysis reaction, while the others formed were unstable and disappeared with increasing incubation time. Ultimately, the main product of the system was octapeptide, which suggests that it represents the thermodynamically favored product of this dynamic library. Its concentration dictated the gelation behavior of the sample, and gels with moduli up to 25 kPa where obtained depending on the initial concentration of tetrapeptide.
Nucleic Acids Research. Jul, 2010 | Pubmed ID: 20460461
Next-generation sequencing has proven an extremely effective technology for molecular counting applications where the number of sequence reads provides a digital readout for RNA-seq, ChIP-seq, Tn-seq and other applications. The extremely large number of sequence reads that can be obtained per run permits the analysis of increasingly complex samples. For lower complexity samples, however, a point of diminishing returns is reached when the number of counts per sequence results in oversampling with no increase in data quality. A solution to making next-generation sequencing as efficient and affordable as possible involves assaying multiple samples in a single run. Here, we report the successful 96-plexing of complex pools of DNA barcoded yeast mutants and show that such 'Bar-seq' assessment of these samples is comparable with data provided by barcode microarrays, the current benchmark for this application. The cost reduction and increased throughput permitted by highly multiplexed sequencing will greatly expand the scope of chemogenomics assays and, equally importantly, the approach is suitable for other sequence counting applications that could benefit from massive parallelization.
Pharmacology & Therapeutics. Aug, 2010 | Pubmed ID: 20546776
Over the past decade, the development and application of chemical genomic assays using the model organism Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of known drugs and novel small molecules in vivo. These assays identify drug target candidates, genes involved in buffering drug target pathways and also help to define the general cellular response to small molecules. In this review, we examine current yeast chemical genomic assays and summarize the potential applications of each approach.
Expert Review of Clinical Immunology. Jul, 2010 | Pubmed ID: 20594132
The pathogenesis of Crohn's disease (CD) has widely been regarded as the consequence of a dysregulated T-cell-mediated response to intestinal microbes, and the majority of the worldwide research effort has focused on characterizing and treating the chronic inflammatory phase of the disease. However, recent molecular biological and clinical investigations indicate that CD is actually a primary immunodeficiency. At first counter-intuitive, the apparent paradox of a pathogenic innate immune defect can be linked mechanistically to the granulomatous chronic inflammation characteristic of the disease. Genome-wide association studies have corroborated the involvement of innate immune dysfunction in the pathogenesis of CD, but less than 20% of the heritable risk is accounted for. By contrast, in vitro and in vivo stimulation of the immune system has highlighted novel areas of interest that may lead to the development of targeted therapeutic and diagnostic tools.
An Investigation of the Conductivity of Peptide Nanotube Networks Prepared by Enzyme-triggered Self-assembly
Nanoscale. Jun, 2010 | Pubmed ID: 20648293
We demonstrate that nanotubular networks formed by enzyme-triggered self-assembly of Fmoc-L3 (9-fluorenylmethoxycarbonyl-tri-leucine) show significant charge transport. FT-IR, fluorescence spectroscopy and wide angle X-ray scattering (WAXS) data confirm formation of beta-sheets that are locked together viapi-stacking interactions. Molecular dynamics simulations confirmed the pi-pi stacking distance between fluorenyl groups to be 3.6-3.8 A. Impedance spectroscopy demonstrated that the nanotubular xerogel networks possess minimum sheet resistances of 0.1 MOmega/sq in air and 500 MOmega/sq in vacuum (pressure: 1.03 mbar) at room temperature, with the conductivity scaling linearly with the mass of peptide in the network. These materials may provide a platform to interface biological components with electronics.
Science (New York, N.Y.). Sep, 2010 | Pubmed ID: 20813934
The Biochemist. Jun, 2010 | Pubmed ID: 20824157
Semiconductor quantum dots (QDs) are tiny light-emitting particles that have emerged as a new class of fluorescent labels for biology and medicine. Compared with traditional fluorescent probes, QDs have unique optical and electronic properties such as size-tuneable light emission, narrow and symmetric emission spectra, and broad absorption spectra that enable the simultaneous excitation of multiple fluorescence colours.
Inflammatory Bowel Diseases. Nov, 2010 | Pubmed ID: 20848466
Crohn's disease and ulcerative colitis are idiopathic chronic inflammatory diseases that primarily affect the gastrointestinal tract. The underlying causes remain poorly understood, but there is a growing body of evidence advocating a likely primary pathogenic role for immunodeficiency in the development of Crohn's lesions. Concordantly, a number of congenital immunodeficiencies disrupting the cellular innate immune system strongly predispose to noninfectious, Crohn's-like inflammatory bowel disease. There are case reports and series suggesting that the same may be true for some of the congenital adaptive and complement immunodeficiencies. This review considers and critiques these potential associations.
JOP : Journal of the Pancreas. 2010 | Pubmed ID: 21068501
Autoimmune pancreatitis is a rare benign disorder that can be confused with pancreatic cancer and the treatment pathway differs dramatically.
Life Sciences. Mar, 2010 | Pubmed ID: 20153755
Restricted somatic growth during fetal or early postnatal periods has been suggested to serve as a predictive indicator for neuroanatomical changes and behavioral impairments during adulthood. Here, the effects of d-amphetamine sulfate (AMPH) exposure during the brain growth spurt period on this potential indicator were evaluated.
Survival After Pancreaticoduodenectomy is Not Improved by Extending Resections to Achieve Negative Margins
Annals of Surgery. Apr, 2010 | Pubmed ID: 20224357
Repeated Nicotine Administration Robustly Increases BPiDDB Inhibitory Potency at Alpha6beta2-containing Nicotinic Receptors Mediating Nicotine-evoked Dopamine Release
Biochemical Pharmacology. Aug, 2010 | Pubmed ID: 20346923
The novel nicotinic receptor (nAChR) antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), and its chemically reduced analog, r-bPiDDB, potently inhibit nicotine-evoked dopamine (DA) release from rat striatal slices. Since tobacco smokers self-administer nicotine repeatedly, animal models incorporating repeated nicotine treatment allow for mechanistic evaluation of therapeutic candidates following neuroadaptive changes. The current study determined the ability of bPiDDB, r-bPiDDB and alpha-conotoxin MII (alpha-CtxMII), a peptide antagonist selective for alpha6beta2-containing nAChRs, to inhibit nicotine-evoked [(3)H]DA release from striatal slices from rats repeatedly administered nicotine (0.4mg/kg for 10 days) or saline (control). Concomitant exposure to maximally effective concentrations of r-bPiDDB (1nM) and alpha-CtxMII (1nM) resulted in inhibition of nicotine-evoked [(3)H]DA release no greater than that produced by either antagonist alone, suggesting that r-bPiDDB inhibits alpha6beta2-containing nAChRs. Repeated nicotine treatment increased locomotor activity, demonstrating behavioral sensitization. Concentration-response curves for nicotine-evoked [(3)H]DA release were not different between nicotine-treated and control groups. Maximal inhibition for alpha-CtxMII was greater following repeated nicotine compared to control (I(max)=90% vs. 62%), with no change in potency. bPiDDB was 3-orders of magnitude more potent in inhibiting nicotine-evoked [(3)H]DA release in nicotine-treated rats compared to control rats (IC(50)=5pM vs. 6nM), with no change in maximal inhibition. Neither a shift to the left in the concentration response nor a change in maximal inhibition was observed for r-bPiDDB following repeated nicotine. Thus, repeated nicotine treatment may differentially regulate the stoichiometry, conformation and/or composition of alpha6beta2-containing nAChRs mediating nicotine-evoked striatal DA release. Therefore, bPiDDB and r-bPiDDB appear to target different alpha6beta2-containing nAChR subtypes.
Delayed Resolution of Acute Inflammation in Ulcerative Colitis is Associated with Elevated Cytokine Release Downstream of TLR4
PloS One. 2010 | Pubmed ID: 20360984
Ulcerative colitis (UC) is widely viewed as a leukocyte-mediated disorder. Although strong evidence implicates an exuberant response to microbial components in its pathogenesis, no intrinsic immune defect has been identified and the underlying pathogenic mechanisms remain obscure.
Accounts of Chemical Research. Feb, 2010 | Pubmed ID: 19827808
Semiconductor nanocrystals are tiny light-emitting particles on the nanometer scale. Researchers have studied these particles intensely and have developed them for broad applications in solar energy conversion, optoelectronic devices, molecular and cellular imaging, and ultrasensitive detection. A major feature of semiconductor nanocrystals is the quantum confinement effect, which leads to spatial enclosure of the electronic charge carriers within the nanocrystal. Because of this effect, researchers can use the size and shape of these "artificial atoms" to widely and precisely tune the energy of discrete electronic energy states and optical transitions. As a result, researchers can tune the light emission from these particles throughout the ultraviolet, visible, near-infrared, and mid-infrared spectral ranges. These particles also span the transition between small molecules and bulk crystals, instilling novel optical properties such as carrier multiplication, single-particle blinking, and spectral diffusion. In addition, semiconductor nanocrystals provide a versatile building block for developing complex nanostructures such as superlattices and multimodal agents for molecular imaging and targeted therapy. In this Account, we discuss recent advances in the understanding of the atomic structure and optical properties of semiconductor nanocrystals. We also discuss new strategies for band gap and electronic wave function engineering to control the location of charge carriers. New methodologies such as alloying, doping, strain-tuning, and band-edge warping will likely play key roles in the further development of these particles for optoelectronic and biomedical applications.
Bright and Compact Alloyed Quantum Dots with Broadly Tunable Near-infrared Absorption and Fluorescence Spectra Through Mercury Cation Exchange
Journal of the American Chemical Society. Jan, 2011 | Pubmed ID: 21142154
We report a new strategy based on mercury cation exchange in nonpolar solvents to prepare bright and compact alloyed quantum dots (QDs) (Hg(x)Cd(1-x)E, where E = Te, Se, or S) with equalized particle size and broadly tunable absorption and fluorescence emission in the near-infrared. The main rationale is that cubic CdE and HgE have nearly identical lattice constants but very different band gap energies and electron/hole masses. Thus, replacement of Cd(2+) by Hg(2+) in CdTe nanocrystals does not change the particle size, but it greatly alters the band gap energy. After capping with a multilayer shell and solubilization with a multidentate ligand, this class of cation-exchanged QDs are compact (6.5 nm nanocrystal size and 10 nm hydrodynamic diameter) and very bright (60-80% quantum yield), with narrow and symmetric fluorescence spectra tunable across the wavelength range from 700 to 1150 nm.
BPiDI: a Novel Selective α6β2* Nicotinic Receptor Antagonist and Preclinical Candidate Treatment for Nicotine Abuse
British Journal of Pharmacology. May, 2011 | Pubmed ID: 21232049
Nicotinic acetylcholine receptors (nAChRs) containing α6β2 subunits expressed by dopamine neurons regulate nicotine-evoked dopamine release. Previous results show that the α6β2* nAChR antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB) inhibits nicotine-evoked dopamine release from dorsal striatum and decreases nicotine self-administration in rats. However, overt toxicity emerged with repeated bPiDDB treatment. The current study evaluated the preclinical pharmacology of a bPiDDB analogue.
Is Response to Allergen Immunotherapy a Good Phenotypic Marker for Differentiating Between Allergic Rhinitis and Mixed Rhinitis?
Allergy and Asthma Proceedings : the Official Journal of Regional and State Allergy Societies. Jan-Feb, 2011 | Pubmed ID: 21262098
This study was designed to determine if patients with allergic rhinitis (AR) and mixed rhinitis (MR) subtypes have similar treatment outcomes after a full course of allergen immunotherapy (AIT). A historical pre- and posttreatment study design was used to identify all AR patients in a large allergy practice who started AIT in 2001 and completed at least a 3-year course. One reviewer compiled data through chart review using a standardized form that recorded allergic and nonallergic irritant triggers, symptoms, and medication requirements before and after completion of ≥3 years of AIT. Other than age, there were no significant differences in baseline demographic characteristics, allergic triggers, or rhinitis symptoms between the AR and MR groups. Both groups experienced reduced episodes of sinusitis after AIT. Although both groups also had a significant decrease in the mean number of rhinitis medications after AIT (p < 0.001), the reduction in number of medications was significantly greater for the AR versus the MR group (1.24 ± 1.09 versus 2.09 ± 1.55; p = 0.0023). AIT is a very effective treatment for AR and MR patients. However, these data support the classification of MR as a distinct rhinitis phenotype because MR patients required significantly more medications after a full course of AIT to control their nonallergic-induced symptoms.
Pancreas. Mar, 2011 | Pubmed ID: 21311313
Indolizidine (-)-235B' and Related Structural Analogs: Discovery of Nicotinic Receptor Antagonists That Inhibit Nicotine-evoked [3H]dopamine Release
European Journal of Pharmacology. May, 2011 | Pubmed ID: 21371454
Although several therapeutic agents are available to aid in tobacco smoking cessation, relapse rates continue to be high, warranting the development of alternative pharmacotherapies. Nicotine-evoked dopamine release from its presynaptic terminals in the central nervous system leads to reward which maintains continued tobacco use. The ability of indolizidine (-)-235B' and a sub-library of structurally related analogs to inhibit nicotine-evoked [(3)H]dopamine release from rat striatal slices was determined in the current study. Indolizidine (-)-235B' inhibited nicotine-evoked [(3)H]dopamine release in a concentration-dependent manner (IC(50)=42 nM, I(max)=55%). Compound (-)-237D, the double bond-reduced analog, afforded the greatest inhibitory potency (IC(50)=0.18 nM, I(max)=76%), and was 233-fold more potent than indolizidine (-)-235B'. The des-8-methyl aza-analog of indolizidine (-)-235B', ZZ-272, also inhibited nicotine-evoked [(3)H]dopamine release (IC(50)=413 nM, I(max)=59%). Concomitant exposure to maximally effective concentrations of indolizidine (-)-235B', ZZ-272 or (-)-237D with a maximally effective concentration of α-conotoxin MII, a selective antagonist for α6β2-containing nicotinic receptors, resulted in inhibition of nicotine-evoked [(3)H]dopamine release no greater than that produced by each compound alone. The latter results suggest that indolizidine (-)-235B', (-)-237D, ZZ-272 and α-conotoxin MII inhibit the same α-conotoxin MII-sensitive nicotinic receptor subtypes. Thus, indolizidine (-)-235B' and its analogs act as antagonists of α6β2-nicotinic receptors and constitute a novel structural scaffold for the discovery of pharmacotherapies for smoking cessation.
G6PC3 Mutations Are Associated with a Major Defect of Glycosylation: a Novel Mechanism for Neutrophil Dysfunction
Glycobiology. Jul, 2011 | Pubmed ID: 21385794
Glucose-6-phosphatase, an enzyme localized in the endoplasmic reticulum (ER), catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. In humans, there are three differentially expressed glucose-6-phosphatase catabolic genes (G6PC1-3). Recently, it has been shown that mutations in the G6PC3 gene result in a syndrome associating congenital neutropenia and various organ malformations. The enzymatic function of G6PC3 is dependent on G6P transport into the ER, mediated by G6P translocase (G6PT). Mutations in the gene encoding G6PT result in glycogen storage disease type-1b (GSD-1b). Interestingly, GSD-1b patients exhibit a similar neutrophil dysfunction to that observed in G6PC3-deficient patients. To better understand the causes of neutrophil dysfunction in both diseases, we have studied the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of patients with G6PC3 and G6PT syndromes. Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments indicated hypo-glycosylation of gp91(phox), the electron-transporting component of the NADPH oxidase, in all of these patients. Rigorous mass spectrometric glycomic profiling showed that most of the complex-type antennae which characterize the neutrophil N-glycome of healthy individuals were severely truncated in the patients' neutrophils. A comparable truncation of the core 2 antenna of the O-glycans was also observed. This aberrant neutrophil glycosylation is predicted to have profound effects on the neutrophil function and merit designation of both syndromes as a new class of congenital disorders of glycosylation.
The Journal of Experimental Biology. May, 2011 | Pubmed ID: 21525316
The terrestrial slug Arion subfuscus secretes a glue that is a dilute gel with remarkable adhesive and cohesive strength. The function of this glue depends on metals, raising the possibility that metal-catalyzed oxidation plays a role. The extent and time course of protein oxidation was measured by immunoblotting to detect the resulting carbonyl groups. Several proteins, particularly one with a relative molecular mass (M(r)) of 165 x 10³, were heavily oxidized. Of the proteins known to distinguish the glue from non-adhesive mucus, only specific size variants were oxidized. The oxidation appears to occur within the first few seconds of secretion. Although carbonyls were detected by 2,4-dinitrophenylhydrazine (DNPH) in denatured proteins, they were not easily detected in the native state. The presence of reversible cross-links derived from carbonyls was tested for by treatment with sodium borohydride, which would reduce uncross-linked carbonyls to alcohols, but stabilize imine bonds formed by carbonyls and thus lead to less soluble complexes. Consistent with imine bond formation, sodium borohydride led to a 20-35% decrease in the amount of soluble protein with a M(r) of 40-165 (x 10³) without changing the carbonyl content per protein. In contrast, the nucleophile hydroxylamine, which would competitively disrupt imine bonds, increased protein solubility in the glue. Finally, the primary amine groups on a protein with a M(r) of 15 x 10³ were not accessible to acid anhydrides. The results suggest that cross-links between aldehydes and primary amines contribute to the cohesive strength of the glue.
BMC Genomics. 2011 | Pubmed ID: 21548937
Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.
Nature Biotechnology. Jun, 2011 | Pubmed ID: 21572441
Dosage suppression is a genetic interaction in which overproduction of one gene rescues a mutant phenotype of another gene. Although dosage suppression is known to map functional connections among genes, the extent to which it might illuminate global cellular functions is unclear. Here we analyze a network of interactions linking dosage suppressors to 437 essential genes in yeast. For 424 genes, we curated interactions from the literature. Analyses revealed that many dosage suppression interactions occur between functionally related genes and that the majority do not overlap with other types of genetic or physical interactions. To confirm the generality of these network properties, we experimentally identified dosage suppressors for 29 genes from pooled populations of temperature-sensitive mutant cells transformed with a high-copy molecular-barcoded open reading frame library, MoBY-ORF 2.0. We classified 87% of the 1,640 total interactions into four general types of suppression mechanisms, which provided insight into their relative frequencies. This work suggests that integrating the results of dosage suppression studies with other interaction networks could generate insights into the functional wiring diagram of a cell.
Digestive Diseases and Sciences. May, 2011 | Pubmed ID: 20936355
Neutrophils are a key part of the innate immune defence against microbes, using the respiratory burst (RB) to optimise killing and digestion. Previous studies of the neutrophil RB in Crohn's disease (CD) have yielded conflicting results.
Immunology and Allergy Clinics of North America. Nov, 2011 | Pubmed ID: 21978850
Much has been learned from epidemiologic studies conducted in the past 4 decades that can be directly applied to the management of workers affected with occupational asthma. Studies have provided information about host factors, environmental exposure, and occupational agents posing the highest risks for development of severe irreversible airway obstruction and asthma disability. Investigators have developed methods for screening workers at risk and novel interventions that may prevent new cases among exposed worker populations. Less is known about the natural history and chronic morbidity associated with work-aggravated asthma and irritant-induced asthma syndromes; more studies are needed in at-risk worker populations.
Musculoskeletal Care. Dec, 2011 | Pubmed ID: 22190538
PURPOSE: There has been a move to reduce and, indeed, stop postoperative appointments for uncomplicated surgical procedures within the National Health Service. The purpose of this study was to measure patient satisfaction with postoperative follow-up conducted by a hand therapist, with no routine postoperative follow-up by the surgeon. METHODS: A total of 124 patients were recruited over two years. Fifty patients post-simple trapeziectomy and 74 patients post-single-digit Dupuytren's fasciectomy were prospectively surveyed for their opinion on their postoperative care and whether or not they would have liked to be reviewed by the surgeon in a routine postoperative follow-up appointment. All patients included in this study had their operations performed by one surgeon in one hospital. All patients were reviewed by a hand therapist within two weeks of surgery. RESULTS: A total of 116 patients completed the study, of whom 106 patients (91%) were satisfied with their postoperative management and 99 patients (85%) did not want to be reviewed by the surgeon in a postoperative outpatient follow-up appointment. DISCUSSION: This study reflects the successful application of postoperative follow-up by a hand therapist. Copyright © 2011 John Wiley & Sons, Ltd.
Pancreatic Lesions in Von Hippel-Lindau Disease? A Systematic Review and Meta-synthesis of the Literature
Journal of Gastrointestinal Surgery : Official Journal of the Society for Surgery of the Alimentary Tract. Jul, 2012 | Pubmed ID: 22370733
von Hippel-Lindau (vHL) disease is a rare condition that leads to characteristic lesions within many different body systems. Pancreatic manifestations of vHL cover a wide spectrum of pathologies, and thus, accurate characterization and management is critical.
Covariation of Learning and "reasoning" Abilities in Mice: Evolutionary Conservation of the Operations of Intelligence
Journal of Experimental Psychology. Animal Behavior Processes. Apr, 2012 | Pubmed ID: 22428547
Contemporary descriptions of human intelligence hold that this trait influences a broad range of cognitive abilities, including learning, attention, and reasoning. Like humans, individual genetically heterogeneous mice express a "general" cognitive trait that influences performance across a diverse array of learning and attentional tasks, and it has been suggested that this trait is qualitatively and structurally analogous to general intelligence in humans. However, the hallmark of human intelligence is the ability to use various forms of "reasoning" to support solutions to novel problems. Here, we find that genetically heterogeneous mice are capable of solving problems that are nominally indicative of inductive and deductive forms of reasoning, and that individuals' capacity for reasoning covaries with more general learning abilities. Mice were characterized for their general learning ability as determined by their aggregate performance (derived from principal component analysis) across a battery of five diverse learning tasks. These animals were then assessed on prototypic tests indicative of deductive reasoning (inferring the meaning of a novel item by exclusion, i.e., "fast mapping") and inductive reasoning (execution of an efficient search strategy in a binary decision tree). The animals exhibited systematic abilities on each of these nominal reasoning tasks that were predicted by their aggregate performance on the battery of learning tasks. These results suggest that the coregulation of reasoning and general learning performance in genetically heterogeneous mice form a core cognitive trait that is analogous to human intelligence.
Defective Tumor Necrosis Factor Release from Crohn's Disease Macrophages in Response to Toll-like Receptor Activation: Relationship to Phenotype and Genome-wide Association Susceptibility Loci
Inflammatory Bowel Diseases. Mar, 2012 | Pubmed ID: 22434667
BACKGROUND: Recent work provides evidence of a failure of acute inflammation in Crohn's disease (CD), and suggests that the primary defect operates at the level of the macrophage and cytokine release. Here we extend the characterization of the innate immune defect in CD by investigating the macrophage response to Toll-like receptor (TLR) agonists and assess potential links between genome-wide association study (GWAS) susceptibility loci, disease phenotype, and therapeutic regimens on tumor necrosis factor α (TNF) release. METHODS: Peripheral blood-derived macrophages were cultured from control subjects and patients with CD, stimulated with TLR ligands, and the release of TNF measured. Genomic DNA was purified from blood and genotyped for 34 single nucleotide polymorphisms (SNPs) identified as being associated with CD by GWAS. RESULTS: All stimuli resulted in a reduction (32%-48%) in TNF release from macrophages derived from CD patients (n = 28-101) compared to those from healthy control (HC) subjects. All phenotypes demonstrated impaired TNF release, with the greatest defect in patients with colonic disease. There was no detectable relationship between the level of TNF released and the presence of GWAS susceptibility loci in CD patients. Reduced TNF levels were not influenced by age, gender, or use of aminosalicylate (5-ASA) medication. CONCLUSIONS: This study supports the hypothesis of defective proinflammatory cytokine secretion and an innate immunodeficiency in CD. Abnormal TNF secretion is evident downstream of multiple TLRs, affects all disease phenotypes, and is unrelated to 34 polymorphisms associated with CD by GWAS. (Inflamm Bowel Dis 2012;).
Hepatobiliary & Pancreatic Diseases International : HBPD INT. Apr, 2012 | Pubmed ID: 22484592
Pancreaticopleural fistula (PPF) are uncommon. Complex multidisciplinary treatment is required due to nutritional compromise and sepsis. This is the first description of long-term follow-up of patients with PPF.
Annals of Allergy, Asthma & Immunology : Official Publication of the American College of Allergy, Asthma, & Immunology. May, 2012 | Pubmed ID: 22541399
The incidence rate of asthma has increased in all age groups in the past 40 years. Asthma in older adults is underdiagnosed and undertreated, resulting in suboptimal asthma control.
Journal of Gastrointestinal Surgery : Official Journal of the Society for Surgery of the Alimentary Tract. Aug, 2012 | Pubmed ID: 22714746
Pancreatoduodenectomy (PD) has always been regarded as one of the most technically demanding abdominal procedures, even when carried out in high-volume centers by experienced surgeons. The reduction in higher surgical trainees working hours has led to reduced exposure, and consequently less experience in operative procedures. Furthermore, trainees have also become victims as health care systems striving for operating room efficiency, have attempted to reduce procedure duration by encouraging consultant led procedures at the expense of training. A strategy therefore needs to be developed to match the ability of the trainee with the complexity of the surgical procedure. As a PD can be deconstructed into a number of different steps, it may indeed be an ideal training operation for varying levels of ability.
Molecular BioSystems. Aug, 2012 | Pubmed ID: 22751784
Fungi rely on regulatory networks to coordinate sensing of environmental stress with initiation of responses crucial for survival. Antifungal drugs are a specific type of environmental stress with broad clinical relevance. Small molecules with antifungal activity are ubiquitous in the environment, and are produced by a myriad of microbes in competitive natural communities. The echinocandins are fungal fermentation products and the most recently developed class of antifungals, with those in clinical use being semisynthetic derivatives that target the fungal cell wall by inhibiting 1,3-β-D-glucan synthase. Recent studies implicate the protein phosphatase calcineurin as a key regulator of cellular stress responses required for fungal survival of echinocandin-induced cell wall stress. Pharmacological inhibition of calcineurin can be achieved using the natural product and immunosuppressive drug cyclosporin A, which inhibits calcineurin by binding to the immunophilin Cpr1. This drug-protein complex inhibits the interaction between the regulatory and catalytic subunits of calcineurin, an interaction necessary for calcineurin function. Here, we report on potent activity of cyclosporin A when combined with the echinocandin micafungin against the model yeast Saccharomyces cerevisiae that is independent of its known mechanism of action of calcineurin inhibition. This calcineurin-independent synergy does not involve any of the 12 immunophilins known in yeast, individually or in combination, and is not mediated by any of the multidrug transporters encoded or controlled by YOR1, SNQ2, PDR5, PDR10, PDR11, YCF1, PDR15, ADP1, VMR1, NFT1, BPT1, YBT1, YNR070w, YOL075c, AUS1, PDR12, PDR1 and/or PDR3. Genome-wide haploinsufficiency profiling (HIP) and homozygous deletion profiling (HOP) strongly implicate the cell wall biosynthesis and integrity pathways as being central to the calcineurin-independent activity of cyclosporin A. Thus, systems level chemical genomic approaches implicate key cellular pathways in a novel mechanism of antifungal drug synergy.
Methods in Molecular Biology (Clifton, N.J.). 2012 | Pubmed ID: 22821592
With the advent of next-generation sequencing (NGS) technology, methods previously developed for microarrays have been adapted for use by NGS. Here we describe in detail a protocol for Barcode analysis by sequencing (Bar-seq) to assess pooled competitive growth of individually barcoded yeast deletion mutants. This protocol has been optimized on two sequencing platforms: Illumina's Genome Analyzer IIx/HiSeq2000 and Life Technologies SOLiD3/5500. In addition, we provide guidelines for assessment of human knockdown cells using short-hairpin RNAs (shRNA) and an Illumina sequencing readout.
Nature. Jul, 2012 | Pubmed ID: 22837002
Current ice loss from the West Antarctic Ice Sheet (WAIS) accounts for about ten per cent of observed global sea-level rise. Losses are dominated by dynamic thinning, in which forcings by oceanic or atmospheric perturbations to the ice margin lead to an accelerated thinning of ice along the coastline. Although central to improving projections of future ice-sheet contributions to global sea-level rise, the incorporation of dynamic thinning into models has been restricted by lack of knowledge of basal topography and subglacial geology so that the rate and ultimate extent of potential WAIS retreat remains difficult to quantify. Here we report the discovery of a subglacial basin under Ferrigno Ice Stream up to 1.5 kilometres deep that connects the ice-sheet interior to the Bellingshausen Sea margin, and whose existence profoundly affects ice loss. We use a suite of ice-penetrating radar, magnetic and gravity measurements to propose a rift origin for the basin in association with the wider development of the West Antarctic rift system. The Ferrigno rift, overdeepened by glacial erosion, is a conduit which fed a major palaeo-ice stream on the adjacent continental shelf during glacial maxima. The palaeo-ice stream, in turn, eroded the 'Belgica' trough, which today routes warm open-ocean water back to the ice front to reinforce dynamic thinning. We show that dynamic thinning from both the Bellingshausen and Amundsen Sea region is being steered back to the ice-sheet interior along rift basins. We conclude that rift basins that cut across the WAIS margin can rapidly transmit coastally perturbed change inland, thereby promoting ice-sheet instability.
Nucleic Acids Research. Jan, 2012 | Pubmed ID: 22080555
The UCSC Archaeal Genome Browser (http://archaea.ucsc.edu) offers a graphical web-based resource for exploration and discovery within archaeal and other selected microbial genomes. By bringing together existing gene annotations, gene expression data, multiple-genome alignments, pre-computed sequence comparisons and other specialized analysis tracks, the genome browser is a powerful aggregator of varied genomic information. The genome browser environment maintains the current look-and-feel of the vertebrate UCSC Genome Browser, but also integrates archaeal and bacterial-specific tracks with a few graphic display enhancements. The browser currently contains 115 archaeal genomes, plus 31 genomes of viruses known to infect archaea. Some of the recently developed or enhanced tracks visualize data from published high-throughput RNA-sequencing studies, the NCBI Conserved Domain Database, sequences from pre-genome sequencing studies, predicted gene boundaries from three different protein gene prediction algorithms, tRNAscan-SE gene predictions with RNA secondary structures and CRISPR locus predictions. We have also developed a companion resource, the Archaeal COG Browser, to provide better search and display of arCOG gene function classifications, including their phylogenetic distribution among available archaeal genomes.