Self-Assembly of Concentric Hexagons and Hierarchical Self-Assembly of Supramolecular Metal-Organic Nanoribbons at the Solid/Liquid Interface.

In an effort to exert more precise control over structural features of supramolecules, a series of giant concentric hexagons were assembled as discrete structures using tetratopic terpyridine (tpy) ligands. In preparation of tetratopic ligand, pyrylium and pyridinium salts chemistry significantly facilitated synthesis. The key compounds were obtained by condensation reactions of pyrylium salts with corresponding primary amine derivatives in good yields. These discrete metallo-supramolecular concentric hexagons were fully characterized by NMR, ESI-MS, TWIM-MS, and TEM, establishing their hexagon-in-hexagon architectures. The combination of different tetratopic ligands also assembled hybrid concentric hexagons with increasing diversity and complexity. Furthermore, these concentric hexagon supramolecules with precisely controlled shapes and sizes were utilized as building blocks to hierarchically self-assemble supramolecular metal-organic nanoribbons (SMON) at solid-liquid interfaces. Ambient STM imaging showed the formation of long 1D SMON rather than 2D assembly on the basal plane of highly oriented pyrolytic graphite (HOPG) surface after simple dropcasting of the solution of preassembled concentric hexagons onto a freshly cleaved surface of HOPG. This wet chemical method based on self-assembly may offer simple, economical, and scalable routes to deliver complex materials.

Association Between Carotid Plaque and Alzheimer's Disease Cerebrospinal Fluid Biomarkers and Cognitive Function in Cognitively Intact Adults: The CABLE Study.

Background: The association between carotid plaque and cognitive decline has recently been reported. However, the current research evidence is insufficient, and the possible causes of cognitive changes are unknown. Objective: This study aims to explore the relationships between carotid plaque and cognition functions, cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers in cognitively intact adults, and try to study the underlying mechanisms. Methods: We enrolled 165 cognitively normal participants from the Chinese Alzheimer's Biomarker and LifestylE (CABLE) study, who had CSF AD biomarker measurements and carotid ultrasound. Linear modeling was used to assess the association of carotid plaque with CSF biomarkers and cognition. Additionally, mediation analysis was conducted through 10,000 bootstrapped iterations to explore potential links between carotid plaque, AD pathology, and cognition. Results: We found that carotid plaque exhibited significant correlations with Aß42 (ß = -1.173, p = 0.022), Aß42/Aß40 (ß = -0.092, p < 0.001), P-tau/Aß42 (ß = 0.110, p = 0.045), and T-tau/Aß42 (ß = 0.451, p = 0.010). A significant correlation between carotid plaque and cognition decline was also found in men (ß = -0.129, p = 0.021), and mediation analyses revealed that the effect of carotid plaque on cognitive function could be mediated by Aß42/Aß40 (proportion of mediation = 55.8%), P-tau/Aß42 (proportion of mediation = 51.6%, p = 0.015) and T-tau/Aß42 (proportion of mediation = 43.8%, p = 0.015) mediated. Conclusions: This study demonstrated the link between carotid plaque and CSF AD biomarkers in cognitively intact adults, and the important role that AD pathology may play in the correlation between carotid plaque and cognitive changes.

Associations of Multimorbidity with Cerebrospinal Fluid Biomarkers for Neurodegenerative Disorders in Early Parkinson's Disease: A Cross-sectional and Longitudinal Study.

OBJECT: The study aims to determine whether multimorbidity status is associated with cerebrospinal fluid (CSF) biomarkers for neurodegenerative disorders. METHODS: A total of 827 patients were enrolled from the Parkinson's Progression Markers Initiative (PPMI) database, including 638 patients with early-stage Parkinson's disease (PD) and 189 healthy controls (HCs). Multimorbidity status was evaluated based on the count of long-term conditions (LTCs) and the multimorbidity pattern. Using linear regression models, cross-sectional and longitudinal analyses were conducted to assess the associations of multimorbidity status with CSF biomarkers for neurodegenerative disorders, including α-synuclein (αSyn), amyloid-ß42 (Aß42), total tau (t-tau), phosphorylated tau (p-tau), glial fibrillary acidic protein (GFAP), and neurofilament light chain protein (NfL). RESULTS: At baseline, the CSF t-tau (p = 0.010), p-tau (p = 0.034), and NfL (p = 0.049) levels showed significant differences across the three categories of LTC counts. In the longitudinal analysis, the presence of LTCs was associated with lower Aß42 (ß < -0.001, p = 0.020), and higher t-tau (ß = 0.007, p = 0.026), GFAP (ß = 0.013, p = 0.022) and NfL (ß = 0.020, p = 0.012); Participants with tumor/musculoskeletal/mental disorders showed higher CSF levels of t-tau (ß = 0.016, p = 0.011) and p-tau (ß = 0.032, p = 0.044) than those without multimorbidity. CONCLUSION: Multimorbidity, especially severe multimorbidity and the pattern of mental/musculoskeletal/ tumor disorders, was associated with CSF biomarkers for neurodegenerative disorders in early-stage PD patients, suggesting that multimorbidity might play a crucial role in aggravating neuronal damage in neurodegenerative diseases.

Transarterial Embolization Enhances Programmed Cell Death Ligand 1 Expression and Influences CD8+T Lymphocytes Cytotoxicity in an Orthotopic Hepatocellular Carcinoma Rat Model.

PURPOSE: To investigate the influence of transarterial embolization (TAE) on programmed cell death-ligand 1(PD-L1) expression and CD8+T tumour infiltrative lymphocyte cytotoxicity in the Sprague-Dawley (SD) rat model of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: An orthotopic HCC model was established in twenty SD rats treated with TAE (lipiodol, n = 10) or sham (normal saline, n = 10) using homologous N1S1 hepatoma cells. Rats were euthanized 1 week after embolization. Flow cytometry was used to assess the proportion of CD4+T, CD8+T and programmed cell death-1+(PD-1+) CD8+T lymphocytes in the spleens and tumours. Distribution of CD8+T, granzyme-B+CD8+T lymphocytes and PD-L1+ cells was assessed by immunohistochemistry (IHC) or multiplex IHC. p value < 0.05 was considered statistically significant. RESULTS: The CD4/CD8 ratio and PD-1+CD8+ T lymphocytes exhibited higher values in TAE-treated tumours compared to sham-treated tumours (p = 0.021 and p = 0.071, respectively). Conversely, the number of CD8+T lymphocytes was decreased in TAE-treated tumours (p = 0.043), especially in the central region (p = 0.045). However, more CD8+T lymphocytes were found infiltrating the marginal region than central region in TAE-treated tumours (p = 0.046). The proportion of granzyme-B+CD8+T lymphocytes and the PD-L1 positive areas was elevated in tumours that treated with TAE (p all < 0.05). There was a negative correlation between PD-L1 expression and the number of infiltration of CD8+ T lymphocytes (p = 0.036). CONCLUSIONS: Immune cells are distributed unevenly in the tumours after TAE. The intrinsic induction state of the tumour after embolization may be insufficient to elicit a maximal response to PD-1/PD-L1 inhibitors.

[Inhibitory effect of paeoniflorin on the collagen production by fibroblasts through IL-13/STAT6 signaling pathway].

OBJECTIVE: To observe the effects of paeoniflorin on 3T3 fibroblast activation, proliferation and collagen production through IL-13/STAT6 signaling pathway. METHODS: 3T3 cell strain was cultured with serum-free medium for 12 h, then stimulated by paeoniflorin (200, 400, 600, 800, and 1000 mg/L) or rIL-13 (6.25, 12.5, 50, 100, and 200 microg/L) for another 24 h. At the same time the blank control group for paeoniflorin or rIL-13 was observed. 3T3 cell proliferation was assayed by Cell Counting Kit-8 (CCK-8), and an appropriate concentration (100 microg/L) of rIL-13 was chosen according to the result of cell proliferation. Subsequently, 3T3 cell cultured with serum-free medium for 12 h was stimulated by 100 microg/L rIL-13 for 12 h, and then was treated with different concentrations of paeoniflorin (200, 400, 600, 800, and 1000 mg/L) for another 24 h. Untreated 3T3 cell served as blank control Cell proliferation was measured by CCK-8. Hydroxyproline content in cell supernatant was determined by alkaline lysis method. IL-13Ralpha1, alpha-SMA and STAT6 protein expression were detected by Western blotting. Col-I, Col-III, IL-13Ralpha1 and STAT6 mRNA expression were analyzed by RT-PCR. RESULTS: Paeoniflorin inhibited 3T3 cell proliferation in a concentration-dependent manner (r = -0.980, P < 0.01), and there was a statistically significant difference among all groups (F = 198.599, P < 0.01). rIL-13 caused a remarkably concentration-dependent increase in proliferation of 3T3 cells (r = 0.538, P < 0.05). Paeoniflorin (200, 400, 600, 800, and 1000 mg/L) inhibited proliferation of 3T3 cell stimulated by rIL-13 in a concentration-dependent manner (1.780 +/- 0.177, 1.636 +/- 0.073, 0.965 +/- 0.066, 0.623 +/- 0.037, 0337 +/- 0.022, r = -0.971, P < 0.01), and among all groups there existed a significant difference (F = 198.537, P < 0.01). Moreover, paeoniflorin also suppressed secretion of hydroxyproline from 3T3 cell stimulated by rIL-13 in a concentration-dependent manner (3.030 +/- 0.094, 2.976 +/- 0.047, 2.814 +/- 0.047, 2.652 +/- 0.124, 2.408 +/- 0.124, r = -0.916, P < 0.01) with a statistical significance among all groups (F = 13.642, P < 0.01). Further investigations showed that paeoniflorin decreased both protein expression of alpha-SMA, IL-13Ralpha1, and STAT6, and mRNA expression of Col-I, Col-III, IL-13Ralpha1, and STAT6 in 3T3 cell stimulated by rIL-13. CONCLUSION: Paeoniflorin inhibits activation, proliferation of fibroblasts and production of collagen from fibroblasts through IL-13/STAT6 signaling pathway, which might be one of mechanisms of anti-hepatic fibrosis of paeoniflorin in schistosomiasis japonica.

Rice straw application with different water regimes stimulate enzymes activity and improve aggregates and their organic carbon contents in a paddy soil.

Soil organic carbon plays considerable roles in binding soil particles together forming aggregates. Carbon (C) incorporated within these aggregates is thought to be microbially processed; thus, investigating changes in microbial activities i.e. dehydrogenase, urease, catalase and phosphatase enzymes may explain, to some extent, the dynamics and probably mechanisms responsible of formation of these aggregates. Since, soil water content (SWC) may take part in stimulating/lessening activities of organic matter decomposers; thus, this study aimed at investigating the effects of rice straw as a source of organic C in combination with variable SWC on bioaccumulation of C within different soil aggregate size fractions (2000-250, 250-53 and < 53 µm) and hence formation of these aggregates. To achieve these objectives, a pot experiment was conducted for 90 days, including five water levels i.e. maintaining a water head 1 cm above the soil surface (W1), 100% of the saturation percentage, SP (W2), 80% of SP (W3), 65% of SP (W4) and 50% of SP (W5), beside of two rates of applied rice straw i.e. 0 and 15 g kg-1 (w/w). Results revealed that application of rice straw at a rate of 15 g kg-1 increased the activities of dehydrogenase, urease, neutral phosphatase and catalase enzymes within the first 60 days after application; thereafter, activities of the first three enzymes decreased considerably. Likewise, formation of soil macro- (2000-250 µm) and micro-aggregates (250-53 µm) increased by the end of the experimental period. The highest concentrations of soil carbon were incorporated within soil macro-aggregate, whereas the least C content was found within the "silt + clay" fraction. Increasing SWC resulted in significant reductions in activities of the aforementioned enzymes and consequent reductions occurred in soil aggregation. Carbon content within aggregates sized <250 µm were significantly correlated with the percentage of these aggregates in soil. Thus, soil aggregation is thought to be the byproduct of an aerobic biosynthetic microbial process in which more stable hydrophobic organic C existed mainly in macropores. This process probably occurred within the first 60 days after RS application.

Comparing rice production systems in China: Economic output and carbon footprint.

In recent years, many rotational and integrated rice production systems coupled with several greenhouse gas (GHG) emissions mitigation practices have been developed and adopted for demand of low carbon production. However, there have been only few studies about comparisons on the balance between high production and mitigation of GHG emissions in different rice production systems. We therefore aimed to evaluate economic output and carbon footprint of different rice production systems, based on several long-term experiments conducted by our lab. CH4 and N2O emission were measured by the same static chamber/gas chromatogram measurement procedure in different rice production systems, including rice-fallow, rice-rapeseed, rice-wheat, double rice, and integrated rice-crayfish production system. Then, we applied the DeNitrification DeComposition model to simulate CH4 and N2O emission over different years under the same condition for comparison. Carbon footprint was calculated following the process-based life cycle assessment (PLCA) methodology. The economic benefit of rice production systems was assessed by cost-benefit analysis. According to the analysis, the double-rice production system exhibited the highest intensity of carbon footprint (ICF = 4.14 kg CO2-eq yuan-1), rain-fed treatment in the rice-rapeseed system had the lowest (ICF = 0.68 kg CO2-eq yuan-1). The intensity of carbon footprint in different treatments in the integrated rice-crayfish production system was around 0.8 kg CO2-eq yuan-1. Overall, the results of this case study suggest: (1) the proposed practices in different rice production systems are no straw returning (rice-fallow), no-tillage without straw returning (rice-wheat), rain-fed farming (rice-rapeseed), no insect and no inoculation (double rice), and feeding with straw returning (rice-crayfish); (2) rotational and integrated systems can achieve high net output with low carbon emission; (3) reducing the amount of nitrogenous fertilizer application is the most important and effective GHG mitigation practice for rotational systems.

Marked thickening of muscularis mucosae and submucosa in the gastric cardia: A histopathological study of 110 surgical resection cases.

OBJECTIVE: To investigate histopathologic changes of muscularis mucosae (MM) and submucosa in the gastric cardia. METHODS: We performed a histopathology study of 50 distal esophagectomies with proximal gastrectomies for esophageal squamous cell carcinoma as the study (non-cancerous cardiac) group and 60 gastrectomies for early gastric cardiac carcinoma as the cancer group. The gastroesophageal junction was defined as the distal end of squamous epithelium, multilayered epithelium, or deep esophageal glands or ducts. Gastric cardia (n = 110) was defined as the presence of cardiac and cardio-oxyntic mucosae distal to the gastroesophageal junction. RESULTS: The average thickness of MM and submucosa in the cardia was 1.04 and 1.41 mm, respectively, which was significantly thicker than that in distal stomach (n = 34) (0.22 and 0.99 mm) or distal esophagus (n = 92) (0.60 and 1.15 mm). In the cardia, thickened MM displayed frayed muscle fibers (93.3%) with a significantly higher prevalence of entrapped glands, cysts, and lymphoid follicles than in the distal stomach or distal esophagus. In the submucosa fatty changes, cysts, and abnormal arteries were significantly more common in the cardia than in the distal stomach or distal esophagus. Compared with the study group, the cardia in the cancer group showed significantly thicker MM (average 1.31 vs 0.72 mm) and submucosa (average 1.61 vs 1.16 mm), more frequent frayed MM (93.3% vs 60.0%), prolapse-like changes (50.0% vs 2.0%), and cysts (26.7% vs 4.0%). CONCLUSION: MM and submucosa of the cardia were significantly thickened, especially in early gastric cardiac carcinomas.

Risk factors of lymph node metastasis in 734 early gastric carcinoma radical resections in a Chinese population.

OBJECTIVE: To investigate the risk factors of lymph node metastasis (LNM) in early gastric carcinoma (EGC) in a Chinese population. METHODS: The data were analyzed to determine risk factors of LNM. The patients' characteristics, the tumor's location, gross features, histological type, differentiation, invasive depth, lymphovascular invasion (LVI), perineural invasion and the numbers of lymph nodes retrieved and involved were statistically analyzed. RESULTS: A total of 734 patients with EGC were finally enrolled in the study, and LNM was present in 14.2% (104/734) of them. By univariate analysis, significant risk factors for LNM included depressed or excavated gross patterns, size ≥1.0 cm, SM2, moderate/poor differentiation, histological type of hepatoid or micropapillary adenocarcinoma, LVI, perineural invasion and tumor necrosis. By multivariate analysis, independent risk factors for LNM were size ≥3.0 cm (odds ratio [OR] 4.9), SM2 (OR 2.4), moderate (OR 3.6) and poor (OR 5.0) differentiation, LVI (OR 3.1) and tumor necrosis (OR 1.7). Early gastric cardiac carcinoma (OR 0.3) had a significantly lower risk than non-cardiac carcinoma. No LNM was identified in 67 EGC of <1.0 cm in size and without poor differentiation, in 142 intramucosal EGC cases of smaller than 2.0 cm and without poor differentiation, in 129 cases of well-differentiated EGC without deep SM2 submucosal invasion, or in 54 intramucosal EGC located in the gastric cardia. CONCLUSION: Independent risk factors for LNM in EGC include tumor size ≥3.0 cm, SM2 invasion, moderate/poor differentiation, LVI and tumor necrosis. Early cardiac carcinoma had a significantly lower risk of LNM than non-cardiac EGC.

The long non-coding RNA NONHSAT062994 inhibits colorectal cancer by inactivating Akt signaling.

The aberrant expression of long noncoding RNAs (lncRNAs) is implicated in cancer development and progression. However, the clinical significance and mechanism by which NONHSAT062994 regulates colorectal cancer (CRC) is unknown. We here reported that NONHSAT062994 was significantly downregulated in human CRC tissues and cell lines. Moreover, its expression was inversely correlated with tumor size and overall survival (OS) time in CRC patients. In CRC cells, the overexpression and knockdown of NONHSAT062994 inhibited and enhanced CRC cell growth, respectively, in vitro and in vivo. Mechanistically, NONHSAT062994 functioned as a tumor suppressor to inhibit CRC cell growth by inactivating Akt signaling. Notably, the NONHSAT062994 expression status was negatively correlated with the Akt downstream targets c-Myc and Cyclin D1 in clinical CRC samples. The current findings suggest that NONHSAT062994 plays a critical role in the development of CRC by regulating Akt signaling, and identified a candidate prognostic biomarker or potential therapeutic target for CRC patients.

Risk factors of lymph node metastasis in 1620 early gastric carcinoma radical resections in Jiangsu Province in China: A multicenter clinicopathological study.

OBJECTIVE: To investigate risk factors of lymph node metastasis (LNM) in early gastric carcinoma (EGC) in four tertiary medical centers in Jiangsu Province, China. METHODS: Among 10 097 consecutive combined gastric cancer radical resections, 1903 EGC were identified and reviewed, 283 excluded and 1620 included in the study. All pathological and some endoscopic reports were reviewed for patients' characteristics, tumor location, gross features, and the number of lymph nodes retrieved and involved. Two pathologists independently investigated the pathological features of tumor type, differentiation, invasion depth, lymphovascular invasion (LVI), and perineural invasion. The data were statistically analyzed to identify risk factors for LNM. RESULTS: The average number of lymph nodes retrieved was 17.5 per patient. LNM was diagnosed in 15.5%. By univariate analysis, significant risk factors for LNM included age ≥ 41 years, female sex, size over 1 cm, submucosal invasion, poor differentiation, poorly cohesive carcinoma, micropapillary adenocarcinoma, adenocarcinoma mixed with signet-ring cell carcinoma, LVI, perineural invasion, and distal gastric location. By multivariate analysis, independent risk factors for LNM were size ≥ 3 cm (odds ratio [OR] 1.9), poor differentiation (OR 2.5), adenocarcinoma mixed with signet-ring cell carcinoma (OR 1.7), LVI (OR 5.8) and submucosal invasion (OR 2.9). In contrast, size < 3 cm and ulcer were not significant risk factors. Early cardiac carcinoma (OR 0.4) had significantly lower risk. CONCLUSIONS: Independent risk factors for LNM in EGC in Chinese patients included tumor size ≥ 3 cm, poor differentiation, submucosal invasion, adenocarcinoma mixed with signet-ring cell carcinoma and LVI. Early cardiac carcinoma had a significantly lower risk for LNM.

Prostaglandin E2 receptor EP2 mediates Snail expression in hepatocellular carcinoma cells.

Prostaglandin E2 (PGE2) has been shown to influence cell invasion and metastasis in several types of cancer, including hepatocellular carcinoma (HCC). however, the molecular mechanisms underlying it remain to be further elucidated. Snail, as one of key inducers of epithelial-mesenchymal transition (EMT), plays pivotal roles in HCC invasion and metastasis. The present study was designed to evaluate the possible signaling pathways through which PGE2 regulates Snail protein expression in HCC cell lines. PGE2 markedly enhanced Huh-7 cell invasion and migration ability by upregulating the expression level of Snail protein, and EP2 receptor played an important role in this process. Src, EGFR, Akt and mTOR were all activated and involved in the regulation of snail protein expression. Our findings suggest that PGE2 could upregulate the expression level of Snail protein through the EP2/Src/EGFR/Akt/mTOR pathway in Huh-7 cells, which promotes HCC cell invasion and migration.

Increased support for linkage of a novel locus on chromosome 5q13 for essential hypertension in the British Genetics of Hypertension Study.

Human hypertension arises from a combination of genetic factors and lifestyle influences. With cardiovascular disease set to become the number 1 cause of death worldwide, it is important to understand the etiologic mechanisms for hypertension, because these might provide new routes to improved treatment. The British Genetics of Hypertension Study has recently published a primary genome screen that identified 4 chromosomal regions of interest. We have now genotyped additional markers to confirm the most promising regions for follow-up studies. Thirty-four additional microsatellites were genotyped in our severely hypertensive affected sibling pair resource (now 1635 families with 2142 affected sibling pairs), leading to a substantial increase in information content in the regions of interest. We found increased support for linkage of chromosome 5q13 to human hypertension (multipoint logarithm of odds=2.50) with 3 adjacent markers yielding single point logarithm of odds scores of 3.22, 2.84, and 2.51. The placement of additional markers on 2q, 6q, and 9q diminished support for linkage in these regions. However, the addition of new data and families identified novel regions of interest on chromosomes 1q and 11q. The 3 positive markers in the chromosome 5 region were also genotyped in 712 distinct parent-offspring trios with the same severe phenotype to replicate linkage and association. Borderline support for replication was found (P=0.07). We found increased evidence for linkage and borderline-significant evidence for association for a hypertension susceptibility locus on chromosome 5q13 that is worthy of detailed fine mapping and assessment of candidate genes.

Linkage analysis using co-phenotypes in the BRIGHT study reveals novel potential susceptibility loci for hypertension.

Identification of the genetic influences on human essential hypertension and other complex diseases has proved difficult, partly because of genetic heterogeneity. In many complex-trait resources, additional phenotypic data have been collected, allowing comorbid intermediary phenotypes to be used to characterize more genetically homogeneous subsets. The traditional approach to analyzing covariate-defined subsets has typically depended on researchers' previous expectations for definition of a comorbid subset and leads to smaller data sets, with a concomitant attrition in power. An alternative is to test for dependence between genetic sharing and covariates across the entire data set. This approach offers the advantage of exploiting the full data set and could be widely applied to complex-trait genome scans. However, existing maximum-likelihood methods can be prohibitively computationally expensive, especially since permutation is often required to determine significance. We developed a less computationally intensive score test and applied it to biometric and biochemical covariate data, from 2,044 sibling pairs with severe hypertension, collected by the British Genetics of Hypertension (BRIGHT) study. We found genomewide-significant evidence for linkage with hypertension and several related covariates. The strongest signals were with leaner-body-mass measures on chromosome 20q (maximum LOD = 4.24) and with parameters of renal function on chromosome 5p (maximum LOD = 3.71). After correction for the multiple traits and genetic locations studied, our global genomewide P value was .046. This is the first identity-by-descent regression analysis of hypertension to our knowledge, and it demonstrates the value of this approach for the incorporation of additional phenotypic information in genetic studies of complex traits.

Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel.

BACKGROUND: The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. METHODS: We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. RESULTS: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of alpha-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G-->T and 1987A-->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M). CONCLUSION: CDCE rapidly detects point mutations in these candidate disease genes.