Two phenolic compound parameters  and 5 antioxidant parameters , FRAP (ferric ion reducing antioxidant power), CUPRAC (cupric ion reducing antioxidant capacity), and MCA  were measured in bulbs and bolts of 43 garlic cultivars. The bulbs of cultivar ‘74-x’ had the highest phenolic content  and the strongest antioxidant capacity , followed by bulbs of cultivar ‘Hanzhong purple’; the bulbs of cultivar ‘Gailiang’ had the lowest phenolic content and antioxidant capacity . The bolts of ‘Hanzhong purple’ also had higher phenolic content. Principal components analysis (PCA) separated the cultivars into 3 groups according to phenolic and flavonoid contents and strength of antioxidant activity. The first group had higher HRSC, FRAP, and flavonoid content; the second group had higher total phenolic content and MCA; some cultivars in the third group had higher HRSC and FRAP. All 8 test garlic bulb extracts successfully prevented Human Vascular Endothelial Cell death and significantly prevented reactive-oxygen species (ROS) formation in oxidative stress model, in which cultivar ‘74-x’ had highest protection capability, following by cultivar ‘Hanzhong purple’, and the bulbs of cultivar ‘No. 105 from Korea’ had the lower protection capability against cell death and ROS formation. The protection capability in vivo of these garlic cultivars was consistent with their phenolic content and antioxidant capacity. Allium sativum L.) is one of the most commonly produced vegetables worldwide. According to the United Nations Food and Agriculture Organization (FAO), approximately 10 million metric tons of garlic is produced annually on approximately 1 million hectares (2.5 million acres) of land. China is by far the largest producer of garlic, producing over 75% of world tonnage. Garlic is a source of various biologically active phytomolecules, including organosulfur compounds, phenolic acids, allyl thiosulfinates, flavonoids, and vitamins. The health properties of garlic depend on its bioactive compounds and especially on phenolic compounds Garlic  were sampled and collected in April 2009 and 2010 and the bulbs of all mature garlic cultivars were sampled in July 2009 and 2010 after air drying in the field. For each cultivar, samples were prepared by mixing equal amounts of garlic powder from 5 bulbs and 10 bolts selected randomly. The garlic cloves or bolts were manually peeled and immediately ground into powder in liquid nitrogen. The powder was stored at −70°C until analysis.Garlic bulbs of eight cultivar were choosed randomly for testing the cytoprotection against oxidative stress cell death and ROS formation after preparation of phenolic extracts, which were cultivar ‘Cangshan’, ‘74-x’, ‘Hanzhong purple’, ‘Suzhou white’, ‘Ningqiang mountain garlic’, ‘Russian garlic’, ‘No. 97’ and ‘No. 105 from Korea’.Folin-Ciocalteu reagent, crystalline aluminum chloride, sodium nitrite, anhydrous sodium carbonate, methanol, hydrochloric acid, and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Tianjin Bodi Chemical Co. . The following standards were purchased from Sigma-Aldrich Chemical Co. : gallic acid, rutin, neocuproine, brown alloxazine, and 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2 -carboxylic acid (Trolox), Dulbecco's modified Eagle's medium (DMEM), Fetal bovine serum (FBS), trypan blue, glyoxal, methylglyoxal or tertiary-butyl hydroperoxide and all other chemicals were of the highest quality commercially available and were purchased from Sigma–Aldrich Chemical Co. .The garlic bulbs and bolts were first treated using the method described by Park et al. For testing cytotoxicity and ROS formation in vitro, the phenolic extracts were prepared from garlic bulbs and the extracts were freeze-dried in the freeze dryer  and then the phenolic extracts concentrations were adjusted to 1 mg/mL.Total phenolic content was determined in the bulbs and bolts of garlic cultivars using the Folin-Ciocalteu method The total flavonoid content (TFAC) was determined according to the method of Sellappan et al. We followed a previously described method for measuring DPPH free radical-scavenging capacity The percentage inhibition of DPPH of the test sample and known solutions of Trolox were calculated by the following formula:Ai is the absorbance of the sample at 515 nm, and Ac is the absorbance of the blank at 515 nm.Where 4 (0.02 M) were added to a microfuge tube. Next, 45 µL H2O2 (0.15%) solution and 1 mL SA (8 M) were added. The final volume of the reaction mixture was then added to 100 µL sample solution and kept in the dark for 50 min at 20°C. The absorbance at 510 nm was recorded, and HRSC was calculated as follows:HRSC was estimated by the methods of Prasad et al. 3 (0.1%) was added to this mixture and the reaction was allowed to proceed for 5 min at room temperature in the dark. The absorbance of the product was measured at 700 nm; FRAP was expressed as this absorbance.FRAP was measured as previously described 4 (5 mM), 1 mL neocuproine (3.75 mM), 1 mL ammonium acetate (1 mM), and 1 mL distilled water and kept in the dark for 30 min in a 37°C water bath. Absorbance was then measured at 450 nm. Results are expressed in milligrams of Trolox per liter of extract.CUPRAC was determined using previously described methods 2⋅4H2O (2 mM), and 50 µL ferrozine (5 mM). The reaction mixture was kept in the dark for 10 min and the Fe2+ concentration was then monitored at 562 nm. The percentage chelating capacity was expressed as follows:Metal chelating capacity was measured using the methods of Prakash et al. A is absorbance at 562 nm.Where ECV-304 viability was tested microscopically by plasma membrane disruption, as determined by the trypan blue (0.1% w/v) exclusion test ECV-304 reactive-oxygen species (ROS) generation induced by tertiary butyl- hydroperoxide was determined by adding dichlorofluorescin diacetate (DCFH-DA) to the Human Vascular Endothelial Cell. DCFH-DA penetrated cell and was hydrolyzed to form non-fluorescent dichlorofluorescin (DCFH). DCFH was then oxidised by ROS to form the highly fluorescent dichlorofluorescein (DCF). After incubation with tertiary butyl hydroperoxide, 1 mL samples of cell were withdrawn at 3 hours and centrifuged at 1000× g for 5 minute. The cells were resuspended in DMEM and 10 µM DCFH-DA was added. Cells were allowed to incubate at 37°C for 20 minutes. The fluorescent intensity of DCF was determined by VictorTM X5 multilabel reader  measuring the excitation and emission wavelengths, which were 490 and 520 nm, respectively.P-values <0.05 were considered significant.All data are expressed as the mean ±SD of 3 replicates, and statistical analyses were subjected to ANOVA procedures (DPS v7.55 for Windows). Significant differences among cultivars were detected by Duncan's multiple range tests. maximum minus the Valueminimum divided by N Frequency distribution histograms of polyphenolic content were constructed according to the distribution interval among cultivars, where the interval was equal to the ValuePrincipal component analysis (PCA) was used to detect clustering and to establish relationships between cultivars and polyphenolic content and antioxidant properties. To eliminate the influence of dimension, data were classified into 10 grades for analysis: grade 1<X−2δ and grade 10>X+2δ, where the interval of each grade was 0.5δ, and δ was the standard deviation.Allium species.The frequency distributions of TPC and TFAC among bulbs and bolts of different garlic cultivars are shown in The TFAC in both bulbs and bolts exhibited an almost normal distribution, ranging from 0.11 to 0.59 mg rutin DW/g FW and 0.075 to 0.675 mg rutin DW/g FW, respectively. There wThe extracts of bulbs from over 40 cultivars were evaluated for total phenolic content and antioxidant activities to determine the extent of genotypic variation. The coefficient of variation in TPC in bulbs and bolts was 3.06%, indicating that the TPC varied significantly among different cultivars . TPC in The TFAC in bulbs also varied significantly (more than 4-fold) among cultivars , with a Variability of TPC and TFAC in bulbs of different cultivars could be attributed to various cultivar characteristics. Clove size must be considered, as it indirectly affects the final concentration of phenolic compounds. In agreement with our results, previous reports have shown that different garlic cultivars had different yields Allium roseum L., where the polyphenol content was 1.23 mg GAE/g aged extract TPC in garlic bolts of 32 cultivars varied from 10.17 mg GAE/g (cultivar ‘Xiangfan ershui early’) to 22.66 mg GAE/g (cultivar ‘Hanzhong purple’), which was much higher than that in leaves of Allium genus A. sativum L. and in fried garlic (60.85%) Normally, organisms have several different antioxidant systems that may be involved in various interactions; thus, the methods used to analyze antioxidant activity should accurately reflect all of the antioxidants in a complex system. In this study, 5 in vitro assays  were used as complementary methods to evaluate the potential antioxidant activity of garlic cultivars. Significant differences were observed among different cultivars in these assays . Among tCultivars ‘No. 97 from Guizhou’, ‘Baihe early’, and ‘Xiangfan ershui early’ had relatively low antioxidant activities, while cultivar ‘74-x’, which had the highest TPC among all the cultivars, showed the highest antioxidant activity. The DPPH-scavenging activity of cultivar ‘74-x’ was 12.68-fold higher than that of cultivar ‘No. 97 from Guizhou’. Significant differences in HRSC were found among cultivars; and the strongest scavenging activity (81.31%) was observed in bulbs of cultivar ‘87-x’ and the lowest •OH scavenging capacity (37.87%) was shown in bulbs of cultivar ‘Pengxian late’.Currently, the most frequently used method for measuring antioxidant activity is FRAP, which is often expressed as absorbance value, i.e., the higher the absorbance, the stronger the reducing capability. Significant differences based on FRAP assays were observed among cultivars. The lowest absorbance value in bulbs was 0.273 (cultivar ‘Gailiang’), and the TPC of this cultivar was also the lowest among all cultivars. The highest absorbance value was 0.613 (cultivar ‘Hanzhong purple’), followed by 0.611 (cultivar ‘74-x’). The reducing capacity of cultivar ‘Gailiang’ was only 44.54% of that of ‘Hanzhong purple’.In contrast to FRAP, which is based on the ferric-ferrous system, the CUPRAC method is based on the potential to reduce copper ions. We found that cultivar ‘74-x’ possessed the highest reducing activity as measured by CUPRAC (8305.43 µg Trolox DW/g), and cultivar ‘Bijie’ had the lowest reducing activity . The metal chelating capacity in bulbs ranged from 1.71% (cultivar ‘Gailiang’) to 22.98% (cultivar ‘Sichuan red’).Antioxidant activities in bolts differed greatly according to the assay used . The DPPPCA allows a large number of variables to be reduced to just a few that accounts for most of the variance in the observed results. Thus, we performed PCA to determine which garlic cultivars were associated with TPC and antioxidant activity in their bulbs . The 43 The higher positive value of PC1 showed that some cultivars  had stronger hydroxyl radical scavenging activity, FRAP, and higher flavonoid contents. The lower negative value of PC1 showed that some cultivars  had higher TPC. The higher positive value of PC2 showed that a number of cultivars, including ‘Wenjiang seven star red’, ‘Jiading No. 2’, ‘Ethiopia white’, ‘87-x’, and ‘Fenggang white’, had stronger hydroxyl radical scavenging activity, FRAP, and higher TPC. The lower negative value of PC2 showed that cultivars, including ‘Xingping white’, ‘Luliang garlic’, and ‘Xuzhou white’, had higher MCA, DPPH, CUPRAC and flavonoid content .Almost all of cultivars in the first group had the higher positive value of PC1 so it indicated that the first group had higher HRSC, FRAP and flavonoid content. The second group had lower negative value of PC1 so it indicated that the second group had higher total phenolic content and MCA. Some cultivars such as ‘74-x’ and ‘Hanzhong purple’ in the third group had higher HRSC, FRAP and flavonoid content.Correlation analyses were performed among the phenolic compounds and antioxidant capacity parameters, and between each phenolic compound and antioxidant capacity measurement for the 43 cultivars. These analyses showed significant positive correlations between TPC and TFAC, and among DPPH, FRAP, and CUPRAC in both bulbs  and boltThe bulb extracts of eight garlic cultivars (extract concentrations 1 mg/mL) were tested and compared for cytoprotection against oxidative stress cell death and ROS formation in Human Vascular Endothelial Cells. It showed that all the bulb extracts tested successfully prevented cell death and the order of protection against cell death was, from most protective to least protective after 3 hours incubation: ‘74-X’ extract > ‘Hanzhong Purple’ extract  =  ‘Suzhou white’ extract > ‘Ningqiang mountain garlic’ extract> ‘Cangshan’ extract  =  ‘Russian garlic’ extract > ‘No. 105 from Korea’ extract  =  ‘No. 97 from Guizhou’ extract . When tein vitro models. One possible mechanism whereby garlic extracts may decrease oxidative stress in cells is in the prevention of lipid peroxidationActually, the order of protection of garlic bulb extracts against oxidative stress cell death was almost the same tendency of TPC and TFAC in these test cultivars even the antioxidant capacity. The TPC order was ‘74-X’> ‘Hanzhong Purple’ >‘Ningqiang mountain garlic’ > ‘Cangshan’ > ‘No. 97 from Guizhou’ > ‘Russian garlic’ > ‘No. 105 from Korea’ > ‘Suzhou white’; and the TFAC of these test cultivars was ‘74-X’> ‘Hanzhong Purple’ > ‘Ningqiang mountain garlic’ > ‘No. 97 from Guizhou’ > ‘Cangshan’ > ‘Suzhou white’ > ‘Russian garlic’ > ‘No. 105 from Korea’. It was showed that protection capability of these garlic bulb extracts against oxidative stress cell death was consistent with their phenolic content and antioxidant capacity. Due to the variety of polyphenolic compounds found in garlic, it is likely that multiple protective mechanisms may act against oxidative and carbonyl induced cytotoxicity in On the basis of PCA, the garlic cultivars examined in this study could be divided into 3 groups. Group 1 contained 23 cultivars with stronger HRSC and FRAP, as well as higher flavonoid contents. The second group consisted of 16 cultivars with a higher TPC and MCA, and the third group consisted of 4 cultivars with stronger HRSC and FRAP. In addition, significant correlations among different antioxidant assays were observed in both bolts and bulbs. These antioxidant properties were highly correlated with the presence of the primary phenolic compounds. It was showed that the bulb extracts of eight test garlic cultivars successfully prevented Human Vascular Endothelial cell death and ROS formation, in which cultivar ‘74-x’ and ‘Hanzhong purple’ had superior protective effects and cultivar ‘No. 105 from Korea’ had the lower protection capability against cell death and ROS formation, which was consistent with their phenolic content and antioxidant capacity.


Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones.  Staphylococcus aureus is a major cause of human disease, made even more notable due to the spread of antibiotic resistance. We used a combination of animal models to study the spread of bacteria between organs during an infection and the resulting effect of antibiotic intervention. We found that S. aureus infection is highly clonal, following a “bottleneck” in which very few bacterial cells found each abscess. Despite previous in vitro research, the effect of antibiotics on S. aureus infection was poorly understood. We utilized our systemic infection models to study intervention with sub-curative antibiotic doses, such as one might encounter upon failing to complete an antibiotic course. We have shown that such doses are able to support the preferential expansion of antibiotic-resistant organisms during a mixed infection. This selection is due to the clonal pattern of infection, occurring despite a lack of effect on growth rate or on the spontaneous generation of resistance. Furthermore, it is generic to multiple pathogen species, including Pseudomonas aeruginosa, and antibiotic classes, such as with methicillin-resistant S. aureus (MRSA) in the presence of oxacillin. Given the current debate in the field, our results have important implications for the design of properly-controlled treatment regimes. Staphylococcus aureus is an opportunistic human pathogen that causes skin and tissue abscesses, occasionally leading to severe systemic illness and death S. aureus ; kanamycin-resistant, KanR (GMSA016); and tetracycline-resistant, TetR (GMSA017). Growth of the three strains in vitro under favorable conditions exactly matched the wild type parent SH1000  lineage USA300, as opposed to the much higher levels of expression seen in the presence of the Newman allele In order to enhance the ability of infection dynamics studies to examine disease progression in both zebrafish embryos and mice, a set of three antibiotic-resistant t SH1000 . Furthert SH1000 . Resistain vivo, leading to lethality, occurs after a biological “bottleneck” that often results in clonal expansion of a single strain post mortem (p>0.1), in agreement with previously published results in vivo and, after mixing with “naïve” bacteria, reintroduced them into the zebrafish model. We found that there was no increase in the selection of these clones relative to their naïve counterparts over three successive passages, suggesting a lack of adaptive mutations, as expected in vivo clonal population expansion of the three strains is likely to be entirely stochastic and, hence, endpoint enumeration of these strains is a suitable and reliable method to investigate in vivo dynamics.Previous work in the zebrafish embryo model suggested that growth of bacteria t mortem . Infectit mortem . In bothS. aureus infection results in kidney abscesses Salmonella entericaBacillus anthracisYersinia pseudotuberculosis7 CFU. At 30 minutes and then at 2, 6, 12, 24, 48 and 72 hours post infection, mice were sacrificed and their visceral organs  were harvested for bacterial enumeration by homogenization, serial dilution and plating on selective media (n = 8–10 mice per time-point). Bacterial numbers in the blood were found to be consistently low, approaching the limit of detection at all time-points, and are therefore not included in the analysis. Pilot data indicated that the bacteria were unlikely to be found at other sites .It is known that intravenous A large fraction of the bacterial inoculum was restricted to the liver and spleen for several hours post infection. Neither extreme clonality nor an overall increase in bacterial numbers were observed at this stage, indicating that little bacterial growth was occurring, or that there was an equilibrium between bacterial growth and killing by the host . As the in vitro MIC. It was hypothesized that a low antibiotic dose, i.e. one that produces no response in fish infected with a drug-sensitive strain, might still offer a preferential advantage to drug-resistant bacteria, as has been observed in vitroClonal expansion of bacteria has important implications during mixed-strain infections, especially when a drug-resistant mutant is present among a population of sensitive organisms, and the zebrafish infection model allowed us to investigate this process. The antibiotic dose required to produce a significant reduction in mortality in the zebrafish embryo model can, in some cases, approach fifty times the p = 0.0003), 10 µg/ml tetracycline showed a trend towards curing (p = 0.0753) and 5 µg/ml was entirely sub-curative (p = 0.2181). We therefore chose 2.5 µg/ml tetracycline for subsequent experiments. Embryos treated with sub-curative 2.5 µg/ml tetracycline contained approximately 106 CFU of EryR bacteria upon death   were immersed in sterile E3 medium containing a range of antibiotic doses for the duration of the experiment .While 50on death , demonst (p>0.9) , indicatp = 0.7412) or in total bacterial numbers per embryo upon death (p = 0.1452). There were, however, significant differences in the ratios between the strains isolated from treated and untreated embryos (p = 0.0143). Lowering the tetracycline dose to 1 µg/ml abolished this effect (p = 0.1008), implying a narrow window (approximately four-fold) in which the antibiotic dose is low enough to be statistically sub-curing but high enough to influence population dynamics. Bacteria recovered from these experiments were invariably found to be resistant to a single antibiotic alone, therefore the change in output ratio is not due to the spontaneous generation of resistance among the sensitive population (as bacteria that had acquired resistance to tetracycline in this way would remain erythromycin-resistant). Phagocyte depletion of infected zebrafish, which has been previously shown to prevent stochastic population variation p = 0.4855), indicating that phagocytes play a role in the antibiotic skewing phenomenon, and again supporting the hypothesis that phagocytes are an important host niche during S. aureus infection.To ascertain whether a sub-curative dose of antibiotic was indeed able to influence bacterial population dynamics, embryos were infected with a total of 1500 CFU SH1000 EryR and TetR (GMSA015 and GMSA017 mixed 1∶1), and treated with sub-curative 2.5 µg/ml tetracycline. Treated and untreated embryos showed no difference in mortality . 2.5 µg/ml tetracycline was again shown in pilot experiments to be non-curative in fish infected with the sensitive strain alone (p = 0.5443). Results of the NewHG mixed population experiment  were consistent with those obtained for SH1000, showing similar embryo mortality between the treated and untreated groups (p = 0.4575), similar total CFU recovered from both groups (p = 0.4068) and a statistically significant difference between the strain ratios (p = 0.0296). As expected, this was abolished in phagocyte-depleted embryos at 10 µg/ml tetracycline (p = 0.2956). Therefore the preferential selection of antibiotic-resistant bacteria at low levels of antibiotic is not limited to a single S. aureus strain.To discover whether the phenomenon is consistent among other Pseudomonas aeruginosa is commonly isolated alongside S. aureus in the lungs of patients with cystic fibrosis S. aureus growth and virulence factor expression during polymicrobial wound infection Staphylococcus, two drug-resistant P. aeruginosa PAO1-L derivatives, gentamicin-resistant GmR (GMPA001) and tetracycline-resistant TetR (GMPA002), were constructed. As expected from previous work by others P. aeruginosa has a naturally high intrinsic resistance to tetracycline and was therefore unaffected by all attempts to cure the infection (p = 0.9449) or terminal CFU load (p = 0.4474) did not prevent the selection phenomenon from occurring (p = 0.0169). It is noteworthy, however, that P. aeruginosa does not reach a specific bacterial load before embryo mortality . Furthermore, in contrast to the S. aureus strains tested above, phagocyte depletion of the host does not abolish the selection phenomenon in P. aeruginosa (p<0.0001). Thus, despite the difference between S. aureus and P. aeruginosa population kinetics (and therefore infection dynamics) in vivo, the two species exhibit the same phenomenon with regards to selection at sub-curative antibiotic doses.The Gram-negative pathogen nfection  using drp = 0.5390) or a 1∶1 mixture of sensitive and resistant strains (p = 0.2087). Although the total bacterial load upon death was marginally but significantly decreased using this dose of oxacillin (p<0.0001), this may be explained by the bactericidal action of oxacillin versus the bacteriostatic nature of tetracycline used in previous experiments. Nonetheless, such a decrease had no effect on the disease state or mortality of the infected host. This sub-curative dose of oxacillin produced a highly significant skew towards the resistant strain (p = 0.0003), indicating that the phenomenon is not restricted to tetracycline and is likely to be an important factor in the clinical treatment of mixed MRSA-MSSA infections. Curiously, the skewing effect was also evident to a minor but significant degree in phagocyte-depleted embryos (p = 0.0018) treated with the same oxacillin dose, suggesting both phagocyte dependent and independent effects. Selection was not significant at 16 µg/ml oxacillin, suggesting a 2-fold concentration range for the effect (p = 0.0964).It was hypothesized that the effect of sub-curative antibiotic doses on strain ratios might not be restricted to tetracycline. To test this, clinical MRSA isolate BH1CC and its methicillin-sensitive, tetracycline-resistant isogenic partner in vivo (katA ahpC double mutant (strain KC043) was not affected by oxacillin relative to its SH1000 parent at all concentrations tested (p>0.74), it is unlikely that ROS play a role in antibiotic skewing in the in vivo infection model.Interestingly, antibiotics have been proposed to share a common killing mechanism involving accumulation of reactive oxygen species (ROS) within bacterial cells, and subsequent death from oxidative stress, irrespective of the antibiotic target or direct mechanism of action in vivo . Since tp = 0.0804, TetR p = 0.9964) or during a mixed infection (p = 0.2695), nor was there a significant difference in terminal bacterial load (p = 0.4438). Yet, again, there was a significant difference in terminal strain ratios (p = 0.0022). Similarly to tetracycline, no cross-resistance was observed in the strains recovered from embryos post mortem. Therefore, preferential selection of resistant strains (not associated with the spontaneous generation of resistant mutants) results from treatment with sub-curative doses of multiple antibiotic classes.Erythromycin is another medically relevant drug which our strain constructs enabled us to test. In the zebrafish embryo model, however, erythromycin proved unable to affect the outcome of infection by immersion alone. Instead, the antibiotic dose was introduced by microinjection not more than two hours after bacterial infection (untreated controls were instead injected with PBS). Results showed no significant difference between treated and untreated groups when comparing mortality during a single-strain infection , which were assayed for bacterial load.p = 0.1965 and p = 0.4194 respectively). Pooled data  showed a trend towards the resistant subpopulation. The variance in the untreated group compared to treated groups makes comparison by Mann-Whitney test highly misleading in this model without using hundreds of animals per group ; therefore, a binomial distribution was used to analyze these data. The underlying assumption of this conservative test is that in an untreated mouse, there is a 50% chance of skewing towards either strain . The comparison showed that whilst 0.1 mg/ml did not give a significantly non-stochastic result , 0.2 mg/ml did . Therefore a sub-curative antibiotic dose is able to cause preferential selection of resistant microorganisms during mammalian infection. Furthermore, as expected from our murine infection kinetics experiment, selection at this low antibiotic concentration was a result of growth in the kidneys (p = 0.0349) rather than liver (p = 0.2188) or spleen (p = 0.2344)  were therefore used in the final experiment alongside an untreated control . There w 0.2344) .Staphylococcus aureus is an important and increasingly antibiotic-resistant human pathogen, little is known about its disease progression or how antibiotic levels affect population dynamics. This is particularly pertinent for the understanding of S. aureus behavior in a hospital environment, in agriculture or even in the community at large, given the failure of some patients to complete prescribed antibiotic courses.Although S. aureus infection Construction of a set of isogenic strains that produce clonal lesions in the host demonstrated stochastic strain variance during zebrafish embryo infection. In the mouse it was shown that kidney abscesses form in the later stages of infection and each likely originates from a single bacterial founder. Interestingly, most of the initial inoculum is localized to the liver and spleen during the early stages of infection (up to approximately one day post intravenous administration). It is unknown whether bacterial cells founding renal abscesses were physically associated with the kidneys during this critical period, or whether individual bacteria “seed” the kidneys after travelling from another organ such as the liver, spleen, lungs, heart or blood at a later stage. Observation of a mouse that showed a dramatic, systemic infection of just two strains strongly implies that spread to secondary organs can occur after an initial period of clonal expansion. The variable speed at which growth occurs in the kidneys may be indicative of lesion founders “seeding” the kidneys at different times, especially in the case where two or more strains reach substantial but unequal numbers, given that no strain has a competitive advantage in terms of growth rate. In a peritoneal infection model, Rauch and colleagues reported parenchymal kidney abscesses that were identical to those observed post-intravenous infection, and that peritoneum-associated kidney surface abscesses were not associated with parenchymal abscesses S. aureus colonization is not directly correlated with mortality Although S. aureus that vancomycin treatment is able to greatly increase a pool of resistant organisms inside a host de novo resistance to tetracycline can arise when the drug is given orally at high levels S. aureus and a Gram-negative pathogen, P. aeruginosa, in vivo. A level of drug so low that it does not alter host mortality or bacterial load (i.e. a dose that is sub-curative) is able to produce a statistically significant skew in strain ratios towards a pre-existing resistant subpopulation. In Escherichia coli, low levels of antibiotics such as β-lactams promote stepwise mutations leading to resistance Pseudomonas aeruginosa, sub-lethal concentrations of several antibiotics increase mutational frequency in vitro experiments The use of antibiotics is the primary driving force behind the development of resistance in vivo growth kinetics experiments reveal a significant difference. Since phagocyte-depleted embryos succumb to even a small bacterial dose within 18 hours S. aureus strain (ahpC katA) defective in oxidative stress resistance ahpC katA strain was no more susceptible to tetracycline in vivo than its isogenic parent, suggesting that ROS may not play a major role in a generalized antibiotic effect. It is well known that the bacterial transcriptome/proteome can fluctuate when exposed to sub-curative antibiotic doses Critically, our result is not dependent upon development of resistance in the sensitive subpopulation. Furthermore, selection of resistant clones is not simply due to retardation of growth of the sensitive bacteria caused by the antibiotic, as phagocyte-depleted zebrafish embryos  was carried out according to guidelines and legislation set out in UK law in the Animals (Scientific Procedures) Act 1986, under Project Licenses PPL 40/3123, PPL 40/3699 and PPL 40/3574. Ethical approval was granted by the University of Sheffield Local Ethical Review Panel.Staphylococcus aureus strains  in S. aureus. This provided a convenient method of screening to ensure that clones retained a wild type phenotype; undesired disruptions of lysA would result in lysine auxotrophy. Growth of clones in chemically defined minimal medium lacking lysine showed that the wild type lysA gene remained intact. After construction by standard PCR and restriction/ligation methods, plasmids were introduced into S. aureus RN4220 by electroporation, whereupon they integrated into the chromosome via homologous recombination. The resulting resistance markers were then transferred into other S. aureus strains as required by Φ11 transduction. pMUTIN4 provided erythromycin/lincomycin resistance (EryR), whereas pAISH1 The suicide vector pMUTIN4 P. aeruginosa PAO1-L derivatives were constructed by integration of mini-Tn7 to a neutral locus according to published protocols pu.1London wild-type (LWT) zebrafish embryos  were used for all experiments and were incubated in E3 medium at 28°C according to standard protocols S. aureus suspension in endotoxin-free PBS (Sigma) corresponding to 1×107 CFU per mouse. Viable bacteria in the inoculum were plated on BHI (plus appropriate antibiotics) after serial decimal dilution to confirm the accuracy of the bacterial dose. Mice were monitored and sacrificed at various time-points according to experimental design.Female BALB/c mice were purchased from Harlan (UK) and maintained at the University of Sheffield using standard husbandry procedures. The 7–8 week old mice were inoculated in the tail vein with 100 µl of S. aureus) or LB (P. aeruginosa) agar supplemented with appropriate antibiotics to determine bacterial numbers.In order to recover bacteria from host tissues, whole zebrafish embryos or mouse organs were individually homogenized in a suitable volume of PBS using the PreCellys 24-Dual (Peqlab). Homogenates were serially diluted in PBS and plated on BHI (t-test was used. For comparisons of strain ratios between two groups (e.g. treated and non-treated) in zebrafish embryos, a (non-parametric) Mann-Whitney U test was used. Analysis was performed using Prism version 6.0 (GraphPad) and statistical significance was assumed at p<0.05. In addition, analyses via generalized linear models, linear models on transformed data and Kruskall Wallace tests were performed and provided the same insights. Strain ratios in mice were analyzed by binomial distribution, as explained in p<0.05) or NS (p≥0.05), and indicate either mean (CFU comparison) or median (ratio comparison) values as appropriate.Survival experiments were evaluated using the Kaplan-Meier method. Comparisons between curves were performed using the log rank test. For comparisons between two CFU groups, a two-tailed, unpaired Student's Figure S1Comparison of fitness of antibiotic resistance-tagged SH1000 strains. (A) Growth in aerated BHI medium at 37°C. (B) Mortality of zebrafish embryos infected with each strain (n = 20–25 per group).(TIF)Click here for additional data file.Figure S2The pattern of clonality observed during a systemic murine infection. Pie charts show the distribution of three strains in each organ, for each mouse at each time point. Numbers inside rings indicate log10(CFU) in each organ . Where total load was <100 CFU, strain distribution is not given, as presenting data this close to the detection limit would be non-representative of true strain dominance. The mouse marked * was culled 6 hours earlier than the other mice in that group due to ill health and was not included in general analyses throughout the paper.(TIF)Click here for additional data file.Figure S3The effect of tetracycline on zebrafish embryos infected with S. aureus SH1000 strains. (A) Mortality of zebrafish infected with SH1000 EryR alone and treated with a range of tetracycline doses (n = 25–30 per group). (B) Terminal EryR bacterial load per EryR-infected embryo, treated with 2.5 µg/ml tetracycline. (C) Terminal EryR/TetR strain ratio per embryo infected with a 1∶1 mixture of SH1000 EryR∶TetR bacteria and treated with 1 µg/ml tetracycline. Solid lines indicate median values.(TIF)Click here for additional data file.Figure S4The effect of tetracycline on growth of S. aureus SH1000 in vivo. Graphs show growth kinetics of (A) EryR and (B) TetR. Zebrafish embryos were either left untreated (circles) or treated with 2.5 µg/ml tetracycline (squares). Bacterial CFU loads in living fish (n = 5 per group per time-point) at 1 and 7 hours post infection and every 12 hours thereafter, were determined (white). Bacterial loads in any dead fish at each time-point were also determined (black).(TIF)Click here for additional data file.Figure S5The effect of tetracycline on zebrafish embryos infected with S. aureus NewHG strains. (A) Mortality of zebrafish infected with NewHG EryR alone, treated with a range of tetracycline doses (n = 30–35 per group). (B) Mortality of zebrafish infected with a 1∶1 mixture of NewHG EryR∶TetR, treated with 2.5 µg/ml tetracycline (n = 70–75 per group). (C) Total terminal CFU load per embryo infected with a 1∶1 mixture of NewHG EryR∶TetR, treated with 2.5 µg/ml tetracycline. (D) Terminal EryR/TetR strain ratio per pu.1 morphant (phagocyte-depleted embryo), treated with 10 µg/ml tetracycline. Solid lines indicate mean (C) and median (D) values.(TIF)Click here for additional data file.Figure S6The effect of tetracycline on zebrafish embryos infected with P. aeruginosa PAO1 strains. (A) Mortality of zebrafish inoculated with either live bacteria, heat-killed bacteria or sterile PBS (n = 30–35 per group). (B) Mortality of zebrafish infected with PAO1 GmR alone, treated with a range of tetracycline doses (n = 55–60 per group). (C) Mortality of zebrafish infected with a 1∶1 mixture of PAO1 GmR∶TetR, treated with 50 µg/ml tetracycline (n = 65–70 per group). (D) Total terminal CFU load per embryo infected with a 1∶1 mixture of PAO1-L GmR∶TetR, treated with 50 µg/ml tetracycline. (E) Terminal GmR/TetR strain ratio per pu.1 morphant (phagocyte-depleted embryo), treated with 50 µg/ml tetracycline. Solid lines indicate mean (D) and median (E) values.(TIF)Click here for additional data file.Figure S7The effect of oxacillin on zebrafish embryos infected with S. aureus strains. (A) Mortality of zebrafish infected with BH1CC ΔmecA::tetR (OxS) alone, treated with a range of oxacillin doses . (B) Mortality of zebrafish infected with a 1∶1 mixture of BH1CC OxS∶OxR, treated with 32 µg/ml oxacillin (n = 65–70 per group). (C) Total terminal CFU load per embryo infected with a 1∶1 mixture of BH1CC OxS∶OxR, treated with 32 µg/ml oxacillin. (D) Terminal BH1CC OxS/OxR strain ratio per pu.1 morphant (phagocyte-depleted embryo), treated with 32 µg/ml oxacillin. (E) Terminal BH1CC OxS∶OxR strain ratio in zebrafish treated with 16 µg/ml oxacillin. (F) Mortality of zebrafish infected with either SH1000 or KC043 (katA ahpC), treated with a range of oxacillin doses (n = 20–22 per group). Solid lines indicate mean (C) and median  values.(TIF)Click here for additional data file.Figure S8The effect of 100 µg/ml (sub-curing) erythromycin on zebrafish embryos infected with S. aureus SH1000 strains. (A) Mortality of zebrafish infected with either SH1000 EryR or TetR individually (n = 80–90 per group). (B) Mortality of zebrafish infected with a 1∶1 mixture of SH1000 EryR∶TetR (n = 80 per group). (C) Total terminal CFU load per embryo infected with a 1∶1 mixture of SH1000 EryR∶TetR. Solid lines indicate mean values.(TIF)Click here for additional data file.Figure S9The effect of sub-curative antibiotic doses on the pattern of S. aureus NewHG infection in different murine organs. EryR/TetR strain ratio is given for (A) kidneys, (B) livers and (C) spleens at two days post infection. Only organs that contained bacterial CFU above the limit of detection are shown. Solid lines indicate median values.(TIF)Click here for additional data file.Table S1Bacterial strains and plasmids used in this study.(DOCX)Click here for additional data file.


Many patients with advanced non-small-cell lung cancer (NSCLC) receive only active supportive care because of poor performance status or presence of several comorbidities. We investigated whether erlotinib improves clinical outcome in these patients.TOPICAL was a double-blind, randomised, placebo-controlled, phase 3 trial, done at 78 centres in the UK. Eligibility criteria were newly diagnosed, pathologically confirmed NSCLC; stage IIIb or IV; chemotherapy naive; no symptomatic brain metastases; deemed unsuitable for chemotherapy because of poor (≥2) Eastern Cooperative Oncology Group performance status or presence of several comorbidities, or both; and estimated life expectancy of at least 8 weeks. Patients were randomly assigned  to receive oral placebo or erlotinib (150 mg per day) until disease progression or unacceptable toxicity. Investigators, clinicians, and patients were masked to assignment. The primary endpoint was overall survival. Analyses were by intention to treat, and prespecified subgroup analyses included development of a rash due to erlotinib within 28 days of starting treatment. This study is registered, number ISRCTN 77383050.vs placebo, 3·6 months, 3·2–3·9; unadjusted hazard ratio [HR] 0·94, 95% CI 0·81–1·10, p=0·46). 59% (178 of 302) of patients assigned erlotinib and who were assessable at 1 month developed first-cycle rash, which was the only independent factor associated with overall survival. Patients with first-cycle rash had better overall survival , compared with placebo. Compared with placebo, overall survival seemed to be worse in the group that did not develop first-cycle rash . Grade 3 or 4 diarrhoea was more common with erlotinib than placebo , as was high-grade rash ; other adverse events were much the same between groups.Between April 14, 2005, and April 1, 2009, we randomly assigned 350 patients to receive erlotinib and 320 to receive placebo. We followed up patients until March 31, 2011. 657 patients died; median overall survival did not differ between groups (erlotinib, 3·7 months, 95% CI 3·2–4·2, Patients with NSCLC who are deemed unsuitable for chemotherapy could be given erlotinib. Patients who develop a first-cycle rash should continue to receive erlotinib, whereas those who do not have a rash after 28 days should discontinue erlotinib, because of the possibility of decreased survival.Cancer Research UK, Roche. Lung cancer, the main cause of cancer-related death, accounts for nearly 1·4 million deaths worldwide every year, and has an annual incidence of more than 41 000 in the UK (age standardised incidence of 48 per 100 000).EGFR mutations, erlotinib significantly improved progression-free survival compared with chemotherapy.8Erlotinib is an oral EGFR inhibitor that is associated with a significant survival benefit among patients with NSCLC when used as maintenance monotherapy after first-line chemotherapy or as second-line or third-line monotherapy.We did a large randomised trial to determine whether erlotinib monotherapy would be beneficial as first-line therapy in unselected patients with advanced NSCLC deemed unsuitable for chemotherapy.TOPICAL was a superiority phase 3, double-blind, randomised, placebo-controlled trial. Eligibility criteria were newly diagnosed, pathologically confirmed NSCLC; stage IIIb or IV disease; chemotherapy naive; no symptomatic brain metastases; deemed unsuitable for chemotherapy because of poor Eastern Cooperative Oncology Group (ECOG) performance status (PS ≥2) or presence of several comorbidities , or both; and estimated life expectancy of at least 8 weeks. Such patients do not normally receive chemotherapy. Other inclusion criteria were age older than 18 years, diagnosis within the past 62 days, able to take oral medication, and using effective contraception if appropriate. Exclusion criteria were previous treatment with any biological anticancer therapy; previous palliative radiotherapy ; pregnant or lactating women; evidence of significant laboratory finding or concurrent uncontrolled medical illness judged to potentially interfere with the trial treatment; and present treatment with a COX-2 inhibitor. We obtained multicentre and local research ethics approvals. All patients provided written informed consent.Randomisation was done by site staff telephoning the Cancer Research UK and University College London Cancer Trials Centre. Patients were randomised in a 1:1 ratio to either once daily oral erlotinib (150 mg tablets) or matching placebo, with a computer generated sequence, stratified by disease stage , performance status , smoking history , and centre (78 sites), with a block size of 10. All investigators, clinicians, and patients were masked to assignment.Patients were to take oral erlotinib or matching placebo daily, 1 h or more before food, or 2 h after food. The dose could be reduced to 100 mg, then 50 mg in cases of substantial toxic effects. Treatment continued until disease progression, adverse side-effects judged by the treating clinician to warrant discontinuation, or patient withdrawal. Patients continued to receive active supportive care, including palliative radiotherapy, at the discretion of their clinician.The primary endpoint was overall survival, measured from the date of randomisation until death from any cause. Secondary endpoints were progression-free survival (time until progression or death from any cause), tumour response , adverse events, and quality of life. Patients who survived or who did not have progression were censored at the date they were last known to be alive.Within 4 weeks before randomisation patients had a physical examination, assessment of comorbidities with the Charlson comorbidity index (CCI), blood count and biochemistry, chest radiograph, and CT of the chest and abdomen. Clinicians did bone and brain scans when clinically indicated. Patients completed quality-of-life assessments  at baseline, monthly during the first year, then 18 and 24 months after randomisation. Presence of several comorbidities was defined as CCI of 3 or more.Clinicians did physical examinations, including assessment of performance status, development of rash, and adverse events , and a chest radiograph every month for the first 12 months, and every 2 months thereafter. We graded rash with the criteria: erythema alone (A), erythema with papules (B), erythema with papules and pustules (C), and erythema with papules and confluent pustules (D). CT scans were done at 3 and 6 months and when clinically indicated—eg, after abnormal chest radiographs.EGFR mutations and KRAS mutations.For translational research, we collected blood samples and diagnostic biopsy material (paraffin blocks) before starting treatment. Purification and assessment of quality and quantity of tumour DNA were done with Wizard genomic DNA purification kit . We used the Sequenom OncoCarta Panel v1.0 service  to do a sensitive  analysis of 238 known cancer mutations in 19 genes, including activating vs supportive careThe target sample size was 664 patients, on the basis of the primary study objective to detect an increase in 1 year overall survival from 10% with placebo to 17·5% with erlotinib  by intention to treat, with Kaplan-Meier curves and Cox proportional hazards regression (hazard ratios [HRs] estimated with maximum likelihood methods), unless otherwise specified . We used the maximum grade for each type of adverse event for each patient. We analysed quality of life with mixed effects models for repeated measures with baseline values as a covariate. We did model adequacy checks for continuous variables, including those for departures from normality assumptions, with residual plots and normal probability plots. We calculated compliance to study treatment by dividing the total number of tablets taken  by the number of days from randomisation to death, progression, or when treatment was stopped early, and expressed results as a percentage. Preplanned subgroup analyses included sex, histological examination, activating This study is registered, number ISRCTN 77383050.Cancer Research UK (C1438/A4147), with an educational grant from Roche for the translational studies, but neither were involved in trial design, data analyses or interpretation, or writing of this report. Additional support came from the University College London and University College London Hospital Biomedical Research Centre, who had no role in study design, data collection, analysis, or interpretation, or writing of the report. The trial sponsor was University College London, who were involved trial design, data collection, analyses, and interpretation, and writing of the report. SML, IK, and AH had access to the full raw data. The corresponding author had the final responsibility for the decision to submit for publication.The trial was funded by We recruited 670 patients from 78 centres from the UK National Cancer Research Network between April 14, 2005, and April 1, 2009. Our randomisation procedure gave 936 cells and by chance the first allocation in each cell for several centres was erlotinib, producing an imbalance in the number randomised to each group; 350 participants were assigned to receive erlotinib and 320 placebo . We foll16 patients assigned erlotinib and seven patients assigned placebo did not start study treatment, because patients had died or progressed beforehand. Compliance to study treatment , was 58% (204 of 350) for erlotinib and 63% (203 of 320) for placebo  of deaths attributed to progressive disease. We identified a significant improvement in progression-free survival with erlotinib ; median progression-free survival was 2·8 months (95% CI 2·6–3·0) with erlotinib versus 2·6 months (2·4–2·9) with placebo . Tumour vs 15% [46 of 313] p<0·0001). 24% (79 of 334) of patients assigned erlotinib had a high-grade (C or D) rash versus 2% (five of 313) of those assigned to placebo (p<0·0001). Significantly more patients assigned erlotinib had diarrhoea of grade 3–4 than did those assigned placebo  with erlotinib and 70% (219 of 313) with placebo p=0·12, . More pa placebo . 21%  and for six of 18 symptoms ; In prespecified subgroup analyses, first-cycle rash among patients assigned erlotinib was the only significant independent factor , overall survival was significantly longer , as was progression-free survival . Hazard ratios for patients assigned erlotinib who did not develop rash, compared with placebo, were 1·30  for overall survival, and 1·09  for progression-free survival, neither of which overlapped the corresponding intervals for patients who were treated with erlotinib and developed rash, indicating that they were significantly different . Median overall survival was 6·2 months (95% CI 4·8–7·2) for the erlotinib and rash group, 2·9 months (2·3–3·7) for the erlotinib without rash group, and 4·1 months (3·7–4·6) for placebo. 1 year overall survival was 24% (95% CI 17–29) for erlotinib and rash, 10% (5–16) for erlotinib without rash, and 18% (12–21) for placebo. For erlotinib and rash compared with placebo, the number needed to treat to save one life at 6 months was seven and at 12 months was 17. Median progression-free survival was 3·4 months (95% CI 3·1–4·0) for erlotinib and rash, 2·5 months (2·2–2·8) for erlotinib without rash, and 2·9 months (2·7–3·2) for placebo.We identified overall survival benefits in patients who developed first-cycle rash in all subgroups including those with the worst characteristics: ECOG performance status 3, overall survival HR 0·58  and progression-free survival HR 0·41 ; stage IV, overall survival HR 0·66  and progression-free survival HR 0·56 ; and age 75 years or older, overall survival HR 0·77  and progression-free survival HR 0·71 . Kaplan-Meier curves for overall survival were much the same for patients assigned placebo who did or did not develop rash .Patients assigned erlotinib who developed first-cycle rash had a higher occurrence of fatigue and diarrhoea than did those assigned erlotinib who did not develop rash . The proEGFR mutation in the study population was only 7% (28 of 390), and 19% (73 of 390) for KRAS. Of the 28 EGFR mutation-positive samples, 11 had exon 19 deletions, ten had a mutation at exon 21 (858Leu→Arg), and seven had other mutations. In these patients, median overall survival was 10·4 months (95% CI 5·5–15·1) for erlotinib (n=17) versus 3·7 months (0·3–49·3) for placebo (n=11). Median progression-free survival was 4·8 months (1·6–8·8) for erlotinib and 2·9 months (0·3–10·1) for placebo. All patients with an EGFR mutation who were assigned erlotinib developed rash. Among patients with wild-type EGFR who were assigned erlotinib and developed rash (n=94), HR for overall survival was 0·86  and for progression-free survival was 0·69  who provided material. Occurrence of the activating KRAS mutation-positivity was not associated with overall survival or progression-free survival. Among those who were KRAS mutation-positive, median overall survival was 4·2 months (95% CI 1·8–6·2) for erlotinib (n=35) versus 3·6 months (1·9–4·4) for placebo (n=38); median progression-free survival was 3·5 months (1·7–4·8) versus 2·7 months (1·8–3·9). Patients with wild-type KRAS had median overall survival of 3·7 months (2·8–4·2) for erlotinib (n=210) versus 3·4 months (2·7–4·3) for placebo (n=180); median progression-free survival was 2·7 months (2·2–2–9) for erlotinib and 2·6 months (2·3–2·9) for placebo. The presence or absence of first-cycle rash did not affect the results (data not shown).First-line treatment with erlotinib did not improve overall survival in all unselected patients with advanced NSCLC deemed unsuitable for chemotherapy treatment, who usually have a poor prognosis  and SATURNEGFR (though BR.21 included some patients with performance status 2–3). We planned TOPICAL in 2002, and did not mandate compulsory tissue collection. In the IPASS study,EGFR mutation data available in 36% (437) of the 1217 patients randomised. This proportion was much the same as in our trial where samples from 58% of patients had sufficient DNA for analysis.A limitation of our study was that the incidence of Another limitation of our study is that we did routine CT scans at 3 and 6 months, whereas some other studies have used CT scans every 6–8 weeks, so our schedule might be deemed suboptimum for assessment of progression-free survival. However, the monthly chest radiographs would trigger a CT scan before the protocol timelines, and overall survival was the primary trial endpoint. Also, we included patients of performance status 2, for whom, on the basis of evidence reported in 2005,When TOPICAL was designed some evidence already suggested that development of a rash during erlotinib therapy could be associated with improved outcomes across several cancers including NSCLC.Drug uptake and cytotoxic T-cell infiltration could be linked, with increased uptake of drug causing increased EGFR blockade and cell killing, resulting in improved host immune response. Future studies could examine whether combining erlotinib and immunomodulatory agents can further fuel a more robust immunological response, increase the severity and incidence of skin rash, and potentially further improve durability of response, progression-free survival, and overall survival in this group of patients who are deemed not suitable for chemotherapy.EGFR tumours, and this notion is supported by the lower than expected proportion who developed rash. Another phase 2 trialA phase 2 studyEGFR mutation tumours should receive erlotinib or gefitinib, in line with the established evidence in patients with good performance status, and supported by the finding that the small number of these patients in TOPICAL all developed rash.14Diarrhoea, hair loss, and fatigue were expected adverse events associated with erlotinib, and their severity was usually mild to moderate. Overall, occurrence and severity of adverse events in TOPICAL were much the same as those in the SATURN and BR.21 trials despite our population being predominantly elderly patients with poor performance status (ECOG 2–3).EGFR wild-type or unknown EGFR status tumours could start erlotinib but they should discontinue if they do not develop rash within 28 days, because these patients had no benefit in overall survival or progression-free survival, and in some subgroups overall survival could be reduced if erlotinib were taken continuously , because at the time there were no other studies of first-line EGFR inhibitors in patients with advanced non-small-cell lung cancer (NSCLC) who were elderly or had poor performance status. These patients are often excluded from first-line chemotherapy trials.Lung cancer is predominantly a disease of elderly people, and elderly patients with advanced NSCLC deemed unsuitable for chemotherapy with poor performance status (2 and 3) or several comorbidities, or both, remain a major challenge. Since TOPICAL, no phase 3 trials of first-line monotherapy for this group have been reported.InterpretationEGFR mutations. However, unlike the TOPICAL patients, most of the patients in other studies had good performance status.The findings of TOPICAL and other trialsEGFR mutation-positive tumours could receive continuous erlotinib; whereas those who have wild-type tumours should discontinue erlotinib after about 28 days if they do not develop a rash because of the possibility of decreased survival.The clinical implications of the TOPICAL trial are that patients who are deemed unsuitable for chemotherapy could be given erlotinib. Those who have Our data suggest patients with inuously . The reaEGFR testing has now become standard of care to select patients who are EGFR mutation-positive for treatment with tyrosine-kinase inhibitors. If erlotinib is to become a standard therapy for patients who have EGFR wild-type tumours and are unsuitable for chemotherapy  it should be in a selected population. Prospective studies are needed to increase our understanding of the biological mechanism linking rash and erlotinib benefit, including dose-escalation studies or studies of the relation between rash and tumour EGFR copy number.


Dicamptodon tenebrosus) in two core regions  versus the species' northern peripheral region  where the species is listed as threatened. Coalescent-based demographic simulations were consistent with a pattern of post-glacial range expansion, with both ancestral and current estimates of effective population size being much larger within the core region relative to the periphery. However, contrary to predictions of recent human-induced population decline in the less genetically diverse peripheral region, there was no genetic signature of population size change. Effects of current demographic processes on genetic structure were evident using a resistance-based landscape genetics approach. Among core populations, genetic structure was best explained by length of the growing season and isolation by resistance (i.e. a ‘flat’ landscape), but at the periphery, topography (slope and elevation) had the greatest influence on genetic structure. Although reduced genetic variation at the range periphery of D. tenebrosus appears to be largely the result of biogeographical history rather than recent impacts, our analyses suggest that inherent landscape features act to alter dispersal pathways uniquely in different parts of the species' geographic range, with implications for habitat management.With predicted decreases in genetic diversity and greater genetic differentiation at range peripheries relative to their cores, it can be difficult to distinguish between the roles of current disturbance versus historic processes in shaping contemporary genetic patterns. To address this problem, we test for differences in historic demography and landscape genetic structure of coastal giant salamanders ( Processes structuring genetic diversity across species' ranges are complex, particularly as populations can vary in connectivity across heterogeneous or fragmented landscapes, or be influenced by geographically variable biogeographical histories that shape current genetic variation eN) and population connectivity at the periphery Peripheral populations generally occur in marginal habitats or areas that are climatically unfavourable, which may limit further expansion and result in genetically isolated populations that have an increased risk of local extinction Hence, disentangling the effects of anthropogenic disturbance versus historical biogeographical processes across species' ranges will help to avoid bias in conservation strategies based on a single study area Dicamptodon tenebrosus) as our focal species, we test whether historical or recent factors have affected population genetic structure at the species' northern periphery versus two core localities using a combination of coalescent modelling and a landscape genetics approach.Amphibians are highly suited to the study of landscape genetic patterns due to their limited dispersal capacity and sensitivity to fine-scale landscape structure D. tenebrosus, which was formed by post-Pleistocene range expansion from the Columbia River Valley in Washington State, USA, up to the northern range limit, which is delineated by the Fraser River in British Columbia eN and genetic variation at range margins D. tenebrosus over the last 100 years are a likely driver of fragmentation effects Our sample regions are located within the northern clade of D. tenebrosus is listed as nationally ‘Threatened’  in Canada and is on the Provincial ‘Red List’ in British Columbia, primarily due to impacts of forest harvest and urban encroachment Although not currently a species of concern in the United States, eN in core and peripheral regions. Current influences on genetic structure are examined using a multiple pathway approach based on circuit theory that identifies how gene flow is limited by landscape resistance in terms of topographical, climatic and land cover features Using a coalescent demographic simulation D. tenebrosusNe, and if so, (2) historical range-expansion processes will have shaped current genetic patterns when the peripheral region shows a stable historic-to-recent population size compared with the core region, which is predicted to show an expansion signature  and the Washington State University Institutional Animal Care and Use Committee.D. tenebrosus) occurs in small streams from sea level to 1830 m elevation in the Pacific Northwest coniferous forests of the United States and south-western Canada 2) in the Chilliwack River watershed of British Columbia. Dicamptodon tenebrosus is highly sedentary The coastal giant salamander : Willapa Hills (WH), (area sampled ∼50 km2), South Cascades (SC) (area sampled ∼40 km2)  (2) was taken from each individual and preserved in 95% ethanol for DNA extraction as described in Steele et al. ∼40 km2) . These s Canada) . For WH  Canada) . A samplDNA extractions were performed using a standard phenol-chloroform ethanol precipitation protocol Individuals with missing genetic data at three or more loci were excluded from the dataset. Individuals from each stream were screened for genetic relatedness in the program Colony 2.0 ST between regions and sites was calculated in Microsatellite Analyser 4.05 (MSA) K = 1–6 were performed with a 50 000 burn in and 1000000 Markov Chain Monte Carlo (MCMC) iterations, under a model of admixture and correlated allele frequencies. The number of genetic clusters (K) was determined using the method of Evanno et al. K method Genetic differentiation between the three study regions  was examined using six loci that were in HW equilibrium across all regions, which excluded D05, D17 and D25 (not in HW equilibrium for WH). Allelic richness within regions was calculated in FSTAT 2.9 We examined whether there were historic or recent changes in effective population sizes and the timing of these changes in core versus peripheral regions, using MSVAR 1.3 0N), ancestral population size at the time of demographic change (1N), and the time in generations since population size change T = aT/0N (aT = number of generations since the beginning of the expansion/decline) D. tenebrosus, yet we used a conservative generation time estimate of 12.5 years for simulations of ‘time since population change’, based on a predicted maximum life-span of 20 yrs r = 0N/1N) to determine population size changes where r = 1 indicates stability, r>1 indicates expansion, and r<1 indicates decline in the effective population size 8 updates and a thinning interval of 10 000 so that 20 000 estimated parameter sets were derived from the posterior distribution The parameters of interest for the current study were: current effective population size  while in CV convergence was supported for 0N (1.23) and 1N (1.10), but less so for T (1.87). The last half of each chain was used to make a combined consensus chain of 50 000 data points, and summary statistics of the marginal posterior distributions for N0, N1 and T were estimated as the mean, 0.025 and 0.975 quantiles.We processed the output from MSVAR 1.3 using the program BOA 1.1.4 for R version 2.3.1 Girod et al. ST) among streams within regions was calculated with Bonferroni correction using MSA. We compared the performance of two different measures of genetic distance between sample sites: G′ST and Dps . G′ST is a standardized measure that is appropriate for examining genetic differentiation between datasets with different numbers of loci, and among loci with different levels of variation ST to be <1 ps between sample sites  because this measure avoids equilibrium assumptions of G′ST and is sensitive to genetic differences while controlling for low variation in allele frequencies among populations Genetic differentiation  and each region was clipped with a minimum buffer of 500 m surrounding all sample sites to minimise ‘edge effects’ associated with calculating resistance values (as suggested by ArcGIS software version 9.3.1 (ESRI) was used to parameterise nine landscape variables, which pertain to topographic characteristics , habitat permeability , or temperature and precipitation  (described in http://www.geobase.ca/geobase/en/index.html (Canadian Council on Geomatics). For WH and SC, land cover and canopy cover data were from the 2001 National Land Cover dataset, stream data were from the National Hydrography Dataset (USGS 1999) and elevation, slope and heat load were derived from USGS digital elevation models. Frost-free period and growing season precipitation for both CV, WH and SC regions were estimated based on a spline model by Rehfeldt Cell values for each landscape variable were converted directly into resistances based on expected linear predictions of suitability ST and Dps). Akaike's Information Criterion (AIC) was applied separately to each region to find the best landscape model for explaining genetic distance between sites To test the relative effects of landscape variables on genetic distance we used multiple matrix regressions using the R statistics package (2.11.0). This analysis included resistance matrices of all landscape variables with genetic distance as the dependent variable in both regions ; therefore we chose to use a model averaging approach. Model-averaged estimators often have a better measure of precision and reduced bias compared to estimators from just the selected best model After the exclusion of full-sibs , and individuals with >1/3 missing data , final sample sizes (n) were: CV, n = 387 (from 20 streams); WH, n = 213 (from 6 streams), and SC, n = 379 (from 13 streams) . The numST: WH vs. SC = 0.04; SC vs. CV = 0.09; WH vs. CV = 0.16; P<0.02 all comparisons)  , S6. AMOST = 0.04. Between SC and CV FST = 0.09, and for WH and CV FST = 0.16. All comparisons were significant after Bonferroni correction. Pairwise site FST comparisons were statistically different (P<0.05) after Bonferroni correction in 32.6% of comparisons for CV (ST (mean ±s.e.) between sites were moderate to low within regions . The peripheral region (CV) had lower allelic diversity in 7/9 loci  compared to the core regions  (Mean pairwise Euclidean distances (km ±s.e.) between sites for WH was 22.35 (±3.92), for SC was 9.5 (±0.75) and for CV was 17.6 (±0.8). Between core regions (WH+SC) pairwise Fs for CV , 37.5% fs for CV . Values r locus) . Allelicr = 0.948), so T (time since change) could not be inferred , which was ∼33% lower than in SC, 0N = 4286 . A slight historic population decline was detected in in SC (r = 0.802), estimated at approximately 849000 years ago  although two sites in SC (15.4%) showed evidence of a mode shift in allele frequencies . At the 2 = 0.00). Furthermore, all variables were highly correlated in WH . Variables with relative importance (RI) scores ≥0.60 were land cover   (2 = 0.20), followed closely by IBR+frost-free period  . A two-v ratios) . Only laed 0.99, , suggestps , but RI values ≥0.60 differed from SC, and included elevation, heat load index, and canopy cover   and RI values were ≥0.60 for slope, heat load index (0.71) and stream vs. other (1∶10 and 1∶100 ratios)  , and the = 0.13) . Two-var ratios) . RI was ad index . All othad index . Howeverad index .ps and G′ST. However the high correlation between these variables means that the additional effect of frost-free period over simple isolation by resistance should be interpreted cautiously. However in CV, the topographical variables elevation (Dps) and slope (G′ST) clearly performed better than IBR alone, with the effect of solar radiation (i.e heat load index) being another possible factor influencing landscape genetic structure in the peripheral region.To summarise the main landscape genetic findings, our results for SC consistently showed an effect of IBR+FFP for DD. tenebrosus. Historic range expansion effects appear to dominate current levels of genetic variation in both regions, with reduced Ne and genetic diversity at the periphery. Despite this, we reveal categorical differences in landscape effects on contemporary gene flow according to core or peripheral location, with stronger evidence for landscape-driven genetic structure at the periphery, in accordance with our hypotheses. Our results suggest that range-wide species conservation, may be better informed by concurrent analyses of historic demography and contemporary landscape genetic patterns that encompass more than one study area.By assessing only contemporary landscape genetic patterns, there is a risk of incorrectly attributing genetic patterns to recent landscape changes that are actually the result of historic biogeographical processes D. tenebrosus had a much smaller founding population than the core, which is in accordance with the previously documented northward range expansion D. tenebrosus conducted in the region eN, and the current stable population signature at the periphery. Smaller populations may be more prone to extinction and have reduced adaptive potential, which can inhibit or slow range expansion into new environments, resulting in a stable population signature In accordance with the ‘central-marginal’ hypothesis eN, potentially due to a loss of genetic diversity during range expansion D. tenebrosus within the early or mid-Pleistocene (1.7 mya ∼800000 ya) eN to be 31563 in the Columbia River Valley refugium. In the core, current landscape effects on genetic structure were evident within SC, but there were no effects within WH. The strong correlations between landscape variables in WH , indicating that census size does not equate to Ne/genetic diversity in these more productive populations.Notably, Dudaniec and Richardson Ascaphus truei) Anthropogenic effects on peripheral landscape genetic structure were not detected explicitly (e.g. via the variables canopy cover or land cover), though solar radiation, which is postively related to forest harvest, was among the top models. However, Dudaniec and Richardson D. tenebrosusD. tenebrosusEcologically dissimilar habitats within a species' range can select for variation in adaptive traits that are likely to reflect landscape resistance to dispersal and genetic patterns D. tenebrosus, it can be expected that metapopulation processes may drive reductions in genetic variation and increased differentiation between streams within regions, while genetic variation at the regional level does not likely decline at the same rate.The finding of greater genetic variation explained within streams than across regions (i.e. via AMOVA) may indicate non-equilibrium processes acting at different temporal and spatial scales, which can cause lower genetic differentiation between regions than that observed at lower hierarchical levels within regions (i.e. the stream level) From a spatial perspective, populations at ecological range limits may have a ‘patchier’ distribution, increasing pairwise genetic distances and landscape resistance. We acknowledge that the study area, and hence pairwise site distances were larger for CV than for SC and WH. However, previous studies suggest that a larger sample size and sampling effort is required in peripheral populations to capture the same proportion of genetic variation as in core populations due to stronger within-population spatial genetic structure D. tenebrosus, but patterns are not consistent across regions with respect to the underlying differences in core and peripheral landscape characteristics. Although additional replicates of core and peripheral regions may help to resolve these disparate regional patterns, this lack of consistency suggests that historical demographic processes strongly influence our observed landscape genetic patterns. Although geologically recent colonisation has evidently shaped the lower genetic diversity at the periphery, these historical effects may act to exacerbate population sensitivity to habitat fragmentation resulting from forest harvest. Therefore, interactions between regional topography and anthropogenic disturbances should be considered for the conservation of threatened D. tenebrosus populations, and potentially other co-occurring, stream-associated amphibians. Our study demonstrates that combining both coalescent and landscape genetic analyses can help to disentangle current from historical processes that influence contemporary patterns of spatial genetic variability.Our results suggest that historic demography influences location-specific landscape genetic processes in core and peripheral populations of Table S1Summary of collection sites (river drainage or label), site codes, final sample sizes (N) and geographical coordinates for British Columbia (BC) and Washington State (WA). Results from BOTTLENECK analyses are presented: ** indicates sites with significant heterozygote excess and a significant mode shift in allele frequency distribution; *indicates sites with just a significant mode shift in allele frequency distribution; † indicates sites with a significant heterozygote deficiency.(DOC)Click here for additional data file.Table S2Genetic diversity indices for each locus across all populations  and the number of populations (percentage in parentheses) not in Hardy-Weinberg equilibrium (HW) after correction for multiple comparisons (significance at the 0.05 level). Total sample sizes for each region are: Chilliwack Valley, n = 387, Willapa Hills, n = 213, South Cascades, n = 379. * locus excluded from analysis.(DOCX)Click here for additional data file.Table S3Parameter hyperpriors used for each simulation ran in MSVAR 1.3, for South Cascades and Chilliwack Valley datasets: ancestral population size (log N0), current population size (log N1), mutation rate (log u), and time since decline/expansion (log T).(DOCX)Click here for additional data file.Table S40), current population size (log N1), mutation rate (log u), and time since decline/expansion (log T). Starting values are the trial values for updating the parameters in the Metropolis-Hastings algorithm used in MSVAR. Locus D05 was excluded from the analysis for regional comparisons.Starting values for parameters for each locus used in all MSVAR simulations: ancestral population size (log N(DOCX)Click here for additional data file.Table S5D. tenebrosus between sampled streams in at the peripheral range in British Columbia, Canada: Chilliwack Valley (n = 387). Bold values were significantly different after Bonferroni correction.Pairwise Fst of (DOCX)Click here for additional data file.Table S6D. tenebrosus between sampled streams in Washington State, USA a) Willapa Hills, b) South Cascades. Bold values were significantly different after Bonferroni correction.Pairwise Fst of (DOCX)Click here for additional data file.Table S7Correlation matrix (Pearson's r) of landscape variables for the Willapa Hills (WH) core region. STR10 = stream vs. terrestrial 1∶10, STR100 = stream vs. terrestrial 1∶100, LC10 = landcover 1∶10, CAN = canopy cover, FFP = frost free period, GSP = growing season precipitation, HLI = heat load index, IBR = isolation by resistance (flat), LC100 = landcover 1∶100, SLP = slope, ELEV = elevation.(DOCX)Click here for additional data file.Table S8Correlation matrix (Pearson's r) of landscape variables for the South Cascades (SC) core region. STR10 = stream vs. terrestrial 1∶10, STR100 = stream vs. terrestrial 1∶100, LC10 = landcover 1∶10, CAN = canopy cover, FFP = frost free period, GSP = growing season precipitation, HLI = heat load index, IBR = isolation by resistance (flat), LC100 = landcover 1∶100, SLP = slope, ELEV = elevation.(DOCX)Click here for additional data file.Table S9Correlation matrix (Pearson's r) of landscape variables for the Chilliwack Valley (CV) peripheral region. STR10 = stream vs. terrestrial 1∶10, STR100 = stream vs. terrestrial 1∶100, LC10 = landcover 1∶10, CAN = canopy cover, FFP = frost free period, GSP = growing season precipitation, HLI = heat load index, IBR = isolation by resistance (flat), LC100 = landcover 1∶100, SLP = slope, ELEV = elevation.(DOCX)Click here for additional data file.


Pseudohypoparathyroidism (PHP) refers to end−organ resistance that primarily impairs the renal actions of parathyroid hormone (PTH). The patients with PHP type Ia (PHP−Ia), one of the 4 types of PHP, show resistance to other peptide hormones as well as clinical features of Albright hereditary osteodystrophy (AHO), a constellation of short stature, obesity, brachydactyly, ectopic ossifications, and/or mental retardation. Here we report clinical follow−up for a long−term period in a PHP−Ia case who had a missense mutation leading to the substitution of proline by serine (Prol115Ser) in exon 5 which has been reported previously in only two patients. An 11−year−old boy applied for hand spasm to our hospital. On physical examination, he had short stature, round−shaped face and brachydactly. Laboratory evaluation revealed PTH and TSH resistance. Molecular genetic analysis of the GNAS gene revealed a P115S substitution. The patient was followed up for 13 years. Normocalcaemia was achieved with reduced doses of calcitriol (0.25 μg/day) and calcium supplements (40 mg/kg/day). Daily requirement for levothyroxine supplementation was still high (2.3 μg/kg) to achieve euthyroidism. His pubertal development was Tanner stage V and he has no gonadotropin resistance. To our knowledge, this is the first report concerning long−term follow−up of this rare mutation. We believe that despite the genetic heterogeneity of AHO, phenotype/genotype correlations of this kind of rare mutations may help to understand progress of the disease.Conflict of interest:None declared. Pseudohypoparathyroidism (PHP), also known as Albright’s hereditary osteodystrophy (AHO), refers to a heterogeneous group of rare metabolic disorders characterized by hypocalcemia and hyperphosphatemia due to parathyroid hormone (PTH) resistance. PHP is associated with a constellation of physical features which include short stature, obesity, round face, brachydactyly, and subcutaneous ossifications. PHP type−I (PHP−I), a term used to describe a condition in patients who show a blunted nephrogenous cAMP response to exogenously administered bioactive PTH, can be further classified into three different subtypes based on the presence of additional endocrine abnormalities, deficiency of the α−subunit of the stimulatory G protein (Gsα), and dysmorphic features of AHO . In addiAlthough several studies investigated the phenotypic heterogeneity of PHP, minimal information exists on the temporal progression of the hormone resistance , 3, 4. Hth percentile) and his body mass index was 18.7 (25−50th percentile). Short stature , round−shaped face (rd percentile) and his palm length was 8 cm (<3rd percentile)   . There wfingers) . Based oLaboratory tests revealed reduced serum calcium , elevated serum phosphorus  and markedly elevated serum parathormone  levels. Serum TSH level was high . Serum free thyroxine (fT4) and free triiodothyronine (fT3) levels were 12 pmol/l  and 4.7 pmol/l , respectively, indicating subclinical hypothyroidism. Anti−thyroid antibodies were absent and thyroid ultrasound findings were normal. Growth hormone (GH) response to insulin tolerance test was normal (14.5 ng/mL). Renal ultrasound findings revealed no nephrocalcinosis. Cranial computerized tomography (CT) scan demonstrated no basal ganglion calcification. These results, including resistance to TSH and PTH, were consistent with a diagnosis of AHO (PHP type−Ia). Genomic DNA was extracted with phenol−chloroform method from peripheral blood leukocytes. Sequence analysis of the GNAS gene identified a missense mutation leading to the substitution of proline by serine (Pro115Ser) in exon 5. The family pedigree is shown on 2D3) in a dose of 1 μg/day and 40 mg/kg/day elementary calcium together with a low−phosphorus diet were started. This treatment regimen resulted in normalization of calcaemia and phosphataemia. The patient also received L−T4 treatment in a dose of 1.6 μg/kg/day.For treatment, vitamin D During the 13−years of follow−up, the patient showed no obesity or cutaneous calcifications. At the age of 24, his height was 146 cm (−4.37 SD), and his weight was 52.4 kg. Body mass index was 24.6. His daily requirement for L−T4 supplementation was still high (2.3 μg/kg). Normal calcaemia was achieved with reduced doses of calcitriol (0.25 μg/day) and calcium supplements (40 mg/kg/day). His pubertal development was at Tanner stage V and he had no gonadotropin resistance .The human Gsα gene (GNAS1) contains 13 exons encoding the Gsα and is located at 20q13.11. Heterozygous loss of function mutations in GNAS1 have been identified in the majority patients with PHP−Ia  and de SLinglart et al  investigThe prevalence of short adult height in PHP−Ia is reported to be as high as 80% . Some ofTo our knowledge, this is the first report of a long−term follow−up in a patient with this rare mutation. We believe that despite the genetic heterogeneity of AHO, investigation of phenotype/genotype correlations in this kind of rare mutations may help to understand the progress of the disease.


Yet left-to-right orientation has been shown to extend to other dimensions, consistent with a general magnitude system wherein different magnitudes share neural and conceptual resources. Such observations raise a fundamental, yet relatively unexplored, question about spatial-numerical associations: What is the nature of the information represented along the mental number line? Here we show that this information is not exclusive to number, simultaneously accommodating numerical and non-numerical magnitudes. Participants completed the classic SNARC  task while sometimes wearing wrist weights. Weighting the left wrist–thereby linking less and more weight to right and left, respectively–worked against left-to-right orientation of number, leaving no behavioral trace of the mental number line. Our findings point to the dynamic integration of magnitude dimensions, with spatial organization instantiating representational currency  shared across magnitudes.The  Of particular interest was whether weight would nevertheless influence the mapping of number to space. The Baseline condition was identical to Dehaene et al.’s We investigated these possibilities by having people make judgments about numbers while exposed to irrelevant information about another magnitude, namely weight The Left condition provided the critical test of numerical exclusivity. With the 5-lb. weight attached to the left wrist and an empty casing around the right, less and more weight were linked to the right and left sides of space, respectively–conflicting with left-to-right orientation of number, and thus allowing us to examine whether the mental number line simultaneously accommodates numerical and non-numerical magnitudes. If the mental number line is fully number-specific and independent of other magnitudes, the conflicting weight-to-space mapping introduced in the Left condition should have no impact on the spatial organization of number. However, if the mental number line instead shows influence from non-numerical magnitudes, weighting the left wrist (but not the right) should work against left-to-right orientation of number, perhaps leaving no behavioral trace of this preexisting mental mapping.Study procedures were approved by the Institutional Review Board (IRB) at Emory University, Atlanta, GA. All participants provided written informed consent.M: 71.4, range: −42.9–100) Twenty-four undergraduates, mostly right-handed as assessed by the Edinburgh Handedness Inventory  and odd responses to the right (“P”), and the other with the reverse assignment . There were 10 practice and 60 test trials in each block, for a total of 120 test trials in each condition. On each trial, a 500-ms fixation cross was presented centrally, followed by an Arabic numeral , which remained onscreen until participants responded . Instructions emphasized speed and accuracy.SD = 105), 582 ms (SD = 87), and 583 ms (SD = 97) in Left, Right, and Baseline conditions, respectively, with no significant differences across conditions, F = .24, p>.7. For each participant, mean RTs were computed for left- and right-side responses separately by number pair  and reaction times (RTs) greater than 2.5 standard deviations (SDs) from individual means (2.8%). Mean RT on remaining trials was 590 ms  = 6.05, p = .005, but no main effect of order or interaction between condition and order (ps>.3). Follow-up analyses revealed that slopes in the Baseline  and Right  conditions differed significantly from zero (Baseline: t(23) = 3.66; Right: t(23) = 3.81; both ps<.002), as predicted, but not from each other (p>.3), indicating left-to-right orientation of number of comparable strength in the two conditions , consistent with previous studies M = 0.25 ms/digit, SD = 12.03) did not differ from zero, t(23) = .10, p>.9, indicating no consistent spatial organization . Critically, slope in the Left condition differed significantly from slopes in the other two conditions (Left vs. Baseline: t(23) = 2.84, p = .009; Left vs. Right: t(23) = 2.71, p = .01), confirming reliably weaker left-to-right orientation of number when the left wrist was weighted.dRT values were regressed on number pairs, producing unstandardized slope coefficients for each condition. A 3 ×6 (order of conditions) analysis of variance (ANOVA) on slopes yielded a main effect of condition, ions see . In bothtion see . ParticiM = 1.06, SD = 78.35) did not differ from zero, t(23) = .07, p>.9, indicating that left responses  were no slower overall than right responses  in this condition. In contrast, intercepts in the Baseline  and Right  conditions were significantly greater than zero (Baseline: t(23) = 3.06; Right: t(23) = 2.26; both ps<.04), indicating faster left than right responses to smaller numbers in these conditions. We also conducted pairwise comparisons of mean RTs by side of response (left hand or right hand) across the three conditions , further suggesting that the differences in left-to-right orientation of number across conditions were not due to overall slower (or faster) manual responses, whether left- or right-side responding.Additional analyses verified that the flat slope in the Left condition was not merely the result of slower (or faster) left-side responses to all numbers when the left wrist was weighted. The regression intercept in the Left condition  with that of participants who were weakly right-handed .  For both the strongly right-handed and weakly right-handed groups, slopes in all three conditions mirrored those for the full sample. Specifically, slopes differed significantly from zero in the Baseline condition, whether participants were strongly right-handed , t(11) = 2.36, p = .04, or weakly right-handed , t(11) = 3.22, p = .01, and in the Right condition, whether participants were strongly right-handed , t(11) = 3.01, p = .01, or weakly right-handed , t(11) = 2.28, p = .04. In contrast, slopes did not differ significantly from zero in the Left condition for either group . Moreover, in all three conditions, slopes for the two groups did not differ significantly from each other (ps>.4), indicating no reliable differences based on handedness.Supplemental analyses revealed that the results did not vary as a function of handedness. We conducted a median split on EHI scores to compare the performance of participants who were strongly right-handed , even when such information is entirely task-irrelevant. An intriguing possibility is that this interaction between numerical and non-numerical magnitudes may occur at a relatively fine-grained level. For example, larger differences in the relative weight associated with the left and right sides of space  might be expected to have a stronger influence on the mental number line than smaller differences , though the latter should still weaken left-to-right orientation relative to baseline (provided that the weight differences are apprehended). Such a manipulation has the potential to provide considerable insight into how different types of magnitude information are mentally combined.Another important issue for future research is whether the mental number line fully integrates numerical and non-numerical magnitudes or, instead, retains some specificity to number. That is, our findings might reflect a single spatial representation of magnitude, with number and weight treated relatively interchangeably , or separate representations for each magnitude, with the weight-to-space mapping distinct from, yet exerting an influence on, the mental number line (see Although weight served as our key experimental manipulation, our findings could reflect the interaction of number with some other magnitude-related dimension, rather than with weight per se. Weighting the wrist may have introduced asymmetries in other non-numerical magnitudes, which in turn came to be associated with left and right sides of space. When the left wrist was weighted, for example, it is possible that participants perceived left-side actions as more effortful see . ImportaPrevious research has highlighted the processing consequences resulting from incongruity between different magnitudes (e.g., slower magnitude judgments when the numbers 3 and 5 are large and small in physical size, respectively, than vice versa