Evaluation of graphical models for multi-group metabolomics data.

Gaussian graphical model is a strong tool for identifying interactions from metabolomics data based on conditional correlation. However, data may be collected from different stages or subgroups of subjects with heterogeneity or hierarchical structure. There are different integrating strategies of graphical models for multi-group data proposed by data scientists. It is challenging to select the methods for metabolism data analysis. This study aimed to evaluate the performance of several different integrating graphical models for multi-group data and provide support for the choice of strategy for similar characteristic data. We compared the performance of seven methods in estimating graph structures through simulation study. We also applied all the methods in breast cancer metabolomics data grouped by stages to illustrate the real data application. The method of Shaddox et al. achieved the highest average area under the receiver operating characteristic curve and area under the precision-recall curve across most scenarios, and it was the only approach with all indicators ranked at the top. Nevertheless, it also cost the most time in all settings. Stochastic search structure learning tends to result in estimates that focus on the precision of identified edges, while BEAM, hierarchical Bayesian approach and birth-death Markov chain Monte Carlo may identify more potential edges. In the real metabolomics data analysis from three stages of breast cancer patients, results were in line with that in simulation study.

Effect and mechanism of "Danggui-kushen" herb pair on ischemic heart disease.

AIMS: The purpose of this study was to investigate the mechanism and effects of "Danggui-kushen" herb pair (DKHP) better than single drug in ischemic heart disease (IHD). METHODS: IHD model was established by left anterior descending branch of coronary artery in rats. Rats were randomized into six groups and oral administration for 7 days: control, model, Danshen dripping pills (DS) (5.103 g/kg), Danggui (DG) (2.7 g/kg), Kushen (KS) (2.7 g/kg) and DKHP (2.7 g/kg). Electrocardiogram (ECG), myocardial infarction and damage assessment, histological inspection analysis, and various biochemical indexes of myocardial tissue were measured to evaluate the myocardial damage and the protective effects of drugs. The inflammatory levels were identified by HE staining and serum cytokine, and the expression of hypoxia-inducible factor 1α (HIF-1α), inhibitor kappa B kinaseß (IKKß) and nuclear transcription factor kappa B (NF-κB) were measured by immunohistochemistry. KEY FINDINGS: The results suggested that: compared with the control group, model group showed significantly myocardial tissue abnormalities, and increased levels of inflammatory cytokine. Treatment with drugs inhibited the increase of α-hydroxybutyrate dehydrogenase (α-HBDH), creatine kinase (CK), creatinekinase isoenzyme (CK-MB), interleukin 1 (IL-1) and interleukin 6 (IL-6). The results of immunohistochemical showed that drugs-treatment inhibited the expression of IKKß and the P-p65, increased the expression of HIF-1α, which demonstrated that the anti-inflammatory effects of DKHP was achieved by suppressing of NF-κB signaling. CONCLUSION: These observations indicated that DKHP can ameliorate myocardial injury better than single. And these are related to the inhibition of NF-κB and actives HIF-1α signaling.

Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans.

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

Gene therapy/bone marrow transplantation in ADA-deficient mice: roles of enzyme-replacement therapy and cytoreduction.

Gene therapy (GT) for adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) can provide significant long-term benefit when patients are given nonmyeloablative conditioning and ADA enzyme-replacement therapy (ERT) is withheld before autologous transplantation of γ-retroviral vector-transduced BM CD34+ cells. To determine the contributions of conditioning and discontinuation of ERT to the therapeutic effects, we analyzed these factors in Ada gene knockout mice (Ada(-/-)). Mice were transplanted with ADA-deficient marrow transduced with an ADA-expressing γ-retroviral vector without preconditioning or after 200 cGy or 900 cGy total-body irradiation and evaluated after 4 months. In all tissues analyzed, vector copy numbers (VCNs) were 100- to 1000-fold greater in mice receiving 900 cGy compared with 200 cGy (P < .05). In mice receiving 200 cGy, VCN was similar whether ERT was stopped or given for 1 or 4 months after GT. In unconditioned mice, there was decreased survival with and without ERT, and VCN was very low to undetectable. When recipients were conditioned with 200 cGy and received transduced lineage-depleted marrow, only recipients receiving ERT (1 or 4 months) had detectable vector sequences in thymocytes. In conclusion, cytoreduction is important for the engraftment of gene-transduced HSC, and short-term ERT after GT did not diminish the capacity of gene-corrected cells to engraft and persist.

Clinicopathological Significance of Mucin 2 Immuno-histochemical Expression in Colorectal Cancer: A Meta-Analysis.

OBJECTIVE: To evaluate the association between mucin 2 (MUC2) expression and clinicopathological characters of colorectal cancer. METHODS: A literature search was performed on December 31, 2010 according to defined selection criteria. We evaluated the correlation between MUC2 (detected by immunohistochemistry) and clinicopathological characters of colorectal cancer. According to the tumor histological type, differentiation, location and TNM staging of colorectal carcinoma, we divided the clinicopathological characteristics into different subgroups. Fixed and random effects models were applied for estimation of the summarized risk ratios (RRs) and 95% confidence intervals (CIs) in different subgroups. Finally, forest plots and funnel plots were created to allow for visual comparison of the results or the effect of publication bias. RESULTS: According with the inclusive criteria, fourteen studies (n=1,558) were eligible for the meta-analysis. We observed a trend towards a correlation of MUC2 higher positivity in mucinous than non-mucinous carcinoma (RR, 2.10; 95% CI, 1.30-3.40; P=0.002) and less positivity in distal than proximal colon (RR, 0.74; 95% CI, 0.64-0.85; P=0.000). There was no statistically significance for the association between MUC2 expression and differentiation or TNM staging of colorectal cancer, but MUC2 overexpression tended to be associated with the presence of T stage tumor (RR, 1.17; P=0.052). CONCLUSION: MUC2 overexpression was associated with the mucinous and proximal colorectal cancer.

Altered expression of MUC2 and MUC5AC in progression of colorectal carcinoma.

AIM: To study the expression profiles of MUC2 and MUC5AC in tumorigenesis of colorectal carcinoma and in its different pathologic types. METHODS: Formalin-fixed, paraffin-embedded human colorectal tissue specimens were immunostained with antibodies against MUC2 and MUC5AC. Six samples of normal mucosa (NM), 12 samples of hyperplastic polyp (HP), 15 samples of tubular adenoma with low-grade dysplasia (LGD), 14 samples of tubular adenoma with high-grade dysplasia (HGD), 26 samples of conventional colorectal adenocarcinoma (CCA), 15 samples of mucinous carcinoma (MC), and 8 samples of signet-ring cell carcinoma (SRCC) were collected. RESULTS: MUC2 was the most widely expressed protein in each study group, although the number of MUC2-positive cases was less in CCA group than in other groups (P < 0.05). The staining score for MUC2 was significantly decreased in the HP-LGD-HGD-CCA sequence (r = -0.73436, P < 0.0001). Among the neoplasms, MC and SRCC were more frequently associated with the high expression of MUC2 (P < 0.05) than with that of CCA. MUC5AC expression was detected in all groups but not in NM group. Furthermore, the staining score for MUC5AC was higher in HP, LGD, HGD, MC and SRCC groups than in NM and CCA groups (P < 0.05). The frequency of simultaneous expression of MUC proteins was significantly higher in MC and SRCC groups than in CCA group (P < 0.05). CONCLUSION: Alterations in MUC expression occur during colorectal tumorigenesis. The transformation process in MC and SRCC may be different from that in the traditional adenoma-carcinoma sequence.

[Establishment of non-parametric probabilistic model for evaluation of Chinese dietary exposure].

OBJECTIVE: To establish a non-parametric probabilistic model for evaluation of Chinese dietary exposure and to improve the assessment accuracy while integrating into the global risk assessment on food safety. METHODS: Contamination data was from the national food contamination monitoring program during 2000 - 2006, including heavy metals, pesticides and mycotoxins, amounting to 135 contaminants with 499 commodities and 487 819 samples. Food consumption data was obtained from the national diet and nutrition survey conducted in 2002 with three consecutive days by 24-hour recall method, and 66 172 consumers were included. Monte Carlo simulation was applied to derive the intake distribution, and the uncertainty of each percentile was estimated using the Bootstrap sampling. RESULTS: Different non-parametric probabilistic models for dietary exposure evaluation on heavy metals, pesticides and some of the toxins were established for Chinese people, and intake distributions with 95% confidence intervals of these contaminants were estimated. Taking acephate as an example, the results of its model shows that, for the 7 - 10 year-old children, the median dietary exposure in urban and rural areas were 1.77 microg x kg(-1) x d(-1) and 2.48 microg x kg(-1) x d(-1) respectively, with a 95% confidence interval of (1.59 - 2.06) microg x kg(-1) x d(-1) and (2.33 - 2.80) microg x kg(-1) x d(-1) respectively. CONCLUSION: The non-parametric probabilistic model can quantify the variability and uncertainty of exposure assessment and improve the assessment accuracy.

[Development and verification of Chinese dietary exposure evaluation model software].

OBJECTIVE: To develop the dietary exposure evaluation model software accredited of Chinese intellectual property rights and to verify the rationality and accuracy of the results from the probabilistic model in Chinese dietary exposure evaluation model software according to international standards. METHODS: The software of SAS was used to build various evaluation model based on the data from Chinese dietary survey and the chemical compound in food surveillance and to design an operation interface. The results from probabilistic dietary exposure model for children 2 - 7 years old were compared with that from duplicate portion study of 2-7 years children dietary exposure in Jinhu, Jiangsu province in order to analyze the rationality of model. The results from probabilistic model of dietary exposure were compared with the results from @Risk software to verify the correction of the probabilistic model by using the same data of randomly selected 10 000 study subjects from national dietary survey. While, the mean drift was used as an internal index to illustrate the accuracy of the computation. RESULTS: Chinese dietary exposure evaluation software was developed successfully. On the rationality, the results from probabilistic model were lower than that from the point estimation (e.g., cucumber: the result of point estimation of acephate was 4.78 microg x kg(-1) x d(-1), while the results of probabilistic model which was 0.39 microg x kg(-1) x d(-1)). Meanwhile the results from probabilistic model were higher than the results of duplicate portion study (on the P95, the result of probabilistic model of Pb exposure in children was 11.08 microg x kg(-1) x d(-1), while the results of duplicate portion study was 5.75 microg x kg(-1) x d(-1)). On accuracy, the results from @Risk and the probabilistic model were highly consistent (on the P95, the result of probabilistic assessment of acephate diet exposure was 4.27 microg x kg(-1) x d(-1), while the results of duplicate portion study was 4.24 microg x kg(-1) x d(-1)), and the mean drift was of random distribution, the drift region varied from 0.05% to 11.9%. CONCLUSION: The results computed by the software of Chinese dietary exposure evaluation model are reliable and reasonable, which is a meaningful step to improve the dietary exposure evaluation technique in China.

Combining previously published studies with current data in Bayesian logistic regression model: an example for identifying susceptibility genes related to lung cancer in humans.

A general analysis method is proposed that utilizes meta-analysis to incorporate similar studies in addition to our current investigation in order to obtain informative prior effect parameters in a logistic regression model. It is common in epidemiological studies that data from similar previous studies are available. The case of gene susceptibility association with increased lung cancer frequency was used to demonstrate this methodology. Results of Markov chain Monte Carlo (MCMC) iterations provided a more precise estimation of the regression coefficient in a logistic model with informative prior distribution compared to the noninformative prior distribution model. In situations where similar historical data are available, it is proposed to include as much relevant information as previously published results in the analysis of current data.

Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion.

Adenosine deaminase (ADA)-deficient severe combined immune deficiency (SCID) may be treated by allogeneic hematopoietic stem cell transplantation without prior cytoreductive conditioning, although the mechanism of immune reconstitution is unclear. We studied this process in a murine gene knockout model of ADA-deficient SCID. Newborn ADA-deficient pups received transplants of intravenous infusion of normal congenic bone marrow, without prior cytoreductive conditioning, which resulted in long-term survival, multisystem correction, and nearly normal lymphocyte numbers and mitogenic proliferative responses. Only 1% to 3% of lymphocytes and myeloid cells were of donor origin without a selective expansion of donor-derived lymphocytes; immune reconstitution was by endogenous, host-derived ADA-deficient lymphocytes. Preconditioning of neonates with 100 to 400 cGy of total body irradiation before normal donor marrow transplant increased the levels of engrafted donor cells in a radiation dose-dependent manner, but the chimerism levels were similar for lymphoid and myeloid cells. The absence of selective reconstitution by donor T lymphocytes in the ADA-deficient mice indicates that restoration of immune function occurred by rescue of endogenous ADA-deficient lymphocytes through cross-correction from the engrafted ADA-replete donor cells. Thus, ADA-deficient SCID is unique in its responses to nonmyeloablative bone marrow transplantation, which has implications for clinical bone marrow transplantation or gene therapy.

Stable gene transfer to human CD34(+) hematopoietic cells using the Sleeping Beauty transposon.

OBJECTIVE: Methods of gene transfer to hematopoietic stem cells that result in stable integration may provide treatments for many inherited and acquired blood diseases. It has been demonstrated previously that a gene delivery system based on the Sleeping Beauty (SB) transposon can be derived where a plasmid transiently expressing the SB transposase can mediate the stable chromosomal integration of a codelivered second plasmid containing a gene expression unit flanked by the inverted repeats derived from the transposon. METHODS: Plasmid DNA containing the elements required for SB transposition was delivered to hematopoietic cells via electroporation. Integrated transgene (enhanced green fluorescent protein [eGFP]) expression was assessed in vitro and in vivo. RESULTS: In the K562 human hematopoietic cell line, we observed stable expression of eGFP in >60% of cells for over 2 months after electroporation of the two plasmids; in contrast, in control cells either not treated with transposase or exposed to a defective mutant transposase, the level of gene expression had fallen to near background (<0.1%) by 2 weeks. In purified human cord blood CD34(+) progenitor cells, the transposase led to stable gene transfer at levels up to 6% for over 4 weeks, but gene transfer to more primitive nonobese diabetic/severe combined immunodeficient repopulating cells or CD34(+)/CD38(-) in long-term culture was low and electroporation of the cells with plasmid DNA caused significant cell death. CONCLUSION: The long-term stable expression highlights the potential of this transposase-based gene delivery method for ameliorating diseases affecting the hematopoietic system, although further improvements in gene transfer efficacy are needed.

Transient gene expression by nonintegrating lentiviral vectors.

Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34(+) hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eGFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (approximately 90% of cells), but expression was transient, diminishing toward background (<0.5%) within less than 1 month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 10(4)-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.

Specific and stable gene transfer to human embryonic stem cells using pseudotyped lentiviral vectors.

Genetic modification of human embryonic stem cells (hESCs) is an important tool for understanding and influencing their biologic properties. At the present time, lentiviral vectors pseudotyped with the vesicular stomatitis virus G protein (VSV-G) have been most effective for stable gene transfer to hESCs. However, they also efficiently transduce murine embryonic fibroblasts (MEF), used to support the undifferentiated state of many commonly used hESC lines. Transduction of both the MEF as well as hESCs complicates analyses of gene transfer and expression. We made lentiviral vectors pseudotyped with envelope glycoproteins from retroviruses that have been shown to have more restricted transduction ranges and evaluated their specificity. Lentiviral vectors pseudotyped by the envelopes from either the gibbon ape leukemia virus (GALV) or the RD114 feline endogenous virus (RD114) specifically transduced hESCs to similar extents as VSV-G pseudotyped vectors, but did not transduce MEF. In addition, gene modfication by these pseudotyped lentiviral vectors was stably maintained throughout differentiation of hESCs in vitro. These pseudotyped lentiviral vectors may be valuable tools for efficient, specific and stable gene modification of hESCs.

Expression from second-generation feline immunodeficiency virus vectors is impaired in human hematopoietic cells.

Vectors based on the feline immunodeficiency virus (FIV) have been developed as an alternative to those based on another lentivirus, human immunodeficiency virus-1 (HIV-1), because of theoretical safety advantages. We compared the efficiency of gene transfer and expression in human and feline hematopoietic progenitors using second-generation HIV-1 and FIV-based vectors. Vector pairs were tested using either human cytomegalovirus or murine phospho-glycerate kinase (PGK) internal promoters and were pseudotyped with the vesicular stomatitis virus G protein (VSV-G). Vector proviral copy numbers were similar in human and feline hematopoietic primary cells and cell lines transduced by HIV-1 or FIV vectors, demonstrating that both vectors are able to transfer genes efficiently to these cell types. HIV-1 vectors were well expressed in human primary hematopoietic cells and cell lines. However, transgene expression from FIV vectors was almost undetectable in human hematopoietic cells. In contrast, the FIV vector was expressed well in primary hematopoietic feline cells and human non-hematopoietic cells, demonstrating that low transgene expression from the FIV vector is a phenomenon specific to human hematopoietic cells. Northern blot analysis demonstrated decreased vector transcript levels in human CEM cells transduced with FIV relative to cells transduced with HIV-1, despite high vector copy numbers. No evidence of vector transcript instability was seen in studies of transduced CEM cells treated with actinomycin D. We conclude that FIV vectors can transfer genes into human hematopoietic cells as effectively as HIV-1 vectors, but that unknown elements in the current FIV backbone inhibit expression from FIV vectors in human hematopoietic cells.