bluuebunny/crossref_metadata_2025 huggingface dataset:
Common Polymorphisms in the CACNA1H Gene Associated with Childhood Absence Epilepsy in Chinese Han Population
Authors: Liang J. , Zhang Y. , Chen Y. , Wang J. , Pan H. , Wu H. , Xu K. , Liu X. , Jiang Y. , Shen Y. , Wu X.
Published: November 2006

Variants with a relatively high frequency in the CACNA1H gene have previously been identified in cases of childhood absence epilepsy (CAE) in the Chinese Han population most of which are located in exons 6 to 12. In present study we attempted to further investigate whether the CACNA1H gene is associated with CAE. Exons 6 to 12 of CACNA1H gene were sequenced in samples of 100 CAE trios recruited consecutively, and 191 normal human controls. Single nucleotide polymorphisms (SNPs) were studied in both single locus and haplotype analyses in 218 CAE trios, of which 118 trios were selected from our previous research. Case‐control comparisons and the transmission disequilibrium test (TDT) both supported a coding SNP (cSNP) rs9934839 (R603R) in exon 9 as being close related to CAE. The carriers of the G allele of rs9934839 had a 3‐fold higher risk of CAE than non‐carriers. Moreover, another cSNP rs8044363 was predicted to be connected directly with CAE in a Bayesian network. In addition, two haplotypes consisting of five cSNPs in the region of CACNA1H were statistically associated with CAE. Our research provides new evidence to further support the hypothesis that CACNA1H may be an important susceptibility gene for CAE in the Chinese Han population.
Farm demand for quality potato seed in Indonesia
Authors: Fuglie Keith O. , Adiyoga Witono , Asmunati Rini , Mahalaya Sukendra , Suherman Rachman
Published: November 2006

Seed is the one of the most costly components of potato production in developing countries. Since potato is a vegetatively reproduced crop, diseases such as viruses build up and yield declines as tubers are saved from one harvest for use as seed the next season. Replacing farm‐saved seed with clean seed is one means to increase yield, but information asymmetry between buyers and sellers on seed quality may restrict market supply of this input. In this article we develop a model of the seed market in which clean seed is treated as a capital good providing benefits over several seasons. To determine farm demand for clean seed, we conducted a survey of 182 potato farmers in the major potato growing areas of Indonesia to elicit their perceptions of seed quality from different sources, and derive farmers' “willingness‐to‐pay” for quality potato seed. Results indicate that the effects of information asymmetry on seed supply may be partially offset by the “reputation” of specialized seed producers. Nevertheless, marginal returns to disease‐free seed appear to significantly exceed marginal costs, indicating that improving supply of quality seed will contribute strongly to productivity growth in potato. We discuss several policy options to encourage supply and utilization of quality potato seed.
Effect of information formats on information services: analysis of four selected agricultural commodities in the USA
Authors: Just David R. , Wolf Steven A. , Zilberman David
Published: November 2006

We analyze sources of information in agricultural markets to understand patterns of demand for decision support services and the division of labor in the supply of these services. Information formatting—qualitative variation that determines accessibility and relevance of information in specific contexts—prohibits universal access to informational content. Information formats give rise to both transaction costs and complementarities reflected in patterns of information exchange in commodity systems. Controlling for actors' structural roles and levels of education, we develop a hedonic model of information demand such that actors seek information that is formatted to meet their needs and capabilities. Employing survey data, we explain elements of observed patterns of information exchange in four commodity systems in the USA. Results point to opportunities to expand our understanding of socioeconomic processes underlying production and consumption of decision support.
SELECTION OF CHEMICAL AND THERMAL PRETREATMENT COMBINATION TO REDUCE THE DEHYDRATION TIME OF SOUR CHERRY (Prunus cerasus L.)
Authors: TARHAN SEFA , ERGUNES GAZANFER , TASER O. FARUK
Published: November 2006

Sour cherry (Prunus cerasus L.) is a good source of anthocyanins showing antioxidant and anti‐inflammatory activities. Contemporary drying processes of fruits and vegetables should be fast and at relatively low drying temperatures to minimize the energy consumption and the thermal degradation of their nutritional composition. Therefore, sour cherries placed into sample bags were dipped in one of eight different pretreatment combinations consisting of chemical solutions (2% ethyl oleate, 2% K2CO3, 1% citric acid or water) and dipping temperature (23 or 60C) for 1 min to accelerate the skin moisture diffusivity by breaking down the waxy cuticular surface of sour cherry. Pretreated sour cherry samples were dehydrated by three different methods (artificial drying, solar drying and open sun drying).

Sour cherries treated with 2% ethyl oleate solution at room temperature (23C) were the fastest to reach the final weight loss percentage (75%). They dried in 24 h in the laboratory tray dryer providing 45.2C drying air. The untreated samples reached the same weight loss percentage of 75% at a drying time of 92 h. A high dipping temperature (60C) eliminated the positive effect of ethyl oleate. Solar drying and open sun drying of sour cherries treated with 2% ethyl oleate at room temperature finished approximately in 149 h and 372 h, respectively. The color of dried sour cherries was used as a quality indicator. The increase of dehydration time decreased redness values as seen in solar drying and open sun drying. The recommended pretreatment and drying processes for sour cherries are dipping them into 2% ethyl oleate solution at room temperature for 1 min and then drying them artificially at 45.2C air temperature to effectively retain their original color.
A New Data Fusion Method for Scanned Models
Authors: Jamshidi Jafar , Owen Geraint Wyn , Mileham Antony Roy
Published: November 2006

This paper introduces a high- and low-resolution data integration method for scanning systems. The high-resolution data captured by a touch probe on a CMM, in the form of geometric features, and the low-resolution laser scanned model, in the form of triangulated mesh, are sent to a CAD software. The two model types are first integrated into a unique coordinate system and then unified into a precise CAD model. Depending on the part complexity, some or all of the modelling process can be done automatically. This data fusion method enhances the flexibility of scanning while maintaining the accuracy of the results.
How large is the “natural” magnetic moment?
Authors: Holstein Barry R.
Published: November 2006

The “natural” magnetic moment of a particle of spin S is generally assumed to be that given by the Belinfante conjecture and has the gyromagnetic ratio g=1∕S. Thus, for the spin 1∕2 electron we find the Dirac value ge=2. However, in the standard model the charged W-boson, a spin-one particle, is found to have gW+=2. We discuss this result and argue that the natural value for any particle of spin S should be g=2, independent of spin.
An analytic solution from a spherical gamma emitter
Authors: Atkinson William J. , Brezovich Ivan A.
Published: November 2006

An analytic solution for the fluence rate of primary (non-scattered) gamma rays emerging from a spherical radioactive source is presented. The solution takes self-attenuation into account and is valid at any distance from the source. It is derived using elementary integral calculus and does not require special functions. The key to the relatively simple solution is a shift of the center of the sphere along the z-axis by a distance equal to the radius, so that the surface of the sphere passes through the origin of the coordinate system.
Novel Alpha Interferon (IFN-α) Variant with Improved Inhibitory Activity against Hepatitis C Virus Genotype 1 Replication Compared to IFN-α2b Therapy in a Subgenomic Replicon System
Authors: Escuret Vanessa , Martin Amaury , Durantel David , Parent Romain , Hantz Olivier , Trépo Christian , Menguy Thierry , Bottius Emmanuel , Dardy Jerome , Maral Jean , Escary Jean Louis , Zoulim Fabien
Published: November 2006

Hepatitis C virus (HCV) treatment is based on the association of pegylated alpha interferon (IFN-α) and ribavirin. To improve the level of sustained virological response to treatment, especially in patients infected with HCV genotype 1, new IFNs with improved efficacy and toxicity profiles may be developed. In this report, we show that, in the BM4-5 cell line harboring an HCV subgenomic replicon, a novel and naturally occurring human IFN-α17 variant, GEA007.1, which was discovered by using an original population genetics-based drug discovery approach, inhibits HCV genotype 1 RNA replication more efficiently than does IFN-α2b. Moreover, we show that complete viral clearance is obtained in BM4-5 cells after long-term treatment with GEA007.1, while HCV subgenomic RNA is still detected in cells treated with other IFN-α variants or with standard IFN-α2b. Eventually, we demonstrate that the better inhibitory activity of GEA007.1 compared to that of standard IFN-α is likely to be due to stronger and faster activation of the JAK-STAT signaling pathway and to broader expression of IFN-α-responsive genes in cells. Our results demonstrate a superior inhibitory activity of GEA007.1 over that of IFN-α2b in the HCV replicon system. Clinical trials are required to determine whether GEA007.1 could be a potent “next generation” IFN for the treatment of HCV infection, especially in nonresponders or relapsing patients infected with HCV genotype 1 who currently represent a clinical unmet need.
Effective Prevention of Microbial Biofilm Formation on Medical Devices by Low-Energy Surface Acoustic Waves
Authors: Hazan Zadik , Zumeris Jona , Jacob Harold , Raskin Hanan , Kratysh Gera , Vishnia Moshe , Dror Naama , Barliya Tilda , Mandel Mathilda , Lavie Gad
Published: November 2006

Low-energy surface acoustic waves generated from electrically activated piezo elements are shown to effectively prevent microbial biofilm formation on indwelling medical devices. The development of biofilms by four different bacteria and
Candida
species is prevented when such elastic waves with amplitudes in the nanometer range are applied. Acoustic-wave-activated Foley catheters have all their surfaces vibrating with longitudinal and transversal dispersion vectors homogeneously surrounding the catheter surfaces. The acoustic waves at the surface are repulsive to bacteria and interfere with the docking and attachment of planktonic microorganisms to solid surfaces that constitute the initial phases of microbial biofilm development. FimH-mediated adhesion of uropathogenic
Escherichia coli
to guinea pig erythrocytes was prevented at power densities below thresholds that activate bacterial force sensor mechanisms. Elevated power densities dramatically enhanced red blood cell aggregation. We inserted Foley urinary catheters attached with elastic-wave-generating actuators into the urinary tracts of male rabbits. The treatment with the elastic acoustic waves maintained urine sterility for up to 9 days compared to 2 days in control catheterized animals. Scanning electron microscopy and bioburden analyses revealed diminished biofilm development on these catheters. The ability to prevent biofilm formation on indwelling devices and catheters can benefit the implanted medical device industry.
Efficacy of High Doses of Levofloxacin in Experimental Foreign-Body Infection by Methicillin-Susceptible
Staphylococcus aureus
Authors: Murillo O. , Doménech A. , Garcia A. , Tubau F. , Cabellos C. , Gudiol F. , Ariza J.
Published: November 2006

Antimicrobial efficacy in orthopedic device infections is diminished because of bacterial biofilms which express tolerance to antibiotics. Recently, the use of high doses of levofloxacin with rifampin has been recommended for staphylococcal infections. In the present study, we evaluated the efficacy of levofloxacin at doses of 50 mg/kg/day and 100 mg/kg/day (mimicking the usual and high human doses of 500 mg/day and 750 to 1,000 mg/day, respectively) and compared it to that of to linezolid, cloxacillin, vancomycin, and rifampin in a rat tissue cage model of experimental foreign-body infection by
Staphylococcus aureus
. The antimicrobial efficacy in vitro (by MIC, minimum bactericidal concentration, and kill curves) for logarithmic- and stationary-phase bacteria was compared with the in vivo efficacy. In vitro bactericidal activity at clinically relevant concentrations was reached by all drugs except rifampin and linezolid in the log-phase studies but only by levofloxacin in the stationary-phase studies. The bacterial count decreases from in vivo tissue cage fluids (means) for levofloxacin at 50 and 100 mg/kg/day, rifampin, cloxacillin, vancomycin, linezolid, and controls, respectively, were: −1.24, −2.26, −2.1, −1.56, −1.47, −1.15, and 0.33 (all groups versus controls,
P
< 0.05). Levofloxacin at 100 mg/kg/day (area under the concentration-time curve/MIC ratio, 234) was the most active therapy (
P
= 0.03 versus linezolid). Overall, in vivo efficacy was better predicted by stationary-phase studies, in which it reached a high correlation coefficient even if the rifampin group was excluded (
r
= 0.96;
P
< 0.05). Our results, including in vitro studies with nongrowing bacteria, pharmacodynamic parameters, and antimicrobial efficacy in experimental infection, provide good evidence to support the use of levofloxacin at high doses (750 to 1,000 mg/day), as recently recommended for treating patients with orthopedic prosthesis infections.
Treatment and Prevention of
Staphylococcus epidermidis
Experimental Biomaterial-Associated Infection by Bactericidal Peptide 2
Authors: Kwakman Paulus H. S. , te Velde Anje A. , Vandenbroucke-Grauls Christina M. J. E. , van Deventer Sander J. H. , Zaat Sebastian A. J.
Published: November 2006

Biomaterial-associated infections (BAI) are the major cause of failure of indwelling medical devices and are predominantly caused by staphylococci, especially
Staphylococcus epidermidis
. We investigated the in vitro microbicidal activity of the synthetic antimicrobial peptide bactericidal peptide 2 (BP2) and its efficacy in a murine model of
S. epidermidis
BAI. BP2 showed potent microbicidal activity at micromolar concentrations against a broad spectrum of microorganisms, including antibiotic-resistant bacteria. The staphylocidal activity of BP2 was not affected by physiological salt concentrations and was only slightly affected by the presence of human plasma. In the BAI model, injection of BP2 (5 mg/kg of body weight) 1 h after challenge with
S. epidermidis
resulted in an 80% reduction in the number of culture-positive implants and a 100-fold reduction in survival of
S. epidermidis
in peri-implant tissue at 24 h postchallenge. When BP2 was injected along implants 3 h prior to bacterial challenge, the median numbers of CFU cultured from biomaterial implants and peri-implant tissue were reduced by 85% and 90%, respectively. In conclusion, BP2 has potent, broad-spectrum in vitro microbicidal activity and showed potent in vivo activity in a murine model of
S. epidermidis
biomaterial-associated infection.
Prevalence and Genetic Analysis of Plasmid-Mediated Quinolone Resistance Determinants QnrA and QnrS in
Enterobacteriaceae
Isolates from a French University Hospital
Authors: Poirel Laurent , Leviandier Cécile , Nordmann Patrice
Published: November 2006

The spread of plasmid-mediated quinolone resistance determinants QnrA and QnrS was evaluated in a collection of 186 extended-spectrum β-lactamase (ESBL)-positive enterobacterial isolates from 2002 to 2005 and 185 nalidixic acid-resistant strains isolated during the first 6 months of 2005 at the Bicêtre hospital, France. Out of these 186 ESBL-positive isolates, 2.2 and 1.6% carried a QnrA1 and a QnrS1 determinant, respectively. The ESBLs associated with QnrA1 were VEB-1, SHV-12, and CTX-M-1, whereas those associated with QnrS1 were TEM-52, SHV-12, and CTX-M-1. Among the 185 nalidixic acid-resistant strains isolated in 2005, 0.5 and 2.7% had a QnrA1 determinant and a QnrS1 determinant, respectively. The genetic environments of the
qnrA1
gene differed but were always associated with
sul1
type integrons. In contrast,
qnrS1
genes were not embedded in class 1 integrons but located often (but not systematically) downstream of the insertion sequence IS
Ecl2
on plasmids that often carried a novel β-lactamase gene,
bla
LAP-1
. This is the first study identifying the QnrS resistance determinant in Europe and indicating that this determinant might also be widespread.
Pharmacokinetics of Itraconazole and Hydroxyitraconazole in Healthy Subjects after Single and Multiple Doses of a Novel Formulation
Authors: Mouton J. W. , van Peer A. , de Beule K. , Van Vliet A. , Donnelly J. P. , Soons P. A.
Published: November 2006

Originally, itraconazole for parenteral administration was licensed in a 40% hydroxypropyl-beta-cyclodextrin (HPBCD) solution for intravenous administration. A novel formulation, the NanoCrystal formulation (NCF), was prepared. NCF consists of drug particles of approximately 200 to 300 nm. The pharmacokinetics of itraconazole and its hydroxy metabolite in healthy subjects were evaluated after single and multiple doses of itraconazole as NCF. In the single-ascending-dose (SAD) study, itraconazole doses were planned to range from 50 to 500 mg, while in the multiple-ascending-dose (MAD) study, itraconazole doses of 100, 200, and 300 mg as NCF were studied, as was one dose level (200 mg) as an HBPCD solution. Samples were collected in heparinized tubes at various time points and were analyzed by high-performance liquid chromatography to allow full pharmacokinetic analysis both after the first dose and on day 7. The results of both the SAD and the MAD studies indicated that there was a dose dependency in the half-life of itraconazole from the novel formulation, increasing from 44 h (100 mg) to more than 150 h (300 mg) once steady state was achieved. Similar dose-dependent effects were observed for the hydroxy metabolite. The areas under the concentration-time curves for itraconazole and hydroxyitraconazole were also dose dependent. The pharmacokinetic profiles after 200-mg doses of itraconazole as NCF and HPBCD formulations were comparable with respect to the terminal half-life, both after a single dose and at steady state. NCF may provide an alternative to the HPBCD solution for the further optimization of antifungal treatment with itraconazole.
LfrR Is a Repressor That Regulates Expression of the Efflux Pump LfrA in
Mycobacterium smegmatis
Authors: Buroni Silvia , Manina Giulia , Guglierame Paola , Pasca Maria Rosalia , Riccardi Giovanna , De Rossi Edda
Published: November 2006

The
lfrA
gene of
Mycobacterium smegmatis
encodes an efflux pump which mediates resistance to different fluoroquinolones, cationic dyes, and anthracyclines. The deletion of the
lfrR
gene, coding for a putative repressor and localized upstream of
lfrA
, increased the
lfrA
expression. In this study, reverse transcription-PCR experiments showed that the two genes are organized as an operon, and
lacZ
reporter fusions were used to identify the
lfrRA
promoter region. The
lfrRA
promoter assignment was verified by mapping the transcription start site by primer extension. Furthermore, we found that some substrates of the multidrug transporter LfrA, e.g., acriflavine, ethidium bromide, and rhodamine 123, enhance
lfrA
expression at a detectable level of transcription. LfrR protein was purified from
Escherichia coli
as a fusion protein with a hexahistidine tag and found to bind specifically to a fragment 143 bp upstream of
lfrR
by gel shift analysis. Furthermore, acriflavine was able to cause the dissociation of the LfrR from the promoter, thus suggesting that this molecule interacts directly with LfrR, inducing
lfrA
expression. These results suggest that the LfrR repressor is able to bind to different compounds, which allows induction of LfrA multidrug efflux pump expression in response to these ones. Together, all data suggest that the LfrA pump is tightly regulated and that the repression and induction can be switched about a critical substrate concentration which is toxic for the cell.
In Vitro Human Immunodeficiency Virus Type 1 Resistance Selections with Combinations of Tenofovir and Emtricitabine or Abacavir and Lamivudine
Authors: Margot N. A. , Waters J. M. , Miller M. D.
Published: November 2006

Human immunodeficiency virus type 1 (HIV-1) resistance development was evaluated in vitro by using combinations of the drugs tenofovir and emtricitabine or abacavir and lamivudine, as well as by using the compounds individually. Emtricitabine- and lamivudine-resistant HIV-1 isolates with the M184I or M184V mutation in reverse transcriptase were readily selected in the cultures with emtricitabine alone, lamivudine alone, and the two drug combinations and conferred high-level resistance to emtricitabine and lamivudine. Tenofovir-resistant HIV-1 isolates with the K65R mutation occurred in both the culture with tenofovir alone and the culture with the combination of emtricitabine and tenofovir. The S68N and S68K mutations were also observed in the tenofovir cultures, with no detectable impact on resistance, suggesting a possible compensatory role in viral fitness. At low concentrations of emtricitabine and tenofovir, the M184I mutation appeared first, followed by the K65R mutation, in a subset of viruses. At intermediate concentrations of emtricitabine and tenofovir, viruses harboring the K65R mutation or a novel K65N and K70R double mutation grew before they gave rise to mutants with K65R and M184V/I double mutations at higher emtricitabine concentrations. Abacavir resistance was characterized by the accumulation of the M184V, Y115F, and K65R mutations in the abacavir culture, while the M184V and L74V mutations were selected in combination with lamivudine. In the presence of the abacavir resistance mutations, viral growth was strong even in the presence of high concentrations of abacavir. In contrast, viral growth was markedly impaired in the cultures with high tenofovir concentrations, even in the presence of K65R. In conclusion, these studies show that HIV-1 mutants with a K65R and M184V genotype are generated under maximum selection pressure from the combination of tenofovir and emtricitabine.
Oxidant Generation by Single Infected Monocytes after Short-Term Fluorescence Labeling of a Protozoan Parasite
Authors: Chang Haeok K. , Thalhofer Colin , Duerkop Breck A. , Mehling Joanna S. , Verma Shilpi , Gollob Kenneth J. , Almeida Roque , Wilson Mary E.
Published: November 2006

Leishmaniaspp. are intracellular protozoa residing in mononuclear phagocytes.Leishmaniaorganisms are susceptible to microbicidal responses generated in response to phagocytosis. Assuming that both phagocyte and parasite populations are heterogeneous, it is advantageous to examine the response of individual cells phagocytosing living parasites. BecauseLeishmaniaspp. lose virulence during the raising of transfectants, we developed a method to label liveLeishmania chagasishort-term with fluorescent dyes. Up to six parasite divisions were detected by flow cytometry after labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE), dioctadecyl-tetramethylindo carbocyanine perchlorate, or chloromethyl tetramethylrhodamine. Labeled parasites entered mononuclear phagocytes as determined by confocal and time-lapse microscopy. Dihydroethidium (DHE) was used to detect macrophage-derived oxidants generated during phagocytosis. PresumablyLeishmaniaorganisms are opsonized with host serum/tissue components such as complement prior to phagocytosis. Therefore, we investigated the effects of opsonization and found that this increased the efficiency of CFSE-labeled parasite entry into monocytes (84.6% ± 8.8% versus 20.2% ± 3.8% monocytes infected;P< 0.001). Opsonization also increased the percentage of phagocytes undergoing a respiratory burst (66.0% ± 6.3% versus 41.0% ± 8.3% of monocytes containing CFSE-labeled parasites;P< 0.001) and the magnitude of oxidant generation by each infected monocyte. Inhibitor data indicated that DHE was oxidized by products of the NADPH oxidase. These data suggest that opsonized serum components such as complement lead to more efficient entry ofLeishmaniainto their target cells but at the same time activate the phagocyte oxidase to generate microbicidal products in infected cells. The parasite must balance these positive and negative survival effects in order to initiate a viable infection.
Mutants of Type II Heat-Labile Enterotoxin LT-IIa with Altered Ganglioside-Binding Activities and Diminished Toxicity Are Potent Mucosal Adjuvants
Authors: Nawar Hesham F. , Arce Sergio , Russell Michael W. , Connell Terry D.
Published: November 2006

The structure and function LT-IIa, a type II heat-labile enterotoxin ofEscherichia coli, are closely related to the structures and functions of cholera toxin and LT-I, the type I heat-labile enterotoxins ofVibrio choleraeand enterotoxigenicEscherichia coli, respectively. While LT-IIa is a potent systemic and mucosal adjuvant, recent studies demonstrated that mutant LT-IIa(T34I), which exhibits no detectable binding activity as determined by an enzyme-linked immunosorbent assay, with gangliosides GD1b, GD1a, and GM1 is a very poor adjuvant. To evaluate whether other mutant LT-IIa enterotoxins that also exhibit diminished ganglioside-binding activities have greater adjuvant activities, BALB/c mice were immunized by the intranasal route with the surface adhesin protein AgI/II ofStreptococcus mutansalone or in combination with LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D). All three mutant enterotoxins potentiated strong mucosal immune responses that were equivalent to the response promulgated by wt LT-IIa. All three mutant enterotoxins augmented the systemic immune responses that correlated with their ganglioside-binding activities. Only LT-IIa and LT-IIa(T14S), however, enhanced expression of major histocompatibility complex class II and the costimulatory molecules CD40, CD80, and CD86 on splenic dendritic cells. LT-IIa(T14I) and LT-IIa(T14D) had extremely diminished toxicities in a mouse Y1 adrenal cell bioassay and reduced abilities to induce the accumulation of intracellular cyclic AMP in a macrophage cell line.
Enteropathogenic
Escherichia coli
,
Shigella flexneri
, and
Listeria monocytogenes
Recruit a Junctional Protein, Zonula Occludens-1, to Actin Tails and Pedestals
Authors: Hanajima-Ozawa Miyuki , Matsuzawa Takeshi , Fukui Aya , Kamitani Shigeki , Ohnishi Hiroe , Abe Akio , Horiguchi Yasuhiko , Miyake Masami
Published: November 2006

Enteropathogenic
Escherichia coli
,
Shigella flexneri
, and
Listeria monocytogenes
induce localized actin polymerization at the cytoplasmic face of the plasma membrane or within the host cytoplasm, creating unique actin-rich structures termed pedestals or actin tails. The process is known to be mediated by the actin-related protein 2 and 3 (Arp2/3) complex, which in these cases acts downstream of neural Wiskott-Aldrich syndrome protein (N-WASP) or of a listerial functional homolog of WASP family proteins. Here, we show that zonula occludens-1 (ZO-1), a protein in the tight junctions of polarized epithelial cells, is recruited to actin tails and pedestals. Immunocytochemical analysis revealed that ZO-1 was stained most in the distal part of the actin-rich structures, and the incorporation was mediated by the proline-rich region of the ZO-1 molecule. The direct clustering of membrane-targeted Nck, which is known to activate the N-WASP-Arp2/3 pathway, triggered the formation of the ZO-1-associated actin tails. The results suggest that the activation of the Arp2/3 complex downstream of N-WASP or a WASP-related molecule is a key to the formation of the particular actin-rich structures that bind with ZO-1. We propose that an analysis of the recruitment on a molecular basis will lead to an understanding of how ZO-1 recognizes a distinctive actin-rich structure under pathophysiological conditions.
Vaccination with Live
Yersinia pestis
Primes CD4 and CD8 T Cells That Synergistically Protect against Lethal Pulmonary
Y. pestis
Infection
Authors: Philipovskiy Alexander V. , Smiley Stephen T.
Published: November 2006

Vaccination with live attenuated
Yersinia pestis
confers protection against pneumonic plague but is not considered safe for general use. Subunit plague vaccines containing the
Y. pestis
F1 and LcrV proteins prime robust antibody responses but may not provide sufficient protection. To aid the development of a safe and effective plague vaccine, we are investigating roles for T cells during defense against
Y. pestis
infection. Here we demonstrate that vaccination of mice with live
Y. pestis
primes specific CD4 and CD8 T cells that, upon purification and direct transfer to naïve mice, synergistically protect against lethal intranasal
Y. pestis
challenge. While not preventing extrapulmonary dissemination, the coadministered T cells promote bacterial clearance and reduce bacteremia. These observations strongly suggest that development of pneumonic plague vaccines should strive to prime both CD4 and CD8 T cells. Finally, we demonstrate that vaccination with live
Y. pestis
primes CD4 and CD8 T cells that respond to
Y. pestis
strains lacking the capacity to express F1, LcrV, and all pCD1/pPCP-encoded proteins, suggesting that protective T cells likely recognize antigens distinct from those previously defined as targets for humoral immunity.
Downregulation of connexin40 and increased prevalence of atrial arrhythmias in transgenic mice with cardiac-restricted overexpression of tumor necrosis factor
Authors: Sawaya Sam E. , Rajawat Yadavendra S. , Rami Tapan G. , Szalai Gabor , Price Robert L. , Sivasubramanian Natarajan , Mann Douglas L. , Khoury Dirar S.
Published: November 2006

Atrial arrhythmias, primarily atrial fibrillation, have been independently associated with structural remodeling and with inflammation. We hypothesized that sustained inflammatory signaling by tumor necrosis factor (TNF) would lead to alterations both in underlying atrial myocardial structure and in atrial electrical conduction. We performed ECG recording, intracardiac electrophysiology studies, epicardial mapping, and connexin immunohistochemical analyses on transgenic mice with targeted overexpression of TNF in the cardiac compartment (MHCsTNF) and on wild-type (WT) control mice (age 8–16 wk). Atrial and ventricular conduction abnormalities were always evident on ECG in MHCsTNF mice, including a shortened atrioventricular interval with a wide QRS duration secondary to junctional rhythm. Supraventricular arrhythmias were observed in five of eight MHCsTNF mice, whereas none of the mice demonstrated ventricular arrhythmias. No arrhythmias were observed in WT mice. Left ventricular conduction velocity during apical pacing was similar between the two mouse groups. Connexin40 was significantly downregulated in MHCsTNF mice. In contrast, connexin43 density was not significantly altered in MHCsTNF mice, but rather dispersed away from the intercalated disks. In conclusion, sustained inflammatory signaling contributed to atrial structural remodeling and downregulation of connexin40 that was associated with an increased prevalence of atrial arrhythmias.
Mapping Geographic Zones of Cancer Risk with Epigenetic Biomarkers in Normal Breast Tissue
Authors: Yan Pearlly S. , Venkataramu Chinnambally , Ibrahim Ashraf , Liu Joseph C. , Shen Rulong Z. , Diaz Nils M. , Centeno Barbara , Weber Frank , Leu Yu-Wei , Shapiro Charles L. , Eng Charis , Yeatman Timothy J. , Huang Tim H.-M.
Published: November 2006

Purpose: Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors.

Experimental Design: First, methylation status of a 4-kb region of RASSF1A promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding RASSF1A promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings.

Results: DNA methylation in the core RASSF1A promoter was low in reduction mammoplasty tissues (P = 0.0001) when compared with primary tumors. The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in RASSF1A promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR.

Conclusions: Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.
Axillary Lymph Node Nanometastases Are Prognostic Factors for Disease-Free Survival and Metastatic Relapse in Breast Cancer Patients
Authors: Querzoli Patrizia , Pedriali Massimo , Rinaldi Rosa , Lombardi Anna Rita , Biganzoli Elia , Boracchi Patrizia , Ferretti Stefano , Frasson Claudia , Zanella Caterina , Ghisellini Sara , Ambrogi Federico , Antolini Laura , Piantelli Mauro , Iacobelli Stefano , Marubini Ettore , Alberti Saverio , Nenci Italo
Published: November 2006

Purpose: Early breast cancer presents with a remarkable heterogeneity of outcomes. Undetected, microscopic lymph node tumor deposits may account for a significant fraction of this prognostic diversity. Thus, we systematically evaluated the presence of lymph node tumor cell deposits ≤0.2 mm in diameter [pN0(i+), nanometastases] and analyzed their prognostic effect.

Experimental Design: Single-institution, consecutive patients with 8 years of median follow-up (n = 702) were studied. To maximize chances of detecting micrometastases and nanometastases, whole-axilla dissections were analyzed. pN0 cases (n = 377) were systematically reevaluated by lymph node (n = 6676) step-sectioning and anticytokeratin immunohistochemical analysis. The risk of first adverse events and of distant relapse of bona fide pN0 patients was compared with that of pN0(i+), pN1mi, and pN1 cases.

Results: Minimal lymph node deposits were revealed in 13% of pN0 patients. The hazard ratio for all adverse events of pN0(i+) versus pN0(i−) was 2.51 (P = 0.00019). Hazards of pN1mi and pN0(i+) cases were not significantly different. A multivariate Cox model showed a hazard ratio of 2.16 for grouped pN0(i+)/pN1mi versus pN0(i−) (P = 0.0005). Crude cumulative incidence curves for metastatic relapse were also significantly different (Gray's test χ2 = 5.54, P = 0.019).

Conclusion: Nanometastases are a strong risk factor for disease-free survival and for metastatic relapse. These findings support the inclusion of procedures for nanometastasis detection in tumor-node-metastasis staging.
FGFR1 Emerges as a Potential Therapeutic Target for Lobular Breast Carcinomas
Authors: Reis-Filho Jorge Sergio , Simpson Pete T. , Turner Nicholas C. , Lambros Maryou Ballo , Jones Chris , Mackay Alan , Grigoriadis Anita , Sarrio David , Savage Kay , Dexter Tim , Iravani Marjan , Fenwick Kerry , Weber Barbara , Hardisson David , Schmitt Fernando Carlos , Palacios Jose , Lakhani Sunil R. , Ashworth Alan
Published: November 2006

Purpose: Classic lobular carcinomas (CLC) account for 10% to 15% of all breast cancers. At the genetic level, CLCs show recurrent physical loss of chromosome16q coupled with the lack of E-cadherin (CDH1 gene) expression. However, little is known about the putative therapeutic targets for these tumors. The aim of this study was to characterize CLCs at the molecular genetic level and identify putative therapeutic targets.

Experimental Design: We subjected 13 cases of CLC to a comprehensive molecular analysis including immunohistochemistry for E-cadherin, estrogen and progesterone receptors, HER2/neu and p53; high-resolution comparative genomic hybridization (HR-CGH); microarray-based CGH (aCGH); and fluorescent and chromogenic in situ hybridization for CCND1 and FGFR1.

Results: All cases lacked the expression of E-cadherin, p53, and HER2, and all but one case was positive for estrogen receptors. HR-CGH revealed recurrent gains on 1q and losses on 16q (both, 85%). aCGH showed a good agreement with but higher resolution and sensitivity than HR-CGH. Recurrent, high level gains at 11q13 (CCND1) and 8p12-p11.2 were identified in seven and six cases, respectively, and were validated with in situ hybridization. Examination of aCGH and the gene expression profile data of the cell lines, MDA-MB-134 and ZR-75-1, which harbor distinct gains of 8p12-p11.2, identified FGFR1 as a putative amplicon driver of 8p12-p11.2 amplification in MDA-MB-134. Inhibition of FGFR1 expression using small interfering RNA or a small-molecule chemical inhibitor showed that FGFR1 signaling contributes to the survival of MDA-MB-134 cells.

Conclusions: Our findings suggest that receptor FGFR1 inhibitors may be useful as therapeutics in a subset of CLCs.
Fluorescence Visualization Detection of Field Alterations in Tumor Margins of Oral Cancer Patients
Authors: Poh Catherine F. , Zhang Lewei , Anderson Don W. , Durham J. Scott , Williams P. Michele , Priddy Robert W. , Berean Ken W. , Ng Samson , Tseng Olivia L. , MacAulay Calum , Rosin Miriam P.
Published: November 2006

Purpose: Genetically altered cells could become widespread across the epithelium of patients with oral cancer, often in clinically and histologically normal tissue, and contribute to recurrent disease. Molecular approaches have begun to yield information on cancer/risk fields; tissue optics could further extend our understanding of alteration to phenotype as a result of molecular change.

Experimental Design: We used a simple hand-held device in the operating room to directly visualize subclinical field changes around oral cancers, documenting alteration to fluorescence. A total of 122 oral mucosa biopsies were obtained from 20 surgical specimens with each biopsy being assessed for location, fluorescence visualization (FV) status, histology, and loss of heterozygosity (LOH; 10 markers on three regions: 3p14, 9p21, and 17p13).

Results: All tumors showed FV loss (FVL). For 19 of the 20 tumors, the loss extended in at least one direction beyond the clinically visible tumor, with the extension varying from 4 to 25 mm. Thirty-two of 36 FVL biopsies showed histologic change (including 7 squamous cell carcinoma/carcinomas in situ, 10 severe dysplasias, and 15 mild/moderate dysplasias) compared with 1 of the 66 FV retained (FVR) biopsies. Molecular analysis on margins with low-grade or no dysplasia showed a significant association of LOH in FVL biopsies, with LOH at 3p and/or 9p (previously associated with local tumor recurrence) present in 12 of 19 FVL biopsies compared with 3 of 13 FVR biopsies (P = 0.04).

Conclusions: These data have, for the first time, shown that direct FV can identify subclinical high-risk fields with cancerous and precancerous changes in the operating room setting.
Synopsis of a Roundtable on Validating Novel Therapeutics for Multiple Myeloma
Authors: Dalton William , Anderson Kenneth C.
Published: November 2006

Purpose: With the identification of new molecular targets and pathways, many new therapeutic approaches are being identified for potential application in the treatment of multiple myeloma. New chemical compounds and biologics have been developed against molecular targets with substantial scientific evidence that these targets are involved in myeloma development, progression, or relapse. To safely and rapidly bring these advances to bear on the disease, new preclinical models in cells and animals need to be established, as well as prioritization and standardization in current preclinical and clinical validation. An experts' roundtable was convened in November 2005 to discuss shortcomings in current preclinical models and discuss what models are needed to best validate therapeutics and combinations of therapies for multiple myeloma.

Conclusions: This exciting event brought together experts in compound validation, preclinical development, and experts in multiple myeloma from academic institutions and the pharmaceutical and biotechnology industries. The goals were to evaluate an algorithm for therapeutic validation and discuss in vitro modeling for target discovery, animal models for preclinical development, and models for testing drug combinations.
Molecular and Cytogenetic Subgroups of Oropharyngeal Squamous Cell Carcinoma
Authors: Perrone Federica , Suardi Simona , Pastore Elisa , Casieri Paola , Orsenigo Marta , Caramuta Stefano , Dagrada Gianpaolo , Losa Marco , Licitra Lisa , Bossi Paolo , Staurengo Samantha , Oggionni Maria , Locati Laura , Cantu Giulio , Squadrelli Massimo , Carbone Antonino , Pierotti Marco A. , Pilotti Silvana
Published: November 2006

Purpose: The aim of this study was to acquire further insights into the pathogenetic pathways of head and neck squamous cell carcinomas (HNSCC) that may be useful for identifying new biomarkers instrumental in developing more specific treatment approaches.

Experimental Design: Cell cycle regulators and epidermal growth factor receptor (EGFR) and BRAF genes were analyzed in a series of 90 oropharyngeal SCCs of a cohort of surgically treated patients from a single institution, and the results were matched with the presence of high-risk human papillomavirus (HR-HPV) DNA and the TP53 status.

Results: At least four distinct groups of tumors were identified sharing a common histology but displaying different molecular/cytogenetic patterns: (a) 19% were HPV-positive SCCs whose lack of alterations of the investigated genes could explain their particular natural history, which requires less aggressive treatment; (b) 37% were HPV-negative SCCs carrying TP53 mutations, which may be more effectively treated by drugs acting through p53-independent apoptosis; (c) 34% were HPV-negative SCCs carrying wild-type TP53 and loss of 9p21 (p16INK4a and p15INK4b) and/or cyclin D1 overexpression that justify treatment with DNA-damaging drugs followed by cell cycle inhibitors; and (d) 10% were HPV-negative lacking tumor suppressor genes and cell cycle alterations. The second, third, and fourth groups also showed an increased copy number of EGFR and chromosome 7 (43%) that might justify the additional or alternative use of EGFR inhibitors.

Conclusions: Our findings suggest that assessing HPV, TP53, 9p21, and EGFR status may be crucial to finding more tailored and beneficial treatments for oropharyngeal SCCs.
Targeting the active β-catenin pathway to treat cancer cells
Authors: Dvory-Sobol Hadas , Sagiv Eyal , Kazanov Diana , Ben-Ze'ev Avri , Arber Nadir
Published: November 2006

The adenomatous polyposis coli or β-catenin genes are frequently mutated in colorectal cancer cells, resulting in oncogenic activation of β-catenin signaling. We tried to establish in vitro and in vivo models for selectively killing human cancer cells with an activated β-catenin/T-cell factor (Tcf) pathway. We used a recombinant adenovirus that carries a lethal gene [p53-up-regulated modulator of apoptosis (PUMA)] under the control of a β-catenin/Tcf–responsive promoter (AdTOP-PUMA) to selectively target human colorectal cancer cells (SW480, HCT116, DLD-1, and LS174T), hepatocellular carcinoma (HepG2), and gastric cancer cells (AGS) in which the β-catenin/Tcf pathway is activated, and compared its efficiency in killing cancer cells in which this pathway is inactive or only weakly active. AdFOP-PUMA, carrying a mutant Tcf-binding site, was used as control virus. The combined effect of AdTOP-PUMA with several chemotherapeutic agents (5-florouracil, doxorubicin, and paclitaxel) was also evaluated. The effect of AdTOP-PUMA on colorectal cancer cells was also examined in nude mice: SW480 cells were infected with the AdTOP-PUMA and AdFOP-PUMA, and then inoculated s.c. into nude mice. The TOP-PUMA adenovirus inhibited cell growth in a dose-dependent fashion, depending on the signaling activity of β-catenin. The growth of cells displaying high levels of active β-catenin/Tcf signaling was inhibited after infection with AdTOP-PUMA, whereas that of cells with low levels of β-catenin signaling was not. Growth inhibition was associated with induction of apoptosis. Chemotherapy synergistically enhanced the effect of AdTOP-PUMA. A combination of the adenovirus system with standard therapy may improve the efficacy and reduce the toxicity of therapy in humans. [Mol Cancer Ther 2006;5(11):2861–71]
Cantharidin-induced mitotic arrest is associated with the formation of aberrant mitotic spindles and lagging chromosomes resulting, in part, from the suppression of PP2Aα
Authors: Bonness Kathy , Aragon Ileana V. , Rutland Beth , Ofori-Acquah Solomon , Dean Nicholas M. , Honkanen Richard E.
Published: November 2006

Cantharidin, a natural vesicant, inhibits the activity of several PPP family phosphatases, displays antitumor activity, and induces apoptosis in many types of tumor cells. However, the molecular mechanisms underlying the antitumor activity of cantharidin are not clear. Here, dose-response studies confirm a strong correlation between the suppression of phosphatase activity and cell death. Flow cytometry analysis indicates that before apoptosis, cantharidin delays cell cycle progression following DNA replication with no apparent effect on G1-S or S-G2 phase progression. In contrast, studies with double thymidine-synchronized populations of cells indicate that cantharidin can rapidly arrest growth when added during G2 or early M phase. Immunostaining indicates that cell cycle arrest occurs before the completion of mitosis and is associated with the appearance of aberrant mitotic spindles. Live cell imaging with time-lapse microscopy shows that cantharidin disrupts the metaphase alignment of chromosomes and produces a prolonged mitotic arrest, with the onset of apoptosis occurring before the onset of anaphase. To explore the contribution of individual phosphatases, antisense oligonucleotides and small interfering RNA were developed to suppress the expression of cantharidin-sensitive phosphatases. The suppression of PP2Aα, but not PP2Aβ, is sufficient to induce metaphase arrest, during which time lagging chromosomes are observed moving between the spindle poles and the metaphase plate. Immunostaining revealed slightly abnormal, yet predominately bipolar, mitotic spindles. Nonetheless, after a 10- to 15-hour delay, the cells enter anaphase, suggesting that an additional cantharidin-sensitive phosphatase is involved in the progression from metaphase into anaphase or to prevent the onset of apoptosis in cells arrested during mitosis. [Mol Cancer Ther 2006;5(11):2727–36]