Escherichia coli cells growing in broth medium and exposed to low concentrations of BLM contain double-strand breaks and require homologous recombination to survive. To a lesser extent, the cells also require the abasic (AP) endonucleases associated with base excision repair, presumably to repair oxidative damage. As expected, there is strong induction of the SOS system in treated cells. In contrast, E. coli cells growing in glucose or glycerol minimal medium are resistant to the lethal action of BLM and do not require either homologous recombination functions or AP-endonucleases for survival. DNA ligase activity, however, is needed for cells growing in minimal medium to resist the lethal effects of BLM. There is weak SOS induction in such treated cells.Bleomycin (BLM) is a glycopeptide antibiotic and anti-tumor agent that targets primarily the furanose rings of DNA and in the presence of ferrous ions produces oxidative damage and DNA strand breaks.  Streptomyces verticillisBleomycin (BLM) is a glycopeptide antibiotic and anti-tumor agent isolated from Escherichia coli, previous studies lexA and recA mutant strains indicating that the SOS response is an important mechanism cells use to resist the toxic effects of the drug. The increased sensitivity of recA and recBC mutant strains to BLM indicates a role for homologous recombination in cell survival recN and recG mutant strains, in an otherwise wildtype background, were also found to be sensitive to BLM exposure, a feature not shared with other common DNA damaging agents In order to more fully understand the mechanism of BLM toxicity in E. coli in glucose minimal medium versus rich medium after exposure to BLM.We have confirmed and extended the above observations to additional mutant strains and show that at the low concentrations of BLM used here, homologous recombination is the principal pathway of repair in cells growing in broth. We also show that there is differential sensitivity of A previous study xthA nfo strain, defective for AP endonucleases, was more sensitive than wildtype after BLM treatment (fpg (mutM) and fpg mutY mutant strains were not sensitive suggesting that some other base damage occurs that requires AP endonuclease action. Bacteria defective for nucleotide excision repair (uvrA) were as resistant as wildtype to the cytotoxic effects of BLM as were dam and mutS strains affected for mismatch repair (data not shown). Two temperature-sensitive mutant strains defective in DNA ligase (lig-4 and lig-7) were more sensitive at the higher temperature when exposed to BLM (see below).An reatment . This rerecA and recBCD strains growing in L broth were more sensitive after BLM exposure than the wild type strain (recN and recG mutants relative to wildtype ruvABC genes (Holliday junction translocation and resolution) were among the most sensitive to BLM action . Double mutants bearing recBCD with recF or recG or recQ were no more sensitive to BLM than the recBCD strain (data not shown). Similarly, the recG ruvC mutant had the same survival to BLM as the recG mutant  and the xthA nfo AP-endonuclease-deficient strain (data not shown). At high BLM concentrations, there was increased killing of the wildtype cells .Only the temperature sensitive DNA ligase-deficient strains showed sensitivity to BLM when cultivated in glucose minimal medium . In L brperature . The lig2SO4 did not alter survival of wildtype or recA bacteria. L broth addition to glucose minimal medium in a 1∶1 ratio only partially reduced survival of wildtype cells (data not shown). Supplementing the glucose minimal medium with casamino acids (0.2%) did not change the survival compared to glucose minimal medium (data not shown). Supplementing L broth with individual components of glucose minimal medium did not produce an increase in survival (data not shown). Replacement of glucose with glycerol in the minimal medium did not alter survival (data not shown).BLM requires ferrous ions to generate radicals but supplementing the glucose minimal medium with 1 µg/ml FeE. coli cells growing in glucose minimal medium were resistant to the lethal effects of BLM, prompted us to test another cytotoxic chemical – N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). MNNG was chosen because its action results in the formation of methylated bases that are subject to base excision repair and that the lability of methylated bases also leads to abasic sites. Furthermore, the modified bases are blocks to replicative polymerases and MNNG exposure leads to the formation of DSBs in DNA of cells exposed to it. Widtype and recA strains were exposed to MNNG in broth and glucose minimal medium and the results are shown in The surprising result that recBCD strain to stabilize such ends. Wildtype or recBCD cells were grown in either L broth or glucose or minimal medium and exposed to various concentrations of BLM for 30 min, centrifuged and washed once to remove BLM, and then incubated for a further 30 and 60 min in respective growth medium. Samples of the washed and incubated cell population were removed, embedded in agarose and subjected to pulse field gel electrophoresis. The results are shown in recBCD strain growing in L broth shows a dose-dependent increased accumulation of low molecular weight DNA after BLM exposure  genes, and its associated cofactor ferridoxin gene, yfaE. These results suggest that there is increased initiation at oriC and increased levels of deoxyribonucleoside triphosphates in BLM-stressed recA cells growing in L broth.Wildtype bacteria exposed to BLM in L broth show a robust SOS regulon induction with the increase . As expesokC gene encodes a small regulatory RNA that indirectly blocks translation of the HokC toxic membrane polypeptide. The mltA gene encodes a membrane-bound murein hydrolase and the napA gene encodes a nitrate reductase. The proV gene produces a high affinity glycine transporter and the fdnH gene encodes a subunit for formate hydrogenase N, an integral inner membrane protein. It is not clear how increased expression of any of these non-SOS genes can produce BLM resistance.In glucose minimal medium, SOS induction occurs in wildtype cells exposed to BLM but the fold induction and the number of expressed SOS genes is less than in L broth . The sokrecA bacteria in glucose minimal medium and exposed to BLM, the gene with the largest fold increase is rygA (omrA), which encodes a small non-coding regulatory RNA  is required for utilization of pyrimidines; b0941 encodes a fimbrin subunit; b2209 (eco) encodes a serine protease inhibitor; the spoT gene product is a ppGpp pyrophosphohydrolase; and sgcC encodes a predicted phosphotransferase . The functions of the yjeT, ygeW, ydfD, yibK genes are not known.In the tory RNA . TogetherecA strains in genes showing increased expression after BLM treatment in glucose minimal medium. Genes showing greater than a two-fold decrease in transcription were few and not informative. The microarray analysis did not, therefore, reveal a transcriptional mechanism responsible for BLM resistance. It should be noted that the array data represent only a single set of mRNA samples.There was no overlap (other than tryptophanase) between wildtype and the E. coli cells growing in broth, exposure to BLM results in the formation of DSBs. We base this conclusion on the detection of such breaks by pulse field gel electrophoresis; by the induction of the SOS regulon; and by the requirement for homologous recombination for cells to survive BLM challenge. The requirement for the RecBCD pathway of homologous recombination, and not the RecF pathway, is consistent with the preferential ability of this pathway to repair DSBs For E. coli phenotypes using the response of the Keio collection strains to various drugs including BLM. These studies were conducted in cells growing in rich (L) media. In general, the results of their study are in agreement with those reported here in terms of the requirement for recombination gene products  and DNA ligase. In addition, the Nichols et al. study identified hfq, fis, rusA and sbcA mutant strains as BLM sensitive as well as a large number of mutant bacteria affecting the structure/function of the cell envelope. The RusA and SbcA proteins are part of a prophage recombination system. The requirement for Hfq likely reflects a requirement for one or more small regulatory non-coding RNA molecules and their study identified istR and gvcB although additional RNA molecules are possible. The Fis protein is important to maintain nucleoid integrity. In sum, both studies indicate the importance of recombination in the resistance of E. coli cells growing in broth to BLM.While this work was in progress, Nichols et al. E. coli with a transposon and selected for mutant strains with greater or less resistance to a panel of antibiotics including BLM. Becket et al. The results of the Nichols et al. study xthA nfo mutant bacteria were sensitive to BLM. This discrepancy is most probably because the single xthA and nfo mutant cells are not as sensitive as the double mutant.The Nichols study did not identify a requirement for gene products involved in repair of oxidative damage to DNA. In this study, however, we showed that recG and recN mutant strains growing in broth to BLM was confirmed. These mutant strains are not sensitive to other commonly used DNA damaging agents such as ultra-violet light or the methylating agents, ethyl- and methyl methane sulfonate recN mutant strains are sensitive to ionizing radiation, but not ultra-violet light, suggesting a role in repair of DSBs and/or oxidative damage through the RecF pathway recN gene is strongly stimulated during the SOS response recN gene product remains unknown although it is a member of the SMC  family of proteins.The sensitivity of recG mutants are only slightly sensitive to ultra-violet light or X-rays but show increased sensitivity in a ruvAB mutant background recG cells exposed to BLM. Alternatively, RecG might unwind persistent recombination intermediates that would otherwise compromise chromosome replication.RecG is a helicase that can translocate Holliday junctions but in the opposite direction to RuvAB E. coli cells growing in minimal medium based on the increased sensitivity to BLM of ligase-deficient strains  were for maltose binding and transport. There were 21 genes in the top 100 associated with Fe binding and transport. A putative zinc protease gene, E. coli in L broth identified a putative protease that when inactivated increased toxicity yfgC) encoding this protein was more sensitive than wildtype when cultivated in L broth but was as resistant as wildtype when tested in glucose minimal medium (data not shown).Previous studies investigating the action of BLM on Further studies could include a screen of the Keio collection for cells sensitive to BLM on glucoase minimal medium. Alternatively, or in addition, mutagenized cultures could be screened for this property.lig-7 (Ts) mutant) and the most important are described in The strains used are derived from GM7330  broth which consists of 20 g tryptone, 10 g yeast extract, 0.5 g NaCl and 4 ml 1 M NaOH per l, and solidified when required with 16 g agar (Difco). Minimal medium contained minimal salts as described by Davis and Mingioli 600 = 0.2–0.3 and exposed to BLM  for 30 min after which they were diluted in minimal salts and plated on L media and incubated overnight at 37°C. There was no difference in viable counts from the cells exposed to BLM in minimal medium when plated on rich medium or minimal medium plates. For the temperature-sensitive (Ts) lig-7 cells, cultures were grown at 30°C and then shifted to 42°C for 30 min before adding BLM. In the absence of BLM, there was no decrease in survival after a 60 min incubation at 42°C.Cells in L broth or minimal medium with glucose or glycerol as carbon source were grown to an OD600 = 0.2–0.3 and exposed to BLM for 30 min after which the cells were harvested by centrifugation, washed once with minimal salts, twice in SE buffer  and resuspended in 160 µl of distilled water. The cells were mixed with an equal volume of 2% low melt agarose (in 0.5×TBE buffer), distributed in 60 µl portions in molds and left for a few minutes at 4°C until set. The plugs were incubated overnight at 56°C in 1 ml LE buffer  with proteinase K (0.5%) Saccharomyces cerevisiae were routinely used as molecular weight standards (New England Biolabs). For each of the PFGE figures, the experiment was done at least three times and a representative figure is shown.We have used the procedure described by Seigneur et al. E. coli Genome 2.0 Array. Cells were cultivated and exposed or not to 0.7 µM BLM as described above and total RNA was isolated using the MasterPure RNA Purification kit (Epicentre Technologies). Further steps in the procedure were carried out at the UMass Medical School Genomics Core. Random primers (Life Technologies) were annealed to 10 µg of the RNA and SuperScript II Reverse Transcriptase used for cDNA synthesis. Residual RNA was removed by alkaline hydrolysis and the cDNA was purified using a Qiagen MinElute PCR Purification Kit. The cDNA was fragmented with DNAse I (Amersham Biosciences) in One-Phor-All buffer (Amersham Biosciences) and end-labeled using Terminal Deoxynucleotidyl Transferase (Promega) and GeneChip DNA Labeling Reagent (Affymetrix). The end-labeled RNA was hybridized to an Affymetrix GeneChip E. coli Genome 2.0 Array in a GeneChip Hybridization Oven 320 at 45°C for 16 hours, then washed in the Affymetrix Fluidics Station 450 and scanned in the Affymetrix GeneChip Scanner 3000. Full details of this procedure are available at the Affymetrix website (www.affymetrix.com). The results from the hybridization were exported as Microsoft Pivot files and imported into Microsoft Excel for sorting. The arrays were done only once as the results were not informative as to the mechanism of BLM resistance Click here for additional data file.Table S2Gene Chip 2.0 values from GM7661 cells exposed or not to BLM in L broth and minimal medium.(XLSX)Click here for additional data file.


Escherichia coli from ambulatory primary care patients in a regional health maintenance organization.Urinary tract infections (UTIs) are one of the most common infections treated in ambulatory care settings, however the epidemiology differs by age and sex. The incidence of UTI is far greater in females than males, and infection in pediatric patients is more often due to anatomical abnormalities. The purpose of this research was to describe age- and sex-specific trends in antibiotic susceptibility to common urinary anti-infectives among urinary isolates of E. coli tested for antibiotic susceptibilities per patient per year was included in the analysis dataset. The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for male and female patients. The Cochrane-Mantel-Haenzel test was used to test for differences in age-stratified susceptibility to each antibiotic between males and females.Clinical microbiology data were collected for all urine cultures from patients with visits to primary care clinics in a regional health maintenance organization between 2005 and 2010. The first positive culture for E. coli isolates from 34,539 unique patients were identified for study inclusion. After stratifying by age, E. coli susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, and nitrofurantoin differed significantly between males and females. However, the magnitude of the differences was less than 10% for all strata except amoxicillin-clavulanate susceptibility in E. coli isolated from males age 18–64 compared to females of the same age.A total of 43,493 E. coli isolated from males versus females. These data suggest that male sex alone should not be used as an indication for empiric use of second-line broad-spectrum antibiotic agents for the treatment of UTIs.We did not observe clinically meaningful differences in antibiotic susceptibility to common urinary anti-infectives among  Urinary tract infections (UTIs) are one of the most commonly treated bacterial infections and account for over 10 million ambulatory care visits annually in the United States . BecauseThe incidence, presentation, and course of infection for UTIs in men and women differ in large part due to anatomical differences. For the treatment of acute uncomplicated cystitis in women, trimethoprim/sulfamethoxazole (TMP/SMX), nitrofurantoin, fosfomycin, and pivmecillinam are recommended first-line empiric therapies ,6. In meEscherichia coli, which accounts for 75-90% of UTIs [E. coli and other Enterobacteriaceae over the past several decades, surveillance data have become critical for appropriate empiric selection of antibiotic therapy. U.S. guidelines specify that TMP/SMX should be avoided for empiric treatment of uncomplicated acute cystitis or pyelonephritis in populations where non-susceptibility to this agent exceeds 20% in uropathogens [While historically it was believed that the causative organism in UTIs differed between men and women, more recent data has shown that for both sexes the primary causative pathogen is  of UTIs ,9. With E. coli urine isolates using six years of data from a large ambulatory primary care patient population.While some surveillance studies have identified significant differences in the frequency of susceptibility to common urinary anti-infectives between isolates collected from male and female patients, this has not been consistently observed ,11. The E. coli isolates from outpatients of Kaiser Permanente Northwest (KPNW) primary care clinics. KPNW is a regional health maintenance organization that serves over 485,000 members in northwest Oregon and southwest Washington. Primary care clinics were defined as non-specialty care clinics within the Family Practice, Internal Medicine, or Pediatrics departments; all primary care clinics were included. Urine cultures positive for E. coli that were drawn from patients with visits in the primary care clinics between January 1, 2005 and December 31, 2010 were eligible for inclusion in the analysis. Cultures in which less than 10,000 colonies/mL were identified or 3 or more organisms were isolated were excluded. The analysis dataset was then limited to the first isolate tested for antibiotic susceptibilities per patient and year to minimize potential bias resulting from repeat culturing [We conducted a cross-sectional study of urinary ulturing .Data were electronically extracted from the virtual data warehouse maintained by the Kaiser Permanente Center for Health Research. Collected data included patient demographics, department in which the clinic visit occurred, and all clinical microbiology data for urine cultures. Approval for this study was obtained from the KPNW institutional review board.E. coli isolates stratified by patient sex and year. The Cochrane-Armitage test for trend was used to identify changes in the frequency of susceptibility to each antibiotic over time independently among males and females. Patient age at the time of culture was categorized as less than 18 years, 18–64 years, and 65 years and older. The frequency of susceptibility to each antibiotic was also compared across age categories among males and females using the Cochran-Mantel-Haenszel test. An alpha level less than or equal to 0.05 was the statistical significance level for all analyses and data were analyzed with SAS . Clinically significant differences were defined as differences of 10% or greater.The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for E. coli and 57,550 of those were tested for antibiotic susceptibilities. After restricting the data to the first isolate per patient per year, 43,493 isolates from 34,539 unique patients remained in the final analysis data set. Table During the study time frame 190,396 urine cultures were performed at primary care clinic visits; 70,180 were positive for E. coli isolated from males and 66.3%  from females were susceptible to ampicillin. Amoxicillin-clavulanate susceptibility was 56.9% (484/850) among males and 67.3%  among females; however, it should be noted that testing for susceptibility to amoxicillin-clavulanate was routinely performed and reported only for ampicillin non-susceptible E. coli. Ciprofloxacin susceptibility for the E. coli isolates was 93.2%  among males and 95.9%  among females, and nitrofurantoin susceptibility was 96.4%  among males and 97.6%  among females. For TMP/SMX, 86.3%  of E. coli isolated from males were susceptible compared to 84.7%  among females.Overall, 66.0%  of E. coli to each antibiotic by sex and year. Susceptibility to amoxicillin-clavulanate and TMP/SMX decreased significantly over time among both males and females. Ciprofloxacin susceptibility also decreased significantly over time among females, but not males. Table E. coli isolated from males and females. The age-specific susceptibilities differed significantly between males and females for all antibiotics except TMP/SMX.Figure E. coli to ampicillin, amoxicillin-clavulanate, ciprofloxacin, and nitrofurantoin. Urinary E. coli isolates from male patients tended to exhibit increased antibiotic resistance than isolates from female patients. Despite the statistical significance of time trends and differences in age-specific susceptibilities, the magnitude of these differences was generally less than 5% and thus may not represent clinically meaningful differences. The exception was susceptibility to amoxicillin-clavulanate, where susceptibility was roughly 10% lower in males age 18 to 64 years than females in the same age group. Yet these differences should be interpreted cautiously because susceptibility to amoxicillin-clavulanate was only provided for ampicillin non-susceptible E. coli isolates in the current analysis. If all ampicillin susceptible isolates are assumed to be amoxicillin-clavulanate susceptible, then the magnitude of the differences between males and females would be less than 5% in all age categories .In this study, we observed statistically significant differences between males and females in the age-specific susceptibilities of E. coli were significantly more resistant to fluoroquinolones, penicillins, nitrofurantoin, and first and second generation cephalosporins among men compared to women [E. coli isolates (not limited to urine isolates) collected from Canadian tertiary medical centers were compared and resistance was also observed to be significantly higher to ciprofloxacin, levofloxacin, and TMP/SMX in isolates collected from male patients versus female patients [While few other studies have explored the differences in antibiotic susceptibility of uropathogens isolated from male and female ambulatory patients, our findings are consistent with the trends observed in the literature. A recent 10-year study of community UTI in Portuguese patients also identified differences in antibiotic susceptibility by patient sex. The authors reported that urinary isolates of to women . Anotherto women . The NAUto women . In the patients .The prevalence and susceptibilities of antibiotic-resistant bacteria varies widely by geographic region, thus these data may not be generalizable to all regions. Local surveillance data of antibiotic susceptibilities would be needed to validate these findings in different patient populations. It should be noted that in our study resistance to TMP/SMX did not exceed 20%, as it does in many other regions of the U.S., thus in this patient setting TMP/SMX may be a viable empiric treatment option in both males and female patients. Additionally, antibiotic susceptibilities may differ by other patient or infection characteristics . These data were not available for analysis in this study and further investigation in this area is needed. Also, because clinical signs and symptoms of infection were not available and because not all suspected infections are cultured, the use of all urinary microbiology from this population cultures may not represent all true infections.E. coli isolated from males and females were neither statistically nor clinically significant and resistance rates are below 20%. Consequently, in this population, there is no evidence that male sex alone should be an indication for empiric selection of a second-line broad-spectrum antibiotic agent for the treatment of UTI. While more research is needed on treatment effectiveness of different regimens used to treat community UTIs in men, population-specific antibiotic susceptibility data are a necessary component in the empiric antibiotic selection process.Currently there exists insufficient data to inform the development of evidence-based guidelines for the treatment of community UTIs in men. UTIs in adult males are considered complicated infections according to most clinical definitions -18. TreaIn the era of increasing antibiotic resistance, prudent use of antibiotics is critical to prolong the clinical effectiveness of existing agents. Excessive use of broad-spectrum agents, such as fluoroquinolones, increases the evolutionary selective pressures that drive the increasing prevalence of resistance. These data suggest that first-line urinary anti-infectives such as TMP/SMX may be effective agents for treating UTIs in men. While more data are needed, clinicians should use local surveillance data to guide the prudent, empiric selection of antibiotic therapy for UTIs.UTI: Urinary Tract Infection; KPNW: Kaiser Permanente Northwest; TMP/SMX: trimethoprim/sulfamethoxazoleThe authors declare that they have no competing interests.JCM participated in the conception and design of the study; the acquisition, statistical analysis, and interpretation of the data; and the drafting of the manuscript. JCM, MRE, DTB, and DHS assisted with the interpretation of the data and participated in the critical revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2296/14/25/prepub


Recent studies suggest simultaneous or metachronous lesions in multiorgans characterized by elevated serum levels of IgG4 and abundant infiltration of IgG4-positive plasma cells with various degrees of fibrosis. Two Japanese research committees for IgG4-RD, one from fibrosclerosis (Okazaki team) and the other from lymph proliferation (Umehara team) supported by the “Research Program for Intractable Disease” of the Ministry of Health, Labor, and Welfare of Japan, have agreed with the unified nomenclature as “IgG4-RD” and proposed the comprehensive diagnostic criteria (CDC) for IgG4-RD. Validation of the CDC demonstrated satisfactory sensitivity for the practical use of general physicians and nonspecialists but low sensitivity in the organs to be difficult in taking biopsy specimens such as type1 autoimmune pancreatitis (IgG4-related AIP), compared with IgG4-related sialadenitis/dacryoadenitis (Mikulicz's disease) and IgG4-related kidney disease. Although the diagnostic criteria covering all IgG4-RD are hard to be established, combination with the CDC and organ-specific diagnostic criteria should improve sensitivity. Recent studies have suggested simultaneous or metachronous lesions in multiorgans characterized by elevated serum levels of IgG4 and abundant infiltration of IgG4-positive plasma cells with various degrees of fibrosis, which lead us to propose the concept of a systemic disease , 23, 24.The two Japanese research committees independently analyzed the clinical features and conditions of IgG4-RD and finally resulted in the following consensus with close collaboration , 24, 25.In addition to MIF, there are many synonyms, such as IgG4-related autoimmune disease , “IgG4-r The patients with IgG-4-related disease show organ enlargement or nodular/hyperplastic lesions in organs in the entire body, synchronously or metachronously, due to the prominent infiltration and fibrosis of lymphocytes and plasmacytes; however, the causes of the disease are still not clear. The organs known to be affected include the central nervous system, lacrimal/salivary glands, thyroid gland, lungs, pancreas, biliary duct, liver, gastrointestinal tracts, kidneys, prostate gland, retroperitoneum, skin, arteries, and lymph nodes. Although it remains unclear whether this disease is the same as multifocal fibrosclerosis, that is a possibility. Clinical symptoms vary depending on the organ in which the lesions are located, which suggests that it is hard to establish criteria covering all patients with IgG4-RD. Therefore, specific diagnostic criteria are required for each involved organ such IgG4-related Mikulicz's disease  curves, and its validity was confirmed in patients with autoimmune pancreatitis  Table 6Table 6. However, elevated IgG4 may be also observed in other diseases , especially in about 10% of malignancy, which suggests that high serum IgG4 is not necessarily specific marker of IgG4-RD . Althougγ-globulin, IgG, and IgE are often, and hypocomplementemia may occur [In addition to increased serum levels of IgG4, high serum levels of polyclonal ay occur . As thesStoriform or swirling fibrosis or obliterative phlebitis is Characteristic of IgG4-RD and may be important in its diagnosis.IgG4/IgG-positive cells more than 40%  or 50%  have beeAlthough tissue biopsies are difficult to obtain from some organs, including the pancreas, retroperitoneum and ocular cavity, histopathological examination is important.Patients with malignant lymphoma or paraneoplastic lesions can sometimes be improved by steroid administration. Therefore, steroid trials should be strictly avoided. Efforts should be made to collect tissue samples for diagnosis. However, patients having disease in organs difficult to biopsy, such as the pancreas, retroperitoneum, and pituitary, and respond to steroids may possibly have IgG4-RD. In accordance with the guidelines for treatment of autoimmune pancreatitis, patients should be started on 0.5-0.6 mg/kg/day/prednisolone. If patients do not respond to the initial steroid therapy, the diagnosis should be reviewed again.In cases of malignancy in the involved organs, it is essential to determine whether malignant cells are present histopathologically.Other similar benign diseases including Sjögren's syndrome, primary sclerosing cholangitis, multicentric Castleman's disease, idiopathic retroperitoneal fibrosis, Wegener's granulomatosis, sarcoidosis, and Churg-Strauss syndrome should be differentially diagnosed using the diagnostic criteria for each disease. It is noted that multicentric Castleman's disease, one of hyper IL-6 syndromes should be excluded from IgG4-RD, even if the CDC for IgG4-RD are fulfilled.The sensitivity of CDC for definitive/probable IgG4-RD is satisfactory in IgG4-related MD  and IgG4Based on these findings, a diagnostic algorithm for IgG4-RD in combination with the CDC and other organ-specific criteria has been proposed, although they have a limitation to the utility of the criteria proposed  Figure . In pati“All Japan Research Team for IgG4-RD” unified the nomenclatures as “IgG4-related disease (IgG4-RD)” and proposed the comprehensive diagnostic criteria (CDC) for IgG4-RD. The CDC for IgG4-RD was made for the practical use and for general physicians to differentiate IgG4-RD from malignancy or similar diseases as much as possible. Although sensitivity of the CDC for definitive IgG4-RD is low in the organs to be difficult in taking biopsy specimens, it can detect possible cases of IgG4-RD. In the probable or possible cases, organ-specific criteria should be used concurrently.


Corrosion of steel bars embedded in reinforced concrete (RC) structures reduces the service life and durability of structures causing early failure of structure, which costs significantly for inspection and maintenance of deteriorating structures. Hence, monitoring of reinforcement corrosion is of significant importance for preventing premature failure of structures. This paper attempts to present the importance of monitoring reinforcement corrosion and describes the different methods for evaluating the corrosion state of RC structures, especially hal-cell potential (HCP) method. This paper also presents few techniques to protect concrete from corrosion. Deterioration of concrete structures due to harsh environmental conditions leads to performance degradation of RC structures, and premature deterioration of structures before completing expected service life is major concern for engineers and researchers. Deterioration rate of structures depends on the exposure conditions and extent of maintenance. Corrosion, a result of chemical or electrochemical actions, is the most common mechanism responsible for deterioration of RC structures which is mainly governed by chloride ingress and carbonation depth of RC structures. Usually, there are two major factors which cause corrosion of rebars in concrete structures, carbonation and ingress of chloride ions. When chloride ions penetrate in concrete more than the threshold value or when carbonation depth exceeds concrete cover, then it initiates the corrosion of RC structures. If the corrosion is initiated in concrete structures, it progresses and reduces service life of the structures and rate of corrosion affects the remaining service life of RC structures. However, these severe environments can cause corrosion of reinforcement only if required amounts of oxygen and moisture are available at the rebar level in concrete structures .Corrosion of steel bars is the major cause of failure of concrete structures and about two tons of concrete is used per capita of the world population every year. Therefore, it has been realized that durable structures will reduce the cement consumption. Corrosion can severely reduce the strength and life of structures and in humid conditions pollutants from atmosphere percolate through the concrete cover and cause corrosion of steel. After the initiation of corrosion in reinforcing steel, products of corrosion expand and occupy a volume of about 6–10 times greater than that of steel resulting in the formation of cracks and finally in the failure of structures as shown in Figures Penetration of corrosion inducing agents such as chloride ions and carbon dioxide increased at the places of cracks, which further increases the corrosion . CorrosiDetection and evaluation of probability of corrosion in RC structures are essential. Proper corrosion monitoring of the concrete structures has been required for planning maintenance and replacement of the concrete structures. The most appropriate repair strategy can be selected for a distressed concrete structure by determining the corrosion status of reinforcing bars . Repair 4 or Ag/AgCl). The metal rod is connected with reinforcement steel by a voltmeter as shown in There are several methods available for detecting and evaluating the corrosion in reinforcement steel as presented in 4 half-cell.Interpretation of results of half-cell potential measurement for reinforced concrete structures required high skills and experience, as this only provides information regarding the probability of corrosion instead of rate and nature of corrosion . AvailabThis half-cell potential is also known as open circuit potential and is measured at several distinct points over a given area to be surveyed. Measured half-cell potential values can be used to plot a potential contour for the surface of reinforced concrete structure and this potential contour map as shown in Several techniques have been reported in previous literatures that can be used for monitoring and evaluating the corrosion of rebars in concrete structures for diagnosing the cause and effect of corrosion. Few such studies performed by different researchers have been presented in To increase the service life of RC structures, it is required to protect reinforcing steel completely from being corroded. Several chemical and mechanical methods are developed to prevent concrete structures from corrosion by retarding the corrosion rate and by controlling corrosion through reducing permeability of concrete and reducing the ingress of harmful ions such as oxygen and moisture, and some protective systems have been used in the form of coating. Different corrosion inhibitors and protecting systems have been discussed in Manually measuring potential values at different points on a large structure is tedious work. Therefore, automatic evaluating method is required. Half-cell potential measurements are widely used in structural engineering to assess the likelihood of corrosion. HCP measurements are found to be associated with several practical limitations such as (1) establishing connection with reinforcement, especially in structures with large concrete cover, (2) properly wetting the concrete cover for establishing proper connection between reference electrode and reinforcement, and (3) availability of oxygen, cover thickness, and concrete resistivity which can influence the results of half-cell potential test.HCP method only provides the evaluation of the point likely to be corroded and no assessment of the corrosion rate. Half-cell potential values are indicative of the probability of corrosion activity of reinforcement located beneath the reference electrode only if the steel rebars are electrically well connected to the voltmeter. Half-cell potential method cannot provide reliable results with epoxy coated reinforcement or with coated concrete surfaces. Moist or wetting condition of concrete can influence the results of half-cell potential method, or it is important to assure the sufficient wetting of concrete to complete the setup for valid half-cell potential measurement. If measured value of the HCP varies with time, prewetting of the concrete is required. It is essential to thoroughly wet the concrete surface and allow sufficient time for the moisture to penetrate the surface layer to stabilize the potential. ASTM C-876 emphasizes that if the measured value of half-cell potential changes with time surface of concrete should be wet for at least 5 min.It has been observed from literature that results of HCP mapping required careful interpretation. To interpret HCP data, factors such as variation in moisture content, chloride content, and concrete electrical resistance are required to be considered as all these parameters have a significant influence on the readings.The major drawback is that HCP requires a localized breakout of the concrete cover to provide an electrical connection to the steel reinforcement. HCP results are highly influenced by the composition of the deteriorated concrete. Therefore, interpretation criteria might be different for different deterioration types. Shortcomings of HCP measurements result from the fact that the potentials are measured not near rebars but on concrete surface. Compensation is required to get more reliable results.Failure of concrete structures due to corrosion of embedded rebars is a major problem causing significant loss of money and time. Hence, there is a need to fully understand the root causes of failure before the repairing for effective remediation. An effective method to measure corrosion is a fundamental requirement for planning maintenance, repairing, and removal for reinforced concrete structures. Information regarding corrosion state required three parameters: half-cell potential, concrete resistivity, and corrosion current density. Corrosion rate in a concrete structure is governed by several parameters such as moisture content, availability of oxygen, and temperature. So, for better results it is necessary to repeat corrosion rate measurement in regular time interval.to assess the corrosion condition of the reinforcement by locating corroded bars,for the condition assessment of a concrete structure,to locate and decide the position of further detailed destructive and nondestructive testing,evaluate the efficiency of repair work through corrosion state monitoring of repaired concreter structures.Half-cell potential measurement is the most widely used technique for the evaluation of corrosion of steel in concrete. However, in interpreting the data environmental factors should be taken into account. For interpretation of half-cell potential readings, it requires precise understanding of corrosion protection mechanisms and good knowledge and experience in half-cell potential mapping. In present research it has been observed that half-cell potential measurements are useful in the following purposes:In concrete with low resistivity potential distribution on surface represents potential at steel concrete interface. For better results interpretation of potential readings can be done in accordance with resistivity. With increase in concrete cover difference between surface and interface potential increases.Content of this paper can be utilized to understand the principal of half-cell potential method, to plan investigation of corroded structures, and to select suitable corrosion monitoring technique.


As we enter the third decade of the AIDS pandemic, numerous texts explore the many aspects of AIDS and its consequences. Mayer and Pizer's premise is that AIDS has transformed many of the disciplines that it has touched. For the most part, this well-written volume supports their thesis. The authors, all established researchers, tackle many of the major issues, including virology, immunology, vaccines, microbicides, and sexually transmitted diseases, as well as the global impact of HIV/AIDS. Each chapter provides a well-referenced overview of its topic with many references as recent as 2003.One of the real strengths of this book is a chapter on quantitative science that explores not only the history of HIV clinical trials, but also the design and importance of clinical trials in general. This chapter should be required reading for those considering clinical research in HIV. The chapters on Africa and Asia ably contrast the differences in these areas of highest prevalence. Another strength is the discussion of HIV in correctional facilities and the challenge of caring for this population, including their coexisting conditions and illicit drug use. Lastly, the discussion of the economics of AIDS is especially welcome in this era of efforts to increase access to drugs worldwide.Overall, this book fills a valuable niche. A relatively concise text, it reviews many aspects of HIV with a focus on how each topic has evolved over the years. A few tables are small, but overall the diagrams and charts are clear and legible. This book would be of interest to infectious disease fellows, HIV caregivers, and those involved in public health and health policy. I heartily recommend this book and plan to keep it handy for future reference.


Recent antibiotic use is a risk factor for infection or colonization with resistant bacterial pathogens. Demand for antibiotics can be affected by consumers’ knowledge, attitudes, and practices. In 1998–1999, the Foodborne Diseases Active Surveillance Network (FoodNet) conducted a population-based, random-digit dialing telephone survey, including questions regarding respondents’ knowledge, attitudes, and practices of antibiotic use. Twelve percent had recently taken antibiotics; 27% believed that taking antibiotics when they had a cold made them better more quickly, 32% believed that taking antibiotics when they had a cold prevented more serious illness, and 48% expected a prescription for antibiotics when they were ill enough from a cold to seek medical attention. These misguided beliefs and expectations were associated with a lack of awareness of the dangers of antibiotic use; 58% of patients were not aware of the possible health dangers. National educational efforts are needed to address these issues if patient demand for antibiotics is to be reduced. Antimicrobial resistance is a rapidly increasing problem in the United States and worldwide. A well-documented risk factor for infection or colonization with resistant bacterial pathogens is recent antibiotic use, particularly within 4 weeks or 1 month before exposure  conducted a telephone-based population survey in Connecticut, Minnesota, and Oregon, and selected counties in California, Georgia, Maryland, and New York . Each month, approximately 150 residents in each state were interviewed. After screening to remove business and nonworking telephone numbers, an outside contractor contacted respondents by telephone using a random-digit-dialing, single-stage sampling method  addressing participants’ knowledge, attitudes, and practices regarding antibiotic use were included in the survey. Recent antibiotic use referred to antibiotic use in the past 4 weeks. Respondents who took an antibiotic were asked whether the antibiotic was prescribed by their physician for a current illness or for a previous illness or if the antibiotic was prescribed for someone else. For the question, “Are you aware of any health dangers to yourself or other people associated with taking antibiotics?” respondents’ knowledge of health dangers associated with taking antibiotics was classified into the following categories: emerging drug resistance, allergies/reactions, antibiotics may kill “friendly”/“good” microbes, it is unhealthy to take drugs/chemicals in general, misuse/overuse of antibiotics, multiple reasons, other, don't know, or refused. Answers to survey items 1 and 5 were yes/no. For statements 2, 3, and 4, participants were asked to respond according to the following 5-point Likert scale: 1=strongly agree, 2=agree somewhat, 3=unsure, 4=disagree somewhat, and 5=strongly disagree. We classified those who answered “strongly agree” or “agree somewhat” to the antibiotic knowledge questions as having agreed and those who answered “strongly disagree” or “disagree somewhat” as having disagreed. Those who refused to answer a question were not included in the analysis.>50,000 residents. Presence of children in the household (yes/no), month of interview, and medical insurance status were also recorded. Respondents were classified as being “with insurance” if they reported any of the following as their type of insurance: health maintenance organization, preferred provider organization, traditional indemnity insurance, Medicaid, Medicare, or other. If respondents reported their type of insurance as “don’t know” or if they refused to answer the question, they were not included in the analysis.In addition to eliciting participants’ responses to these questions, the survey also recorded demographic characteristics of the participants, including their sex, age, income level, education, race, state, and place of residence. Respondents’ place of residence was categorized as urban if they reported living in a city or town of To simplify our analysis, we coded persons indicating Hispanic ethnicity as Hispanic, even if they also identified themselves by race . For our multivariable analysis, we grouped persons identified as Asian, Pacific Islander, American Indian, or Alaskan Native into the category called “other.” We also added those who responded “don’t know” or “unsure” to the attitude questions to the “agree” group to divide respondents into two groups: those who responded correctly (disagree) and those who did not (agree or don’t know). For our multivariable logistic regression, we grouped respondents who answered “don’t know” to the question, “Are you aware of dangers associated with antibiotics?” with those who answered “no.” Persons responding “don’t know” to the question, “In the past 4 weeks, have you taken antibiotics?” were not included in the analysis. We evaluated respondents’ education and income levels as continuous variables.To compensate for respondents’ unequal probability of selection and allow population estimates to be made, we weighted the data following procedures from the Behavioral Risk Factor Surveillance System , a specialized statistical procedure for analyzing complex sample survey data, and ran the analysis using SAS   This software adjusts for the complexity of the sampling design  and uses Taylor series linearization methods to estimate variances. Because the ratio of sample size to population size was small, we approximated the sample design by a “with-replacement” design for purposes of variance estimation in SUDAAN. Any bias resulting from such replacement sampling will be in the conservative direction.We examined respondents’ attitudes toward, and awareness of, antibiotic use by their age, sex, race, income level, education, state, place of residence, medical insurance status, presence of children in household, and month of the interview. We then tested the relationships between respondents’ demographic characteristics and their responses to the questions and statements about antibiotics using chi-square tests for independence. We used the results of the bivariate analyses to develop two multivariable logistic regression models: 1) a model assessing the effects of respondents’ awareness of antibiotic dangers on their attitudes toward and expectations of antibiotics; and 2) a model assessing the influence of respondents’ attitudes on their recent antibiotic use.Because of the complexity of the analyses, we used only second-degree product terms to assess interaction effects. Results of the logistic regression models are reported as odds ratios (ORs) with 95% confidence intervals (CIs). The level of significance is p=0.05.The sample consisted of 12,755 respondents: 7,254 females and 5,501 males. Of these 12,755, a total of 1,975 were <18 years old or of an unknown age and thus were excluded from the analysis . Of the Of the 10,780 respondents, 27% believed taking antibiotics when they had a cold prevented more serious illness  and each of the three different attitude statements as the outcome. Each of these relationships was significant in the univariate and multivariable analyses .Participants not aware of adverse effects of antibiotic use were 2.5 times more likely to agree with the statement, “When I have a cold, I should take antibiotics to prevent getting a more serious illness” (95% CI 2.14 to 2.92). In addition, the demographic variables of age, sex, race, income level, education level, and state were all significant predictors of agreement. We also found significant interactions between the awareness variable and race and education, as well as interactions between age and gender.We also found a significant association between participants agreeing with the statement, “When I have a cold, antibiotics help me to get better more quickly,” and their being aware of health dangers associated with indiscriminate use of antibiotics . Those agreeing with this statement were more likely to be older .Participants not aware of dangers were 1.96 times more likely to agree with the statement, “By the time I am sick enough to talk to or visit a doctor because of a cold, I usually expect a prescription for antibiotics” (95% CI 1.72 to 2.23). The other demographic variables in the model significantly associated with participants’ responses to this statement were age, sex, income level, education level, insurance, state, and place of residence.Using another multivariable model, we examined the association between respondents’ taking antibiotics in the prior 4 weeks and their attitudes toward and knowledge of the adverse effects of antibiotic use . The oveParadoxically, participants aware of dangers related to antibiotic use were 1.37 times more likely to have taken antibiotics in the previous 4 weeks (95% CI 1.11 to 1.69) even though awareness of these dangers was not a univariate predictor of antibiotic use . Of note, only one attitude statement was significant in predicting antibiotic use, suggesting that all of the statements are measuring similar things .The results of this FoodNet survey showed that 12% of adult respondents had used antibiotics during the prior month, most (91%) of which were prescribed for a current infection. Extrapolating from the survey data, we estimate that every adult in the United States in 1998 used antibiotics an average of 1.4 times and that approximately 1 in 10 adults who used antibiotics did so without seeing a physician.The results also suggest that peoples’ knowledge and attitudes regarding antibiotic use can be substantially improved and that improved knowledge may be important for efforts to reduce the misconceptions and misguided expectations contributing to inappropriate antibiotic use. Overall, 53% of respondents to this population-based survey reported at least one misconception that may put them at unnecessary risk for infection with resistant bacterial pathogens, and 58% were not aware of the health dangers associated with antibiotic use. Nearly half (48%) of the respondents indicated that they expected an antibiotic when they visit a doctor.This survey identified persons in demographic groups who had both higher levels of misconceptions and lower levels of knowledge about the potential adverse impact of antibiotics. These groups included persons of lower socioeconomic status, lower educational status, males, those in younger age groups, and the elderly. Efforts to reach these groups must be a part of any educational efforts to change patient expectations and to reduce the corresponding pressure on providers to prescribe antibiotics inappropriately.The results of this study did not show a consistent direct link between misguided expectations and higher levels of recent antibiotic use. In part, this lack may have been due to the design of the survey, which focused on collecting frequency data and did not aim to define the reasons for antibiotic use. In addition, in our analysis, we found that the three attitude statements were similar measures of a person’s opinions on antibiotic use. The statements have the same demographic predictors and association with the knowledge variable and, in reality, they appear to measure the same thing .We did not find an association between recent antibiotic use and lower knowledge levels. Before the analysis, we assumed that persons lacking knowledge about the dangers associated with antibiotic use would be more likely to take antibiotics. However, we found that study participants aware of these health dangers were actually more likely to have taken antibiotics in the prior 4 weeks. Persons of higher socioeconomic status (higher education and income) have better access to health care and are more likely to use antibiotics in general; we did find that people who took an antibiotic recently were more likely to have medical insurance. Another possible explanation is that those who recently took antibiotics may have learned about the adverse effects of antibiotic use from their physician or pharmacist or from their personal experience with antibiotic side effects. Future epidemiologic studies of antibiotic use in diverse populations should be designed to collect information on why participants use antibiotics to distinguish between appropriate and inappropriate antibiotic use.This type of study has several other important limitations. A telephone survey creates the possibility of selection bias because it may not reflect the population being surveyed (Another limitation is the cross-sectional nature of this study. Each participant was assessed only once, and the study was not designed to detect recent changes in opinion. Furthermore, the indicators used measured self-reported behavior not actual behavior. We did not attempt to validate responses on the basis of actual observation, and the survey did not determine whether the antibiotic use was appropriate.Additionally, respondents may have misunderstood the statements about colds and antibiotics. For example, if they had previous experience with what they thought was a cold, and a physician diagnosed a bacterial ear infection, they may have responded that antibiotics help them get better more quickly when they have a cold (This study focused only on antibiotic use among adults. Antibiotic use is, however, highest among children, as is the potential for its misuse. In fact, we found that respondents with children in the household were more likely to agree with the attitude statements, demonstrating that it is often parents who influence their children’s perceptions of antibiotic use.The results of this analysis demonstrate that population-based surveys can contribute to efforts to monitor and reduce inappropriate antibiotic use. The magnitude of recent antibiotic use among adults, as well as widespread lack of awareness about and inappropriate attitudes toward such use indicate that continued population-based surveys could be useful in efforts to monitor trends in antibiotic use. Furthermore, such surveys have the potential to effectively monitor antibiotic knowledge, attitudes, and practices among demographic subgroups of concern. Knowing the magnitude of the problem and the groups who misuse antibiotics most frequently will help public health officials develop and fund intervention efforts, including public information campaigns.However, our findings also point out some important issues that need to be addressed if this surveillance tool is to be used to full effect. First, additional population-based studies are needed not only to measure antibiotic use but also to determine the reasons that people use them. Such studies should explore the motivations, expectations, and incentives that lead persons to use or not use antibiotics. Second, future studies should include more clearly defined measures of patients’ knowledge. Better measures of knowledge may involve asking respondents to differentiate between antibiotics and other types of prescription medicine and to identify types of infections requiring antibiotics. A more thorough evaluation of respondents’ attitudes may also be useful. To this end, focus groups may help develop questions that better monitor the general population’s attitudes toward antibiotics. Finally, longitudinal tracking of these types of studies will provide important information for the assessment of public health programs.


Our aim was to investigate both the prevalence of MET amplification in gastric cancer as well as the potential of this genetic alteration to serve as a therapeutic target in gastric cancer. MET amplification was assessed by initial screening with a PCR-based copy number assay followed by confirmatory FISH analysis in formalin-fixed, paraffin-embedded specimens of gastric cancer obtained at surgery. The effects of MET tyrosine kinase inhibitors (MET-TKIs) in gastric cancer cells with or without MET amplification were also examined. The median MET copy number in 266 cases of gastric cancer was 1.7, with a range of 0.41 to 21.3. We performed FISH analysis for the 15 cases with the highest MET copy numbers. MET amplification was confirmed in the four assessable cases with a MET copy number of at least 4, whereas MET amplification was not detected in those with a gene copy number of <4. The prevalence of MET amplification was thus 1.5% (4 out of 266 cases). Inhibition of MET by MET-TKIs resulted in the induction of apoptosis accompanied by attenuation of downstream MET signaling in gastric cancer cell lines with MET amplification but not in those without this genetic change. MET amplification identifies a small but clinically important subgroup of gastric cancer patients who are likely to respond to MET-TKIs. Furthermore, screening with a PCR-based copy number assay is an efficient way to reduce the number of patients requiring confirmation of MET amplification by FISH analysis. Gastric cancer is the third most common cause of death from malignant disease in men (fifth in women) worldwide . The proMET proto-oncogene encodes the receptor tyrosine kinase c-MET. The binding of its ligand, hepatocyte growth factor, to MET results in tyrosine phosphorylation of the receptor and activation of downstream signaling molecules. Oncogenic activation of MET suppresses apoptosis and promotes cell survival, proliferation, migration, and differentiation as well as gene transcription and angiogenesis [MET has been attributed to gene amplification [MET amplification has varied among studies [MET amplification in 266 formalin-fixed, paraffin-embedded (FFPE) specimens of gastric cancer obtained during surgery. To ensure the efficient detection of MET amplification, we adopted a sequential approach involving PCR-based determination of gene copy number followed by confirmatory FISH analysis. Moreover, to assess the potential of MET amplification as a therapeutic target in gastric cancer, we investigated its impact on cell survival and signal transduction.The ogenesis . In gastfication -15. Howe studies -21, possMET copy number in gastric cancer cell lines whose MET amplification status was previously determined [MET amplification, including KATO III, SNU1, SNU216, MKN1, MKN7, HSC39, MKN28, and NUGC3, the copy number of MET as determined by the PCR-based assay ranged between 1.3 and 3.3. In contrast, cell lines positive for MET amplification, including Hs746T, MKN45, and SNU5, showed MET copy numbers of 21.3, 21.3, and 17.9, respectively. The PCR-based assay thus revealed a high copy number for MET only in gastric cancer cell lines previously shown to be positive for MET amplification by FISH.We first applied FISH Figure  and a retermined . In gastMET amplification in advanced gastric cancer, we examined 266 FFPE specimens of surgically resected primary gastric tumors. Most of the patients were male (68.8%) and had undifferentiated-type gastric cancer (62.8%), including mucinous adenocarcinoma, signet ring cell adenocarcinoma, and poorly differentiated adenocarcinoma , which had a MET copy number of at least 4, whereas six cases  with a copy number of less than 4 did not exhibit MET amplification .We thus identified four out of 266 gastric cancer patients (1.5%) as having MET amplification in gastric cancer, we first examined the effects of two highly selective MET receptor tyrosine kinase inhibitors (MET-TKIs), JNJ38877605 and SGX523, on the growth of gastric cancer cell lines positive or negative for MET amplification. The IC50 values of JNJ3887605 and SGX523 for inhibition of cell growth were 0.02 to 0.05 μM and 0.06 to 0.07 μM, respectively, for cells positive for MET amplification, whereas they were >10 μM for MET amplification–negative cells  was not assessable by FISH because of a lack of hybridization signals. MET amplification was not detected in the cases with a gene copy number of <4. We therefore identified MET amplification at a frequency of 1.5% (4 out of 266 cases), consistent with values determined by FISH analysis in recent studies of gastric cancer [MET amplification with a PCR-based assay is an efficient means with which to reduce the number of specimens requiring evaluation by FISH analysis. As mentioned above, one specimen (G331) in the present study showed a high MET copy number (6.14 copies) but could not be confirmed positive for MET amplification because of the lack of a FISH result. The issue of how to identify MET amplification status in such cases remains to be resolved.FISH analysis is a semiquantitative method that can be performed with two probes for determination of the number of signals for a target gene and for the centromeric portion of the corresponding chromosome. Given that the number of centromeric signals directly reflects the copy number of the chromosome, FISH analysis yields the copy number gain for the target gene from the ratio of the copy number of the gene to that of the chromosome. FISH is thus the gold standard for detection of gene amplification. However, the identification and counting of FISH signals are hampered by many factors including cutting artifacts, nuclear overlap, and heterogeneity of tumor specimens. Moreover, FISH is expensive and time-consuming, and it requires technical expertise . The effc cancer , 20. OurMET amplification in gastric cancer cells by comparing the effects of the MET-TKIs JNJ38877605 and SGX523 between gastric cancer cell lines positive for MET amplification and those negative for this genetic alteration. In gastric cancer cells with MET amplification, the MET-TKIs markedly inhibited AKT, ERK, and STAT3 signaling and triggered apoptosis, whereas such effects were not evident in cells without MET amplification. To investigate whether attenuation of MET signaling by the MET-TKIs is related to the induction of apoptosis, we transfected gastric cancer cell lines with an siRNA specific for MET mRNA. Such transfection inhibited MET signal transduction as well as induced apoptosis in gastric cancer cell lines with MET amplification but not in those without it (data not shown). Our observations thus indicate that gastric cancer cell lines positive for MET amplification depend predominantly on constitutive activation of the encoded growth factor receptor for their survival and thus show high sensitivity to cell killing by MET-TKIs. Targeting of MET signaling by MET-TKIs is therefore a potentially valuable therapeutic approach for patients with MET amplification–positive gastric cancer. Indeed, the MET-TKI crizotinib (PF-02341066) was recently found to induce a radiographic response  and rapid clinical improvement in patients with advanced gastric cancer who were found to be positive for MET amplification by FISH [MET amplification is thus warranted.We examined the biological impact of  by FISH . FurtherMET amplification, it is important to determine the prevalence of such gene amplification in patients with unresectable advanced gastric cancer, most of whom are currently treated with systemic chemotherapy. Our present study was limited to gastric cancer patients who underwent gastrectomy, and so further studies will be needed for patients with unresectable advanced tumors. Given the apparent low prevalence of MET amplification in gastric cancer, implementation of a sequential approach including screening with a PCR-based copy number assay followed by confirmatory FISH analysis should facilitate the identification of MET amplification in a large cohort of patients with unresectable advanced gastric cancer.Given the potential of MET-targeted therapy for gastric cancer with 2 at 37°C in RPMI 1640 medium  supplemented with 10% heat-inactivated FBS , penicillin, and streptomycin.The human gastric cancer cell lines SNU1, SNU5 and Hs746T were obtained from American Type Culture Collection ; MKN1, MKN7, MKN45, and NUGC3 were from the Health Science Research Resources Bank ; KATO III, MKN28, and HSC39 were from Immuno-Biological Laboratories ; and SNU216 was from the Korean Cell Line Bank . All of the cell lines were maintained under a humidified atmosphere of 5% COA total of 267 patients with histologically confirmed gastric cancer who had undergone surgery at the National Cancer Center Hospital  between 1996 and 2006 were included in the study. All the patients had an Eastern Cooperative Oncology Group performance status of 0 to 2. One patient was subsequently excluded as a result of an insufficient quantity of DNA extracted from the corresponding tissue specimen. The specimens from the remaining 266 patients were thus analyzed. The present study was approved by the Institutional Review Board of the National Cancer Center Hospital, and informed consent was obtained from all subjects.Macrodissection of the surgical specimens preserved as FFPE tissue was performed after removal of paraffin in order to select a region of cancer tissue. Genomic DNA was extracted from the cancer tissue with the use of a QIAamp DNA Micro Kit . The DNA concentration of the extracts was determined with a NanoDrop 2000 spectrophotometer .MET was determined with the use of a TaqMan Copy Number Assay [TERT locus was used as the internal reference, and DNA from noncancerous FFPE tissue was used as a normal control. Real-time PCR was performed in a total volume of 20 μL per well containing 10 μL of TaqMan genotyping master mix, 20 ng of genomic DNA, and each primer. The amplification protocol included an initial incubation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The resulting products were detected with the use of ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Data were analyzed with SDS 2.2 software and Copy Caller software (Applied Biosystems).The copy number of er Assay  and the MET copy number per cell was determined by FISH with the use of the c-met / CEN7p Dual Color FISH Probe  [MET/CEN7p copy number ratio of >2.2, corresponding to a previous definition of MET amplification [MET/CEN7p ratio (1.8 to 2.2) were thus scored as negative for amplification., Japan) , where Cfication . The preImmunoblot analysis was performed as described previously . Rabbit 50 values of JNJ38877605 and SGX523 for inhibition of cell growth were determined.Cells were transferred to 96-well flat-bottomed plates and cultured for 24 h before exposure to various concentrations of JNJ38877605  or SGX523  for 72 h. Tetra Color One  was then added to each well, and the cells were incubated for 3 h at 37°C before measurement of absorbance at 490 nm with a Multiskan Spectrum instrument . Absorbance values were expressed as a percentage of that for nontreated cells, and the ICg for 5 min. The cell pellets were resuspended in 100 μL of Annexin-V-FLUOS labeling solution, incubated for 10 to 15 min at 15° to 25°C, and then analyzed for fluorescence with a flow cytometer  and Cell Quest software .The binding of annexin V to cells was measured with the use of an Annexin-V-FLUOS Staining Kit . Cells were harvested by exposure to trypsin-EDTA, washed with PBS, and centrifuged at 200 × t test or the chi-square test. A P value of <0.05 was considered statistically significant.Overall survival (OS) curves were estimated with the Kaplan-Meier method and compared with the log-rank test. Other statistical analysis was performed with Student's two-tailed


We contrasted the predictive power of three measures of semantic richness—number of features (NFs), contextual dispersion (CD), and a novel measure of number of semantic neighbors (NSN)—for a large set of concrete and abstract concepts on lexical decision and naming tasks. NSN (but not NF) facilitated processing for abstract concepts, while NF (but not NSN) facilitated processing for the most concrete concepts, consistent with claims that linguistic information is more relevant for abstract concepts in early processing. Additionally, converging evidence from two datasets suggests that when NSN and CD are controlled for, the features that most facilitate processing are those associated with a concept's physical characteristics and real-world contexts. These results suggest that rich linguistic contexts (many semantic neighbors) facilitate early activation of abstract concepts, whereas concrete concepts benefit more from rich physical contexts (many associated objects and locations). The majority of experimental evidence guiding our knowledge of lexical semantic representation comes from research using concrete words. Concrete words are the dominant stimuli in the literatures on semantic priming, property generation, and single-item recognition . In contrast, a larger proportion of the human lexicon may actually be composed of abstract words. For example, of the 500 most frequent words in the TASA corpus  effects have established the importance of semantic richness in concrete word representation. Investigating whether NF effects are obtained for abstract words—and if so, for what types of features—can yield insight into their representations and the information sources used to learn those representations.If semantic features are important to abstract concept representations, one might expect to find feature-based effects similar to those observed for concrete words. For concrete concepts, being associated with many semantic features facilitates lexical processing  as words that carry information about internal states (“introspective features”), and many elements of situations were observed in descriptions of abstract concepts in the present study, including mentions of persons (DANGER → “a policeman may face this in his job”), objects (SUCCESS → “great house”), and events (MISCHIEF → “crimes at night”). When information of this sort is not ignored, apparent differences in richness between concrete and abstract concepts disappear or become far less extreme , and if so, what kinds of properties are most facilitative.On some accounts, the situation-relevant and introspective utterances that participants use to describe abstract concepts in feature generation tasks are conceived of as properties in a strong sense, playing a central role in abstract concept representations  have shown the latter to have a relatively weak or undetectable impact . Using these measures, they found that neighborhood density, CD, and NF all accounted for unique variance above and beyond that accounted for by lexical-level variables in the lexical decision task, whereas number of senses and lexical ambiguity did not. Yap et al. also found that CD and NF, but not NSN, predicted unique variance in speeded pronunciation times, although the effects were less robust. This is consistent with previous findings of facilitation for words with many features  and concreteness . However, courthouse, crime, and justice are all concepts that possess a meaningful semantic relationship to law, and may play a role in its semantic representation, even if it is difficult to produce a three- or four-word sentence that encapsulates the nature of this relationship. We risk missing important insights about the nature of abstract concept representation if we exclude such concepts from analysis simply because participants do not express them as predicates. On the other hand, if we interpret all frequent responses as “features,” we risk being too inclusive. We do not pretend to have a solution to this dilemma, and believe there may be value in both broad and constrained notions of what constitutes a property. For this reason, we restricted our definition of NF to the number of {concept → word} pairs that matched a subset of predefined semantic relations identified in the literature as being of likely importance to concrete and abstract concept representations. However, we also created an additional variable, NW, which is simply a count of all words produced by at least six of the 30 subjects who generated descriptions for that word. Details on how each of these measures was calculated appear in the Methods section.However, predicates are not the basic units of semantic analysis, but are rather only one way of expressing underlying semantic relationships. Seven hundred and sixty six participants  participated via the Indiana University Psychology Department subject pool for partial course credit. An additional 208 participants recruited via Amazon Mechanical Turk who completed the study for a payment of $1 per session. All participants resided in the United States and reported English as their first language.After surveying the literature on feature generation studies and abstract word representation, 593 English nouns were selected to be normed. Items used in the feature generation studies of Barsalou and Wiemer-Hastings , McRae eProperty generation. Participants were asked to participate in an online game in which they would be required to describe various words, and were informed that a future participant would be responsible for guessing the words from their descriptions. Participants were asked to provide 10 short descriptive properties for each of 20 words that would help their partner guess the target word. Participants were instructed to describe the concept, not the word itself; i.e., clues about the letter that a word starts with or words that it rhymed with were not permitted. Participants were asked to fill in all 10 blanks, but the online application did not require all 10 blanks to be filled in order to move on to the next word. Word type was alternated for each participant , and word type that began the task was balanced across participants. While McRae et al.  produced by at least sixis made of), while others exhibited semantic relationships that were not necessarily expressed as predicates but were captured by Wu and Barsalou's (event). Yet others matched categories not covered in the Wu and Barsalou taxonomy, but which have been hypothesized to be of particular importance to abstract concept representations, such as communicative acts and social institutions/artifacts  or associated entities? Systematic disagreements of this nature between raters were generally solved either by collapsing multiple categories into one or omitting a category entirely. However, when fine-grained distinctions could be preserved while retaining high reliability, we generally did so. Agreement between two raters on a 500-feature subset of the data was quite good (Cohen's κ = 0.78), and so the remainder of the data was coded by a single rater. Each {concept, word} pair that was produced by at least six participants and which matched one of these codes was considered a feature and was included in the NF measure.Although Wu and Barsalou  reportedA common trade-off in the development of a coding scheme is between reliability and comprehensiveness; our criteria clearly lean toward reliability. Our exclusion of some feature categories means that some valid features will have escaped our NF measure, but the feature categories that are coded for are consistent between raters. Thus, our NW and NF measures represent broad and narrow ends of the spectrum of definitions of what constitutes a “feature.” As with any measure of NF, it is important to keep in mind that exactly how features are defined is critically important to the interpretation of NF effects (or the absence thereof).WordMine2 application. According to this measure, a word's neighborhood consisted of all words occurring within a specific radius of the high-dimensional space defined by HAL  represents the probability of observing word x if a random window of text is selected from the corpus, P(y) the probability of observing word y, and P(xy) the probability of observing x and y together. PMI has been shown to be a good predictor of human semantic similarity and synonymy judgments , a measure of association frequently used in computational linguistics to contrast the actual probability of observing two items together  with the probability of having observed them together if they had been independently distributed . Thus, the control variables entered into the regression were log-frequency (SUBTLEX corpus), number of morphemes, number of syllables, orthographic Levenshtein distance 20  even after variance from control variables had been accounted for. Table p-values from the regression analysis. Consistent with Yap et al. , but NF was not (p = 0.15). In contrast, for the set of the 147 most concrete items, NF (p < 0.01) and CD (p < 0.001) were found to be significant predictors of LDT, but NSN was not (p = 0.14). When LDT regressions were repeated with one or neither Levenshtein distance predictor, there were no changes in which semantic richness variables remained significant and non-significant predictors. With Levenshtein variables omitted, variance inflation factors (VIFs) were acceptably low for both abstract concepts  and concrete concepts (3.3 and 3.0). We considered the possibility that NSN was not a significant predictor for the most concrete words merely because the variance accounted for by NF and NSN overlapped in such a way that NSN would have been a significant predictor for the most concrete words had NF not been part of the analysis. However, NSN was still not a significant predictor for the set of the most concrete words even when NF was omitted from the regression (p = 0.2). Similarly, NF was not a significant predictor for the set of abstract words even when NSN was omitted from the regression (p = 0.4).Table p = 0.07), but not for abstract concepts (p = 0.28).For NTs, CD was the only significant semantic richness predictor for abstract and concrete concepts. NF was a marginally significant predictor for concrete concepts  have been hypothesized to play a special role in grounding abstract concepts, as have complex situations involving multiple actors, particularly social actors , and approached significance in the NT regressionp = 0.07), implies a stronger role for features in concrete concept representations.The fact that abstract concepts were so frequently described in terms of internal and social experiences hints that these may indeed be important aspects of abstract concept representation. However, the present analyses suggest that being rich in these kinds of features likely does not facilitate early processing of abstract concepts in the same way that being feature-rich facilitates early processing of highly concrete concepts. Of course, it is certainly possible that annotating participants' descriptions for other kinds of features would yield different results. It is also possible that 147 abstract concepts was simply too few to detect a NF effect. However, the fact that NF was a significant predictor of the 147 most concrete concepts' LDT  detailed in Recchia and Jones , and theAlthough we coded our own data according to our own feature taxonomy, we also wished to take advantage of the fact that the McRae et al.  feature locations , associated entities , and larger continuous wholes . No other feature classes were a significant predictor of unique variance in our data. Betas and significance levels for this analysis are reported in Recchia and Jones , the number of the following kinds of features accounted for unique variance: nd Jones .locations (p < 0.05) and associated entities (p < 0.001) were the only two variables that explained significant unique variance. No other feature classes accounted for unique variance in this dataset. The full set of betas and significance levels for this analysis are listed in Recchia and Jones  were more frequent than locations.Our replications of Analysis 2 with specific feature types proved inconclusive: When restricting the regressions to 147-concept subsets and dividing NF into 19 variables, none of the variables accounted for significant levels of variance in LDTs, although this could be due to data sparsity. However, on the two replications of Analysis 1 using our data and the data from McRae et al. , there wlocations and associated entities were predictive in both datasets is especially striking, considering the substantial differences in the process according to which features were coded in each. As previously described, the NF measure in the McRae et al. norms is a count of the number of distinct predicates used to describe each concept, whereas our measure of NF was obtained for each concept by counting the number of distinct {concept → word} pairs that matched a set of predefined semantic relations. Although an investigation of three publicly available sets of properties . This is likely due to differences in the way the category of associated entity was defined in our coding categories vs. the Wu and Barsalou . The fact that number of locations accounts for unique variance across multiple datasets and coding schemas suggests that being associated with many physical contexts facilitates lexical decision latencies for concrete concepts.The finding that Barsalou  categorinumber of visual properties did not predict LDT, even though shared visual form/surface properties have predicted LDT in at least one previous study , and other situation properties. See Wu and Barsalou , introspective properties , taxonomic properties (19), concrete situation properties , and other situation properties . Means and standard deviations for the NFs in each supercategory are reported in Table All regressions performed in Analysis 3a were repeated, with two differences: First, the 27 NF variables corresponding to feature subtypes in the McRae norms were grouped into five supercategories representing the number of Barsalou  for the Barsalou  as entit2, 3, 4, , 6, 7, inumber of entity properties (p < 0.05), number of concrete situation properties (p < 0.01), and number of other situation properties (p < 0.05). Predictive supercategory variables for the McRae subset were number of entity properties (p < 0.05) and number of concrete situation properties (p < 0.01). The correlation between number of entity properties variables calculated using the McRae data/codes vs. our own data/codes was 0.41. Tables In our own norms, the supercategory variables that predicted lexical decision latency were number of entity properties predicted lexical decision latencies for the set of the 147 most concrete nouns, b = −0.167, p = 0.002.As before, no supercategory variables predicted lexical decision latencies for the set of 147 abstract stimuli. In contrast, concrete situation properties (associated entities and locations) was a strong predictor of lexical decision latency in both datasets. In addition, the number of entity properties facilitated lexical decision, a fact obscured by the many subcategories in the previous analysis. Entity properties are properties of the concept itself, both physical  and systemic ; they are extremely infrequent for abstract concepts–see the General Discussion and Recchia and Jones . Overall, entity and concrete situation properties appear to drive NF effects, consistent with the finding of Analysis 2 that NF predicted LDT for concrete but not abstract words.As expected from the results of Analysis 3a, the number of nd Jones , categorli Table . In contWe replicated the general findings from Pexman et al.  and Yap entity properties and concrete situation properties (locations and associated entities) that were attributed to a concept predicted its lexical decision latency. Entity properties generally refer to physical characteristics of objects, and are far more frequent for concrete words  than for abstract words . Generally speaking, entity properties were only attributed to abstract words that were capable of being visualized or audiated despite being rated as abstract , or which had structured components (story → plot). Locations are references to places in which the concept might be located; examples from our data include helicopter → air, raisin → box, pigeon → city, cancer → lung, etc. These, too, were quite frequent for concrete nouns . Therefore, we did not attempt to distinguish the two types of relation, but collapsed them both under a single category: as noted in Recchia and Jones  facilitates lexical decision for concrete concepts. It is inconclusive whether this is the case for abstract concepts. One intriguing similarity between locations and associated entities is that each picks out concrete entities (places or objects) that co-occur with the concept in day-to-day situations. This suggests that the features that facilitate concrete concept processing include those that pick out a concept's real-world contexts  appear to facilitate early activation of abstract concepts, as demonstrated in Analysis 2. This may be due to the fact that we acquire and use abstract words primarily in social situations in which language is highly salient  and entities with a communicative purpose , as this seemed likely to be a type of feature that might participate in abstract concept representations. As many abstract concepts are themselves communicative terms, this category often overlapped with code 19: taxonomic superordinates/subordinates. Due to the taxonomic ambiguity of these terms (is an inquiry a kind of request?) and the relatively low theoretical relevance of taxonomic relationships to abstract concept representations, such conflicts were resolved by defining code 19 as “hypernyms and hyponyms not otherwise coded.” These were seemingly sensible decisions that resulted in high interrater reliability due to ease of coding: All words that described communicative acts or entities with a communicative purpose were tagged with code 1. However, it also meant that the communication category became populated with a mishmash of synonyms (yell → holler), hypernyms (rule → decree), functions (phone → communicate), terms that occur when participants describe situations relevant to the concept or its opposite (truth → lie), etc. This example alone should make it clear that many possible alternative coding schemes are possible. Although we found no influence of NF on lexical decision for abstract words, alternative methods of subdividing the feature space may reveal feature categories for which a higher NFs does facilitate abstract LDTs.Any classification scheme that attempts to shoehorn a rich conceptual space into a set of discrete and non-overlapping categories faces significant limitations, and ours is no exception. For example, in the property generation task conducted by Barsalou and Wiemer-Hastings , utteranWhile it is possible that we did not detect NF effects for abstract concepts due to the wrong task or the wrong codes, it is also possible that counting features is simply not a useful method for uncovering the structure of abstract concepts. Indeed, our finding that rich linguistic contexts facilitate LDT moreso for abstract words than for highly concrete words is consistent with theoretical claims that language plays a central role in abstract concept representations Paivio, , as wellOther approaches include the work of Schmid , who conentity properties and concrete situation properties  primarily drove our NF effects. A broad interpretation of these results consistent with some theories of concept representation is that while rich language contexts facilitate abstract concept recognition, rich physical characteristics, and contexts facilitate concrete concept recognition. Similar investigations using different tasks are likely to add further nuance to our understanding of different forms of semantic richness and the conceptual representations they support.Questions about the role of context in abstract concept representation go back at least as far as Schwanenflugel and Shoben . UltimatThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As HIV/AIDS continues to pose a public health challenge in Africa, the pandemic cut-across borders. It affects all the age groups including old persons, despite engagement in risky sexual activities which increases HIV/AIDS infection. However, limited attention is paid to this sub-group in mitigating the pandemic. This study therefore examined condom-use and perceived HIV/AIDS infection among old people in Nigeria.The study was cross-sectional in design. A multi-stage sampling procedure was used to select 400-geriatrics. Pre-tested questionnaire developed, using information obtained from 10 Focus Group Discussion (FGD), was used to collect information. FGD data were analyzed thematically, while questionnaire data were analyzed using descriptive and statistically.Twenty-five percent of the participants had extra-marital sex since they attained elderly age. However, among this subgroup that had extra-marital sex, few (6.8%) used a condom. More males (5.3%) than females (1.5%) used condom during the last extramarital sex. Low level of condom-use was attributed to condom not worthwhile (34.5%) and opinion (50.0%) condom not made for the elderly. Moreover, FGD participants viewed sex could not lead to pregnancy and majority (60.3%) posited patronizing traditional healers and few (10.3%) use of herbs/concussion could prevent HIV/AIDS. Similarly, non-condom use was due to confidence in traditional herbs, perceived to protect against STIs including HIV/AIDS.Engagement in risky activities among elderly is a growing HIV/AIDS challenge. Condom-use is misconstrued probably due to knowledge gap. Without urgent measures to enable them protect themselves, development efforts will be in jeopardy. Investing in geriatric SRH is cost-effective intervention in mitigating HIV/AIDS pandemic.None declared