Infants with perinatal subtype of BA have higher tissue levels of PROM1 expression than those with embryonic subtype. Conclusion: Expansion of collagen-producing PROM1pos cells within regions of

periportal fibrosis is associated with activated FGF and TGFβ pathways in both experimental and human BA. PROM1pos cells may therefore play an important role in the biliary LY294002 mw fibrosis of BA. (Hepatology 2014;60:941–953) “
“This chapter contains sections titled: Epidemiology Histopathology Etiology and pathophysiology Clinical features and diagnosis One or two diseases? Treatment of microscopic colitis References “
“The reported prevalence of cancer associated with a primary choledochal cyst ranges from 2.5%[1]to 26%.[2] Total cyst excision is the standard procedure to prevent malignant changes. An essential part of this surgery is the termination of the Staurosporine in vivo reflux of pancreatic juice into the bile duct through the anomalous pancreaticobiliary duct union, so as to avoid activation of pancreatic enzymes involved in the pathogenesis of bile duct carcinoma. Theoretically, biliary malignancy should not develop after excision of the choledochal cyst because the presumed cause was abrogated by separation-operation. However, although the incidence of biliary malignancy after excision of choledochal cyst was 0.6% in Korean multicenter

study[3] and 0.7% in a Japanese nationwide study,[4] far lower than that associated with unresected choledochal cysts, it is still higher (about 200 times) than the incidence of biliary cancer in the general population in Japan. The main issues about the biliary malignancy after cyst excision are the perspective it might give on risk factors for oncogenesis, the relevance of cyst excision as a standard treatment and risk factors. In this issue of the Journal of Gastroenterology and Hepatology, Ohashi et al.[5] provided cumulative incidence data on subsequent biliary malignancy increase

more than 15 years after choledochal cyst excision, describe unfavorable outcomes, and provide a comprehensive review of biliary malignancy after cyst excision. However, several questions mentioned remained unanswered. The authors searched only the English literature. Because choledochal cyst is a relatively rare disease in the West and until more than half of the reported cases have occurred in Japan, we searched both English and Japanese language literature. Between 1970 and 2011, 58 cases were identified, and among them, data of site of malignancy can be available in 54 cases[3, 6-47] (Table 1). According to Todani’s classification, 24 of 41 patients (59%) were classified into type IVa, and 17 (42%) were into type I. The most common site of involvement in 54 cases was the hepatic duct, at or near the choledocho-enteric anastomosis (43%) followed by the intrahepatic duct (41%) and distal choledochus (17%).

The combined antiproliferative and metabolism-altering properties of rapamycin may therefore be important in preventing tumor regrowth post-transplantation and may explain the lower incidence of HCC and skin malignancies observed in transplant recipients taking this drug. Calorie restriction is known to extend lifespan21 and its effect is apparently mediated Copanlisib through mTOR,22 with superoxide-based signals playing a role.23 The effect

of calorie restriction on longevity is highly conserved, because rapamycin also increases murine lifespan, even when administered late in life.24 However, as we have noted above, rapamycin treatment is also associated with insulin resistance (IR), hyperlipidemia, and IS, thus making it important to identify competing downstream mechanisms of the rapamycin/mTOR interaction that may affect aging,

as some groups check details are all ready doing with some success.25, 26 As well as suppressing graft rejection, rapamycin and its analogs have multiple effects with exciting implications for their therapeutic use. By inducing Tregs, rapamycin may prevent the reemergence of autoimmune disease post-transplantation. It may also prevent weight gain, reduce the incidence of malignancy, and increase longevity. However, the negative effect of rapamycin treatment on metabolism, including induction of glucose intolerance and IR, also need to be considered. Regulatory processes are critical for deciding on the balance of efficacy and side effects required for approval of any drug. Occasionally, data prove to be inaccurate or incomplete, and drugs may need to be removed from the market. However, mistaken assumptions and poor study design may also lead to an incorrect interpretation of a drug’s potential benefit and result in its failure to be approved or correctly utilized. Regulatory agencies should be just as eager to identify these oversights and have mechanisms in place to resurrect drugs once new supportive evidence for their beneficial use is 3-mercaptopyruvate sulfurtransferase found. The potential for rapamycin to prevent hepatoma recurrence

affects over 1,000 patients in the United States every year (almost one fifth of liver transplant recipients), and promotes graft tolerance in thousands of patients, if the Levitsky hypothesis bears out. To demand stringent new double-blind registration trials is unrealistic, because the drug’s patent life is about to expire. A new paradigm must be developed by the U.S. Food and Drug Administration, together with the physician and scientific communities, to realize the extended therapeutic benefit of this and other drugs for the benefit of all patients. “
“Hepatocellular carcinoma (HCC) is a highly invasive tumor with frequent intrahepatic or pulmonary metastasis, which is the main reason for high recurrence and poor survival of HCC after liver resection.

In addition, somatic copy numbers of 661 and 206 genes were also significantly associated with DSS and DFS in our cohort, respectively (P < 1 × 10−4), whereas by chance one could expect only two and one genes, respectively, at the same P value cutoff (Supporting Fig. 1B). Hence, somatic CNAs in HCC are clinically relevant and may provide novel prognostic

markers. We also observed a nonrandom distribution of CNA-to-CNA correlations where unlinked loci were frequently correlated to each other (Supporting Fig. 2). As expected, adjacent loci were highly correlated, whereas check details at a higher level some chromosome arms became either unlinked (e.g., 6p versus 6q and 17p versus 17q) or anticorrelated (e.g., 1p versus 1q and 8p versus 8q). In addition, numerous correlations between unlinked loci were observed, suggesting coselection of these genomic regions (e.g., 1p versus 16p, 1q versus 4q, and 5q versus 19q) as previously reported.[14]

Although the overall CNA pattern is broadly consistent with the literature on HCC,[5, 9, 10, 14] the size and quality of our dataset should provide greater power to accurately localize and identify both large-scale and focal chromosomal alterations. To identify regions of copy number changes that may be responsible for driving tumorigenesis, we applied the GISTIC2 algorithm,[11] which incorporates both amplitude and frequency of CNAs to determine their statistical significance. Amplification or deletion click here peaks identified by GISTIC2 represent recurrent overlapping CNAs among multiple tumors, thus providing a finer resolution for mapping putative oncogenes Palmatine and tumor-suppressor genes. Our GISTIC2 analysis identified 146 focal events,

including 99 amplification peaks and 47 deletion peaks (Fig. 1B; Supporting Table 3). The median size of amplification peaks is 0.24 Mb (ranging from 1.5 kb to 11.6 Mb), containing an average of ∼5 genes per peak (excluding peaks that contain no genes, or “gene-less” hereafter). The median size of deletion peaks is 2.8 Mb (ranging from 46 kb to 122 Mb), containing an average of ∼100 genes per peak. We found that amplification peaks were significantly smaller than deletion peaks (P = 2.6 × 10−7; Supporting Fig. 3), and that genes under the amplification peaks tended to have stronger cis-correlation than those under deletion peaks, whereas both showed stronger cis-correlation compared to genes not located within any peak (Supporting Fig. 3). These observations support the disease relevance of the CNA peaks and are consistent with the assumption that oncogene activation is more locus specific than tumor-suppressor inactivation in cancer. We also thoroughly examined the association of GISTIC2 peaks to clinical and outcome variables (summarized in Supporting Table 4). We next focused on higher confidence peaks with residue Q value (by GISTIC2) ≤0.

This study aims to understand the viability of diatoms exhibiting teratological frustules and their reproduction capacities within a Cd-impacted population to predict their return selleck to normal diatom forms. We worked on a frequently encountered species in French hydrosystems: Planothidium frequentissimum (Lange-Bertalot) Round & L. Bukhtiyarova. First, a 21-d contamination phase highlighted increasing inductionof different teratological

types in response to two levels of Cd contamination: 20 and 100 μg · L−1. The deformity counting indicated that Cd firstly generated striae and mixed teratologies, then affected the central area and the valves. Second, a 28-d decontamination www.selleckchem.com/products/DAPT-GSI-IX.html phase demonstrated the Cd depuration capacity of Planothidium frequentissimum. Cd half-lives appeared relatively low, ~6 d for the 100 μg · L−1 condition. Moreover, the decontamination phase showed a decrease in teratology abundances, but

a still incomplete recovery after 28 d. Deformations of the striae appeared to be the most sustainable phenotype since they were still significantly higher than in reference cultures at the end of the decontamination phase for both Cd cultures. “
“Benthic diatoms form a particularly important community in oligotrophic lakes, but factors influencing their distribution are not well known. This study reports the depth distribution of living motile and total diatoms (living plus dead diatoms) on both natural (from sand to fine organic mud) and artificial substrates in an oligotrophic

lake. On artificial substrates, motile diatom densities peaked in abundance (24–30 cells · mm−2) between 0.6 and 1.9 m depth; on natural sediment surfaces, motile diatoms were generally more numerous and peaked in abundance (925 cells · mm−2) at 1.3 m depth. Total diatom densities on artificial substrates were highest (1260 valves · mm−2) at 0.6 m depth, with very low values below 3 m depth; on natural sediment surfaces, total diatom abundances were generally much higher (21600 valves · mm−2) at 3 m depth and declined gradually with depth. Significant relationships were found between light and diatom densities on the artificial substrate. Ordination MG-132 analysis indicated that substrate type significantly correlated with the variation of diatom composition on artificial and natural substrates. Our results suggest that in oligotrophic lakes, light influences benthic diatom abundance, whereas substrate type has more influence on benthic diatom composition. “
“Few members of the well-studied marine phytoplankton taxa have such a complex and polymorphic life cycle as the genus Phaeocystis. However, despite the ecological and biogeochemical importance of Phaeocystis blooms, the life cycle of the major bloom-forming species of this genus remains illusive and poorly resolved.

[1] To eliminate HCV, which establishes chronic infection in ~80% of infected individuals, interferon (IFN)-based treatments have been developed. The treatment of first choice at present for IFN-naïve patients of HCV genotype 1 with high viral load in Japan is pegylated interferon (PEG IFN), ribavirin

(RBV) and telaprevir (TVR) triple therapy, if it can be tolerated.[2] Although the sustained virological response (SVR) rate is much improved by the triple therapy, it is poorly tolerated due to a number of adverse events. Anemia is often a critical barrier to successful treatment for chronic hepatitis C patients on IFN therapy with RBV, forcing reduction or discontinuation of RBV administration. To overcome this obstacle, several groups reported employment see more of human recombinant erythropoietin (EPO) administration to alleviate anemia and thereby complete the therapy without RBV reduction in patients under IFN/RBV or PEG IFN/RBV combination therapy.[3-8] Most of the reports describe the successful role of EPO as an alternative to RBV dose reduction, and meta-analysis demonstrates that EPO administration can considerably enhance SVR with no adverse event due to EPO.[9] In triple therapy, Decitabine research buy anemia develops more frequently than in PEG IFN/RBV combination therapy, consequently resulting in poor adherence to RBV.[10,

11] Thus far, little is known about the efficacy of EPO to RBV-induced anemia in the triple therapy. In the present study, we examined whether EPO administration can alleviate anemia in hepatitis C patients on IFN therapy receiving both RBV and TVR, as observed in the PEG IFN/RBV combination therapy. The patients were given human recombinant epoetin-α at a dose of 12 000 or 24 000 IU/week, which is a relatively low dose compared with those used in previous reports, determined according

to the hemoglobin (Hb) decline from the baseline. The average adherence of the patients with EPO administration to RBV during the triple therapy phase was 97.5%, which was clearly higher than that of the phase III study of triple therapy,[10, 11] and no adverse event was observed. These findings indicate that low-dose EPO administration facilitates RBV adherence and can Rebamipide be a favorable alternative to RBV dose reduction. THE OBJECTIVE OF this study was to determine the safety of EPO administration and find whether it could prevent dose reduction of RBV due to anemia in triple therapy. Twenty-two patients (15 men and seven women, mean age of 56 years [range, 31–70]) with HCV genotype 1 infection and 5.0 log10 IU/mL or higher HCV RNA level were enrolled. All patients gave their informed consent before participating in the study. The study was approved by the ethics committee of Osaka National Hospital and conducted in accordance with good clinical practice and the Declaration of Helsinki. All patients received PEG IFN-α-2b (PegIntron; MSD, Tokyo, Japan) at a dose of 1.5 μg/kg/week s.c.

002, 0.0001, and 0.03 for non-Hispanic whites, non-Hispanic blacks, and Mexican Americans, respectively). Allele frequencies of the selected candidate gene polymorphisms were similar to those reported in comparable HapMap populations33 (Table 1). For 65 of the 67 variants (97%), allele frequencies were significantly different (P < 0.001) across racial/ethnic groups. The extent of this differentiation varied greatly between SNPs and between racial/ethnic populations (Table 1 and Supporting Table 2).

As several SNPs differed substantially between populations (e.g., DA = 0.250 for CYP3A4 rs2740574 between non-Hispanic whites and non-Hispanic blacks), each genetic variant was tested for associations with anti-HAV seropositivity in univariate and multivariable regression models stratified by the three racial/ethnic groups. This study identified significant genetic associations with anti-HAV seropositivity BMN 673 concentration among Mexican

Americans, but not among non-Hispanic whites or non-Hispanic blacks, under an additive genetic model using both univariate (data not shown) and multivariable regression models (Table 3). Since age and birthplace were the most important determinants of HAV infection between 1988 and 1994 in the United States,17 and since both were significantly associated in each racial/ethnic group (Table 2), we adjusted for age and country of origin (birthplace) in multivariable regression models. Two variants were found

associated with susceptibility to anti-HAV seropositivity among Mexican Americans (Table 3 and Supporting Table 3). PI3K inhibitor Specifically, for TGFB1 rs1800469 and XRCC rs1799782, the T allele was associated with an increased risk of anti-HAV seropositivity (FDR-P = 0.017 and 0.0007, respectively). The prevalence odds ratio of seropositivity calculated by multivariable regression was 1.38 (95% CI, 1.14-1.68) for TGFB1 rs1800469 and 1.57 (95% CI, 1.27-1.94) for XRCC1 rs1799782. These two minor alleles are common in the Mexican American population (TGFB1 rs1800469 [44.8%] and XRCC1 rs1799782 [14.8%]), but are less frequent among non-Hispanic whites (TGFB1 rs1800469 [31.4%] and XRCC1 rs1799782 [5.0%]) and non-Hispanic Chloroambucil blacks (TGFB1 rs1800469 [44.0%] and XRCC1 rs1799782 [6.2%]) (P < 0.001) (Table 1). CYP2E1 rs2031920 was marginally associated with increased odds of anti-HAV seropositivity (OR, 1.46; 95% CI, 1.12-1.91; FDR-P = 0.043) (Table 3). The minor allele (T) of ABCB1 rs1045642 was associated with lower risk for anti-HAV seropositivity among Mexican Americans (OR, 0.79; 95% CI, 0.71-0.89; FDR-P = 0.0007) (Table 3 and Supporting Table 3). Another variant, CAT rs769214, was marginally associated with decreased odds of anti-HAV seropositivity (OR, 0.82; 95% CI, 0.71-0.94; FDR-P = 0.043) (Table 3).

In addition, United States Centers for Disease Control and Prevention (CDC) laboratory-confirmed cases of PAM, B mandrillaris GAE, and AK will be analyzed statistically to determine significant risk factors for exposure and infection; and to recommend strategies for the management and prevention of these increasingly described free-living amebic CNS infections. Initially, Medline, Pub Med, Google®, and Google Scholar® search engines were queried for references using all of the key words MLN0128 purchase as medical subject headings terms. The only cases of free-living amebic meningoencephalitis included in the case analyses

were cases with CDC laboratory-confirmed detection of N fowleri, Acanthamoeba spp, or B mandrillaris life forms or DNA as detected by polymerase chain reaction (PCR) in cerebrospinal fluid (CSF), brain biopsy, or brain necropsy tissue. Sources of US cases of PAM came from the registry of the CDC’s Naegleria Workgroup, which ultimately confirmed 121 cases of PAM in the United States BGB324 solubility dmso during the period 1937 to 2007.2 Similar analyses were conducted for all CDC laboratory-confirmed cases of GAE caused by B mandrillaris (N = 15) in the United States during the period, 1999 to 2007. Sources of US cases of Balmuthia GAE, or balamuthiasis, came from state departments of public health and the California Encephalitis Project, a joint project launched in 1998 by the California Department of Public

Health and the CDC. Similar analyses were conducted for CDC laboratory-confirmed cases of AK during the period, 1987 to 2007 (N = 73). Significant behavioral, demographic, and recreational risk factors for PAM, Balamuthia GAE, and AK were identified over the study period to make recommendations for the Cyclic nucleotide phosphodiesterase early diagnosis, management, and prevention of these infections. All categorical variables were analyzed for statistically significant differences by Yates-corrected, chi-square analyses that compared

patients with potential risk factors for free-living amebic infections to patients with meningoencephalitis or infectious keratitis of undetermined causes or to other cases of free-living amebic meningoencephalitis or infectious keratitis without risk factors reported during the same time periods. Statistical significance was indicated by p-values ≤0.05. As this investigation was a comparative statistical analysis of previously reported CDC-confirmed cases, institutional review board approval was not required. Table 1 compares and contrasts the prominent epidemiological, pathological, clinical, and diagnostic features of four free-living amebic infections in humans, and outlines some of their successful treatment strategies. Table 2 presents a step-wise approach for selecting and sending appropriate diagnostic laboratory specimens to the CDC Division of Parasitic Diseases for free-living ameba testing.

In addition, sensitive strain S2 and the CRVs 2X and 2Y did not differ significantly in terms of accumulation of CIP with CCCP. The antioxidant capacity of P. mirabilis determined by FRAP, was significantly higher in CRVs showing greater MICs (1X and 2X), revealing a close correlation between CIP resistance and FRAP (Fig. 3). Lipid oxidation to MDA increased with CIP in both sensitive parental strains and decreased in CRVs (Fig. 4a). Additionally, in absence of antibiotic, MDA was higher in S1, the strain with a lower MIC. Moreover, the Selleck Everolimus oxidization of proteins to carbonyls and AOPP in the presence of CIP increased more

in S1 and S2 than in the CRVs 1X, 1Y, 2X and 2Y (Fig. 4b,c). Table 2 shows that the incorporation of GSH

or AA to culture media reduced the susceptibility of all P. mirabilis CRVs to CIP, as there was an evident increase of MIC in isolates S1, S2 and in all the CRVs after incubation AZD6244 with both antioxidants. The mechanisms involved in the resistance to CIP can be best interpreted by considering the different aspects that may be implicated in the antibacterial mechanism of action. The molecular mechanisms underlying resistance to fluoroquinolones in P. mirabilis include mutations in the target enzymes DNA gyrase and topoisomerase IV (Ser-83 in GyrA, Ser-464 in GyrB and Ser-80 in ParC) and over-expression of endogenous multidrug efflux pumps (Weigel et al., 2002; Saito et al., 2006). Therefore, the

results obtained, indicated that MICs of up to 16 μg mL−1 were displayed in the P. mirabilis CRVs, without typical mutations in DNA gyrase or topoisomerase IV genes. In addition, accumulation studies with CCCP indicated that the influx/efflux mechanisms could contribute to the increase 5-Fluoracil ic50 in the resistance of the CRVs to CIP only in 1X. In this work, an increase in FRAP was proposed as another factor involved in resistance. Previous results of elevated superoxide dismutase and GSH in CRVs (Aiassa et al., 2010) led to the investigation of the antioxidant capacity, as FRAP involves the combined or total reducing power of electron-donating antioxidants (Benzie & Strain, 1996; Litescu et al., 2011). FRAP is also an assay employed in different cellular extracts to measure the antioxidant capacity of different compounds, including antioxidant peptides (Nilsson et al., 2005; Di Bernardini et al., 2011), alpha-lipoic acid and vitamins that can be found in bacteria (Schlesier et al., 2002; Piechota & Goraca, 2009), as validated by several studies (Huang et al., 2005; Thaipong et al., 2006; Magalhães et al., 2008). These antecede even more the investigation of CIP action on biofilm (Aiassa et al., 2007), which indicated that enzymatic and non-enzymatic antioxidant systems may have a role in the defensive reaction against the oxidative stress caused by CIP in P. mirabilis.

Implementation of pooling of RNA for acute HIV screening presents several challenges. The need to provide rapid turnaround of test results

in a clinically meaningful time frame to ensure patient follow-up makes it difficult to accumulate a large number of specimens for pooling [15]; this barrier may be overcome by pooling specimens from dried blood spots [24]. Optimal pool size depends on the prevalence of acute HIV infection in the population and the skill of the laboratory personnel if a manual pooling technique is required. The failure of rapid HIV tests in this study to identify all cases of chronic HIV infection led to an increased number of positive pools requiring additional testing that highlighted chronic rather than acute HIV cases. Patients with a negative or discordant rapid HIV test had ∼2% probability of having chronic HIV infection in this setting. From this study, we are unable to evaluate Caspase inhibitor whether this relatively high false negative rate, higher than reported by the test kit manufacturers, was the result of operator error, faulty test kits/storage, or characteristics of the patient population. There was no apparent change during the study period in the rate of false negative results, despite retraining the HIV counsellors and changing the test kits. A recently reported

South African field study also noted challenges in HIV rapid test sensitivity compared with enzyme-linked immunosorbent assay and pooled HIV RNA PCR. In that Vemurafenib order study, which also used the SD Bioline kit, 5% of participants, all of whom were pregnant, had false negative results [25]. A high rate of false negative rapid tests was also reported in a study from South Africa among children on antiretroviral therapy, however, the test kits evaluated were different from those used in Avelestat (AZD9668) the current study [26]. The performance

of rapid test kits has been disappointing in other contexts [22,27,28], suggesting that inaccurate rapid tests may not be a setting- or test-specific problem. Other than the Abbott Determine HIV 1/2 rapid test, none of the rapid kits used during the study period has been extensively validated against gold standard tests in Africa in published studies; the World Health Organization recommends that individual countries evaluate each assay used to determine its performance characteristics and suitability for use within a given setting [29,30]. To the extent that this is not practised, many false negatives are probably occurring, as the settings using pooled HIV RNA are extremely limited. Rapid HIV testing has been an essential element in improving diagnostic capacity and treatment opportunities for patients in resource-limited settings [31]. It is important to counsel patients and providers, however, that there is a small but real risk of a false negative test due to both chronic and acute infection and to encourage retesting; country-wide guidelines should recommend a retesting frequency to guide counsellors’ efforts.

We thank Dr Fuminobu Yoshimura and Ms Mikie Sato for help with PMF analysis. This work was supported

by Grants-in-Aid for Scientific Research (to K.S. and K.N.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Global COE Program at Nagasaki University (to K.N.). “
“The iron-regulated surface determinant LY294002 proteins (Isd) of Staphylococcus aureus are expressed during iron limitation and have been proposed to be involved in the scavenging of iron from heme. In this study, the genes encoding the surface proteins IsdA, IsdB, and IsdH were inactivated in order to determine their combined role. The triple mutant was found to have no defect in growth under any conditions of iron limitation tested. Also using a mouse septic arthritis model of S. aureus systemic disease, no significant difference in bacterial load was observed for the triple mutant, compared

with its otherwise isogenic parent. The Gram-positive pathogen Staphylococcus aureus is the most commonly identified antibiotic-resistant cause of infection in many parts of the world including East Asia, America, and Europe (Foster, 2004). The natural niche for S. aureus, however, is as a commensal in the human nose, being carried by approximately 30% of the population (Wenzel & Perl, 1995). Thus, it is extremely selleck products prevalent in the human environment making its eradication more difficult and contributing to potential infections. As well as being a commensal of humans,

S. aureus can cause a variety of life-threatening diseases (Emori & PAK5 Gaynes, 1993). Thus, the organism is very adaptable colonizing a wide range of niches. Success of S. aureus requires the ability to respond to the host environment in order to grow and survive. A key nutritional factor that can limit the growth of bacteria in vivo is iron availability (Bullen, 1985). In fact, the sequestration of iron by mammalian hosts is a mechanism to stop the invasion of pathogens. Thus, iron deprivation is an important signal to which S. aureus responds using such regulatory systems as Fur (Horsburgh et al., 2001a). Fur responds to the lack of iron (as a marker of host interaction) by the derepression of a number of iron acquisition systems, including siderophore production and a heme iron uptake system (Heinrichs et al., 1999; Horsburgh et al., 2001a). Also negatively regulated by Fur is the expression of several surface proteins (Dryla et al., 2003). These iron-regulated surface determinants (Isd) are found covalently bound to the cell wall peptidoglycan, by the action of sortases, and thus interface with the external milieu. There are four cell wall–bound Isd proteins (IsdA, IsdB, IsdC, and IsdH) in S. aureus, and all have varying numbers of NEAT domains, which have been proposed to be involved in iron acquisition (Mazmanian et al., 2003).