Aberrant GATA2 epigenetic dysregulation induces a GATA2/GATA6 switch in human gastric cancer.

Six GATA transcription factors play important roles in eukaryotic development. Among these, GATA2, an essential factor for the hematopoietic cell lineage, exhibits low expression in human gastric tissues, whereas GATA6, which is crucial for gastrointestinal development and differentiation, is frequently amplified and/or overexpressed in human gastric cancer. Interestingly, we found that GATA6 was overexpressed in human gastric cancer cells only when GATA2 expression was completely absent, thereby showing an inverse correlation between GATA2 and GATA6. In gastric cancer cells that express high GATA6 levels, a GATA2 CpG island is hypermethylated, repressing expression in these cells. In contrast, GATA6 expression is undetectable in GATA2-overexpressing gastric cancer cells, which lack GATA2 DNA methylation. Furthermore, PRC2 complex-mediated transcriptional silencing of GATA6 was observed in the GATA2-overexpressing cells. We also show that the GATA2 and PRC2 complexes are enriched within the GATA6 locus, and that the recruitment of the PRC2 complex is impaired by disrupting GATA2 expression, resulting in GATA6 upregulation. In addition, ectopic GATA2 expression significantly downregulates GATA6 expression, suggesting GATA2 directly represses GATA6. Furthermore, GATA6 downregulation showed antitumor activity by inducing growth arrest. Finally, we show that aberrant GATA2 methylation occurs early during the multistep process of gastric carcinogenesis regardless of Helicobacter pylori infection. Taken together, GATA2 dysregulation by epigenetic modification is associated with unfavorable phenotypes in human gastric cancer cells by allowing GATA6 expression.

Effects of eosinophilic oesophagitis on quality of life in an adult UK population: a case control study.

Eosinophilic oesophagitis (EoE) is a chronic immune-mediated esophageal disease, characterized by symptoms related to esophageal dysfunction and histologically by eosinophil predominant inflammation. Current evidence for an adverse impact on quality of life (QoL) is conflicting and there are no data from a UK population regarding QoL. We conducted a prospective cross-sectional observational study using the Short Form-36 Health Survey, Hospital Dysphagia/Odynophagia Questionnaire, and the EoE Adult Quality of Life Questionnaire to assess QoL and severity of dysphagia in EoE patients, compared to age and gender matched healthy control subjects. Data were also collected on comorbidity and medication use. Eighty-eight subjects were recruited (44 patients). Patients had higher rates of antihistamine and topical (swallowed) corticosteroid use. Physical QoL did not differ between patients and controls, although patients did report a statistically significant lower mental QoL, with small absolute magnitude of difference. Patients reported higher dysphagia scores and these were negatively correlated with both physical and mental QoL. Higher rates of dysphagia and medication use in patients may among other things account for lower mental QoL. However, a higher rate of dysphagia in patients is not associated with a reduced physical QoL. Our findings are of clinical value, particularly when a new diagnosis of EoE is made, as clinicians can reassure patients that their general physical health should not be greatly affected by the diagnosis. Moreover, it may also be useful for patients to be aware that EoE may have an impact on their mental health, but this effect is likely to be small. We therefore advocate education and reassurance in this respect for all patients at diagnosis.

Improved p-type conductivity in Al-rich AlGaN using multidimensional Mg-doped superlattices.

A novel multidimensional Mg-doped superlattice (SL) is proposed to enhance vertical hole conductivity in conventional Mg-doped AlGaN SL which generally suffers from large potential barrier for holes. Electronic structure calculations within the first-principle theoretical framework indicate that the densities of states (DOS) of the valence band nearby the Fermi level are more delocalized along the c-axis than that in conventional SL, and the potential barrier significantly decreases. Hole concentration is greatly enhanced in the barrier of multidimensional SL. Detailed comparisons of partial charges and decomposed DOS reveal that the improvement of vertical conductance may be ascribed to the stronger pz hybridization between Mg and N. Based on the theoretical analysis, highly conductive p-type multidimensional Al0.63Ga0.37N/Al0.51Ga0.49N SLs are grown with identified steps via metalorganic vapor-phase epitaxy. The hole concentration reaches up to 3.5 × 10(18) cm(-3), while the corresponding resistivity reduces to 0.7 Ω cm at room temperature, which is tens times improvement in conductivity compared with that of conventional SLs. High hole concentration can be maintained even at 100 K. High p-type conductivity in Al-rich structural material is an important step for the future design of superior AlGaN-based deep ultraviolet devices.

Asymmetric light reflectance from metal nanoparticle arrays on dielectric surfaces.

Asymmetric light reflectance associated with localized surface plasmons excited in metal nanoparticles on a quartz substrate is observed and analyzed. This phenomenon is explained by the superposition of two waves, the wave reflected by the air/quartz interface and that reflected by the metal nanoparticles, and the resulting interference effects. Far field behavior investigation suggests that zero reflection can be achieved by optimizing the density of metal nanoparticles. Near field behavior investigation suggests that the coupling efficiency of localized surface plasmon can be additionally enhanced by separating the metal NPs from substrates using a thin film with refractive index smaller than the substrate. The latter behavior is confirmed via surface-enhanced Raman spectroscopy studies using metal nanoparticles on Si/SiO2 substrates.

Lifestyle measures in the management of gastro-oesophageal reflux disease: clinical and pathophysiological considerations.

Several lifestyle and dietary factors are commonly cited as risk factors for gastro-oesophageal reflux disease (GORD) and modification of these factors has been advocated as first-line measures for the management of GORD. We performed a systematic review of the literature from 2005 to the present relating to the effect of these factors and their modification on GORD symptoms, physiological parameters of reflux as well as endoscopic appearances. Conflicting results existed for the association between smoking, alcohol and various dietary factors in the development of GORD. These equivocal findings are partly due to methodology problems. There is recent good evidence that weight reduction and smoking cessation are beneficial in reducing GORD symptoms. Clinical and physiological studies also suggest that some physical measures as well as modification of meal size and timing can also be beneficial. However, there is limited evidence for the role of avoiding alcohol and certain dietary ingredients including carbonated drinks, caffeine, fat, spicy foods, chocolate and mint.

Disruption of CTCF/cohesin-mediated high-order chromatin structures by DNA methylation downregulates PTGS2 expression.

The CCCTC-binding factor (CTCF)/cohesin complex regulates gene transcription via high-order chromatin organization of the genome. De novo methylation of CpG islands in the promoter region is an epigenetic hallmark of gene silencing in cancer. Although the CTCF/cohesin complex preferentially targets hypomethylated DNA, it remains unclear whether the CTCF/cohesin-mediated high-order chromatin structure is affected by DNA methylation during tumorigenesis. We found that DNA methylation downregulates the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), which is an inducible, rate-limiting enzyme for prostaglandin synthesis, by disrupting CTCF/cohesin-mediated chromatin looping. We show that the CTCF/cohesin complex is enriched near a CpG island associated with PTGS2 and that the PTGS2 locus forms chromatin loops through methylation-sensitive binding of the CTCF/cohesin complex. DNA methylation abolishes the association of the CTCF/cohesin complex with the PTGS2 CpG island. Disruption of chromatin looping by DNA methylation abrogates the enrichment of transcriptional components, such as positive elongation factor b, at the transcriptional start site of the PTGS2 locus. These alterations result in the downregulation of PTGS2. Our results provide evidence that CTCF/cohesin-mediated chromatin looping of the PTGS2 locus is dynamically influenced by the DNA methylation status.

Conducting multinational, cross-cultural research in the functional gastrointestinal disorders: issues and recommendations. A Rome Foundation working team report.

BACKGROUND: Cross-cultural, multinational research can advance the field of functional gastrointestinal disorders (FGIDs). Cross-cultural comparative research can make a significant contribution in areas such as epidemiology, genetics, psychosocial modulators, symptom reporting and interpretation, extra-intestinal co-morbidity, diagnosis and treatment, determinants of disease severity, health care utilisation, and health-related quality of life, all issues that can be affected by geographical region, culture, ethnicity and race. AIMS: To identify methodological challenges for cross-cultural, multinational research, and suggest possible solutions. METHODS: This report, which summarises the full report of a working team established by the Rome Foundation that is available on the Internet, reflects an effort by an international committee of FGID clinicians and researchers. It is based on comprehensive literature reviews and expert opinion. RESULTS: Cross-cultural, multinational research is important and feasible, but has barriers to successful implementation. This report contains recommendations for future research relating to study design, subject recruitment, availability of appropriate study instruments, translation and validation of study instruments, documenting confounders, statistical analyses and reporting of results. CONCLUSIONS: Advances in study design and methodology, as well as cross-cultural research competence, have not matched technological advancements. The development of multinational research networks and cross-cultural research collaboration is still in its early stages. This report is intended to be aspirational rather than prescriptive, so we present recommendations, not guidelines. We aim to raise awareness of these issues and to pose higher standards, but not to discourage investigators from doing what is feasible in any particular setting.

Isolation and characterization of novel EST-SSRs in the showy dendrobium, Dendrobium nobile (Orchidaceae).

Expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers could enrich the current resource of molecular markers. In this study, microsatellite markers were developed for Dendrobium nobile Lindl. by mining the ESTs. Twenty-eight EST-SSRs amplified 2 to 6 nucleotide repeats with a mean number of 2.82 alleles per locus. The observed and expected heterozygosities per locus ranged from 0.158 to 0.579 and 0.422 to 0.752, respectively. These novel EST-SSRs enriched the current resource of molecular markers for the Dendrobium genus and would facilitate further applications in germplasm appraisal, evolution and genetic diversity studies, genetic mapping, and plant breeding of D. nobile and other congeneric species.

Comparison of alfuzosin 10 mg with or without propiverine 10 mg, 20 mg in men with lower urinary tract symptom and an overactive bladder: randomised, single-blind, prospective study.

PURPOSE: The efficacy and safety of treatment with alfuzosin 10 mg plus propiverine 10 or 20 mg in men with lower urinary tract symptoms (LUTS) and an overactive bladder were investigated. MATERIALS AND METHODS: In this parallel-arm, prospective, multicentre, single-blind study, men who were ≥ 40 years old, had an International Prostate Symptom Score (IPSS) of ≥ 8, an Overactive Bladder Symptom Score (OABSS) of ≥ 3 and an OABSS urgency item score of ≥ 2 were randomised in a 1 : 1 :1 ratio to receive alfuzosin 10 mg alone (Group A) or with propiverine 10 mg (Group B) or 20 mg (Group C) for 8 weeks. Four and 8 weeks after commencing treatment, OABSS was measured along with IPSS, maximal urinary flow rate (Qmax ) and postvoid residual volume (PVR). Adverse events were recorded. RESULTS: A total of 135 men, including 43 in Group A, 48 in Group B and 44 in Group C, completed the study. Relative to baseline, all groups demonstrated significant reductions in OABSS and the IPSS after eight treatment weeks (p < 0.005). The improvement of OABSS in Group C was significantly greater than Group A and B (Group A: 0.70 ± 1.94; Group B: 2.50 ± 2.98; Group C: 4.30 ± 3.40; p < 0.005). An observed improvement of Qmax and PVR in the three groups did not achieve statistical significance. Overall adverse event rates were higher in Group C but not significant compared with others. CONCLUSION: In patients with LUTS and overactive bladder, combined therapy with alfuzosin 10 mg plus propiverine 20 mg was significantly more effective than alfuzosin monotherapy and propiverine 10 mg combined therapy in terms of improving OABSS while not significantly affecting Qmax or PVR.

Phenotypic traits, virulence-associated gene profile and genetic relatedness of Edwardsiella tarda isolates from Japanese eel Anguilla japonica in Korea.

UNLABELLED: Edwardsiella tarda is the predominant bacterium in farm-cultured eel in Korea. Here, we evaluated the heterogeneity of 37 E. tarda isolates derived from Japanese eel with various origins (olive flounder, common carp and ornamental fish) between 2003 and 2010. Regardless of origins, the biochemical characteristics of E. tarda isolates were homogenous except hydrogen sulfide production, citrate utilization and mannitol fermentation. Based on the phylogenetic analysis of 16S rRNA, E. tarda isolates could be classified into two subgroups and displayed a close relation with Edwardsiella ictaluri and Edwardsiella hosinae lineages, suggesting that the subgroup I has been a predominant type in the Jeonnam and Jeonbuk provinces. I-CeuI-based pulsed-field gel electrophoresis (PFGE) typing showed that the isolates from Japanese eels belonged to 11 pulsotypes, indicating that the presence of highly genomic diversity. Additionally, two isolates, ET-060 and ET-191, showed a high frequency of virulence genes (100%) and caused 90% and 60% mortality in Japanese eel, respectively. This finding suggests a substantial congruence of virulence gene profiles and pathogenicity. Our results demonstrate that the intraspecific diversity within E. tarda strains from Japanese eel has been in prior existence. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the biochemical characteristics, the phylogenetic property of the 16S rRNA gene and PFGE types of Edwardsiella tarda, we could identify the intraspecific diversity of isolates from Japanese eel, Anguilla japonica in Korea. In addition, this study describes the strong congruence of virulence-related genes and pathogenicity, suggesting that the virulence profile may be useful tool for prediction of pathogenicity.

Systematic review: worldwide variation in the frequency of coeliac disease and changes over time.

BACKGROUND: Coeliac disease (CD), originally thought to be largely confined to Northern Europe and Australasia and uncommon in North America and the Middle East, is now recognised to be equally common in all these countries. It is still thought to be rare in the Orient and Sub-Saharan Africa. AIM: To assess geographical differences and time trends in the frequency of CD. METHODS: Medline and Embase searches were conducted on 10 November 2012, from 1946 and 1980 respectively, using the key words: coeliac disease or celiac disease + prevalence or incidence or frequency. RESULTS: There were significant intra- and inter-country differences in the prevalence and incidence of CD. Only 24 ethnic Chinese and Japanese patients have been reported in the English literature. Of CD-associated HLA DQ antigens, DQ2 occurs in 5-10% of Chinese and sub-Saharan Africans, compared to 5-20% in Western Europe. DQ8 occurs in 5-10% of English, Tunisians and Iranians, but in <5% of Eastern Europeans, Americans and Asians. The prevalence and incidence of both clinically and serologically diagnosed CD increased in recent years. These geographical and temporal differences seem genuine, although variable indices of suspicion and availability of diagnostic facilities are confounding factors. CONCLUSIONS: Coeliac disease is increasing in frequency, with significant geographical differences. Although few cases have been described to date in the Orient and Sub-Saharan Africa, there is a significant prevalence of HLA DQ2 and wheat consumption is of the same order as that in Western Europe. CD may therefore become more common in the future in these countries.

A retrospective study of clinical outcomes of α-blocker or finasteride monotherapy followed by combination therapy: determination of the period of combination therapy of α-blocker and finasteride.

AIMS: We investigated outcomes after discontinuing alpha-blockers or finasteride in patients who initially received combination therapy and compared differences by duration of combination therapy. METHODS: Patients with international prostate symptom score ≥ 8, serum prostatic-specific antigen (PSA) < 4 ng/ml, prostate volume > 25 cm(3) and combination therapy of alpha-blockers and finasteride for more than 6 months were classified into three groups. Group 1 continued combination therapy; group 2, alpha-blockers monotherapy; and group 3, finasteride monotherapy. All parameters were evaluated before and after changing to monotherapy. Patients who received combination therapy for 6-9 months and those who received combination therapy for longer were comparatively analysed. RESULTS: Mean age of the 106 patients was 66.9 ± 7.8 years. No significant differences in baseline symptom scores, PSA or prostate volume were found. Following combination therapy, symptom scores, and quality of life (QoL) decreased for all groups. Group 1 maintained decreased PSA and prostate volume, and improved uroflowmetric profiles. No differences in uroflowmetric parameters were found after 6 months. Group 2 maintained improved symptoms, QoL and uroflowmetric profiles, although PSA and prostate volume returned to baseline. Group 3 maintained lowered PSA and prostate volume, whereas Qmax returned to baseline. QoL scores showed no change. Patients who received combination therapy for ≥9 months improved more in symptoms and QoL than those who received shorter combination therapy. CONCLUSIONS: Discontinuation of alpha-blockers or finasteride after combination therapy for ≥ 6 months maintained improvements in symptoms. The appropriate period of combination therapy was ≥ 9 months.

Reliability and validity of the Korean version of the manual ability classification system for children with cerebral palsy.

BACKGROUND: To investigate the inter- and intra-rater reliability of the Korean version of the Manual Ability Classification System (MACS) for children with cerebral palsy. METHODS: After a two-step forward and one-step backward translation, the inter-rater reliability of the Korean version of the MACS was assessed separately by parents, occupational therapists and physicians. A second assessment for intra-rater reliability was performed 4 weeks later. RESULTS: Sixty-nine children were enrolled. The intra-class correlation coefficients were 0.956 between occupational therapists and physicians, 0.927 between parents and physicians, and 0.960 between parents and occupational therapists. Intra-rater reliability ranged from 0.965 to 0.987. CONCLUSIONS: The Korean version of the MACS is reliable and valid and is suitable for assessing manual ability in Korean children with cerebral palsy.

Upper gastrointestinal haemorrhage in the acute cardiac care setting: antiplatelets and endoscopy.

Upper gastrointestinal haemorrhage (UGIH) in cardiac patients receiving antiplatelets presents a difficult management problem. The aim of this study was to describe a series of cardiac inpatients receiving antiplatelets who underwent endoscopy for an acute UGIH. Cardiac inpatients receiving antiplatelets and requiring endoscopy for UGIH over an 18-month period were followed up. Forty-one patients were studied. Most patients (25 [61%]) presented with melaena. Antiplatelets were withheld in 34 (83%) patients; predominantly in those with higher pre-endoscopy Rockall scores (median, 4; interquartile range [IQR], 3-5 versus median, 3; IQR, 2-4; P < 0.05). Positive findings were identified at endoscopy in 80%. Duodenal ulcers were the most common lesion and adrenaline the most common method of haemostasis. Median time to first endoscopy was 0 (IQR, 0-1) days. Seven (17%) patients re-bled, median Rockall score was six (IQR, 4-8). Three (7%) patients experienced procedural complications, two patients became hypoxic and one patient died. Following endoscopy, antiplatelets were restarted after a median of three (IQR, 3-5) days. On discharge, 27/28 (96%) patients continued with antiplatelet and proton-pump inhibitor therapy. Thirty-day inpatient mortality was 7% (3 patients). One patient re-bled within six months of discharge. Endoscopy helped assess the risk of re-bleeding and timing of antiplatelet re-introduction in cardiac inpatients experiencing UGIH.

National-wide data on the treatment of BPH in Korea.

The healthcare system in Korea provides coverage to all the people who are residing in Korea, so the data of the Korea healthcare system are national-wide and relatively accurate. We obtained the recent 5-year data (2004-2008) on the treatment of BPH from the national health insurance system. We tried to determine the trends or changes of BPH treatments in Korea. Over 3.8 million men visited clinics and were prescribed one or more BPH medications, and more than 44 000 men underwent surgical treatment during 2004-2008. Compared with the year 2004, two times the patients were prescribed BPH medications in 2008. With respect to the surgical treatment, the number of cases was increased 1.6 times in 2006 compared with the previous years. The most commonly used surgical option was TURP before 2006, but laser therapy was carried out as much as TURP in 2006 and in the following years. The relative risk of laser therapy in the 50 s is 1.53 (95% CI is 1.47-1.59). In conclusion, our national-wide data for the Korean BPH patients show that these patients' medical treatment increased during the 5 years from 2004 to 2008. Laser treatment had increased and it might replace TURP in several years.

Uncomplicated diverticular disease is not a common cause of colonic symptoms.

BACKGROUND: Colonic diverticular disease is common among older individuals whereas colonic symptoms, such as those of irritable bowel syndrome, are frequent in the general population. AIM: To determine among patients in secondary care, if uncomplicated diverticular disease is a common cause of colonic symptoms. METHODS: Patients aged ≥50 years attending gastroenterology out-patient clinics or scheduled for colonoscopy or barium enema in a secondary care hospital were invited to take part. Those with structural gastrointestinal diseases were excluded. Participants completed a locally validated Rome II questionnaire on colonic symptoms. Patients with diverticular disease were compared with those without. RESULTS: Seven hundred and eighty four patients with no structural pathology other than diverticular disease or benign colonic polyps completed the study. A total of 744 patients underwent colonoscopy, 40 barium enema. Of these, 281 patients had diverticular disease. Among patients with and without diverticular disease, the frequency of abdominal pain, diarrhoea, constipation and irritable bowel syndrome were 123 (44%) and 226 (46%), 44 (16%) and 80 (17%), 38 (14%) and 80 (17%) and 66 (25%) and 119 (25%), respectively (N.S.). CONCLUSION: Uncomplicated colonic diverticular disease is not a common cause of colonic symptoms among patients in secondary care.

The increasing hospital disease burden of haemochromatosis in England.

BACKGROUND: Hereditary haemochromatosis is a preventable cause of liver disease with an increasing disease burden. AIMS: To investigate time trends for hospital admission ascribed to haemochromatosis in England during the period from 1989/1990 to 2002/2003 and mortality from 1979 to 2005. METHODS: Hospital admission data, relating to both in-patients and day-cases, were obtained from the Hospital Episodes Statistics service. Mortality rates for England and Wales were provided by the Office for National Statistics. RESULTS: Haemochromatosis is an uncommon cause for hospital admission. Age-standardized in-patient admission rates increased over the study period by 269% in men and by 290% in women: (from 0.64 to 2.36 and from 0.21 to 0.81 per year per 100 000). The increase in age-standardized day-case admission rates was even higher (men: from 2.78 to 34.9 per year per 100 000, 1155%; women: from 0.58 to 11.67 per year per 100 000, 1924%). Haemochromatosis was recorded as an uncommon cause of death. CONCLUSIONS: Hospital in-patient and day case admissions for haemochromatosis increased markedly over the study period while mortality remained low. Both admission rates and mortality were higher in men than in women. The increase in admission rate may reflect improved recognition and diagnosis of iron overload disorders following identification of the HFE gene.

Comparison of desaturation and resaturation response times between transmission and reflectance pulse oximeters.

BACKGROUND: In general, there is a response time between actual arterial hypoxemia and its detection by pulse oximeters. We compared the desaturation and resaturation response times between two types of pulse oximeters, transmission and reflectance pulse oximeters, to find out which oximeter has a more rapid response time. METHODS: Thirty-three ASA 1 or 2 patients were enrolled in this study. A transmission pulse oximeter was placed on the index finger and a reflectance pulse oximeter was placed on the forehead and monitored simultaneously. After the induction of general anesthesia without pre-oxygenation, we waited until the oxygen saturation value of any of two pulse oximeters declined to 90%, and then mask ventilation was started with 100% oxygen. Oxygen saturation was recorded at an interval of 2 s during this time. RESULTS: The desaturation response time of SpO(2) to 95% after apnea was 82.0 s (interquartile range: 67.0-98.5 s) vs. 94.0 s (interquartile range: 84.0-106.5 s) (P<0.001) and SpO(2) to 90% was 94.0 s (interquartile range: 75.5-109.5 s) vs. 100.0 s (interquartile range: 84.5-114.5 s) (P<0.001) in the reflectance and transmission oximeters, respectively. The resaturation response time from mask ventilation to 100% SpO(2) was 23.2+/-5.6 vs. 28.9+/-7.6 s (P<0.001) in the reflectance and transmission oximeters, respectively. CONCLUSION: In clinical situations in which rapid changes in oxygen saturation are expected, we recommend the forehead reflectance pulse oximeter because it responds more quickly in detecting oxygen desaturation and resaturation compared with the transmission pulse oximeter.

A multicenter phase II study of belotecan, new camptothecin analogue, in patients with previously untreated extensive stage disease small cell lung cancer.

Belotecan (Camtobell, CKD602) is a new camptothecin derivative antitumor agent that belongs to the topoisomerase inhibitors. The aim of this phase II study was to evaluate the efficacy and safety of single agent belotecan in patients with small cell lung cancer (SCLC). Patients with previously untreated extensive stage disease (ED) SCLC were entered into the study. Belotecan was given by daily intravenous infusion at 0.5mg/m(2)/day for 5 consecutive days, every 3 weeks. 62 patients were enrolled in this study. The overall response rate to chemotherapy on an intention-to-treat basis was 53.2%. The median overall survival was 10.4 months, the median time to progression 4.6 months, and the 1-year survival rate 49.9%. The most common toxicity was hematologic. Grade 3/4 neutropenia occurred in 71.0% of patients and grade 3/4 thrombocytopenia 12.9%. Non-hematologic toxicity of grade 3 or 4 was low. The results suggest that belotecan is relatively active and well tolerable as single agent in patients with ED SCLC. Further investigations with platinum or other active agents are needed.