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					ITI faculty Publications for December 2007, January 2008



Cancer Res. 2008 Jan 1;68(1):257-65. Links
Homologous recombination is the principal pathway for the repair of DNA damage
induced by tirapazamine in mammalian cells.Evans JW, Chernikova SB, Kachnic LA,
Banath JP, Sordet O, Delahoussaye YM, Treszezamsky A, Chon BH, Feng Z, Gu Y,
Wilson WR, Pommier Y, Olive PL, Powell SN, Brown JM.
Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford
University, Stanford, California 94305-5152, USA.

Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a promising hypoxia-selective
cytotoxin that has shown significant activity in advanced clinical trials in combination
with radiotherapy and cisplatin. The current study aimed to advance our understanding of
tirapazamine-induced lesions and the pathways involved in their repair. We show that
homologous recombination plays a critical role in repair of tirapazamine-induced damage
because cells defective in homologous recombination proteins XRCC2, XRCC3,
Rad51D, BRCA1, or BRCA2 are particularly sensitive to tirapazamine. Consistent with
the involvement of homologous recombination repair, we observed extensive sister
chromatid exchanges after treatment with tirapazamine. We also show that the
nonhomologous end-joining pathway, which predominantly deals with frank double-
strand breaks (DSB), is not involved in the repair of tirapazamine-induced DSBs. In
addition, we show that tirapazamine preferentially kills mutants both with defects in
XPF/ERCC1 (but not in other nucleotide excision repair factors) and with defects in base
excision repair. Tirapazamine also induces DNA-protein cross-links, which include stable
DNA-topoisomerase I cleavable complexes. We further show that gamma H2AX, an
indicator of DNA DSBs, is induced preferentially in cells in the S phase of the cell cycle.
These observations lead us to an overall model of tirapazamine damage in which DNA
single-strand breaks, base damage, and DNA-protein cross-links (including
topoisomerase I and II cleavable complexes) produce stalling and collapse of replication
forks, the resolution of which results in DSB intermediates, requiring homologous
recombination and XPF/ERCC1 for their repair.

PMID: 18172318 [PubMed - in process]

Gastroenterology. 2008 Jan;134(1):306-23. Links
Host-bacterial interactions in Helicobacter pylori infection.Amieva MR, El-Omar EM.
Department of Microbiology and Immunology, Stanford University School of Medicine,
Stanford, California, USA.

Helicobacter pylori are spiral-shaped gram-negative bacteria with polar flagella that live
near the surface of the human gastric mucosa. They have evolved intricate mechanisms to
avoid the bactericidal acid in the gastric lumen and to survive near, to attach to, and to
communicate with the human gastric epithelium and host immune system. This
interaction sometimes results in severe gastric pathology. H pylori infection is the
strongest known risk factor for the development of gastroduodenal ulcers, with infection
being present in 60%-80% of gastric and 95% of duodenal ulcers.(1)H pylori is also the
first bacterium to be classified as a definite carcinogen by the World Health
Organization's International Agency for Research on Cancer because of its epidemiologic
relationship to gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue
lymphoma.(2) In the last 25 years, since H pylori was first described and cultured, a
complete paradigm shift has occurred in our clinical approach to these gastric diseases,
and more than 20,000 scientific publications have appeared on the subject. From the
medical point of view, H pylori is a formidable pathogen responsible for much morbidity
and mortality worldwide. However, H pylori infection occurs in approximately half of the
world population, with disease being an exception rather than the rule. Understanding
how this organism interacts with its host is essential for formulating an intelligent
strategy for dealing with its most important clinical consequences. This review offers an
insight into H pylori host-bacterial interactions.

PMID: 18166359 [PubMed - in process]

J Biomed Opt. 2007 Nov-Dec;12(6):064025. Links
Multimodality imaging of T-cell hybridoma trafficking in collagen-induced arthritic
mice: image-based estimation of the number of cells accumulating in mouse
paws.Yaghoubi SS, Creusot RJ, Ray P, Fathman CG, Gambhir SS.
Stanford University, Department of Radiology, Bio-X, Molecular Imaging Program,
Stanford, California 94305-5427.

Appropriate targeting of therapeutic cells is essential in adoptive cellular gene therapy
(ACGT). Imaging cell trafficking in animal models and patients will guide development
of ACGT protocols. Collagen type II (C-II)-specific T cell hybridomas are transduced
with a lentivirus carrying a triple fusion reporter gene (TFR) construct consisting of a
fluorescent reporter gene (RG), a bioluminescent RG (hRluc), and a positron emission
tomography (PET) RG. Collagen-induced arthritic (CIA) mice are scanned with a
bioluminescence imaging camera before and after implantation of various known cell
quantities in their paws. Linear regression analysis yields equations relating two
parameters of image signal intensity in mice paws to the quantity of hRluc expressing
cells in the paws. Afterward, trafficking of intravenously injected cells is studied by
quantitative analysis of bioluminescence images. Comparison of the average cell
numbers does not demonstrate consistently higher accumulation of T-cell hybridomas in
the paws with higher inflammation scores, and injecting more cells does not cause
increased accumulation. MicroPET images illustrate above background signal in the
inflamed paws and chest areas of CIA mice. The procedures described in this study can
be used to derive equations for cells expressing other bioluminescent RGs and in other
animal models.

PMID: 18163841 [PubMed - in process]

Nat Chem Biol. 2007 Dec 23 [Epub ahead of print] Links
High-content single-cell drug screening with phosphospecific flow cytometry.Krutzik
PO, Crane JM, Clutter MR, Nolan GP.
Department of Microbiology and Immunology, Baxter Laboratory in Genetic
Pharmacology, Stanford University, 269 Campus Drive, Stanford, California 94305,
USA.

Drug screening is often limited to cell-free assays involving purified enzymes, but it is
arguably best applied against systems that represent disease states or complex
physiological cellular networks. Here, we describe a high-content, cell-based drug
discovery platform based on phosphospecific flow cytometry, or phosphoflow, that
enabled screening for inhibitors against multiple endogenous kinase signaling pathways
in heterogeneous primary cell populations at the single-cell level. From a library of small-
molecule natural products, we identified pathway-selective inhibitors of Jak-Stat and
MAP kinase signaling. Dose-response experiments in primary cells confirmed pathway
selectivity, but importantly also revealed differential inhibition of cell types and new
druggability trends across multiple compounds. Lead compound selectivity was
confirmed in vivo in mice. Phosphoflow therefore provides a unique platform that can be
applied throughout the drug discovery process, from early compound screening to in vivo
testing and clinical monitoring of drug efficacy.

PMID: 18157122 [PubMed - as supplied by publisher]

J Clin Oncol. 2007 Dec 17 [Epub ahead of print] Links
LMO2 Protein Expression Predicts Survival in Patients With Diffuse Large B-Cell
Lymphoma Treated With Anthracycline-Based Chemotherapy With and Without
Rituximab.Natkunam Y, Farinha P, Hsi ED, Hans CP, Tibshirani R, Sehn LH, Connors
JM, Gratzinger D, Rosado M, Zhao S, Pohlman B, Wongchaowart N, Bast M, Avigdor
A, Schiby G, Nagler A, Byrne GE, Levy R, Gascoyne RD, Lossos IS.
Department of Pathology and Department of Medicine, Division of Oncology, Stanford
University School of Medicine; Departments of Health Research and Policy and
Statistics, Stanford University, Stanford, CA; Departments of Clinical Pathology and
Hematologic Oncology and Blood Disorders, Cleveland Clinic Foundation, Cleveland,
OH; Departments of Pathology and Medicine, University of Nebraska Medical Center,
Omaha, NE; Department of Medicine, Division of Hematology-Oncology and Molecular
and Cellular Pharmacology, Sylvester Comprehensive Cancer Center, and Department of
Pathology, University of Miami, Miami, FL; Department of Pathology and Division of
Medical Oncology, British Columbia Cancer Agency, Vancouver, British Columbia,
Canada; and Chaim-Sheba Medical Center, Tel-Aviv, Israel.

PURPOSE: The heterogeneity of diffuse large B-cell lymphoma (DLBCL) has prompted
the search for new markers that can accurately separate prognostic risk groups. We
previously showed in a multivariate model that LMO2 mRNA was a strong predictor of
superior outcome in DLBCL patients. Here, we tested the prognostic impact of LMO2
protein expression in DLBCL patients treated with anthracycline-based chemotherapy
with or without rituximab. PATIENTS AND METHODS: DLBCL patients treated with
anthracycline-based chemotherapy alone (263 patients) or with the addition of rituximab
(80 patients) were studied using immunohistochemistry for LMO2 on tissue microarrays
of original biopsies. Staining results were correlated with outcome. RESULTS: In
anthracycline-treated patients, LMO2 protein expression was significantly correlated with
improved overall survival (OS) and progression-free survival (PFS) in univariate analyses
(OS, P = .018; PFS, P = .010) and was a significant predictor independent of the clinical
International Prognostic Index (IPI) in multivariate analysis. Similarly, in patients treated
with the combination of anthracycline-containing regimens and rituximab, LMO2 protein
expression was also significantly correlated with improved OS and PFS (OS, P = .005;
PFS, P = .009) and was a significant predictor independent of the IPI in multivariate
analysis. CONCLUSION: We conclude that LMO2 protein expression is a prognostic
marker in DLBCL patients treated with anthracycline-based regimens alone or in
combination with rituximab. After further validation, immunohistologic analysis of
LMO2 protein expression may become a practical assay for newly diagnosed DLBCL
patients to optimize their clinical management.

PMID: 18086797 [PubMed - as supplied by publisher]

Hepatology. 2007 Dec 14 [Epub ahead of print] Links
The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular
transformation and tumor formation without Ha-ras co-transfection.Einav S, Sklan EH,
Moon HM, Gehrig E, Liu P, Hao Y, Lowe AW, Glenn JS.
Division of Infectious Diseases and Geographic Medicine, Stanford University School of
Medicine, Stanford, California.

Hepatitis C virus (HCV) is an important cause of chronic liver disease and is complicated
by hepatocellular carcinoma (HCC). Mechanisms whereby the virus promotes cellular
transformation are poorly understood. We hypothesized that the guanosine triphosphatase
activity encoded in the HCV NS4B protein's nucleotide binding motif (NBM) might play
a role in the transformation process. Here we report that NS4B can transform NIH-3T3
cells, leading to tumor formation in vivo. This transformation was independent of co-
transfection with activated Ha-ras. Detailed analyses of NS4B mutants revealed that this
transforming activity could be progressively inhibited and completely abrogated by
increasing genetic impairment of the NS4B nucleotide binding motif. Conclusion: NS4B
has in vitro and in vivo tumorigenic potential, and the NS4B transforming activity is
indeed mediated by its NBM. Moreover, our results suggest that pharmacological
inhibition of the latter might inhibit not only HCV replication but also the associated
HCC. (HEPATOLOGY 2008.).

PMID: 18081150 [PubMed - as supplied by publisher]

Nat Protoc. 2007;2(12):3270-7. Links
Purification and characterization of transcribed RNAs using gel filtration
chromatography.McKenna SA, Kim I, Puglisi EV, Lindhout DA, Aitken CE, Marshall
RA, Puglisi JD.
Department of Structural Biology, Stanford University School of Medicine, Stanford,
California, USA.
RNA synthesis using in vitro transcription by phage T7 RNA polymerase allows
preparation of milligram quantities of RNA for biochemical, biophysical and structural
investigations. Previous purification approaches relied on gel electrophoretic or gravity-
flow chromatography methods. We present here a protocol for the in vitro transcription of
RNAs and subsequent purification using fast-performance liquid chromatography. This
protocol greatly facilitates production of RNA in a single day from transcription to
purification.

PMID: 18079727 [PubMed - in process]


Med Phys. 2007 Nov;34(11):4359-67.Links
Design and evaluation of a variable aperture collimator for conformal radiotherapy of
small animals using a microCT scanner.Graves EE, Zhou H, Chatterjee R, Keall PJ,
Gambhir SS, Contag CH, Boyer AL.
Department of Radiology Oncology, Molecular Imaging Program at Stanford, Stanford
University, Stanford, California 94305, USA. egraves@stanford.edu

Treatment of small animals with radiation has in general been limited to planar fields
shaped with lead blocks, complicating spatial localization of dose and treatment of deep-
seated targets. In order to advance laboratory radiotherapy toward what is accomplished
in the clinic, we have constructed a variable aperture collimator for use in shaping the
beam of microCT scanner. This unit can image small animal subjects at high resolution,
and is capable of delivering therapeutic doses in reasonable exposure times. The
proposed collimator consists of two stages, each containing six trapezoidal brass blocks
that move along a frame in a manner similar to a camera iris producing a hexagonal
aperture of variable size. The two stages are offset by 30 degrees and adjusted for the
divergence of the x-ray beam so as to produce a dodecagonal profile at isocenter. Slotted
rotating driving plates are used to apply force to pins in the collimator blocks and effect
collimator motion. This device has been investigated through both simulation and
measurement. The collimator aperture size varied from 0 to 8.5 cm as the driving plate
angle increased from 0 to 41 degrees. The torque required to adjust the collimator varied
from 0.5 to 5 N x m, increasing with increasing driving plate angle. The transmission
profiles produced by the scanner at isocenter exhibited a penumbra of approximately 10%
of the collimator aperture width. Misalignment between the collimator assembly and the
x-ray source could be identified on the transmission images and corrected by adjustment
of the collimator location. This variable aperture collimator technology is therefore a
feasible and flexible solution for adjustable shaping of radiation beams for use in small
animal radiotherapy as well as other applications in which beam shaping is desired.

PMID: 18072501 [PubMed - indexed for MEDLINE]

Infect Immun. 2007 Dec 10 [Epub ahead of print] Links
Evasion of Innate Immune Responses: Evidence for Mannose Binding Lectin Inhibition
of TNF-{alpha} Production by Macrophages in Response to Blastomyces
dermatitidis.Koneti A, Linke MJ, Brummer E, Stevens DA.
California Institute for Medical Research and Division of Infectious Diseases,
Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA; Veterans
Administration Medical Center, Cincinnati, OH, and Department of Medicine, Stanford
University School of Medicine, Stanford, CA.

Serum factors, including mannose binding lectins (MBL), influence innate responses to
microbes. Little is known about the effect of serum factor(s) or MBL on Blastomyces
dermatitidis (Bd), a pulmonary fungal pathogen, interaction with macrophages, and
tumor necrosis factor-alpha (TNF-alpha) production. Since macrophage production of
TNF-alpha is an important innate immune response, we examined a mouse peritoneal
macrophage (PM) cell line (RAW) and resident PM from CD-1 mice to study TNF-alpha
production by PM stimulated with heat killed (HK) or live (L) Bd yeast cells. Mouse
serum and heat inactivated (HI) mouse serum inhibited TNF-alpha production by 94%
when macrophages were stimulated by Bd, whereas mouse IgG did not have this effect.
HK-Bd incubated with serum, and washed, also failed to stimulate significant TNF-alpha
production by PM. By the sandwich immunoflourescent antibody (IFA) method with
anti-mouse mannose binding lectin (MBL-A or C) we showed that serum MBL bound to
Bd. When serum was absorbed with HK-Bd or L-Bd, absorbed serum failed to
significantly inhibit TNF-alpha production by RAW plus Bd; immunoblotting showed
absorbed serum was depleted of MBL-C. If serum was absorbed with L-Bd, unbound
serum eluted, and bound serum factor(s) (BS) released with guanidine buffer, BS
inhibited TNF-alpha production by PM + Bd in a concentration-dependent manner. BS
contained MBL-C, which bound Bd, as shown by IFA. 1,3-beta-glucan stimulated TNF-
alpha production by PM and this was inhibited by mouse serum. Treatment of Bd with
anti-1,3-beta-glucan antibody inhibited TNF-alpha production by PM. With anti-1,3-beta-
glucan antibody we showed by IFA that Bd contained 1,3-beta-glucan. In IFA study with
Bd, serum with anti-mouse IgG conjugate did not result in fluorescence, yet serum
blocked IFA staining of Bd by anti-1,3-beta-glucan IgG antibody. This indicated that
non-IgG serum factors binding to Bd prevented access to 1,3-beta-glucan by anti-1,3-
beta-glucan antibody. These results suggest the mechanism for inhibition of the innate
proinflammatory immune response of PM to Bd is mediated by serum MBL binding to
Bd at 1,3-beta-glucan sites, or sterically masking 1,3-beta-glucan sites, thus preventing
1,3-beta-glucan stimulation of PM for TNF-alpha production.

PMID: 18070904 [PubMed - as supplied by publisher]

Genome Biol. 2007 Dec 11;8(12):R261 [Epub ahead of print] Links
Gene-expression patterns reveal underlying biological processes in Kawasaki
disease.Popper SJ, Shimizu C, Shike H, Kanegaye JT, Newburger JW, Sundel RP, Brown
PO, Burns JC, Relman DA.
Departments of Microbiology and Immunology, and Medicine, Stanford University
School of Medicine, Stanford, CA 94305, USA. spopper@stanford.edu.
ABSTRACT: BACKGROUND: Kawasaki disease (KD) is an acute self-limited
vasculitis and the leading cause of acquired heart disease in children in developed
countries. No etiologic agent(s) has been identified, and the processes that mediate
formation of coronary artery aneurysms and abatement of fever following treatment with
intravenous immunoglobulin (IVIG) remain poorly understood. RESULTS: In an initial
survey, we used DNA microarrays to examine patterns of gene expression in peripheral
whole blood from 20 children with KD; each was sampled during the acute, subacute,
and convalescent phases of the illness. Acute KD was characterized by increased relative
abundance of gene transcripts associated with innate immune and proinflammatory
responses and decreased abundance of transcripts associated with natural killer cells and
CD8+ lymphocytes. There was significant temporal variation in transcript levels during
the acute disease phase and stabilization thereafter. We confirmed these temporal patterns
in a second cohort of 64 patients, and identified additional inter-individual differences in
transcript abundance. Notably, higher levels of transcripts of the gene for
carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) were associated
with an increased percentage of unsegmented neutrophils, fewer days of illness, higher
levels of C-reactive protein, and subsequent non-response to IVIG; this last association
was confirmed by quantitative reverse transcription PCR in a third cohort of 33 patients,
and was independent of day of illness. CONCLUSION: Acute KD is characterized by
dynamic and variable gene-expression programs that highlight the importance of
neutrophil activation state and apoptosis in KD pathogenesis. Our findings also support
the feasibility of extracting biomarkers associated with clinical prognosis from gene-
expression profiles of individuals with systemic inflammatory illnesses.

PMID: 18067656 [PubMed - as supplied by publisher]

Bull Math Biol. 2007 Dec 1 [Epub ahead of print] Links
Modeling Imatinib-Treated Chronic Myelogenous Leukemia: Reducing the Complexity
of Agent-Based Models.Kim PS, Lee PP, Levy D.
Department of Mathematics, Stanford University, Stanford, CA, 94305-2125, USA.

We develop a model for describing the dynamics of imatinib-treated chronic
myelogenous leukemia. Our model is based on replacing the recent agent-based model of
Roeder et al. (Nat. Med. 12(10):1181-1184, 2006) by a system of deterministic difference
equations. These difference equations describe the time-evolution of clusters of
individual agents that are grouped by discretizing the state space. Hence, unlike standard
agent-base models, the complexity of our model is independent of the number of agents,
which allows to conduct simulation studies with a realistic number of cells. This
approach also allows to directly evaluate the expected steady states of the system. The
results of our numerical simulations show that our model replicates the averaged behavior
of the original Roeder model with a significantly reduced computational cost. Our
general approach can be used to simplify other similar agent-based models. In particular,
due to the reduced computational complexity of our technique, one can use it to conduct
sensitivity studies of the parameters in large agent-based systems.

PMID: 18060460 [PubMed - as supplied by publisher]
J Clin Invest. 2007 Dec;117(12):3633-41. Links
Therapeutic application of RNAi: is mRNA targeting finally ready for prime
time?Grimm D, Kay MA.
Departments of Pediatrics and Genetics, Stanford University School of Medicine,
Stanford, California, USA.

With unprecedented speed, RNA interference (RNAi) has advanced from its basic
discovery in lower organisms to becoming a powerful genetic tool and perhaps our single
most promising biotherapeutic for a wide array of diseases. Numerous studies document
RNAi efficacy in laboratory animals, and the first clinical trials are underway and thus far
suggest that RNAi is safe to use in humans. Yet substantial hurdles have also surfaced
and must be surmounted before therapeutic RNAi applications can become a standard
therapy. Here we review the most critical roadblocks and concerns for clinical RNAi
transition, delivery, and safety. We highlight emerging solutions and concurrently discuss
novel therapeutic RNAi-based concepts. The current rapid advances create realistic
optimism that the establishment of RNAi as a new and potent clinical modality in humans
is near.

PMID: 18060021 [PubMed - in process]

J Immunol. 2007 Dec 15;179(12):8225-34. Links
Latent Membrane Protein 1 of EBV Activates Phosphatidylinositol 3-Kinase to Induce
Production of IL-10.Lambert SL, Martinez OM.
Program in Immunology and Department of Surgery, Division of Transplantation,
Stanford University, Stanford, CA 94305.

EBV is a B lymphotrophic gamma-herpesvirus that is associated with multiple human
malignancies, including posttransplant lymphoproliferative disorder. The EBV-encoded
protein, latent membrane protein 1 (LMP1), is required for oncogenic transformation of
human B cells by EBV. An important consequence of LMP1 expression in EBV-infected
B cells is the induction of cellular IL-10, which acts as an autocrine growth factor for B
cell lymphomas. However, the mechanisms by which LMP1 induces IL-10 are
incompletely understood. We previously showed that rapamycin, a clinically relevant
immunosuppressant and mammalian target of rapamycin inhibitor, could suppress IL-10
production by EBV-infected B cell lines. To test the hypothesis that PI3K, which acts
upstream of mammalian target of rapamycin, might also be involved in LMP1-dependent
IL-10 production, we generated B cell lines expressing signaling-inducible chimeric
LMP1. Our results show that induced LMP1 signaling elicits both p38- and PI3K-
dependent IL-10 production in EBV(-) B cells. Moreover, distinct regions of the LMP1
signaling tail are associated with p38- vs PI3K-dependent IL-10 induction. We also
demonstrate that the LMP1-dependent p38 and PI3K activation regulates IL-10 induction
through discrete mechanisms. Whereas p38 activation is critical for the phosphorylation
of the transcription factor CREB, PI3K activation is required for the inactivation of
glycogen synthase kinase 3beta (GSK3beta), an inhibitory kinase that can regulate CREB
function. We find that GSK3beta regulates LMP1-dependent IL-10 induction, with
GSK3beta inhibition by pharmacologic or small interfering RNA strategies enhancing
LMP1-induced IL-10 induction. These findings demonstrate that LMP1 uses both p38
and PI3K activation for maximal up-regulation of IL-10.

PMID: 18056366 [PubMed - in process]

Antivir Ther. 2007;12(7):1015-25.Links
Future treatment of chronic hepatitis C.Keeffe EB.
Division of Gastroenterology and Hepatology, Department of Medicine, Stanford
University School of Medicine, Stanford, CA, USA. ekeeffe@stanford.edu

The current standard therapy for chronic hepatitis C is peginterferon plus ribavirin and
yields a sustained virological response rate of approximately 50% overall. Over the past
2-3 years, many new therapeutic agents directed at a number of different viral targets
have entered into development for the treatment of patients with chronic hepatitis C.
Many of these agents exhibit high levels of potency against the hepatitis C virus and have
a rapid onset of activity. Some agents have been abandoned because of lack of efficacy or
toxicity, but many others have shown promise and are undergoing further testing.
Although debated, new therapies in the immediate future will most likely be used in
combination with peginterferon, either alone or with ribavirin. This concise review is
focused on new drugs undergoing development for the treatment of patients with chronic
hepatitis C, and on drugs that have shown efficacy in preliminary investigations and
progressed to Phase II or III trials. This information should allow physicians involved in
the care of patients with chronic hepatitis C to provide realistic expectations of what
types of drugs are progressing in clinical development, the likelihood that new treatment
will include peginterferon with or without ribavirin, and when these novel therapies
might become available.

PMID: 18018759 [PubMed - indexed for MEDLINE]

Cell Host Microbe. 2007 Nov 15;2(5):284-5. Links
Comment on:
Cell Host Microbe. 2007 Nov 15;2(5):295-305.
How viruses avoid stress.Schütz S, Sarnow P.
Department of Microbiology and Immunology, Stanford University School of Medicine,
Stanford, CA 94305, USA.

Cellular responses to counter virus infection lead to the induction of cytoplasmic stress
granules, which are composed of translationally stalled mRNAs. In this issue of Cell Host
& Microbe, White and colleagues elucidate a mechanism where a poliovirus protease
specifically cleaves a host cell factor involved in assembly of stress granules. This
strategy ensures viral access to the limiting amounts of translation factors and interferes
with host cell mRNA sorting.

PMID: 18005747 [PubMed - indexed for MEDLINE]
Gastroenterology. 2007 Nov;133(5):1579-91. Epub 2007 Aug 28. Links
Wnt/beta-catenin signaling in murine hepatic transit amplifying progenitor cells.Hu M,
Kurobe M, Jeong YJ, Fuerer C, Ghole S, Nusse R, Sylvester KG.
Department of Surgery, Stanford University School of Medicine, Stanford, California,
USA.

BACKGROUND & AIMS: Oval cells are postnatal hepatic progenitors with high
proliferative potential and bipotent differentiation ability to become hepatocytes and
cholangiocytes. Because Wnt/beta-catenin signaling is a known regulatory pathway for
liver development and regeneration, we studied the role of Wnt signaling in oval cells
using a mouse model of chronic liver injury. METHODS: A 3,5-diethoxycarbonyl-1,4-
dihydrocollidine (DDC)-enriched diet was used to stimulate oval cell proliferation. Livers
were harvested for histologic analysis and determination of Wnt family gene expression
by quantitative reverse transcription-polymerase chain reaction and in situ hybridization.
The transgenic beta-catenin reporter mouse (TOPGAL) was use to confirm canonical
Wnt/beta-catenin signal transduction in proliferating oval cells within atypical ductal
proliferations (ADPs). Confocal fluorescence microscopy and immunohistochemistry
was used to confirm colocalization of beta-catenin with the oval cell antigen A-6.
RESULTS: Several Wnt ligands were significantly induced in the liver of DDC-fed mice
and localized to proliferating cells in and adjacent to the ADPs. Oval cells isolated from
DDC-fed mouse livers showed the presence of active beta-catenin in the nucleus along
with cell-cycle entry in response to purified Wnt3a in vitro. Moreover, Wnt3a-induced
beta-catenin/T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activation
was quantified by TCF/LEF luciferase reporter assays. CONCLUSIONS: From these
data, we conclude that oval cells respond to Wnt ligands (Wnt3a) in vitro with an
increase in amino-terminus dephosphorylated beta-catenin and cell-cycle entry and that
canonical Wnt/beta-catenin/TCF signaling is active in proliferating facultative hepatic
progenitor cells in vivo. These findings may lend insight to the consequences of increased
canonical Wnt signaling during periods of chronic liver injury.

PMID: 17983805 [PubMed - indexed for MEDLINE]

J Immunol. 2007 Nov 15;179(10):6547-54. Links
Naive and memory T cells induce different types of graft-versus-host disease.Dutt S,
Tseng D, Ermann J, George TI, Liu YP, Davis CR, Fathman CG, Strober S.
Department of Medicine, Stanford University School of Medicine, CA 94305, USA.

The goal of this study was to compare the ability of donor naive and alloantigen-primed
effector memory T cells to induce graft-vs-host disease after bone marrow transplantation
in MHC-mismatched irradiated host mice. Purified CD4(+) naive
(CD62L(high)CD44(low)) T cells and CD4(+) effector memory
(CD62L(low)CD44(high)) T cells obtained from unprimed donors and donors primed to
host alloantigens, respectively, were injected into host mice, and the rapidity, severity,
and pattern of tissue injury of graft-vs-host disease was assessed. Unexpectedly, the naive
T cells induced a more acute and severe colitis than the primed memory cells. Whereas
the naive T cells expressing CD62L and CCR7 lymph node homing receptors vigorously
expanded in mesenteric lymph nodes and colon by day 6 after transplantation, the primed
memory T cells without these receptors had 20- to 100-fold lower accumulation at this
early time point. These differences were reflected in the significantly more rapid decline
in survival and weight loss induced by naive T cells. The primed memory T cells had a
greater capacity to induce chronic colitis and liver injury and secrete IL-2 and IFN-
gamma in response to alloantigenic stimulation compared with memory T cells from
unprimed donors. Nevertheless, the expected increase in potency as compared with naive
T cells was not observed due to differences in the pattern and kinetics of tissue injury.

PMID: 17982043 [PubMed - indexed for MEDLINE]

Science. 2007 Nov 2;318(5851):806-9. Links
Comment in:
Science. 2007 Nov 2;318(5851):729.
Menin controls growth of pancreatic beta-cells in pregnant mice and promotes gestational
diabetes mellitus.Karnik SK, Chen H, McLean GW, Heit JJ, Gu X, Zhang AY, Fontaine
M, Yen MH, Kim SK.
Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA.

During pregnancy, maternal pancreatic islets grow to match dynamic physiological
demands, but the mechanisms regulating adaptive islet growth in this setting are poorly
understood. Here we show that menin, a protein previously characterized as an endocrine
tumor suppressor and transcriptional regulator, controls islet growth in pregnant mice.
Pregnancy stimulated proliferation of maternal pancreatic islet beta-cells that was
accompanied by reduced islet levels of menin and its targets. Transgenic expression of
menin in maternal beta-cells prevented islet expansion and led to hyperglycemia and
impaired glucose tolerance, hallmark features of gestational diabetes. Prolactin, a
hormonal regulator of pregnancy, repressed islet menin levels and stimulated beta-cell
proliferation. These results expand our understanding of mechanisms underlying diabetes
pathogenesis and reveal potential targets for therapy in diabetes.

PMID: 17975067 [PubMed - indexed for MEDLINE]

FEBS Lett. 2007 Nov 13;581(27):5307-14. Epub 2007 Oct 24. Links
Probing the conformation of human tRNA(3)(Lys) in solution by NMR.Puglisi EV,
Puglisi JD.
Department of Structural Biology, D105A Fairchild Building, 299 Campus Drive West,
Stanford University School of Medicine, Stanford, CA 94305-5126, USA.

Human tRNA(3)(Lys) acts as a primer for the reverse transcription of human
immunodeficiency virus genomic RNA. To form an initiation complex with genomic
RNA, tRNA(3)(Lys) must reorganize its secondary structure. To provide a starting point
for mechanistic studies of the formation of the initiation complex, we here present
solution NMR investigations of human tRNA(3)(Lys). We use a straightforward set of
NMR experiments to show that tRNA(3)(Lys) adopts a standard transfer ribonucleic acid
tertiary structure in solution, and that Mg(2+) is required for this folding. The results
underscore the power of NMR to reveal rapidly the conformation of RNAs.

PMID: 17963705 [PubMed - indexed for MEDLINE]

J Immunol. 2007 Nov 1;179(9):5907-15. Links
Posttranslational regulation of I-Ed by affinity for CLIP.Rinderknecht CH, Belmares MP,
Catanzarite TL, Bankovich AJ, Holmes TH, Garcia KC, Nanda NK, Busch R, Kovats S,
Mellins ED.
Program in Immunology, Department of Pediatrics, Stanford University, Stanford, CA
94305, USA.

Several MHC class II alleles linked with autoimmune diseases form unusually low
stability complexes with CLIP, leading us to hypothesize that this is an important feature
contributing to autoimmune pathogenesis. To investigate cellular consequences of
altering class II/CLIP affinity, we evaluated invariant chain (Ii) mutants with varying
CLIP affinity for a mouse class II allele, I-E(d), which has low affinity for wild-type
CLIP and is associated with a mouse model of spontaneous, autoimmune joint
inflammation. Increasing CLIP affinity for I-E(d) resulted in increased cell surface and
total cellular abundance and half-life of I-E(d). This reveals a post-endoplasmic reticulum
chaperoning capacity of Ii via its CLIP peptides. Quantitative effects on I-E(d) were less
pronounced in DM-expressing cells, suggesting complementary chaperoning effects
mediated by Ii and DM, and implying that the impact of allelic variation in CLIP affinity
on immune responses will be highest in cells with limited DM activity. Differences in the
ability of cell lines expressing wild-type or high-CLIP-affinity mutant Ii to present Ag to
T cells suggest a model in which increased CLIP affinity for class II serves to restrict
peptide loading to DM-containing compartments, ensuring proper editing of antigenic
peptides.

PMID: 17947664 [PubMed - indexed for MEDLINE]

Nature. 2007 Oct 18;449(7164):811-8. Links
An ecological and evolutionary perspective on human-microbe mutualism and
disease.Dethlefsen L, McFall-Ngai M, Relman DA.
Department of Microbiology and Immunology, Stanford University, Stanford, California
94305, USA.

The microbial communities of humans are characteristic and complex mixtures of
microorganisms that have co-evolved with their human hosts. The species that make up
these communities vary between hosts as a result of restricted migration of
microorganisms between hosts and strong ecological interactions within hosts, as well as
host variability in terms of diet, genotype and colonization history. The shared
evolutionary fate of humans and their symbiotic bacteria has selected for mutualistic
interactions that are essential for human health, and ecological or genetic changes that
uncouple this shared fate can result in disease. In this way, looking to ecological and
evolutionary principles might provide new strategies for restoring and maintaining human
health.

PMID: 17943117 [PubMed - indexed for MEDLINE]

Mol Cell Biol. 2007 Dec;27(24):8824-33. Epub 2007 Oct 15. Links
Postintegrative gene silencing within the Sleeping Beauty transposition system.Garrison
BS, Yant SR, Mikkelsen JG, Kay MA.
Stanford University School of Medicine, Department of Pediatrics, Stanford, CA 94305-
5208, USA.

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene
delivery because it can efficiently and stably integrate into mammalian genomes. In this
report, we examined transposon expression in human cells using a novel nonselective
fluorescence-activated cell sorter-based method and discovered that SB integrates
approximately 20 times more frequently than previously reported within systems that
were dependent on transgene expression and likely subject to postintegrative gene
silencing. Over time, phenotypic analysis of clonal integrants demonstrated that SB
undergoes additional postintegrative gene silencing, which varied based on the promoter
used for transgene expression. Molecular and biochemical studies suggested that
transposon silencing was influenced by DNA methylation and histone deacetylation
because both 5-aza-2'-deoxycytidine and trichostatin A partially rescued transgene
silencing in clonal cell lines. Collectively, these data reveal the existence of a
multicomponent postintegrative gene silencing network that efficiently targets invading
transposon sequences for transcriptional silencing in mammalian cells.

PMID: 17938204 [PubMed - indexed for MEDLINE]

Clin Gastroenterol Hepatol. 2007 Nov;5(11):1300-5. Epub 2007 Oct 23.Links
Comment in:
Clin Gastroenterol Hepatol. 2007 Nov;5(11):1259-60.
Functional imaging of colonic mucosa with a fibered confocal microscope for real-time
in vivo pathology.Wang TD, Friedland S, Sahbaie P, Soetikno R, Hsiung PL, Liu JT,
Crawford JM, Contag CH.
Division of Gastroenterology, Stanford University School of Medicine, Stanford,
California, USA. tdwang@stanford.edu

BACKGROUND & AIMS: Histologic interpretation of disease currently is performed
with static images of excised tissues, and is limited by processing artifact, sampling error,
and interpretive variability. The aim of this study was to show the use of functional
optical imaging of viable mucosa for quantitative evaluation of colonic neoplasia in real
time. METHODS: Fluorescein (5 mg/mL) was administered topically in 54 human
subjects undergoing screening colonoscopy. Fluorescence images were collected with
488-nm excitation at 12 frames/s with the confocal microendoscopy system. Movement
of fluorescein in the transient period (<5 s) and the lamina propria:crypt contrast ratio in
the steady-state phase (>5 s) were quantified. RESULTS: Normal mucosa showed
circular crypts with uniform size, hyperplasia revealed proliferative glands with serrated
lumens, and adenomas displayed distorted elongated glands. For t less than 5 seconds,
fluorescein passed through normal epithelium with a peak speed of 1.14 +/- 0.09
microm/s at t = 0.5 seconds, and accumulated into lamina propria as points of
fluorescence that moved through the interglandular space with an average speed of 41.7
+/- 3.4 microm/s. Passage of fluorescein through adenomatous mucosa was delayed
substantially. For t greater than 5 seconds, high sensitivity, specificity, and accuracy was
achieved using a discriminant function to evaluate the contrast ratio to distinguish normal
from lesional mucosa (91%, 87%, and 89%, respectively; P < .001), hyperplasia from
adenoma (97%, 96%, and 96%, respectively; P < .001), and tubular from villous adenoma
(100%, 92%, and 93%, respectively; P < .001). CONCLUSIONS: Confocal imaging can
be performed in vivo to assess the functional behavior of tissue in real time for providing
pathologic interpretation, representing a new method for histologic evaluation.

PMID: 17936692 [PubMed - indexed for MEDLINE]

Bioinformatics. 2007 Nov 1;23(21):2910-7. Epub 2007 Oct 5. Links
Evaluation and integration of 49 genome-wide experiments and the prediction of
previously unknown obesity-related genes.English SB, Butte AJ.
Department of Medicine, Stanford Medical Informatics, Stanford University School of
Medicine, Lucile Packard Children's Hospital, Stanford, CA 94305, USA.

MOTIVATION: Genome-wide experiments only rarely show resounding success in
yielding genes associated with complex polygenic disorders. We evaluate 49 obesity-
related genome-wide experiments with publicly available findings including microarray,
genetics, proteomics and gene knock-down from human, mouse, rat and worm, in terms
of their ability to rediscover a comprehensive set of genes previously found to be causally
associated or having variants associated with obesity. RESULTS: Individual experiments
show poor predictive ability for rediscovering known obesity-associated genes. We show
that intersecting the results of experiments significantly improves the sensitivity,
specificity and precision of the prediction of obesity-associated genes. We create an
integrative model that statistically significantly outperforms all 49 individual genome-
wide experiments. We find that genes known to be associated with obesity are
significantly implicated in more obesity-related experiments and use this to provide a list
of genes that we predict to have the highest likelihood of association for obesity. The
approach described here can include any number and type of genome-wide experiments
and might be useful for other complex polygenic disorders as well.

PMID: 17921495 [PubMed - indexed for MEDLINE]

Medinfo. 2007;12(Pt 1):550-4.Links
Extracting subject demographic information from abstracts of randomized clinical trial
reports.Xu R, Garten Y, Supekar KS, Das AK, Altman RB, Garber AM.
Biomedical Informatics Training Program, Stanford University School of Medicine,
USA.
In order to make more informed healthcare decisions, consumers need information
systems that deliver accurate and reliable information about their illnesses and potential
treatments. Reports of randomized clinical trials (RCTs) provide reliable medical
evidence about the efficacy of treatments. Current methods to access, search for, and
retrieve RCTs are keyword-based, time-consuming, and suffer from poor precision.
Personalized semantic search and medical evidence summarization aim to solve this
problem. The performance of these approaches may improve if they have access to study
subject descriptors (e.g. age, gender, and ethnicity), trial sizes, and diseases/symptoms
studied. We have developed a novel method to automatically extract such subject
demographic information from RCT abstracts. We used text classification augmented
with a Hidden Markov Model to identify sentences containing subject demographics, and
subsequently these sentences were parsed using Natural Language Processing techniques
to extract relevant information. Our results show accuracy levels of 82.5%, 92.5%, and
92.0% for extraction of subject descriptors, trial sizes, and diseases/symptoms descriptors
respectively.

PMID: 17911777 [PubMed - indexed for MEDLINE]

Medinfo. 2007;12(Pt 1):311-5.Links
Knowledge-level querying of temporal patterns in clinical research systems.O'Connor
MJ, Shankar RD, Parrish DB, Das AK.
Stanford Medical Informatics, Stanford University, Stanford, CA 94305, USA.
martin.oconnor@stanford.edu

Managing time-stamped data is essential to clinical research activities and often requires
the use of considerable domain knowledge. Adequately representing this domain
knowledge is difficult in relational database systems. As a result, there is a need for
principled methods to overcome the disconnect between the database representation of
time-oriented research data and corresponding knowledge of domain-relevant concepts.
In this paper, we present a set of methodologies for undertaking knowledge level
querying of temporal patterns, and discuss its application to the verification of temporal
constraints in clinical-trial applications. Our approach allows knowledge generated from
query results to be tied to the data and, if necessary, used for further inference. We show
how the Semantic Web ontology and rule languages, OWL and SWRL, respectively, can
support the temporal knowledge model needed to integrate low-level representations of
relational data with high-level domain concepts used in research data management. We
present a scalable bridge-based software architecture that uses this knowledge model to
enable dynamic querying of time-oriented research data.

PMID: 17911729 [PubMed - indexed for MEDLINE]

Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15864-9. Epub 2007 Sep 27. Links
Detection of endogenous biomolecules in Barrett's esophagus by Fourier transform
infrared spectroscopy.Wang TD, Triadafilopoulos G, Crawford JM, Dixon LR, Bhandari
T, Sahbaie P, Friedland S, Soetikno R, Contag CH.
Division of Gastroenterology, Stanford University School of Medicine, Palo Alto
Veterans Affairs Health Care System, Palo Alto, CA 94304, USA. thomaswa@umich.edu

Fourier transform infrared (FTIR) spectroscopy provides a unique molecular fingerprint
of tissue from endogenous sources of light absorption; however, specific molecular
components of the overall FTIR signature of precancer have not been characterized. In
attenuated total reflectance mode, infrared light penetrates only a few microns of the
tissue surface, and the influence of water on the spectra can be minimized, allowing for
the analyses of the molecular composition of tissues. Here, spectra were collected from
98 excised specimens of the distal esophagus, including 38 squamous, 38 intestinal
metaplasia (Barrett's), and 22 gastric, obtained endoscopically from 32 patients. We show
that DNA, protein, glycogen, and glycoprotein comprise the principal sources of infrared
absorption in the 950- to 1,800-cm(-1) regime. The concentrations of these biomolecules
can be quantified by using a partial least-squares fit and used to classify disease states
with high sensitivity, specificity, and accuracy. Moreover, use of FTIR to detect
premalignant (dysplastic) mucosa results in a sensitivity, specificity, positive predictive
value, and total accuracy of 92%, 80%, 92%, and 89%, respectively, and leads to a better
interobserver agreement between two gastrointestinal pathologists for dysplasia (kappa =
0.72) versus histology alone (kappa = 0.52). Here, we demonstrate that the concentration
of specific biomolecules can be determined from the FTIR spectra collected in attenuated
total reflectance mode and can be used for predicting the underlying histopathology,
which will contribute to the early detection and rapid staging of many diseases.

PMID: 17901200 [PubMed - indexed for MEDLINE]

PLoS Biol. 2007 Oct;5(10):e257. Links
Nanosensor detection of an immunoregulatory tryptophan influx/kynurenine efflux
cycle.Kaper T, Looger LL, Takanaga H, Platten M, Steinman L, Frommer WB.
Department of Plant Biology, Carnegie Institution, Stanford, California, United States of
America.

Mammalian cells rely on cellular uptake of the essential amino acid tryptophan.
Tryptophan sequestration by up-regulation of the key enzyme for tryptophan degradation,
indoleamine 2,3-dioxygenase (IDO), e.g., in cancer and inflammation, is thought to
suppress the immune response via T cell starvation. Additionally, the excreted tryptophan
catabolites (kynurenines) induce apoptosis of lymphocytes. Whereas tryptophan transport
systems have been identified, the molecular nature of kynurenine export remains
unknown. To measure cytosolic tryptophan steady-state levels and flux in real time, we
developed genetically encoded fluorescence resonance energy transfer nanosensors
(FLIPW). The transport properties detected by FLIPW in KB cells, a human oral cancer
cell line, and COS-7 cells implicate LAT1, a transporter that is present in proliferative
tissues like cancer, in tryptophan uptake. Importantly, we found that this transport system
mediates tryptophan/kynurenine exchange. The tryptophan influx/kynurenine efflux cycle
couples tryptophan starvation to elevation of kynurenine serum levels, providing a two-
pronged induction of apoptosis in neighboring cells. The strict coupling protects cells that
overproduce IDO from kynurenine accumulation. Consequently, this mechanism may
contribute to immunosuppression involved in autoimmunity and tumor immune escape.

PMID: 17896864 [PubMed - indexed for MEDLINE]

Biochem Biophys Res Commun. 2007 Nov 16;363(2):438-43. Epub 2007 Sep 12. Links
Caenorhabditis elegans pgp-5 is involved in resistance to bacterial infection and heavy
metal and its regulation requires TIR-1 and a p38 map kinase cascade.Kurz CL, Shapira
M, Chen K, Baillie DL, Tan MW.
Department of Genetics and Department of Microbiology and Immunology, M337
Always Building, 300 Pasteur Drive, Stanford University School of Medicine, Stanford,
CA 94305-5120, USA.

Animals and plants respond to bacterial infections and environmental stresses by
inducing overlapping repertoires of defense genes. How the signals associated with
infection and abiotic stresses are differentially integrated within a whole organism
remains to be fully addressed. We show that the transcription of a Caenorhabditis elegans
ABC transporter, pgp-5 is induced by both bacterial infection and heavy metal stress, but
the magnitude and tissue distribution of its expression differs, depending on the type of
stressor. PGP-5 contributes to resistance to bacterial infection and heavy metals. Using
pgp-5 transcription as a read-out, we show that signals from both biotic and abiotic
stresses are integrated by TIR-1, a TIR domain adaptor protein orthologous to human
SARM, and a p38 MAP kinase signaling cassette. We further demonstrate that not all the
TIR-1 isoforms are necessary for nematode resistance to infection, suggesting a
molecular basis for the differential response to abiotic and biotic stress.

PMID: 17888400 [PubMed - indexed for MEDLINE]

Anal Chem. 2007 Nov 1;79(21):8316-22. Epub 2007 Sep 21. Links
Free-solution oligonucleotide separation in nanoscale channels.Pennathur S, Baldessari F,
Santiago JG, Kattah MG, Steinman JB, Utz PJ.
Mechanical Engineering Department, Stanford University, Stanford, California 94305,
USA.

In this paper, we report an experimental study of electrokinetic transport and separation
of double-stranded deoxyribonucleic acid (dsDNA) oligonucleotides in custom-fabricated
fused-silica nanochannels filled with a gel-free sodium borate aqueous buffer. Mixtures
of fluorescently labeled dsDNA molecules in the range of 10-100 base pair (bp),
fluorescein, and fluorescein-12-UTP (UTP) were separated in less than 120 s in channels
of depth ranging from 40 to 1560 nm. We varied the channel depth and background
buffer concentration to achieve a 0.006-0.2 range of Debye length-to-channel-half-depth
ratio (lambdaD/h), and a 0.004-1.7 range of the ratio of length of dsDNA molecule to
channel half-depth (l/h). We find observed oligonucleotide migration times depend on
both l/h and lambdaD/h. Electrophoretic mobility estimates agree well with published
(micrometer-scale channel) values for background electrolyte (BGE) concentrations
greater than approximately 10 mM. At BGE concentrations of 1 and 5 mM, mobility
estimates in our nanochannels are higher than published values. Of the cases studied, the
highest separation sensitivities were achieved in 100 nm channels with 1-10 mM ion
density buffers. Potential applications of this technology include rapid small-scale
sequencing and other fluorescence-based oligonucleotide separation and detection assays.

PMID: 17883279 [PubMed - indexed for MEDLINE]

PLoS Biol. 2007 Sep;5(9):e247. Links
Comment on:
PLoS Biol. 2007 Sep;5(9):e238.
How and why does a fly turn its immune system off?Schneider DS.
Department of Microbiology and Immunology, Stanford University, Stanford, California,
United States of America. dschneider@stanford.edu

PMID: 17880266 [PubMed - indexed for MEDLINE]

J Microsc. 2007 Aug;227(Pt 2):140-56. Links
Comparison of quantitative methods for cell-shape analysis.Pincus Z, Theriot JA.
Program in Biomedical Informatics, and Department of Biochemistry, Stanford
University School of Medicine, Stanford, CA 94305, USA.

Morphology is an important large-scale manifestation of the global organizational and
physiological state of cells, and is commonly used as a qualitative or quantitative measure
of the outcome of various assays. Here we evaluate several different basic representations
of cell shape - binary masks, distance maps and polygonal outlines - and different
subsequent encodings of those representations - Fourier and Zernike decompositions, and
the principal and independent components analyses - to determine which are best at
capturing biologically important shape variation. We find that principal components
analysis of two-dimensional shapes represented as outlines provide measures of
morphology which are quantitative, biologically meaningful, human interpretable and
work well across a range of cell types and parameter settings.

PMID: 17845709 [PubMed - indexed for MEDLINE]

Clin Transplant. 2007 Sep-Oct;21(5):597-608. Links
Transplant reno-vascular stenoses associated with early erythropoietin use.Nagarajan S,
Mansfield E, Hsieh S, Liu R, Hsieh F, Li L, Salvatierra O Jr, Sarwal MM.
Department of Pediatrics (Nephrology) Stanford University, Palo Alto, CA 94305-5208,
USA.

BACKGROUND AND OBJECTIVES: This report describes an unusual presentation of
severe hypertension (HTN) in a subset of pediatric kidney recipients treated with a
steroid avoidance pediatric renal transplantation protocol. The HTN was secondary to
atypical, reno-vascular abnormalities (RVA) of the transplanted vasculature, temporally
associated with erythropoietin (EPO) use. DESIGN, SETTING, PARTICIPANTS, AND
MEASUREMENTS: To investigate the clinical significance underlying this event, a
retrospective clinical study of 100 pediatric renal transplants was undertaken (50 steroid-
free and 50 matched steroid-based controls), with peripheral blood transcriptional
analysis of four RVA patients and controls. RESULTS: Regardless of a higher observed
incidence of anemia (p < 0.001) and greater overall EPO usage in the first post-transplant
year in steroid-free patients, the incidence of new-onset HTN at one yr was significantly
less in the steroid-free cohort (p = 0.03). Nevertheless, early EPO (first week post-
transplant) was significantly associated with the combinatory findings of new-onset HTN
(p = 0.03) and RVA (p = 0.007). Molecular mechanisms of RVA injury were investigated
further by peripheral blood cDNA microarray gene expression profiling. A panel of 42
transcripts differentiated patients with RVA and HTN from three sets of matched
controls, with and without HTN and EPO use, with 100% concordance (p < 0.001). The
biological processes governed by these significant genes suggest a role for EPO
regulation of growth factor receptor ubiquitination as a putative mechanism for renal
vascular injury. CONCLUSION: This study cautions against the use early post-transplant
use of EPO in immunosuppression regimens with steroid minimization/avoidance, which
may have an increased incidence of post-transplant anemia.

PMID: 17845633 [PubMed - indexed for MEDLINE]

Nat Chem Biol. 2007 Oct;3(10):668-77. Epub 2007 Sep 9. Links
Noninvasive optical imaging of cysteine protease activity using fluorescently quenched
activity-based probes.Blum G, von Degenfeld G, Merchant MJ, Blau HM, Bogyo M.
Department of Pathology, Stanford University School of Medicine, 300 Pasteur Dr.,
Stanford, California 94305, USA.

We have generated a series of quenched near-infrared fluorescent activity-based probes
(qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown
previously to be important in multiple stages of tumorigenesis. These 'smart' probes emit
a fluorescent signal only after covalently modifying a specific protease target. After
intravenous injection of NIRF-ABPs into mice bearing grafted tumors, noninvasive,
whole-body imaging allowed direct monitoring of cathepsin activity. Importantly, the
permanent nature of the probes also allowed secondary, ex vivo biochemical profiling to
identify specific proteases and to correlate their activity with whole-body images. Finally,
we demonstrate that these probes can be used to monitor small-molecule inhibition of
protease targets both biochemically and by direct imaging methods. Thus, NIRF-ABPs
are (i) potentially valuable new imaging agents for disease diagnosis and (ii) powerful
tools for preclinical and clinical testing of small-molecule therapeutic agents in vivo.

PMID: 17828252 [PubMed - indexed for MEDLINE]

J Vasc Interv Radiol. 2007 Sep;18(9):1177-82. Links
Emergency retrieval of a G2 filter after complete migration into the right ventricle.Kuo
WT, Loh CT, Sze DY.
Division of Vascular and Interventional Radiology, Department of Radiology Stanford
University Medical Center, 300 Pasteur Dr, H-3651, Stanford, CA 94305-5642, USA.
wkuo@stanford.edu
A G2 inferior vena cava filter migrated completely into the right ventricle, resulting in
chest pain, ventricular tachycardia, and hypotension in a 63-year-old man. Due to the
filter's position, the patient was at high risk for further life-threatening cardiopulmonary
complications. Percutaneous filter retrieval was successfully performed as a less-invasive
alternative to open cardiothoracic surgery.

PMID: 17804782 [PubMed - indexed for MEDLINE]

Aliment Pharmacol Ther. 2007 Sep 15;26(6):839-46. Links
Long-term survival of patients with unresectable hepatocellular carcinoma treated with
transcatheter arterial chemoinfusion.Ha BY, Ahmed A, Sze DY, Razavi MK, Simpson N,
Keeffe EB, Nguyen MH.
Division of GI and Hepatology, Stanford University School of Medicine, Stanford, CA
94304-1509, USA.

BACKGROUND: Transcatheter arterial chemoembolization (TACE) has become one of
the most common treatments for unresectable hepatocellular carcinoma. Published
studies of TACE report a 5-16% risk of serious complications. Compared with TACE,
transcatheter arterial chemoinfusion (TACI) may have similar efficacy and fewer side
effects. AIM: To examine the clinical outcomes of TACI. METHODS: We performed a
retrospective cohort study of 345 consecutive TACI cases in 165 patients performed at a
single United States medical center between 1998 and 2002. Primary outcomes were
tumour response and survival rates. RESULTS: Only seven patients were hospitalized for
more than 24 h after the procedure, and only three patients had worsening of liver
function within 30 days of TACI. Survival was significantly poorer for patients with
tumour-node-metastasis (TNM) IV compared to those with TNM I-III and also for
patients with Child's class B/C vs. A. Following adjustment for age, gender, ethnicity and
aetiology of liver diseases, independent predictors of poor survival were Child's class B/C
[Hazard Ratio (HR) = 1.69, P = 0.024] and TNM IV staging (HR = 1.63, P = 0.014).
CONCLUSIONS: TACI appears to be safe and effective for unresectable hepatocellular
carcinoma with TNM stage I-III; randomized controlled trials are needed to compare
TACI to TACE.

PMID: 17767468 [PubMed - indexed for MEDLINE]

Nat Immunol. 2007 Oct;8(10):1095-104. Epub 2007 Sep 2. Links
Mast cell-derived interleukin 10 limits skin pathology in contact dermatitis and chronic
irradiation with ultraviolet B.Grimbaldeston MA, Nakae S, Kalesnikoff J, Tsai M, Galli
SJ.
Department of Pathology, Stanford University School of Medicine, Stanford, California
94305-5176, USA.

Allergic contact dermatitis, such as in response to poison ivy or poison oak, and chronic
low-dose ultraviolet B irradiation can damage the skin. Mast cells produce
proinflammatory mediators that are thought to exacerbate these prevalent acquired
immune or innate responses. Here we found that, unexpectedly, mast cells substantially
limited the pathology associated with these responses, including infiltrates of leukocytes,
epidermal hyperplasia and epidermal necrosis. Production of interleukin 10 by mast cells
contributed to the anti-inflammatory or immunosuppressive effects of mast cells in these
conditions. Our findings identify a previously unrecognized function for mast cells and
mast cell-derived interleukin 10 in limiting leukocyte infiltration, inflammation and tissue
damage associated with immunological or innate responses that can injure the skin.

PMID: 17767162 [PubMed - indexed for MEDLINE]

PLoS Biol. 2007 Sep;5(9):e233. Links
Emergence of large-scale cell morphology and movement from local actin filament
growth dynamics.Lacayo CI, Pincus Z, VanDuijn MM, Wilson CA, Fletcher DA, Gertler
FB, Mogilner A, Theriot JA.
Department of Biochemistry, Stanford University, Stanford, California, United States of
America.

Variations in cell migration and morphology are consequences of changes in underlying
cytoskeletal organization and dynamics. We investigated how these large-scale cellular
events emerge as direct consequences of small-scale cytoskeletal molecular activities.
Because the properties of the actin cytoskeleton can be modulated by actin-remodeling
proteins, we quantitatively examined how one such family of proteins,
enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), affects the migration and
morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently
while exhibiting a characteristic smooth-edged "canoe" shape, but may also exhibit less
regular morphologies and less persistent movement. When we observed that the smooth-
edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the
leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this
led to changes in the morphology and movement persistence of cells within a population.
Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused
changes in cell morphology, which is coupled to the motile behavior of keratocytes. We
also characterized the range of natural cell-to-cell variation within a population by using
measurable morphological and behavioral features--cell shape, leading-edge shape,
filamentous actin (F-actin) distribution, cell speed, and directional persistence--that we
have found to correlate with each other to describe a spectrum of coordinated phenotypes
based on Ena/VASP enrichment at the leading edge. This spectrum stretched from
smooth-edged, canoe-shaped keratocytes--which had VASP highly enriched at their
leading edges and migrated fast with straight trajectories--to more irregular, rounder cells
migrating slower with less directional persistence and low levels of VASP at their leading
edges. We developed a mathematical model that accounts for these coordinated cell-
shape and behavior phenotypes as large-scale consequences of kinetic contributions of
VASP to actin filament growth and protection from capping at the leading edge. This
work shows that the local effects of actin-remodeling proteins on cytoskeletal dynamics
and organization can manifest as global modifications of the shape and behavior of
migrating cells and that mathematical modeling can elucidate these large-scale cell
behaviors from knowledge of detailed multiscale protein interactions.
PMID: 17760506 [PubMed - indexed for MEDLINE]

FEMS Microbiol Lett. 2007 Oct;275(2):199-206. Epub 2007 Aug 14. Links
vpsA- and luxO-independent biofilms of Vibrio cholerae.Müller J, Miller MC, Nielsen
AT, Schoolnik GK, Spormann AM.
Department of Civil & Environmental Engineering, Stanford University, Stanford, CA,
USA.

The natural life cycle of Vibrio cholerae involves the transitioning of cells between
different environmental surfaces such as the chitinous shell of Crustaceae and the
epithelial layer of the human intestine. Previous studies using static biofilm systems
showed a strict dependence of biofilm formation on the vps and lux genes, which are
essential for exopolysaccharide formation and cell-cell signaling, respectively. The
authors' report here that in biofilms grown under hydrodynamic conditions, DeltavpsA
and DeltaluxO mutants of V. cholerae do form pronounced, three-dimensional biofilms
that resemble all aspects of wild-type biofilms. By genetic experiments, it was shown that
in hydrodynamically grown biofilms this independence of vpsA is due to the expression
of rpoS, which is a negative regulator of vpsA expression. Biofilms also underwent
substantial dissolution after 96 h that could be induced by a simple stop of medium flow.
The studies indicate that metabolic conditions control the reversible attachment of cells to
the biofilm matrix and are key in regulating biofilm cell physiology via RpoS.
Furthermore, the results redefine the roles of vps and quorum-sensing in V. cholerae
biofilms.

PMID: 17697110 [PubMed - indexed for MEDLINE]

Nat Genet. 2007 Sep;39(9):1092-9. Epub 2007 Aug 12. Links
Unusual selection on the KIR3DL1/S1 natural killer cell receptor in Africans.Norman PJ,
Abi-Rached L, Gendzekhadze K, Korbel D, Gleimer M, Rowley D, Bruno D, Carrington
CV, Chandanayingyong D, Chang YH, Crespí C, Saruhan-Direskeneli G, Fraser PA,
Hameed K, Kamkamidze G, Koram KA, Layrisse Z, Matamoros N, Milà J, Park MH,
Pitchappan RM, Ramdath DD, Shiau MY, Stephens HA, Struik S, Verity DH, Vaughan
RW, Tyan D, Davis RW, Riley EM, Ronaghi M, Parham P.
Department of Structural Biology, Stanford University School of Medicine, Stanford,
California 94305, USA. paul.norman@stanford.edu

Interactions of killer cell immunoglobulin-like receptors (KIRs) with major
histocompatibility complex (MHC) class I ligands diversify natural killer cell responses
to infection. By analyzing sequence variation in diverse human populations, we show that
the KIR3DL1/S1 locus encodes two lineages of polymorphic inhibitory KIR3DL1
allotypes that recognize Bw4 epitopes of protein">HLA-A and HLA-B and one lineage
of conserved activating KIR3DS1 allotypes, also implicated in Bw4 recognition.
Balancing selection has maintained these three lineages for over 3 million years.
Variation was selected at D1 and D2 domain residues that contact HLA class I and at two
sites on D0, the domain that enhances the binding of KIR3D to HLA class I. HLA-B
variants that gained Bw4 through interallelic microconversion are also products of
selection. A worldwide comparison uncovers unusual KIR3DL1/S1 evolution in modern
sub-Saharan Africans. Balancing selection is weak and confined to D0, KIR3DS1 is rare
and KIR3DL1 allotypes with similar binding sites predominate. Natural killer cells
express the dominant KIR3DL1 at a high frequency and with high surface density,
providing strong responses to cells perturbed in Bw4 expression.

PMID: 17694054 [PubMed - indexed for MEDLINE]

Biophys J. 2007 Nov 15;93(10):3575-82. Epub 2007 Aug 10. Links
Fluctuations of transfer RNAs between classical and hybrid states.Kim HD, Puglisi JD,
Chu S.
Department of Physics, Stanford University, Stanford, California 94305, USA.

Adjacent transfer RNAs (tRNAs) in the A- and P-sites of the ribosome are in dynamic
equilibrium between two different conformations called classical and hybrid states before
translocation. Here, we have used single-molecule fluorescence resonance energy transfer
to study the effect of Mg(2+) on tRNA dynamics with and without an acetyl group on the
A-site tRNA. When the A-site tRNA is not acetylated, tRNA dynamics do not depend on
[Mg(2+)], indicating that the relative positions of the substrates for peptide-bond
formation are not affected by Mg(2+). In sharp contrast, when the A-site tRNA is
acetylated, Mg(2+) lengthens the lifetime of the classical state but does not change the
lifetime of the hybrid state. Based on these findings, the classical state resembles a state
with direct stabilization of tertiary structure by Mg(2+) ions whereas the hybrid state
resembles a state with little Mg(2+)-assisted stabilization. The antibiotic viomycin, a
translocation inhibitor, suppresses tRNA dynamics, suggesting that the enhanced
fluctuations of tRNAs after peptide-bond formation drive spontaneous attempts at
translocation by the ribosome.

PMID: 17693476 [PubMed - indexed for MEDLINE]

Anal Chem. 2007 Sep 15;79(18):7027-35. Epub 2007 Aug 8. Links
Employing two different quartz crystal microbalance models to study changes in
viscoelastic behavior upon transformation of lipid vesicles to a bilayer on a gold
surface.Cho NJ, Kanazawa KK, Glenn JS, Frank CW.
Department of Materials Science, Stanford University School of Medicine, Stanford
University, Stanford, California 94305, USA.

By analyzing the viscoelastic properties of two distinct layers, a layer of "soft" vesicles
and a "rigid" bilayer, we have created a model system to permit the study of film
behavior in the region of nonlinear mass and frequency change (non-Sauerbrey). The
structural transformation of lipid vesicles to a bilayer is shown to be accompanied by
significant changes in their physical properties. After the adsorption and saturation of
intact vesicles on gold surfaces, the adsorbed vesicle layer exhibits a soft, water-rich,
viscoelastic state. The AH peptide, a vesicle-destabilizing agent, is then added to trigger
the formation of a much thinner (approximately 5 nm), compact, and rigid bilayer. In this
study, we used the quartz crystal microbalance with dissipation technique. Large non-
Sauerbrey frequency and energy dissipation changes characterize the viscoelastic nature
of adsorbed intact vesicle films thicker than approximately 10 nm. Once the
transformation is complete, the frequency changes along with zero energy dissipation for
sufficiently thin films (t approximately 5 nm) were effectively modeled with the
Sauerbrey equation. Furthermore, we checked the validity of the Voigt-Voinova model in
which the quartz substrate is treated as a Voigt element, which is beyond the Sauerbrey
description. The calculations treating the film as having a constant viscosity agreed well
with the Voigt-Voinova model. These results were compared to calculations done using
the electromechanical (EM) model, which does not require a series expansion. The Voigt-
Voinova results were in excellent agreement with the EM model, providing evidence that
the expansion used in their study is quite accurate.

PMID: 17685547 [PubMed - indexed for MEDLINE]

J Mol Diagn. 2007 Sep;9(4):530-7. Epub 2007 Jul 25. Links
''Minor'' BCL2 breakpoints in follicular lymphoma: frequency and correlation with grade
and disease presentation in 236 cases.Weinberg OK, Ai WZ, Mariappan MR, Shum C,
Levy R, Arber DA.
Stanford University, Department of Pathology, 300 Pasteur Dr., Room L235, Stanford,
CA 94305, USA. okw@stanford.edu

Follicular lymphomas are frequently associated with the t(14;18)(q32;q21). This
translocation can be detected by karyotype, polymerase chain reaction (PCR), and
fluorescence in situ hybridization (FISH). In addition to the breakpoints currently used
for diagnosis located in the major breakpoint region (MBR) and the minor cluster region
(mcr), recent studies have reported the existence of other breakpoints (3' BCL2, 5'mcr,
and icr). In this study, we examined the frequency of all five breakpoints in 236 cases of
follicular lymphomas by real-time PCR analysis. The distribution of breakpoint sites
consisted of MBR in 118 cases (50%), mcr in 11 (5%), icr in 32 (13%), 3' BCL2 in 13
(6%), and 5' mcr in three cases (1%). These findings illustrate significantly higher
frequency of the icr breakpoint as compared with the more frequently studied mcr.
Correlation of breakpoints with histology showed that MBR breakpoints occur more
frequently in grade 2 lymphomas (P = 0.042). A majority of the PCR-negative cases
(75%) contained an IGH/BCL2 translocation with FISH methods, suggesting the
presence of other BCL2 breakpoints. Correlation of breakpoints with survival did not
reveal significant differences. Diagnostic laboratories should consider expanding their
PCR methods to include other BCL2 breakpoints and correlating with FISH methods
when appropriate.

PMID: 17652637 [PubMed - indexed for MEDLINE]

Clin Gastroenterol Hepatol. 2007 Aug;5(8):890-7. Epub 2007 Jul 13.Links
Report of an international workshop: Roadmap for management of patients receiving oral
therapy for chronic hepatitis B.Keeffe EB, Zeuzem S, Koff RS, Dieterich DT, Esteban-
Mur R, Gane EJ, Jacobson IM, Lim SG, Naoumov N, Marcellin P, Piratvisuth T, Zoulim
F.
Stanford University School of Medicine, Stanford, California 94304-1509, USA.
ekeeffe@stanford.edu

An international group of experienced hepatologists and virologists conducted a single-
day workshop to review the management of patients with chronic hepatitis B receiving
treatment with oral nucleosides or nucleotides. Guidelines regarding on-treatment
management and available published data on the importance of serum hepatitis B virus
(HBV) DNA as a marker of outcomes were reviewed. On-treatment monitoring strategies
to define early virologic responses that might be predictive of better outcomes and a
reduced risk of viral resistance were proposed for further study. This treatment plan,
labeled the roadmap concept, recommends monitoring of serum HBV DNA levels to
identify outcomes of therapy. Primary treatment failure was defined as a reduction of
serum HBV DNA levels by less than 1 log10 IU/mL from baseline at week 12.
Measurement of the HBV DNA level at week 24 was considered essential to characterize
virologic responses as complete, partial, or inadequate. Complete virologic response was
defined as negative HBV DNA by a sensitive assay (<60 IU/mL or <300 copies/mL);
partial virologic response was defined as HBV DNA levels less than 2000 IU/mL (4
log10 copies/mL), and inadequate virologic response was defined as HBV DNA levels of
2000 IU/mL or greater (4 log10 copies/mL). Strategies are proposed for managing
patients in each of these categories, depending in part on the rapidity with which HBV
DNA suppression is achieved and the emergence of genotypic mutations that reduce the
effectiveness of a specific drug. Future studies of the use of the roadmap concept in
improving outcomes of chronic hepatitis B are warranted.

PMID: 17632041 [PubMed - indexed for MEDLINE]

Stem Cells. 2007 Oct;25(10):2677-84. Epub 2007 Jul 12. Links
Molecular imaging of bone marrow mononuclear cell homing and engraftment in
ischemic myocardium.Sheikh AY, Lin SA, Cao F, Cao Y, van der Bogt KE, Chu P,
Chang CP, Contag CH, Robbins RC, Wu JC.
Department of Cardiothoracic Surgery, Stanford University School of Medicine, Edwards
Building R354, Stanford, California 94305-5344, USA.

Bone marrow mononuclear cell (BMMC) therapy shows promise as a treatment for
ischemic heart disease. However, the ability to monitor long-term cell fate remains
limited. We hypothesized that molecular imaging could be used to track stem cell homing
and survival after myocardial ischemia-reperfusion (I/R) injury. We first harvested donor
BMMCs from adult male L2G85 transgenic mice constitutively expressing both firefly
luciferase (Fluc) and enhanced green fluorescence protein reporter gene. Fluorescence-
activated cell sorting analysis revealed approximately 0.07% of the population to consist
of classic hematopoietic stem cells (lin-, thy-int, c-kit+, Sca-1+). Afterward, adult female
FVB recipients (n = 38) were randomized to sham surgery or acute I/R injury. Animals in
the sham (n = 16) and I/R (n = 22) groups received 5 x 10(6) of the L2G85-derived
BMMCs via tail vein injection. Bioluminescence imaging (BLI) was used to track cell
migration and survival in vivo for 4 weeks. BLI showed preferential homing of BMMCs
to hearts with I/R injury compared with sham hearts within the first week following cell
injection. Ex vivo analysis of explanted hearts by histology confirmed BLI imaging
results, and quantitative real-time polymerase chain reaction (for the male Sry gene)
further demonstrated a greater number of BMMCs in hearts with I/R injury compared
with the sham group. Functional evaluation by echocardiography demonstrated a trend
toward improved left ventricular fractional shortening in animals receiving BMMCs.
Taken together, these data demonstrate that molecular imaging can be used to
successfully track BMMC therapy in murine models of heart disease. Specifically, we
have demonstrated that systemically delivered BMMCs preferentially home to and are
retained by injured myocardium. Disclosure of potential conflicts of interest is found at
the end of this article.

PMID: 17628019 [PubMed - indexed for MEDLINE]

Biochim Biophys Acta. 2007 Oct;1768(10):2421-31. Epub 2007 May 22. Links
Biophysical analysis of the interaction of granulysin-derived peptides with
enterobacterial endotoxins.Chen X, Howe J, Andrä J, Rössle M, Richter W, da Silva AP,
Krensky AM, Clayberger C, Brandenburg K.
Division of Immunology and Transplantation Biology, Department of Pediatrics, CCSR
2105, Stanford University School of Medicine, Stanford, CA 94305, USA.

To combat infections by Gram-negative bacteria, it is not only necessary to kill the
bacteria but also to neutralize pathogenicity factors such as endotoxin
(lipopolysaccharide, LPS). The development of antimicrobial peptides based on
mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial
infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin
(Gra-pep) were investigated in microbiological and biophysical assays to understand their
interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl
chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform
spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and
cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration
calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-
LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes
was monitored. Our findings demonstrate a characteristic change in the aggregate
structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no
change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a
scavenging effect in solution, but rather proceeds after incorporation into target
membranes, suggesting a requisite membrane-bound step.

PMID: 17555705 [PubMed - indexed for MEDLINE]

Eukaryot Cell. 2007 Jun;6(6):940-8. Epub 2007 Apr 27. Links
Functional characterization of spliceosomal introns and identification of U2, U4, and U5
snRNAs in the deep-branching eukaryote Entamoeba histolytica.Davis CA, Brown MP,
Singh U.
Department of Medicine, Division of Infectious Diseases, Stanford University School of
Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.

Pre-mRNA splicing is essential to ensure accurate expression of many genes in
eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote,
approximately 30% of the annotated genes are predicted to contain introns; however, the
accuracy of these predictions has not been tested. In this study, we mined an expressed
sequence tag (EST) library representing 7% of amoebic genes and found evidence
supporting splicing of 60% of the testable intron predictions, the majority of which
contain a GUUUGU 5' splice site and a UAG 3' splice site. Additionally, we identified
several splice site misannotations, evidence for the existence of 30 novel introns in
previously annotated genes, and identified novel genes through uncovering their spliced
ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5
snRNAs. These data lay the foundation for further dissection of the role of RNA
processing in E. histolytica gene expression.

PMID: 17468393 [PubMed - indexed for MEDLINE]

J Exp Med. 2007 May 14;204(5):987-94. Epub 2007 Apr 23. Links
Type I interferon signaling is required for activation of the inflammasome during
Francisella infection.Henry T, Brotcke A, Weiss DS, Thompson LJ, Monack DM.
Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305,
USA.

Francisella tularensis is a pathogenic bacterium whose virulence is linked to its ability to
replicate within the host cell cytosol. Entry into the macrophage cytosol activates a host-
protective multimolecular complex called the inflammasome to release the
proinflammatory cytokines interleukin (IL)-1beta and -18 and trigger caspase-1-
dependent cell death. In this study, we show that cytosolic F. tularensis subspecies
novicida (F. novicida) induces a type I interferon (IFN) response that is essential for
caspase-1 activation, inflammasome-mediated cell death, and release of IL-1beta and -18.
Extensive type I IFN-dependent cell death resulting in macrophage depletion occurs in
vivo during F. novicida infection. Type I IFN is also necessary for inflammasome
activation in response to cytosolic Listeria monocytogenes but not vacuole-localized
Salmonella enterica serovar Typhimurium or extracellular adenosine triphosphate. These
results show the specific connection between type I IFN signaling and inflammasome
activation, which are two sequential events triggered by the recognition of cytosolic
bacteria. To our knowledge, this is the first example of the positive regulation of
inflammasome activation. This connection underscores the importance of the cytosolic
recognition of pathogens and highlights how multiple innate immunity pathways interact
before commitment to critical host responses.

PMID: 17452523 [PubMed - indexed for MEDLINE]

Ann N Y Acad Sci. 2007 Sep;1111:442-54. Epub 2007 Mar 7. Links
Azole therapy of clinical and experimental coccidioidomycosis.Stevens DA, Clemons
KV.
Department of Medicine, Santa Clara Valley Medical Center, 751 So. Bascom Avenue,
San Jose, CA 95128-2699, USA. stevens@stanford.edu

The therapy of coccidioidomycosis has been an early target, both experimentally and
clinically, for study of new members of the azole class of drugs, because of the
recognition that coccidioidomycosis is one of the most difficult mycoses to treat, and
because our research group and our collaborators have been eager to pioneer new
therapies for this problem pathogen. There have been steady advances in the
pharmacologic and antimicrobial properties of this class since the initial introduction of
miconazole, and many patients with coccidioidomycosis have benefited. Perhaps the
greatest contribution has been the development of well-tolerated oral drugs that make
possible prolonged courses of a conveniently administered agent, and perhaps the most
impressive advance has been the utility of the agents in coccidioidal meningitis, at least
as an adjunct to the polyenes. More potent agents are still required, so that complete
biological cure can be attained in meningeal and nonmeningeal coccidioidomycosis.

PMID: 17344535 [PubMed - indexed for MEDLINE]

Cell Microbiol. 2007 Jun;9(6):1426-44. Epub 2007 Jan 22. Links
Identification of developmentally regulated genes in Entamoeba histolytica: insights into
mechanisms of stage conversion in a protozoan parasite.Ehrenkaufer GM, Haque R,
Hackney JA, Eichinger DJ, Singh U.
Department of Microbiology and Immunology, Stanford University School of Medicine,
Stanford, CA 94305, USA.

Developmental switching between life-cycle stages is a common feature among many
pathogenic organisms. The protozoan parasite Entamoeba histolytica converts between
cysts (essential for disease transmission) and trophozoites (responsible for tissue
invasion). Identification of genes involved in the developmental pathway has been
severely hindered by the inability to generate E. histolytica cysts in vitro. Using parasite
strains derived from recent human infections and whole-genome transcriptional profiling,
we determined that 1439 genes (approximately 15% of annotated genes) were potentially
developmentally regulated. Genes enriched in cysts (672 in total) included cysteine
proteinases and transmembrane protein kinases, which may be involved in signal
transduction. Genes enriched in trophozoites (767 in total) included genes typically
thought of as important in tissue invasion by trophozoites, including the Gal/GalNAc
lectin light subunit and cysteine protease 1. Putative regulators of differentiation
including possible G-protein coupled receptors, signal transduction proteins and
transcription factors were identified. A number of E. histolytica stage-specific genes were
also developmentally regulated in the reptilian parasite E. invadens, indicating that they
likely have conserved functions in Entamoeba development. These advances lay the
groundwork for dissection of the molecular signals that initiate stage conversion and
development of novel diagnostic and therapeutic measures targeting E. histolytica cysts.
PMID: 17250591 [PubMed - indexed for MEDLINE]

				
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