Jiawei guomin decoction regulates the degranulation of mast cells in atopic dermatitis mice via the HIS/PAR-2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Guomin decoction (GMD) is a traditional Chinese medicine commonly used in clinical practice. It has traditionally been used to treat all allergic diseases. Currently, Jiawei Guomin Decoction (JWGMD) is used to treat sensitive skin after initial therapy. Although it has a significant clinical therapeutic effect, the exact role of mast cell degranulation in treating atopic dermatitis (AD) is still unclear. AIM OF THE STUDY: GMD and JWGMD can both treat allergic diseases, while JWGMD focuses on skin allergies. This study aims to explore the potential effect of JWGMD on the degranulation of mast cells in an AD mouse model induced by 2,4-dinitrofluorobenzene (DNFB) and investigate the effectiveness of JWGMD in alleviating disease progression to further provide specific therapeutic targets for treating AD. MATERIALS AND METHODS: The scratching times and skin lesions of model mice induced by DNFB were observed, and skin tissues were collected for subsequent measurement. Histopathological changes in the back skin of mice were observed by haematoxylin eosin (H&E) staining, Toluidine blue staining was used to detect the degranulation of mouse skin mast cells, and the relationship between the expression of histamine (HIS), mast cell tryptase (MCT) and mast cell degranulation was analysed by enzyme-linked immunosorbent assay (ELISA). The expression of protease-activated receptor-2 (PAR-2), histamine 1 receptor (H1R), H2R, H4R and MCT proteins in AD mice was detected by Western blot (WB). Immunofluorescence assay (IFA) further confirmed the localization of PAR-2, H1R, H2R, H4R, and MCT proteins in the skin. Quantitative real-time PCR (qPCR) was used to determine PAR-2, H1R, H2R and H4R mRNA levels in skin lesions to further clarify the mechanism by which JWGMD amplifies mast cell degranulation in AD. In addition, a reliable ultrahigh-performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry (UPLC-QE-MS) nontargeted metabolomics analysis was performed to analyse the differences in metabolite abundance between GMD and JWGMD, and these results were used to identify the active components in JWGMD that may have antipruritic and anti-inflammatory properties and inhibit mast cell degranulation. RESULTS: After intermittent stimulation with DNFB, the skin lesions showed extensive desquamation, dryness, scabbing, skin thickening, and slight bleeding. Both treatments alleviated this phenomenon and reduced the number of scratches, with JWGMD being the most effective. JWGMD can significantly reduce inflammatory cell infiltration, oedema, and some capillary neogenesis in mice and reduce the degranulation of mast cells. The ELISA results showed that JWGMD can increase the levels of MCT and HIS proteins. The WB and IFA results demonstrated that JWGMD reduced the expression levels of PAR-2, H1R, H4R, and MCT proteins in skin lesions, with protein localization mainly in the epidermal layer, while H2R protein levels were increased and mainly localized in the dermis. In addition, JWGMD downregulates the mRNA expression of PAR-2, H1R, H2R, and H4R. Interestingly, through UPLC-QE-MS nontargeted metabolomic analysis, we detected the anti-inflammatory and antiallergy active substances in JWGMD, such as methyl eugenol, dictamnine and sinapine. CONCLUSIONS: JWGMD may alleviate itching through methyl syringol, dictamnine, sinapine and other substances, and its mechanism may be related to inhibiting the HIS/PAR-2 pathway in AD model mice and further regulating the self-amplification of mast cell degranulation. JWGMD is a potential drug for treating AD. Therefore, it deserves continuous attention and research.

LY303511 displays antiproliferation potential against oral cancer cells in vitro and in vivo.

LY303511 was developed as a negative control of LY294002 without pan-phosphoinositide 3-kinase (PI3K) inhibition. We hypothesize LY303511 generate reactive oxygen species (ROS) to induce apoptosis for killing oral cancer cells. In MTS assay, LY303511 dose-responsively decreases survival in three kinds of oral cancer cells but little damage to normal oral cells (HGF-1). Two oral cancer cells (CAL 27 and SCC-9) with highly sensitivity to LY303511 were used. In 7-aminoactinomycin D (7AAD) assay, LY303511 slightly increases subG1 population in oral cancer cells. In annexin V/7AAD and/or pancaspase assays, LY303511 induces apoptosis in oral cancer cells but HGF-1 cells remains in basal level. In oxidative stress, LY303511 induces ROS and mitochondrial superoxide in oral cancer cells. In 8-oxo-2'-deoxyguanosine assay, LY303511 induces oxidative DNA damage in oral cancer cells. In zebrafish model, LY303511 inhibits CAL 27-xenografted tumor growth. Therefore, LY303511 displays antiproliferation potential against oral cancer cells in vitro and in vivo.

[Effects of moxibustion on the structure and function of blood brain barrier in Alzheimer's disease model rats].

OBJECTIVE: To investigate the effects of moxibustion on the structure and function of blood-brain barrier in Alzheimer's disease (AD) model rats. METHODS: Forty-eight SD rats were randomly divided into 4 groups: normal control group, sham operation group, model group, moxibustion group. Model group and moxibustion group rats were injected with aggregated Aß25-35 by bilateral hippocampus. In the rat model, the sham-operated group was injected with the same amount of normal saline in the bilateral hippocampus, and the normal group was not treated. After successful modeling, the moxibustion treatment was given at 2～3 cm above the Baihui, Shenshu and Yintang points of the moxibustion group rats, each time for 10 min, once a day, continuous treatment for 21 d. The Morris water maze test was used to evaluate the learning and memory ability of rats in each group. The Evans blue method was used to detect the permeability of blood-brain barrier. The ultrastructure of blood-brain barrier was observed under electron microscope. The matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) positive cells in hippocampus were detected by immunohistochemistry. RESULTS: Compared with the sham operation group, the escape latency was significantly increased (P＜0.01), and the space exploration time was decreased (P＜0.01), the learning and memory function in model group was impaired seriously, the Evans blue content in the brain was increased significantly (P＜0.01), the perivascular edema became larger, and the blood-brain barrier structure function was impaired. At the same time, the positive expressions of MMP-2 and MMP-9 in hippocampus were increased significantly (P＜0.01). Compared with model group, the learning and memory ability in moxibustion group rats was enhanced (P＜ 0.05), the content of Evans blue in the brain was decreased (P＜0.05), the degree of perivascular edema was reduced, and the damage of blood-brain barrier was improved. Positive expressions of MMP-2 and MMP-9 in hippocampus were decreased (P＜0.05 or P＜ 0.01). CONCLUSION: Moxibustion can decrease the expressions of MMP-2 and MMP-9, and reduce the damage of the structure and function of blood-brain barrier, thereby improving the learning and memory ability of AD model rats, and exerting therapeutic effects.

Understanding the mechanisms of dormancy in an invasive alien Sycamore lace bug, Corythucha ciliata through transcript and metabolite profiling.

The sycamore lace bug, Corythucha ciliata, is a pest of sycamore trees. In China, it is found in the most northern border where it has been known to become dormant during harsh winters. But the molecular and metabolic basis for dormancy in this insect is still unknown. In this study, we analyzed the transcript and metabolite profiles of this bug to identify key genes and metabolites that are significantly regulated during dormancy in adult females and males. In total, 149 differentially expressed genes (DEGs) were significantly up-regulated and 337 DEGs were significantly down-regulated in dormant adults (both females and males). We found major differences in heat shock protein (HSPs), immunity-responsive genes, NAD-dependent deacetylase sirtuin-1 (SIRT1) and genes involved in the spliceosome pathway that is known to regulate stress. Among the 62 metabolites identified by GC-MS, 12 metabolites including glycerol, trehalose, and alanine were significantly increased during C. ciliata dormancy. By integrating the transcriptome and metabolite datasets, we found that the metabolites in glycolysis/gluconeogenesis and citrate cycle (TCA) were significantly reduced. This study is the first to report both transcript and metabolite profiles of the overwintering responses of C. ciliata to cold stress at the molecular level.

Multifactorial theory applied to the neurotoxicity of paraquat and paraquat-induced mechanisms of developing Parkinson's disease.

Laboratory studies involving repeated exposure to paraquat (PQ) in different animal models can induce many of the pathological features of Parkinson's disease (PD), such as the loss of dopaminergic neurons in the nigrostriatal dopamine system. Epidemiological studies identify an increased risk of developing PD in human populations living in areas where PQ exposure is likely to occur and among workers lacking appropriate protective equipment. The mechanisms involved in developing PD may not be due to any single cause, but rather a multifactorial situation may exist where PQ exposure may cause PD in some circumstances. Multifactorial theory is adopted into this review that includes a number of sub-cellular mechanisms to explain the pathogenesis of PD. The theory is placed into an environmental context of chronic low-dose exposure to PQ that consequently acts as an oxidative stress inducer. Oxidative stress and the metabolic processes of PQ-inducing excitotoxicity, α-synuclein aggregate formation, autophagy, alteration of dopamine catabolism, and inactivation of tyrosine hydroxylase are positioned as causes for the loss of dopaminergic cells. The environmental context and biochemistry of PQ in soils, water, and organisms is also reviewed to identify potential routes that can lead to chronic rates of low-dose exposure that would replicate the type of response that is observed in animal models, epidemiological studies, and other types of laboratory investigations involving PQ exposure. The purpose of this review is to synthesize key relations and summarize hypotheses linking PD to PQ exposure by using the multifactorial approach. Recommendations are given to integrate laboratory methods to the environmental context as a means to improve on experimental design. The multifactorial approach is necessary for conducting valid tests of causal relations, for understanding of potential relations between PD and PQ exposure, and may prevent further delay in solving what has proven to be an evasive etiological problem.

[Study on the risk factors of tuberculosis in four cities and provinces in China].

OBJECTIVE: To investigate the influencing factors on tuberculosis (TB) in four provinces in the eastern and central parts of China. METHODS: From Nov. 2009 to Feb. 2011, three population-based field surveys were conducted among the resident population in several townships/streets in Guangdong, Hunan and Jiangsu provinces and Shanghai municipality to collect TB-related information. 474 sputum smear positive TB patients and 1896 controls were randomly selected from the population under study and each case was matched by province, age and sex using a frequency matching method. Single-variable and multiple non-conditional logistic regression modeling were applied for data analysis, and odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were estimated. RESULTS: Data from Single-variable analysis showed that TB history, history of exposure to TB, DM history, immigrant population and per-capita living space were risk factors for TB, and high level of education was protective factors. RESULTS: from multiple logistic regression showed that the risk factors of TB would include the following items: history of having had TB (OR = 52.356, 95%CI: 18.956 - 144.607), living space over 50 m(2)per-capita (OR = 8.742, 95%CI: 1.107 - 69.064), history of exposure to TB (OR = 6.083, 95%CI: 2.336 - 15.839) and being immigrants (OR = 3.306, 95%CI: 1.907 - 5.734), while having had high degree of education as the protective factor of TB (OR = 0.284, 95%CI: 0.110 - 0.733). CONCLUSION: Control programs targeting those ever having TB patients and contacts to TB patients as well as immigrants should be strengthened.

[Construction of surveillance and early-warning-index-system on tuberculosis in China].

OBJECTIVE: To establish the surveillance and warning index system corresponding with the epidemic characteristics of tuberculosis, in China. METHODS: Literature review and expert meeting were conducted to formulate the preliminary index system frame. Delphi method was used for screening the index and determining the weight of each index. RESULTS: Two-round Delphi consultations were performed. The activity coefficients were 87.0%, 90.0% respectively, with means of authority coefficient as 0.850±0.055 and 0.917±0.017, respectively. Mean scores of the index were 7.063±1.435, 8.156±0.611 respectively, with the means of coefficient of variation as 0.352±0.161 and 0.170±0.057 respectively. The harmony coefficients were 0.322 (χ2=499.472, P<0.05) and 0.393 (χ2=241.126, P<0.05) respectively. After the two-round consultation, the tuberculosis monitoring and warning index system was developed, including 4 first-class indicators, 9 second-class indicators and 48 third-class indicators. CONCLUSION: An index system was established for tuberculosis monitoring and early warning that could provide evidence for tuberculosis prevention and control as well as for the forecasting and warning model of the disease.

[The influence of T lymphocyte activation on HIV-1 susceptibility of Han Chinese].

OBJECTIVE: To explore the influence of T lymphocyte activation on HIV-1 susceptibility of Han Chinese. METHODS: In 2008, 37 HIV-1 highly exposed persistently seronegative individuals (ESNs) and 101 healthy controls were screened from Shenzhen. Flow cytometer was used to assay the expression difference of HIV-1 infection related co-receptor, the difference between the two groups were analyzed by Mann-Whitney U statistics methods. RESULTS: T cell HLA-DR(+) CD4 T cells and HLA-DR(+) expression of ESNs (12.64 (5.94 - 21.90), 21.12 (10.74 - 30.21)) were all significantly lower than that of healthy controls (22.52 (7.91 - 58.60), 32.28 (14.72 - 67.82)) (P values all < 0.05). T cell CD45RA-RO(+), CCR5(+)CD4 expression of ESNs (58.68 (49.06 - 72.44), 21.93 (15.84 - 25.89)) were all significantly higher than that of healthy controls (53.17 (42.63 - 63.21), 16.14 (11.94 - 21.98)) (P values all < 0.05). T cell CXCR4(+)CD4 T cells expression of ESNs (93.67 (92.17 - 94.96)) was significantly lower than that of healthy controls (95.16 (92.99 - 96.77)) (P values all < 0.05). Healthy controls and ESNs could be divided into low expression group and high expression group according to HLA-DR(+)CD8 T cells bimodal distribution. A total of 89.2% (33/37) ESNs fell into HLA-DR + CD8 low expression group, and 58.4% (59/101) of the healthy controls located in low expression group (P < 0.05). CONCLUSION: To Han Chinese, the low activation status of T lymphocyte has significant correlation with HIV-1 low susceptibility.

Interactions between genetic variants in the adiponectin, adiponectin receptor 1 and environmental factors on the risk of colorectal cancer.

BACKGROUND: Metabolic syndrome traits play an important role in the development of colorectal cancer. Adipokines, key metabolic syndrome cellular mediators, when abnormal, may induce carcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: To investigate whether polymorphisms of important adipokines, adiponectin (ADIPOQ) and its receptors, either alone or in combination with environmental factors, are implicated in colorectal cancer, a two-stage case-control study was conducted. In the first stage, we evaluated 24 tag single nucleotide polymorphisms (tag SNPs) across ADIPOQ ligand and two ADIPOQ receptors (ADIPOR1 and ADIPOR2) among 470 cases and 458 controls. One SNP with promising association was then analyzed in stage 2 among 314 cases and 355 controls. In our study, ADIPOQ rs1063538 was consistently associated with increased colorectal cancer risk, with an odds ratio (OR) of 1.94 (95%CI: 1.48-2.54) for CC genotype compared with TT genotype. In two-factor gene-environment interaction analyses, rs1063538 presented significant interactions with smoking status, family history of cancer and alcohol use, with ORs of 4.52 (95%CI: 2.78-7.34), 3.18 (95%CI: 1.73-5.82) and 1.97 (95%CI: 1.27-3.04) for smokers, individuals with family history of cancer or drinkers with CC genotype compared with non-smokers, individuals without family history of cancer or non-drinkers with TT genotype, respectively. Multifactor gene-environment interactions analysis revealed significant interactions between ADIPOQ rs1063538, ADIPOR1 rs1539355, smoking status and BMI. Individuals carrying one, two and at least three risk factors presented 1.18-fold (95%CI:0.89-fold to 1.58-fold), 1.87-fold (95%CI: 1.38-fold to 2.54-fold) and 4.39-fold (95%CI: 2.75-fold to 7.01-fold) increased colorectal cancer risk compared with those who without risk factor, respectively (P(trend) <0.0001). CONCLUSIONS/SIGNIFICANCE: Our results suggest that variants in ADIPOQ may contribute to increased colorectal cancer risk in Chinese and this contribution may be modified by environmental factors, such as smoking status, family history of cancer and BMI.

Predicting the incidence risk of ischemic stroke in a hospital population of southern China: a classification tree analysis.

OBJECTIVE: To determine the major risk factors and their interactions of ischemic stroke (IS) and to develop a classification tree model to predict the incidence risk of IS for a Chinese population. METHODS: Exhaustive Chi-squared Automatic Interaction Detection (Exhaustive CHAID) algorithm of classification tree method was applied to build a prediction model for the incidence risk of IS under the design of 1:1 matched case-control study. The statistics of misclassification risk was used to evaluate the fitness of the model. RESULTS: In the prediction model, six variables of physical exercise, history of hypertension, tea drinking, HDL-c level, smoking status and educational level were in turn selected as the predictors of IS incidence risk. In the subgroup of lacking of physical exercise, individuals who had history of hypertension would have a significantly higher IS risk (92%) than that of the ones who had no history of hypertension (64%). The misclassification risk estimate of the prediction model was 0.21 with the standard error of 0.02, indicating that 79% of the cases could be classified correctly based on current prediction model. CONCLUSIONS: Lacking of physical exercise and history of hypertension are identified to be the prominent predicting variables of IS risk for a hospital population of southern China. Although CHAID analysis could provide detailed information and insight about interactions among risk factors of IS, we still need to validate our model and improve the vascular risk prediction for Chinese subjects in further studies.

Ventilator-associated pneumonia in intensive care units in Hubei Province, China: a multicentre prospective cohort survey.

A multicentre prospective cohort study was performed in 17 intensive care units (ICUs) in tertiary care hospitals in Hubei Province, China. Ventilator-associated pneumonia (VAP) was defined according to modified criteria from the published literature. Among 4155 ventilated patients, the crude incidence and incidence rate of VAP were 20.9% and 28.9 cases per 1000 ventilator-days. Multivariate analysis using logistic regression revealed risk factors including male sex [risk ratio (RR): 1.5; P<0.001], coma (RR: 2.1; P<0.001), chronic obstructive pulmonary disease (RR: 1.4; P<0.001), infections at other sites (RR: 1.6; P=0.001), serious disease predating the onset of VAP (RR: 1.6; P<0.001) and interventions including antacid treatment (RR: 1.4; P<0.001), antimicrobial treatment (RR: 5.1; P<0.001), bronchoscopy (RR: 1.5; P=0.041) and tracheostomy (RR: 1.4; P=0.014). The most frequently isolated causative pathogens were Pseudomonas aeruginosa and Acinetobacter baumannii. Of all Staphylococcus aureus isolates, 45.7% were meticillin resistant. Rates, risk factors and causal pathogens of VAP in ICUs in Hubei differ from those reported from developed countries. These data show the need for more effective infection control interventions in Hubei, China.

Point prevalence surveys of healthcare-associated infection in 13 hospitals in Hubei Province, China, 2007-2008.

Successive point prevalence surveys were conducted in November 2007 and 2008 to monitor the prevalence of healthcare-associated infection (HCAI) in 13 grade III, 1st class hospitals in Hubei Province of China, using the case definition criteria established by the Ministry of Health in the People's Republic of China. In total, of 20 350 patients surveyed, 833 (4.09%) HCAIs were observed in 790 (3.88%) patients. There was no significant difference between the overall prevalence of HCAI in 2007 (4.14%) and 2008 (3.72%). Respiratory tract infection was the most common HCAI (63.15%), followed by surgical site infection (9.60%) and urinary tract infection (8.64%). Only 35.29% (294/833) of HCAI patients had positive microbiology results. Gram-negative bacteria were isolated most frequently and the most frequent organism was Pseudomonas aeruginosa, followed by Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus. Antibiotic use was documented for 10,344 (50.83%) patients, and cephalosporins, penicillins, and quinolones were the most commonly used agents for treatment or prophylaxis.

Association between two functional polymorphisms of insulin-like growth factor binding protein 3 and colorectal cancer risk in a Chinese population.

Circulating levels of insulin-like growth factor binding protein 3 (IGFBP3) are modulated by functional variants of IGFBP3 and therefore may be associated with higher risk of colorectal cancer development. However, few studies have investigated the role of IGFBP3 polymorphisms in colorectal cancer in Chinese individuals. In this study, two common polymorphisms of IGFBP3 were determined by the Taqman genotyping platform in 202 Chinese colorectal cancer cases diagnosed between 2006 and 2008 and 212 cancer-free population controls. Data showed that the genotype distribution of G2133C (rs2864746), but not A-202C (rs2864744), was significantly different between cancer cases and controls. Unconditional logistic regression analyses revealed that participants carrying the G2133C GC heterozygote or CC homozygote had a significant 1.55-fold increased risk of colorectal cancer development in an allele dose-responsive manner. However, there was no evidence of a dose-effect relationship between number of variants and risk for CRC occurrence. Data suggest that the exon 1 G2133C missense variant of IGFBP3 may be a susceptibility factor for colorectal cancer in Chinese subjects. Larger studies are warranted to validate our findings in a Chinese population.

[The application of artificial neural networks to predict individual risk of essential hypertension].

OBJECTIVE: To establish models to predict individual risk of essential hypertension and to evaluate and explore new forecasting methods. METHODS: To select data of 3054 community residents from an epidemiological survey and divided them into 4:1 (2438 cases and 616 cases) ratio in accordance with the balance of age and sex to filter variables, and to establish, test and evaluate the prediction models. Using artificial neural network (ANN) and logistic regression analysis to establish models while applying ROC to evaluate the prediction models. RESULTS: Forecast results of the models applying to the test set proved that ANN had lower specificity but better veracity and sensitivity than logistic regression. In particular, the Youden's index of the ANN2 came up to 0.8399 which was distinctly higher than the other two models. When the area was under the ROC curve of logistic regression, the ANN, and ANN2 models equaled to 0.732 +/- 0.026, 0.900 +/- 0.014 and 0.918 +/- 0.013 respectively, which proved that the ANN model was better in the prediction about individual health risk of essential hypertension. CONCLUSION: Our results showed that ANN method seemed better than logistic regression in terms of predicting the individual risk from hypertension thus supplied a new method to solve the forecast of individual risk.

[Evaluation on the highly active anti-retroviral therapy in 181 AIDS patients].

OBJECTIVE: To study the clinical effect under highly active anti-retroviral therapy (HAART) in AIDS patients and for improving the curative effect and prognosis. METHODS: Epidemiological method was used from five aspects to describe the post-treatment clinical symptoms of 181 AIDS patients in Suizhou, and to evaluate the change of virus load and immune function of 79 AIDS patients. Data was doubly recorded by Epi Data and database was set up by SPSS 13.0 for analysis. RESULTS: The effective powers of anomal-fever, cough, diarrhoea, lymphadenectasis, weight drop, erythra, mycotic infection were 81.39%, 85.00%, 84.62%, 81.89%, 82.86%, 66.07% and 45.45% respectively. CD4+ T lymphocyte count rose obviously after treatment, with an averag of 276 x 10(6) cells/ml (65 x 10(6)-824 x 10(6) cells/ml), an 129 x 10(6) cells/ml increase in three months and was 294 x 10(6) cells/ml (102 x 10(6)-750 x 10(6) cells/ml) in six months. The count change of CD4+ T lymphocyte between 3 months and 6 months did not show sigificant difference. The number of deaths among drug withdrawals was 14, with a case fatality rate as 29.79%; while the number of deaths among non-drug withdrawals was 3, with the case fatality rate as 2.24%. CONCLUSION: Results through this study showed that HAART could obviously improve the clinical symptom of AIDS patients, and to increase the number of virus load. Improving the compliance could also reduce the case fatality rate.

CpG PatternFinder: a Windows-based utility program for easy and rapid identification of the CpG methylation status of DNA.

The bisulfite genomic sequencing technique is one of the most widely used techniques to study sequence-specific DNA methylation because of its unambiguous ability to reveal DNA methylation status to the order of a single nucleotide. One characteristic feature of the bisulfite genomic sequencing technique is that a number of sample sequence files will be produced from a single DNA sample. The PCR products of bisulfite-treated DNA samples cannot be sequenced directly because they are heterogeneous in nature; therefore they should be cloned into suitable plasmids and then sequenced. This procedure generates an enormous number of sample DNA sequence files as well as adding extra bases belonging to the plasmids to the sequence, which will cause problems in the final sequence comparison. Finding the methylation status for each CpG in each sample sequence is not an easy job. As a result CpG PatternFinder was developed for this purpose. The main functions of the CpG PatternFinder are: (i) to analyze the reference sequence to obtain CpG and non-CpG-C residue position information. (ii) To tailor sample sequence files (delete insertions and mark deletions from the sample sequence files) based on a configuration of ClustalW multiple alignment. (iii) To align sample sequence files with a reference file to obtain bisulfite conversion efficiency and CpG methylation status. And, (iv) to produce graphics, highlighted aligned sequence text and a summary report which can be easily exported to Microsoft Office suite. CpG PatternFinder is designed to operate cooperatively with BioEdit, a freeware on the internet. It can handle up to 100 files of sample DNA sequences simultaneously, and the total CpG pattern analysis process can be finished in minutes. CpG PatternFinder is an ideal software tool for DNA methylation studies to determine the differential methylation pattern in a large number of individuals in a population. Previously we developed the CpG Analyzer program; CpG PatternFinder is our further effort to create software tools for DNA methylation studies.

Three-dimensional reconstruction from serial sections in PC-Windows platform by using 3D_Viewer.

Three-dimensional (3D) reconstruction from serial sections allows identification of objects of interest in 3D and clarifies the relationship among these objects. 3D_Viewer, developed in our laboratory for this purpose, has four major functions: image alignment, movie frame production, movie viewing, and shift-overlay image generation. Color images captured from serial sections were aligned; then the contours of objects of interest were highlighted in a semi-automatic manner. These 2D images were then automatically stacked at different viewing angles, and their composite images on a projected plane were recorded by an image transform-shift-overlay technique. These composition images are used in the object-rotation movie show. The design considerations of the program and the procedures used for 3D reconstruction from serial sections are described. This program, with a digital image-capture system, a semi-automatic contours highlight method, and an automatic image transform-shift-overlay technique, greatly speeds up the reconstruction process. Since images generated by 3D_Viewer are in a general graphic format, data sharing with others is easy. 3D_Viewer is written in MS Visual Basic 6, obtainable from our laboratory on request.

An improved stereologic method for three-dimensional estimation of particle size distribution from observations in two dimensions and its application.

Single enzyme-altered hepatocytes; altered hepatic foci (AHF); and nodular lesions have been implicated, respectively in the processes of initiation, promotion, and progression in rodent hepatocarcinogenesis. Qualitative and quantitative analyses of such lesions have been utilized both to identify and to determine the potency of initiating, promoting, and progressor agents in rodent liver. Of a number of possible parameters determined in the study of such lesions, estimation of the number of foci or nodules in the liver is very important. The method of Saltykov has been used for estimating the number of AHF in rat liver. However, in practice, the Saltykov calculation has at least two weak points: (a) the size class range is limited to 12, which in many instances is too narrow to cover the range of AHF data obtained; and (b) under some conditions, the Saltykov equation generates negative values in several size classes, an obvious impossibility in the real world. In order to overcome these limitations in the Saltykov calculations, a study of the particle size distribution in a wide-range, polydispersed sphere system was performed. A stereologic method, termed the 25F Association method, was developed from this study. This method offers 25 association factors that are derived from the frequency of different-sized transections obtained from transecting a spherical particle, thus expanding the size class range to be analyzed up to 25, which is sufficiently wide to encompass all rat AHF found in most cases. This method exhibits greater flexibility, which allows adjustments to be made within the calculation process when NA((k,k)), the net number of transections from the same size spheres, was found to be a negative value, which is not possible in real situations. The reliability of the 25F Association method was tested thoroughly by computer simulation in both monodispersed and polydispersed sphere systems. The test results were compared with the original Saltykov method. We found that the 25F Association method yielded a better estimate of the total number of spheres in the three-dimensional tissue sample as well as the detailed size distribution information. Although the 25F Association method was derived from the study of a polydispersed sphere system, it can be used for continuous size distribution sphere systems. Application of this method to the estimation of parameters of preneoplastic foci in rodent liver is presented as an example of its utility. An application software program, 3D_estimation.exe, which uses the 25F Association method to estimate the number of AHF in rodent liver, has been developed and is now available at the website of this laboratory.