bluuebunny/crossref_metadata_2025 huggingface dataset:
Point-Counterpoint: Molecular Testing for Infectious Diseases Should Be Done in the Clinical Microbiology Laboratory
Authors: Mosammaparast Nima , McAdam Alexander J. , Nolte Frederick S.
Published: March 2012

Over the past decade, there has been an explosion in the use of molecular tests to diagnose and manage infectious diseases. HIV is a prime example of an infectious agent whose diagnosis at least in the acute stage, susceptibility testing, and management are all dependent on molecular diagnostics. The ability to accurately diagnose a plethora of respiratory pathogens quickly, simply, and relatively inexpensively compared to traditional methods is becoming a reality. Direct sequencing and microarray analysis holds great promise for directly detecting a wide variety of organisms from clinical specimens. The question is where this testing should be done in the clinical laboratory. There are at least four models that have emerged:

Molecular infectious disease testing as an arm of the clinical microbiology laboratory

Molecular infectious disease testing done in a central molecular pathology laboratory under the leadership of a clinical microbiologist

Molecular infectious disease testing done in a central molecular pathology laboratory under the leadership of an individual whose primary interest is in another area of molecular pathology

Molecular infectious disease testing sent to a reference laboratory and not done on site or within the institution's health care system.

IMPORTANCE
We have asked three individuals who have thought about this very complex issue to share their rationale for supporting one of these models. Frederick Nolte is the Director of Clinical Laboratories and Director of Molecular Pathology at the Medical University of South Carolina, is active in and held several positions of responsibility in AMP (Association of Molecular Pathology) and is Chair of the CLSI's Area Committee for Molecular Methods, Alex McAdam is the Director of the Infectious Diseases Diagnostic Division at Children's Hospital Boston and an editor of this journal, and his colleague, Nima Mosammaparast, is the Assistant Director of the Infectious Diseases Diagnostic Laboratory at Children's Hospital Boston.
Direct Detection of Mycobacterium tuberculosis Complex in Clinical Samples by a Molecular Method Based on GenoQuick Technology
Authors: Moure Raquel , Torres Miriam , Martín Rogelio , Alcaide Fernando
Published: March 2012

Several molecular systems for direct detection of
Mycobacterium tuberculosis
complex (MTBC) have recently been developed. The GenoQuick MTB assay (GQ-MTB) used in this study detected 82 of the 96 (85.4%) samples with MTBC, including 50 of 64 (78.1%) samples with negative acid-fast bacillus smears. Fifteen samples containing nontuberculous mycobacteria were also studied: 13 were GQ-MTB negative, one was positive, and one was indeterminate. GQ-MTB showed good effectiveness for the direct detection of MTBC from clinical samples.
The Small Membrane Filter Method of Microscopy to Diagnose Pulmonary Tuberculosis
Authors: Fennelly Kevin P. , Morais Carlos Gustavo , Hadad David Jamil , Vinhas Solange , Dietze Reynaldo , Palaci Moises
Published: March 2012

We modified microscopy for acid-fast bacilli to diagnose tuberculosis (TB) using small membrane filters (SMFs) after special processing and prefiltration. With the first specimen obtained from each of 335 persons suspected of having TB, the sensitivity of the new SMF method using fluorescence microscopy (FM) was 89% (95% confidence interval [CI]: 80%, 95%). This was significantly better (
P
= 0.0001) than the sensitivity of routine FM of centrifuged specimens of 60% (95% CI: 49%, 71%) or that of direct sputum smears of 56% (95% CI: 40%, 72%).
HIV-2 Genome Dimerization Is Required for the Correct Processing of Gag: a Second-Site Reversion in Matrix Can Restore Both Processes in Dimerization-Impaired Mutant Viruses
Authors: L'Hernault Anne , Weiss Eva U. , Greatorex Jane S. , Lever Andrew M.
Published: March 2012

A unique feature of retroviruses is the packaging of two copies of their genome, noncovalently linked at their 5′ ends.
In vitro
, dimerization of human immunodeficiency virus type 2 (HIV-2) RNA occurs by interaction of a self-complementary sequence exposed in the loop of stem-loop 1 (SL-1), also termed the dimer initiation site (DIS). However, in virions, HIV-2 genome dimerization does not depend on the DIS. Instead, a palindrome located within the packaging signal (Psi) is the essential motif for genome dimerization. We reported previously that a mutation within Psi decreasing genome dimerization and packaging also resulted in a reduced proportion of mature particles (A. L'Hernault, J. S. Greatorex, R. A. Crowther, and A. M. Lever, Retrovirology 4:90, 2007). In this study, we investigated further the relationship between HIV-2 genome dimerization, particle maturation, and infectivity by using a series of targeted mutations in SL-1. Our results show that disruption of a purine-rich (
392
-GGAG-
395
) motif within Psi causes a severe reduction in genome dimerization and a replication defect. Maintaining the extended SL-1 structure in combination with the
392
-GGAG-
395
motif enhanced packaging. Unlike that of HIV-1, which can replicate despite mutation of the DIS, HIV-2 replication depends critically on genome dimerization rather than just packaging efficiency. Gag processing was altered in the HIV-2 dimerization mutants, resulting in the accumulation of the MA-CA-p2 processing intermediate and suggesting a link between genome dimerization and particle assembly. Analysis of revertant SL-1 mutant viruses revealed that a compensatory mutation in matrix (70TI) could rescue viral replication and partially restore genome dimerization and Gag processing. Our results are consistent with interdependence between HIV-2 RNA dimerization and the correct proteolytic cleavage of the Gag polyprotein.
Virus Inhibition Activity of Effector Memory CD8
+
T Cells Determines Simian Immunodeficiency Virus Load in Vaccinated Monkeys after Vaccine Breakthrough Infection
Authors: Yamamoto Takuya , Johnson Matthew J. , Price David A. , Wolinsky David I. , Almeida Jorge R. , Petrovas Constantinos , Nason Martha , Yeh Wendy W. , Shen Ling , Roederer Mario , Rao Srinivas S. , McDermott Adrian B. , Lefebvre Francois , Nabel Gary J. , Haddad Elias K. , Letvin Norman L. , Douek Daniel C. , Koup Richard A.
Published: March 2012

The goal of an effective AIDS vaccine is to generate immunity that will prevent human immunodeficiency virus 1 (HIV-1) acquisition. Despite limited progress toward this goal, renewed optimism has followed the recent success of the RV144 vaccine trial in Thailand. However, the lack of complete protection in this trial suggests that breakthroughs, where infection occurs despite adequate vaccination, will be a reality for many vaccine candidates. We previously reported that neutralizing antibodies elicited by DNA prime-recombinant adenovirus serotype 5 (rAd5) boost vaccination with simian immunodeficiency virus strain mac239 (SIVmac239) Gag-Pol and Env provided protection against pathogenic SIVsmE660 acquisition after repeated mucosal challenge. Here, we report that SIV-specific CD8
+
T cells elicited by that vaccine lowered both peak and set-point viral loads in macaques that became infected despite vaccination. These SIV-specific CD8
+
T cells showed strong virus-inhibitory activity (VIA) and displayed an effector memory (EM) phenotype. VIA correlated with high levels of CD107a mobilization and perforin expression in SIV-specific CD8
+
T cells. Remarkably, both the frequency and the number of Gag CM9-specific public clonotypes were strongly correlated with VIA mediated by EM CD8
+
T cells. The ability to elicit such virus-specific EM CD8
+
T cells might contribute substantially to an efficacious HIV/AIDS vaccine, even after breakthrough infection.
The Autographa californica M Nucleopolyhedrovirus
ac79
Gene Encodes an Early Gene Product with Structural Similarities to UvrC and Intron-Encoded Endonucleases That Is Required for Efficient Budded Virus Production
Authors: Wu Wenbi , Passarelli A. Lorena
Published: March 2012

The
Autographa californica
M nucleopolyhedrovirus (AcMNPV)
orf79
(
ac79
) gene is a conserved gene in baculoviruses and shares homology with genes in ascoviruses, iridoviruses, and several bacteria. Ac79 has a conserved motif and structural similarities to UvrC and intron-encoded endonucleases. Ac79 is produced at early times during infection and concentrates in the nucleus of infected cells at late times, suggesting a cellular compartment-specific function. To investigate its function, an
ac79
-knockout bacmid was generated through homologous recombination in
Escherichia coli
. Titration assays showed that budded virus (BV) production was reduced in the
ac79
-knockout virus compared to control viruses, following either virus infection or the transfection of bacmid DNA. The
ac79
-knockout virus-infected cells produced plaques smaller than those infected with control
ac79
-carrying viruses. No obvious differences were observed in viral DNA synthesis, viral protein accumulation, or the formation of occlusion bodies in
ac79
-knockout and control viral DNA-transfected cells, indicating progression into the late and very late phases of viral infection. However, comparative analyses of the amounts of BV genomic DNA and structural proteins in a given quantity of infectious virions suggested that the
ac79
-knockout virus produced more noninfectious BV in infected cells than the control virus. The structure of the
ac79
-knockout BV determined by transmission electron microscopy appeared to be similar to that of the control virus, although aberrant capsid protein-containing tubular structures were observed in the nuclei of
ac79
-knockout virus-infected cells. Tubular structures were not observed for
ac79
viruses with mutations in conserved endonuclease residues. These results indicate that Ac79 is required for efficient BV production.
Analysis of Clinical Ostreid Herpesvirus 1 (Malacoherpesviridae) Specimens by Sequencing Amplified Fragments from Three Virus Genome Areas
Authors: Renault Tristan , Moreau Pierrick , Faury Nicole , Pepin Jean-François , Segarra Amélie , Webb Stephen
Published: March 2012

Although there are a number of ostreid herpesvirus 1 (OsHV-1) variants, it is expected that the true diversity of this virus will be known only after the analysis of significantly more data. To this end, we analyzed 72 OsHV-1 “specimens” collected mainly in France over an 18-year period, from 1993 to 2010. Additional samples were also collected in Ireland, the United States, China, Japan, and New Zealand. Three virus genome regions (open reading frame 4 [ORF4], ORF35, -36, -37, and -38, and ORF42 and -43) were selected for PCR analysis and sequencing. Although ORF4 appeared to be the most polymorphic genome area, distinguishing several genogroups, ORF35, -36, -37, and -38 and ORF42 and -43 also showed variations useful in grouping subpopulations of this virus.
Removal of Vaccinia Virus Genes That Block Interferon Type I and II Pathways Improves Adaptive and Memory Responses of the HIV/AIDS Vaccine Candidate NYVAC-C in Mice
Authors: Gómez Carmen Elena , Perdiguero Beatriz , Nájera Jose Luis , Sorzano Carlos Oscar S. , Jiménez Victoria , González-Sanz Rubén , Esteban Mariano
Published: March 2012

Poxviruses encode multiple inhibitors of the interferon (IFN) system, acting at different levels and blocking the induction of host defense mechanisms. Two viral gene products, B19 and B8, have been shown to act as decoy receptors of type I and type II IFNs, blocking the binding of IFN to its receptor. Since IFN plays a major role in innate immune responses, in this investigation we asked to what extent the viral inhibitors of the IFN system impact the capacity of poxvirus vectors to activate immune responses. This was tested in a mouse model with single and double deletion mutants of the vaccine candidate NYVAC-C, which expresses the HIV-1 Env, Gag, Pol, and Nef antigens. When deleted individually or in double, the type I (B19) and type II (B8) IFN binding proteins were not required for virus replication in cultured cells. Studies of immune responses in mice after DNA prime/NYVAC boost revealed that deletion of
B8R
and/or
B19R
genes improved the magnitude and quality of HIV-1-specific CD8
+
T cell adaptive immune responses and impacted their memory phase, changing the contraction, the memory differentiation, the effect magnitude, and the functionality profile. For B cell responses, deletion of the viral gene
B8R
and/or
B19R
had no effect on antibody levels to HIV-1 Env. These findings revealed that single or double deletion of viral factors (B8 and B19) targeting the IFN pathway is a useful approach in the design of improved poxvirus-based vaccines.
Transformation by E1A Oncoprotein Involves Ubiquitin-Mediated Proteolysis of the Neuronal and Tumor Repressor REST in the Nucleus
Authors: Guan Hancheng , Ricciardi Robert P.
Published: March 2012

The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by mediating the activities of key cellular regulators. The repressor element 1-silencing transcription factor (REST), which is a major neuronal and tumor suppressor, was previously found mainly in the cytoplasm rather than in the nuclei of adenovirus-transformed rodent cells (22). We now demonstrate that the loss of REST in the nucleus is due to its rapid degradation by the ubiquitin-proteasome system. Only nuclear REST, but not its cytoplasmic counterpart, was ubiquitinated and degraded. REST degradation was blocked by the ubiquitination inhibitor PYR-41 and the proteasome inhibitor MG-132 but not by the nuclear export inhibitor leptomycin B. REST degradation required both of its two C-terminal degrons that are recognized by the ubiquitin ligase SCF
β-TrCP
, since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and degradation by upregulating β-TrCP. Knockdown of E1A in virus-transformed cells reduced both β-TrCP and ubiquitination of nuclear REST. In contrast, when expressed in HeLa cells, E1A enhanced the degradation of nuclear REST. Reconstitution of REST in virus-transformed cells negatively affected E1A-mediated cell proliferation and anchorage-independent growth. These data strongly indicate that E1A stimulates ubiquitination and proteolysis of REST in the nucleus, thereby abolishing the tumor suppressor functions of REST.
The Amino Terminus of the Vaccinia Virus E3 Protein Is Necessary To Inhibit the Interferon Response
Authors: White Stacy D. , Jacobs Bertram L.
Published: March 2012

Vaccinia virus (VACV) encodes a multifunctional protein, E3L, that is necessary for interferon (IFN) resistance in cells in culture. Interferon resistance has been mapped to the well-characterized carboxy terminus of E3L, which contains a conserved double-stranded RNA binding domain. The amino terminus of E3L has a Z-form nucleic acid binding domain, which has been shown to be dispensable for replication and IFN resistance in HeLa and RK13 cells; however, a virus expressing E3L deleted of the amino terminus has reduced pathogenicity in an animal model. In this study, we demonstrate that the pathogenicity of a virus expressing E3L deleted of the amino terminus was fully rescued in type I IFN receptor knockout (IFN-α/βR−/−) mice. Furthermore, this virus was IFN sensitive in primary mouse embryo fibroblasts (MEFs). This virus induced the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) in MEFs in an IFN-dependent manner. The depletion of double-stranded RNA-dependent protein kinase (PKR) from these MEFs restored the IFN resistance of this virus. Furthermore, the virus expressing E3L deleted of the amino terminus was also IFN resistant in PKR−/−MEFs. Thus, our data demonstrate that the amino terminus of E3L is necessary to inhibit the type I IFN response both in mice and in MEFs and that in MEFs, the amino terminus of E3L functions to inhibit the PKR pathway.
Abortive Replication of Influenza Virus in Mouse Dendritic Cells
Authors: Ioannidis Lisa J. , Verity Erin E. , Crawford Simon , Rockman Steven P. , Brown Lorena E.
Published: March 2012

The interaction between influenza virus and dendritic cells (DCs) remains poorly defined and controversial. Here we show that influenza virus replication in mouse bone marrow-derived DCs is abortive, despite viral genome transcription and replication occurring for each gene segment and viral hemagglutinin and nucleoprotein, at least, being produced. Electron microscopy reveals that virus assembly, rather than release of virus from the cell surface, is defective.
MicroRNA Targeting of Neurotropic Flavivirus: Effective Control of Virus Escape and Reversion to Neurovirulent Phenotype
Authors: Heiss Brian L. , Maximova Olga A. , Thach Dzung C. , Speicher James M. , Pletnev Alexander G.
Published: March 2012

Neurotropic flaviviruses can efficiently replicate in the developing and mature central nervous systems (CNS) of mice causing lethal encephalitis. Insertion of a single copy of a target for brain-expressed microRNAs (miRNAs) in the 3′ noncoding region (3′NCR) of the flavivirus genome (chimeric tick-borne encephalitis virus/dengue virus) abolished virus neurovirulence in the mature mouse CNS. However, in the developing CNS of highly permissive suckling mice, the miRNA-targeted viruses can revert to a neurovirulent phenotype by accumulating deletions or mutations within the miRNA target sequence. Virus escape from miRNA-mediated suppression in the developing CNS was markedly diminished by increasing the number of miRNA target sites and by extending the distance between these sites in the virus genome. Insertion of multiple miRNA targets into the 3′NCR altered virus neuroinvasiveness, decreased neurovirulence and neuroinflammatory responses, and prevented neurodegeneration without loss of immunogenicity. Although the onset of encephalitis was delayed, a small number of suckling mice still succumbed to lethal intracerebral infection with the miRNA-targeted viruses. Sequence analysis of brain isolates from moribund mice revealed that the viruses escaped from miRNA-mediated suppression exclusively through the deletion of miRNA targets and viral genome sequence located between the two miRNA targets separated by the greatest distance. These findings offer a general strategy to control the reversion of virus to a virulent phenotype: a simultaneous miRNA targeting of the viral genome at many different functionally important regions could prevent virus escape from miRNA-based attenuation, since a deletion of the targeted genomic sequences located between the inserted miRNA binding sites would be lethal for the virus.
Identification of Host-Chromosome Binding Sites and Candidate Gene Targets for Kaposi's Sarcoma-Associated Herpesvirus LANA
Authors: Lu Fang , Tsai Kevin , Chen Horng-Shen , Wikramasinghe Priyankara , Davuluri Ramana V. , Showe Louise , Domsic John , Marmorstein Ronen , Lieberman Paul M.
Published: March 2012

LANA is essential for tethering the Kaposi's sarcoma-associated herpesvirus (KSHV) genome to metaphase chromosomes and for modulating host-cell gene expression, but the binding sites in the host-chromosome remain unknown. Here, we use LANA-specific chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to identify LANA binding sites in the viral and host-cell genomes of a latently infected pleural effusion lymphoma cell line BCBL1. LANA bound with high occupancy to the KSHV genome terminal repeats (TR) and to a few minor binding sites in the KSHV genome, including the LANA promoter region. We identified 256 putative LANA binding site peaks with
P
< 0.01 and overlap in two independent ChIP-Seq experiments. We validated several of the high-occupancy binding sites by conventional ChIP assays and quantitative PCR. Candidate cellular LANA binding motifs were identified and assayed for binding to purified recombinant LANA protein
in vitro
but bound with low affinity compared to the viral TR binding site. More than half of the LANA binding sites (170/256) could be mapped to within 2.5 kb of a cellular gene transcript. Pathways and Gene Ontogeny (GO) analysis revealed that LANA binds to genes within the p53 and tumor necrosis factor (TNF) regulatory network. Further analysis revealed partial overlap of LANA and STAT1 binding sites in several gamma interferon (IFN-γ)-regulated genes. We show that ectopic expression of LANA can downmodulate IFN-γ-mediated activation of a subset of genes, including the TAP1 peptide transporter and proteasome subunit beta type 9 (PSMB9), both of which are required for class I antigen presentation. Our data provide a potential mechanism through which LANA may regulate several host cell pathways by direct binding to gene regulatory elements.
Entanglement, Kronecker Product, Pauli Spin Matrices and a Nonlinear Eigenvalue Problem
Authors: Steeb Willi-Hans , Hardy Yorick
Published: March 2012

Entanglement for the eigenvectors of a nonlinear eigenvalue problem given by the Kronecker product of the Pauli spin matrices is investigated. Fully and partially entangled eigenvectors are found.
Who Has Seen a Free Photon?
Authors: Ojima Izumi , Saigo Hayato
Published: March 2012

While the notion of the position of photons is indispensable in quantum optical situations, it has been known in mathematical physics that any position operator cannot be defined for a massless free particle with a non-zero finite spin. This dilemma is resolved by introducing the "effective mass" of a photon due to the interaction with matter. The validity of this interpretation is confirmed in reference to the picture of "polariton", a basic notion in optical and solid physics. In this connection, we discuss the long-standing controversy between Minkowski's and Abraham's definitions of the momenta of a photon in media from the general viewpoint adopted in the Appendix.
A prospective Neoproterozoic-Cambrian hydrocarbon/exploration play in the Kirthar Fold Belt, Pakistan
Authors: Siddiqui Nusrat Kamal
Published: March 2012

During the Late Neoproterozoic to Early Mesozoic the Indian Plate was part of Pangaea, the Palaeo supercontinent, with the African Plate to the west, as interpreted by various authors. At the same time, the Indian Plate was juxtaposed with the Central Iranian Plate, which separated it from the Arabian Plate in the vicinity of present-day Oman,
c.
800 Ma ago. Between 612 and 200 Ma (Late Neoproterozoic to the end of the Triassic) these plates remained firmly attached. The changing configuration of the plates from 200 Ma to the present are provided in this paper. The Neoproterozoic–Cambrian rocks are recorded along the eastern border of Pakistan with India, extending into the Indian Territory and comprising part of the Neoproterozoic–Cambrian basin (Lower Indus Basin). The southern part of the Lower Indus Basin, from the Thar Platform in the east, deepens westwards towards Karachi (embayment) and the Kachhi Foredeep, where the Neoproterozoic–Cambrian succession is likely to be deep-seated. However,
c.
5 km uplift of the Kirthar Fold Belt (KFB) across the Western Boundary Thrust (WBT) and the subsequent erosion have exposed the Middle Jurassic Chiltan Limestone, particularly in the central region of the KFB (Landsat images and domal structures with Jurassic outcrops are described in this paper) The limestone rests over older strata, with a possible presence of that of Neoproterozoic–Cambrian age, which is interpreted to be at drillable depth.
Stratigraphy and correlation of the Neoproterozoic deposits of central and western India: an overview
Authors: Kumar S.
Published: March 2012

In the central and western part of India, the Neoproterozoic deposits are identified in the Vindhyan and Marwar Basins. The Vindhyan Basin consists of two sub-basins; one in the eastern part and the other in the western part. The basic problem with the Vindhyan Basin is the correlation of the eastern part with the western part, as the two areas show different stratigraphic successions and the outcrops in the eastern part are not traceable in the western part. In this paper, an attempt is made to suggest intrabasinal correlation within the Vindhyan Basin on the basis of stromatolites, carbon isotope data, microbial mats, fossils and lithology. The Marwar Supergroup is developed in the western Rajasthan and unconformably overlies the Malani Igneous Suite, which has previously been dated as 779–681 Ma. On the basis of the available fossil records, the Jodhpur Group has been assigned an Ediacaran age and the Precambrian–Cambrian boundary is suggested within the Bilara Group. As both the Maihar Sandstone of the eastern part of the Vindhyan Basin and the Jodhpur Sandstone of the Marwar Supergroup have been assigned an Ediacaran age, these have been correlated.
Expression and function of KV2-containing channels in human urinary bladder smooth muscle
Authors: Hristov Kiril L. , Chen Muyan , Afeli Serge A. Y. , Cheng Qiuping , Rovner Eric S. , Petkov Georgi V.
Published: March 2012

The functional role of the voltage-gated K+ (KV) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of KV2.1, KV2.2, and the electrically silent KV9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of KV2.1, KV2.2, and KV4.2 homotetrameric channels and of KV2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca2+ imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of KV2.1, KV2.2, and KV9.3 (but not KV4.2) channel subunits in human isolated DSM cells. KV2.1 and KV2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced KV current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca2+ level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5–30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive KV2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.
Activation state of stromal inflammatory cells in murine metastatic pancreatic adenocarcinoma
Authors: Benson Douglas D. , Meng Xianzhong , Fullerton David A. , Moore Ernest E. , Lee Joon H. , Ao Lihua , Silliman Christopher C. , Barnett Carlton C.
Published: March 2012

The histologic presence of macrophages (tumor-associated macrophages, TAMs) and neutrophils (tumor-associated neutrophils, TANs) has been linked to poor clinical outcomes for solid tumors. The exact mechanism for this association with worsened prognosis is unclear. It has been theorized that TAMs are immunomodulated to an alternatively activated state and promote tumor progression. Similarly, TANs have been shown to promote angiogenesis and tumor detachment. TAMs and TANs were characterized for activation state and production of prometastatic mediators in an immunocompetent murine model of pancreatic adenocarcinoma. Specimens from liver metastases were evaluated by immunofluorescence and immunoblotting. TAMS have upregulated expression of CD206 and CD163 markers of alternative activation, (4.14 ± 0.55-fold and 7.36 ± 1.13-fold over control, respectively, P < 0.001) but do not have increased expression of classically activated macrophage markers CCR2 and CCR5. TAMs also express oncostatin M (OSM). We found that TANs, not TAMs, predominantly produce matrix metalloproteinase-9 (MMP-9) in this metastatic tumor microenvironment, while MMP-2 production is pan-tumoral. Moreover, increased expression of VEGF colocalized with TAMs as opposed to TANs. TAMs and TANs may act as distinct effector cells, with TAMs phenotypically exhibiting alternative activation and releasing OSM and VEGF. TANs are localized at the invasive front of the metastasis, where they colocalize with MMP-9. Improved understanding of these interactions may lead to targeted therapies for pancreas adenocarcinoma.
In utero glucocorticoid exposure reduces fetal skeletal muscle mass in rats independent of effects on maternal nutrition
Authors: Gokulakrishnan Ganga , Estrada Irma J. , Sosa Horacio A. , Fiorotto Marta L.
Published: March 2012

Maternal stress and undernutrition can occur together and expose the fetus to high glucocorticoid (GLC) levels during this vulnerable period. To determine the consequences of GLC exposure on fetal skeletal muscle independently of maternal food intake, groups of timed-pregnant Sprague-Dawley rats ( n = 7/group) were studied: ad libitum food intake (control, CON); ad libitum food intake with 1 mg dexamethasone/l drinking water from embryonic day (ED)13 to ED21 (DEX); pair-fed (PF) to DEX from ED13 to ED21. On ED22, dams were injected with [3H]phenylalanine for measurements of fetal leg muscle and diaphragm fractional protein synthesis rates (FSR). Fetal muscles were analyzed for protein and RNA contents, [3H]phenylalanine incorporation, and MuRF1 and atrogin-1 (MAFbx) mRNA expression. Fetal liver tyrosine aminotransferase (TAT) expression was quantified to assess fetal exposure to GLCs. DEX treatment reduced maternal food intake by 13% ( P < 0.001) and significantly reduced placental mass relative to CON and PF dams. Liver TAT expression was elevated only in DEX fetuses ( P < 0.01). DEX muscle protein masses were 56% and 70% than those of CON ( P < 0.01) and PF ( P < 0.05) fetuses, respectively; PF muscles were 80% of CON ( P < 0.01). Muscle FSR decreased by 35% in DEX fetuses ( P < 0.001) but were not different between PF and CON. Only atrogin-1 expression was increased in DEX fetus muscles. We conclude that high maternal GLC levels and inadequate maternal food intake impair fetal skeletal muscle growth, most likely through different mechanisms. When combined, the effects of decreased maternal intake and maternal GLC intake on fetal muscle growth are additive.
Vascular perfusion limits mesenteric lymph flow during anaphylactic hypotension in rats
Authors: Zhang Wei , Shibamoto Toshishige , Kuda Yuhichi , Kurata Yasutaka , Shinomiya Shohei , Kida Masahiro , Tsuchida Hideaki
Published: March 2012

To determine fluid extravasation in the splanchnic vascular bed during anaphylactic hypotension, the mesenteric lymph flow (Qlym) was measured in anesthetized rats sensitized with ovalbumin, along with the systemic arterial pressure (Psa) and portal venous pressure (Ppv). When the antigen was injected into the sensitized rats ( n = 10), Psadecreased from 125 ± 4 to 37 ± 2 mmHg at 10 min with a gradual recovery, whereas Ppvincreased by 16 mmHg at 2 min and returned to the baseline at 10 min. Qlymincreased 3.3-fold from the baseline of 0.023 ± 0.002 g/min to the peak levels of 0.075 ± 0.009 g/min at 2 min and returned to the baseline within 12 min. The lymph protein concentrations increased after antigen, a finding indicating increased vascular permeability. To determine the role of the Ppvincrease in the antigen-induced increase in Qlym, Ppvof the nonsensitized rats ( n = 10) was mechanically elevated in a manner similar to that of the sensitized rats by compressing the portal vein near the hepatic hilus. Unexpectedly, Ppvelevation alone produced a similar increase in Qlym, with the peak comparable to that of the sensitized rats. This finding aroused a question why the antigen-induced increase in Qlymwas limited despite the presence of increased vascular permeability. Thus the changes in splanchnic vascular surface area were assessed by measuring the mesenteric arterial flow. The mesenteric arterial flow was decreased much more in the sensitized rats (75%; n = 5) than the nonsensitized Ppvelevated rats (50%; n = 5). In conclusion, mesenteric lymph flow increases transiently after antigen presumably due to increased capillary pressure of the splanchnic vascular bed via downstream Ppvelevation and perfusion and increased vascular permeability in anesthetized rats. However, this increased extravasation is subsequently limited by decreases in vascular surface area and filtration pressure.
Rapid stimulus-bound suppression of intake in response to an intraduodenal nonnutritive sweetener after training with nutritive sugars predicting malaise
Authors: Schier Lindsey A. , Davidson Terry L. , Powley Terry L.
Published: March 2012

In a previous report (Schier et al., Am J Physiol Regul Integr Comp Physiol 301: R1557–R1568, 2011), we demonstrated with a new behavioral procedure that rats exhibit stimulus-bound suppression of intake in response to an intraduodenal (ID) bitter tastant predicting subsequent malaise. With the use of the same modified taste aversion procedure, the present experiments evaluated whether the sweet taste properties of ID stimuli are likewise detected and encoded. Thirsty rats licked at sipper spouts for hypotonic NaCl for 30 min and received brief (first 6 min) yoked ID infusions of either the same NaCl or an isomolar lithium chloride (LiCl) solution in each session. An intestinal taste cue was mixed directly into the LiCl infusate for aversion training. Results showed that rats failed to detect intestinal sweet taste alone (20 mM Sucralose) but clearly suppressed licking in response to a nutritive sweet taste stimulus (234 mM sucrose) in the intestine that had been repeatedly paired with LiCl. Rats trained with ID sucrose in LiCl subsequently generalized responding to ID Sucralose alone at test. Replicating this, rats trained with ID Sucralose in compound with 80 mM Polycose rapidly suppressed licking to the 20 mM Sucralose alone in a later test. Furthermore, ID sweet taste signaling did not support the rapid negative feedback of sucrose or Polycose on intake when their digestion and transport were blocked. Together, these results suggest that other signaling pathways and/or transporters engaged by caloric carbohydrate stimuli potentiate detection of sweet taste signals in the intestine.
In mpkCCD cells, long-term regulation of aquaporin-2 by vasopressin occurs independent of protein kinase A and CREB but may involve Epac
Authors: Kortenoeven Marleen L. A. , Trimpert Christiane , van den Brand Michiel , Li Yuedan , Wetzels Jack F. M. , Deen Peter M. T.
Published: March 2012

Urine concentration involves the hormone vasopressin (AVP), which stimulates cAMP production in renal principal cells, resulting in translocation and transcription of aquaporin-2 (AQP2) water channels, greatly increasing the water permeability, leading to a concentrated urine. As cAMP levels decrease shortly after AVP addition, whereas AQP2 levels still increase and are maintained for days, we investigated in the present study the mechanism responsible for the AQP2 increase after long-term 1-desamino-8-d-arginine vasopressin (dDAVP) application using mouse collecting duct (mpkCCD) cells. While 30 min of dDAVP incubation strongly increased cAMP, cAMP was lower with 1 day and was even further reduced with 4 days of dDAVP, although still significantly higher than in control cells. One day of dDAVP incubation increased AQP2 promoter-dependent transcription, which was blocked by the protein kinase A (PKA) inhibitor H89. Moreover, phosphorylation of the cAMP-responsive element binding protein (CREB) and CRE-dependent transcription was observed after short-term dDAVP stimulation. With 4 days of dDAVP, AQP2 transcription remained elevated, but this was not blocked by H89, and CRE-dependent transcription and CREB phosphorylation were not increased. Exchange factor directly activated by cAMP (Epac) 1 and 2 were found to be endogenously expressed in mpkCCD cells. Application of dDAVP increased the expression of Epac1, while Epac2 was reduced. Incubation with a specific Epac activator after dDAVP pretreatment increased both AQP2 abundance and transcription compared with cells left unstimulated the last day. In conclusion, the PKA-CRE pathway is involved in the initial rise in AQP2 levels after dDAVP stimulation but not in the long-term effect of dDAVP. Instead, long-term regulation of AQP2 may involve the activation of Epac.
Renal tubular fluid shear stress facilitates monocyte activation toward inflammatory macrophages
Authors: Miravète Mathieu , Dissard Romain , Klein Julie , Gonzalez Julien , Caubet Cécile , Pecher Christiane , Pipy Bernard , Bascands Jean-Loup , Mercier-Bonin Muriel , Schanstra Joost P. , Buffin-Meyer Bénédicte
Published: March 2012

Modified urinary fluid shear stress (FSS) induced by variations of urinary fluid flow and composition is observed in early phases of most kidney diseases. Recently, we reported that renal tubular FSS promotes endothelial cell activation and subsequent adhesion of human monocytes, thereby suggesting that changes in urinary FSS can induce the development of inflammation (Miravète M, Klein J, Besse-Patin A, Gonzalez J, Pecher C, Bascands JL, Mercier-Bonin M, Schanstra JP, Buffin-Meyer B, BBRC 407: 813–817, 2011). Here, we evaluated the influence of tubular FSS on monocytes as they play an important role in the progression of inflammation in nephropathies. Human renal tubular cells (HK-2) were exposed to FSS 0.01 Pa for 30 min or 5 h. Treatment of human THP-1 monocytes with the resulting conditioned medium (FSS-CM) modified the expression of macrophage differentiation markers, suggesting differentiation toward the inflammatory M1-type macrophage. The effect was confirmed in freshly isolated human monocytes. In contrast to endothelial cells, the activation of monocytes by FSS-CM did not require TNF-α. Cytokine array analysis of FSS-CM showed that FSS modified secretion of cytokines by HK-2 cells, particularly by increasing secretion of TGF-β and by decreasing secretion of C-C chemokine ligand 2 (CCL2). Neutralization of TGF-β or CCL2 supplementation attenuated the effect of FSS-CM on macrophage differentiation. Finally, FSS-injured HK-2 cells expressed and secreted early biomarkers of tubular damage such as kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. In conclusion, changes in urinary FSS should now also be considered as potential insults for tubular cells that initiate/perpetuate interstitial inflammation.
Sustained-release prostacyclin analog ONO-1301 ameliorates tubulointerstitial alterations in a mouse obstructive nephropathy model
Authors: Nasu Tatsuyo , Kinomura Masaru , Tanabe Katsuyuki , Yamasaki Hiroko , Htay Su Le , Saito Daisuke , Hinamoto Norikazu , Watatani Hiroyuki , Ujike Haruyo , Suzuki Yoshinori , Sugaya Takeshi , Sugiyama Hitoshi , Sakai Yoshiki , Matsumoto Kunio , Maeshima Yohei , Makino Hirofumi
Published: March 2012

Tubulointerstitial injuries are crucial histological alterations that predict the deterioration of renal function in chronic kidney disease. ONO-1301, a novel sustained-release prostacyclin analog, accompanied by thromboxane synthase activity, exerts therapeutic effects on experimental pulmonary hypertension, lung fibrosis, cardiomyopathy, and myocardial ischemia, partly associated with the induction of hepatocyte growth factor (HGF). In the present study, we examined the therapeutic efficacies of ONO-1301 on tubulointerstitial alterations induced by unilateral ureteral obstruction (UUO). After inducing unilateral ureteral obstruction in C57/BL6J mice, a single injection of sustained-release ONO-1301 polymerized with poly (d,l-lactic-co-glycolic acid) sustained-release ONO-1301 (SR-ONO) significantly suppressed interstitial fibrosis, accumulation of types I and III collagen, increase in the number of interstitial fibroblast-specific protein-1 (FSP-1)+cells, and interstitial infiltration of monocytes/macrophages (F4/80+) in the obstructed kidneys (OBK; day 7). Treatment with SR-ONO significantly suppressed the increase of the renal levels of profibrotic factor TGF-β and phosphorylation of Smad2/3, and elevated the renal levels of HGF in the OBK. In cultured mouse proximal tubular epithelial cells (mProx24), ONO-1301 significantly ameliorated the expression of fibroblast-specific protein-1 and α-smooth muscle actin as well as phosphorylation of Smad3 and increased the expression of zonula occludens-1 and E-cadherin in the presence of TGF-β1 as detected by immunoblot and immunocytochemistry, partly dependent on PGI2receptor-mediated signaling. Administration of rabbit anti-HGF antibodies, but not the control IgG, partly reversed the suppressive effects of SR-ONO on tubulointerstitial injuries in the OBK. Taken together, our findings suggest the potential therapeutic efficacies of ONO-1301 in suppressing tubulointerstitial alterations partly mediated via inducing HGF, an antifibrotic factor counteracting TGF-β.
Addition of endothelial progenitor cells to renal revascularization restores medullary tubular oxygen consumption in swine renal artery stenosis
Authors: Ebrahimi Behzad , Li Zilun , Eirin Alfonso , Zhu Xiang-Yang , Textor Stephen C. , Lerman Lilach O.
Published: March 2012

Renal artery stenosis (RAS) promotes microvascular rarefaction and fibrogenesis, which may eventuate in irreversible kidney injury. We have shown that percutaneous transluminal renal angioplasty (PTRA) or endothelial progenitor cells (EPC) improve renal cortical hemodynamics and function in the poststenotic kidney. The renal medulla is particularly sensitive to hypoxia, yet little is known about reversibility of medullary injury on restoration of renal blood flow. This study was designed to test the hypothesis that PTRA, with or without adjunct EPC delivery to the stenotic kidney, may improve medullary remodeling and tubular function. RAS was induced in 21 pigs using implantation of irritant coils, while another group served as normal controls ( n = 7 each). Two RAS groups were then treated 6 wk later with PTRA or both PTRA and EPC. Four weeks later, medullary hemodynamics, microvascular architecture, and oxygen-dependent tubular function of the stenotic kidneys were examined using multidetector computed tomography, microcomputed tomography, and blood oxygenation level-dependent MRI, respectively. Medullary protein expression of vascular endothelial growth factor, endothelial nitric oxide synthase, hypoxia-inducible factor-1α, and NAD(P)H oxidase p47 were determined. All RAS groups showed decreased medullary vascular density and blood flow. However, in RAS+PTRA+EPC animals, EPC were engrafted in tubular structures, oxygen-dependent tubular function was normalized, and fibrosis attenuated, despite elevated expression of hypoxia-inducible factor-1α and sustained downregulation of vascular endothelial growth factor. In conclusion, EPC delivery, in addition to PTRA, restores medullary oxygen-dependent tubular function, despite impaired medullary blood and oxygen supply. These results support further development of cell-based therapy as an adjunct to revascularization of RAS.
NMDAR antagonist action in thalamus imposes delta oscillations on the hippocampus
Authors: Zhang Yuchun , Yoshida Takashi , Katz Donald B. , Lisman John E.
Published: March 2012

Work on schizophrenia demonstrates the involvement of the hippocampus in the disease and points specifically to hyperactivity of CA1. Many symptoms of schizophrenia can be mimicked by N-methyl-d-aspartate receptor (NMDAR) antagonist; notably, delta frequency oscillations in the awake state are enhanced in schizophrenia, an abnormality that can be mimicked by NMDAR antagonist action in the thalamus. Given that CA1 receives input from the nucleus reuniens of the thalamus, we sought to determine whether an NMDAR antagonist in the thalamus can affect hippocampal processes. We found that a systemic NMDAR antagonist (ketamine; 50 mg/kg) increased the firing rate of cells in the reuniens and CA1 in awake rats. Furthermore, ketamine increased the power of delta oscillations in both structures. The thalamic origin of the change in hippocampal properties was demonstrated in three ways: 1) oscillations in the two structures were coherent; 2) the hippocampal changes induced by systematic ketamine were reduced by thalamic injection of muscimol; and 3) the hippocampal changes could be induced by local injection of ketamine into the thalamus. Lower doses of ketamine (20 mg/kg) did not evoke delta oscillations but did increase hippocampal gamma power, an effect not dependent on the thalamus. There are thus at least two mechanisms for ketamine action on the hippocampus: a low-dose mechanism that affects gamma through a nonthalamic mechanism and a high-dose mechanism that increases CA1 activity and delta oscillations as a result of input from the thalamus. Both mechanisms may be important in producing symptoms of schizophrenia.
Ability of primary auditory cortical neurons to detect amplitude modulation with rate and temporal codes: neurometric analysis
Authors: Johnson Jeffrey S. , Yin Pingbo , O'Connor Kevin N. , Sutter Mitchell L.
Published: March 2012

Amplitude modulation (AM) is a common feature of natural sounds, and its detection is biologically important. Even though most sounds are not fully modulated, the majority of physiological studies have focused on fully modulated (100% modulation depth) sounds. We presented AM noise at a range of modulation depths to awake macaque monkeys while recording from neurons in primary auditory cortex (A1). The ability of neurons to detect partial AM with rate and temporal codes was assessed with signal detection methods. On average, single-cell synchrony was as or more sensitive than spike count in modulation detection. Cells are less sensitive to modulation depth if tested away from their best modulation frequency, particularly for temporal measures. Mean neural modulation detection thresholds in A1 are not as sensitive as behavioral thresholds, but with phase locking the most sensitive neurons are more sensitive, suggesting that for temporal measures the lower-envelope principle cannot account for thresholds. Three methods of preanalysis pooling of spike trains (multiunit, similar to convergence from a cortical column; within cell, similar to convergence of cells with matched response properties; across cell, similar to indiscriminate convergence of cells) all result in an increase in neural sensitivity to modulation depth for both temporal and rate codes. For the across-cell method, pooling of a few dozen cells can result in detection thresholds that approximate those of the behaving animal. With synchrony measures, indiscriminate pooling results in sensitive detection of modulation frequencies between 20 and 60 Hz, suggesting that differences in AM response phase are minor in A1.