Of Gastric Carcinoma (JCGC) [16].Evaluation of Monoclonal Antibodies for MUCCells and
Of Gastric Carcinoma (JCGC) [16].Evaluation of Monoclonal Antibodies for MUCCells and culture conditions. Human gastric cancer cell lines (SNU-16 and NCI-N87) and pancreatic cancer cell lines (PANC1 and CAPAN1) were purchased from the American Type Culture Collection (Manassas, VA). Both gastric cancer cells were maintained in RPMI-1640 (Sigma-Aldrich, St Louis, MO); PANC1 cells were maintained in DMEM (Sigma-Aldrich);MUC4 and MUC1 Expression in Early Gastric CancersMUC4 and MUC1 Expression in Early Gastric CancersFigure 2. Expression patterns of MUC4/8G7, MUC4/1G8 and MUC1/DF3 in each histological type of gastric carcinoma. Hematoxylineosin (HE) (A), MUC4/8G7 (B), MUC4/1G8 (C) and MUC1/DF3 (D) in papillary adenocarcinoma (pap). HE (E), MUC4/8G7 (F), MUC4/1G8 (G) and MUC1/ DF3 (H) in well differentiated tubular adenocarcinoma (tub1). HE (I), MUC4/8G7 (J), MUC4/1G8 (K) and MUC1/DF3 (L) in moderately differentiated tubular adenocarcinoma (tub2). HE (M), 
MUC4/8G7 (N), MUC4/1G8 (O) and MUC1/DF3 (P) in mucinous carcinomas (muc). HE (Q), MUC4/8G7 (R), MUC4/1G8 (S) and MUC1/DF3 (T) in solid type poorly differentiated adenocarcinoma (por1). HE (U), MUC4/8G7 (V), MUC4/1G8 (W) and MUC1/DF3 (X) in non-solid type poorly differentiated adenocarcinoma (por2). HE (Y), MUC4/8G7 (Z), MUC4/1G8 (a) and MUC1/DF3 (b) in signet-ring cell carcinoma (sig). MUC4/8G7 was Microcystin-LR expressed in the cytoplasm of pap (B), tub1 (F) and tub2 (J), but not in muc (N), por1 (R), por2 (V) nor sig (Z). MUC4/1G8 was expressed mainly at the cell apexes of pap (C), tub1 (G) and tub2 (K), but not in muc (O), por1 (S) nor por2 (W). MUC4/1G8 expression was seen in the intracytoplasmic mucin substance of sig (a). MUC1/DF3 was expressed mainly at the cell apexes tub2 (L), but not expressed in the cases shown in this figure (D, H, P, T, X 
and b). Original magnification 6200 (A , M ), 6400 (I , U ). doi:10.1371/journal.pone.0049251.gCapan1 cells were maintained in 15755315 DMEM/F-12 (Sigma-Aldrich). All media were supplemented with 10 fetal bovine serum (GIBCO, Breda, The Netherlands) and 100 U/mL penicillin/ 100 mg/mL streptomycin (Sigma-Aldrich). All cells were incubated in 5 CO2 at 37uC and maintained at sub-confluent levels. RNA extraction and RT-PCR. Total RNA was extracted from the cells using the RNeasy mini kit (Qiagen, Hilden, Germany) and quantified by NanoDrop ND-1000 spectrophotometer. The obtained mRNA (2ug) was reverse transcribed to cDNA with the High Capacity RNA to cDNA kit (Applied Biosystems, Foster City, CA). The following primers were designed for the subsequent PCR: MUC4, 59- TGGGACGATGCTGACTTCTC-39, 59-CCCCGTTGTTTGTCATCTTTC-39; ACTB, 59-CTCTTCCAGCCTTCCTTCCTG-39, 59-GAAGCATTTGCGGTGGACGAT-39. PCR was performed with the MedChemExpress 58543-16-1 AmpliTaq Gold Fast PCR Master Mix (Applied Biosystems) following the manufacturer’s protocol. Protein extraction and western blotting. Total cell lysates were prepared using RIPA buffer containing protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The protein concentration was measured by the BCA assay (Thermo Scientific, Rockford, IL). An equal amount of protein lysate was resolved on 2 agarose gel containing SDS and passively transferred onto PVDF membrane overnight at room temperature. Membraneswere blocked with 1 skim milk/PBST over 2 hours and subjected to the standard immunodetection procedure using specific primary antibodies. The primary antibodies are as follows: anti-human MUC4 MAb 8G7 (1:1000, generated by Dr. Surinder K. Batra, University o.Of Gastric Carcinoma (JCGC) [16].Evaluation of Monoclonal Antibodies for MUCCells and culture conditions. Human gastric cancer cell lines (SNU-16 and NCI-N87) and pancreatic cancer cell lines (PANC1 and CAPAN1) were purchased from the American Type Culture Collection (Manassas, VA). Both gastric cancer cells were maintained in RPMI-1640 (Sigma-Aldrich, St Louis, MO); PANC1 cells were maintained in DMEM (Sigma-Aldrich);MUC4 and MUC1 Expression in Early Gastric CancersMUC4 and MUC1 Expression in Early Gastric CancersFigure 2. Expression patterns of MUC4/8G7, MUC4/1G8 and MUC1/DF3 in each histological type of gastric carcinoma. Hematoxylineosin (HE) (A), MUC4/8G7 (B), MUC4/1G8 (C) and MUC1/DF3 (D) in papillary adenocarcinoma (pap). HE (E), MUC4/8G7 (F), MUC4/1G8 (G) and MUC1/ DF3 (H) in well differentiated tubular adenocarcinoma (tub1). HE (I), MUC4/8G7 (J), MUC4/1G8 (K) and MUC1/DF3 (L) in moderately differentiated tubular adenocarcinoma (tub2). HE (M), MUC4/8G7 (N), MUC4/1G8 (O) and MUC1/DF3 (P) in mucinous carcinomas (muc). HE (Q), MUC4/8G7 (R), MUC4/1G8 (S) and MUC1/DF3 (T) in solid type poorly differentiated adenocarcinoma (por1). HE (U), MUC4/8G7 (V), MUC4/1G8 (W) and MUC1/DF3 (X) in non-solid type poorly differentiated adenocarcinoma (por2). HE (Y), MUC4/8G7 (Z), MUC4/1G8 (a) and MUC1/DF3 (b) in signet-ring cell carcinoma (sig). MUC4/8G7 was expressed in the cytoplasm of pap (B), tub1 (F) and tub2 (J), but not in muc (N), por1 (R), por2 (V) nor sig (Z). MUC4/1G8 was expressed mainly at the cell apexes of pap (C), tub1 (G) and tub2 (K), but not in muc (O), por1 (S) nor por2 (W). MUC4/1G8 expression was seen in the intracytoplasmic mucin substance of sig (a). MUC1/DF3 was expressed mainly at the cell apexes tub2 (L), but not expressed in the cases shown in this figure (D, H, P, T, X and b). Original magnification 6200 (A , M ), 6400 (I , U ). doi:10.1371/journal.pone.0049251.gCapan1 cells were maintained in 15755315 DMEM/F-12 (Sigma-Aldrich). All media were supplemented with 10 fetal bovine serum (GIBCO, Breda, The Netherlands) and 100 U/mL penicillin/ 100 mg/mL streptomycin (Sigma-Aldrich). All cells were incubated in 5 CO2 at 37uC and maintained at sub-confluent levels. RNA extraction and RT-PCR. Total RNA was extracted from the cells using the RNeasy mini kit (Qiagen, Hilden, Germany) and quantified by NanoDrop ND-1000 spectrophotometer. The obtained mRNA (2ug) was reverse transcribed to cDNA with the High Capacity RNA to cDNA kit (Applied Biosystems, Foster City, CA). The following primers were designed for the subsequent PCR: MUC4, 59- TGGGACGATGCTGACTTCTC-39, 59-CCCCGTTGTTTGTCATCTTTC-39; ACTB, 59-CTCTTCCAGCCTTCCTTCCTG-39, 59-GAAGCATTTGCGGTGGACGAT-39. PCR was performed with the AmpliTaq Gold Fast PCR Master Mix (Applied Biosystems) following the manufacturer’s protocol. Protein extraction and western blotting. Total cell lysates were prepared using RIPA buffer containing protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The protein concentration was measured by the BCA assay (Thermo Scientific, Rockford, IL). An equal amount of protein lysate was resolved on 2 agarose gel containing SDS and passively transferred onto PVDF membrane overnight at room temperature. Membraneswere blocked with 1 skim milk/PBST over 2 hours and subjected to the standard immunodetection procedure using specific primary antibodies. The primary antibodies are as follows: anti-human MUC4 MAb 8G7 (1:1000, generated by Dr. Surinder K. Batra, University o.
of the three capsid VPs, VP1, VP2, and VP3. Seventeen of these (251, 329, 330, 454, 455, 491, 503, 550, 581, 592, 597, 671, 659, 660, 701, 713, 716) are surface-exposed [20]. Each of the 17 T residues was substituted with valine (V) by site-directed mutagenesis as described previously [12,13]. Most mutants could be generated at titers similar to the WT AAV2 vectors, with the exception of T329V and T330V which were produced at ,10-fold lower titers, and T713V and T716V, which produced no detectable levels of DNase I-resistant vector particles. Each of the T-V mutant vectors was evaluated for transduction efficiency in HEK293 cells. These results, shown in Fig. 1a and b, indicate that of the 17 mutants, the T491V mutant transduced HEK293 cells ,4-fold more efficiently than its WT counterpart. 
vectors and increases the number of the viral genomes translocated to the nucleus [13,25]. In the present studies, we wished to examine whether the addition of the T491V mutant to the tyrosine triple-mutant vector augmented the transduction efficiency by further increasing nuclear transport of these vectors. To this end, we first evaluated the kinetics of transgene expression in H2.35 cells mediated by the Y444+500+730F+T491V quadruple-mutant and compared it with the Y444+500+730F tri.
pathogenic mechanism of obesity-related disorders such as type 2 diabetes, 
target tissues. Inflammation plays a crucial role in the pathophysiology of obesity-related disorders such as metabolic syndrome and atherosclerosis. However, to our knowledge, there have been no previous studies to examine circulating progranulin levels in subjects with metabolic syndrome and its relationship with carotid intima media thickness (CIMT), a useful surrogate marker for atherosclerosis. C1q/TNF-related protein-3 (CTRP3) is a novel adipokine that is a structural and functional adiponectin paralog [9]. Peterson et al. demonstrated that administration of recombinant CTRP3 to ob/ob mice significantly lowered blood glucose levels by activation of the Akt signaling pathway and suppression of gluconeogenic enzymes in the liver [10]. Furthermore, CTRP3 exhibited potent anti-inflammatory properties by inhibiting the binding of lipopolysaccharides (LPS) to toll-like receptor 4 (TLR4) [11] and reducing TNF-a and IL-6 secretion in monocytic cells [12]. Recently, we developed an enzyme-linked immunosorbent assay (ELISA) for CTRP3 and reported that CTRP3 concentrations were significantly higher in subjects with type 2 diabetes or prediabetes than subjects in a normal glucose tolerance group [13]. In the present study, we aimed to clarify the clinical significance of progranulin and CTRP-3 in the context of metabolic syndrome and atherosclerosis.Erformed the experiments: TS AU. Analyzed the data: TS AU TN. Contributed reagents/materials/analysis tools: MH NA. Wrote the paper: TS TN.
cirrhosis is characterized by disturbances in the systemic circulation, including marked arterial vasodilation that occurs principally in the splanchnic circulation, reduces the total peripheral vascular resistance and arterial pressure, and causes a secondary increase in the cardiac output. These abnormalities are central to the development of several major complications 
in patients with cirrhosis, such as the hepatorenal syndrome, ascites, spontaneous bacterial peritonitis, dilutional hyponatremia, and hepatopulmonary syndrome. Renal failure is the most clinically relevant condition among these conditions because its appearance generally indicates a very poor prognosis [1?0].We developed the MBRS scoring system, a simple prognostic model that 
of 
these mechanisms hinder the growth, penetration, reproduction and transmission of a pathogen. QR in plants slows down but does not prevent epidemics, thus effective disease control may require supplementary applications of fungicides. Triazoles represent a major category of fungicides used widely in agriculture and medicine. This group of fungicides inhibits cytochrome P450 sterol 14 alpha-demethylase, an enzyme required for the biosynthesis of ergosterol in many fungi [31]. Resistance to triazoles is thought to be polygenic [32] and mediated by several mechanisms including mutations in the target protein gene CYP51 and increased active efflux by ABC transporters [33], [34], [35], [36]. Cyproconazole is a triazole fungicide that has been used for many years to control M. graminicola [32].The genotype data were published earlier [21]. Only isolates with a distinct multi-locus RFLP haplotype and DNA fingerprint were chosen for virulence and fungicide resistance tests. A total of 141 genetically distinct isolates were included in the experiment. Each population was represented by 25?0 isolates.Measurement of cyproconazole toleranceM. graminicola isolates retrieved from silica gel long-term storage were grown on potato dextrose agar (PDA) amended with 50 mg/ L kanamycin and placed at 18uC for seven days. Blastospores formed on these plates were transferred into 50 mL Falcon tubes containing 30 ml 
P = 0.07; Figure S3A). The presence of KRAS mutations was associated with poor survival in tumor stage III (HR = 1.94, P = 0.03; Table S2). Risk factors such as 
or diabetes had no effect on patient survival either with or without KRAS mutations. A multivariate Cox regression model that included age, gender, TNM, tumor grade and tumor histology as co-variants confirmed KRAS mutational status as a potential independent prognostic marker with a hazard ratio (HR) of 1.87 (95 CI 0.99?.51, P = 0.05; Table 2). Analysis with specific types of KRAS mutati.Experiments, showed that point mutations in tumor samples up to 5 tumor content were detectable. This provided confidence that our inclusion of tumor samples, only if those had at least 10 tumor content (n = 171), would more than adequately enable the detection of mutations. Another criterion applied for mutation detection was reproducibility. Mutations were scored only when band shifts were reproducible in at least two independent experiments. Repeat experiments using SSCP followed by DNA sequencing were used for confirmation and identification of mutations (Figure S2). We also obtained independent confirmation of KRAS mutations in a random sub-set (n = 6) analyzed blindly in the reference laboratory of the Institute of Pathology, University Hospital of Heidelberg. In the KRAS gene, we detected 134 mutations in 171 tumors (78 ), with 131 mutations in exon 2 and 3 mutations in exon 3 (Table 1). Mutations in exon 2 in all tumors were localized to codon 12. Out of 131 tumors that carried mutation at codon 12, 61 tumors had GGT.GAT (G12D, 80 of 131) mutation, followed by GGT.CGT (G12R, 23 of 131, 18 ), GGT.GTT (G12V, 22 of 131, 17 ), GGT.TGT (G12C, 4 of 131, 3 ), GGT.GCT (G12A, 1 of 131) and GGT.GTC (G12V, 1 of 131). Three tumors carried mutations in exon 3 that were confined to codon 61 featuring the Q61H mutation due to CAA.CAC base change. The mutation frequency in ductal adenocarcinomas was 82 (117 of 143) including adenosquamous and anaplastic undifferentiated tumors. All 4 of the ampulla of Vater tumors showed KRAS mutation, while 7 of 9 IPMN-malignant types harbored mutation (Table 1 and Table S3). A total of 43 tumors (25 ) showed 
GT JW. Performed the experiments: GT SF CN CD. Analyzed the data: GT SF CN. Contributed reagents/materials/analysis tools: PS BC DB. Wrote the paper: GT CN SF CD PS BC DB JW.
the present assay system actually measures not the active BNP level, but the total BNP (BNP+proBNP) level [8]. It is important to know the proBNP level and/or proBNP/total BNP ratio in heart failure, because proBNP has much less ability to induce cGMP production (about 13?7 ) than BNP, and higher levels of the low-activity proBNP may be associated with the development of heart failure [7]. Consistent with that idea, we recen.Was similar to a previous study involving 301 healthy individuals (0.25 cm2) [58] and areas ranging from 0.28?.35 cm2 have been previously reported in healthy young adults [31]. The higher area of echogenicity in the current study is likely due to differences in the ultrasound manufacturer, transducer properties (1? MHz versus 2.5 MHz), greater propensity for the ultrasound beam to penetrate bone (97.5 versus 77?2 ) [24,52,53,58], and improvements in ultrasound resolution over time. Such factors did not contribute to the between group difference observed in the current study because all subjects were tested with a Philips iU22 system and s5? transducer. The between group difference in substantia nigra echogenicity is also unlikely due to the ultrasound operator. All subjects were tested by one operator and the measurements collected by this operator were consistent with those collected immediately after by a second operator. The reliability and reproducibility statistics were comparable to those published previously [63,64] and a subset of images was viewed by a third person for confirmation of image quality. However, a limitation of the current study is that the operator was not blinded to the individual’s drug history.ConclusionsThe results of the current study suggest that some individuals with a history of illicit stimulant use exhibit abnormal substantia nigra morphology. Substantia nigra hyperechogenicity is a strong risk factor for developing Parkinson’s disease later in life [36] and our result supports recent epidemiological data suggesting that methamphetamine use is associated with increased risk (hazard ratio = 2.65) of developing Parkinson’s disease [65]. Further research is required to determine if the observed abnormality in stimulant users is associated with subtle movement dysfunction.AcknowledgmentsThe authors would like to thank Ms Verity Pearson-Dennett for assistance with data collection and Dr Eva Betz for assistance with recruitment of volunteers.Author ContributionsConceived and designed the experiments: GT JW. Performed the experiments: GT SF CN CD. Analyzed the data: GT SF CN. Contributed reagents/materials/analysis tools: PS BC DB. Wrote the paper: GT CN SF CD PS BC DB JW.
date, Materials and Methods Animals and Experimental Procedures Chicken Broilers were reared together in a closed building at the ex For accurate cell division, an exact copy of the genome must be equally transmitted from a mother cell to two d
be regulated at the level of DNA, by mutation or epigenetic modifications, as well as post-transcriptionally, mRNA levels are considered a legitimate measure of gene expression, and analysis of expression microarrays is a valid method for analysis of changes in cellular functions. There are several ways to analyze microarray data, as described in [8?0]. One of the 
main hurdles in microarray analysis is the heterogeneity between biological replicates. In most cases, the analyst attempts to smooth over the heterogeneity, and looks at averagedexpression changes that are significant in most or all of the replicates [11,12]. Cluster analysis then delineates groups with significant differences. Although for many purposes this average analysis is appropriate, heterogenic data reflect real differences between biological replicates. These differences, which are minimized when looking at average expression, can have profound phenotypic effects. In recent years, the concept of personalized therapy has gained popularity [13?5]. Two fundamental principles that underlie the concept of personalized cancer therapy are that significant genomic heterogeneity exists among tumors, even those derived from the same tissue of origin, and that these differences can play an important role in determining the likelihood of a clinical response to treatment with particular agents. Such genomic heterogeneity can involve differences in the spectrum of coding sequence mutations,