Transcriptome profiling reveals the genetic basis of alkalinity tolerance in wheat.

BACKGROUND: Soil alkalinity shows significant constraints to crop productivity; however, much less attention has been paid to analyze the effect of soil alkalinity on plant growth and development. Shanrong No. 4 (SR4) is an alkalinity tolerant bread wheat cultivar selected from an asymmetric somatic hybridization between the bread wheat cultivar Jinan 177 (JN177) and tall wheatgrass (Thinopyrum ponticum), which is a suitable material for studying alkalinity tolerant associate genes. RESULTS: The growth of SR4 plant seedlings was less inhibited than that of JN177 when exposed to alkalinity stress conditions. The root cytosolic Na+/K+ ratio in alkalinity stressed SR4 was lower than in JN177, while alkalinity stressed SR4 contained higher level of nutrient elements than in JN177. SR4 plant seedlings accumulated less malondialdehyde (MDA) and reactive oxygen species (ROS), it also showed higher activity of ROS scavenging enzymes than JN177 under alkalinity stress. The root intracellular pH decreased in both alkalinity stressed JN177 and SR4, however, it was much lower in SR4 than in JN177 under alkalinity stress. The transcriptomes of SR4 and JN177 seedlings exposed to alkalinity stress were analyzed by digital gene expression tag profiling method. Alkalinity stress conditions up- and down-regulated a large number of genes in the seedling roots that play the functions in the categories of transcription regulation, signal transduction and protein modification. CONCLUSIONS: SR4 expresses a superior tolerance to alkaline stress conditions which is due to its strong absorbing ability for nutrient ions, a strong regulating ability for intracellular and rhizosphere pH and a more active ROS scavenging ability.

Isolation and characterization of novel Glu-St1 alleles from Pseudoroegneria spicata and Pd. strigosa.

Pseudoroegneria is a small genus of the Triticeae tribe; its St genome is present in over half of allopolyploid Triticeae species. The high molecular weight (HMW) subunits of glutenin (GS) encoded by the St genome are not well described. In this paper, we report the characterization of fourteen alleles of HMW-GS genes from the two species Pd. spicata and Pd. strigosa. Analysis shows that all fourteen sequences possess a typical primary structure shared by other known HMW-GS, but with some unique modifications. All fourteen Glu-St1 alleles are significantly smaller than normal Glu-1 genes due to fewer repeat motifs in a repetitive region with no indication of large deletion in other conserved regions. Thus, the small size is a common feature of HMW-GS encoded by Glu-St1 loci of Pseudoroegneria species. Sequence analysis indicated that all fourteen Glu-St1 alleles were intermediate type between x- and y-type, which represent an intermediate stage in the evolutionary divergence of x- and y-type subunits.

Acquisition of thin coronal sectional dataset of cadaveric liver.

PURPOSE: To obtain the thin coronal sectional anatomic dataset of the liver by using digital freezing milling technique. METHODS: The upper abdomen of one Chinese adult cadaver was selected as the specimen. After CT and MRI examinations verification of absent liver lesions, the specimen was embedded with gelatin in stand erect position and frozen under profound hypothermia, and the specimen was then serially sectioned from anterior to posterior layer by layer with digital milling machine in the freezing chamber. The sequential images were captured by means of a digital camera and the dataset was imported to imaging workstation. RESULTS: The thin serial section of the liver added up to 699 layers with each layer being 0.2 mm in thickness. The shape, location, structure, intrahepatic vessels and adjacent structures of the liver was displayed clearly on each layer of the coronal sectional slice. CT and MR images through the body were obtained at 1.0 and 3.0 mm intervals, respectively. CONCLUSION: The methodology reported here is an adaptation of the milling methods previously described, which is a new data acquisition method for sectional anatomy. The thin coronal sectional anatomic dataset of the liver obtained by this technique is of high precision and good quality.

Pulmonary intersegmental planes: imaging appearance and possible reasons leading to their visualization.

PURPOSE: To describe the normal imaging appearance of pulmonary intersegmental planes on thoracic computed tomographic (CT) scans and determine the possible reasons related to their visualization in terms of aging and anatomy. MATERIALS AND METHODS: The study was approved by the internal ethics review board. Informed consent was obtained. A retrospective review was undertaken of 104 thoracic multidetector CT scans of an older group (>65 years) and younger group (<55 years). The number, location, and appearance rate of intersegmental planes were assessed. Group comparisons were made, and linear regression analysis was used to assess relationships between age and visualization of intersegmental planes. Thirty lung samples (10 × 10 × 10 mm(3)) from autopsy were scanned by using micro-CT. Thicknesses of intersegmental planes were measured. Significant differences of the thickness between visible and invisible intersegmental planes were assessed with the independent t test. In five fetal specimens (17-21 weeks in gestational age), 7.0-T magnetic resonance (MR) imaging was performed to determine the congenital difference of thickness of intersegmental planes. RESULTS: Within the right lung, appearance rates of visible intersegmental planes were 71.2% at S1-S3, 54.8% at S4-S5, and 70.2% at S7-S10. Within the left lung, appearance rates of visible intersegmental planes were 39.4% at S1+2 to S3, 64.4% at S4-S5, 18.3% at S7-S8, and 89.4% at S7-S10. Appearance rates of visible intersegmental planes on thoracic CT scans were not significantly different (P ≥ .38) between younger and older groups. Mean thicknesses of visible and invisible intersegmental planes were 681.3 µm ± 75.3 (standard deviation) and 221.7 µm ± 54.1, respectively. Visible intersegmental planes were significantly thicker than invisible intersegmental planes (P < .05). Visible intersegmental planes were also seen on fetal lung 7.0-T MR images. CONCLUSION: The thickness of pulmonary intersegmental planes and variation of intersegmental veins were closely related to visualization of intersegmental planes on thoracic CT scans. Aging was excluded as the possible reason.

Spindle and kinetochore associated complex subunit 1 regulates the proliferation of oral adenosquamous carcinoma CAL-27 cells in vitro.

BACKGROUND: The prognosis of oral squamous cell carcinoma is very poor due to local recurrence and metastasis. This study explores the molecular events involved in oral carcinoma with the goal of developing novel therapeutic strategies. The mitotic spindle is a complex mechanical apparatus required for the accurate segregation of sister chromosomes during mitosis. Spindle and kinetochore associated complex subunit 1 (SKA1) is a microtubule-binding subcomplex of the outer kinetochore that is essential for proper chromosome segregation. In recent years, much attention has been focused on determining how SKA proteins interact with each other, as well as their biological role in cancer cells. However, the precise role of SKA1 in oral carcinoma remains unknown. METHODS: In order to investigate the role of SKA1 in oral cancer, we employed lentivirus-mediated shRNA to silence SKA1 expression in the CAL-27 human oral adenosquamous carcinoma cell line. RESULTS: Depletion of SKA1 in CAL-27 cells significantly decreased cell proliferation, as determined by MTT and colony formation assays. These results strongly demonstrate that reduced SKA1 protein levels may cause inhibition of tumor formation. The shRNA-mediated depletion of SKA1 also led to G2/M phase cell cycle arrest and apoptosis. CONCLUSION: This is the first report to show that SKA1 plays an important role in the progression of oral adenosqamous carcinoma. Thus, silencing of SKA1 by RNAi might be a potential therapy for this disease.

Lentivirus-based RNA silencing of Nemo-like kinase (NLK) inhibits the CAL 27 human adenosquamos carcinoma cells proliferation and blocks G0/G1 phase to S phase.

BACKGROUND: The Nemo-like kinase (NLK) is a serine/threonine-protein kinase that involved in a number of signaling pathways regulating cell fate. Variation of NLK has been shown to be associated with the risk of cancer. However, the function of NLK in oral adenosquamous carcinoma cells line CAL-27 is unknown. METHODS: In this study, we evaluated the function of NLK in CAL-27 cells by using lentivirus-mediated RNA silence. The targeted gene expression, cell proliferation and cell cycle are investigated by RT-PCR, western-blot, MTT method, colony forming assay and flow cytometry analysis respectively. RESULTS: After NLK silencing, the number of colonies was significantly reduced (54 ± 5 colonies/well compared with 262 ± 18 colonies/well in non-infected or 226 ± 4 colonies/well in negative control group (sequence not related to NLK sequence with mismatched bases). Using crystal violet staining, we also found that the cell number per colony was dramatically reduced. The RNA silencing of NLK blocks the G0/G1 phase to S phase progression during the cell cycle. CONCLUSIONS: These results suggest that NLK silencing by lentivirus-mediated RNA interference would be a potential therapeutic method to control oral squamous carcinoma growth.

[Mapping fetal brain development in the second trimester with 7.0T magnetic resonance imaging].

OBJECTIVE: To obtain the three dimensional visualization model with normal measurements of fetal brain in the second trimester and analyze the developmental changes with gestational age (GA), sexual dimorphisms and cerebral asymmetries. METHODS: The brains of 69 fetal specimens of 12 - 22 weeks GA were scanned by 7.0T magnetic resonance imaging (MRI). The developing structures were analyzed and a three dimensional visualization model was rebuilt with Amira 4.1 software. RESULTS: Most sulci, except for postcentral and intraparietal sulcus, were present until 22 weeks GA. And none developed secondary branches. Laminar organization, described as early as 12 weeks GA, was delineated as layers with different signal intensities and became typical after 16 weeks GA. Basal nuclei was distinctly visible. Brains had different growth rates linearly increasing with GA. But neither sexual dimorphisms nor cerebral asymmetries was detected. CONCLUSIONS: The initial developmental stage of fetal brain occurs at 12 - 22 weeks GA. The developing structures may be distinctly visualized on 7.0T post-mortem MRI. And the three dimensional visualization model aids greatly in the precise cognition of immature brain.

[Effects of functional connectivity between anterior cingulate cortex and dorsolateral prefrontal cortex on executive control of attention in healthy individuals].

OBJECTIVE: To explore the presence of functional connectivity between anterior cingulate cortex (ACC) and dorsolateral prefrontal cortex (DLPFC) during the manipulation of attentional network test (ANT) and its relationship with behavioral performance. METHODS: Functional magnetic resonance imaging was performed on 25 healthy subjects aged 17 - 20 years. And ANT was used as a paradigm. Functional connectivity between ACC-DLPFC was tested and correlation analysis conducted between functional connectivity coefficients and behavioral scores of ANT. RESULTS: Significant functional connectivity between the dorsal ACC (dACC) with bilateral DLPFC was found. Furthermore, event-related functional connectivity coefficients between left dACC and left DLPFC were negatively associated with the behavioral scores of executive control (r = -0.63; P < 0.01). CONCLUSION: Our findings provide new evidence that ACC and DLPFC are functionally connected and such functional connectivity has advantageous influence on executive control function of attention so as to contribute to our understanding of the integrated role of these brain regions in attentional network.

A smartphone-based support system coupled with a bluetooth breathalyzer in the treatment of alcohol dependence: A 12-week randomized controlled trial.

Background: Our prior open trial showed the feasibility of a smartphone-based support system coupled with a Bluetooth breathalyzer (SoberDiary) in assisting recovery for patients with alcohol dependence (AD). In this 24-week follow-up study, we further explored the efficacy of supplementing SoberDiary to treatment as usual (TAU) over 12 weeks of intervention and whether the efficacy persisted in the post-intervention 12 weeks. Methods: 51 patients who met the DSM-IV criteria of AD were randomly assigned to the technological intervention group (TI group, receiving technology intervention of SoberDiary plus TAU, n = 25) or those receiving only TAU (TAU group, n = 26). After 12 weeks of intervention (Phase I), all participants were followed for another post-intervention 12 weeks (Phase II). We collected the drinking variables and psychological assessment data every 4 weeks (i.e., weeks 4, 8, 12, 16, 20, and 24). In addition, the cumulative abstinence days and retention rates were recorded. We used mixed-model analysis to compare the difference in outcomes between groups. Results: In Phase I or Phase II, we did not find differences in drinking variables, alcohol craving, depression, or anxiety severity between the two groups. However, the TI group showed greater self-efficacy for drinking refusal in Phase II than the TAU group. Conclusions: Although our system (SoberDiary) did not demonstrate benefits in drinking or emotional outcomes, we found the system holds promise to enhance self-efficacy on drinking refusal. Whether the benefit in promoting self-efficacy persists longer than 24 weeks requires further investigation.

Quantitative assessment of gait and neurochemical correlation in a classical murine model of Parkinson's disease.

BACKGROUND: Gait deficits are important clinical symptoms of Parkinson's disease (PD). However, existing behavioral tests for the detection of motor impairments in rodents with systemic dopamine depletion only measure akinesia and dyskinesia, and data focusing on gait are scarce. We evaluated gait changes in the methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced C57BL/6 murine model of PD by using a computer-assisted CatWalk system. Correlations of gait parameters with tyrosine hydroxylase (TH) protein levels in the substantia nigra (SN) were also investigated. RESULTS: The gait readouts, including the walking duration, variation of walking speed, step cycle, duty cycle, stance, initial dual stance, terminal dual stance, three- and four-point supports, and the base of support between hind limbs was noted to increase significantly one week after MPTP injection. In contrast, values of the stride length, cadence, swing speed, and diagonal dual support decreased substantially following MPTP treatment (p < 0.05). All of these changes lasted for three weeks after the last MPTP administration. Except for the stance in the fore limbs and the swing speed in the hind limbs, the gait variability in the PD mice showed a closer correlation with the protein levels of TH in the SN than the walking distances in the conventional open field test. Coordination parameters of the regularity index and step pattern were not affected in mice treated with MPTP. CONCLUSION: Data of the study suggest that the computer-assisted CatWalk system can provide reliable and objective criteria to stratify gait changes arising from MPTP-induced bilateral lesions in C57/BL6 mice. The extent of gait changes was noted to correlate with the expression of the biomarker for dopaminergic neurons. This novel analytical method may hold promise in the study of disease progression and new drug screening in a murine PD model.

The Visible Human Projects in Korea and China with improved images and diverse applications.

PURPOSE: The Visible Human Projects, which were launched in the United States, have also been developed in Korea and China during the past decade. This article includes the new trials to promote a variety of their applications. METHODS: In a Korean laboratory, whole bodies of two cadavers were serially sectioned (Visible Korean), while two Chinese institutes have sectioned nine cadavers (Chinese Visible Human and Virtual Chinese Human). For acquiring sectioned images and stereoscopic models of better quality, appropriate cadavers were chosen; equipments and techniques for embedding, sectioning, photography, and computer processing were continuously improved in the two countries. To facilitate the research, Korean and Chinese scientists have visited each other. RESULTS: The sectioned images with thinner intervals (0.2 mm or less) and higher resolution were obtained. From the advanced data, the segmented images of comprehensive structures were prepared to construct three-dimensional models. Then, cross-sectional images and models were offered for medical education and clinical practice such as electronic anatomy atlas and virtual lumbar puncture. CONCLUSION: Every project has its strengths and weaknesses with regard to the image data; users in the world can choose the project that best suits their needs.

Hybridization affects the structure and function of root microbiome by altering gene expression in roots of wheat introgression line under saline-alkali stress.

The mutually beneficial relationship between plants and their root microbiota is essential for plants to adapt to unfavorable environments. However, the molecular mechanism of wheat regulating the structure of root microbiome and the influence of distant hybridization on this process are poorly understood. In this study, we systematically compared the root transcriptome and microbiome between a saline-alkali tolerant wheat introgression line SR4 (derived from somatic hybridization between wheat and tall wheatgrass) and its parent wheat variety JN177. The results indicated that root microorganisms were key factor maintaining better homeostasis of the sodium and potassium ion contents in SR4 than in JN177 under saline-alkali stress. Through systematic comparisons, we identified SR4-specific root bacterial and fungal taxa under saline-alkali stress. Through a weighted gene correlation network analysis (WGCNA) combining microbiome and transcriptome data, key functional genes and pathways, which were strongly related to root bacteria and fungi with differential abundance between JN177 and SR4, were identified. These results suggest that somatic hybridization has altered the key genes regulating root microbiome in wheat, further improving the saline-alkali tolerance of wheat introgression line. These findings provide the key bacterial and fungal taxa and functional target genes for wheat root microbiome engineering under saline-alkali stress.

Prevalence and Correlates of Prescription Drug Misuse in a Nationwide Population Survey in Taiwan.

BACKGROUND: Prescription drug misuse (PDM) is a critical mental health issue relating to psychiatric morbidity. This study investigated the prevalence of PDM and its associated psychopathology and psychosocial factors in the general population in Taiwan. METHODS: The survey randomly selected a representative sample >15 year-olds using the stratified proportional randomization method. The measurements included demographic variables, previous experience with PDM, self-rated physical and mental health, health self-efficacy, risk factors for suicidality, and psychological distress. RESULTS: The weighted one-year prevalence of PDM was 8.5% (n = 180) among 2126 participants. Those with psychological distress and lifetime suicide ideation (23.3%) or suicide attempts (5.0%) were significantly associated with PDM. PDM was also prevalent among those with poorer self-rated health and lower self-efficacy. Insomnia (OR = 1.52), depression (OR = 1.77), and low self-efficacy (OR = 2.29) had higher odds of PDM after adjustment in the logistic regression model. CONCLUSIONS: Individuals who misused prescription drugs had a higher prevalence of psychological distress and suicidality and lower levels of self-rated health. Prescription drug misuse problems should be screened for early prevention when prescribing medications for people with insomnia, depression, or lower perceived health beliefs or conditions.

High frequency of HMW-GS sequence variation through somatic hybridization between Agropyron elongatum and common wheat.

A symmetric somatic hybridization was performed to combine the protoplasts of tall wheatgrass (Agropyron elongatum) and bread wheat (Triticum aestivum). Fertile regenerants were obtained which were morphologically similar to tall wheatgrass, but which contained some introgression segments from wheat. An SDS-PAGE analysis showed that a number of non-parental high-molecular weight glutenin subunits (HMW-GS) were present in the symmetric somatic hybridization derivatives. These sequences were amplified, cloned and sequenced, to deliver 14 distinct HMW-GS coding sequences, eight of which were of the y-type (Hy1-Hy8) and six x-type (Hx1-Hx6). Five of the cloned HMW-GS sequences were successfully expressed in E. coli. The analysis of their deduced peptide sequences showed that they all possessed the typical HMW-GS primary structure. Sequence alignments indicated that Hx5 and Hy1 were probably derived from the tall wheatgrass genes Aex5 and Aey6, while Hy2, Hy3, Hx1 and Hy6 may have resulted from slippage in the replication of a related biparental gene. We found that both symmetric and asymmetric somatic hybridization could promote the emergence of novel alleles. We discussed the origination of allelic variation of HMW-GS genes in somatic hybridization, which might be the result from the response to genomic shock triggered by the merger and interaction of biparent genomes.

The thin sectional anatomy of the sellar region with MRI correlation.

The purpose of this study was to explore the anatomical complexity and adjacent relationships of the sellar region in thin continuous sections so as to provide intimate morphological data for imaging diagnosis and surgical operations of the diseases in this region. After CT and MR examination verifying no brain lesions, one normal cadaver head was selected for this study from four Chinese adult male cadavers. After being embedded and frozen, the head was sliced into serial sections at 0.1 mm intervals in the transverse plane with SKC 500 computerized freezing milling machine. Then the serial transverse sections were photographed by a high-resolution digital camera and saved in the computer. Subsequently, the anatomic structures of the sellar region on the thin transverse sections were investigated and correlated with the MR images of the specimen as well as in vivo MR images, which were obtained from 20 normal Chinese male adult volunteers by a 3.0 T GE MR scanner. The base lines of the sectioning and the MR scan were all parallel to the AC-PC line. A total of 320 transverse sections and 10-12 transverse MR images related with the sellar region were obtained, respectively. We investigated the sectional anatomy of the sellar region and divided it into three parts: supra hypophysial area, hypophysial area and infra hypophysial area. The cavernous sinus was a venous passage full of blood and it could be divided into four interspaces according to its position relation with the internal carotid artery. The third, fourth, sixth cranial nerves and trigeminal branches ophthalmic nerve, maxillary nerve displayed from the anterior to the posterior in the lateral wall of cavernous sinus in transverse planes. Comparing continuous thin sections with MR images offers a better understanding of the complex anatomical structures and provides practical submillimeter anatomical data for imaging diagnosis and clinical treatment in this region.

[Comparison of Floating Chamber and Diffusion Model Methods for Measuring Methane Emissions from Inland Fish-Aquaculture Ponds].

Freshwater aquaculture ponds collectively cover a large area in southeast China. They are an important anthropogenic source of CH4 emissions, however current knowledge of CH4 emissions from aquaculture ponds is limited. During September 2017 and August 2018, two independent sampling campaigns of CH4 flux measurements over annual cycles were carried out in a fish-aquaculture pond in southeast China using a floating chamber and diffusion model methods for comparison. The purpose of this study is to investigate the variation of CH4 fluxes obtained from the fish-aquaculture pond, analyze the emission mechanisms, and amplify the CH4 emissions inventory. The diffusion model method can further quantify the contribution of diffusion transmission to CH4 emissions. The seasonal variations of CH4 fluxes measured by the two methods were similar, with higher CH4 fluxes appearing in summer and fall and lower CH4 fluxes appearing in spring and winter. CH4 fluxes measured by the floating chamber method and diffusion model method ranged from 0.14-3.13 mg·(m2·h)-1 and 0.04-1.41 mg·(m2·h)-1, respectively, and the respective average values were (0.86±0.30) mg·(m2·h)-1and (0.45±0.08) mg·(m2·h)-1. The CH4 fluxes were positively related to water temperature, dissolved organic carbon (DOC) and chemical oxygen demand (COD), but negatively related to the water dissolved oxygen (DO) concentration in the fish-aquaculture pond. CH4 fluxes calculated by diffusion model methods were 45% lower than those determined by the floating chamber methods over annual cycles. The result suggested that the floating chamber method is more suitable than the diffusion model method for measurements of CH4 fluxes in freshwater aquaculture ecosystems.

Segmentation and reconstruction of hepatic veins and intrahepatic portal vein based on the coronal sectional anatomic dataset.

Three-dimensional (3D) reconstruction of intrahepatic vessels is very useful in visualizing the complex anatomy of hepatic veins and intrahepatic portal vein. It also provides a 3D anatomic basis for diagnostic imaging and surgical operation on the liver. In the present study, we built a 3D digitized model of hepatic veins and intrahepatic portal vein based on the coronal sectional anatomic dataset of the liver. The dataset was obtained using the digital freezing milling technique. The pre-reconstructed structures were identified and extracted, and then were segmented by the method of manual intervention. The digitized model of hepatic veins and intrahepatic portal vein was established using 3D medical visualization software. This model facilitated a continuous and dynamic displaying of the hepatic veins and intrahepatic portal vein at different orientations, which demonstrated the complicated relationship of adjacent hepatic veins and intrahepatic portal vein realistically in the 3D space. This study indicated that high-quality 2D images, precise data segmentation, and suitable 3D reconstruction methods ensured the reality and accuracy of the digital visualized model of hepatic veins and intrahepatic portal vein.

Characterization of the genes coding for the high molecular weight glutenin subunits in Lophopyrum elongatum.

In order to reveal the evolution of Glu-E1 loci of Lophopyrum elongatum (Host) A. Löve and find novel alleles for wheat quality breeding, four Glu-1 alleles were isolated and characterized via genomic PCR, from this wheat-related species. Of them, 1Ex2 and 1Ey2 were novel alleles, which differ from all the previously known HMW-GS alleles of L. elongatum. Two alleles 1Ex1 and 1Ey2, which contain intact open reading frames, have been successfully expressed in E. coli. The expressed proteins showed similar electrophoresis mobility with the candidate high molecular weight glutenin subunit bands found in seeds. Sequence alignment indicated that proteins encoded by the novel alleles showed similar primary structure with those of wheat and other wheat-related grasses, however, they possess some unique modifications in their own structure. For example, the number of residues in the N-terminal domain is different from those of wheat, an irregular tripeptide present between two nonapeptide motifs and a unique cysteine position in the repetitive region. Phylogenetic analyses using N-terminal conserved sequences showed that 1Ex2 was homologous to those from the D genome; but 1Ey2 was homologous to a y-type allele 1Ky from the K genome. The evolution relationship of Glu-E1 alleles and the possible utilization of the alleles in wheat breeding are discussed.

Lead as a legendary pollutant with emerging concern: Survey of lead in tap water in an old campus building using four sampling methods.

In this study, a sampling campaign with a total of nine sampling events investigating lead in drinking water was conducted at 7 sampling locations in an old building with lead pipes in service in part of the building on the National Taiwan University campus. This study aims to assess the effectiveness of four different sampling methods, namely first draw sampling, sequential sampling, random daytime sampling and flush sampling, in lead contamination detection. In 3 out of the 7 sampling locations without lead pipe, lead could not be detected (<1.1 µg/L) in most samples regardless of the sampling methods. On the other hand, in the 4 sampling locations where lead pipes still existed, total lead concentrations >10 µg/L were consistently observed in 3 locations using any of the four sampling methods while the remaining location was identified to be contaminated using sequential sampling. High lead levels were consistently measured by the four sampling methods in the 3 locations in which particulate lead was either predominant or comparable to soluble lead. Compared to first draw and random daytime samplings, although flush sampling had a high tendency to reduce total lead in samples in lead-contaminated sites, the extent of lead reduction was location-dependent and not dependent on flush durations between 5 and 10 min. Overall, first draw sampling and random daytime sampling were reliable and effective in determining lead contamination in this study. Flush sampling could reveal the contamination if the extent is severe but tends to underestimate lead exposure risk.