oeg/software_benchmark_v2 huggingface dataset:
The first-round multiplex PCR amplification was performed in a 70-ml volume containing 5 ml of template DNA, 10 mM Tris-HCl (pH 8.3), 2 mM MgCl2, 200 mM each of dATP, dCTP, dGTP, and dTTP, 0.7-1 mM of each primer (0.7 mM for microsporidia and 1 mM for Cyclospora and Cryptosporidium), and 5 U Taq DNA polymerase (Promega).
Both amplifications were performed using a C-1000 DNA thermal cycler (BioRad, Hercules, California, USA) with initial denaturation at 94 ℃ for 5 min, followed by 35 cycles at 94℃ for 30 sec, 53℃ for 30 sec, and 72℃ for 90 sec and a final extension at 72℃ for 10 min.
Although there was a high overlap ratio of annexin V staining with DiI (0.63, confirming the presence of apoptosis at the level of the RGCs), there was a very low ratio (0.072) of DiI cells also positive for annexin V. This demonstrates either that only some RGCs were ever annexin V positive and that apoptosis is not the only pathway responsible for cell death in this model, or that annexin V positivity is transient (as shown in Figure 3).
These findings show that PMA stimulates a higher rate of early-phase apoptosis (annexin V staining alone) in 3xTg-AD animals, and are consistent with the data in Figure 4 showing a similar effect of PMA in the model of acute RGC death mediated by Ab.To assess the magnitude of late apoptosis, we studied the co-localisation of PI and annexin V using ImageJ software with the Intensity Correlation Analysis plugin.
Different lasers and filters were used for different fluorochromes: for excitation of Alexa Fluor 488, an argon laser wavelength of 488 nm and a solid state photodetector with a 521 nm cut-off filter and a 550 nm cut-on filter were used; for DiI, a diode-pumped crystal green laser with a wavelength of 532 nm was used with 550 nm cut-off and 600 nm cut-on filters; the same laser was used for PI visualisation, but with a 650 nm cut-off.
Microglia tightly contact specific brain structures in their resting state (Wake et al., 2009), often making it difficult to clearly separate them from their surroundingWhile the red cell remains in resting state, the green cell is activated through an induced injury and migrates along a trajectory (orange line, trajectory obtained by ct3d blue line, trajectory obtained from manual annotation) toward a site of injury (purple dot); see Supplementary Video 1 for a rendered animation of the same data.
Cells were then suspended in lysis buffer (50 mM HEPES, 50 mM NaCl, 5 mM EDTA, 10 mM sodium pyrophosphate, 50 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride and 1× complete protease inhibitor cocktail).
We determined the equilibrium dissociation constant for TDG binding to unlabeled DNA using equilibrium competition anisotropy experiments, where TDG binding to TR-labeled 16U F 11 was monitored in the presence of varying concentrations of unlabeled DNA.DynaFit was used for global fitting of the data to a model involving one or two TDG binding sites for the unlabeled DNA and two binding sites for TR-labeled 16U F 11.
For the total protein extract preparation, tissue samples or cell pellets were homogenized in ice-cold buffer (H-buffer) containing 20 mM Tris (pH 7.5), 2 mM EDTA, 2 mM EGTA, 250 mM sucrose, 5 mM dithiothreitol, leupeptin at 200 mg/ml, Aprotinin at 10 mg/ml, 1 mM phenylmethylsulfonyl fluoride, and 0.1% Triton X-100 (all from Sigma-Aldrich, St. Louis, MO), as previously described (Zappelli et al., 1996).
Nuclei were counterstained with Hoechst 33342 (Fluka) or with TO-PRO-3 (Invitrogen), and the samples were analyzed under an epifluorescence Zeiss Axioskop 2 Plus microscope (Carl Zeiss, Oberkochen, Germany) or a Leica Leitz DMRB microscope fitted with a DFC300FX camera (Leica, Wetzlar, Germany).
Single channel currents evoked by pressure-applied 5-HT (10 M) were recorded in an extracellular solution (E1) consisting of 142.8 mM NaCl, 0.1 mM MgCl 2 , 0.1 mM CaCl 2 , 10 mM glucose, 10 mM HEPES, pH 7.2 (adjusted by 1 M NaOH; final [Na ϩ ] o ϭ 146 mM).
Whole-cell recording was used to determine the reversal potentials (E 5-HT ) of macroscopic currents elicited by 5-HT (10 M) by means of a voltageramp protocol using WinWCP V3 9.6 electrophysiology software (J. Dempster, Department of Physiology and Pharmacology, University of Strathclyde, UK; available on the World Wide Web).
In order to determine the permeability of Na ϩ relative to Cs ϩ (P Na /P Cs ), E 5-HT was determined in solution E1, and the permeability ratio was calculated from the Goldman-HodgkinKatz (GHK) (voltage) equation,For simplicity, we routinely refer to ion concentrations rather than activities throughout, although the latter were employed in the calculations of relative permeabilities.
(Fig. 1B), GEC (median: range, 3.58: 3.33-4.11vs 3.65: 3.46-3.93; P = 0.285) (Fig. 1C), β (median: range, 1.85: 1.59-2.39 vs 1.80: 1.43-2.25; P = 0.575) (Fig. 1D) and κ (median: range, 0.61: 0.54-0.81 vs 0.62: 0.54-0.71; P = 0.959) Fig. 1E), between the control and chewing gum groups.
The amount of transcript accumulated for the SiDREB2 gene (SiDREB2 RT_F, 5#-GCCTTGTAGTCATTTGTGGTTT-3#; and SiDREB2 RT_R, 5#-CCTCACAACTCCTTTTCTCAAGCT-3#) normalized to the internal control actin was analysed using the 2-DD Ct method (Livak and Schmittgen, 2001).
To determine further the relatedness of SiDREB2 to the other AP2/EREBP proteins, a systematic phylogenetic analysis was carried out based on the similarities of AP2 domains in the proteins isolated from Arabidopsis, rice, maize, wheat, sorghum, barley, Festuca, Phyllostachys, tomato, tobacco, cotton, Catharanthus, and Brassica species using MEGA 4.0 by the NJ method (Fig. 1C).
The functional differences in trait performance mainly caused by SNPs has also been reported in previous studies on several cloned genes (Peng et al., 1999; Takahashi et al., 2001; Liu et al., 2002; Jin et al., 2003; Toshiyuki et al., 2003; Kim et al., 2005; Garcés- Claver et al., 2007; Fan et al., 2009).
Two main clades of DEG7 proteases can be distinguished: clade A contains DEG7 proteases from fungi, whereas DEG7 proteins from green, heterokont and haptophyte algae, mosses and higher plants form clade B. Whereas most species investigated possess only one DEG7-like gene, the genomes of the yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, the ascomycete Giberella zeae, the oomycete Phytophtora soja, the haptophyte alga Emiliania huxleyi and the moss Physcomitrella patens contain two, whereas the higher plant Populus trichocarpa possesses three DEG7 paralogues (Figure 2).
In the first approach, gene models (http:// augustus.gobics.de/predictions/chlamydomonas/augustus.u5.aa) from the Augustus update 5 (Au5) of C. reinhardtii genome assembly version JGI v4.0 were functionally annotated by assigning enzyme classification (EC) terms using BLASTP results against UniProt (http:// www.uniprot.org/) and AraCyc (http://www.arabidopsis.org/biocyc/) enzyme protein sequences and their EC annotations as the basis.
Three primers (N069, N076, and N078; see Supplementary Table S2 at JXB online) were designed to differentiate between the two nucleotides for each of the three haplotype tagging SNPs (htSNPs) detected by HAP in haplotype block 2. All 248 accessions were genotyped at these htSNP positions by PCR with primer combinations N069-N020, N053-N076, and N053-N078.
Gene expression was analysed in leaves, stems, floral buds, and flowers of three different B. napus accessions, the winter type Express, the semi-winter type Ningyou7 (with or without prior vernalization), and the spring type Haydn (with or without prior vernalization), using primer combinations specific for either the gene pair BnaA.FRI.a/BnaX.FRI.d or BnaX.FRI.b/BnaX.FRI.c.
Three common haplotypes (represented by more than two accessions each) were identified in block 1, while four common haplotypes were found in block 2. Three polymorphic sites (InDel99, SNP282, and SNP348) in block 1 and three polymorphic sites (SNP1224, SNP1615, and SNP1790) in block 2 were identified as haplotype tagging SNPs (htSNPs) or InDels (Table 4; Taking population structure into account, block 2 showed association with flowering time for both years (P <0.05).
Consistent with the phase relationships in the QTL population, the BnaA.FRI.a allele in the parental accession Ningyou7 corresponds to the earliest flowering haplotype 4, whereas the Tapidor allele corresponds to the latest flowering haplotype 3. Although there are several nonsynonymous SNPs within haplotype block 2, including the htSNP at position 1790 which shows a SNP-trait association in one year (P¼0.017 in 2008; Table 2) and could possibly affect protein function, the protein sequence in this region is identical between the two extreme haplotypes 3 and 4. However, the two haplotypes differ at two positions at the nucleotide sequence level, i.e. the htSNP at position 1615 and the SNP at position 1702, which are both located within the coding sequence.
Interestingly, a glutamate to glycine substitution in the winter type allele in Tapidor (in an otherwise unchanged, theoretical molecule) would reduce the probability of a coiled-coil domain in the N-terminal region from ;50% to <5%, and the probability of a coiled-coil domain in the semi-winter type allele in Ningyou7 is also low (<10%; predictions according to COILS for a window size of 28 residues; http://www.ch.embnet.org/software/coils/ COILS_doc.html; data not shown).
Both the RNAand DNA-dependent DNA synthesis reactions were primed with the same 5 0 -end-32 P-labeled 18-nt DNAC for 30 min (DNA-dependent DNA synthesis) or 60 min (RNA-dependent DNA synthesis) and then quenched by addition of 20 ml of gel loading buffer (98% deionized formamide containing 1 mg/ml each of bromophenol blue and xylene cyanol).
(ii) Inhibition of HIV-1 RT DNA synthesis was also evaluated to determine IC 50 values using a 5 0 -end-32 P-labeled 19-nt DNA primer (5 0 -TTGTAG CACCATCCAAAGG-3 0 ) that was annealed to oneof three 36-nt DNA templates that contained five consecutive thymine, cytosine or adenosine bases.
The median CRP was 1.8 mg/L (interquartile range 0.8-4.1) overall; 1.7 mg/L (0.8-3.6) in men and 2 mg/L (0 8- 4.5) in women (Wilcoxon test, P , 0.0001); and 3.2 mg/L (1.5-6.2) and 1.8 mg/L (0.8-4.0), respectively, in participants with and without diabetes (P , 0.0001).
Molecular Modeling of the Arf1-AMF-26-Sec7 Domain Complex-To obtain the initial structure of the Arf1-AMF-26-Sec7 domain complex, the sampled conformers of AMF-26 were superposed onto BFA in the X-ray structure of the Arf-BFA-Sec7 domain complex (PDB code 1r8q) (8) using the parallel version of the SUPERPOSE program (30).
4, D and E, supplemental Table SEffects of AMF-26 on the cis-Golgi, TGN, and Recycling Endosomes-To clarify the mechanism of Golgi disruption by AMF-26, we first examined the effect of AMF-26 on localization of cis-Golgi-associated proteins, ␤COPI, GBF1, and p58/ ERGIC53.
ICRs were PCR amplified using a GeneAmp 2720 thermal Frontiers in Genetics | Epigenomics cycler (Applied Biosystems, Kotoku, Tokyo, Japan) in a final volume of 50 μl containing 0.2 mM dNTPs, 1 × PCR Gold Buffer, 1.5 mM MgCl 2 , 1 U AmpliTaq Gold (Applied Biosystems, Kotoku, Tokyo, Japan), 2 μl DNA (100 ng/μl), 10 μM of each forward (F) and reverse (R) primer according to the following conditions: 95˚C 5 min (95˚C 30 s, Ta˚C 1 min, 72˚C 1 min) for 50 cycles, 72˚C 5 min.
The annealing temperatures and primer sequences for amplification of the ICRs from bisulfite modified DNA were as follows: H19 55˚C (F 5 -GAGTATTTAGGAGGTATAAGAATT-3 , R 5 -ATCAAAAACTAACATAAACCCCT-3 ); Rasgrf1 59˚C (F 5 -TAGAGAGTTTATAAAGTTAG-3 , R 5 -ACTAAAACAAAAACAA CA-3 ); Snrpn 59˚C (F 5 -TATGTAATATGATATAGTTTAGAAATT AG-3 , R 5 -AATAAACCCAAATCTAAAATATTTTAATC-3 ).
Sequencing (S) primers were designed using the PSQ Assay Design Software (Biotage, Uppsala, Uppsala County, Sweden) as follows: H19 (CTCF 1; S 5 -GGAATTAGTTGTGGGGTT-3 ); H19 (CTCF 2; S 5 -GTGTAAAGATTAGGGTTGTT-3 ); Snrpn (S 5 -GTGTAGTTATTGTTTGGGA-3 ); Rasgrf1 (S 5 -CCAAATACCACAACAACAA-3 ).
The real-time confocal microscope imaging system used in this study was composed of an inverted epifluorescence microscope Eclipse Ti-U (Nikon, Tokyo, Japan), a CSU-X1 laserscanning unit with a 520612.5 nm band-pass filter (Yokogawa, Tokyo, Japan), and an ImagEM EM-CCD camera (Hamamatsu Photonics, Hamamatsu, Japan).
The wells were sequentially injected with 20 mM 2-deoxyglucose (2-DG) to inhibit glycolysis, 100 nM oligomycin to inhibit ATP-synthase, 500-1000 nM carbonylcyanide-4-trifluorometho-xyphenylhydrazone (FCCP) to uncouple the respiratory chain, and 200 nM rotenone to inhibit complex I.
Annealing was performed in 8 ml aliquot containing 200 mM NH 4 OAc, 40 mM Tris-HCl (pH 7.6), 3 mM MgCl 2 , 6 mM b-mercaptoethanol, 2 pmol transcript 8, 2 pmol transcript 9 and 5 0 -32 P-labeled transcript 8 (220 000 cpm) as a tracer.
After 3 min pre-incubation at 30 C, 0.8 ml of the mixture was mixed with 19.2 ml solution containing 200 mM NH 4 OAc, 70 mM Tris-HCl (pH 7.6), 3 mM MgCl 2 , 60 mM KCl, 6 mM b-mercaptoethanol, 0.6 mM dithiothreitol (DTT) 1 mM Ado-Met, 4 pmol non-labeled transcript 8 and 10 mg of RlmKL (100 pmol) or acetylated bovine serum albumin (BSA) (B2518 Sigma-Aldrich).
Polycrystalline samples of the A-site-ordered double-perovskite solid solution Ca 1-x La x Cu 3 Fe 4 O 12 (x 5 0.0, 1/4, 1/2, 3/4 and 1.0) were prepared, at 15 GPa and 1300 K, from stoichiometric amounts of Ca 2 Fe 2 O 5 , La 2 O 3 , CuO, Fe 2 O 3 , and the oxidizing agent KClO 4 by using a multianvil press.
We conducted two-tailed t tests and considered results significant when P was ,0.05.Insurance coverage was associated with both frequency of health care use and routine place to go for health care; uninsured persons less frequently than the insured visited a doctor or other health care professional (e.g., 12.2% of uninsured persons reported not visiting a doctor during the past 12 months compared with only 2.1% of insured falling into this category).
Those who did not report a health care visit had poorer diabetes control outcomes than those who reported health care visits four or more times in the past year: mean A1C 9.1 vs. 7.4%, P = 0.014; mean systolic blood pressure 127.6 vs. 126.2 mmHg, P = 0.56; mean diastolic blood pressure 79.6 vs. 72.4 mmHg, P = 0.002; and mean non-HDL cholesterol 171.3 vs. 146.6 mg/dL, P , 0.001, respectively.
PBMNCs were washed twice with PBS (Invitrogen Life Technologies) and resuspended in endotoxin-free high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 2 mmol/L L-glutamine and 100 units/mL penicillin/ streptomycin (both from Invitrogen Life Technologies), and 10% human AB serum (PAA Laboratories, New Bedford, MA).
For intracellular cytokine staining, PBMNCs were cultured in the presence or absence of 100 ng/mL ultrapurified lipopolysaccharide (LPS; O111:B4; InvivoGen, San Diego, CA), 50 mg/mL polyinosinic: polycytidylic acid (Poly[I:C]; Sigma-Aldrich, St. Louis, MO), and 10 mg/mL R848 (Axxora, San Diego, CA) for 4 h, followed by staining for DC and monocyte surface markers, followed by staining for IL-1b and IL-6, as previously published (11).
Twenty-five microliters of serum were used for measuring levels of IL-1 receptor antagonist (IL-1RA), interferon (IFN)-a, IFN-g, IL-1a, IL-1b, IL-6, IL-17A, chemokine (C-X-C motif) ligand (CXCL-10), and monocyte chemoattractant protein (MCP)-1 using the Meso Scale Discovery (MSD) cytokine multiplex kit according to the manufacturer's instruction (MSD, Gaithersburg, MD).
Statistical differences in frequencies of freshly drawn mDCs, pDCs, and monocytes, as well as frequencies of monocytes and mDCsexpressing cytokines after TLR ligation in positive versus negative subjects, frequencies of monocytes and mDCs expressing cytokines in at-risk subjects positive for one versus more than one autoantibodies, cytokine responses based on age group, cytokine expression levels, and serum cytokine levels in subjects with one versus more than one autoantibodies were evaluated using the nonparametric Mann-Whitney U test.
After 24 h, serial dilutions of lapatinib (GlaxoSmithKline, Stevenage, UK), doxorubicin (Pharmachemie B.V., Haarlem, The Netherlands), 5-fluorouracil (EBEWE Pharma, Unterach, Austria), methotrexate (Emthexate PF, Pharmachemie B.V.), ifosfamide (Holoxan, Baxter B.V., Utrecht, The Netherlands), or paclitaxel (Paclitaxel, EBEWE Pharma) were added.
For each experiment, four cultures of the normal cell line RI-EBV, established from lymphocytes from a normal male peripheral blood sample (Karyotype 46XY), grown in RPMI supplemented with 15% FBS, 1% L-glutamine and 1% Pen/Strep were set up and exposed to the drugs at varying concentrations: 10 À3 M, 10 À4 M, 10 À5 M and 0 M. Freshly prepared 5azaC-dR or GuaUre-dR was added to separate 10 ml cell cultures and incubated at 37 C for 7 h.
To show the direct targeting of E2F7 by miR-26a, we constructed luciferase reporters containing the wild type (WT) 3 0 -UTR of E2F7 (WT), the E2F7 3 0 -UTR mutated at the miR-26a binding site I (Mut1), the E2F7 3 0 -UTR mutated at the miR-26a binding site II (Mut2) or the E2F7 3 0 -UTR mutated at both miR-26a binding sites (Mut1-2).
For cell cycle analysis, 2 Â 10 5 cells were resuspended in PBS/50% FCS, fixed in 70% ethanol for 24 h, incubated with 50 Ag/ml propidium iodide (Sigma-Aldrich) and 50 U/ml DNase-free RNase A (Sigma-Aldrich), and analyzed after 3 h (10 000 events) using an Epics XL Cytometer (Beckman Coulter, Brea, CA, USA).
M-CSF, M-CSFr and MafB were detected with TaqMan oligonucleotides Hs00166144_m1, Hs00234622_m1 and Hs00534343_s1, respectively, and normalized by the amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Hs99999905_m1 (Applied Biosystems, Life Technologies Italia, Monza MB, Italy).
16 17 Group comparison and covariate effect size calculation, OR (with 95% CIs and p values) were estimated using repeated measures of ordinal logistic regression, with covariates selected for inclusion by backward stepwise regression ( p-value for exclusion 0.22) from maternal age, Hb, ferritin, Socio-Economic Indexes for Areas ( based on the Collector District of residence of mothers), quality of sleep, use and duration of breastfeeding, hospitalisation of the baby, baby gender and mode of delivery.
To this, we used GSEA of genes induced or repressed by DNA damage in a p53-dependent manner 32. Our results indicated that although p53-activated genes are very significantly underexpressed in the tumors, the p53-repressed genes do not display overexpression in tumors (Table II)carcinomas displayed partial inhibition of p53 function as transcriptional regulator, corroborating the luciferase experiments (Fig. 2b).
For the antibodies against active Cdc42 and Rac1 (NewEast Biosciences), cells were fixed in 3% PFA for 20 min at room temperature or methanol for 5 min at 20°C followed by a 3-min incubation in 0.3% Triton X-100 in blocking buffer (PBS containing 0.5% BSA, 10 mM glycine, and 0.1% sodium azide).
Next, the samples were further permeabilized and blocked for 8-10 h at room temperature in a humidified chamber in BSA permeabilization buffer (3% BSA, 1% Triton X-100, 0.5% Na-deoxycholate, 0.2% Na-dodecylsulfate, 150 mM NaCl, and 10 mM Hepes, pH 7.4) containing 5% donkey serum (Sigma-Aldrich).
For FRET experiments, siRNA-transfected cells were plated into eight multiwell chamber slides (ibidi), transfected with pRaichu-Cdc42, and analyzed using a microscope (SP2; 63×/1.4 NA objective, 37°C, in medium with 10 mM Hepes, pH 7.4) and LCS FRET software using the donor recovery after acceptor bleaching protocol (YFP was bleached to 30%; Yoshizaki et al., 2003; Terry et al., 2011).
Cell culture, cell lines, 3D morphogenesis, calcium switch, and permeability assays Human adenocarcinoma colon cells (Caco-2), human epithelial carcinoma cell line (A431), and HCE cells were cultured in DME containing 20% (Caco-2) or 10% (A431 and HCE) heat-inactivated FCS with 100 µg/ml streptomycin and 100 µg/ml penicillin (PAA Laboratories) at 37°C in a 5% CO 2 atmosphere.
Here, we use a coding scheme x 1 = -1, 0, 1 and x 2 = -0.5, 0.5, -0.5 for genotypes homozygous for major alleles, heterozygous, and homozygous for minor alleles (for APOE , 4 was assumed to be the minor allele, and 2 and 3 were assumed to be major alleles simultaneously), respectively, to represent an additive effect with x 1 and a dominant/recessive effect with x 2 .
Flow Cytometry Analysis-Cell surface markers were analyzed using specific antibodies against Ly6G (APC, 1A8; BioLegend), GR1 (PercP-Cy5.5, RB6 -8C5; BioLegend), and CD11b (phycoerythrin, M1/70; eBioscience) for identification of neutrophils or against CD4 (eFluor450, RM4 -5; eBioscience) for identification of CD4 ϩ T cells.
Interestingly, we found a striking coregulation of Oct2 and BOB.1/OBFInterestingly, EMSA studies (Supplementary Figure S1) revealed that the NF-kB inhibitor Bay11-7082 inhibits additionally NFAT binding to DNA and, vice versa, the CN inhibitors CsA and FK506 interfere with NFAT binding but also with the NF-kB signaling pathway, as it was described recently (44,45).
However, treatment of murine primary CD4 + T cells or transfected Jurkat cells with specific NFAT and NF-kB signaling pathway inhibitors, like the NFAT inhibitory peptide 11 R-VIVIT and the NF-kB essential modulator (NEMO)-binding peptide, respectively, clearlyThe BOB.1/OBF.1 promoter contains several binding sites for NFAT and NF-iB Previously, the BOB.1/OBF.1 promoter was analysed to identify regulatory elements responsible for its activity in B cells (34-36).
The positions of the coronal plane on which the measurements were taken were defined as the AE (anterior wall of bulla ethmoidalis that was removed first during anterior ethmoidectomy), BL (the basal lamella through which posterior ethmoidectomy was performed), and PE (the midportion of the posterior ethmoid sinus in the sagittal plane) in order to evaluate the intraorbital structures in view of the ethmoidectomy [11].
The tissue was then cut into small pieces, washed with 100 mM sodium cacodylate, pH 7.4, cut into small blocks (2-5 mm in size), and then postfixed in 1.0% (wt/vol) OsO 4 and 1.0% (wt/vol) K 4 Fe(CN) 6 , in 100 mM sodium cacodylate, pH 7.4.
For each organism, we first retrieved its 16S rDNA sequence using the 'Browsers' on the Ribosomal Database Project (http://rdp .cme.msu.edu/index.jsp) website, and then created MSA of these 16S rDNAs and built a tree using the 'Tree Builder tools (http://rdp.cme.msu.edu/treebuilderpub/index.jsp) (25).
Our findings seemingly conflict with earlier observations made in yeast, which suggest a failure of disulfide bond formation 2007), with a 63× oil immersion lens (NA 1.4) coupled to a microscope incubator, maintaining standard tissue culture conditions (Okolab), using a pulsed (sub-10 ps, 40 MHz) supercontinuum (430-2,000 nm) light source (SC 450; Fianium Ltd.).
Anchorage-independent growth and cell proliferation assays For measurement of anchorage-independent cell growth, 50 000 cells (HCC4006, control or gefitinib resistant) were suspended in 1.5 ml RPMI 1640 containing 10% fetal bovine serum and 0.35% Difco agar noble (Becton Dickinson and Co., Sparks, MD, USA) and overlaid on base layers containing 1.5 ml RPMI 1640 containing 10% fetal bovine serum and 0.5% agar noble in six-well plates.
2. In large ATC groups and ATC groups with drugs used for multiple heterogeneous indications, that is, cardiac therapy (C01), agents acting on the reninangiotensin system (C09), sex hormones (G03), urologicals (G04), analgesics (N02), psycholeptics (N05), psychoanaleptics (N06), ophthalmologicals (S01), a subdivision was done to the ATC third or fourth level to attain a more clinically relevant description of the utilisation.
These anatomical regions, defined using Anatomy ToolBox included the premotor cortices (Brodmann area 6 in Anatomy ToolBox (Gazzola and Keysers, 2009; Caspers et al., 2010), the primary and secondary somatosensory cortices (SI = BA3a + b + BA1 + BA2 and SII = OP1 + OP2 + OP3 + OP4) (Keysers et al., 2010).
pMECs (2 × 10 5 /genotype) from B1 f/f Luc f/+ , KLuc f/+ , and KB1 f/f Luc f/+ mice were resuspended 1:1 in 1× PBS/Matrigel (Matrigel Matrix High Concentration; BD) and injected into precleared fat pads of 21-d-old FVB recipient mice.