First, just

in the river, the Arun, and later, having joi

First, just

in the river, the Arun, and later, having joined the local sea angling club, occasionally out into the Channel when I could persuade my old Mum to part with the hire fee or as a freebie when boat owners were persuaded to keep us kids off the street and out fishing during summer holidays. On one of the latter trips, there was the skipper, his pimply callow mate and four of us boys all around twelve. Getting the rods organised on the way out Torin 1 supplier of harbour, we noticed that the mate was gearing up with a steel trace and huge hook. We asked what he was fishing for and, smirkingly, he said ‘congers’ that were known to live in an old sewerage pipe between our home port of Littlehampton and Worthing. On arrival, baited hooks were lowered, the mate trying to tempt a conger out of its pipe with a whole mackerel. After about an hour’s fishing, the mate’s rod suddenly bent so ferociously that I thought it was going to snap. But, he persevered and another half an hour later he had a gaffed conger at the surface, which he and the skipper now, possibly unwisely in hindsight, eventually managed to get into the boat. It was not so big as the Torquay

individual but, to my young eyes, it PD-0332991 mw was big enough. I would say well over a metre and a half in length, as thick as my waist and, most importantly, not yet dead. Suddenly, it awoke from its torpor and begin thrashing around the boat’s well, snapping at anything and everything. Us boys, me in my brother’s cut-down trousers and plimsolls, were up on the boat’s gunnel before you could say ‘knife’, hanging onto the bits of safety rigging any way we could and cautiously making our way onto the cabin’s roof where the spectacle in the well beneath us could be better enjoyed. The mate was trying to beat the conger

to death with the boat’s club but, not to be outdone, the conger bit him on the toe of his Wellington boot and refused to let go. We now had the spectacle of the wildly thrashing conger shaking the leg of the mate who, in turn, while hopping around on the other one, was still trying to club it senseless but was acutely aware of his own predicament. No more smirking Non-specific serine/threonine protein kinase either, I noted with quiet satisfaction. Eventually, the skipper grabbed hold of the club and was trying to also achieve the conger’s demise, but with a wide-eyed mate now even more frightened of the nightmarish consequences of his catch. It seems like an age later but, ultimately, by dint of discarding his boot to the conger, the beast was overcome and we boys descended to inspect a dead fish in a well of blood and gore and two badly shaken fishermen. It was thereupon decided that that was enough fun for one day’s angling and the boat turned for home. On the return trip, us boys returned to the cabin’s roof to gigglingly mull over the spectacle we had just experienced while the mate tried to clean up the blood-soaked deck.

, 1995) After incubation, cells were washed twice with FACS buff

, 1995). After incubation, cells were washed twice with FACS buffer and were either used for intracellular staining or fixed with a solution of 2% paraformaldehyde in PBS. Incubation with primary antibodies to MHC I (Salomonsen et al., 1987) and MHC II (Kaufman et al., 1990) was followed by Alexa-647 conjugated goat anti-mouse antibody (Life Technologies). Secondary antibody alone or unconjugated goat anti-mouse antibody (Life Technologies) was used as an unstained control for surface MHC staining. Intracellular staining high throughput screening was carried out as described previously (Ariaans et al., 2008). Briefly, splenocytes from challenged birds or non-infected controls were seeded in a 96-well round-bottom plates (Nunc)

at 106 cells/well in a final volume of 200 μl of culture media or culture media supplemented with the different stimuli at the concentration described in the ELISpot technique (except PMA which was used at 50 ng/ml). Cells

were cultured using the conditions described above for ELISpot assays (24 h culture). Volasertib price For intracellular staining, during the last 2 h of culture, cells were treated with Brefeldin A according to the manufacturer’s instructions (Cytofix/Cytoperm™ Plus Fixation/Permeabilization kit, BD Biosciences). To avoid non-specific binding signal, we preincubated cells with Cytofix/Cytoperm™ buffer containing 2% normal mouse serum and further staining steps involved Cytofix/Cytoperm™ washing buffer containing 1% normal mouse serum (Biosource). To confirm the specificity of the anti-IFNγ antibody EH9 we also employed a validated anti-IFNγ [mAb80 (Ariaans et al., 2008)]. Purified fractions of both antibodies were conjugated using Alexa Fluor® 647 monoclonal antibody labeling kit (Molecular Probes) according to the manufacturer’s 4��8C instructions. A mouse isotype matched control antibody IgG1 Alexa Fluor® 647 (Life Technologies) was employed at the same concentration as EH9 and Mab80. For analysis, a gate on the FSC/SSC region of lymphocytes was selected and a minimum of 10,000 events were acquired on a FACSCalibur instrument using Cell Quest software (BD Becton Dickinson). Flow cytometry

analysis indicates that non-adherent CKC were not present at significant levels (data not shown). FlowJo software (TreeStar) was used to analyze flow cytometry data. A paired or unpaired t-student test or one-way ANOVA was performed using GraphPad Prism (version 6.0 for Windows, GraphPad Software, San Diego, California, USA). Screening identified two anti-chicken IFNγ antibodies (clones EH9 and AF10) which were shown by ELISA to bind recombinant chicken IFNγ and to work effectively as an antibody pair in capture ELISA (Supplementary Fig. 2A–C). We subsequently compared this antibody pair with commercially available antibodies [from Life Technologies (Ariaans et al., 2008 and Reemers et al., 2012)] in ELISpot assays.

The MI value of AWW showed a decline from 44 7 to 37 8 to the ext

The MI value of AWW showed a decline from 44.7 to 37.8 to the extent of 15.4% whereas the presence of mannitol in the treatment tubes brought it upto 41.2 exhibiting a recovery of 8.25%. Chromosomal aberration based on its index showed its value for AWW treated A.cepa to be 11.2% compared to 2.6%

for the aquaguard water. The aberration index showed 52% reduction in the presence of mannitol. Interestingly, lead nitrate also exhibited the MI value, the chromosomal aberrations and aberration index very close to those of AWW. Water pollution has attracted a lot of interest in recent years due to its multidimensional hazardous effects. Disposal of the treated as well as untreated disposal of industrial waste material leads to serious problems for human health and survival. Oil refineries as well BMN 673 purchase as other industries generate huge amount of sludge containing both organic and inorganic toxicants which pollute the nearby sites. Many of the constituents in the wastes are carcinogenic and potent immunotoxicants [15]. To monitor the harmful effects of pollutants, a number of toxicity bioassays have been developed. Allium cepa test introduced by Levan [16] has been used frequently and validated by several workers for testing chemical pollutants posing hazardous

environmental effects ( [13], CHIR-99021 order [17], [18] and [19]). Root growth inhibition of Allium cepa was used as an indicator of toxicity of the refinery waste water and Aligarh waste water. The IC50 value in A. cepa system was recorded to be 0.14 X for RWW and 0.10 X for Aligarh waste water in the year 2008. Since water bodies represent a highly dynamic system, the degree of toxicity induced by industrial effluents can surely change over a period of time. Recent study in mafosfamide our lab [20] on the phytotoxic potential

of RWW suggested the IC50 value to be 0.75X. Thus, it can be concluded that from the year 2008 to 2011, there was a definite hike in the IC50 value from 0.14 X to 0.75X. This increase in the IC50 value from 2008 to 2012 signifies a reduced toxic potential of RWW, which might be the outcome of the installation of treatment plants in the refinery. These treatment plants must have detoxified or blocked the release of certain toxicants into water bodies. Toxicity of several other industrial waste samples have been determined in terms of IC50 values employing A. cepa system [21]. In addition to significant phytotoxicity of the RWW, present study also establishes its genotoxic potential in terms of significantly decreased survival of the DNA repair defective mutants of the E. coli K12 ( Figure 2, 3). The efficacy of the E. coli 12 repair defective mutants of E.coli K12 in assaying the genotoxicity of waste water has been well established (IGGE 1990; [22] and [23]).

In the early spermatids ( Fig 7A) the cytoplasm symmetrically en

In the early spermatids ( Fig. 7A) the cytoplasm symmetrically encircles the nucleus, which displays diffuse homogenous chromatin and has a circular outline. The centriolar complex lies laterally to the nucleus and is anchored to the plasma membrane. The proximal centriole is anterior and perpendicular to the distal centriole. The distal centriole differentiates into the basal body and forms the single flagellum. The nucleus rotates toward the centriolar complex ( Fig. 7B) with nuclear rotation of 90° considered complete. A depression is newly formed in the nuclear outline at the level of the centriolar complex that penetrates it ( Fig. 7C). Simultaneous to nuclear rotation, the cytoplasm projects in the direction

of the initial segment of the flagellum forming the cytoplasmic canal and midpiece

( Fig. 7A–C). The midpiece contains the mitochondria, forming vesicles and cytoplasmic canal housing the initial segment of the flagellum ( Fig. Afatinib 7B and C). In the spermatozoon of O. kneri the spherical nucleus (about 1.5 μm in diameter) contains highly condensed homogeneous chromatin interspersed by electron-lucent areas, and is surrounded by a narrow strip of cytoplasm with no organelles ( Fig. 7D buy KU-57788 and E). In the nuclear outline that faces the midpiece there is a medial and moderately deep depression, the nuclear fossa ( Fig. 7D–F). The proximal centriole, initially anterior and perpendicular to distal one, attains an oblique acute angle to the distal centriole. The centrioles are covered by electron dense material and are fastened to one another, to the

nuclear envelope at the nuclear fossa, Cediranib (AZD2171) and to the plasma membrane by stabilization fibrils. The proximal centriole and most of the distal centriole are inside the nuclear fossa ( Fig. 7F and G). The midpiece contains the mitochondria, abundant vesicles and the cytoplasmic canal in which lies the initial segment of the flagellum. The midpiece is slightly asymmetric due to the unequal distribution of mitochondria and vesicles. The mitochondria are elongated and mainly accumulated in the larger portion of the midpiece. Vesicles are elongated and mainly concentrated at the periphery and at the terminal regions of the midpiece ( Fig. 7H–K). The single flagellum contains a classic axoneme (9 + 2) ( Fig. 7L). Information on spermiogenesis in A. cataphractus is not available. In P. granulosus and R. dorbignyi, as in O. kneri, spermatogenesis is cystic and spermiogenesis is Type I. In the spermatozoa of A. cataphractus, P. granulosus and R. dorbignyi the nucleus contains highly condensed homogeneous chromatin and is surrounded by a narrow strip of cytoplasm with no organelles ( Fig. 8A, E, I). The nucleus is flattened at the tip and assumes an ovoid shape in P. granulosus (about 1.2 μm in height by 1.8 μm in width) vs. almost spherical in A. cataphractus (about 1.2 μm in height by 1.3 μm in width) and in R. dorbignyi (about 1.4 μm in height by 1.3 μm in width).

An average score of resistance of all lines to NCLB, SCLB, CLS, G

An average score of resistance of all lines to NCLB, SCLB, CLS, GLS, common rust, and southern rust was calculated, respectively, for each year.

For each disease the average score between the two years was Selleckchem Nutlin-3a insignificantly different (Table 1). A wide range of reactions to NCLB, SCLB, CLS, GLS, common rust, and southern rust was observed in the 152 inbred lines tested (Table 2). The proportions of lines that showed HR, R, or MR reactions to inoculation of different pathogens varied (Fig. 1). The percentage of lines resistant to NCLB was 53.3%, but all of them exhibited resistant or moderately resistant reactions and none was highly resistant. Most lines that were resistant to SCLB showed a moderately resistant reaction. Two lines, P138 and D Huang 212, were resistant with an IT of 3. None of the lines was highly resistant to SCLB (Fig. 1). The majority of lines (97.4%) were susceptible or highly susceptible to CLS. Only 4 lines, Shen 137, Qi 318, 77, and Nan 60-1, displayed a MR reaction. The percentage of lines that exhibited R or MR reactions to GLS was 14.4%. Approximately 85% of the Daporinad concentration lines were susceptible to GLS. Although the proportion

of lines resistant to common rust was 80.7%, only two lines (i.e., CS 339 and Ji 412) showed an HR reaction. Most lines (90.8%) were susceptible to southern rust. Lines C8605-2, Qi 319, Shen 136, Dan 3130, and Jinhuang 55 were highly resistant

to southern Bay 11-7085 rust. Lines OH 43, X178, Qi 318, Za C546, 8065, 81565, 313, CAL99, and B 151 were resistant or moderately resistant to southern rust. A small percentage of lines was resistant to several diseases simultaneously. Four lines Shen 137, Qi 318, Qi 319, and 313 were resistant to 5 diseases tested. Lines Shen 136, Zhongzi 01, Dan 9046, CN165, Chang 7-2, 8065, Nan 60-1, and C8605-2 were resistant to 4 diseases. Most of these multiple-disease-resistant lines were derived from the U.S. hybrids, except for Chang 7-2, which falls into the heterotic group SPT, based on pedigree information (Table 3) [27], [28], [29], [30], [31] and [32]. These lines had been used in developing commercial hybrids widely used in maize production. Approximately 60% of the lines tested were resistant to 2 or 3 diseases. The percentages of lines resistant to NCLB in different heterotic subgroups ranged from 41.4% to 63.2%. Over 50% of the lines in subgroups BSSS, LRC, PB, Lan, and SPT were resistant to NCLB (Fig. 2). Subgroup SPT consisted of 70% lines resistant to SCLB, which included Huangzaosi and Chang 7-2, the most important parental lines in many popular hybrids throughout the country.

While consideration should be given to the individual capabilitie

While consideration should be given to the individual capabilities of diagnostic laboratories, the testing Cabozantinib price of these additional samples may lead to an increase in the number of successful mutation results, enabling a greater number of patients to be accurately diagnosed, and receive the most effective and personalized therapy. This work was supported by AstraZeneca, UK. J.C.-H. Yang has received advisory fees from AstraZeneca, Roche, Genentech, Pfizer, and Clovis, and has been an uncompensated advisor to Boehringer Ingelheim and Eli Lilly. Y.-L. Wu and K. Nakagawa have received speaker fees from

AstraZeneca. G. McWalter and R. McCormack are employees of AstraZeneca and hold shares in AstraZeneca. T.S. Mok has received research funding from AstraZeneca and advisory fees from AstraZeneca, Roche, Eli Lilly, Boehringer Ingelheim, Merck Serono, and Pfizer. M. Fukuoka, N. Saijo, V. Chan, and J. Kurnianda have no conflicts of see more interest to disclose. The authors would like to thank the patients and investigators for their participation in the IPASS study. Sample analysis

was performed by Dr Guanshan Zhu, Dr Li Zheng, and Dr Peter Lu at Innovation Center China (China cohort) and Genzyme genetics (non-China samples). Statistical analysis was performed by Dr Rosie Taylor from AstraZeneca, UK. Editing support funded by AstraZeneca was provided by Sarah Lewis, from Complete Medical Communications. “
“Non-small cell lung cancer (NSCLC) is the most common

type of lung cancer, accounting for approximately 80% of lung cancers. NSCLC is attributed in part to somatic mutations of the epidermal growth factor receptor gene (EGFR) [1]. The most common mutations are an in-frame E746-A750 deletion in exon 19 and a single-point substitutional L858R mutation in exon 21, both of which are located in the tyrosine kinase domain of EGFR. These two mutations are observed in approximately 90% of EGFR mutations and are termed “activating mutations” [2]. EGFR-TKIs, such as gefitinib and erlotinib, block autophosphorylation of EGFR with subsequent inhibition of the downstream signaling Loperamide pathways involving RAS/extracellular signal regulated kinase (ERK)1/2 and phosphoinositide 3-kinase (PI3K)/AKT, and show favorable activity in NSCLC patients with activating mutations of EGFR [3]. However, almost all patients eventually develop acquired resistance to EGFR-TKIs within several years [4]. Two genetic mechanisms of acquired resistance to EGFR-TKIs have been identified in EGFR-mutated NSCLC. A secondary mutation of T790M in exon 20 of EGFR and amplification of the MET oncogene are observed in approximately 50% and 5% of resistant cases, respectively [5], [6], [7] and [8]. Moreover, Yano et al. showed that overexpression of hepatocyte growth factor (HGF), a ligand for MET, induces acquired resistance by activating MET signals [9].

Values of K = 2 to 10 are reported here and represent the average

Values of K = 2 to 10 are reported here and represent the average probability of 20 runs. The appropriate lengths of the program’s burn-in (initiation) period and run time (actual number of simulations) were 20,000 and 100,000, respectively. The default model of the program that uses admixture and correlated allele frequencies was applied to SNP data. In addition to the estimated log probability calculated by STRUCTURE, the ad hoc statistics of Evanno et al. [38] were used to determine the most likely population structure. The hypothesis of association of molecular markers with phenotypic

data was tested using the software program TASSEL 3.0.1 [39] and [40]. First, a single factor analysis (SFA) of variance

that does not consider population structure was performed using each marker as the independent variable. The mean performance of each allelic class was compared using the general linear model (GLM) function in TASSEL. Next, a Q GLM analysis was carried out using the same software. This analysis applies population structure detected by STRUCTURE (Q matrix) as co-factors. To obtain an empirical threshold for marker significance and an experiment-wise P-value, 10,000 permutations of data were performed. The final analysis was performed using the Q + K MLM method. This approach considers both the kinship matrix and the population structure Q matrix in Fluorouracil mw the marker-trait association test. The K matrix of pairwise kinship coefficients for all pairs of lines was calculated from SNP data by the SPAGeDi software [41]. Genotyping with the LSGermOPA panel provided high-quality SNP markers for the tested lettuce accessions. For the 384 tested SNPs, 363 (94.5%) had a GenCall score (a designability rank score, which theoretically ranges from 0 to 1.0 as determined by GenomeStudio ver 1.0) greater than 0.6, and

old 41 SNPs were discarded because they were monomorphic, had more than 1% missing data points, or had more than 1% heterozygous genotype calls. For the remaining 322 SNPs, 189 distributed across all nine linkage groups each with 9 (on LG9) to 32 (on LG2) markers. The remaining 133 SNPs have not yet been placed on any molecular linkage map. A detailed description of the marker distribution is shown in Kwon et al. [30]. Of the 384 plants, 82 had more than 1% missing data points or were heterozygous at more than 1% of the 322 targeted loci; four plants were control duplicates used for checking reproducibility. To avoid potential negative effects of the missing data points and heterozygous genotypes on genetic differentiation and marker-trait association, we analyzed only the plants with more than 99% homozygosity using the SNPs with more than 99% of the data points. As a result, the final data set contained 298 homozygous plants, including 122 butterhead, 53 romaine, 63 crisphead, 53 leaf and 7 stem-type lines, genotyped with 322 SNPs.

GNN was

as effective as placebo in achieving therapeutic

GNN was

as effective as placebo in achieving therapeutic success in constipated children [8]. In the second, multicenter, 2-nation (The Netherlands and Poland) trial (n = 159) [9], children aged 3–16 years with functional constipation according to the Rome III criteria were randomly allocated to receive a fermented dairy product with Bifidobacterium lactis I-2494 (B. lactis) twice daily for 3 weeks or a comparable placebo. The effectiveness of the experimental treatment was comparable to that of the placebo [9]. Follow-up data were collected using a standardized questionnaire at 24 months after completion of the GNN study and at 36 months after completion of the B. lactis study. Participants were contacted by phone or regular mail. The questions asked related to the frequency, size, and consistency of stools defecated into the toilet, the presence of abdominal pain, and CHIR-99021 datasheet the need for laxative therapy. The primary outcome measure was treatment success, defined as ≥3 spontaneous bowel movements with no episodes of soiling during the last week, no abdominal pain, and no need for laxative treatment. The secondary outcomes were functional constipation according to the Rome III criteria and the need for laxative treatment. The computer software Stats Direct [version 2.7.9.(2012-07-09)] was used to calculate the relative risk (RR) and mean difference (MD), both with a 95%

this website CI. The difference between study groups was considered significant when the p value was <0.05, when the 95% CI for RR did not include 1.0, or when the 95% CI for MD did not include 0. All statistical tests were two tailed and performed at the 5% level of significance. The baseline characteristics of the 2 included populations [8] and [9] are summarized in Table I. The primary and secondary outcomes are summarized in Table II. In the GNN study, follow-up data at 24 months were obtained from 63 of 72 (87.5%) of the children. Overall, treatment success was reported in 36 of 63 (57%) of the children, and there was Hydroxychloroquine in vitro no difference in treatment success rates between the GNN and placebo groups (RR 1.08, 95% CI 0.70 to 1.66).

Functional constipation was reported in 17 of 63 (27%) of the children; the rate did not differ between groups (RR 0.86, 95% CI 0.38 to 1.94). The need for laxatives was reported in 13 of 63 (21%) of the children; the rate was similar in both groups (RR 0.83, 95% CI 0.31 to 2.20). The mean age of children with constipation was higher than that of children with treatment success, although the difference was of borderline statistical significance (9.7 ± 3.19 vs. 7.83 ± 3.4 years; MD 1.87, 95% CI −0.01 to 3.75). In the B. lactis study, only a subset of 76 children enrolled in Poland was invited to participate in the present follow-up. Follow-up data at 36 months were obtained from 57 of 82 (70%) of the children ( Table II). Treatment success was achieved in 26 of 57 (46%) of the children, and the rate did not differ between the B.

These hypotheses will be demonstrated below In the Southern Ocea

These hypotheses will be demonstrated below. In the Southern Ocean, CM5_piStart Alectinib is generally too cold around 50°S and too warm south of 60°S in the surface as compared to observations (Fig. 8 top left). The warm surface anomalies do not extend at depth though, where CM5_piStart is generally too cold over

the whole water column (Fig. 9 top left). This surface warm bias remains relatively unchanged in CM5_RETRO. Yet it extends down to almost 1000 m as well as along the oceanic floor (except for weak anomalies of the opposite sign between 50 m and 100 m, suggesting a modification of the thermocline). This is consistent with the forced simulations (compare F5_CMIP5 and F1_CMIP3, Fig. 2). The suite of sensitivity experiments in forced mode suggests that this effect is due to the implementation of the partial steps (F2). Bottom waters along the Antarctic continental shelf are colder in CM5_piStart as compared to CM5_RETRO. This is indicative of an intensified AABW formation, in agreement with forced

simulations, and confirmed by deeper mixed layers (not shown) and meridional streamfunctions (below). Furthermore, along the Antarctic continent, surface water masses are saltier Everolimus in CM5_piStart, while they are fresher north of 50°S (Fig. 9 bottom right). Fig 10 shows that these salinity anomalies in the Southern Hemisphere are responsible for an increase of the density gradient across Gefitinib in vitro the Southern Ocean (80°S–50°S) in CM5_piStart by roughly 15% as compared to CM5_RETRO. This consistent with intensified ACC in CM5_piStart, as described below. Regarding the tropical regions, Fig. 8 (bottom) shows that surface waters are colder by up to 1 °C and saltier by more than 1.5 psu in CM5_piStart as compared to CM5_RETRO in the southern part of the Indonesian Archipelago

(IA). This results from the implementation of tidal mixing, consistent with coupled simulations from Koch-Larrouy et al. (2009). Further north, offshore of southeastern Asia, CM5_piStart displays a strong fresh anomaly compared to observations while this anomaly was much weaker in CM5_RETRO. This difference between the two simulations can be partly tracked down to changes in atmospheric freshwater flux, as shown in Fig. 12, with larger precipitation into the ocean (blue colour) in CM5_piStart along 5°N and weaker along the Equator and 5°S in the Indian Ocean. These changes are the signature of a northward shift of the ITCZ, and induce the SSS anomalies seen in Fig. 8 (bottom right). Note that from Fig. 12, atmospheric freshwater changes are also very strong in the tropical Atlantic, similarly characterised by a northward shift of the mean ITCZ position (around 10°N). Stronger precipitation are also found along 10°S.

312 mg/ml) of Alamar Blue (Resazurin, Sigma Aldrich Co St Louis

312 mg/ml) of Alamar Blue (Resazurin, Sigma Aldrich Co. St. Louis, MO, USA) was added to each Ion Channel Ligand Library screening well. The absorbance was measured using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter®), and the drug effect was quantified as the percentage of control absorbance at 570 and 595 nm. The absorbance of Alamar Blue in culture medium is measured at a higher wavelength and lower wavelength. The absorbance of the medium is also measured at the higher and lower wavelengths. The absorbance of the medium alone is subtracted from the absorbance of medium plus Alamar

Blue at the higher wavelength. This value is called AOHW. The absorbance of the medium alone is subtracted from the absorban‘ce of medium plus Alamar Blue at the lower wavelength. This value is called AOLW. A correction factor R0 can be calculated from AOHW and AOLW, where R0 = AOLW/AOHW. The percent Alamar Blue reduced is then expressed as follows: % reduced = ALW − (AHW × R0) × 100. Cultured human lymphocytes were plated at a concentration of 0.3 × 106 cells/ml and incubated for 24 h with different concentrations of PHT (0.25, 0.5, 1.0, 2.0, and 4.0 μM) and then mixed with low-melting point agarose. Doxorubicin (0.5 μM) was used as a positive

control. The alkaline version Selleck RG7422 of the comet assay (single cell gel electrophoresis) was performed as described by Singh et al. (1988) with minor modifications (Hartmann and Speit, 1997). Slides were prepared in duplicate, and 100 cells were screened per sample (50 cells from each duplicate slide), using a fluorescence microscope (Zeiss) equipped with a 515–560 nm excitation filter, a 590 nm barrier filter, and a 40× objective. Cells were scored visually according to tail length into five

classes: (1) class 0: undamaged, without a tail; (2) class 1: with a tail shorter than the diameter of the head (nucleus); (3) class 2: with a tail length 1–2× the diameter of the head; (4) class 3: with a tail longer than 2× the diameter of the head; (5) class 4: comets with no heads. Two different but complementary parameters were employed: Damage index (DI) and damage frequency (DF). DI is based on migration length and on the amount Oxymatrine of DNA in the tail, and it is considered a sensitive DNA measure. A value (DI) was assigned to each comet according to its class, using the formula: DI = (0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4), where n = number of cells in each class analyzed. The damage index ranged from 0 (completely undamaged: 100 cells × 0) to 400 (with maximum damage: 100 cells × 4). On the other hand, DF represents the percentage of cells (tailed cells) with DNA damage ( Speit and Hartmann, 1999). Naturally synchronized human peripheral blood lymphocytes were used with more than 95% of cells in the G0 phase (Bender et al., 1988 and Wojcik et al., 1996). Short-term lymphocyte cultures, at a concentration of 0.3 × 106 cells/ml, were initiated according to a standard protocol (Preston et al., 1987).