bluuebunny/crossref_metadata_2025 huggingface dataset:
Early decline of hemoglobin can predict progression of hemolytic anemia during pegylated interferon and ribavirin combination therapy in patients with chronic hepatitis C
Authors: Hiramatsu Naoki , Kurashige Nao , Oze Tsugiko , Takehara Tetsuo , Tamura Shinji , Kasahara Akinori , Oshita Masahide , Katayama Kazuhiro , Yoshihara Harumasa , Imai Yasuharu , Kato Michio , Kawata Sumio , Tsubouchi Hirohito , Kumada Hiromitsu , Okanoue Takeshi , Kakumu Shinichi , Hayashi Norio
Published: August 2007

Aim:  Ribavirin, used to treat chronic hepatitis C, can induce hemolytic anemia, forcing the discontinuance of treatment. To establish a predictive measure to help circumvent this, we evaluated the relationship of hemoglobin (Hb) decline with the discontinuance of treatment during the progression of ribavirin‐induced anemia.

Methods:  One hundred and sixteen patients (71% male) with genotype 1 chronic hepatitis C were treated with pegylated interferon (PegIFN) α‐2b and ribavirin. The mean age was 50.6 years and 55% were IFN naïve. A decline of Hb concentration by 2 g/dL at two weeks from the start of the treatment (“2 by 2” standard) was adopted as the predictive factor for the progression of anemia.

Results:  By applying the “2 by 2” standard, with ΔHb ≥ 2 g/dL (34%, n = 39), treatment was discontinued in 12 cases (31%), three of which (8%) because of severe anemia. ForΔHb < 2 g/dL (64%, n = 76), treatment was discontinued in 11 (14%) cases; none due to severe anemia. Ten percent (4/39) of patients showed the minimum Hb ≤ 8.5 g/dL in the ΔHb ≥ 2 g/dL group, with none in the ΔHb < 2 g/dL group (P = 0.001). Furthermore, the patients with minimum Hb ≤ 8.5 g/dL were found only in the “2 by 2” standard‐positive and low CL/F (<15) group (4/29, 14%).

Conclusion:  Monitoring the Hb decline using the “2 by 2” standard can identify patients who are prone to developing severe anemia. Further prospective studies are needed using ribavirin reduction based on the “2 by 2” standard.
Hepatocyte growth factor promotes remodeling of murine liver fibrosis, accelerating recruitment of bone marrow‐derived cells into the liver
Authors: Asano Yasukane , Iimuro Yuji , Son Gakuhei , Hirano Tadamichi , Fujimoto Jiro
Published: August 2007

Aim:  Hepatocyte growth factor (HGF) ameliorates liver fibrosis/cirrhosis in animal models, while the participation of bone marrow‐derived cells (BMC) in the repair process of injured organs has recently been reported. In this study we investigated the roles of HGF and BMC in a remodeling process of liver fibrosis.

Methods:  C57BL/6 J mice were treated with carbon tetrachloride (CCl4) for 10 weeks. At week six, the mice underwent whole body irradiation and transplantation with bone marrow cells from syngenic LacZ‐transgenic mice. After the transplantation, gene transfer of HGF into skeletal muscles was performed once a week for four weeks. In the control group, sterile saline was injected.

Results:  HGF gene transfer ameliorated the CCl4‐induced liver fibrosis, accelerating recruitment of LacZ‐expressing cells into the liver. This phenomenon was accompanied byincreased gelatinase activity in the liver. A large number of the LacZ‐positive cells expressed markers of vascular endothelial cells, while some of them had a marker of macrophages. Expression of stromal cell‐derived factor (SDF)‐1 in the liver was upregulated around the central veins, especially in the HGF gene‐transferred animals, recruiting chemokine (C‐X‐C motif) receptor (CXCR) 4‐positive cells in this area.

Conclusion:  Transplanted BMC participate in the HGF‐induced remodeling process of liver fibrosis. The roles of HGF in this process include the recruitment of BMC, possibly through increased expression of SDF‐1 in part, as well as anti‐apoptotic, mitogenic and antifibrotic activities on liver cells.
Candidate genes responsible for human hepatocellular carcinoma identified from differentially expressed genes in hepatocarcinogenesis of the tree shrew (Tupaia belangeri chinesis)
Authors: Li Yuan , Wan Dafang , Wei Wei , Su Jianjia , Cao Ji , Qiu Xiaokun , Ou Chao , Ban Kecheng , Yang Chun , Yue Huifen
Published: August 2007

Aim:  To explore gene expression profiles during hepatocarcinogenesis of the tree shrew, and to find the genes responsible for human hepatocellular carcinoma (HCC).

Methods:  Tree shrews were used as an animal model for HCC induction employing aflatoxin B1 (AFB1) alone or AFB1 plus hepatitis B virus (HBV) as etiological factors. Gene expression profiles from the tissues of HCC, HCC‐surrounding liver tissues (para‐HCC) and the corresponding biopsies taken from the same animals before HCC had developed (pre‐HCC) were analyzed by cDNA microarray assay to identify differentially expressed genes. Two genes, CuZn‐superoxide dismutase (SOD1) and glutathione S‐transferase A1 (GSTA1), were further investigated by reverse transcriptase–polymerase chain reaction (RT–PCR) and immunohistochemical (IHC) assays were done on tree shrew and human HCC samples.

Results:  Results from the cDNA microarray analysis indicated that the gene expression profiles of HCC between AFB1and AFB1 + HBV treatment groups were markedly different. A total of 11 genes, including SOD1 and GSTA1, were found changing in expression levels in all detected samples from both groups. Results from RT–PCR and IHC assays indicated that mRNA and protein levels of SOD1 and GSTA1 were markedly downregulated in both tree shrew and human HCC, and downregulation of SOD1 and GSTA1 proteins in human HCC samples was closely correlated with the histopathological grading (P < 0.05).

Conclusion:  The differentially expressed genes found in all HCC cases induced by different etiological factors among different species should be considered as good candidate genes responsible for HCC. Downregulation of SOD1 and GSTA1 might play an important role in hepatocarcinogenesis.
An Adaptive Machining Fixture Design System for Automatically Dealing With Design Changes
Authors: Fathianathan Mervyn , Kumar A. Senthil , Nee A. Y. C.
Published: August 2007

Integrated product and process design involves a dynamic environment where various design changes are made when the requirements of the different domains are not sufficiently met. Dealing with changes is a tedious and cumbersome process. To deal with design changes automatically, this paper presents the development of an adaptive fixture design system based on an evolutionary search algorithm. The system senses a change made to the workpiece model and automatically deals with the change. Conducted experiments reveal that the evolutionary search algorithm is efficient and effective in dealing with design changes adaptively.
A Hybrid Analytical, Solid Modeler and Feature-Based Methodology for Extracting Tool-Workpiece Engagements in Turning
Authors: Zhou Jing , Yip-Hoi Derek , Huang Xuemei
Published: August 2007

In order to optimize turning processes, cutting forces need to be accurately predicted. This in turn requires accurate extraction of the geometry of tool-workpiece engagements (TWE) at critical points during machining. TWE extraction is challenging because the in-process workpiece geometry is continually changing as each tool pass is executed. This paper describes research on a hybrid analytical, solid modeler, and feature-based methodology for extracting TWEs generated during general turning. Although a pure solid modeler-based solution can be applied, it will be shown that because of the ability to capture different cutting tool inserts with similar geometry and to model the process in 2D, an analytical solution can be used instead of the solid modeler in many instances. This solution identifies features in the removal volumes, where the engagement conditions are not changing or changing predictably. This leads to significant reductions in the number of Boolean operations that are executed during the extraction of TWEs and associated parameters required for modeling a turning process. TWE extraction is a critical component of a virtual turning system currently under development.
Automatic Subdivision and Refinement of Large Components for Rapid Prototyping Production
Authors: Medellín H. , Lim T. , Corney J. , Ritchie J. M. , Davies J. B. C.
Published: August 2007

The aim of the work presented in this paper is to enable production of large, complex components on rapid prototyping machines whose build volume is less than the size of the desired component. Such large components can be produced as fabrications if a suitable subdivision can be generated. In general, any component can be subdivided into smaller parts by an array of orthogonal planes, but the resulting shapes could have geometries that are difficult to produce accurately on many rapid prototyping systems. The system presented here creates a decomposition designed for both rapid prototyping and assembly. The proposed method considers potential manufacturing problems, and modifies the boundaries of individual parts, where necessary. Additionally, the system also generates complementary male/female (i.e., matching protrusion/depression) assembly features at the interface between the component parts in order to improve the integrity and assemblability of the final component. To prove the functionality of the system, three components are analyzed at the end of this paper.
Optimization of Aircraft Fuselage Assembly Process Using Digital
Manufacturing
Authors: Butterfield J. , Crosby S. , Curran R. , Price M. , Armstrong C. G. , Raghunathan S. , McAleenan D. , Gibson C.
Published: August 2007

The work demonstrates the benefits of using digital methods for the development and optimization of large assembly manufacturing networks. Although an aircraft assembly has been used for this work, the methods and advantages of digital manufacturing techniques, which are demonstrated here, are equally applicable to any large assembly process, such as those used in the automotive, railway, and shipbuilding industries. The introduction of manufacturability into the design arena using advanced computer aided methods means that manufacturing engineers can operate more directly in assembly planning and concurrent engineering design. Network analyses carried out on the final assembly operations for a regional jet fuselage section using a methodical, step by step approach, shows that the process efficiency for workers carrying out fitting operations can be more than doubled when compared to existing shop floor performance figures. The more efficient use of operator time results in a simulated 19% improvement in financial efficiency, as the actual working hours required for assembly are reduced to below budgeted levels. The simulation predicts that these results can be achieved with one final assembly station. With two stations currently in use for the fuselage section, this means that a significant financial saving is possible in tooling expenditure.
Efficient Simulation of a Dynamic System with LuGre Friction
Authors: Do Nguyen B. , Ferri Aldo A. , Bauchau Olivier A.
Published: August 2007

Friction is a difficult phenomenon to model and simulate. One promising friction model is the LuGre model, which captures key frictional behavior from experiments and from other friction models. While displaying many modeling advantages, the LuGre model of friction can result in numerically stiff system dynamics. In particular, the LuGre friction model exhibits very slow dynamics during periods of sticking and very fast dynamics during periods of slip. This paper investigates the best simulation strategies for application to dynamic systems with LuGre friction. Several simulation strategies are applied including the explicit Runge–Kutta, implicit Trapezoidal, and implicit Radau-IIA schemes. It was found that both the Runge–Kutta and Radau-IIA methods performed well in simulating the system. The Runge–Kutta method had better accuracy, but the Radau-IIA method required less integration steps.
Passive, Nonlinear, Mechanical Structures for Seismic Attenuation
Authors: DeSalvo Riccardo
Published: August 2007

Gravitational wave detectors aim to detect strain perturbations of space-time on the order of 10−21–10−22 at frequencies between 1Hz and a few kHz. This space-time strain, integrated over kilometer scale interferometers, will induce movements of suspended mirrors on the order of 10−18–10−19m. Seismic motion in this frequency band varies between 10−6m and 10−12m. Required seismic attenuation factors, as large as 10−12, by far exceed the performance of motion sensors, and are only obtained by means of a chain of passive attenuators. High quality springs in configurations yielding nonlinear response are used to generate attenuation at low frequency. Similarly, nonlinear mechanisms are used in the horizontal direction. A description of some of these systems and some of the technical challenges that they involve is presented.
Validation of a Numerical Model for the Simulation of Tramcar Vehicle Dynamics by Means of Comparison With Experimental Data
Authors: Cheli Federico , Corradi Roberto , Diana Giorgio , Facchinetti Alan
Published: August 2007

Tramcar vehicles significantly differ from traditional railway vehicles for both the adopted structural configuration and design solutions and the operating conditions. For this reason, a new numerical model specific for the analysis of tramcar dynamics has been developed at Politecnico di Milano. Before the numerical model can be adopted as a means to analyze tramcar dynamics in typical operating conditions, the capability of the model to reproduce the actual tramcar behavior has to be verified. This paper deals with the validation of the proposed numerical model by means of a comparison with experimental data.
The Simulation of Dual Layer Fuel Tank During the Impact With the Ground Based on Parallel Computing
Authors: Zheng Li , Xiang-long Jin , Xiang-dong Chen
Published: August 2007

The crashworthiness of a dual layer fuel tank, with the outer layer made of metal and the inner layer made of woven fabric composite material, is fundamental for the survivability of an impact with the ground in emergency. In this research, the simulation of a three-dimensional dual layer fuel tank in the impact with the ground is achieved through the multimaterial arbitrary Lagrangian-Eulerian (ALE) finite element method because of its ability to control mesh geometry independently of geometry. At the same time, the naked flexible tank in the impact with the ground is simulated for the evaluation of the outer metal tank. The ALE description is adopted for the fluid domain, while for the structural domain the Lagrangian formulation is considered. The computation of the fluid-structure interaction and the impact contact between the tank and the ground are realized by the penalty-based coupling method. Then, the dynamic behaviors of the dual layer fuel tank and the naked flexible tank in the impact are analyzed. In the meantime, the parallelism of the dual layer fuel tank is discussed because the computation of the fluid-structure interaction and the impact contact is quite time consuming. Based on domain decomposition, the recursive coordinate bisection (RCB) is improved according to the time-consuming characteristics of fluid-filled tank in the impact. The result indicates, comparing with the RCB algorithm, that the improved recursive coordinate bisection algorithm has improved the speedup and parallel efficiency.
Recognition of User-Defined Turning Features for Mill/Turn Parts
Authors: Li Shiqiao , Shah Jami J.
Published: August 2007

This paper focuses on efficient algorithms for automatic recognition of user-defined turning features on mill/turn parts. As with other domains, recognition of interacting features is a difficult issue because feature interaction removes faces and alters the topology of the isolated turning features. This paper presents a method for efficiently recognizing both noninteracting and interacting rotational features from CAD model of mill/turn parts. Additionally, the method supports user-defined turning features that are represented using N-REP, a neutral feature representation language. First, the profiles of the revolved faces on a mill/turn part are obtained and the unturnable portions of these profiles are detected. These profiles then are used to construct the part graph and to solve feature interactions between coaxial turning features. Finally, graph-based and rule-based feature recognition are combined to recognize user-defined features.
Automated Drill Modeling for Drilling Process Simulation
Authors: Vijayaraghavan Athulan , Dornfeld David A.
Published: August 2007

Accurate models of two-flute conical twist drills are needed for finite element simulations of the drilling process. Existing drill designing methods rely extensively on discretized analytical equations to describe the drill and different sets of equations that need to be formulated as the drill design changes. This paper presents a method to create accurate models of two-flute conical twist drills using solid-modeling techniques, which addresses some of these shortcomings. Boolean operations are used to mimic the drill manufacturing steps and generate the fully designed drill. The drills generated by this method have been used in finite element simulations to study the effect of drill point geometry on burr formation in drilling.
Utilizing Diverse Feature Data for Reconstruction of Scanned Object as a Basis for Inspection
Authors: Miropolsky A. , Fischer A.
Published: August 2007

Inspection of machined objects is one of the most important quality control tasks in the manufacturing industry. Ideally, inspection processes should be able to work directly on scan point data. Scan data, however, are typically very large scale (i.e., many points), unorganized, noisy, and incomplete. Therefore, direct processing of scanned points is problematic. Many of these problems may be reduced if reconstruction methods exploit diverse scan data, that is, information about the properties of the scanned object. This paper describes this concept and proposes new methods for extraction and processing of diverse scan data: (1) extraction (detection of a scanned object’s sharp features by the sharp feature detection method) and (2) processing (scan data reduction by the geometric bilateral filter method). The proposed methods are applied directly on the scanned points and are completely automatic, fast, and straightforward to implement. Finally, this paper demonstrates the integration of the proposed methods into the computational inspection process.
Comparison of β-Lactam and Macrolide Combination Therapy versus Fluoroquinolone Monotherapy in Hospitalized Veterans Affairs Patients with Community-Acquired Pneumonia
Authors: Lodise Thomas P. , Kwa Andrea , Cosler Leon , Gupta Reetu , Smith Raymond P.
Published: August 2007

Data comparing the treatment outcomes of the two most frequently recommended empirical antibiotic regimens for community-acquired pneumonia (CAP)—combination therapy with an extended-spectrum β-lactam and a macrolide (BL+M) or fluoroquinolone (F) monotherapy—for patients with severe CAP are sparse. The purpose of this study was to compare empirical BL+M combination therapy with F monotherapy for Veterans Affairs (VA) patients with severe CAP. This retrospective study included patients with CAP who received empirical therapy with BL+M or F between October 1999 and May 2003 in the Upstate New York VA Network. Outcome measures were 14-day mortality, 30-day mortality, and length of hospital stay (LOS). Severe CAP was defined as a class V pneumonia severity index (PSI). During the study period, 261 patients received BL+M and 254 received F. Disease severity was similar for the two treatment groups at admission, and the presence of tachycardia was the only difference noted. For PSI class V patients, there were lower 14-day and 30-day mortality rates with BL+M than with F (14-day rates, 8.2% versus 26.8% [P= 0.02]; 30-day rates, 18.4% versus 36.6% [P= 0.05]). No differences in mortality between treatment groups were noted for the lower PSI classes. The overall median LOS was significantly longer for the BL+M combination group than for the F monotherapy group (6.0 days versus 5.0 days, respectively [P= 0.01]), but no difference in LOS was noted among PSI class V patients. Our study showed that improved outcomes may be realized with BL+M in cases of severe CAP. A randomized clinical study is warranted based on these results.
Quinuclidine Derivatives as Potential Antiparasitics
Authors: Cammerer Simon B. , Jimenez Carmen , Jones Simon , Gros Ludovic , Lorente Silvia Orenes , Rodrigues Carlos , Rodrigues Juliany C. F. , Caldera Aura , Ruiz Perez Luis Miguel , da Souza Wanderley , Kaiser Marcel , Brun Reto , Urbina Julio A. , Gonzalez Pacanowska Dolores , Gilbert Ian H.
Published: August 2007

There is an urgent need for the development of new drugs for the treatment of tropical parasitic diseases such as Chagas' disease and leishmaniasis. One potential drug target in the organisms that cause these diseases is sterol biosynthesis. This paper describes the design and synthesis of quinuclidine derivatives as potential inhibitors of a key enzyme in sterol biosynthesis, squalene synthase (SQS). A number of compounds that were inhibitors of the recombinantLeishmania majorSQS at submicromolar concentrations were discovered. Some of these compounds were also selective for the parasite enzyme rather than the homologous human enzyme. The compounds inhibited the growth of and sterol biosynthesis inLeishmaniaparasites. In addition, we identified other quinuclidine derivatives that inhibit the growth ofTrypanosoma brucei(the causative organism of human African trypanosomiasis) andPlasmodium falciparum(a causative agent of malaria), but through an unknown mode(s) of action.
Novel Small-Molecule Inhibitors of Transmissible Gastroenteritis Virus
Authors: Yang Cheng-Wei , Yang Yung-Ning , Liang Po-Huang , Chen Chi-Min , Chen Wei-Liang , Chang Hwan-You , Chao Yu-Sheng , Lee Shiow-Ju
Published: August 2007

We used swine testicle (ST) cells infected with transmissible gastroenteritis virus (TGEV) and an indirect immunofluorescent assay with antibodies against TGEV spike and nucleocapsid proteins to screen small-molecule compounds that inhibit TGEV replication. Analogues of initial hits were collected and subjected to a 3CL protease (3CL
pro
) inhibition assay with recombinant 3CL
pro
and a fluorogenic peptide substrate. A series of benzothiazolium compounds were found to have inhibitory activity against TGEV 3CL
pro
and to exert anti-TGEV activities in terms of viral protein and RNA replication in TGEV-infected ST cells, with consequent protection of TGEV-infected ST cells from cytopathic effect by blocking the activation of caspase-3.
In Vitro and In Vivo Activities of Novel Fluoroquinolones Alone and in Combination with Clarithromycin against Clinically Isolated
Mycobacterium avium
Complex Strains in Japan
Authors: Kohno Yoshihisa , Ohno Hideaki , Miyazaki Yoshitsugu , Higashiyama Yasuhito , Yanagihara Katsunori , Hirakata Yoichi , Fukushima Kiyoyasu , Kohno Shigeru
Published: August 2007

The recommended treatments for
Mycobacterium avium
complex (MAC) infectious disease are combination regimens of clarithromycin (CLR) or azithromycin with ethambutol and rifamycin. However, these chemotherapy regimens are sometimes unsuccessful. Recently developed antimicrobial agents, such as newer fluoroquinolones (FQs) containing C-8 methoxy quinolone (moxifloxacin [MXF] and gatifloxacin [GAT]), are expected to be novel antimycobacterial agents. Here, we evaluated the in vitro and in vivo antimycobacterial activities of three FQs (MXF, GAT, and levofloxacin) and CLR against clinically isolated MAC strains. Subsequently, the in vitro and in vivo synergic activities of FQ-CLR combinations against MAC strains were investigated. CLR and the individual FQs alone showed promising activity against MAC strains in vitro, and the bacterial counts in organs (lungs, liver, and spleen) of MAC-infected mice treated with single agents were significantly reduced compared to control mice. CLR showed the best anti-MAC effect in vivo. When the three FQs were individually combined with CLR in vitro, mild antagonism was observed for 53 to 57% of the tested isolates. Moreover, mice were infected with MAC strains showing mild antagonism for FQ-CLR combinations in vitro, and the anti-MAC effects of the FQ-CLR combinations were evaluated by counting the viable bacteria in their organs and by histopathological examination after 28 days of treatment. Several FQ-CLR combinations exhibited bacterial counts in organs significantly higher than those in mice treated with CLR alone. Our results indicate that the activity of CLR is occasionally attenuated by combination with an FQ both in vitro and in vivo and that this effect seems to be MAC strain dependent. Careful combination chemotherapy using these agents against MAC infectious disease may be required.
Polymyxin B Induces Lysis of Marine Pseudoalteromonads
Authors: Krupovič Mart , Daugelavičius Rimantas , Bamford Dennis H.
Published: August 2007

Polymyxin B (PMB) is a cationic antibiotic that interacts with the envelopes of gram-negative bacterial cells. The therapeutic use of PMB was abandoned for a long time due to its undesirable side effects; however, the spread of resistance to currently used antibiotics has forced the reevaluation of PMB for clinical use. Previous studies have used enteric bacteria to examine the mode of PMB action, resulting in a somewhat limited understanding of this process. This study examined the effects of PMB on marine pseudoalteromonads and demonstrates that the frequently accepted view that “what is true for
Escherichia coli
is true for all bacteria” does not hold true. We show here that in contrast to the growth inhibition observed for enteric bacteria, PMB induces lysis of pseudoalteromonads, which is not prevented by high concentrations of divalent cations. Furthermore, we demonstrate that a high membrane voltage is required for the interaction of PMB with the cytoplasmic membranes of pseudoalteromonads, further elucidating the mechanisms by which PMB interacts with the cell envelopes of those gram-negative bacteria.
Antiprion Activity of Cholesterol Esterification Modulators: a Comparative Study Using Ex Vivo Sheep Fibroblasts and Lymphocytes and Mouse Neuroblastoma Cell Lines
Authors: Pani Alessandra , Norfo Claudia , Abete Claudia , Mulas Claudia , Putzolu Marirosa , Laconi Sergio , Orrù Christina Doriana , Cannas M. Dolores , Vascellari Sarah , La Colla Paolo , Dessì Sandra
Published: August 2007

Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC
50
] range, 1.4 to 40 μM) were comparable to those of antiprion reference compounds (EC
50
range, 0.6 to 10 μM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.
Functional Characterization of TcaA: Minimal Requirement for Teicoplanin Susceptibility and Role in
Caenorhabditis elegans
Virulence
Authors: McCallum Nadine , Brassinga Ann Karen C. , Sifri Costi D. , Berger-Bächi Brigitte
Published: August 2007

The inactivation of TcaA contributes to intrinsic teicoplanin resistance in experimental and clinical isolates of glycopeptide-intermediate resistant
Staphylococcus aureus
. PhoA fusions confirmed that TcaA is a transmembrane protein with a short intracellular N-terminal domain containing a C-4 zinc finger binding motif, a single membrane-spanning domain, and a large extracellular C-terminal domain. The region conferring teicoplanin susceptibility was narrowed down to the transmembrane part and the first third of the extracellular domain of TcaA, suggesting that neither the C-4 zinc finger binding motif nor the C terminus contributed to teicoplanin susceptibility. TcaA belongs to the cell wall stress stimulon, which comprises a set of genes universally upregulated by cell wall damage. Induction of
tcaA
was shown to be fully dependent on the two-component regulatory system VraSR. A 66-bp region upstream of the transcriptional start site, which contained an inverted repeat partially covering the promoter box, was shown to be essential for VraSR-mediated induction by cell wall stress. Interestingly, the induction or overexpression of
tcaA
did not contribute further to teicoplanin susceptibility, suggesting that small amounts of TcaA, such as those present under normal uninduced conditions, were sufficient for TcaA-mediated teicoplanin susceptibility. The strong attenuation of
tcaA
deletion mutants in a
Caenorhabditis elegans
survival assay suggested that TcaA may, in addition to affecting glycopeptide susceptibility, also play a role in virulence.
Growth and Drug Resistance Phenotypes Resulting from Cytomegalovirus DNA Polymerase Region III Mutations Observed in Clinical Specimens
Authors: Chou Sunwen , Marousek Gail I. , Van Wechel Laura C. , Li Shaobing , Weinberg Adriana
Published: August 2007

Recombinant phenotyping of cytomegalovirus (CMV)
pol
region III mutations from clinical specimens showed that T813S and G841A each conferred foscarnet resistance and approximately threefold increased ganciclovir resistance; adding the UL97 mutation C592G increased ganciclovir resistance to approximately sixfold. Bacterial artificial chromosome CMV clones containing
pol
mutation L845P were nonviable unless repaired with the wild-type sequence.
Composite Structure of
Streptococcus pneumoniae
Containing the Erythromycin Efflux Resistance Gene
mef
(I) and the Chloramphenicol Resistance Gene
catQ
Authors: Mingoia Marina , Vecchi Manuela , Cochetti Ileana , Tili Emily , Vitali Luca A. , Manzin Aldo , Varaldo Pietro E. , Montanari Maria Pia
Published: August 2007

In recent years
mef
genes, encoding efflux pumps responsible for M-type macrolide resistance, have been investigated extensively for streptococci.
mef
(I) is a recently described
mef
variant detected in particular isolates of
Streptococcus pneumoniae
instead of the more common
mef
(E) and
mef
(A). This study shows that
mef
(I) is located in a new composite genetic element, whose sequence was completely analyzed and the left and right junctions determined, demonstrating a unique genetic organization. The new composite structure (30,505 bp), designated the 5216IQ complex, consists of two halves: a left one (15,316 bp) formed by parts of the known transposons Tn
5252
and Tn
916
, and a right one (15,115 bp) formed by a new fragment, designated the IQ element. While the defective Tn
916
contained a silent
tet
(M) gene, the IQ element, ending with identical transposase genes on both sides and containing the
mef
(I) gene with an adjacent new
msr
(D) gene variant and a
catQ
chloramphenicol acetyltransferase gene, was completely different from the genetic elements carrying other
mef
genes in pneumococci. This is the first report demonstrating
catQ
in
S. pneumoniae
and showing its linkage with a
mef
gene. Analysis of the chromosomal region beyond the left junction revealed an organization more similar to that of
S. pneumoniae
strain TIGR4 than to that of strain R6. The 5216IQ complex was apparently nonmobile, with no detectable transfer of erythromycin resistance being obtained in repeated transformation and conjugation assays.
Cyclic Tetrapyrrole Sulfonation, Metals, and Oligomerization in Antiprion Activity
Authors: Caughey Winslow S. , Priola Suzette A. , Kocisko David A. , Raymond Lynne D. , Ward Anne , Caughey Byron
Published: August 2007

Cyclic tetrapyrroles are among the most potent compounds with activity against transmissible spongiform encephalopathies (TSEs; or prion diseases). Here the effects of differential sulfonation and metal binding to cyclic tetrapyrroles were investigated. Their potencies in inhibiting disease-associated protease-resistant prion protein were compared in several types of TSE-infected cell cultures. In addition, prophylactic antiscrapie activities were determined in scrapie-infected mice. The activity of phthalocyanine was relatively insensitive to the number of peripheral sulfonate groups but varied with the type of metal bound at the center of the molecule. The tendency of the various phthalocyanine sulfonates to oligomerize (i.e., stack) correlated with anti-TSE activity. Notably, aluminum(III) phthalocyanine tetrasulfonate was both the poorest anti-TSE compound and the least prone to oligomerization in aqueous media. Similar comparisons of iron- and manganese-bound porphyrin sulfonates confirmed that stacking ability correlates with anti-TSE activity among cyclic tetrapyrroles.
Two Domains of Cytotoxic Necrotizing Factor Type 1 Bind the Cellular Receptor, Laminin Receptor Precursor Protein
Authors: McNichol Beth A. , Rasmussen Susan B. , Carvalho Humberto M. , Meysick Karen C. , O'Brien Alison D.
Published: August 2007

Cytotoxic necrotizing factor type 1 (CNF1) and CNF2 are highly homologous toxins that are produced by certain pathogenic strains ofEscherichia coli. These 1,014-amino-acid toxins catalyze the deamidation of a specific glutamine residue in RhoA, Rac1, and Cdc42 and consist of a putative N-terminal binding domain, a transmembrane region, and a C-terminal catalytic domain. To define the regions of CNF1 that are responsible for binding of the toxin to its cellular receptor, the laminin receptor precursor protein (LRP), a series of CNF1 truncated toxins were characterized and assessed for toxin binding. In particular, three truncated toxins, ΔN63, ΔN545, and ΔC469, retained conformational integrity and in vitro enzymatic activity and were immunologically reactive against a panel of anti-CNF1 monoclonal antibodies (MAbs). Based on a comparison of these truncated toxins with wild-type CNF1 and CNF2 in LRP and HEp-2 cell binding assays and in MAb and LRP competitive binding inhibition assays and based on the results of confocal microscopy, we concluded that CNF1 contains two major binding regions: one located within the N terminus, which contained amino acids 135 to 164, and one which resided in the C terminus and included amino acids 683 to 730. The data further indicate that CNF1 can bind to an additional receptor(s) on HEp-2 cells and that LRP can also serve as a cellular receptor for CNF2.
Expression of
Helicobacter pylori
Virulence Factors and Associated Expression Profiles of Inflammatory Genes in the Human Gastric Mucosa
Authors: Wen Sicheng , Velin Dominique , Felley Christian P. , Du Likun , Michetti Pierre , Pan-Hammarström Qiang
Published: August 2007

Helicobacter pylori
virulence factors have been suggested to be important in determining the outcome of infection. The
H. pylori
adhesion protein BabA2 is thought to play a crucial role in bacterial colonization and in induction of severe gastric inflammation, particularly in combination with expression of CagA and VacA. However, the influence of these virulence factors on the pathogenesis of
H. pylori
infection is still poorly understood. To address this question, the inflammatory gene expression profiles for two groups of patients infected with triple-negative strains (lacking expression of
cagA
,
babA2
, and
vacAs1
but expressing
vacAs2
) and triple-positive strains (expressing
cagA
,
vacAs1
, and
babA2
but lacking expression of
vacAs2
) were investigated. The gene expression patterns in the antrum gastric mucosa from patients infected with different
H. pylori
strains were very similar, and no differentially expressed genes could be identified by pairwise comparisons. Our data thus suggest that there is a lack of correlation between the host inflammatory responses in the gastric mucosa and expression of the
babA2
,
cagA
, and
vacAs1
genes.
Brucella abortusRequires the Heme Transporter BhuA for Maintenance of Chronic Infection in BALB/c Mice
Authors: Paulley James T. , Anderson Eric S. , Roop R. Martin
Published: August 2007

The gene annotated BAB2_1150 in theBrucella abortus2308 genome sequence is predicted to encode a homolog of the well-characterized heme transporter ShuA ofShigella dysenteriaeand accordingly has been given the designationbhuA(Brucellahemeutilization). Phenotypic analysis of an isogenicbhuAmutant derived fromB. abortus2308 verified that there is a link between BhuA and the ability of the parent strain to use heme as an iron source in in vitro assays. Maximum expression ofbhuAinB. abortus2308 is observed during stationary phase when this strain in cultivated in low-iron minimal medium, and a comparison of the growth characteristics of theB. abortus bhuAmutant and 2308 in this medium suggested that heme serves as an important iron source for the parent strain during stationary phase. TheB. abortus bhuAmutant HR1703 exhibits significant attenuation in cultured murine macrophages compared to strain 2308, and unlike its parent strain, theB. abortus bhuAmutant is unable to maintain a chronic spleen infection in experimentally infected BALB/c mice. These experimental findings suggest that heme and/or heme-containing proteins represent important iron sources forB. abortus2308 during its residence in the mammalian host and that BhuA is required for efficient utilization of these iron sources.
In Vitro and In Vivo Characterization of Anthrax Anti-Protective Antigen and Anti-Lethal Factor Monoclonal Antibodies after Passive Transfer in a Mouse Lethal Toxin Challenge Model To Define Correlates of Immunity
Authors: Staats Herman F. , Alam S. Munir , Scearce Richard M. , Kirwan Shaun M. , Zhang Julia Xianzhi , Gwinn William M. , Haynes Barton F.
Published: August 2007

Passive transfer of antibody may be useful for preexposure prophylaxis against biological agents used as weapons of terror, such asBacillus anthracis. Studies were performed to evaluate the ability of anthrax antiprotective antigen (anti-PA) and antilethal factor (anti-LF) neutralizing monoclonal antibodies (mAbs) to protect against an anthrax lethal toxin (LeTx) challenge in a mouse model and to identify correlates of immunity to LeTx challenge. Despite having similar affinities for their respective antigens, anti-PA (3F11) and anti-LF (9A11), passive transfer of up to 1.5 mg of anti-PA 3F11 mAb did not provide significant protection when transferred to mice 24 h before LeTx challenge, while passive transfer of as low as 0.375 mg of anti-LF 9A11 did provide significant protection. Serum collected 24 h after passive transfer had LeTx-neutralizing activity when tested using a standard LeTx neutralization assay, but neutralization titers measured using this assay did not correlate with protection against LeTx challenge. However, measurement of LeTx-neutralizing serum responses with an LeTx neutralization assay in vitro employing the addition of LeTx to J774A.1 cells 15 min before the addition of the serum did result in neutralization titers that correlated with protection against LeTx challenge. Our results demonstrate that only the LeTx neutralization titers measured utilizing the addition of LeTx to J774A.1 cells 15 min before the addition of sample correlated with protection in vivo. Thus, this LeTx neutralization assay may be a more biologically relevant neutralization assay to predict the in vivo protective capacity of LeTx-neutralizing antibodies.
Contribution of thestgFimbrial Operon ofSalmonella entericaSerovar Typhi during Interaction with Human Cells
Authors: Forest Chantal , Faucher Sébastien P. , Poirier Katherine , Houle Sébastien , Dozois Charles M. , Daigle France
Published: August 2007

Salmonellaserovars contain a wide variety of putative fimbrial systems that may contribute to colonization of specific niches.Salmonella entericaserovar Typhi is the etiologic agent of typhoid fever and is a pathogen specific to humans. In a previous study, we identified a gene, STY3920 (stgC), encoding the predicted usher of thestgfimbrial operon, that was expressed by serovar Typhi during infection of human macrophages. Thestggenes are located in theglmS-pstSintergenic region in serovar Typhi and certainEscherichia colistrains, but they are absent in otherS. entericaserovars. We cloned thestgfimbrial operon into a nonfimbriateE. coliK-12 strain and intoS. entericaserovar Typhimurium. We demonstrated that thestgfimbrial operon contributed to increased adherence to human epithelial cells. Transcriptional fusion assays with serovar Typhi suggested thatstgis preferentially expressed in minimal medium. Deletion ofstgreduced adherence of serovar Typhi to epithelial cells. However, deletion ofstgincreased uptake of serovar Typhi by human macrophages, and overexpression ofstgin serovar Typhi and serovar Typhimurium strains reduced phagocytosis by human macrophages. These strains survived inside macrophages as well as the wild-type parent. Although thestgCgene contains a premature stop codon that disrupts the expected open reading frame encoding the usher and is therefore considered a pseudogene, our results show that thestgoperon may encode a functional fimbria. Thus, this serovar Typhi-specific fimbrial operon contributes to interactions with host cells, and further characterization is important for understanding the role of thestgfimbrial cluster in typhoid fever pathogenesis.
Identification and Isolation of
Brucella suis
Virulence Genes Involved in Resistance to the Human Innate Immune System
Authors: Liautard Janny , Ouahrani-Bettache Safia , Jubier-Maurin Véronique , Lafont Virginie , Köhler Stephan , Liautard Jean-Pierre
Published: August 2007

Brucella
strains are facultative intracellular pathogens that induce chronic diseases in humans and animals. This observation implies that
Brucella
subverts innate and specific immune responses of the host to develop its full virulence. Deciphering the genes involved in the subversion of the immune system is of primary importance for understanding the virulence of the bacteria, for understanding the pathogenic consequences of infection, and for designing an efficient vaccine. We have developed an in vitro system involving human macrophages infected by
Brucella suis
and activated syngeneic γ9δ2 T lymphocytes. Under these conditions, multiplication of
B. suis
inside macrophages is only slightly reduced. To identify the genes responsible for this reduced sensitivity, we screened a library of 2,000 clones of transposon-mutated
B. suis
. For rapid and quantitative analysis of the multiplication of the bacteria, we describe a simple method based on Alamar blue reduction, which is compatible with screening a large library. By comparing multiplication inside macrophages alone and multiplication inside macrophages with activated γ9δ2 T cells, we identified four genes of
B. suis
that were necessary to resist to the action of the γ9δ2 T cells. The putative functions of these genes are discussed in order to propose possible explanations for understanding their exact role in the subversion of innate immunity.
Chicken Cecum Immune Response toSalmonella entericaSerovars of Different Levels of Invasiveness
Authors: Berndt Angela , Wilhelm Anne , Jugert Christiane , Pieper Jana , Sachse Konrad , Methner Ulrich
Published: August 2007

Day-old chicks are very susceptible to infections withSalmonella entericasubspecies. The gut mucosa is the initial site of host invasion and provides the first line of defense against the bacteria. To study the potential of differentS. entericaserovars to invade the gut mucosa and trigger an immune response, day-old chicks were infected orally withSalmonella entericaserovar Enteritidis,S. entericaserovar Typhimurium,S. entericaserovar Hadar, orS. entericaserovar Infantis, respectively. The localization ofSalmonellaorganisms in gut mucosa and the number of immune cells in cecum were determined by immunohistochemistry in the period between 4 h and 9 days after infection. Using quantitative real-time reverse transcription (RT)-PCR, mRNA expression of various cytokines, chemokines, and inducible nitric oxide synthase (iNOS) was examined in cecum. As a result, allS. entericaserovars were able to infect epithelial cells and the lamina propria. Notably, serovar Enteritidis showed the highest invasiveness of lamina propria tissue, whereas serovars Typhimurium and Hadar displayed moderate invasiveness and serovar Infantis hardly any invasion capabilities. Only a limited number of bacteria of all serovars were found within intestinal macrophages. Elevated numbers of granulocytes, CD8+cells, and TCR1+cells and mRNA expression rates for interleukin 12 (IL-12), IL-18, tumor necrosis factor alpha factor, and iNOS in cecum correlated well with the invasiveness of serovars in the lamina propria. In contrast, changes in numbers of TCR2+and CD4+cells and IL-2 mRNA expression seemed to be more dependent on infection of epithelial cells. The data indicate that the capability ofSalmonellaserovars to enter the cecal mucosa and invade lower regions affects both the level and character of the immune response in tissue.
l-Fucose Stimulates Utilization ofd-Ribose byEscherichia coliMG1655 ΔfucAOandE. coliNissle 1917 ΔfucAOMutants in the Mouse Intestine and in M9 Minimal Medium
Authors: Autieri Steven M. , Lins Jeremy J. , Leatham Mary P. , Laux David C. , Conway Tyrrell , Cohen Paul S.
Published: August 2007

Escherichia coliMG1655 uses several sugars for growth in the mouse intestine. To determine the roles ofl-fucose andd-ribose, anE. coliMG1655 ΔfucAOmutant and anE. coliMG1655 ΔrbsKmutant were fed separately to mice along with wild-typeE. coliMG1655. TheE. coliMG1655 ΔfucAOmutant colonized the intestine at a level 2 orders of magnitude lower than that of the wild type, but theE. coliMG1655 ΔrbsKmutant and the wild type colonized at nearly identical levels. Surprisingly, anE. coliMG1655 ΔfucAOΔrbsKmutant was eliminated from the intestine by either wild-typeE. coliMG1655 orE. coliMG1655 ΔfucAO, suggesting that the ΔfucAOmutant switches to ribose in vivo. Indeed, in vitro growth experiments showed thatl-fucose stimulated utilization ofd-ribose by theE. coliMG1655 ΔfucAOmutant but not by anE. coliMG1655 ΔfucKmutant. Since the ΔfucKmutant cannot convertl-fuculose tol-fuculose-1-phosphate, whereas the ΔfucAOmutant accumulatesl-fuculose-1-phosphate, the data suggest thatl-fuculose-1-phosphate stimulates growth on ribose both in the intestine and in vitro. AnE. coliNissle 1917 ΔfucAOmutant, derived from a human probiotic commensal strain, acted in a manner identical to that ofE. coliMG1655 ΔfucAOin vivo and in vitro. Furthermore,l-fucose at a concentration too low to support growth stimulated the utilization of ribose by the wild-typeE. colistrains in vitro. Collectively, the data suggest thatl-fuculose-1-phosphate plays a role in the regulation of ribose usage as a carbon source byE. coliMG1655 andE. coliNissle 1917 in the mouse intestine.