ࡱ>   5bjbjVV 4<<?=D v!v!v!$?\v!v!v!v!v! u^^^v!G ^v!^^ @9tiJ0{&HBDv!v!^v!v!v!v!v!8}&v!v!v!v!v!v!v!v!v!v!v!v!v!v!v!v! : ITI PUBLICATIONS NOVEMBER 2011 (77) 1)J Heart Lung Transplant. 2011 Nov 1. [Epub ahead of print] Heart transplantation and cardiac amyloidosis: Approach to screening and novel management strategies. Varr BC, Liedtke M, Arai S, Lafayette RA, Schrier SL, Witteles RM. Department of Internal Medicine, Stanford University School of Medicine, Stanford, California. Limited data exist regarding screening methods and outcomes for orthotopic heart transplantation (OHT) in cardiac amyloidosis. As a result, uncertainty exists over the best approach to OHT for cardiac amyloidosis and for the timing of critical post-transplant therapies. This article reviews 6 patients who underwent OHT for cardiac amyloidosis at the Stanford University Amyloid Center from 2008 to present. All patients with light-chain amyloidosis received chemotherapy in the interval between OHT and autologous hematopoietic stem cell transplant. Five patients remain alive up to 25 months after OHT, without evidence of recurrent cardiac amyloid deposition. A novel strategy of OHT, followed by light-chain suppressive chemotherapy before autologous hematopoietic stem cell transplant, is feasible for patients with light-chain amyloidosis.Copyright © 2011 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved. PMID: 22051505 [PubMed - as supplied by publisher] -- 2) J Biol Chem. 2011 Nov 7. [Epub ahead of print] Nuclear localized antisense small RNAs with 5'-polyphosphate termini regulate long-term transcriptional gene silencing in entamoeba histolytica G3 strain. Zhang H, Alramini H, Tran V, Singh U. Stanford University, United States. Recent studies have linked the RNAi pathway to transcriptional gene silencing in many eukaryotic systems from yeast to mammals. In the deep-branching eukaryotic parasite Entamoeba histolytica, transcriptional gene silencing of the Amoebapore A gene (Ap- A) in the G3 strain has been reported with subsequent development of this parasite strain for gene silencing. However, the mechanisms underlying this gene silencing approach are poorly understood. Here we report that antisense small RNAs specific to the silenced Ap-A gene can be identified in G3 parasites. Furthermore, when additional genes are silenced in the G3 strain, antisense small RNAs to the newly silenced genes can be detected. Characterization of these antisense small RNAs demonstrate that they are ~27nt in size, have 5'-polyphosphate termini, and persist even after removal of the silencing plasmid. Immunofluorescence analysis and fluorescence in situ hybridization show that both the Argonaute protein EhAGO2-2 and antisense small RNAs are enriched in the parasite nucleus. Finally, chromatin immunoprecipitation assays demonstrate that the loci of the silenced genes are enriched for both histone H3 and EhAGO2-2 indicating that both chromatin modification and the RNA-induced transcriptional silencing complex are involved for permanent gene silencing in G3 parasites. In conclusion, our data demonstrate that G3-based gene silencing in E. histolytica is mediated by a siRNA pathway. To our knowledge, this is the first study to show that siRNA-mediated transcriptional gene silencing is functional in protozoan parasites.PMID: 22049083 [PubMed - as supplied by publisher] --- 3) Blood. 2011 Nov 1. [Epub ahead of print] In situ vaccination against mycosis fungoides by intratumoral injection of a TLR9 agonist combined with radiation: a phase I/II study. Kim YH, Gratzinger D, Harrison C, Brody JD, Czerwinski DK, Ai WZ, Morales A, Abdulla F, Xing L, Navi D, Tibshirani RJ, Advani RH, Lingala B, Shah S, Hoppe RT, Levy R. Department of Dermatology, Stanford University School of Medicine, Stanford, CA, United States; We have developed and previously reported on a therapeutic vaccination strategy for indolent B-cell lymphoma that combines local radiation to enhance tumor immunogenecity with the injection into the tumor of a TLR9 agonist. As a result, antitumor CD8 T-cells are induced and systemic tumor regression was documented. Since the vaccination occurs in situ, there is no need to manufacture a vaccine product. We have now explored this strategy in a second disease- mycosis fungoides (MF). We treated 15 patients. Clinical responses were assessed at the distant, untreated sites as a measure of systemic antitumor activity. Five clinically meaningful responses were observed. The procedure was well tolerated and adverse effects consisted mostly of mild and transient injection site or flu-like symptoms. The immunized sites showed a significant reduction of CD25+, Foxp3+ T-cells that could be either MF cells or tissue Tregs and a similar reduction in S100+, CD1a+ dendritic cells (DCs). There was a trend towards greater reduction of CD25+ T-cells and skin DCs in clinical responders vs. non-responders. Our in situ vaccination strategy is feasible also in MF and the clinical responses that occurred in a subset of patients warrant further study with modifications to augment these therapeutic effects. This study is registered at www.clinicaltrials.gov as NCT00226993.PMID: 22045986 [PubMed - as supplied by publisher -- 4) Semin Immunol. 2011 Oct 27. [Epub ahead of print] The coin toss of B cell in rejection and tolerance: Danger versus defense. Zarkhin V, Sarwal MM. Department of Pediatrics, Stanford University, Stanford, CA, USA. Transplantation is the preferred therapy for the end stage organ disease. Since the introduction of organ transplantation into medical practice in 1953 [1], significant progress has been achieved in patient and graft survival rates due to improvements in surgical techniques and more targeted immunosuppressive medications [2]. Nevertheless, current gaps in the management of the transplant patient stem from an incomplete understanding about the heterogeneity of the injury response in organ transplantation, at different rates and different time points after transplantation, as well as our inability to monitor the immunologic threshold of risk versus safety in each individual patient. Recent advances in immunology/transplantation biology with the advent of high throughput "omic" assays such as gene microarrays, proteomics, metabolomics, antibiomics, chemical genomics and functional imaging with nanoparticles, offers us unique methods to interrogate and decipher the variability and unpredictability of the immune response in organ transplantation (Fig. 1) [3]. Recent studies using these applications [3-8] have uncovered a critical and pivotal role for specific B cell lineages in organ injury [9] and organ acceptance [10,11] (Fig. 2). The availability of specific therapies against some of these defined B cell populations provides for an exciting new field of B cell targeted manipulation that can both abrogate the allospecific injury response, as well as promote allospecific graft accommodation and health.Copyright © 2011 Elsevier Ltd. All rights reserved. PMID: 22035649 [PubMed - as supplied by publisher] --- 5)Arterioscler Throm Basc Biol. 2011 Oct 27. [Epub ahead of print] In Vivo Functional and Transcriptional Profiling of Bone Marrow Stem Cells After Transplantation Into Ischemic Myocardium. Sheikh AY, Huber BC, Narsinh KH, Spin JM, van der Bogt K, de Almeida PE, Ransohoff KJ, Kraft DL, Fajardo G, Ardigo D, Ransohoff J, Bernstein D, Fischbein MP, Robbins RC, Wu JC. From the Departments of Cardiothoracic Surgery (A.Y.S., K.v.d.B., D.A., M.P.F., R.C.R.), Medicine, Division of Cardiology (B.C.H., J.C.W.), Radiology (B.C.H., K.H.N., J.M.S., P.E.d.A., K.J.R., J.R., J.C.W.), Pathology (D.L.K.), and Pediatrics, Division of Cardiology (G.F., D.B.) and the Institute of Stem Cell Biology and Regenerative Medicine (R.C.R., J.C.W.), Stanford University School of Medicine, Stanford, CA. OBJECTIVE-: Clinical trials of bone marrow-derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlies their potential efficacy remains unknown. In the present study, we evaluated the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in murine myocardial infarction model. METHODS AND RESULTS-: We used molecular-genetic bioluminescence imaging and high-throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks posttransplantation. Moreover, transcriptomic analysis of BMMCs revealed nonspecific upregulation of various cell regulatory genes, with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography. Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis. CONCLUSION-: Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy. PMID: 22034515 [PubMed - as supplied by publisher] --- 6) Gatrointest Endosc. 2011 Nov;74(5):959-60. Emmet B. Keeffe, MD. Ahmed A, Esquivel CO. Stanford University School of Medicine, Stanford, California. PMID: 22032312 [PubMed - in process -- 7) Ann Neurol. 2011 Oct;70(4):606-15. doi: 10.1002/ana.22476. Significance of marrow-derived nicotinamide adenine dinucleotide phosphate oxidase in experimental ischemic stroke. Tang XN, Zheng Z, Giffard RG, Yenari MA. Department of Neurology, University of California, San Francisco and San Francisco Veterans Affairs Medical Center, San Francisco; Department of Anesthesia, Stanford University School of Medicine, Stanford, CA. OBJECTIVE:Reperfusion after stroke leads to infiltration of inflammatory cells into the ischemic brain. Nicotinamide adenine dinucleotide phosphate oxidase (NOX2) is a major enzyme system that generates superoxide in immune cells. We studied the effect of NOX2 derived from the immune cells in the brain and in blood cells in experimental stroke. METHODS:To establish whether NOX2 plays a role in brain ischemia, strokes were created in mice, then mice were treated with the NOX2 inhibitor apocynin or vehicle and compared to mice deficient in NOX2's gp91 subunit and their wild-type littermates. To determine whether NOX2 in circulating cells versus brain resident cells contribute to ischemic injury, bone marrow chimeras were generated by transplanting bone marrow from wild-type or NOX2-deficient mice into NOX2 or wild-type hosts, respectively. RESULTS:Apocynin and NOX2 deletion both significantly reduced infarct size, blood-brain barrier disruption, and hemorrhagic transformation of the infarcts, compared to untreated wild-type controls. This was associated with decreased matrix metalloproteinase 9 expression and reduced loss of tight junction proteins. NOX2-deficient mice receiving wild-type marrow had better outcomes compared to the wild-type mice receiving wild-type marrow. Interestingly, wild-type mice receiving NOX2-deficient marrow had even smaller infarct sizes and less hemorrhage than NOX2-deficient mice receiving wild-type marrow. INTERPRETATION:This indicates that NOX2, whether present in circulating cells or brain resident cells, contributes to ischemic brain injury and hemorrhage. However, NOX2 from the circulating cells contributed more to the exacerbation of stroke than that from brain resident cells. These data suggest the importance of targeting the peripheral immune system for treatment of stroke. Ann Neurol 2011;70:606-615.Copyright 2011 American Neurological Association. PMID: 22028221 [PubMed - in process] PMCID: PMC3205431 [Available on 2012/10/1] --- 8) Proc Natl Acad Sci USA. 2011 Nov 8;108(45):18412-7. Epub 2011 Oct 24. Structure-function analysis of varicella-zoster virus glycoprotein H identifies domain-specific roles for fusion and skin tropism. Vleck SE, Oliver SL, Brady JJ, Blau HM, Rajamani J, Sommer MH, Arvin AM. Departments of Pediatrics and Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305. Enveloped viruses require membrane fusion for cell entry and replication. For herpesviruses, this event is governed by the multiprotein core complex of conserved glycoproteins (g)B and gH/gL. The recent crystal structures of gH/gL from herpes simplex virus 2, pseudorabies virus, and Epstein-Barr virus revealed distinct domains that, surprisingly, do not resemble known viral fusogens. Varicella-zoster virus (VZV) causes chicken pox and shingles. VZV is an -herpesvirus closely related to herpes simplex virus 2, enabling prediction of the VZV gH structure by homology modeling. We have defined specific roles for each gH domain in VZV replication and pathogenesis using structure-based site-directed mutagenesis of gH. The distal tip of domain (D)I was important for skin tropism, entry, and fusion. DII helices and a conserved disulfide bond were essential for gH structure and VZV replication. An essential (724)CXXC(727) motif was critical for DIII structural stability and membrane fusion. This assignment of domain-dependent mechanisms to VZV gH links elements of the glycoprotein structure to function in herpesvirus replication and virulence. PMID: 22025718 [PubMed - in process] -- 9) Req Anesth Pain Med. 2011 Nov;36(6):632. Epidural catheter removal in patients on warfarin thromboprophylaxis: a more cautious interpretation of results required? Carvalho B, Mariano ER, Butwick AJ. Department of Anesthesia Stanford University School of Medicine Stanford, CA, Anesthesiology and Perioperative Care Service VA Palo Alto Health Care System Palo Alto, CA. PMID: 22024706 [PubMed - in process] -- 10) Epigenetics Chromatin. 2011 Oct 24;4:19. A proteomic approach for the identification of novel lysine methyltransferase substrates. Levy D, Liu CL, Yang Z, Newman AM, Alizadeh AA, Utz PJ, Gozani O. Department of Biology, Stanford University, Stanford, CA 94305, USA. danl@stanford.edu. ABSTRACT: BACKGROUND:Signaling via protein lysine methylation has been proposed to play a central role in the regulation of many physiologic and pathologic programs. In contrast to other post-translational modifications such as phosphorylation, proteome-wide approaches to investigate lysine methylation networks do not exist. RESULTS:In the current study, we used the ProtoArray platform, containing over 9,500 human proteins, and developed and optimized a system for proteome-wide identification of novel methylation events catalyzed by the protein lysine methyltransferase (PKMT) SETD6. This enzyme had previously been shown to methylate the transcription factor RelA, but it was not known whether SETD6 had other substrates. By using two independent detection approaches, we identified novel candidate substrates for SETD6, and verified that all targets tested in vitro and in cells were genuine substrates. CONCLUSIONS:We describe a novel proteome-wide methodology for the identification of new PKMT substrates. This technological advance may lead to a better understanding of the enzymatic activity and substrate specificity of the large number (more than 50) PKMTs present in the human proteome, most of which are uncharacterized.PMID: 22024134 [PubMed] PMCID: PMC3212905 11) PLoS One. 2011;6(10):e26369. Epub 2011 Oct 13. Strain-dependent host transcriptional responses to toxoplasma infection are largely conserved in Mammalian and avian hosts. Ong YC, Boyle JP, Boothroyd JC. Stanford University, Department of Microbiology and Immunology, Stanford, California, United States of America. Toxoplasma gondii has a remarkable ability to infect an enormous variety of mammalian and avian species. Given this, it is surprising that three strains (Types I/II/III) account for the majority of isolates from Europe/North America. The selective pressures that have driven the emergence of these particular strains, however, remain enigmatic. We hypothesized that strain selection might be partially driven by adaptation of strains for mammalian versus avian hosts. To test this, we examine in vitro, strain-dependent host responses in fibroblasts of a representative avian host, the chicken (Gallus gallus). Using gene expression profiling of infected chicken embryonic fibroblasts and pathway analysis to assess host response, we show here that chicken cells respond with distinct transcriptional profiles upon infection with Type II versus III strains that are reminiscent of profiles observed in mammalian cells. To identify the parasite drivers of these differences, chicken fibroblasts were infected with individual F1 progeny of a Type II x III cross and host gene expression was assessed for each by microarray. QTL mapping of transcriptional differences suggested, and deletion strains confirmed, that, as in mammalian cells, the polymorphic rhoptry kinase ROP16 is the major driver of strain-specific responses. We originally hypothesized that comparing avian versus mammalian host response might reveal an inversion in parasite strain-dependent phenotypes; specifically, for polymorphic effectors like ROP16, we hypothesized that the allele with most activity in mammalian cells might be less active in avian cells. Instead, we found that activity of ROP16 alleles appears to be conserved across host species; moreover, additional parasite loci that were previously mapped for strain-specific effects on mammalian response showed similar strain-specific effects in chicken cells. These results indicate that if different hosts select for different parasite genotypes, the selection operates downstream of the signaling occurring during the beginning of the host's immune response.PMID: 22022607 [PubMed - in process] PMCID: PMC3192797 -- 12) Eukaryot Cell. 2011 Oct 21. [Epub ahead of print] Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma bradyzoites. Buchholz KR, Fritz HM, Chen X, Durbin-Johnson B, Rocke DM, Ferguson DJ, Conrad PA, Boothroyd JC. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, 94305, USA. The Toxoplasma gondii bradyzoite is essential to establish persistent infection yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes of in vitro tachyzoites 2 days post-infection, in vitro bradyzoites 4 days post-infection, and in vivo bradyzoites 21 days post-infection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One-hundred-and-two putative bradyzoite-secreted effectors were identified with this approach. Two candidates, bradyzoite pseudokinase 1 (BPK1) and microneme adhesive repeat (MAR)-domain containing protein 4 (MCP4), were chosen for further investigation and confirmed to be induced and secreted by bradyzoites in vitro and in vivo. Thus, we report the first analysis of the transcriptomes of in vitro and in vivo bradyzoites and identify two new protein components of the Toxoplasma tissue cyst wall.PMID: 22021236 [PubMed - as supplied by publisher] -- 13)Cell Host Microbe. 2011 Oct 4;10(4):410-9. The Phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii Reveal Unusual Adaptations Within and Beyond the Parasites' Boundaries. Treeck M, Sanders JL, Elias JE, Boothroyd JC. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. Plasmodium falciparum and Toxoplasma gondii are obligate intracellular apicomplexan parasites that rapidly invade and extensively modify host cells. Protein phosphorylation is one mechanism by which these parasites can control such processes. Here we present a phosphoproteome analysis of peptides enriched from schizont stage P. falciparum and T. gondii tachyzoites that are either "intracellular" or purified away from host material. Using liquid chromatography-tandem mass spectrometry, we identified over 5,000 and 10,000 previously unknown phosphorylation sites in P. falciparum and T. gondii, respectively, revealing that protein phosphorylation is an extensively used regulation mechanism both within and beyond parasite boundaries. Unexpectedly, both parasites have phosphorylated tyrosines, and P. falciparum has unusual phosphorylation motifs that are apparently shaped by its A:T-rich genome. This data set provides important information on the role of phosphorylation in the host-pathogen interaction and clues to the evolutionary forces operating on protein phosphorylation motifs in both parasites. Copyright 2011 Elsevier Inc. All rights reserved.PMID: 22018241 [PubMed - in process] -- 14) Obstet Gynecol. 2011 Nov;118(5):1090-4. Labor room setting compared with the operating room for simulated perimortem cesarean delivery: a randomized controlled trial. Lipman S, Daniels K, Cohen SE, Carvalho B. From the Departments of Anesthesia and Obstetrics & Gynecology, Stanford University School of Medicine, Stanford, California. OBJECTIVE: To compare the labor room and operating room for perimortem cesarean delivery during simulated maternal arrests occurring outside the operating room. We hypothesized transport to the operating room for perimortem cesarean delivery would delay incision and other important resuscitation milestones. METHODS: We randomized 15 teams composed of obstetricians, nurses, anesthesiologists, and neonatal staff to perform perimortem cesarean delivery in the labor room or operating room. A manikin with an abdominal model overlay was used for simulated cesarean delivery. The scenario began in the labor room with maternal cardiopulmonary arrest and fetal bradycardia. The primary outcome was time to incision. Secondary outcomes included times to important milestones, percentage of tasks completed, and type of incision. RESULTS: The median (interquartile range) times from time zero to incision were 4:25 (3:59-4:50) and 7:53 (7:18-8:57) minutes in the labor room and operating room groups, respectively (P=.004). Fifty-seven percent of labor room teams and 14% of operating room teams achieved delivery within 5 minutes. Contacting the neonatal team, placing the defibrillator, resuming compressions after analysis, and endotracheal intubation all occurred more rapidly in the labor room group. CONCLUSION: Perimortem cesarean delivery performed in the labor room was significantly faster than perimortem cesarean delivery performed after moving to the operating room. Delivery within 5 minutes was challenging in either location despite optimal study conditions (eg, the manikin was light and easily moved; teams knew the scenario mandated perimortem cesarean delivery and were aware of being timed). Our findings imply that perimortem cesarean delivery during actual arrest would require more than 5 minutes and should be performed in the labor room rather than relocating to the operating room. LEVEL OF EVIDENCE: I. PMID: 22015877 [PubMed - in process] -- 15) Nat Immunol. 2011 Oct 19;12(11):1035-44. doi: 10.1038/ni.2109. Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils. Galli SJ, Borregaard N, Wynn TA. 1] Department of Pathology, Stanford University School of Medicine, Stanford, California, USA. [2] Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA. Hematopoietic cells, including lymphoid and myeloid cells, can develop into phenotypically distinct 'subpopulations' with different functions. However, evidence indicates that some of these subpopulations can manifest substantial plasticity (that is, undergo changes in their phenotype and function). Here we focus on the occurrence of phenotypically distinct subpopulations in three lineages of myeloid cells with important roles in innate and acquired immunity: macrophages, mast cells and neutrophils. Cytokine signals, epigenetic modifications and other microenvironmental factors can substantially and, in some cases, rapidly and reversibly alter the phenotype of these cells and influence their function. This suggests that regulation of the phenotype and function of differentiated hematopoietic cells by microenvironmental factors, including those generated during immune responses, represents a common mechanism for modulating innate or adaptive immunity.PMID: 22012443 [PubMed - in process] -- 16) Transplantation. 2011 Oct 19;12(11):1035-44. doi: 10.1038/ni.2109. Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils. Galli SJ, Borregaard N, Wynn TA. 1] Department of Pathology, Stanford University School of Medicine, Stanford, California, USA. [2] Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA. Hematopoietic cells, including lymphoid and myeloid cells, can develop into phenotypically distinct 'subpopulations' with different functions. However, evidence indicates that some of these subpopulations can manifest substantial plasticity (that is, undergo changes in their phenotype and function). Here we focus on the occurrence of phenotypically distinct subpopulations in three lineages of myeloid cells with important roles in innate and acquired immunity: macrophages, mast cells and neutrophils. Cytokine signals, epigenetic modifications and other microenvironmental factors can substantially and, in some cases, rapidly and reversibly alter the phenotype of these cells and influence their function. This suggests that regulation of the phenotype and function of differentiated hematopoietic cells by microenvironmental factors, including those generated during immune responses, represents a common mechanism for modulating innate or adaptive immunity.PMID: 22012443 [PubMed - in process] --- 17) ISME J. 2011 Oct 20. doi: 10.1038/ismej.2011.142. [Epub ahead of print] Hydrogen production in photosynthetic microbial mats in the Elkhorn Slough estuary, Monterey Bay. Burow LC, Woebken D, Bebout BM, McMurdie PJ, Singer SW, Pett-Ridge J, Prufert-Bebout L, Spormann AM, Weber PK, Hoehler TM. 1] Department of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA [2] Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA. Hydrogen (H(2)) release from photosynthetic microbial mats has contributed to the chemical evolution of Earth and could potentially be a source of renewable H(2) in the future. However, the taxonomy of H(2)-producing microorganisms (hydrogenogens) in these mats has not been previously determined. With combined biogeochemical and molecular studies of microbial mats collected from Elkhorn Slough, Monterey Bay, California, we characterized the mechanisms of H(2) production and identified a dominant hydrogenogen. Net production of H(2) was observed within the upper photosynthetic layer (0-2 mm) of the mats under dark and anoxic conditions. Pyrosequencing of rRNA gene libraries generated from this layer demonstrated the presence of 64 phyla, with Bacteriodetes, Cyanobacteria and Proteobacteria dominating the sequences. Sequencing of rRNA transcripts obtained from this layer demonstrated that Cyanobacteria dominated rRNA transcript pyrotag libraries. An OTU affiliated to Microcoleus spp. was the most abundant OTU in both rRNA gene and transcript libraries. Depriving mats of sunlight resulted in an order of magnitude decrease in subsequent nighttime H(2) production, suggesting that newly fixed carbon is critical to H(2) production. Suppression of nitrogen (N(2))-fixation in the mats did not suppress H(2) production, which indicates that co-metabolic production of H(2) during N(2)-fixation is not an important contributor to H(2) production. Concomitant production of organic acids is consistent with fermentation of recently produced photosynthate as the dominant mode of H(2) production. Analysis of rRNA % transcript:% gene ratios and H(2)-evolving bidirectional [NiFe] hydrogenase % transcript:% gene ratios indicated that Microcoelus spp. are dominant hydrogenogens in the Elkhorn Slough mats.The ISME Journal advance online publication, 20 October 2011; doi:10.1038/ismej.2011.142. PMID: 22011721 [PubMed - as supplied by publisher] -- 18)Ann Intern Med. 2011 Oct 18;155(8):550-3. The bedside evaluation: ritual and reason. Verghese A, Brady E, Kapur CC, Horwitz RI. Stanford University School of Medicine and Stanford University, Stanford, California; Western Kentucky University, Bowling Green, Kentucky; and GlaxoSmithKline, King of Prussia, Pennsylvania. The bedside evaluation, consisting of the history and physical examination, was once the primary means of diagnosis and clinical monitoring. The recent explosion of imaging and laboratory testing has inverted the diagnostic paradigm. Physicians often bypass the bedside evaluation for immediate testing and therefore encounter an image of the patient before seeing the patient in the flesh. In addition to risking delayed or missed diagnosis of readily recognizable disease, physicians who forgo or circumvent the bedside evaluation risk the loss of an important ritual that can enhance the physician-patient relationship. Patients expect that some form of bedside evaluation will take place when they visit a physician. When physicians complete this evaluation in an expert manner, it can have a salutary effect. If done poorly or not at all, in contrast, it can undermine the physician-patient relationship. Studies suggest that the context, locale, and quality of the bedside evaluation are associated with neurobiological changes in the patient. Recognizing the importance of the bedside evaluation as a healing ritual and a powerful diagnostic tool when paired with judicious use of technology could be a stimulus for the recovery of an ebbing skill set among physicians.PMID: 22007047 [PubMed - in process] --- 19) Appl Clin Inform. 2011;2(4):406-419. Sociotechnical Challenges of Developing an Interoperable Personal Health Record: Lessons Learned. Gaskin GL, Longhurst CA, Slayton R, Das AK. Program in Science, Technology and Society, School of Humanities and Sciences, Stanford University, Stanford, CA. OBJECTIVES:To analyze sociotechnical issues involved in the process of developing an interoperable commercial Personal Health Record (PHR) in a hospital setting, and to create guidelines for future PHR implementations. METHODS:This qualitative study utilized observational research and semi-structured interviews with 8 members of the hospital team, as gathered over a 28 week period of developing and adapting a vendor-based PHR at Lucile Packard Children's Hospital at Stanford University. A grounded theory approach was utilized to code and analyze over 100 pages of typewritten field notes and interview transcripts. This grounded analysis allowed themes to surface during the data collection process which were subsequently explored in greater detail in the observations and interviews. RESULTS:Four major themes emerged: (1) Multidisciplinary teamwork helped team members identify crucial features of the PHR; (2) Divergent goals for the PHR existed even within the hospital team; (3) Differing organizational conceptions of the end-user between the hospital and software company differentially shaped expectations for the final product; (4) Difficulties with coordination and accountability between the hospital and software company caused major delays and expenses and strained the relationship between hospital and software vendor. CONCLUSIONS:Though commercial interoperable PHRs have great potential to improve healthcare, the process of designing and developing such systems is an inherently sociotechnical process with many complex issues and barriers. This paper offers recommendations based on the lessons learned to guide future development of such PHRs. PMID: 22003373 [PubMed] PMCID: PMC3191536 --- 20) Surg Endosc. 2011 Oct 15. [Epub ahead of print] Transgastric versus laparoendoscopic single-site peritoneoscopy in a rat model: effects on motility, inflammation, and nociception. Guo J, Pasricha NP, Shenoy MM, Liu L, Mehta K, Pasricha PJ. Division of Gastroenterology and Hepatology, Stanford University Medical Center, M211 Alway Building, 300 Pasteur Drive, MC: 5187, Stanford, CA, 94305, USA. BACKGROUND:Natural orifice translumenal endoscopic surgery (NOTES) and laparoendoscopic single-port surgery (LESS) are emerging approaches to abdominal surgery that have been advocated as potentially causing fewer physiologic derangements and less pain. This study aimed to compare these procedures in a novel rat model by assessing peritoneal inflammation, gastric motility, and nociception in response to peritoneoscopy performed via NOTES and LESS. METHODS:Adult male rats underwent peritoneoscopy via either transgastric NOTES or LESS using the same type of endoscope and were allowed to recover for 2 to 4 h. Liquid gastric emptying was assessed using phenol red, and cytokine levels were analyzed in peritoneal washings. Thoracic spinal cord segments were stained for Finkel-Biskins-Jinkins osteosarcoma gene (FOS) to assess activation of nociceptive pathways. RESULTS:The NOTES procedure significantly delayed both postsurgical recovery time compared with LESS (115 25 vs. 82 20 min, respectively; P = 0.04) and liquid gastric emptying (26.7 11.1% vs. 57 10.5%; P = 0.004). Several cytokines such as interleukin-1 (IL-1), IL-6, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1beta (MIP-1) were significantly elevated in the NOTES group compared with the LESS group. However, the two groups did not differ significantly in spinal FOS activation. CONCLUSIONS:The NOTES approach is feasible in an experimental rat model, facilitating a scientific approach to hypothesis testing through specific methods and instruments. The transgastric NOTES approach in rats is associated with a worse physiologic outcome in terms of gastric motility and peritoneal inflammation but does not differ significantly from LESS in activation of pain pathways.PMID: 22002201 [PubMed - as supplied by publisher] --- 21) Blood. 2011 Oct 14. [Epub ahead of print] Reduced mast cell and basophil numbers and function in Cpa3-Cre; Mcl-1fl/fl mice. Lilla JN, Chen CC, Mukai K, Benbarak MJ, Franco CB, Kalesnikoff J, Yu M, Tsai M, Piliponsky AM, Galli SJ. Department of Pathology, Stanford University School of Medicine, Stanford, CA, United States; It has been reported that the intracellular anti-apoptotic factor Mcl-1 is required for mast cell survival in vitro and genetic manipulation of Mcl-1 can be used to delete individual hematopoietic cell populations in vivo. Here we report the generation of C57BL/6 mice in which Cre recombinase is expressed under the control of a segment of the Cpa3 (carboxypeptidase A3) promoter. C57BL/6-Cpa3-Cre; Mcl-1(fl/fl) mice are severely deficient in mast cells (92-100% reduced in various tissues analyzed) and also have a marked deficiency in basophils (58-78% reduced in the compartments analyzed), whereas the numbers of other hematopoietic cell populations exhibit little or no changes. Moreover, Cpa3-Cre; Mcl-1(fl/fl) mice exhibited marked reductions in the tissue swelling and leukocyte infiltration associated with both mast cell- and IgE-dependent passive cutaneous anaphylaxis (except at sites engrafted with in vitro-derived mast cells) and a basophil- and IgE-dependent model of chronic allergic inflammation, and they do not develop IgE-dependent passive systemic anaphylaxis. Our findings support the conclusion that Mcl-1 is required for normal mast cell and basophil development/survival in vivo in mice, and also suggest that Cpa3-Cre; Mcl-1(fl/fl) mice may be useful in analyzing the roles of mast cells and basophils in health and disease.PMID: 22001390 [PubMed - as supplied by publisher] -- 22) Circ Res. 2011 Nov 11;109(11):1290-301. Epub 2011 Oct 13. CD4+ T Cells and Complement Independently Mediate Graft Ischemia in the Rejection of Mouse Orthotopic Tracheal Transplants. Khan MA, Jiang X, Dhillon G, Beilke J, Holers VM, Atkinson C, Tomlinson S, Nicolls MR. Associate Professor of Medicine, Division of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, VA Palo Health Care System, Medical Service 111P, 3801 Miranda Ave., Palo Alto, CA 94304. mnicolls@stanford.edu. Rationale: While microvascular injury is associated with chronic rejection, the cause of tissue ischemia during alloimmune injury is not yet elucidated. Objective: We investigated the contribution of T lymphocytes and complement to microvascular injury-associated ischemia during acute rejection of mouse tracheal transplants. Methods and Results: Using novel techniques to assess microvascular integrity and function, we evaluated how lymphocyte subsets and complement specifically affect microvascular perfusion and tissue oxygenation in MHC-mismatched transplants. To characterize T cell effects on microvessel loss and recovery, we transplanted functional airway grafts in the presence and absence of CD4(+) and CD8(+) T cells. To establish the contribution of complement-mediated injury to the allograft microcirculation, we transplanted C3-deficient and C3-inhibited recipients. We demonstrated that CD4(+) T cells and complement are independently sufficient to cause graft ischemia. CD8(+) T cells were required for airway neovascularization to occur following CD4-mediated rejection. Activation of antibody-dependent complement pathways mediated tissue ischemia even in the absence of cellular rejection. Complement inhibition by CR2-Crry attenuated graft hypoxia, complement/antibody deposition on vascular endothelium and promoted vascular perfusion by enhanced angiogenesis. Finally, there was a clear relationship between the burden of tissue hypoxia (ischemiatime duration) and the development of subsequent airway remodeling. Conclusions: These studies demonstrated that CD4(+) T cells and complement operate independently to cause transplant ischemia during acute rejection and that sustained ischemia is a precursor to chronic rejection.PMID: 21998328 [PubMed - in process] -- 23) Stem Cells. 2011 Dec;29(12):2018-2029. doi: 10.1002/stem.757. Nonintegrating Knockdown and Customized Scaffold Design Enhances Human Adipose-Derived Stem Cells in Skeletal Repair. Levi B, Hyun JS, Nelson ER, Li S, Montoro DT, Wan DC, Jia FJ, Glotzbach JC, James AW, Lee M, Huang M, Quarto N, Gurtner GC, Wu JC, Longaker MT. Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery DivisionStanford University School of Medicine, Stanford, California, USA. An urgent need exists in clinical medicine for suitable alternatives to available techniques for bone tissue repair. Human adipose-derived stem cells (hASCs) represent a readily available, autogenous cell source with well-documented in vivo osteogenic potential. In this article, we manipulated Noggin expression levels in hASCs using lentiviral and nonintegrating minicircle short hairpin ribonucleic acid (shRNA) methodologies in vitro and in vivo to enhance hASC osteogenesis. Human ASCs with Noggin knockdown showed significantly increased bone morphogenetic protein (BMP) signaling and osteogenic differentiation both in vitro and in vivo, and when placed onto a BMP-releasing scaffold embedded with lentiviral Noggin shRNA particles, hASCs more rapidly healed mouse calvarial defects. This study therefore suggests that genetic targeting of hASCs combined with custom scaffold design can optimize hASCs for skeletal regenerative medicine. STEM Cells 2011;29:2018-2029. Copyright 2011 AlphaMed Press.PMID: 21997852 [PubMed - as supplied by publisher] -- 24) ISME J. 2011 Oct 13. doi: 10.1038/ismej.2011.136. [Epub ahead of print] The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities. Marshall IP, Berggren DR, Azizian MF, Burow LC, Semprini L, Spormann AM. Department of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA. We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 -2 coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H(2)-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H(2) production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.PMID: 21993396 [PubMed - as supplied by publisher] -- 25) J Neurointerv Surg. 2011 Sep 20. [Epub ahead of print] Cerebral proliferative angiopathy. Marks MP, Steinberg GK. Department of Radiology and Neurosurgery, Stanford University School of Medicine, Stanford, California, USA. Cerebral proliferative angiopathy is a rare lesion marked by diffuse intravascular shunting, which should be differentiated from brain arteriovenous malformations. A patient is presented with cerebral proliferative angiopathy and documented progressive development of hypervascular shunting involving extensive portions of the left hemisphere. The patient had angiographic and laboratory evidence of angiogenesis and a progressive neurologic deterioration which corresponded to the development of her lesion. This is the first case which documents the progressive proliferative changes seen with this abnormality. PMID: 21990497 [PubMed - as supplied by publisher] -- 26) Immunol Invest. 2011;40(7-8):751-66. Aspergillosis in the 'nonimmunocompromised' host. Stevens DA, Melikian GL. Department of Medicine, Santa Clara Valley Medical Center, 751 So. Bascom Ave., San Jose, CA 95128-2699, USA. stevens@stanford.edu Invasive aspergillosis has been classically associated with certain risk factors: cytotoxic chemotherapy, prolonged neutropenia, corticosteroids, transplantation, AIDS. However, the literature is growing that this mycosis, particularly pulmonary aspergillosis, can be seen in patients lacking these factors. Many of the latter patients are in the intensive care unit. Other associated conditions include influenza, nonfungal pneumonia, chronic obstructive lung disease, immaturity, sepsis, liver failure, alcoholism, chronic granulomatous disease and surgery. Certain focal sites, such as sinusitis or cerebral aspergillosis, have additional risk factors. This emphasizes the potential importance of a positive culture for Aspergillus in the critically ill, the need for awareness about possible aspergillosis in patients lacking the classical risk factors, and readiness to proceed with appropriate diagnostic maneuvers. PMID: 21985304 [PubMed - in process] -- 27) J Virol Methods. 2011 Sep 29. [Epub ahead of print] Digital PCR provides absolute quantitation of viral load for an occult RNA virus. White RA 3rd, Quake SR, Curr K. Department of Bioengineering at Stanford University and Howard Hughes Medical Institute, Stanford, CA 94305, USA; Department of Biology at California State University-East Bay, Hayward, CA 94544, USA. Using a multiplexed LNA-based Taqman assay, RT-digital PCR (RT-dPCR) was performed in a prefabricated microfluidic device that monitored absolute viral load in native and immortalized cell lines, overall precision of detection, and the absolute detection limit of an occult RNA virus GB Virus Type C (GBV-C). RT-dPCR had on average a 10% lower overall coefficient of variation (CV, a measurement of precision) for viral load testing than RT-qPCR and had a higher overall detection limit, able to quantify as low as three 5'-UTR molecules of GBV-C genome. Two commercial high-yield in vitro transcription kits (T7 Ribomax Express by Promega and Ampliscribe T7 Flash by Epicentre) were compared to amplify GBV-C RNA genome with T7-mediated amplification. The Ampliscribe T7 Flash outperformed the T7 Ribomax Express in yield of full-length GBV-C RNA genome. THP-1 cells (a model of monocytic derived cells) were transfected with GBV-C, yielding infectious virions that replicated over a 120h time course and could be infected directly. This study provides the first evidence of GBV-C replication in monocytic derived clonal cells. Thus far, it is the only study using a microfluidic device that measures directly viral load of mammalian RNA virus in a digital format without need for a standard curve.Copyright 2011 Elsevier B.V. All rights reserved. PMID: 21983150 [PubMed - as supplied by publisher] -- 28) Stroke. 2011 Nov;42(11):3304-10. Epub 2011 Oct 6. Neurosurgical advances in the treatment of moyamoya disease. Pandey P, Steinberg GK. R281, Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5327. gsteinberg@stanford.edu. BACKGROUND AND PURPOSE: Moyamoya disease is characterized by chronic stenoocclusive vasculopathy involving the distal supraclinoid internal carotid arteries and presents with ischemic or hemorrhagic symptoms. We review advances in the understanding and management of moyamoya disease. Summary of Review- Cerebral revascularization, either direct or indirect, is the cornerstone of treatment for moyamoya disease. Recent advances have been made in understanding the molecular biology and pathophysiology of moyamoya disease, and new genetic mutations and deletions have been identified. Imaging for moyamoya disease is also rapidly improving with new sequences of MRI and better methods of assessing ischemia and cerebrovascular reserve. Positron emission tomography has emerged as an important tool to measure cerebrovascular reserve. Novel surgical techniques assess patency and ischemia during superficial temporal to middle cerebral artery bypass, including indocyanine green videoangiography to evaluate anastomosis patency, and various methods to monitor intraoperative blood flow. Newer methods of indirect revascularization have been described with placement of more tissues supplied by the external carotid artery on the brain surface. Postoperative hyperperfusion to the chronically ischemic brain tissue is a recently identified causative factor of complications. Interestingly, complications from hyperperfusion mimic those caused by ischemia, although they have different treatments, making the role of postoperative blood flow assessment important in distinguishing between the two. Awareness has also increased that even asymptomatic patients can experience significant cognitive decline attributable to chronic ischemia. Whether this reverts after successful revascularization requires investigation. CONCLUSIONS: Surgical revascularization with direct, indirect, and combined methods remains the preferred procedure for patients with moyamoya disease. PMID: 21980214 [PubMed - in process] -- 29) Pediatr Infect Dis J. 2011 Oct 5. [Epub ahead of print] Timing of Antiretroviral Therapy Initiation and its Impact on Disease Progression in Perinatal Human Immunodeficiency Virus-1 Infection. Sturt AS, Halpern MS, Sullivan B, Maldonado YA. From the *Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, CA; Department of Medicine, Division of AIDS Medicine, Santa Clara Valley Medical Center, San Jose, CA; and Department of Pediatrics, Infectious Diseases Division, Santa Clara Valley Medical Center, San Jose, CA. Abstract OBJECTIVE:Treatment with highly active antiretroviral therapy (HAART) reduces overall perinatal human immunodeficiency virus (HIV) type 1-related mortality. The effect of timing of HAART initiation on reduction of morbidity is not well defined. We evaluated the association of timing of HAART initiation on progression to moderate or severe disease. METHODS:Retrospective, population-based study of 196 perinatally HIV-infected children followed from birth in northern California from 1988 to 2009. RESULTS:Of 196 children, 58% received HAART and were followed for a median of 6.2 years after HAART initiation. HAART use was associated with improved survival to the age of 5 years: no HAART, 50% versus HAART, 88%; P < 0.0001. However, the advantage of initial HAART over mono or dual therapy transitioning to HAART was small and not statistically significant (P = 0.23). Starting HAART before the development of moderate or severe disease delayed the median age of diagnosis of moderate disease from 0.4 years (interquartile range, [0.3-0.8]) without HAART to 3.0 years ([interquartile range, 1.9-5.8]; P < 0.0001) with HAART. HAART initiation after progression to moderate or severe disease was associated with decreased progression to severe disease or death, respectively (moderate to severe: 8% [3/36] with HAART vs. 84% [70/83] with no HAART, P < 0.0001; severe to death: 9% [6/68] with HAART vs. 73% [49/67] with no HAART, P < 0.0001). CONCLUSIONS:In perinatal HIV infection, HAART is associated with delayed progression and reduced mortality regardless of disease severity at HAART initiation. This finding reinforces US guidelines regarding HAART initiation at >1 year of age if children present with most clinical category B diagnoses, regardless of CD4 measurements or plasma HIV RNA level. PMID: 21979798 [PubMed - as supplied by publisher] -- 30) Aliment Pharmacol Ther. 2011 Nov;34(10):1145-58. doi: 10.1111/j.1365-2036.2011.04869.x. Epub 2011 Oct 7. Review article: current antiviral therapy of chronic hepatitis B. Ayoub WS, Keeffe EB. Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University Medical Center, Stanford, CA, USA. Aliment Pharmacol Ther 2011; 34: 1145-1158 SUMMARY: Background The indications and endpoints for treatment of chronic hepatitis B continue to evolve. The aim of the therapy for chronic hepatitis B is to achieve a long-term continued suppression of the hepatitis B virus (HBV) DNA to prevent disease progression leading to the development of cirrhosis and hepatocellular carcinoma. Aim To summarise current literature on therapy of chronic hepatitis B, with a focus on indications for therapy, preferred treatment options, and management of resistance and partial responders. Methods A systematic review of the literature, with a focus on international guidelines, was performed. Results Seven drugs are licensed for the treatment of chronic hepatitis B in many countries. The selection of a drug with high potency and low rate of resistance is essential to achieve rapid and long-term viral suppression. The prevention of the sequelae of antiviral drug resistance and appropriate management of viral breakthrough are major goals of current management. The addition or change to an antiviral agent that is not cross-resistant is critical to restore suppression of viral replication for patients with breakthrough resistance. Patient adherence to medication is essential to achieve adequate HBV DNA suppression. Conclusions The current treatment strategy of chronic hepatitis B is now standard: initial selection of entecavir, tenofovir, or peginterferon alfa-2a. Future studies are required to determine if combination therapy using two oral agents or peginterferon with an oral agent with a high genetic barrier to resistance might be superior to standard current monotherapy. 2011 Blackwell Publishing Ltd.PMID: 21978243 [PubMed - in process] -- 31) Bioinformatics. 2011 Oct 3. [Epub ahead of print] ProfileChaser: searching microarray repositories based on genome-wide patterns of differential expression. Engreitz JM, Chen R, Morgan AA, Dudley JT, Mallelwar R, Butte AJ. Division of Systems Medicine, Department of Pediatrics, Department of Bioengineering, Lucile Packard Children's Hospital, and Biomedical Informatics Training Program, Stanford University School of Medicine, Stanford, CA, USA; and Optra Systems Pvt. Ltd, 1, Dnyanesh, CTS No. 1179/3, Modern College Road, Shivajinagar, Pune, 411 005, India. SUMMARY:We introduce ProfileChaser, a web server that allows for querying the Gene Expression Omnibus (GEO) based on genome-wide patterns of differential expression. Using a novel, content-based approach, ProfileChaser retrieves expression profiles that match the differentially regulated transcriptional programs in a user-supplied experiment. This analysis identifies statistical links to similar expression experiments from the vast array of publicly available data on diseases, drugs, phenotypes, and other experimental conditions. AVAILABILITY:http://profilechaser.stanford.edu CONTACT:abutte@stanford.edu SUPPLEMENTARY INFORMATION:Supplementary methods and figures are available at Bioinformatics online.PMID: 21967760 [PubMed - as supplied by publisher] -- 32)Neucleic Acids Res. 2011 Sep 30. [Epub ahead of print] Simplified RNA secondary structure mapping by automation of SHAPE data analysis. Pang PS, Elazar M, Pham EA, Glenn JS. Department of Medicine, Stanford University Medical Center and Palo Alto Veterans Administration Medical Center, Palo Alto, CA, USA. SHAPE (Selective 2'-hydroxyl acylation analysed by primer extension) technology has emerged as one of the leading methods of determining RNA secondary structure at the nucleotide level. A significant bottleneck in using SHAPE is the complex and time-consuming data processing that is required. We present here a modified data collection method and a series of algorithms, embodied in a program entitled Fast Analysis of SHAPE traces (FAST), which significantly reduces processing time. We have used this method to resolve the secondary structure of the first <"900 nt of the hepatitis C virus (HCV) genome, including the entire core gene. We have also demonstrated the ability of SHAPE/FAST to detect the binding of a small molecule inhibitor to the HCV internal ribosomal entry site (IRES). In conclusion, FAST allows for high-throughput data processing to match the current high-throughput generation of data possible with SHAPE, reducing the barrier to determining the structure of RNAs of interest. PMID: 21965531 [PubMed - as supplied by publisher] -- 33) Nat Biotechnol. 2011 Oct 2;29(10):886-91. doi: 10.1038/nbt.1991. Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE. Qiu P, Simonds EF, Bendall SC, Gibbs KD Jr, Bruggner RV, Linderman MD, Sachs K, Nolan GP, Plevritis SK. 1] Department of Radiology, Stanford University, Stanford, California, USA. [2] Department of Bioinformatics and Computational Biology, University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA. The ability to analyze multiple single-cell parameters is critical for understanding cellular heterogeneity. Despite recent advances in measurement technology, methods for analyzing high-dimensional single-cell data are often subjective, labor intensive and require prior knowledge of the biological system. To objectively uncover cellular heterogeneity from single-cell measurements, we present a versatile computational approach, spanning-tree progression analysis of density-normalized events (SPADE). We applied SPADE to flow cytometry data of mouse bone marrow and to mass cytometry data of human bone marrow. In both cases, SPADE organized cells in a hierarchy of related phenotypes that partially recapitulated well-described patterns of hematopoiesis. We demonstrate that SPADE is robust to measurement noise and to the choice of cellular markers. SPADE facilitates the analysis of cellular heterogeneity, the identification of cell types and comparison of functional markers in response to perturbations. PMID: 21964415 [PubMed - in process] PMCID: PMC3196363 [Available on 2012/4/2] -- 34) Nat Biotechnol. 2011 Oct 2;29(10):928-33. doi: 10.1038/nbt.1977. Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding. Lu R, Neff NF, Quake SR, Weissman IL. Institute for Stem Cell Biology and Regenerative Medicine and the Ludwig Center, School of Medicine, Stanford University, Stanford, California, USA. Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single-cell transplantation studies but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggest that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse after irradiation. This technique can be applied to any virus-accessible cell type for both in vitro and in vivo processes.PMID: 21964413 [PubMed - in process] PMCID: PMC3196379 [Available on 2012/4/2] -- 35) J Vasc Interv Radiol. 2011 Oct;22(10):1364-1371.e1. Consolidation of hepatic arterial inflow by embolization of variant hepatic arteries in preparation for yttrium-90 radioembolization. Abdelmaksoud MH, Louie JD, Kothary N, Hwang GL, Kuo WT, Hofmann LV, Hovsepian DM, Sze DY. Division of Interventional Radiology, Stanford University Medical Center, Stanford, CA 94305-5642, USA. PURPOSE:Before yttrium-90 ((90)Y) radioembolization administration, the authors consolidated arterial inflow by embolizing variant hepatic arteries (HAs) to make microsphere delivery simpler and safer. The present study reviews the technical and clinical success of these consolidation procedures. MATERIALS AND METHODS:Preparatory and treatment angiograms were retrospectively analyzed for 201 patients. Variant HAs were coil-embolized during preparatory angiography to simplify arterial anatomy. Collateral arterial perfusion of territories previously supplied by variant HAs was evaluated by digital subtraction angiography (DSA), C-arm computed tomography (CT), and technetium-99m ((99m)Tc)-macroaggregated albumin (MAA) scintigraphy, and by follow-up evaluation of regional tumor response. RESULTS:A total of 47 variant HAs were embolized in 43 patients. After embolization of variant HAs, cross-perfusion into the embolized territory was depicted by DSA and by C-arm CT in 100% of patients and by (99m)Tc-MAA scintigraphy in 92.7%. Uniform progressive disease prevented evaluation in 33% of patients, but regional tumor response in patients who responded supported successful delivery of microspheres to the embolized territories in 95.5% of evaluable patients. CONCLUSIONS:Embolization of variant HAs for consolidation of hepatic supply in preparation for (90)Y radioembolization promotes treatment of affected territories via intrahepatic collateral channels.Copyright 2011 SIR. Published by Elsevier Inc. All rights reserved. PMID: 21961981 [PubMed - in process] -- 37) J Vasc Interv Radiol. 2011 Oct;22(10):1355-62. Embolization of parasitized extrahepatic arteries to reestablish intrahepatic arterial supply to tumors before yttrium-90 radioembolization. Abdelmaksoud MH, Louie JD, Kothary N, Hwang GL, Kuo WT, Hofmann LV, Hovsepian DM, Sze DY. Division of Interventional Radiology, H-3646, Stanford University Medical Center, Stanford, CA 94305-5642, USA. PURPOSE:To perform embolization of parasitized extrahepatic arteries (EHAs) before radioembolization to reestablish intrahepatic arterial supply to large, peripheral tumors, and to evaluate the technical and clinical outcomes of this intervention. MATERIALS AND METHODS:Among 201 patients retrospectively analyzed, embolization of 73 parasitized EHAs in 35 patients was performed. Most embolization procedures were performed during preparatory angiography using large particles and coils. Digital subtraction angiography (DSA), C-arm computed tomography (CT), and technetium-99m macroaggregated albumin ((99m)TcMAA) scintigraphy were used to evaluate the immediate perfusion via intrahepatic collateral channels of target tumor areas previously supplied by parasitized EHAs. Follow-up imaging of differential regional tumor response was used to evaluate microsphere distribution and clinical outcome. RESULTS:After embolization, reestablishment of intrahepatic arterial supply was confirmed by both DSA and C-arm CT in 94% of territories and by scintigraphy in 96%. In 32% of patients, the differential response of treatment could not be evaluated because of uniform disease progression. However, symmetric regional tumor response in 94% of evaluable patients indicated successful delivery of microspheres to the territories previously supplied by parasitized EHAs. CONCLUSIONS:Reestablishment of intrahepatic arterial inflow to hepatic tumors by embolization of parasitized EHAs is safe and effective and results in successful delivery of yttrium-90 microspheres to tumors previously perfused by parasitized EHAs. Copyright 2011 SIR. Published by Elsevier Inc. All rights reserved. PMID: 21961979 [PubMed - in process] -- 37) Circulation. 2011 Sep 13;124(11 Suppl):S46-54. Double knockdown of prolyl hydroxylase and factor-inhibiting hypoxia-inducible factor with nonviral minicircle gene therapy enhances stem cell mobilization and angiogenesis after myocardial infarction. Huang M, Nguyen P, Jia F, Hu S, Gong Y, de Almeida PE, Wang L, Nag D, Kay MA, Giaccia AJ, Robbins RC, Wu JC. Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305-5454, USA. BACKGROUND:Under normoxic conditions, hypoxia-inducible factor (HIF)-1 is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1 mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure. METHODS AND RESULTS:PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1 protein and angiogenesis genes, respectively. CONCLUSIONS:We demonstrated that HIF-1 upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.PMID: 21911818 [PubMed - indexed for MEDLINE] PMCID: PMC3181087 [Available on 2012/9/13] -- 38) Circulation. 2011 Sep 13;124(11 Suppl):S3-9. Human leukocyte antigen I knockdown human embryonic stem cells induce host ignorance and achieve prolonged xenogeneic survival. Deuse T, Seifert M, Phillips N, Fire A, Tyan D, Kay M, Tsao PS, Hua X, Velden J, Eiermann T, Volk HD, Reichenspurner H, Robbins RC, Schrepfer S. Department of Cardiovascular Surgery, Stanford University, Stanford, CA, USA. BACKGROUND:Although human embryonic stem cells (hESC) have enormous potential for cell replacement therapy of heart failure, immune rejection of hESC derivatives inevitably would occur after transplantation. We therefore aimed to generate a hypoantigeneic hESC line with improved survival characteristics. METHODS AND RESULTS:Using various in vivo, nonischemic, hindlimb xenotransplant models (immunocompetent and defined immunodefective mouse strains) as well as human in vitro T-cell and natural killer (NK)-cell assays, we revealed a central role for T cells in mediating hESC rejection. The NK-cell susceptibility of hESC in vivo was found to be low, and the NK response to hESC challenge in vitro was negligible. To reduce the antigenicity of hESC, we successfully generated human leukocyte antigen (HLA) I knockdown cells (hESC(siRNA+IB)) using both HLA I RNA interference (siRNA) and intrabody (IB) technology. HLA I expression was H"99% reduced after 7 days and remained low for weeks. Cellular immune recognition of these hESC(siRNA+IB) was strongly reduced in both xenogeneic and allogeneic settings. Immune rejection was profoundly mitigated after hESC(siRNA+IB) transplantation into immunocompetent mice, and even long-term graft survival was achieved in one third of the animals without any immunosuppression. The survival benefit of hESC(siRNA+IB) was further confirmed under ischemic conditions in a left anterior descending coronary artery ligation model. CONCLUSIONS:HLA I knockdown hESC(siRNA+IB) provoke T-cell ignorance and experience largely mitigated xenogeneic rejection. By generating hypoantigeneic hESC lines, the generation of acceptable hESC derivatives may become a practical concept and push cell replacement strategies forward. PMID: 21911816 [PubMed - indexed for MEDLINE] -- 39) Circulation. 2011 Sep 13;124(11 Suppl):S27-34. Novel microRNA prosurvival cocktail for improving engraftment and function of cardiac progenitor cell transplantation. Hu S, Huang M, Nguyen PK, Gong Y, Li Z, Jia F, Lan F, Liu J, Nag D, Robbins RC, Wu JC. Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305-5454, USA. BACKGROUND:Although stem cell therapy has provided a promising treatment for myocardial infarction, the low survival of the transplanted cells in the infarcted myocardium is possibly a primary reason for failure of long-term improvement. Therefore, the development of novel prosurvival strategies to boost stem cell survival will be of significant benefit to this field. METHODS AND RESULTS:Cardiac progenitor cells (CPCs) were isolated from transgenic mice, which constitutively express firefly luciferase and green fluorescent protein. The CPCs were transduced with individual lentivirus carrying the precursor of miR-21, miR-24, and miR-221, a cocktail of these 3 microRNA precursors, or green fluorescent protein as a control. After challenge in serum free medium, CPCs treated with the 3 microRNA cocktail showed significantly higher viability compared with untreated CPCs. After intramuscular and intramyocardial injections, in vivo bioluminescence imaging showed that microRNA cocktail-treated CPCs survived significantly longer after transplantation. After left anterior descending artery ligation, microRNA cocktail-treated CPCs boost the therapeutic efficacy in terms of functional recovery. Histological analysis confirmed increased myocardial wall thickness and CPC engraftment in the myocardium with the microRNA cocktail. Finally, we used bioinformatics analysis and experimental validation assays to show that Bim, a critical apoptotic activator, is an important target gene of the microRNA cocktail, which collectively can bind to the 3'UTR region of Bim and suppress its expression. CONCLUSIONS:We have demonstrated that a microRNA prosurvival cocktail (miR-21, miR-24, and miR-221) can improve the engraftment of transplanted cardiac progenitor cells and therapeutic efficacy for treatment of ischemic heart disease. PMID: 21911815 [PubMed - indexed for MEDLINE] PMCID: PMC3181082 [Available on 2012/9/13] -- 40) Circulation. 2011 Sep 13;124(11 Suppl):S187-96. Interleukin-17 accelerates allograft rejection by suppressing regulatory T cell expansion. Itoh S, Kimura N, Axtell RC, Velotta JB, Gong Y, Wang X, Kajiwara N, Nambu A, Shimura E, Adachi H, Iwakura Y, Saito H, Okumura K, Sudo K, Steinman L, Robbins RC, Nakae S, Fischbein MP. Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA. BACKGROUND:Interleukin-17 (IL-17), which is predominantly produced by T helper 17 cells distinct from T helper 1 or T helper 2 cells, participates in the pathogenesis of infectious, autoimmune, and allergic disorders. However, the precise role in allograft rejection remains uncertain. In the present study, we investigated the role of IL-17 in acute allograft rejection using IL-17-deficient mice. METHODS AND RESULTS:Donor hearts from FVB mice were heterotopically transplanted into either C57BL/6J-IL-17-deficient (IL-17(-/-)) or -wild-type mice. Allograft survival was significantly prolonged in IL-17(-/-) recipient mice due to reduced local inflammation accompanied by decreased inflammatory cell recruitment and cytokine/chemokine expression. IL-17(-/-) recipient mice exhibited decreased IL-6 production and reciprocally enhanced regulatory T cell expansion, suggesting a contribution of regulatory T cells to prolonged allograft survival. Indeed, allografts transplanted into anti-CD25 mAb-treated IL-17(-/-) recipient mice (regulatory T cell-depleted) developed acute rejection similar to wild-type recipient mice. Surprisingly, we found that gamma delta T cells rather than CD4(+) and CD8(+) T cells were key IL-17 producers in the allografts. In support, equivalent allograft rejection was observed in Rag-2(-/-) recipient mice engrafted with either wild-type or IL-17(-/-) CD4(+) and CD8(+) T cells. Finally, hearts transplanted into gamma delta T cell-deficient mice resulted in decreased allograft rejection compared with wild-type controls. CONCLUSIONS:During heart transplantation, (1) IL-17 is crucial for acceleration of acute rejection; (2) IL-17-deficiency enhances regulatory T cell expansion; and (3) gamma delta T cells rather than CD4(+) and CD8(+) T cells are a potential source of IL-17. IL-17 neutralization may provide a potential target for novel therapeutic treatment for cardiac allograft rejection. PMID: 21911812 [PubMed - indexed for MEDLINE] -- 41) Science. 2011 Oct 7;334(6052):89-94. Epub 2011 Aug 25. The shaping of modern human immune systems by multiregional admixture with archaic humans. Abi-Rached L, Jobin MJ, Kulkarni S, McWhinnie A, Dalva K, Gragert L, Babrzadeh F, Gharizadeh B, Luo M, Plummer FA, Kimani J, Carrington M, Middleton D, Rajalingam R, Beksac M, Marsh SG, Maiers M, Guethlein LA, Tavoularis S, Little AM, Green RE, Norman PJ, Parham P. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. Whole genome comparisons identified introgression from archaic to modern humans. Our analysis of highly polymorphic human leukocyte antigen (HLA) class I, vital immune system components subject to strong balancing selection, shows how modern humans acquired the HLA-B*73 allele in west Asia through admixture with archaic humans called Denisovans, a likely sister group to the Neandertals. Virtual genotyping of Denisovan and Neandertal genomes identified archaic HLA haplotypes carrying functionally distinctive alleles that have introgressed into modern Eurasian and Oceanian populations. These alleles, of which several encode unique or strong ligands for natural killer cell receptors, now represent more than half the HLA alleles of modern Eurasians and also appear to have been later introduced into Africans. Thus, adaptive introgression of archaic alleles has significantly shaped modern human immune systems. PMID: 21868630 [PubMed - indexed for MEDLINE] -- 43) Cell. 2011 Aug 19;146(4):621-32. Structural linkage between ligand discrimination and receptor activation by type I interferons. Thomas C, Moraga I, Levin D, Krutzik PO, Podoplelova Y, Trejo A, Lee C, Yarden G, Vleck SE, Glenn JS, Nolan GP, Piehler J, Schreiber G, Garcia KC. Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA. Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFN2 and IFN reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns. Copyright 2011 Elsevier Inc. All rights reserved. Comment in * Nat Rev Immunol. 2011 Oct;11(10):640. PMID: 21854986 -- 43) Am Heart J. 2011 Aug;162(2):324-30. Epub 2011 Jul 18. Angiotensin-converting enzyme inhibitors and cardiovascular outcomes in patients on maintenance hemodialysis. Chang TI, Shilane D, Brunelli SM, Cheung AK, Chertow GM, Winkelmayer WC. Division of Nephrology, Stanford University School of Medicine, Palo Alto, CA 94304, USA. BACKGROUND:Persons with end-stage renal disease (ESRD) on hemodialysis carry an exceptionally high burden of cardiovascular disease. Angiotensin-converting enzyme inhibitors (ACEIs) are recommended for patients on dialysis, but there are few data regarding their effectiveness in ESRD. METHODS:We conducted a secondary analysis of results of the HEMO study, a randomized trial of dialysis dose and membrane flux in patients on maintenance hemodialysis. We focused on the nonrandomized exposure of ACEI use, using proportional hazards regression and a propensity score analysis. The primary outcome was all-cause mortality. Secondary outcomes examined in the present analysis were cardiovascular hospitalization, heart failure hospitalization, and the composite outcomes of death or cardiovascular hospitalization and death or heart failure hospitalization. RESULTS:In multivariable-adjusted analyses, there were no significant associations among ACEI use and mortality (hazard ratio 0.97, 95% CI 0.82-1.14), cardiovascular hospitalization, and either composite outcome. Angiotensin-converting enzyme inhibitor use was associated with a higher risk of heart failure hospitalization (hazard ratio 1.41, 95% CI 1.11-1.80). In the propensity score-matched cohort, ACEI use was not significantly associated with any outcomes, including heart failure hospitalization. CONCLUSIONS:In a well-characterized cohort of patients on maintenance hemodialysis, ACEI use was not significantly associated with mortality or cardiovascular morbidity. The higher risk of heart failure hospitalization associated with ACEI use may not only reflect residual confounding but also highlights gaps in evidence when applying treatments proven effective in the general population to patients with ESRD. Our results underscore the need for definitive trials in ESRD to inform the treatment of cardiovascular disease. Copyright 2011 Mosby, Inc. All rights reserved.PMID: 21835294 [PubMed - indexed for MEDLINE] -- 44) Proc Natl Acad Sci USA. 2011 Aug 9;108(32):13287-92. Epub 2011 Jul 26. Systemic augmentation of alphaB-crystallin provides therapeutic benefit twelve hours post-stroke onset via immune modulation. Arac A, Brownell SE, Rothbard JB, Chen C, Ko RM, Pereira MP, Albers GW, Steinman L, Steinberg GK. Department of Neurosurgery, Stanford Institute for Neuro-Innovation and Translational Neurosciences, Stanford Stroke Center, Stanford University School of Medicine, Stanford, CA 94305, USA. Tissue plasminogen activator is the only treatment option for stroke victims; however, it has to be administered within 4.5 h after symptom onset, making its use very limited. This report describes a unique target for effective treatment of stroke, even 12 h after onset, by the administration of B-crystallin (Cryab), an endogenous immunomodulatory neuroprotectant. In Cryab(-/-) mice, there was increased lesion size and diminished neurologic function after stroke compared with wild-type mice. Increased plasma Cryab was detected after experimental stroke in mice and after stroke in human patients. Administration of Cryab even 12 h after experimental stroke reduced both stroke volume and inflammatory cytokines associated with stroke pathology. Cryab is an endogenous anti-inflammatory and neuroprotectant molecule produced after stroke, whose beneficial properties can be augmented when administered therapeutically after stroke. PMID: 21828004 [PubMed - indexed for MEDLINE] PMCID: PMC3156222 [Available on 2012/2/9] -- 45) Arch Intern Med. 2011 Aug 8;171(15):1371-8. Predialysis nephrology care of older patients approaching end-stage renal disease. Winkelmayer WC, Liu J, Chertow GM, Tamura MK. Division of Pharmacoepidemiology and Pharmacoeconomics, Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts, USA. wcw1@stanford.edu BACKGROUND:Little is known about trends in the timing of first nephrology consultation and associated outcomes among older patients initiating dialysis. METHODS:Data from patients aged 67 years or older who initiated dialysis in the United States between January 1, 1996, and December 31, 2006, were stratified by timing of the earliest identifiable nephrology visit. Trends of earlier nephrology consultation were formally examined in light of concurrently changing case mix and juxtaposed with trends in 1-year mortality rates after initiation of dialysis. RESULTS:Among 323,977 older patients initiating dialysis, the proportion of patients receiving nephrology care less than 3 months before initiation of dialysis decreased from 49.6% (in 1996) to 34.7% (in 2006). Patients initiated dialysis with increasingly preserved kidney function, from a mean estimated glomerular filtration rate of 8 mL/min/1.73 m(2) in 1996 to 12 mL/min/1.73 m(2) in 2006. Patients were less anemic in later years, which was partly attributable to increased use of erythropoiesis-stimulating agents, and fewer used peritoneal dialysis as the initial modality. During the same period, crude 1-year mortality rates remained unchanged (annual change in mortality rate, +0.2%; 95% confidence interval, 0% to +0.4%). Adjustment for changes in demographic and comorbidity patterns yielded estimated annual reductions in 1-year mortality rates of 0.9% (95% confidence interval, 0.7% to 1.1%), which were explained only partly by concurrent trends toward earlier nephrology consultation (annual mortality reduction after accounting for timing of nephrology care was attenuated to 0.4% [0.2% to 0.6%]). CONCLUSIONS:Despite significant trends toward earlier use of nephrology consultation among older patients approaching maintenance dialysis, we observed no material improvement in 1-year survival rates after dialysis initiation during the same time period. Comment in * Arch Intern Med. 2011 Aug 8;171(15):1317-8. * Arch Intern Med. 2011 Aug 8;171(15):1378. PMID: 21824952 [PubMed - indexed for MEDLINE] -- 46) J Clin Invest. 2011 Sep 1;121(9):3517-27. doi: 10.1172/JCI46387. Epub 2011 Aug 1. Plasma carboxypeptidase B downregulates inflammatory responses in autoimmune arthritis. Song JJ, Hwang I, Cho KH, Garcia MA, Kim AJ, Wang TH, Lindstrom TM, Lee AT, Nishimura T, Zhao L, Morser J, Nesheim M, Goodman SB, Lee DM, Bridges SL Jr; Consortium for the Longitudinal Evaluation of African Americans with Early Rheumatoid Arthritis (CLEAR) Registry, Gregersen PK, Leung LL, Robinson WH. Collaborators (7) Moreland LW, Howard G, Conn DL, Jonas BL, Callahan LF, Smith EA, Brasington RD Jr. Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California, USA. The immune and coagulation systems are both implicated in the pathogenesis of rheumatoid arthritis (RA). Plasma carboxypeptidase B (CPB), which is activated by the thrombin/thrombomodulin complex, plays a procoagulant role during fibrin clot formation. However, an antiinflammatory role for CPB is suggested by the recent observation that CPB can cleave proinflammatory mediators, such as C5a, bradykinin, and osteopontin. Here, we show that CPB plays a central role in downregulating C5a-mediated inflammatory responses in autoimmune arthritis. CPB deficiency exacerbated inflammatory arthritis in a mouse model of RA, and cleavage of C5a by CPB suppressed the ability of C5a to recruit immune cells in vivo. In human patients with RA, genotyping of nonsynonymous SNPs in the CPB-encoding gene revealed that the allele encoding a CPB variant with longer half-life was associated with a lower risk of developing radiographically severe RA. Functionally, this CPB variant was more effective at abrogating the proinflammatory properties of C5a. Additionally, expression of both CPB and C5a in synovial fluid was higher in patients with RA than in those with osteoarthritis. These findings suggest that CPB plays a critical role in dampening local, C5a-mediated inflammation and represents a molecular link between inflammation and coagulation in autoimmune arthritis. Comment in * Nat Rev Rheumatol. 2011;7(10):558. PMID: 21804193 [PubMed - indexed for MEDLINE] PMCID: PMC3163960 -- 47) Stroke. 2011 Sep;42(9):2485-91. Epub 2011 Jul 28. Arterial spin-labeling MRI can identify the presence and intensity of collateral perfusion in patients with moyamoya disease. Zaharchuk G, Do HM, Marks MP, Rosenberg J, Moseley ME, Steinberg GK. Stanford University Medical Center, 1201 Welch Road, Mailcode 5488, Stanford, CA 94305-5488, USA. gregz@stanford.edu BACKGROUND AND PURPOSE:Determining the presence and adequacy of collateral blood flow is important in cerebrovascular disease. Therefore, we explored whether a noninvasive imaging modality, arterial spin labeling (ASL) MRI, could be used to detect the presence and intensity of collateral flow using digital subtraction angiography (DSA) and stable xenon CT cerebral blood flow as gold standards for collaterals and cerebral blood flow, respectively. METHODS:ASL and DSA were obtained within 4 days of each other in 18 patients with Moyamoya disease. Two neurointerventionalists scored DSA images using a collateral grading scale in regions of interest corresponding to ASPECTS methodology. Two neuroradiologists similarly scored ASL images based on the presence of arterial transit artifact. Agreement of ASL and DSA consensus scores was determined, including kappa statistics. In 15 patients, additional quantitative xenon CT cerebral blood flow measurements were performed and compared with collateral grades. RESULTS:The agreement between ASL and DSA consensus readings was moderate to strong, with a weighted kappa value of 0.58 (95% confidence interval, 0.52-0.64), but there was better agreement between readers for ASL compared with DSA. Sensitivity and specificity for identifying collaterals with ASL were 0.83 (95% confidence interval, 0.77-0.88) and 0.82 (95% confidence interval, 0.76-0.87), respectively. Xenon CT cerebral blood flow increased with increasing DSA and ASL collateral grade (P<0.05). CONCLUSIONS:ASL can noninvasively predict the presence and intensity of collateral flow in patients with Moyamoya disease using DSA as a gold standard. Further study of other cerebrovascular diseases, including acute ischemic stroke, is warranted. PMID: 21799169 [PubMed - indexed for MEDLINE] PMCID: PMC3164217 [Available on 2012/9/1] -- 48) J Virol. 2011 Oct;85(19):10101-8. Epub 2011 Jul 27. Escape from transcriptional shutoff during poliovirus infection: NF-B-responsive genes IBa and A20. Doukas T, Sarnow P. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. It has been known for a long time that infection of cultured cells with poliovirus results in the overall inhibition of transcription of most host genes. We examined whether selected host genes can escape transcriptional inhibition by thiouridine marking newly synthesized host mRNAs during viral infection. Using cDNA microarrays hybridized to cDNAs made from thiolated mRNAs, a small set of host transcripts was identified and their expression verified by quantitative PCR and Northern and Western blot analyses. These transcripts were synthesized from genes that displayed enrichment for NF-B binding sites in their promoter regions, suggesting that some NF-B-regulated promoters can escape the virus-induced inhibition of transcription. In particular, two negative regulators of NF-B, IBa and A20, were upregulated during viral infection. Depletion of A20 enhanced viral RNA abundance and viral yield, arguing that cells respond to virus infection by counteracting NF-B-induced proviral effects. PMID: 21795344 [PubMed - indexed for MEDLINE] PMCID: PMC3196431 [Available on 2012/4/1] -- 51) Circulation. 2011 Sep 13;124(11 Suppl):S187-96. Interleukin-17 accelerates allograft rejection by suppressing regulatory T cell expansion. Itoh S, Kimura N, Axtell RC, Velotta JB, Gong Y, Wang X, Kajiwara N, Nambu A, Shimura E, Adachi H, Iwakura Y, Saito H, Okumura K, Sudo K, Steinman L, Robbins RC, Nakae S, Fischbein MP. Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA. BACKGROUND:Interleukin-17 (IL-17), which is predominantly produced by T helper 17 cells distinct from T helper 1 or T helper 2 cells, participates in the pathogenesis of infectious, autoimmune, and allergic disorders. However, the precise role in allograft rejection remains uncertain. In the present study, we investigated the role of IL-17 in acute allograft rejection using IL-17-deficient mice. METHODS AND RESULTS:Donor hearts from FVB mice were heterotopically transplanted into either C57BL/6J-IL-17-deficient (IL-17(-/-)) or -wild-type mice. Allograft survival was significantly prolonged in IL-17(-/-) recipient mice due to reduced local inflammation accompanied by decreased inflammatory cell recruitment and cytokine/chemokine expression. IL-17(-/-) recipient mice exhibited decreased IL-6 production and reciprocally enhanced regulatory T cell expansion, suggesting a contribution of regulatory T cells to prolonged allograft survival. Indeed, allografts transplanted into anti-CD25 mAb-treated IL-17(-/-) recipient mice (regulatory T cell-depleted) developed acute rejection similar to wild-type recipient mice. Surprisingly, we found that gamma delta T cells rather than CD4(+) and CD8(+) T cells were key IL-17 producers in the allografts. In support, equivalent allograft rejection was observed in Rag-2(-/-) recipient mice engrafted with either wild-type or IL-17(-/-) CD4(+) and CD8(+) T cells. Finally, hearts transplanted into gamma delta T cell-deficient mice resulted in decreased allograft rejection compared with wild-type controls. CONCLUSIONS:During heart transplantation, (1) IL-17 is crucial for acceleration of acute rejection; (2) IL-17-deficiency enhances regulatory T cell expansion; and (3) gamma delta T cells rather than CD4(+) and CD8(+) T cells are a potential source of IL-17. IL-17 neutralization may provide a potential target for novel therapeutic treatment for cardiac allograft rejection. PMID: 21911812 [PubMed - indexed for MEDLINE] -- 50) Plast Reconstr Surg. 2011 Aug;128(2):373-86. Differences in osteogenic differentiation of adipose-derived stromal cells from murine, canine, and human sources in vitro and in vivo. Levi B, Nelson ER, Brown K, James AW, Xu D, Dunlevie R, Wu JC, Lee M, Wu B, Commons GW, Vistnes D, Longaker MT. Hagey Pediatric Regenerative Medicine Research Laboratory, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA 94305-5148, USA. BACKGROUND:Given the diversity of species from which adipose-derived stromal cells are derived and studied, the authors set out to delineate the differences in the basic cell biology that may exist across species. Briefly, the authors found that significant differences exist with regard to proliferation and osteogenic potentials of adipose-derived stromal cells across species. METHODS:Adipose-derived stromal cells were derived from human, mouse, and canine sources as previously described. Retinoic acid, insulin-like growth factor-1, and bone morphogenetic protein-2 were added to culture medium; proliferation and osteogenic differentiation were assessed by standardized assays. In vivo methods included seeding 150,000 adipose-derived stromal cells on a biomimetic scaffold and analyzing healing by micro-computed tomography and histology. RESULTS:Adipose-derived stromal cells from all species had the capability to undergo osteogenic differentiation. Canine adipose-derived stromal cells were the most proliferative, whereas human adipose-derived stromal cells were the most osteogenic (p < 0.05). Human cells, however, had the most significant osteogenic response to osteogenic media. Retinoic acid stimulated osteogenesis in mouse and canine cells but not in human adipose-derived stromal cells. Insulin-like growth factor-1 enhanced osteogenesis across all species, most notably in human- and canine-derived cells. CONCLUSIONS:Adipose-derived stromal cells derived from human, mouse, and canine all have the capacity to undergo osteogenic differentiation. Canine adipose-derived stromal cells appear to be the most proliferative, whereas human adipose-derived stromal cells appear to be the most osteogenic. Different cytokines and chemicals can be used to modulate this osteogenic response. These results are promising as attempts are made to optimize tissue-engineered bone using adipose-derived stromal cells.PMID: 21788829 [PubMed - indexed for MEDLINE] -- 50) Proc Natl Acad Sci USA. 2011 Aug 9;108(32):13299-304. Epub 2011 Jul 25. Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1). Hoover PJ, Lewis RS. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA. Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of <"2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.PMID: 21788510 [PubMed - indexed for MEDLINE] PMCID: PMC3156176 [Available on 2012/2/9] -- 51) J Clin Invest. 2011 Aug 1;121(8):3109-19. doi: 10.1172/JCI57834. Epub 2011 Jul 25. Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies. Sasaki S, Sullivan M, Narvaez CF, Holmes TH, Furman D, Zheng NY, Nishtala M, Wrammert J, Smith K, James JA, Dekker CL, Davis MM, Wilson PC, Greenberg HB, He XS. Department of Immunology and Microbiology, Stanford University School of Medicine, Stanford, California, USA. During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies. Comment in * J Clin Invest. 2011 Aug 1;121(8):2981-3. PMID: 21785218 [PubMed - indexed for MEDLINE] PMCID: PMC3148747 -- 52) J Clin Invest. 2011 Aug 1;121(8):3109-19. doi: 10.1172/JCI57834. Epub 2011 Jul 25. Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies. Sasaki S, Sullivan M, Narvaez CF, Holmes TH, Furman D, Zheng NY, Nishtala M, Wrammert J, Smith K, James JA, Dekker CL, Davis MM, Wilson PC, Greenberg HB, He XS. Department of Immunology and Microbiology, Stanford University School of Medicine, Stanford, California, USA. During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies. Comment in * J Clin Invest. 2011 Aug 1;121(8):2981-3. PMID: 21785218 [PubMed - indexed for MEDLINE] PMCID: PMC3148747 -- 52) J Immunol. 2011 Sep 1;187(5):2442-52. Epub 2011 Jul 20. An insertion mutant in DQA1*0501 restores susceptibility to HLA-DM: implications for disease associations. Hou T, Macmillan H, Chen Z, Keech CL, Jin X, Sidney J, Strohman M, Yoon T, Mellins ED. Department of Pediatrics, Program in Immunology, Stanford University, Stanford, CA 94305, USA. HLA-DM (DM) catalyzes CLIP release, stabilizes MHC class II molecules, and edits the peptide repertoire presented by class II. Impaired DM function may have profound effects on Ag presentation events in the thymus and periphery that are critical for maintenance of self-tolerance. The associations of the HLA-DQ2 (DQ2) allele with celiac disease and type 1 diabetes mellitus have been appreciated for a long time. The explanation for these associations, however, remains unknown. We previously found that DQ2 is a poor substrate for DM. In this study, to further characterize DQ2-DM interaction, we introduced point mutations into DQ2 on the proposed DQ2-DM interface to restore the sensitivity of DQ2 to DM. The effects of mutations were investigated by measuring the peptide dissociation and exchange rate in vitro, CLIP and DQ2 expression on the cell surface, and the presentation of -II-gliadin epitope (residues 62-70) to murine, DQ2-restricted T cell hybridomas. We found that the three -chain mutations (+53G, +53R, or Y22F) decreased the intrinsic stability of peptide-class II complex. More interestingly, the +53G mutant restored DQ2 sensitivity to DM, likely due to improved interaction with DM. Our data also suggest that -II-gliadin 62-70 is a DM-suppressed epitope. The DQ2 resistance to DM changes the fate of this peptide from a cryptic to an immunodominant epitope. Our findings elucidate the structural basis for reduced DQ2-DM interaction and have implications for mechanisms underlying disease associations of DQ2. PMID: 21775680 [PubMed - indexed for MEDLINE] PMCID: PMC3159820 [Available on 2012/9/1] -- 53) PLoS Genet. 2011 Jun;7(6):e1002153. Epub 2011 Jun 30. Dynamic chromatin localization of Sirt6 shapes stress- and aging-related transcriptional networks. Kawahara TL, Rapicavoli NA, Wu AR, Qu K, Quake SR, Chang HY. Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, United States of America. The sirtuin Sirt6 is a NAD-dependent histone deacetylase that is implicated in gene regulation and lifespan control. Sirt6 can interact with the stress-responsive transcription factor NF-B and regulate some NF-B target genes, but the full scope of Sirt6 target genes as well as dynamics of Sirt6 occupancy on chromatin are not known. Here we map Sirt6 occupancy on mouse promoters genome-wide and show that Sirt6 occupancy is highly dynamic in response to TNF-. More than half of Sirt6 target genes are only revealed upon stress-signaling. The majority of genes bound by NF-B subunit RelA recruit Sirt6, and dynamic Sirt6 relocalization is largely driven in a RelA-dependent manner. Integrative analysis with global gene expression patterns in wild-type, Sirt6-/-, and double Sirt6-/- RelA-/- cells reveals the epistatic relationships between Sirt6 and RelA in shaping diverse temporal patterns of gene expression. Genes under the direct joint control of Sirt6 and RelA include several with prominent roles in cell senescence and organismal aging. These data suggest dynamic chromatin relocalization of Sirt6 as a key output of NF-B signaling in stress response and aging.PMID:21738489[PubMed - indexed for MEDLINE] PMCID: PMC3128103 -- 54) J Clin Invest. 2011 Aug 1;121(8):3133-43. Identification of an IFN-/mast cell axis in a mouse model of chronic asthma. Yu M, Eckart MR, Morgan AA, Mukai K, Butte AJ, Tsai M, Galli SJ. Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5176, USA. Asthma is considered a Th2 cellassociated disorder. Despite this, both the Th1 cell associated cytokine IFN- and airway neutrophilia have been implicated in severe asthma. To investigate the relative contributions of different immune system components to the pathogenesis of asthma, we previously developed a model that exhibits several features of severe asthma in humans, including airway neutrophilia and increased lung IFN-. In the present studies, we tested the hypothesis that IFN- regulates mast cell function in our model of chronic asthma. Engraftment of mast cell deficient KitW(-sh/W-sh) mice, which develop markedly attenuated features of disease, with wild-type mast cells restored disease pathology in this model of chronic asthma. However, disease pathology was not fully restored by engraftment with either IFN- receptor 1 null (Ifngr1 / ) or Fc receptor 1 null (Fcer1g / ) mast cells. Additional analysis, including gene array studies, showed that mast cell expression of IFN-R contributed to the development of many FcRI-dependent and some FcRI-independent features of disease in our model, including airway hyperresponsiveness, neutrophilic and eosinophilic inflammation, airway remodeling, and lung expression of several cytokines, chemokines, and markers of an alternatively activated macrophage response. These findings identify a previously unsuspected IFN-/mast cell axis in the pathology of chronic allergic inflammation of the airways in mice. PMID: 21737883 [PubMed - indexed for MEDLINE] PMCID: PMC3148724 -- 55) PLoS One. 2011;6(6):e21211. Epub 2011 Jun 22. An information theoretic, microfluidic-based single cell analysis permits identification of subpopulations among putatively homogeneous stem cells. Glotzbach JP, Januszyk M, Vial IN, Wong VW, Gelbard A, Kalisky T, Thangarajah H, Longaker MT, Quake SR, Chu G, Gurtner GC. Department of Surgery, Stanford University School of Medicine, Stanford, California, United States of America. An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly 'purified' cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation. PMID: 21731674 [PubMed - indexed for MEDLINE] PMCID: PMC3120839 -- 56) J Biol Chem. 2011 Aug 19;286(33):29366-75. Epub 2011 Jun 27. A conserved non-canonical motif in the pseudoactive site of the ROP5 pseudokinase domain mediates its effect on Toxoplasma virulence. Reese ML, Boothroyd JC. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5124, USA. The ROP5 family is a closely related set of polymorphic pseudokinases that are critical to the ability of Toxoplasma to cause disease. Polymorphisms in ROP5 also make it a major determinant of strain-specific differences in virulence. ROP5 possesses all of the major kinase motifs required for catalysis except for a substitution at the catalytic Asp. We show that this substitution in the catalytic loop of ROP5 is part of a motif conserved in other pseudokinases of both Toxoplasma and human origin, and that this motif is required for the full activity in vivo of ROP5. This suggests evolutionary selection at this site for a biochemical function, rather than simple drift away from catalysis. We present the crystal structures of a virulent isoform of ROP5 both in its ATP-bound and -unbound states and have demonstrated that despite maintaining the canonical ATP-binding motifs, ROP5 binds ATP in a distorted conformation mediated by unusual magnesium coordination sites that would not be predicted from the primary sequence. In addition, we have mapped the polymorphisms spread throughout the primary sequence of ROP5 to two major surfaces, including the activation segment of ROP5. This suggests that the pseudoactive site of this class of pseudokinases may have evolved to use the canonical ATP-binding motifs for non-catalytic signaling through allostery. PMID: 21708941 [PubMed - indexed for MEDLINE] PMCID: PMC3190742 [Available on 2012/8/19] -- 57) Methods Mol Biol. 2011;748:169-82. Probing the plasma membrane structure of immune cells through the analysis of membrane sheets by electron microscopy. Lillemeier BF, Davis MM. Howard Hughes Medical Institute and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. This chapter describes a method to generate plasma membrane sheets that are large enough to visualize the membrane architecture and perform quantitative analyses of protein distributions. This procedure places the sheets on electron microscopy grids, parallel to the imaging plane of the microscope, where they can be characterized by transmission electron microscopy. The basic principle of the technique is that cells are broken open ("ripped") through mechanical forces applied by the separation of two opposing surfaces sandwiching the cell, with one of the surfaces coated onto an EM grid. The exposed inner membrane surfaces can then be visualized with electron dense stains and specific proteins can be detected with gold conjugated probes. PMID: 21701974 [PubMed - indexed for MEDLINE] -- 59) Chem Biol. 2011 Jun 24;18(6):722-32. Development of small molecule inhibitors and probes of human SUMO deconjugating proteases. Albrow VE, Ponder EL, Fasci D, Bks M, Deu E, Salvesen GS, Bogyo M. Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA. Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compound therefore represents promising new reagents to study the process of SUMO deconjugation.Copyright 2011 Elsevier Ltd. All rights reserved. PMID: 21700208 [PubMed - indexed for MEDLINE]PMCID: PMC3131534 [Available on 2012/6/24] -- 60) Chem Biol. 2011 Jun 24;18(6):711-21. Functional characterization of a SUMO deconjugating protease of Plasmodium falciparum using newly identified small molecule inhibitors. Ponder EL, Albrow VE, Leader BA, Bks M, Mikolajczyk J, Fonovi UP, Shen A, Drag M, Xiao J, Deu E, Campbell AJ, Powers JC, Salvesen GS, Bogyo M. Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA. Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum.Copyright 2011 Elsevier Ltd. All rights reserved. PMID: 21700207 [PubMed - indexed for MEDLINE] PMCID: PMC3131532 [Available on 2012/6/24] -- 61) J Pediatr Gastroenterol Nutr. 2011 Jul;53(1):40-7. Immunophenotyping of peripheral eosinophils demonstrates activation in eosinophilic esophagitis. Nguyen T, Gernez Y, Fuentebella J, Patel A, Tirouvanziam R, Reshamwala N, Bass D, Berquist WE, Cox KL, Kerner JA, Nadeau KC. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA. BACKGROUND AND AIM:Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder characterized by upper gastrointestinal symptoms and the presence of high numbers of eosinophils in the esophagus. Although eosinophils in the esophagus have been found to be activated in subjects with EoE, detailed studies of intracellular signaling pathways involved in the mechanism of activation of eosinophils in EoE have heretofore been limited. The aim of the study was to assess whether any surface molecules or transcription factors are activated in peripheral eosinophils in subjects with EoE. METHODS:Eosinophils and CD3+ lymphocytes were identified directly from 50 L of whole blood of EoE and control subjects. Using Hi-FACS, levels of surface activation markers, including CD66b, and intracellular phosphoepitopes, including phosphorylated forms of signal transducer and activator of transcription (phospho-STAT) 1 and 6, were measured within each cell subset. RESULTS:Levels of surface CD66b as well as levels of intracellular phospho-STAT1 and phospho-STAT6 in peripheral blood eosinophils were significantly higher for untreated subjects with EoE vs healthy controls (P < 0.05). Levels of phospho-STAT1 and phospho-STAT6 in peripheral blood eosinophils were lower in subjects with EoE on therapy versus untreated subjects with EoE (P < 0.05). CONCLUSIONS:Levels of phospho-STAT1 and phospho-STAT6, transcription factors involved in inflammatory processes, were both significantly higher in peripheral eosinophils from untreated (ie, newly diagnosed) subjects with EoE versus subjects with EoE on therapy, healthy controls. Blood-based measurements of CD66b and phospho-STAT levels in peripheral eosinophils may be beneficial for identifying EoE.PMID: 21694534 [PubMed - indexed for MEDLINE] -- 62) Compr Psychiatry. 2011 Jul-Aug;52(4):405-12. Epub 2010 Oct 30. Medical students as patients: implications of their dual role as explored in a vignette-based survey study of 1027 medical students at nine medical schools. Roberts LW, Warner TD, Smithpeter M, Rogers M, Horwitz R. Department of Psychiatry and Behavioral Sciences, Stanford University, CA 94305-5717, USA. OBJECTIVE:Medical students experience a range of mental and physical illnesses during training and may encounter significant barriers in seeking health care. Little is known about the issues surrounding the dual role as both learner and patient when a medical student seeks care at his or her training institution. METHOD:A confidential survey examining medical students' health care needs, practices, and concerns was administered at 9 US medical schools. One part of the survey focused on responses to 4 medical student-patient vignettes. The vignettes systematically varied preexisting student vs preexisting patient status before assuming a medical student-patient role, and the vignettes also varied illness situations that were more vs less stigmatizing. Responses were analyzed using (2) and multivariate analysis of variance tests. RESULTS:A total of 1027 students participated. We found that students were more likely to accept the dual role as medical student-patient in vignettes depicting a preexisting patient role than a preexisting student role. Students sought to avoid the dual role as student-patient in the context of stigmatizing health concerns. Women students were more likely than men to reject the dual role in all cases. CONCLUSION:Medical students appear to be sensitive to the conflicts that may be associated with the dual role as both medical student and patient when seeking care at their training institution. Our data suggest the importance of substantive efforts to promote the health, interests, and well-being of medical student-patients.Copyright 2011 Elsevier Inc. All rights reserved.PMID: 21683176 [PubMed - indexed for MEDLINE] -- 63) Gastroenterology. 2011 Aug;141(2):707-18, 718.e1-5. Epub 2011 May 4. Wnt--catenin signaling protects against hepatic ischemia and reperfusion injury in mice. Lehwald N, Tao GZ, Jang KY, Sorkin M, Knoefel WT, Sylvester KG. Divison of Pediatric Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148, USA. BACKGROUND & AIMS:Ischemia and reperfusion injury are common causes of oxidative tissue damage associated with many liver diseases and hepatic surgery. The Wnt--catenin signaling pathway is an important regulator of hepatic development, regeneration, and carcinogenesis. However, the role of Wnt signaling in the hepatocellular response to ischemia-reperfusion (I/R) injury has not been determined. METHODS:Hepatic injury following ischemia or I/R was investigated in hepatocyte-specific, -catenin-deficient mice, as well as Wnt1-overexpressing and wild-type (control) mice. RESULTS:Wnt--catenin signaling was affected by the cellular redox balance in hepatocytes. Following ischemia or I/R, mice with -catenin-deficient hepatocytes were significantly more susceptible to liver injury. Conversely, mice that overexpressed Wnt1 in hepatocytes were resistant to hepatic I/R injury. Hypoxia inducible factor (HIF)-1 signaling was reduced in -catenin-deficient liver but increased in hepatocytes that overexpressed Wnt1 under hypoxia and following I/R, indicating an interaction between -catenin and HIF-1 signaling in the liver. The mechanism by which Wnt signaling protects against liver injury involves the role of -catenin as a transcriptional coactivator of HIF-1 signaling, which promotes hepatocyte survival under hypoxic conditions. CONCLUSIONS:Cellular redox balance affects Wnt--catenin signaling, which protects against hypoxia and I/R injury. These findings might be used to develop strategies for protection of hepatocytes, regeneration of liver, and inhibition of carcinogenesis. Copyright 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.PMID: 21679710 [PubMed - indexed for MEDLINE] -- 64) Am J Physiol Heart Circ Physiol. 2011 Sep;301(3):H663-71. Epub 2011 Jun 10. In vivo bioluminescence for tracking cell fate and function. de Almeida PE, van Rappard JR, Wu JC. Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5454, USA. Tracking the fate and function of cells in vivo is paramount for the development of rational therapies for cardiac injury. Bioluminescence imaging (BLI) provides a means for monitoring physiological processes in real time, ranging from cell survival to gene expression to complex molecular processes. In mice and rats, BLI provides unmatched sensitivity because of the absence of endogenous luciferase expression in mammalian cells and the low background luminescence emanating from animals. In the field of stem cell therapy, BLI provides an unprecedented means to monitor the biology of these cells in vivo, giving researchers a greater understanding of their survival, migration, immunogenicity, and potential tumorigenicity in a living animal. In addition to longitudinal monitoring of cell survival, BLI is a useful tool for semiquantitative measurements of gene expression in vivo, allowing a better optimization of drug and gene therapies. Overall, this technology not only enables rapid, reproducible, and quantitative monitoring of physiological processes in vivo but also can measure the influences of therapeutic interventions on the outcome of cardiac injuries.PMID: 21666118 [PubMed - indexed for MEDLINE] PMCID: PMC3191083 [Available on 2012/9/1] -- 65) PLoS One. 2011;6(5):e20634. Epub 2011 May 31. Heme oxygenase-1 deletion affects stress erythropoiesis. Cao YA, Kusy S, Luong R, Wong RJ, Stevenson DK, Contag CH. Department of Pediatrics, Stanford University School of Medicine, Stanford, California, United States of America. BACKGROUND:Homeostatic erythropoiesis leads to the formation of mature red blood cells under non-stress conditions, and the production of new erythrocytes occurs as the need arises. In response to environmental stimuli, such as bone marrow transplantation, myelosuppression, or anemia, erythroid progenitors proliferate rapidly in a process referred to as stress erythropoiesis. We have previously demonstrated that heme oxygenase-1 (HO-1) deficiency leads to disrupted stress hematopoiesis. Here, we describe the specific effects of HO-1 deficiency on stress erythropoiesis. METHODOLOGY/PRINCIPAL FINDINGS:We used a transplant model to induce stress conditions. In irradiated recipients that received hmox(+/-) or hmox(+/+) bone marrow cells, we evaluated (i) the erythrocyte parameters in the peripheral blood; (ii) the staining intensity of CD71-, Ter119-, and CD49d-specific surface markers during erythroblast differentiation; (iii) the patterns of histological iron staining; and (iv) the number of Mac-1(+)-cells expressing TNF-. In the spleens of mice that received hmox(+/-) cells, we show (i) decreases in the proerythroblast, basophilic, and polychromatophilic erythroblast populations; (ii) increases in the insoluble iron levels and decreases in the soluble iron levels; (iii) increased numbers of Mac-1(+)-cells expressing TNF-; and (iv) decreased levels of CD49d expression in the basophilic and polychromatophilic erythroblast populations. CONCLUSIONS/SIGNIFICANCE:As reflected by effects on secreted and cell surface proteins, HO-1 deletion likely affects stress erythropoiesis through the retention of erythroblasts in the erythroblastic islands of the spleen. Thus, HO-1 may serve as a therapeutic target for controlling erythropoiesis, and the dysregulation of HO-1 may be a predisposing condition for hematologic diseases.PMID: 21655188 [PubMed - indexed for MEDLINE] PMCID: PMC3105104 -- 66) Nat Rev Rheumatol. 2011 Jun 7;7(7):416-26. doi: 10.1038/nrrheum.2011.68. Pathogenesis of systemic juvenile idiopathic arthritis: some answers, more questions. Mellins ED, Macaubas C, Grom AA. Department of Pediatrics, Stanford University, 300 Pasteur Drive, Stanford, CA 94305, USA. Systemic juvenile idiopathic arthritis (sJIA) has long been recognized as unique among childhood arthritides, because of its distinctive clinical and epidemiological features, including an association with macrophage activation syndrome. Here, we summarize research into sJIA pathogenesis. The triggers of disease are unknown, although infections are suspects. Once initiated, sJIA seems to be driven by innate proinflammatory cytokines. Endogenous Toll-like receptor ligands, including S100 proteins, probably synergize with cytokines to perpetuate inflammation. These and other findings support the hypothesis that sJIA is an autoinflammatory condition. Indeed, IL-1 is implicated as a pivotal cytokine, but the source of excess IL-1 activity remains obscure and the role of IL-1 in chronic arthritis is less clear. Another hypothesis is that a form of hemophagocytic lymphohistiocytosis underlies sJIA, with varying degrees of its expression across the spectrum of disease. Alternatively, sJIA with MAS might be a genetically distinct subtype. Yet another hypothesis proposes that inadequate downregulation of immune activation is central to sJIA, supporting evidence for which includes 'alternative activation' of monocyte and macrophages and possible deficiencies in IL-10 and T regulatory cells. Some altered immune phenotypes persist during clinically inactive disease, which suggests that this stage might represent compensated inflammation. Despite much progress being made, many questions remain, providing fertile ground for future research.PMID: 21647204 [PubMed - indexed for MEDLINE] -- 67) Cancer Res. 2011 Jul 15;71(14):5030-9. Epub 2011 Jun 6. Dissecting the oncogenic and tumorigenic potential of differentiated human induced pluripotent stem cells and human embryonic stem cells. Ghosh Z, Huang M, Hu S, Wilson KD, Dey D, Wu JC. Departments of Medicine, Stanford University School of Medicine, Stanford, CA 94305-5454, USA. Pluripotent stem cells, both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC), can give rise to multiple cell types and hence have tremendous potential for regenerative therapies. However, the tumorigenic potential of these cells remains a great concern, as reflected in the formation of teratomas by transplanted pluripotent cells. In clinical practice, most pluripotent cells will be differentiated into useful therapeutic cell types such as neuronal, cardiac, or endothelial cells prior to human transplantation, drastically reducing their tumorigenic potential. Our work investigated the extent to which these differentiated stem cell derivatives are truly devoid of oncogenic potential. In this study, we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells, and compared their transcriptomes with those of five different types of cancer. Our analysis revealed a significant gene expression overlap of the hiPSC- and hESC-derivatives with cancer, whereas the corresponding primary cells showed minimum overlap. Real-time quantitative PCR analysis of a set of cancer-related genes (selected on the basis of rigorous functional and pathway analyses) confirmed our results. Overall, our findings suggested that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation, and additional stringent functional assays to purify these cells should be done before they can be used for regenerative therapy.2011 AACR.PMID: 21646469 [PubMed - indexed for MEDLINE] PMCID: PMC3138859 [Available on 2012/7/15] -- 68) Int J Obstet Anesth. 2011 Jul;20(3):240-5. Epub 2011 Jun 2. Non-invasive measurement of hemoglobin during cesarean hysterectomy: a case series. Butwick AJ, Hilton G, Riley ET, Carvalho B. Department of Anesthesiology, Stanford University School of Medicine, Stanford, CA 94305, USA. Obstetric patients diagnosed with abnormal placentation (placenta accreta, increta or percreta) are at increased risk of major postpartum hemorrhage and cesarean hysterectomy. Obstetric anesthesiologists are primarily involved in intraoperative transfusion management in these cases. Hemoglobin assessment is invaluable for assisting transfusion decision-making during the acute period of obstetric hemorrhage. However, laboratory and point-of-care tests of hemoglobin concentration are time-dependent and intermittent, and do not provide a real-time assessment of change during the acute phase of blood loss. A new non-invasive hemoglobin monitor has been introduced recently, which provides real-time measurement of hemoglobin values (SpHb) using multi-wavelength pulse co-oximetry. We present a review of five patients with suspected abnormal placentation who received SpHb monitoring during cesarean hysterectomy at our institution. We discuss the potential clinical utility of non-invasive hemoglobin monitoring for pregnant patients at high risk of obstetric hemorrhage, and the potential role of SpHb in guiding transfusion therapy.Copyright 2011 Elsevier Ltd. All rights reserved.PMID: 21640577 [PubMed - indexed for MEDLINE] -- 69) J Endourol. 2011 Jun;25(6):917-21. Epub 2011 May 13. Comparison of 2.6- and 1.4-mm imaging probes for confocal laser endomicroscopy of the urinary tract. Adams W, Wu K, Liu JJ, Hsiao ST, Jensen KC, Liao JC. Department of Urology and Bio-X Program, Stanford University School of Medicine, Stanford, California 94305-5118, USA. INTRODUCTION:Probe-based confocal laser endomicroscopy (pCLE) is an emerging technology for dynamic, in vivo imaging of the urinary tract with micron-scale resolution. We conducted a comparative analysis of pCLE with a 2.6-mm probe and a 1.4-mm probe that is compatible with flexible endoscopes. MATERIALS AND METHODS:Sixty-seven patients scheduled for bladder tumor resection were recruited. pCLE imaging was performed using 2.6- and 1.4-mm probes. Image quality with the different probes was examined and further compared with standard histopathology. RESULTS:Images with the 2.6-mm probe have better resolution of cell morphology. The 1.4-mm probe has a wider field of view and better view of microarchitecture. While image quality with the 2.6-mm probe is superior, the 1.4-mm probe is compatible with flexible cystoscopy and maneuverability is maintained, enabling imaging of areas of the bladder that were previously challenging to access with the larger probe. CONCLUSIONS:The optical specifications of the 2.6-mm probe are more suitable for distinguishing urinary tract histopathology. Further design optimization to improve resolution and additional validation of the diagnostic accuracy of the smaller probe are needed to help extend application of pCLE for optical biopsy of the upper and lower urinary tract. PMID: 21568756 [PubMed - indexed for MEDLINE] -- 70) Mol Microbiol. 2011 Jul;81(2):368-94. doi: 10.1111/j.1365-2958.2011.07698.x. Epub 2011 May 26. Mutations in the nucleotide binding pocket of MreB can alter cell curvature and polar morphology in Caulobacter. Dye NA, Pincus Z, Fisher IC, Shapiro L, Theriot JA. Department of Biochemistry and Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA. The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. Another subset have variable cell widths, with wide cell bodies and actively growing thin extensions of the cell poles that concentrate fluorescent MreB. We found that the extent to which MreB localization is perturbed is linearly correlated with the development of pointed cell poles and variable cell widths. Further, we find that a mutation to glycine of two conserved aspartic acid residues that are important for nucleotide hydrolysis in other members of the actin superfamily abolishes robust midcell recruitment of MreB but supports a normal rate of growth. These mutant strains provide novel insight into how MreB's protein structure, subcellular localization, and activity contribute to its function in bacterial cell shape. 2011 Blackwell Publishing Ltd. PMID: 21564339 [PubMed - indexed for MEDLINE] PMCID: PMC3137890 [Available on 2012/1/1] -- 71) J Neurosurg. 2011 Aug;115(2):328-36. Epub 2011 Apr 29. Outcome of repeat revascularization surgery for moyamoya disease after an unsuccessful indirect revascularization. Clinical article. Pandey P, Steinberg GK. Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA. OBJECT:Revascularization for moyamoya disease, either by direct anastomosis or indirect procedures, is an accepted and effective form of treatment for prevention of future ischemic events. Indirect procedures do not provide sufficient collateral vessels in a subset of patients, who then have persistent or new symptoms. Repeat revascularization procedures may be recommended for these patients. METHODS:Sixteen patients underwent repeat revascularization after undergoing an indirect procedure in the same hemisphere. These patients were included in the study, and a retrospective review of their clinical details, neuroimaging results, surgical details, and outcome was performed. Direct revascularization was the procedure of choice; however, in patients with no acceptable recipient vessel (> 0.6 mm) the authors added a second indirect procedure for further revascularization. RESULTS:Over the last 19 years, 16 patients (8 male and 8 female patients, age range 5-48 years, mean 16.7 years, 10 pediatric and 6 adult patients) underwent repeat revascularization for moyamoya disease. Initially all patients presented with ischemic symptoms (4 transient ischemic attacks [TIAs] and 12 strokes; 2 patients had bilateral symptoms). Angiography revealed that 13 patients had bilateral disease, and 3 had unilateral disease. Initial surgery was bilateral encephaloduroarteriosynangiosis (EDAS) in 9, unilateral EDAS alone in 3, unilateral EDAS with contralateral superficial temporal artery-middle cerebral artery (STA-MCA) bypass in 2, bilateral encephalomyosynangiosis (EMS) in 1, and unilateral EMS in 1. Thirteen of the 16 patients continued to have TIAs in the hemisphere ipsilateral to surgery, whereas 1 patient had seizures and cognitive deficit, 1 had asymptomatic infarct on MR imaging, and 1 had visual symptoms. Poor revascularization was seen on angiography studies in all patients. The median duration between the surgeries was 24 months (3 months-10 years). Repeat revascularization was performed in 23 hemispheres (16 patients). Direct revascularization was performed in 14 hemispheres (60.9%): STA-MCA bypass in 10, external carotid artery-MCA vein bypass in 2, occipital artery (OA)-MCA in 1, and OA-posterior cerebral artery in 1 hemisphere. Indirect revascularization was performed for patients without an acceptable recipient vessel, and was done in 9 hemispheres. The procedures included EMS (4 hemispheres), repeat EDAS (2), and omental transposition (3). There was 1 postoperative death in a patient undergoing a high-flow vein graft implan$%&'Bd 7 p w A2QYh####S$,--1----f.c/d/;7[77 8z>>>Ϻشش~~~~~hch>5B*aJphh>h>aJ h>aJhchoA5B*aJphhoAhoAaJ hoAaJ hcaJhch25B*aJphh2h2aJ h2aJh5CJ$aJ$hchc5CJ$aJ$hch25CJ$aJ$/&'d  m n 8 Agh2`aSgd>gdoAgd2$a$gdc/0X####S$%%&&,-1-G-H-----f...c/d/0256gd>6<77 8U8V888P>>$?H?I???P@@@@EAFAPABD:FLFFFGGgd>>$?w??@>@P@@KFZFFFO P0PPUUU~V[[\\)]*]dd"eeojjj%kpppXppqqzzzzz{{#$%ۈrsٓ;;;ָ⯸hah">5B*aJphh">h">aJ h">aJhah"5B*aJphh"h"aJ h"aJ h>aJ haaJh>h>aJhah>5B*aJph?GGG0PP Q QQQU~VVVWWs[\\\\)]*]_^d`@bdd"eeegd>eeffj%kFkGkllXpp5q6qqqMzNzzzz {{{{#gd"gd>$%=bԉՉrs7֍Αٓ+3wx£gd">gd"ٓ+u3|£8#?b³׳ kqcisڿ^abd|'e~/R⻲hEhEOJQJ^JaJh hE5B*aJphhEhEaJ hEaJ h"aJh h">5B*aJph h aJ haaJ h">aJh">h">aJhah">5B*aJph:£8ȤɤTABb r׳ "#Algdagd">+ڿ34b'WXUegd">9:~.]yROP=^gdEgd"> =-^.467Mkop/5Df0! " 5FGW2 3 ''''R(s)t)3191ǸǸǸǸЯǸǩǚǩǩhYlhVd5B*aJph hYlaJhVdh aJh hVd5B*aJphhVdhVdaJ hVdaJ h">aJh hE5B*aJph hEaJ h aJhEhEaJ<^kKLophf0gdVdgdE0! " > 5FGVuvW2 3 !s&^''R(gd gdVdR( ) )s)t)+/1r1122;3<36.77!8"888N>>>?v???@gd "0gdVd91D1r1177.7788>>>>>?v?@@GGH1HHNNNObO-P.PYX_XpXX Y```aaQbRbiiiijjqrr6rrssr{x{{{/|e}f}#=m  dƗhVdhVdOJQJ^JaJ h "0aJh "0hVd5B*aJphhYlhVd5B*aJphhVdhVdaJ hVdaJK@@:AuCnE}G1HHIIII{NObOOO-P.PP]RVWW&XX Y;ZMZNZgdVdNZZZ[[e``aaaaQbRbdGf;h3iijjjj`kbkX 6s    . g 7 9&& '_',,-h-..r333 h">aJ hpTaJ hVdaJhpTh"o5B*aJph h\aJhTh"o5B*aJph hTaJ hva`aJhva`h"o5B*aJphh"oh"oaJ h"oaJ<2+,p nGH* T4no4gdTgd"o46<>^`6s. g   V7XYgdpTgd"o9T U  '_'''''-h---..>/@01?33J4~444gdpTgd"o3J4::G::D;E;HE":a56Cd،l#abnp۠z[}Ԥ>ͥΥyU45ſh2h"oaJ h\aJ hT9aJhT9h"o5B*aJphU hV>aJ h"oaJh"oh"oaJhV>h"o5B*aJph.449 :G::::D;E;<>a56;/G،ABgd"otation. None of the other patients experienced any neurological worsening after surgery. Follow-up was available in all patients, ranging from 3 to 144 months (mean 34 months, median 12 months). Of the 15 patients who survived repeat revascularization surgery, 12 (80%) were free from any TIA, stroke, or any other neurological symptoms. Two patients had occasional TIAs, less frequent than before, whereas 1 patient had frequent TIAs and underwent revision of the revascularization. Angiographic studies were available in 11 patients, and showed improved flow in the hemispheres in 10 patients. Follow-up MR imaging performed at 6 months did not reveal a new infarct in any patient. CONCLUSIONS:Repeat revascularization procedures are effective for patients who are clinically symptomatic and have inadequate collateral vessels following indirect procedures. Although direct procedures are preferred, the choice of procedure depends on the operative findings and the status of donor and recipient vessels. PMID: 21529138 [PubMed - indexed for MEDLINE] -- 72) J Pediatr Urol. 2011 Jun;7(3):294-8. Epub 2011 Apr 27. Epidemiologic trends in penile anomalies and hypospadias in the state of California, 1985-2006. Elliott CS, Halpern MS, Paik J, Maldonado Y, Shortliffe LD. Stanford University, Stanford, CA, USA. chrsuz@aol.com PURPOSE:Using statewide data, we evaluated whether the changing incidence of penile anomalies and hypospadias is reflected in the diverse California population of newborn males over the past 20 years. METHODS:Discharge data from all California hospitals, prepared by the OSHPD (Sacramento, CA) was reviewed for the years 1985-2006 for male infant births with an ICD-9 code (752.6) for hypospadias, epispadias or other penile anomalies. Trends were examined by Generalized Estimation Equations for Poisson regression. RESULTS:From 1985 to 2006, the birth incidence of newborn penile anomalies increased in California from 47 to 57 cases per 10,000 newborn discharges, yet the trend for hypospadias alone appears stable from 1997. The rates for penile anomalies in newborns increased 1.4% annually (p < 0.001). All racial/ethnic groups analyzed showed this increase (p < 0.001 for each). During the study period there was a 2% increase per year in plural births (p < 0.001). Interestingly, the rate of change in penile anomaly incidence was greater in males of plural births compared to their singleton cohorts (2% vs 1% annually) (p < 0.001). The birth incidence of cleft palate, another congenital anomaly known to be stable over time, remained unchanged over this period. CONCLUSIONS:From 1985 to 2006 in California the incidence of penile anomalies increased in a statistically significant manner, but the incidence of hypospadias appears stable for the last decade. Our data support the notion that different racial/ethnic groups have distinct incidences of penile anomaly formation and that an association with plural births appears to be present. Copyright 2011 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.PMID: 21527236 [PubMed - indexed for MEDLINE] -- 73) J Heart Lung Transplant. 2011 Jul;30(7):761-9. Epub 2011 Mar 31. Prevention of transplant coronary artery disease by prenylation inhibitors. Stein W, Schrepfer S, Itoh S, Kimura N, Velotta J, Palmer O, Bartos J, Wang X, Robbins RC, Fischbein MP. Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California 94305, USA. BACKGROUND:In this study we systematically dissect the prenylation pathway to better define the mechanism behind statin inhibition in chronic allograft rejection in heart transplants, or transplant coronary artery disease (TCAD). METHODS:Utilizing a murine heterotopic heart transplant model, animals received daily treatments of either statin or selective isoprenoid blockade inhibitors to block the four major downstream branches of the mevalonate pathway. TCAD was assessed by morphometric analysis at Day 52. Graft-infiltrating cells, cytokine production, smooth muscle cell proliferation and migration and endothelial cell MHC II expression were detected on Day 7. RESULTS:Atorvastatin and two prenylation inhibitors, NE-10790 and manumycin A, significantly reduced TCAD lesions compared with untreated animals. Perillyl alcohol treatment resulted in a trend toward decreased luminal narrowing. Finally, zaragozic acid (cholesterol blockade only) did not alter TCAD severity. Statins and prenylation inhibitors reduced inflammatory cell allograft recruitment, but did not always correlate with TCAD reduction. Cytokine production was decreased in recipient spleens in all treatment groups. Both in vitro and in vivo IFN--stimulated MHC II expression was decreased in a dose-dependent manner in the atorvastatin, perillyl alcohol and NE-10790 groups. In vitro smooth muscle cell proliferation was decreased in all treatment groups. Finally, in vitro smooth muscle cell migration was decreased in the atorvastatin, NE-10790 and manumycin A groups only. CONCLUSIONS:FPT and GGPT-2 (inhibition) are the key enzymes in the HGM-CoA reductase pathway and most influential in TCAD prevention. TCAD reduction is most closely related to smooth muscle cell migration, but not its anti-inflammatory properties. Copyright 2011 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.PMID: 21458297 [PubMed - indexed for MEDLINE] -- 74) Clin Gastroenterol Hepatol. 2011 Jul;9(7):567-76.e1-4. Epub 2011 Mar 11. Characteristics of patients with chronic unexplained nausea and vomiting and normal gastric emptying. Pasricha PJ, Colvin R, Yates K, Hasler WL, Abell TL, Unalp-Arida A, Nguyen L, Farrugia G, Koch KL, Parkman HP, Snape WJ, Lee L, Tonascia J, Hamilton F. Division of Gastroenterology and Hepatology, Stanford University School of Medicine, 300 Pasteur Drive, Palo Alto, California 94305-5187, USA. Pasricha@stanford.edu BACKGROUND & AIMS:Chronic nausea and vomiting with normal gastric emptying is a poorly understood syndrome; we analyzed its characteristics. METHODS:We collected and analyzed data from 425 patients with chronic nausea and vomiting, enrolled at 6 centers by the Gastroparesis Clinical Research Consortium in the National Institute of Diabetes and Digestive and Kidney Diseases Gastroparesis Registry. RESULTS:Among the patients, 319 (75%) had delayed emptying, defined by the results of a standardized, low-fat meal, and 106 had normal gastric emptying. Patients with or without delayed emptying did not differ in age, sex, or race, although those with normal gastric emptying were less likely to be diabetic. Symptom severity indexes were similar between groups for nausea, retching, vomiting, stomach fullness, inability to complete a meal, feeling excessively full after meals, loss of appetite, bloating, and visibly larger stomach. There were no differences in health care utilization, quality of life indexes, depression, or trait anxiety scores. However, state anxiety scores were slightly higher among patients with delayed gastric emptying. Total gastroparesis cardinal symptom index scores were not correlated with gastric retention after 2 or 4 hours in either group. Patients with the syndrome were not adequately captured by the stand-alone criteria for the Rome III diagnoses of chronic idiopathic nausea and functional vomiting. With rare exceptions, the diagnosis remained stable after a 48-week follow-up period. CONCLUSIONS:Patients with nausea and vomiting with normal gastric emptying represent a significant medical problem and are, for the most part, indistinguishable from those with gastroparesis. This syndrome is not categorized in the medical literature--it might be a separate clinical entity.Copyright 2011 AGA Institute. Published by Elsevier Inc. All rights reserved. Comment in * Clin Gastroenterol Hepatol. 2011 Jul;9(7):534-5. PMID: 21397732 [PubMed - indexed for MEDLINE] PMCID: PMC3123425 [Available on 2012/7/1] -- 75) Transpl Infect Dis. 2011 Jun;13(3):312-7. doi: 10.1111/j.1399-3062.2010.00595.x. Epub 2011 Jan 16. West Nile virus encephalitis acquired via liver transplantation and clinical response to intravenous immunoglobulin: case report and review of the literature. Rhee C, Eaton EF, Concepcion W, Blackburn BG. Department of Internal Medicine, Stanford University School of Medicine, Palo Alto, California, USA. A patient developed West Nile virus (WNV) encephalitis 2 weeks after receiving a liver transplant and recovered fully, following treatment with intravenous immunoglobulin (IVIg). Laboratory testing documented transmission from the organ donor. Clinicians should be suspicious for organ-transmitted WNV in any post-transplant patient who develops fever and neurological symptoms. We review previous cases of organ-transmitted WNV, the use of IVIg for WNV encephalitis, and the issue of organ donor screening. 2011 John Wiley & Sons A/S.PMID: 21235711 [PubMed - indexed for MEDLINE] -- 76) Neurogastroenterol Motil. 2011 Apr;23(4):356-61, e160-1. doi: 10.1111/j.1365-2982.2010.01649.x. Epub 2011 Jan 4. The analgesic effects of the GABAB receptor agonist, baclofen, in a rodent model of functional dyspepsia. Liu LS, Shenoy M, Pasricha PJ. Division of Gastroenterology and Hepatology, Stanford University Medical Center, Stanford, CA 94305-5187, USA. BACKGROUND:The amino acid -aminobutyric acid (GABA) is an important modulator of pain but its role in visceral pain syndromes is just beginning to be studied. Our aims were to investigate the effect and mechanism of action of the GABA(B) receptor agonist, baclofen, on gastric hypersensitivity in a validated rat model of functional dyspepsia (FD). METHODS:10-day-old male rats received 0.2 mL of 0.1% iodoacetamide in 2% sucrose daily by oral gavages for 6 days. Control group received 2% sucrose. At 8-10 weeks rats treated with baclofen (0.3, 1, and 3 mg kg(-1) bw) or saline were tested for behavioral and electromyographic (EMG) visceromotor responses; gastric spinal afferent nerve activity to graded gastric distention and Fos protein expression in dorsal horn of spinal cord segments T8-T10 to noxious gastric distention. KEY RESULTS:Baclofen administration was associated with a significant attenuation of the behavioral and EMG responses (at 1 and 3 mg kg(-1)) and expression of Fos in T8 and T9 segments in neonatal iodoacetamide sensitized rats. However, baclofen administration did not significantly affect splanchnic nerve activity to gastric distention. Baclofen (3 mg kg(-1)) also significantly reduced the expression of spinal Fos in response to gastric distention in control rats to a lesser extent than sensitized rats. CONCLUSIONS & INFERENCES:Baclofen is effective in attenuating pain associated responses in an experimental model of FD and appears to act by central mechanisms. These results provide a basis for clinical trials of this drug in FD patients. 2011 Blackwell Publishing Ltd.PMID: 21199535 [PubMed - indexed for MEDLINE] -- 77) Genome Res. 2011 Feb;21(2):265-75. Epub 2010 Dec 22. Multimodal RNA-seq using single-strand, double-strand, and CircLigase-based capture yields a refined and extended description of the C. elegans transcriptome. Lamm AT, Stadler MR, Zhang H, Gent JI, Fire AZ. Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324, USA. We have used a combination of three high-throughput RNA capture and sequencing methods to refine and augment the transcriptome map of a well-studied genetic model, Caenorhabditis elegans. The three methods include a standard (non-directional) library preparation protocol relying on cDNA priming and foldback that has been used in several previous studies for transcriptome characterization in this species, and two directional protocols, one involving direct capture of single-stranded RNA fragments and one involving circular-template PCR (CircLigase). We find that each RNA-seq approach shows specific limitations and biases, with the application of multiple methods providing a more complete map than was obtained from any single method. Of particular note in the analysis were substantial advantages of CircLigase-based and ssRNA-based capture for defining sequences and structures of the precise 5' ends (which were lost using the double-strand cDNA capture method). Of the three methods, ssRNA capture was most effective in defining sequences to the poly(A) junction. Using data sets from a spectrum of C. elegans strains and stages and the UCSC Genome Browser, we provide a series of tools, which facilitate rapid visualization and assignment of gene structures. 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