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1.  Biospecimens and Biorepositories for the Community Pathologist 
Pathologists have long served as custodians of human biospecimens collected for diagnostic purposes. Rapid advancements in diagnostic technologies require that pathologists change their practices to optimize patient care. The proper handling of biospecimens creates opportunities for pathologists to improve their diagnoses while assessing prognosis and treatment. In addition, the growing need for high-quality biorepositories represents an opportunity for community pathologists to strengthen their role within the health care team, ensuring that clinical care is not compromised while facilitating research. This article provides a resource to community pathologists learning how to create high-quality biorepositories and participating in emerging opportunities in the biorepository field. While a variety of topics are covered to provide breadth of information, the intent is to facilitate a level of understanding that permits community pathologists to make more informed choices in identifying how best their skills and practice may be augmented to address developments in this field.
PMCID: PMC3679406  PMID: 22646276
2.  Dexamethasone induces apoptosis in the developing rat amygdala in an age, region, and sex specific manner 
Neuroscience  2011;199:535-547.
Exposure to glucocorticoids (GCs) in early development can lead to long-term changes in brain function and behavior although little is known about the underlying neural mechanisms. Perinatal exposure to GCs alters adult anxiety and neuroendocrine responses to stress. Therefore, we investigated the effects of either late gestational or neonatal exposure to the GC receptor agonist dexamethasone (DEX), on apoptosis within the amygdala, a region critical for emotional regulation. DEX was administered to timed-pregnant rat dams from gestational day 18 until parturition, or postnatal day 4-6. Offspring were sacrificed the day following the last DEX treatment and tissue was processed for immunohistochemical detection of cleaved caspase-3, a marker for apoptotic cells. Prenatal DEX treatment significantly increased the number of cleaved caspase-3 positive cells in the amygdala of both sexes, largely due to increases within the medial and basomedial sub-regions. Postnatal DEX treatment also increased cleaved caspase-3 immunoreactivity within the amygdala, although effects reached significance only in the central nucleus of females. Overall, DEX induction of cleaved caspase-3 in the amygdala was greater following prenatal compared to postnatal treatment, yet in both instances elevations in cleaved caspase-3 correlated with an increase in pro-apoptotic Bax mRNA expression. Dual-label immunohistochemistry of cleaved caspase-3 and the neuronal marker NeuN confirmed that virtually all cleaved caspase-3 positive cells in the amygdala were neurons and a subset of these cells (primarily following postnatal treatment) expressed a GABAergic calcium binding protein phenotype (calbindin or calretinin). Together these results indicate that early developmental GC exposure induces neuronal apoptosis within the amygdala in an age, sex, and region dependent manner.
PMCID: PMC3237835  PMID: 22008524
dexamethasone; glucocorticoid; apoptosis; prenatal; neonatal; sex difference
3.  Circulating Osteogenic Precursor Cells in Heterotopic Bone Formation 
Stem cells (Dayton, Ohio)  2009;27(9):2209-2219.
Cells with osteogenic potential can be found in a variety of tissues. Here we show that circulating osteogenic precursor (COP) cells, a bone marrow-derived type I collagen+/CD45+ subpopulation of mononuclear adherent cells, are present in early pre-osseous fibroproliferative lesions in patients with fibrodysplasia ossificans progressiva (FOP) and nucleate heterotopic ossification (HO) in a murine in vivo implantation assay. Blood samples from FOP patients with active episodes of HO contain significantly higher numbers of clonally-derived COP cell colonies than patients with stable disease or unaffected individuals. The highest level of COP cells was found in a patient just prior to the clinical onset of an HO exacerbation. Our studies show that even COP cells derived from an unaffected individual can contribute to HO in genetically susceptible host tissue. The possibility that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites has tremendous implications and, to our knowledge, represents the first example of their involvement in clinical HO. Thus, bone formation is not limited to cells of the mesenchymal lineage, and circulating cells of hematopoietic origin can also serve as osteogenic precursors at remote sites of tissue inflammation.
PMCID: PMC3496263  PMID: 19522009
Heterotopic ossification; mesenchymal progenitor cells; fibrodysplasia ossificans progressive; bone-marrow transplantation
4.  A Theoretically Based Behavioral Nutrition Intervention for Community Elders at High Risk: The B-NICE Randomized Controlled Clinical Trial 
We conducted a study designed to evaluate the efficacy and feasibility of a multilevel self-management intervention to improve nutritional intake in a group of older adults receiving Medicare home health services who were at especially high risk for experiencing undernutrition. The Behavioral Nutrition Intervention for Community Elders (B-NICE) trial used a prospective randomized controlled design to determine whether individually tailored counseling focused on social and behavioral aspects of eating resulted in increased caloric intake and improved nutrition-related health outcomes in a high-risk population of older adults. The study was guided by the theoretical approaches of the Ecological Model and Social Cognitive Theory. The development and implementation of the B-NICE protocol, including the theoretical framework, methodology, specific elements of the behavioral intervention, and assurances of the treatment fidelity, as well as the health policy implications of the trial results, are presented in this article.
PMCID: PMC3495161  PMID: 22098180
aging; care transitions; community health; home health care; nutrition; randomized controlled trial; self management
5.  New covalent capture probes for imaging and therapy, based on a combination of binding affinity and disulfide bond formation 
Bioconjugate chemistry  2011;22(8):1479-1483.
We describe the synthesis and development of new reactive DOTA-metal complexes for covalently targeting engineered receptors in vivo, which have superior tumor uptake and clearance properties for biomedical applications. These probes are found to clear efficiently through the kidneys and minimally through other routes, but bind persistently in the tumor target. We also explore the new technique of Cerenkov luminescence imaging to optically monitor radiolabeled probe distribution and kinetics in vivo. Cerenkov luminescence imaging uniquely enables sensitive noninvasive in vivo imaging of a β− emitter such as 90Y with an optical imager.
PMCID: PMC3158659  PMID: 21755984
6.  Social Cognition in Schizophrenia: An Overview 
Schizophrenia Bulletin  2008;34(3):408-411.
The purpose of this column is to provide an overview of social cognition in schizophrenia. The column begins with a short introduction to social cognition. Then, we describe the application of social cognition to the study of schizophrenia, with an emphasis on key domains (i.e., emotion perception, Theory of Mind, and attributional style). We conclude the column by discussing the relationship of social cognition to neurocognition, negative symptoms, and functioning, with an eye toward strategies for improving social cognition in schizophrenia.
PMCID: PMC2632430  PMID: 18375928
social cognition; attributions; emotion perception; theory of mind; functional outcome
7.  The Functional Significance of Social Cognition in Schizophrenia: A Review 
Schizophrenia Bulletin  2006;32(Suppl 1):S44-S63.
Deficits in a wide array of functional outcome areas (eg, social functioning, social skills, independent living skills, etc) are marked in schizophrenia. Consequently, much recent research has attempted to identify factors that may contribute to functional outcome; social cognition is one such domain. The purpose of this article is to review research examining the relationship between social cognition and functional outcome. Comprehensive searches of PsycINFO and MEDLINE/PUBMED were conducted to identify relevant published manuscripts to include in the current review. It is concluded that the relationship between social cognition and functional outcome depends on the specific domains of each construct examined; however, it can generally be concluded that there are clear and consistent relationships between aspects of functional outcome and social cognition. These findings are discussed in light of treatment implications for schizophrenia.
PMCID: PMC2632537  PMID: 16916889
social functioning; emotion perception; social perception; theory of mind
8.  Database resources of the National Center for Biotechnology Information 
Nucleic Acids Research  2005;34(Database issue):D173-D180.
In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups, Retroviral Genotyping Tools, HIV-1, Human Protein Interaction Database, SAGEmap, Gene Expression Omnibus, Entrez Probe, GENSAT, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized datasets. All of the resources can be accessed through the NCBI home page at: .
PMCID: PMC1347520  PMID: 16381840
9.  Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor–BI mediate HDL-initiated signaling 
Journal of Clinical Investigation  2005;115(4):969-977.
The binding of HDL to scavenger receptor–BI (SR-BI) mediates cholesterol movement. HDL also induces multiple cellular signals, which in endothelium occur through SR-BI and converge to activate eNOS. To determine the molecular basis of a signaling event induced by HDL, we examined the proximal mechanisms in HDL activation of eNOS. In endothelial cells, HDL and methyl-β-cyclodextrin caused comparable eNOS activation, whereas cholesterol-loaded methyl-β-cyclodextrin had no effect. Phosphatidylcholine-loaded HDL caused greater stimulation than native HDL, and blocking antibody against SR-BI, which prevents cholesterol efflux, prevented eNOS activation. In a reconstitution model in COS-M6 cells, wild-type SR-BI mediated eNOS activation by both HDL and small unilamellar vesicles (SUVs), whereas the SR-BI mutant AVI, which is incapable of efflux to SUV, transmitted signal by only HDL. In addition, eNOS activation by methyl-β-cyclodextrin was SR-BI dependent. Studies of mutant and chimeric class B scavenger receptors revealed that the C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains of SR-BI are both necessary for HDL signaling. Furthermore, we demonstrated direct binding of cholesterol to the C-terminal transmembrane domain using a photoactivated derivative of cholesterol. Thus, HDL signaling requires cholesterol binding and efflux and C-terminal domains of SR-BI, and SR-BI serves as a cholesterol sensor on the plasma membrane.
PMCID: PMC1069105  PMID: 15841181
10.  Bioterrorism-Related Anthrax Surveillance, Connecticut, September–December, 2001 
Emerging Infectious Diseases  2002;8(10):1078-1082.
On November 19, 2001, a case of inhalational anthrax was identified in a 94-year-old Connecticut woman, who later died. We conducted intensive surveillance for additional anthrax cases, which included collecting data from hospitals, emergency departments, private practitioners, death certificates, postal facilities, veterinarians, and the state medical examiner. No additional cases of anthrax were identified. The absence of additional anthrax cases argued against an intentional environmental release of Bacillus anthracis in Connecticut and suggested that, if the source of anthrax had been cross-contaminated mail, the risk for anthrax in this setting was very low. This surveillance system provides a model that can be adapted for use in similar emergency settings.
PMCID: PMC2730303  PMID: 12396919
11.  Direct Renin Inhibition Prevents Cardiac Dysfunction in a Diabetic Mouse Model: Comparison with an Angiotensin Receptor Antagonist and Angiotensin Converting Enzyme Inhibitor 
Hyperglycemia upregulates intracellular angiotensin II production in cardiac myocytes, effects of which are blocked more effectively by renin inhibition than angiotensin receptor blockers (ARBs) or ACE inhibitors. Here we determined whether renin inhibition is more effective at preventing diabetic cardiomyopathy than an ARB or ACE inhibitor. Diabetes was induced in adult mice for 10 wks by streptozotocin. Diabetic mice were treated with insulin, aliskiren (renin inhibitor), benazeprilat (ACE inhibitor), or valsartan (ARB) via subcutaneous minipumps. Significant impairment in diastolic and systolic cardiac function was observed in diabetic mice, which was completely prevented by all three RAS inhibitors. Hyperglycemia significantly increased cardiac oxidative stress and circulating inflammatory cytokines, which were blocked by aliskiren and benazeprilat, while valsartan was partially effective. Diabetes increased cardiac (pro)renin receptor (PRR) expression and nuclear translocation of promyelocytic zinc finger protein (PLZF), which was completely prevented by aliskiren and valsartan, and partially by benazeprilat. Renin inhibition provided similar protection of cardiac function as ARBs and ACE inhibitors. Activation of PLZF by PRR represented a novel mechanism in diabetic cardiomyopathy. Differential effects of the three agents on oxidative stress, cytokines, and PRR expression suggested subtle differences in their mechanism of action.
PMCID: PMC3982397  PMID: 23116220
Diabetic cardiomyopathy; intracrine; prorenin receptor
12.  In vivo efficacy of enabling formulations based on hydroxypropyl-β-cyclodextrins, micellar preparation and liposomes for the lipophilic cannabinoid CB2 agonist, MDA7. 
Journal of pharmaceutical sciences  2012;102(2):352-364.
Enabling formulation based on hydroxypropyl-β-cyclodextrins (HPβCD), micellar preparation and liposomes have been designed to deliver the racemic mixture of a lipophilic CB2 agonist, MDA7. The antiallodynic effects of MDA7 formulated in these three different systems were compared after intravenous administration in rats. Stoichiometry of the inclusion complex formed by MDA7 in HPβCD was determined by continuous variation plot, ESI-MS analysis, phase solubility and NMR studies and indicate formation of exclusively 1:1 adduct. Morphology and particle sizes determined by DLS and TEM show the presence of a homogenous population of closed round-shaped oligolamellar MDA7 containing-liposomes, with average size of 117nm [polydispersity index (PDI) <0.1]. Monodisperse micelles exhibited average size of 15 nm (PDI 0.1). HPβCD based formulation administrated in vivo was composed of two discrete particles populations with a narrow size distribution of 3 nm (PDI<0.1) and 510 nm (PDI<0.1). HPβCD based formulation dramatically improved antiallodynic effect of MDA7 in comparison to the liposomes preparation. Through inclusion complexation and possibly formation of aggregates, HPβCD can enhance the aqueous solubility of lipophilic drugs thereby improving their bioavailability for i.v. administration.
PMCID: PMC3981967  PMID: 23192786
Chirality; Complexation; Cyclodextrins; Micelle; Bioavailability; CNS; Liposomes
13.  Interleukin-19 Impairment in Active Crohn’s Disease Patients 
PLoS ONE  2014;9(4):e93910.
The exact function of interleukin-19 (IL-19) on immune response is poorly understood. In mice, IL-19 up-regulates TNFα and IL-6 expression and its deficiency increases susceptibility to DSS-induced colitis. In humans, IL-19 favors a Th2 response and is elevated in several diseases. We here investigate the expression and effects of IL-19 on cells from active Crohn’s disease (CD) patient. Twenty-three active CD patients and 20 healthy controls (HC) were included. mRNA and protein IL-19 levels were analyzed in monocytes. IL-19 effects were determined in vitro on the T cell phenotype and in the production of cytokines by immune cells. We observed that unstimulated and TLR-activated monocytes expressed significantly lower IL-19 mRNA in active CD patients than in HC (logFC = −1.97 unstimulated; −1.88 with Pam3CSK4; and −1.91 with FSL-1; p<0.001). These results were confirmed at protein level. Exogenous IL-19 had an anti-inflammatory effect on HC but not on CD patients. IL-19 decreased TNFα production in PBMC (850.7±75.29 pg/ml vs 2626.0±350 pg/ml; p<0.01) and increased CTLA4 expression (22.04±1.55% vs 13.98±2.05%; p<0.05) and IL-4 production (32.5±8.9 pg/ml vs 13.5±2.9 pg/ml; p<0.05) in T cells from HC. IL-10 regulated IL-19 production in both active CD patients and HC. We observed that three of the miRNAs that can modulate IL-19 mRNA expression, were up-regulated in monocytes from active CD patients. These results suggested that IL-19 had an anti-inflammatory role in this study. Defects in IL-19 expression and the lack of response to this cytokine could contribute to inflammatory mechanisms in active CD patients.
PMCID: PMC3981722  PMID: 24718601
14.  53BP1 is limiting for nonhomologous end-joining in ATM-deficient cells subjected to oncogenic stress or radiation 
Molecular cancer research : MCR  2013;11(10):1223-1234.
DNA Damage Response (DDR) factors Ataxia Telangiectasia Mutated (ATM) and p53 Binding Protein 1 (53BP1) function as tumor suppressors in humans and mice, but the significance of their mutual interaction to the suppression of oncogenic translocations in vivo has not been investigated. To address this question, we have examined here phenotypes of mice lacking 53BP1 and ATM (Trp53bp1−/−/Atm−/−), relative to single mutants. These analyses reveal that loss of 53BP1 markedly decreases the latency of T lineage lymphomas driven by RAG-dependent oncogenic translocations in Atm−/− mice (average survival, 23 and 14 weeks for Atm−/− and Trp53bp1−/−/Atm−/− mice, respectively). Mechanistically, 53BP1 deficiency aggravates the deleterious effect of ATM deficiency on nonhomologous end-joining (NHEJ)-mediated double-strand break (DSB) repair. Analysis of V(D)J recombinase-mediated coding joints (CJs) and signal joints (SJs) in Trp53bp1−/−/Atm−/− primary thymocytes is, however, consistent with canonical NHEJ-mediated repair. Together, these findings indicate that the greater NHEJ defect in double mutants results from a decrease in the efficiency of rejoining rather than a switch to alternative NHEJ-mediated repair. Complementary analyses of irradiated primary cells indicate that defects in cell cycle checkpoints subsequently function to amplify the NHEJ defect, resulting in more frequent chromosomal breaks and translocations in double mutants cells throughout the cell cycle. Finally, we find that 53BP1 is dispensable for the formation of RAG-mediated hybrid joints (HJs) in Atm−/− thymocytes, but is required to suppress large deletions in a subset of HJs. Collectively, these results uncover novel ATM-independent functions for 53BP1 in the suppression of oncogenic translocations and in radioprotection.
PMCID: PMC3975592  PMID: 23858098
53BP1; ATM; lymphoma; translocation; double-strand break
15.  Electrophysiological insights into conceptual disorganization in schizophrenia 
Schizophrenia research  2007;92(0):225-236.
Disorganized speech, or thought disorder, in schizophrenia may reflect abnormal processing of meaningful concepts. To examine whether schizophrenia involves abnormalities in how a meaningful context influences processing of concepts strongly, weakly, or not related to it, we used the N400, an event-related brain potential (ERP) index of semantic relatedness. ERPs were recorded from schizophrenia patients (n=18) and normal controls (n=18) while they viewed category definitions (e.g., a type of fruit), each followed by a target word that was either a high-typicality category exemplar (apple), low-typicality exemplar (cherry), or non-exemplar (clamp). Participants' task was to indicate via button-press whether or not the target belonged to the category. In both patients and controls, N400 amplitude was largest (most negative) for non-exemplars, intermediate for low-typicality exemplars, and smallest (least negative) for high-typicality exemplars. Compared to controls, patients showed a trend toward reduced N400 amplitude differences between non-exemplars and low-typicality exemplars. Most importantly, within patients, reduced N400 amplitude differences between high- and low-typicality exemplars were correlated with psychotic symptoms. In schizophrenia patients, an N400 index of semantic processing was associated with psychotic symptoms. Psychosis may be associated with greater similarity in how concepts strongly and weakly meaningfully related to their context are processed.
PMCID: PMC3974604  PMID: 17383161
Schizophrenia; Semantic priming; Thought disorder; Psychosis; Event-related potentials; N400
16.  Structure, Dynamics, Evolution, and Function of a Major Scaffold Component in the Nuclear Pore Complex 
The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings. Nup192 is a major component of the NPC’s inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2–960 [ScNup192(2–960)], which adopts an α-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2–960) could undergo long-range transition between “open” and “closed” conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2–960), and homology modeling. Evolutionary analyses using the ScNup192(2–960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.
PMCID: PMC3755625  PMID: 23499021
17.  Single cell molecular recognition of migrating and invading tumor cells using a targeted fluorescent probe to receptor PTPmu 
Detection of an extracellular cleaved fragment of a cell-cell adhesion molecule represents a new paradigm in molecular recognition and imaging of tumors. We previously demonstrated that probes that recognize the cleaved extracellular domain of PTPmu label human glioblastoma brain tumor sections and the main tumor mass of intracranial xenograft gliomas. In this manuscript, we examine whether one of these probes, SBK2, can label dispersed glioma cells that are no longer connected to the main tumor mass. Live mice with highly dispersive glioma tumors were injected intravenously with the fluorescent PTPmu probe to test the ability of the probe to label the dispersive glioma cells in vivo. Analysis was performed using a unique 3-D cryo-imaging technique to reveal highly migratory and invasive glioma cell dispersal within the brain and the extent of co-labeling by the PTPmu probe. The PTPmu probe labeled the main tumor site and dispersed cells up to 3.5 mm away. The cryo-images of tumors labeled with the PTPmu probe provide a novel, high-resolution view of molecular tumor recognition, with excellent 3-D detail regarding the pathways of tumor cell migration. Our data demonstrate that the PTPmu probe recognizes distant tumor cells even in parts of the brain where the blood-brain barrier is likely intact. The PTPmu probe has potential translational significance for recognizing tumor cells to facilitate molecular imaging, a more complete tumor resection and to serve as a molecular targeting agent to deliver chemotherapeutics to the main tumor mass and distant dispersive tumor cells.
PMCID: PMC3558593  PMID: 22987116
receptor protein tyrosine phosphatase; proteolytic cleavage; glioma; molecular imaging; targeting agent; migration; invasion
18.  The Native Cell Population Does Not Contribute to Central-Third Graft Healing at 6, 12 or 26 Weeks in the Rabbit Patellar Tendon 
Investigators do not yet understand the role of intrinsic tendon cells in healing at the tendon-to-bone enthesis. Therefore, our first objective was to understand how the native cell population influences tendon autograft incorporation in the central-third patellar tendon defect site. To do this, we contrasted the histochemical and biomechanical properties of de-cellularized patellar tendon autograft (dcPTA) and patellar tendon autograft (PTA) repairs in the skeletally mature New Zealand white rabbit. Recognizing that soft tissues in many animal models require up to 26 weeks to incorporate into bone, our second objective was to investigate how recovery time affects enthesis formation and graft tissue biomechanical properties. Thus, we examined graft structure and mechanics at 6, 12, and 26 weeks post-surgery. Our results showed that maintaining the native cell population produced no histochemical or biomechanical benefit at 6, 12 or 26 weeks. These findings suggest that PTA healing is mediated more by extrinsic rather than intrinsic cellular mechanisms. Moreover, while repair tissue biomechanical properties generally increased from 6 to 12 weeks after surgery, no further improvements were noted up to 26 weeks.
PMCID: PMC3586979  PMID: 23138453
Patellar tendon; intrinsic healing; enthesis
19.  Analysis of ER Resident Proteins in S. cerevisiae: Implementation of H/KDEL Retrieval Sequences 
Traffic (Copenhagen, Denmark)  2013;14(4):365-381.
An elaborate quality control system regulates ER homeostasis by ensuring the fidelity of protein synthesis and maturation. In budding yeast, genomic analyses and high-throughput proteomic studies have identified ER resident proteins that restore homeostasis following local perturbations. Yet, how these folding factors modulate stress has been largely unexplored. In this study, we designed a series of PCR-based modules including codon-optimized epitopes and FP variants complete with C-terminal H/KDEL retrieval motifs. These conserved sequences are inherent to most soluble ER resident proteins. To monitor multiple proteins simultaneously, H/KDEL cassettes are available with six different selection markers, providing optimal flexibility for live-cell imaging and multicolor labeling in vivo. A single pair of PCR primers can be used for the amplification of these 26 modules, enabling numerous combinations of tags and selection markers. The versatility of pCY H/KDEL cassettes was demonstrated by labeling BiP/Kar2p, Pdi1p, and Scj1p with all novel tags, thus providing a direct comparison among FP variants. Furthermore, to advance in vitro studies of yeast ER proteins, Strep-tag II was engineered with a C-terminal retrieval sequence. Here, an efficient purification strategy was established for BiP under physiological conditions.
PMCID: PMC3594122  PMID: 23324027
BiP; Scj1p; Pdi1p; H/KDEL retrieval sequences; GFP variants; mCherry; pFA6a plasmid; UPR
20.  Connective tissue spectrum abnormalities associated with spontaneous cerebrospinal fluid leaks: a prospective study 
We aimed to assess the frequency of connective tissue abnormalities among patients with cerebrospinal fluid (CSF) leaks in a prospective study using a large cohort of patients. We enrolled a consecutive group of 50 patients, referred for consultation because of CSF leak. All patients have been carefully examined for the presence of connective tissue abnormalities, and based on findings, patients underwent genetic testing. Ancillary diagnostic studies included echocardiography, eye exam, and histopathological examinations of skin and dura biopsies in selected patients. We identified nine patients with heritable connective tissue disorders, including Marfan syndrome, Ehlers–Danlos syndrome and other unclassified forms. In seven patients, spontaneous CSF leak was the first noted manifestation of the genetic disorder. We conclude that spontaneous CSF leaks are associated with a spectrum of connective tissue abnormalities and may be the first noted clinical presentation of the genetic disorder. We propose that there is a clinical basis for considering spontaneous CSF leak as a clinical manifestation of heritable connective tissue disorders, and we suggest that patients with CSF leaks should be screened for connective tissue and vascular abnormalities.
PMCID: PMC3598315  PMID: 22929030
hereditary disorders of connective tissue; spontaneous cerebrospinal fluid leak; positional headache screening
21.  Quality Assessment for Clinical Proteomics 
Clinical biochemistry  2012;46(6):411-420.
Proteomics has emerged from the labs of technologists to enter widespread application in clinical contexts. This transition, however, has been hindered by overstated early claims of accuracy, concerns about reproducibility, and the challenges of handling batch effects properly. New efforts have produced sets of performance metrics and measurements of variability that establish sound expectations for experiments in clinical proteomics. As researchers begin incorporating these metrics in a quality by design paradigm, the variability of individual steps in experimental pipelines will be reduced, regularizing overall outcomes. This review discusses the evolution of quality assessment in 2D gel electrophoresis, mass spectrometry-based proteomic profiling, tandem mass spectrometry-based protein inventories, and proteomic quantitation. Taken together, the advances in each of these technologies are establishing databases that will be increasingly useful for decision-making in clinical experimentation.
PMCID: PMC3602213  PMID: 23246537
Quality Assessment; Quality Control; Quality by Design; Repeatability; Reproducibility; Proteomics; Selected Reaction Monitoring; Mass Spectrometry Profiling; Polyacrylamide Gel Electrophoresis
22.  Demonstration of a Sucrose-derived Contrast Agent for Magnetic Resonance Imaging of the GI Tract 
A scaffold bearing eight terminal alkyne groups was synthesized from sucrose, and copies of an azide-terminated Gd-DOTA complex were attached via copper(I)-catalyzed azide-alkyne cycloaddition. The resulting contrast agent (CA) was administered by gavage to C3H mice. Passage of the CA through the gastrointestinal (GI) tract was followed by T1-weighted magnetic resonance imaging (MRI) over a period of 47 hours, by which time the CA had exited the GI tract. No evidence for leakage of the CA from the GI tract was observed. Thus, a new, orally administered CA for MRI of the GI tract has been developed and successfully demonstrated.
PMCID: PMC3602218  PMID: 23481651
Magnetic resonance imaging; Contrast agent; Gadolinium; DOTA; Gastrointestinal tract
23.  Antigen-specific memory Treg control memory responses to influenza virus infection 
Regulatory CD4+FoxP3+ T cells (Treg) are key regulators of inflammatory responses and control the magnitude of cellular immune responses to viral infections. However, little is known about how Treg contribute to immune regulation during memory responses to previously-encountered pathogens. Here we utilized influenza NP311-325/IAb Class II tetramers to track the antigen-specific Treg response to primary and secondary influenza virus infections. During secondary infections, antigen-specific memory Treg showed accelerated accumulation in the lung-draining lymph node and lung parenchyma relative to a primary infection. Memory Treg effectively controlled the in vitro proliferation of memory CD8+ cells in an antigen-specific fashion that was MHC class II dependent. When memory Treg were depleted prior to secondary infection, the magnitude of the antigen-specific memory CD8+ T cell response was increased, as was pulmonary inflammation and airway cytokine/chemokine expression. Replacement of memory Treg with naïve Treg failed to restore the regulation of the memory CD8 T cell response during secondary infection. Together, these data demonstrate the existence of a previously undescribed population of antigen-specific memory Treg that shape the cellular immune response to secondary influenza virus challenges and offer an additional parameter to consider when determining the efficacy of vaccinations.
PMCID: PMC3608733  PMID: 23467933
24.  Chromatin Organization and Transcriptional Regulation 
Cell type specific transcriptional regulation must be adhered to in order to maintain cell identity throughout the lifetime of an organism, yet flexible enough to allow for responses to endogenous and exogenous stimuli. This regulation is mediated not only by molecular factors (e.g. cell type specific transcription factors, histone and DNA modifications), but also on the level of chromatin and genome organization. In this review we focus on recent findings that have contributed to our understanding of higher order chromatin structure and genome organization within the nucleus. We highlight new findings on the dynamic positioning of genes relative to each other, as well as to their chromosome territory and the nuclear lamina, and how the position of genes correlates with their transcriptional activity.
PMCID: PMC3612554  PMID: 23270812
25.  Autophagy in Antarctica 
Autophagy  2013;9(4):629-631.
The midge Belgica antarctica is the only insect endemic to Antarctica and has the southernmost range of any insect. In its natural environment, B. antarctica frequently faces desiccating conditions, as environmental water is frozen for up to 9 months annually. The molecular mechanisms by which B. antarctica tolerates extreme dehydration are poorly understood, but recent work from our laboratory reports genome-wide expression changes in response to extreme dehydration (~40% water loss), the first genome-scale transcriptome reported for an Antarctic animal. Among transcripts differentially regulated during dehydration, there is coordinated upregulation of numerous genes involved in autophagy, including genes responsible for autophagosome synthesis and autophagy-associated transcription factors. Also, several genes and pathways that interact with and regulate autophagy, e.g., sestrins and proteasomal genes, are concurrently upregulated. This suggests that autophagy and related processes are key elements regulating stress tolerance in this extreme environment.
PMCID: PMC3627684  PMID: 23380735
antarctic midge; dehydration; environmental stress; RNA-seq; autophagy; sestrin

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