In terms of targeting signaling pathways in TISCs, ON123300, an alkaline triphosphate (ATP) mimetic kinase inhibitor,72, 73 inhibits tumor cell proliferation and induces

apoptosis, primarily through inhibition of CDK4 and AMPK-related protein kinase 5, without causing broader hepatotoxicity. This ATP kinase inhibitor may provide alternative TISC targeting. Novel chemoprevention strategies aimed at targeting TISC through inhibition of the MAPK pathway and/or vitamin D supplementation have been proposed.74-76 Key insights include the marked vitamin D deficiency observed in cirrhotic patients who develop HCC. In the setting of cirrhosis, vitamin D supplementation may act as a chemoprevention strategy by restoring TGF-β signaling, because vitamin D

up-regulates β2-spectrin in cirrhotic patients. CH5424802 datasheet Within alcoholic liver disease, a sensitization of liver macrophages to portal endotoxin, which activates TLR4 on macrophages, results in the production of inflammatory cytokines and activation of p38 MAPK, both contributing to the activation of the nuclear factor kappa light-chain enhancer of activated B cell signaling cascade.42, 77 Liver cirrhosis beta-catenin inhibitor results from increased sensitivity of hepatic stellate cells to TGF-β, leading to increased proliferation and production of extracellular matrix via activation of p38 MAPK signaling. New work reinforces that the TGF-β and β-catenin pathways are central to the process of TISC transformation and maintenance. Transcriptome profiling confirms poor prognosis of TISC-based HCC. At the conference, several issues were identified as areas of focus for future work. One unresolved issue is whether liver TISCs have reduced rates of proliferation, compared to the bulk tumor population. A quiescent state is proposed for TISCs, but strong evidence 上海皓元医药股份有限公司 is lacking. A second outstanding issue is the origin of TISCs. Although the hierarchic cancer model proposes that TISCs are derived from stem cells, they may also originate from hepatocyte dedifferentiation through loss of β2-Spectrin or up-regulation of β-catenin and resultant up-regulation of Oct-4 and Nanog. A third issue is the need for agreement

on the phenotype of TISCs. For example, in the field of hematopoietic malignancy, CD34+CD38− is a standard immune phenotype for TISCs, whereas CD44high/+CD24low/− is used to identify TISCs in breast cancer. As reviewed above, surface markers, such as EpCAM, CD133, CD49f, CD44, and others, have all been proposed for identifying TISCs, as have functional traits, such as efflux of Hoechst 33342, associated with the side population. A final unanswered question is the effect of TISC-targeted therapy on the LPC population of the regenerating liver. As LPCs and TISCs share many common pathways for proliferation and maintenance of stemness, targeting TGF-β or β-catenin may reduce the effectiveness of LPCs to regenerate the liver during cirrhosis.

The most common mutation in cirrhosis patients was located in the c-terminus of the TERT gene (the c.3325G>A mutation resulting in the amino acid change p.G1109R) (Fig. 2B, Table 1, Supporting Fig. 1G). This mutation is located next to an amino acid change (p.T1110M), which has previously

been associated with pulmonary fibrosis.21 The mean age of the patients with telomerase mutation was 55.8 years (Table 2) compared to a mean age of www.selleckchem.com/products/azd2014.html 58.7 years in the group of cirrhosis patients without telomerase mutations. There was no obvious overrepresentation of a specific disease etiology or ethnicity in the mutation carriers compared to the controls (see above). However, many of the mutation carriers

showed a rapid progression of chronic liver disease toward cirrhosis of liver cancer development (Table 2). Specifically, the frequency of endstage liver disease (defined as Child B or C cirrhosis, hepatocellular carcinoma [HCC] occurrence, and conduction or evaluation for liver transplantation) was significantly higher in the group of cirrhosis patients harboring telomerase mutations (93%, 13 out of 14) compared to cirrhosis patients without Selleckchem ZVADFMK telomerase mutations (62%, 327 out of 507, P = 0.042), indicating that telomerase mutations may induce a more aggressive progression of chronic liver disease—a hypothesis that should be addressed in future prospective trials. Cirrhosis patients carrying telomerase gene mutations had significantly shorter telomeres in peripheral blood cells compared to control patients without telomerase gene mutations (Fig.

3A, P = 0.0004). A significant reduction of telomerase activity was detectable by TRAP assay for three of the investigated, cirrhosis-associated telomerase mutations (p.P65A: P < 0.0001, p.G1109R: P = 0.0035, and p.P380S: P < 0.0001), including the most frequent mutation p.G1109R (Fig. 3B,C). Studies on telomerase-negative, human fibroblasts revealed that the cirrhosis-associated TERT mutations (p.P65A and p.G1109R) did not lose immortalization capacity when overexpressed (Fig. 3D). However, proliferation rates of fibroblast lines expressing these TERT mutants were significantly 上海皓元医药股份有限公司 reduced compared to cells expressing wildtype TERT (Fig. 3D). Similarly, Epstein-Barr virus-immortalized, primary lymphocytes from two patients with a homozygous p.G1109R TERT mutation showed an impaired proliferation capacity (Fig. 3E) and shortened telomeres (Fig. 3F,G) compared to immortalized lymphocytes from cirrhosis patients with wildtype TERT. γH2Ax staining of liver sections of six cirrhosis mutations carriers and eight liver cirrhosis patients without telomerase mutation did not reveal any significant difference in the percentage of γH2Ax-positive hepatocytes, a marker for the induction of DNA double-strand breaks (data not shown).

This new

subdomain structure was used for IRT-based scoring. Unlike traditional CLDQ scores, IRT-based scores closely approximated a normal distribution. With traditional CLDQ scoring, LD severity significantly influenced both general QOL scores and subdomain scores (ANOVA p<0.05). While LD severity significantly influenced IRT-based general QOL scores it did not significantly influence IRT-based subdo-mains see more scores. Traditional subdomain scores correlated highly with general QOL scores (r>0.7), while IRT-based subdomain scores did not. When the effect of general QOL on traditional subdomain scores was accounted for, the influence of non-LD-related factors on traditional subdomain scores was greatly reduced. Interestingly, when the effect of general QOL was accounted for, the influence of LD severity on traditional subdo-main scores was no longer significant. CONCLUSIONS: The strong influence of general QOL on “LD-specific” subdomain scores from the CLDQ renders them susceptible to the influence of non-liver disease-related factors and causes their clinical significance to be overestimated. This study questions the clinical utility of CLDQ subdomain

scores. Disclosures: Serine Protease inhibitor Robert Gibbons – Stock Shareholder: Psychiatric Assessments Inc. The following people have nothing to disclose: Sujit V. Janardhan, Andrew Aron-sohn, Jason Morris, David G. Beiser Background: Of the 3 million Americans chronically infected with Hepatitis C virus (HCV), it is estimated that only 25-50% are aware of their diagnosis. In August 2012, the CDC released recommendations for one-time HCV testing in persons born between 1945-1965 to improve screening in individuals at highest risk for chronic HCV infection. Aims: This study aims to assess the rate of HCV screening, follow-up testing, and linkage to care in an academic

primary care practice, MCE公司 before and after the CDC birth cohort guidelines. Methods: All patients born 1945-1965 and seen in the primary care clinic at the University of Chicago from July 1st, 2010 to May 15th, 2013 were included in the study. Key demographics, results of HCV-related laboratory testing, and completion of hepatology follow-up were obtained from the electronic medical record database. All patients seen from July 2012 to May 2013 were identified as the post-guideline group. MedCalc software (medcalc.org) was used for statistical analysis as appropriate. Results: A total of 24,947 patients in the eligible birth cohort were identified; 16,811 and 8,066 fell in the pre- and post-guideline groups, respectively. Only one third of patients in the birth-cohort had screening by HCV antibody testing and this rate continued to be low in the post-guideline group (Table 1). For patients screening positive, rates of confirmatory testing with viral load, genotype analysis and referral to hepatology were high in both the pre- and post-guideline group (Table 1).

Patients in the low replicative phase are believed to have good prognosis. There is increasing evidence that a fourth phase, the immune escape phase, is also common in Asian patients in association with evolution of HBeAg negative mutant forms of HBV.21 These patients have elevated HBV DNA with intermittent elevated ALT levels. Similar to the reports in Europe, HBeAg-negative patients with persistent viremia and biochemical activity have a higher risk of cirrhotic complications and HCC.22 The presence of viral mutations and immune escape has cast doubt on the importance

of HBeAg seroconversion. Previous reports suggested that approximately one-third of patients would develop HBeAg reversion or disease reactivation within 6 months after HBeAg seroconversion.23,24 PD98059 nmr HBV DNA usually falls to below 20 000 IU/mL after HBeAg seroconversion, but no clear HBV DNA level can predict viral reactivation.25,26 With long-term follow-up studies, we now learn that the long-term prognosis is better if the age ABT-263 of HBeAg seroconversion is younger. In a long-term follow-up of

64 untreated Caucasian pediatric chronic hepatitis B patients who cleared HBeAg without liver cirrhosis, 59 (92%) of them had stable disease.27 Among 408 Taiwanese patients who had no evidence of cirrhosis at the time of HBeAg seroconversion, the 15-year cumulative incidences of HBeAg-negative hepatitis, cirrhosis and HCC among patients who seroconverted at age younger than 30 versus those seroconverted after age 40 were 31.2% vs 66.7% (P < 0.001), 3.7% vs 42.9% (P < 0.001) and 2.1% vs 7.7% (P = 0.29), respectively.28 The age of HBeAg seroconversion is influenced by the HBV genotype. Patients infected with HBV genotype A, B, D and F tend to undergo HBeAg seronconversion at a much earlier age than those infected with genotype C HBV.29 上海皓元 Furthermore, patients infected with genotype C HBV also tend to have more frequent hepatitis B reactivation after HBeAg seroconversion than those infected

with genotype B HBV.30 All these findings have provided supportive evidence on the higher rate of HBeAg-negative active hepatitis,31 worse liver histology32,33 and higher risk of HCC34,35 among patients infected with genotype C HBV. In the last decade, HBV DNA could only be measured by the relatively insensitive non-polymerase chain reaction (PCR) based assays with a lower limit of detection at approximately 20 000 IU/mL.36 The lack of sensitivity of the HBV DNA assays precluded accurate assessment of the viral load among HBeAg-negative patients who tend to have lower viremia than their HBeAg-positive counterparts.37 The development of real-time PCR based assays has brought the sensitivity of HBV DNA measurement down to lower than 20 IU/mL (or 100 copies/mL). In several recent histologic series, HBV DNA lower than 2000 IU/mL were associated with mild histologic necroinflammation and fibrosis among HBeAg-negative patients.

This would establish a complex pathogenesis involving cross-talk between the hypothalamus and visceral organs (gut, liver, pancreas and white adipose tissue [WAT]).28 Hence, hypothalamic storage of particular types of fat

such as free fatty acids or ceramide, might also damage hypothalamic neurons secondary to lipotoxicity.29 Complementing experimental studies, evidence for a role of the hypothalamus in the development of human Trichostatin A manufacturer non-alcoholic steatohepatitis (NASH) has recently been reviewed.12 Recent demonstrations indicate that while germ-free mice consume more calories than their wild-type lean littermates, the latter gain significantly more weight.30,31 Furthermore, feeding rodents a high-fat diet will alter the intestinal bacterial flora and can impair

the host defense by affecting the innate immune function negatively.31 Several studies in Toll-like receptor knockout (TLR-KO) mice demonstrate that high-fat feeding increases the bacterial load in the intestines of the mice and change the respective check details bacteria flora. Upon transferring gut flora derived from the TLR-KO mice to germ-free mice, all of the findings associated with MS including weight gain, IR, hyperglycemia, NAFLD and hypertriglyceridemia were also transferred.32 The change in the bacterial flora or gut microbiome, and impaired host defenses to combat such changes, results in increased paracellular intestinal permeability which may be the etiologic factor in inflammation, production of lipopolysaccharide and tumor necrosis factor (TNF)-α and impaired function in adipose tissue, skeletal muscle and the liver (Fig. 1).32–34 Increased free fatty acid (FFA) released from WAT is also a well-established contributing factor in hepatocyte triglyceride and fatty acid storage which contributes to the development and exacerbation of hepatic IR. In the 1990s, the seminal discovery of leptin,35–37 derived from the ancient Greek word λεπτóς (leptòs) meaning “thin”, by Friedman, initiated a

decade-long study of visceral WAT, where this 16-kDa protein is synthesized. Soon, adiponectin,38 resistin,39 TNF-α40 and interleukin (IL)-641 were all recognized as being synthesized by WAT.42 The endocrine community 上海皓元医药股份有限公司 recognized that obesity was not just a consequence of diet and impaired insulin sensitivity, but was also a chronic inflammatory disease.43 As a target organ of chronic inflammatory processes, WAT is bombarded with the recruitment of macrophages; and when taken together, WAT releases not only adipocytokines, noted above, but paradoxically releases FFA into the circulation. Because most WAT is in the abdominal cavity – and not in subcutaneous tissue – the delivery of FFA as well as adipokines are destined directly to the liver via the portal vein.

In ‘high-dose protocols’, the aim of treatment is that the individual should be able to live as normal life as possible, which usually translates into guidelines to try to Selleckchem LDE225 maintain FVIII >1% of normal at all times. ‘Intermediate-dose protocols’ are exemplified by standards in The Netherlands, where infusion levels are usually 15–25 IU kg−1 two to three times per week and doses are often adjusted according to clinical needs (frequency of breakthrough bleeds). The Canadian ‘Hemophilia-Dose-Escalation Prophylaxis’ protocol is another attempt to tailor the dose interval individually according to bleeding frequency [5]. The work (E.B. and R.L.) has been

supported by grants from Lund University and Region of Skåne (ALF, regionalt forskningsstöd). VWD PN is supported by an unrestricted grant from CSL Behring. The authors stated that they had no interests selleck chemicals which might be perceived as posing a conflict or bias. “
“Haemophilia is a rare disease. To improve knowledge, prospective studies of large numbers of subjects are needed. To establish a large well-documented birth cohort of patients with haemophilia

enabling studies on early presentation, side effects and outcome of treatment. Twenty-one haemophilia treatment centres have been collecting data on all children with haemophilia with FVIII/IX levels up to 25% born from 2000 onwards. Another eight centres collected data on severe haemophilia A only. At baseline, details on delivery and diagnosis, gene mutation, family history of haemophilia and inhibitors are collected. For the first 75 exposure days, date, reason, dose and product are recorded for each infusion. Clinically relevant inhibitors are defined as follows: at least two positive inhibitor titres and a FVIII/IX recovery <66% of expected. For inhibitor patients, results of all inhibitor- and recovery tests are collected. For continued treatment, data on bleeding, surgery, prophylaxis and clotting factor consumption are collected annually. Data are downloaded

for analysis annually. In May 2013, a total of 1094 patients were included: 701 with severe, 146 with moderate and 247 with mild haemophilia. Gene defect data were available for 87.6% of patients with severe haemophilia A. The first analysis, performed in May MCE 2011, lead to two landmark publications. The outcome of this large collaborative research confirms its value for the improvement of haemophilia care. High-quality prospective observational cohorts form an ideal source to study natural history and treatment in rare diseases such as haemophilia. “
“Summary.  Previously treated patients are the first patients to receive novel factor VIII products during clinical investigations under the rationale that a product with increased antigenicity is more likely to be detected in this population because of a low baseline risk of inhibitor formation compared with previously untreated patients.

Descriptive statistics Roscovitine in vitro such as the mean plus standard deviation and percentages were used to characterize the cohort. Categorical variables were tested for statistical significant differences by way of the chi-square or Fisher’s exact test and continuous variables by way of the Student t test; P < 0.05 was considered statistically significant. Out of 207 eligible compounds, 149 belonged to the significant hepatic metabolism group and 55 to the nonsignificant hepatic metabolism group (Supporting Table 1). There were three compounds for which the details of their metabolism could

not be identified (docusate, dicyclomine, nitrofurantoin). The mean number of prescriptions written for the significant hepatic metabolism group was 7,954,705 and was not statistically different from the nonsignificant buy MG-132 hepatic metabolism group (9,068,470, P = 0.5). Thirty-six percent of the compounds

in the significant hepatic metabolism group had an average daily dose of ≥50 mg versus 51% of the compounds in the nonsignificant hepatic metabolism group (P = 0.03). Compared with compounds without significant hepatic metabolism, compounds in the significant hepatic metabolism group were significantly more likely to have reports of ALT ≥3 times the ULN (35% versus 11%, P = 0.001), liver failure (28% versus 9%, P = 0.004), and fatal DILI (23% versus 4%, P = 0.001), but not liver transplantation (9% versus 2%, P = 0.11) or jaundice (46% versus 35%, P = 0.2) (Table 1). Compared with compounds metabolized only through phase I reactions, compounds metabolized through both phase I and II reactions

did not have greater frequency of jaundice (P = 0.74), liver failure (P = 0.36), liver transplantation (P = 0.36), or fatal DILI (P = 0.56), but had significantly higher reports of ALT >3 times the ULN (45% versus 28%, P = 0.03) (Table 2). There were nine compounds with metabolism only through phase II reactions; of these, one had ALT >3 times the ULN, four had jaundice, two had liver failure, one caused liver transplantation, and two caused fatal DILI (Table 2). These nine compounds were levothyroxine, MCE telmisartan, metoclopramide, hydralazine, prednisolone, topiramate, labetalol, and niacin. There were 50 compounds with documented biliary excretion of the parent compound or its active metabolite. When compared with those without biliary excretion, compounds with biliary excretion had significantly higher frequency of jaundice (74% versus 40%, P = 0.0001) but not other hepatic adverse events (Table 3). Table 4 shows the relationship between hepatic adverse events and metabolism through four common CYPs. There are potentially significant differences among different CYP pathways and reports of liver failure and fatal DILI. In general, CYP2C9 and CYP2C19 pathways appeared more toxic than CYP3A and CYP2D6 (Table 4). There were 12 compounds without any hepatic metabolism (Table 5).

Generally, hypercaloric diet, especially rich in trans/saturated fat and cholesterol,

and fructose-sweetened beverages seem to increase visceral adiposity and stimulate hepatic lipid accumulation and progression www.selleckchem.com/products/PF-2341066.html into non-alcoholic steatohepatitis, whereas reducing caloric intake, increasing soy protein and whey consumption, and supplement of monounsaturated fatty acids, omega-3 fatty acids, and probiotics have preventive and therapeutic effects. In addition, choline, fiber, coffee, green tea, and light alcohol drinking might be protective factors for NAFLD. Based on available data, at least 3–5% of weight loss, achieved by hypocaloric diet alone or in conjunction with exercise and behavioral modification, generally

reduces hepatic steatosis, and up to 10% weight loss may be needed to improve hepatic necroinflammation. A sustained AZD3965 price adherence to diet rather than the actual diet type is a major predictor of successful weight loss. Moreover, a healthy diet has benefits beyond weight reduction on NAFLD patients whether obese or of normal weight. Therefore, nutrition serves as a major route of prevention and treatment of NAFLD, and patients with NAFLD should have an individualized diet recommendation. Non-alcoholic fatty liver disease (NAFLD) is an acquired metabolic stress-related liver disease sharing histological similarities to alcoholic liver disease in the absence of substantial alcohol consumption.[1, 2] The spectrum of NAFLD is from 上海皓元 simple steatosis to non-alcoholic steatohepatitis (NASH), and eventually cirrhosis and hepatocellular carcinoma.[1, 2] NAFLD is strongly associated with obesity, dyslipidemia, hypertension, type 2 diabetes mellitus (T2DM), and metabolic syndrome.[1-3] With the rising incidence of obesity and metabolic syndrome in adults and children worldwide, NAFLD is developing into a new and major health problem.[1-3] Currently, NAFLD/NASH is the most common cause of liver disease worldwide and the third most common indication for liver

transplantation in North America.[1] The management of patients with NAFLD consists of treating steatohepatitis and the associated metabolic comorbidities.[1, 2] However, patient with simple steatosis is only needed to treat the associated conditions to prevent hepatic and metabolic complications.[1, 2] Based on available data, most patients with NAFLD have excessive body weight or recently, weight gain; obesity is a common and well-documented risk factor for metabolic syndrome and NAFLD.[1-3] Although promising pharmacological agents and bariatric surgery are emerging, gradually and maintaining weight loss by lifestyle intervention is safe and the most effective treatment for NAFLD and metabolic disorders.[1, 2, 4-9] On one hand, diet alone or in conjunction with increased physical activity and behavior modification is the important measure for successful weight loss.

pylori status (adjusted hazard ratio 4.88, 95% confidence interval [CI] 1.32–18.2, P = 0.018, Table 3). When we reduced the covariates to age and intestinal metaplasia in the lesser curvature of the corpus, which were significant predictors by univariate analysis, the independent significance of open-type, atrophic fundic gastritis, demonstrated by AFI, remained. In the present study, we found that a considerable number of metachronous EGCs developed in patients

who received ESD for EGC after successful eradication of H. pylori. This shows that early recognition and management of multiple EGCs is one of the keys to obtaining good prognosis after treatment of EGC with ESD. Moreover, a wide area of atrophic fundic gastritis diagnosed by AFI was an independent predictor for development of metachronous EGC. Our previous PF-562271 order study has shown that atrophy in biopsy specimens of the lesser curvature of the corpus is associated strongly with gastric cancer risk.11 In the current study, intestinal metaplasia at the lesser curvature of the corpus was associated with development of metachronous EGC, by univariate PF-6463922 price analysis; however, it was not statistically significant in multivariate analysis after adjusting for the extent of atrophic fundic gastritis

diagnosed by AFI. Biopsy evaluates only a narrow area in the whole gastric mucosa, therefore it cannot evaluate the actual extent or distribution of the gastric mucosal changes related to gastric carcinogenesis, such as mucosal atrophy or intestinal metaplasia, and it might cause sampling error. This suggests that the evaluation of gastritis by endoscopy is more suitable for the assessment of risk of development of metachronous EGC compared with point evaluation by biopsy. To diagnose the extent of chronic atrophic fundic gastritis by endoscopy, we have developed the endoscopic Congo red test and have demonstrated that the 上海皓元 method can

identify patients at high risk for development of gastric cancer, in a long-term cohort study.16,17 However, the endoscopic Congo red test requires gastrin injection to stimulate acid secretion, dye spraying over the entire gastric mucosa, and observation of the color change in the Congo red dye for several minutes. Therefore, it has not been used widely in clinical practice because of its complicated and time-consuming procedure, and potential adverse effects of drug or dye administration. AFI is a new endoscopic imaging technology that uses illumination of different wavelength light through a filter in a light source, and it can be performed subsequently to white light endoscopy by simply pressing a small button on the videoendoscope. The method demands neither drug injection nor dye spraying.

pylori status (adjusted hazard ratio 4.88, 95% confidence interval [CI] 1.32–18.2, P = 0.018, Table 3). When we reduced the covariates to age and intestinal metaplasia in the lesser curvature of the corpus, which were significant predictors by univariate analysis, the independent significance of open-type, atrophic fundic gastritis, demonstrated by AFI, remained. In the present study, we found that a considerable number of metachronous EGCs developed in patients

who received ESD for EGC after successful eradication of H. pylori. This shows that early recognition and management of multiple EGCs is one of the keys to obtaining good prognosis after treatment of EGC with ESD. Moreover, a wide area of atrophic fundic gastritis diagnosed by AFI was an independent predictor for development of metachronous EGC. Our previous selleck chemical study has shown that atrophy in biopsy specimens of the lesser curvature of the corpus is associated strongly with gastric cancer risk.11 In the current study, intestinal metaplasia at the lesser curvature of the corpus was associated with development of metachronous EGC, by univariate RXDX-106 solubility dmso analysis; however, it was not statistically significant in multivariate analysis after adjusting for the extent of atrophic fundic gastritis

diagnosed by AFI. Biopsy evaluates only a narrow area in the whole gastric mucosa, therefore it cannot evaluate the actual extent or distribution of the gastric mucosal changes related to gastric carcinogenesis, such as mucosal atrophy or intestinal metaplasia, and it might cause sampling error. This suggests that the evaluation of gastritis by endoscopy is more suitable for the assessment of risk of development of metachronous EGC compared with point evaluation by biopsy. To diagnose the extent of chronic atrophic fundic gastritis by endoscopy, we have developed the endoscopic Congo red test and have demonstrated that the 上海皓元医药股份有限公司 method can

identify patients at high risk for development of gastric cancer, in a long-term cohort study.16,17 However, the endoscopic Congo red test requires gastrin injection to stimulate acid secretion, dye spraying over the entire gastric mucosa, and observation of the color change in the Congo red dye for several minutes. Therefore, it has not been used widely in clinical practice because of its complicated and time-consuming procedure, and potential adverse effects of drug or dye administration. AFI is a new endoscopic imaging technology that uses illumination of different wavelength light through a filter in a light source, and it can be performed subsequently to white light endoscopy by simply pressing a small button on the videoendoscope. The method demands neither drug injection nor dye spraying.