An Integrated Approach of Learning Genetic Networks From Genome-Wide Gene Expression Data Using Gaussian Graphical Model and Monte Carlo Method.

Global genetic networks provide additional information for the analysis of human diseases, beyond the traditional analysis that focuses on single genes or local networks. The Gaussian graphical model (GGM) is widely applied to learn genetic networks because it defines an undirected graph decoding the conditional dependence between genes. Many algorithms based on the GGM have been proposed for learning genetic network structures. Because the number of gene variables is typically far more than the number of samples collected, and a real genetic network is typically sparse, the graphical lasso implementation of GGM becomes a popular tool for inferring the conditional interdependence among genes. However, graphical lasso, although showing good performance in low dimensional data sets, is computationally expensive and inefficient or even unable to work directly on genome-wide gene expression data sets. In this study, the method of Monte Carlo Gaussian graphical model (MCGGM) was proposed to learn global genetic networks of genes. This method uses a Monte Carlo approach to sample subnetworks from genome-wide gene expression data and graphical lasso to learn the structures of the subnetworks. The learned subnetworks are then integrated to approximate a global genetic network. The proposed method was evaluated with a relatively small real data set of RNA-seq expression levels. The results indicate the proposed method shows a strong ability of decoding the interactions with high conditional dependences among genes. The method was then applied to genome-wide data sets of RNA-seq expression levels. The gene interactions with high interdependence from the estimated global networks show that most of the predicted gene-gene interactions have been reported in the literatures playing important roles in different human cancers. Also, the results validate the ability and reliability of the proposed method to identify high conditional dependences among genes in large-scale data sets.

Pattern of Radiotherapy Treatment in Low-Risk, Intermediate-Risk, and High-Risk Prostate Cancer Patients: Analysis of National Cancer Database.

Background: In this study, the utilization rates and survival outcomes of different radiotherapy techniques are compared in prostate cancer (PCa) patients stratified by risk group. Methods: We analyzed an extensive data set of N0, M0, non-surgical PCa patients diagnosed between 2004 and 2015 from the National Cancer Database (NCDB). Patients were grouped into six categories based on RT modality: an intensity-modulated radiation therapy (IMRT) group with brachytherapy (BT) boost, IMRT with/without IMRT boost, proton therapy, stereotactic body radiation therapy (SBRT), low-dose-rate brachytherapy (BT LDR), and high-dose-rate brachytherapy (BT HDR). Patients were also stratified by the National Comprehensive Cancer Network (NCCN) guidelines: low-risk (clinical stage T1−T2a, Gleason Score (GS) ≤ 6, and Prostate-Specific Antigen (PSA) < 10), intermediate-risk (clinical stage T2b or T2c, GS of 7, or PSA of 10−20), and high-risk (clinical stage T3−T4, or GS of 8−10, or PSA > 20). Overall survival (OS) probability was determined using a Kaplan−Meier estimator. Univariate and multivariate analyses were performed by risk group for the six treatment modalities. Results: The most utilized treatment modality for all PCa patients was IMRT (53.1%). Over the years, a steady increase in SBRT utilization was observed, whereas BT HDR usage declined. IMRT-treated patient groups exhibited relatively lower survival probability in all risk categories. A slightly better survival probability was observed for the proton therapy group. Hormonal therapy was used for a large number of patients in all risk groups. Conclusion: This study revealed that IMRT was the most common treatment modality for PCa patients. Brachytherapy, SBRT, and IMRT+BT exhibited similar survival rates, whereas proton showed slightly better overall survival across the three risk groups. However, analysis of the demographics indicates that these differences are at least in part due to selection bias.

Role of radiation in extensive stage small cell lung cancer: a National Cancer Database registry analysis.

The role of prophylactic cranial irradiation (PCI) and thoracic radiation therapy (TRT) in extensive-stage small cell lung cancer remains controversial. The authors examined the National Cancer Database and identified patients with extensive-stage small cell lung cancer with no brain metastasis. Patients were excluded if they died 30 days from diagnosis, did not receive polychemotherapy, had other palliative radiation or had missing information. A propensity score-matched analysis was also performed. A total of 21,019 patients were identified. The majority of patients did not receive radiation (69%), whereas 10% received PCI and 21% received TRT. The addition of PCI and TRT improved median survival and survival at 1 and 2 years (p ≤ 0.05). The propensity score-matched analysis confirmed the same overall survival benefit with both PCI and TRT. This registry-based analysis of >1500 accredited cancer programs shows that PCI and TRT are not commonly utilized for extensive-stage small cell lung cancer patients who are treated with multiagent chemotherapy. The addition of PCI and TRT significantly improves overall survival in this otherwise poor prognostic group. Further research is needed to confirm the role of PCI and TRT, especially in the era of improved systemic therapy.

Lay abstract The role of radiation therapy in patients with metastatic small cell lung cancer remains controversial. The authors examined the National Cancer Database and identified patients with metastatic small cell lung cancer without brain metastasis. Patients were excluded if they died 30 days from diagnosis, did not receive multiagent chemotherapy, had other palliative radiation or had missing information regarding treatment. A total of 21,019 patients were identified. The majority of patients did not receive radiation (69%), whereas 10% received radiation to the brain and 21% received radiation to their lungs. The addition of brain and lung radiation therapy improved median survival and survival at 1 and 2 years. The addition of prophylactic cranial irradiation and thoracic radiation therapy improves survival in extensive-stage small cell lung cancer. Future research is needed to evaluate the role of radiation in the era of chemoimmunotherapy.

TSP-1 (Thrombospondin-1) Deficiency Protects ApoE-/- Mice Against Leptin-Induced Atherosclerosis.

OBJECTIVE: Hyperleptinemia, hallmark of obesity, is a putative pathophysiologic trigger for atherosclerosis. We previously reported a stimulatory effect of leptin on TSP-1 (thrombospondin-1) expression, a proatherogenic matricellular protein implicated in atherogenesis. However, a causal role of TSP-1 in leptin-driven atherosclerosis remains unknown. Approach and Results: Seventeen-weeks-old ApoE-/- and TSP-1-/-/ApoE-/- double knockout mice, on normocholesterolemic diet, were treated with or without murine recombinant leptin (5 µg/g bwt, IP) once daily for 3 weeks. Using aortic root morphometry and en face lesion assay, we found that TSP-1 deletion abrogated leptin-stimulated lipid-filled lesion burden, plaque area, and collagen accumulation in aortic roots of ApoE-/- mice, shown via Oil red O, hematoxylin and eosin, and Masson trichrome staining, respectively. Immunofluorescence microscopy of aortic roots showed that TSP-1 deficiency blocked leptin-induced inflammatory and smooth muscle cell abundance as well as cellular proliferation in ApoE-/- mice. Moreover, these effects were concomitant to changes in VLDL (very low-density lipoprotein)-triglyceride and HDL (high-density lipoprotein)-cholesterol levels. Immunoblotting further revealed reduced vimentin and pCREB (phospho-cyclic AMP response element-binding protein) accompanied with augmented smooth muscle-myosin heavy chain expression in aortic vessels of leptin-treated double knockout versus leptin-treated ApoE-/-; also confirmed in aortic smooth muscle cells from the mice genotypes, incubated ± leptin in vitro. Finally, TSP-1 deletion impeded plaque burden in leptin-treated ApoE-/- on western diet, independent of plasma lipid alterations. CONCLUSIONS: The present study provides evidence for a protective effect of TSP-1 deletion on leptin-stimulated atherogenesis. Our findings suggest a regulatory role of TSP-1 on leptin-induced vascular smooth muscle cell phenotypic transition and inflammatory lesion invasion. Collectively, these results underscore TSP-1 as a potential target of leptin-induced vasculopathy.

Parenteral administration of L-arginine enhances fetal survival and growth in sheep carrying multiple fetuses.

The frequency of multiple fetuses has increased in human pregnancies due to assisted reproductive technologies. This translates into a greater proportion of premature and low-birth weight infants in the United States and worldwide. In addition, improvements in sheep breeding have resulted in new breeds with increased litter size but reduced fetal survival and birth weight. Currently, there are no treatments for preventing fetal growth restriction in humans or sheep (an established model for studying human fetal physiology) carrying multiple fetuses. In this work, Booroola Rambouillet ewes (FecB+/-) with 2-4 fetuses were fed a diet providing 100% of NRC-recommended nutrient requirements. Between d 100 and 121 of gestation, ewes received an i.v. bolus injection of either saline solution or 345 µmol arginine-HCl/kg body weight 3 times daily. The arginine treatment reduced (P < 0.05) the percentage of lambs born dead by 23% while increasing (P = 0.05) the percentage of lambs born alive by 59%. The i.v. administration of arginine enhanced (P < 0.05) the birth weights of quadruplets by 23% without affecting maternal body weight. The improved pregnancy outcome was associated with an increase in maternal plasma concentrations of arginine, ornithine, cysteine, and proline, as well as a decrease in circulating levels of ammonia and ß-hydroxybutyrate. These novel results indicate that parenteral administration of arginine to prolific ewes ameliorated fetal mortality and growth retardation. Our findings provide support for experiments to assess the clinical use of arginine to enhance fetal growth and survival in women gestating multiple fetuses.

Bayesian Modeling of MPSS Data: Gene Expression Analysis of Bovine Salmonella Infection.

Massively Parallel Signature Sequencing (MPSS) is a high-throughput counting-based technology available for gene expression profiling. It produces output that is similar to Serial Analysis of Gene Expression (SAGE) and is ideal for building complex relational databases for gene expression. Our goal is to compare the in vivo global gene expression profiles of tissues infected with different strains of Salmonella obtained using the MPSS technology. In this article, we develop an exact ANOVA type model for this count data using a zero-inflated Poisson (ZIP) distribution, different from existing methods that assume continuous densities. We adopt two Bayesian hierarchical models-one parametric and the other semiparametric with a Dirichlet process prior that has the ability to "borrow strength" across related signatures, where a signature is a specific arrangement of the nucleotides, usually 16-21 base-pairs long. We utilize the discreteness of Dirichlet process prior to cluster signatures that exhibit similar differential expression profiles. Tests for differential expression are carried out using non-parametric approaches, while controlling the false discovery rate. We identify several differentially expressed genes that have important biological significance and conclude with a summary of the biological discoveries.

Parenteral administration of L-arginine prevents fetal growth restriction in undernourished ewes.

Intrauterine growth restriction (IUGR) is a major health problem worldwide that currently lacks an effective therapeutic solution. This study was conducted with an ovine IUGR model to test the hypothesis that parenteral administration of l-arginine (Arg) is effective in enhancing fetal growth. Beginning on d 28 of gestation, ewes were fed a diet providing 100% (control-fed) or 50% (underfed) of NRC-recommended nutrient requirements. Between d 60 of gestation and parturition, underfed ewes received i.v. infusions of saline or 155 micromol Arg-HCl/kg body weight 3 times daily, whereas control-fed ewes received only saline. The birth weights of lambs from saline-infused underfed ewes were 23% lower (P < 0.01) than those of lambs from control-fed dams. Administration of Arg to underfed ewes increased (P < 0.01) concentrations of Arg (69%), ornithine (55%), proline (29%), methionine (37%), leucine (36%), isoleucine (35%), cysteine (19%), and FFA (43%) in maternal serum, decreased maternal circulating levels of ammonia (18%) and triglycerides (32%), and enhanced birth weights of lambs by 21% compared with saline-infused underfed ewes. There was no difference in birth weights of lambs between the control-fed and the Arg-infused underfed ewes. These novel results indicate that parenteral administration of Arg to underfed ewes prevented fetal growth restriction and provide support for its clinical use to ameliorate IUGR in humans. The findings also lay a new framework for studying cellular and molecular mechanisms responsible for the beneficial effects of Arg in regulating conceptus growth and development.

Functional relationships between genes associated with differentiation potential of aged myogenic progenitors.

Aging is accompanied by considerable heterogeneity with possible co-expression of differentiation pathways. The present study investigates the interplay between crucial myogenic, adipogenic, and Wnt-related genes orchestrating aged myogenic progenitor differentiation (AMPD) using clonal gene expression profiling in conjunction with Bayesian structure learning (BSL) techniques. The expression of three myogenic regulatory factor genes (Myogenin, Myf-5, MyoD1), four genes involved in regulating adipogenic potential (C/EBPα, DDIT3, FoxC2, PPARγ), and two genes in the Wnt signaling pathway (Lrp5, Wnt5a) known to influence both differentiation programs were determined across 34 clones by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Three control genes were used for normalization of the clonal expression data (18S, GAPDH, and B2M). Constraint-based BSL techniques, namely (a) PC Algorithm, (b) Grow-shrink (GS) algorithm, and (c) Incremental Association Markov Blanket (IAMB) were used to model the functional relationships (FRs) in the form of acyclic networks from the clonal expression profiles. A novel resampling approach that obviates the need for a user-defined confidence threshold is proposed to identify statistically significant FRs at small sample sizes. Interestingly, the resulting acyclic network consisted of FRs corresponding to myogenic, adipogenic, Wnt-related genes and their interaction. A significant number of these FRs were robust to normalization across the three house-keeping genes and the choice of the BSL technique. The results presented elucidate the delicate balance between differentiation pathways (i.e., myogenic as well as adipogenic) and possible cross-talk between pathways in AMPD.

Dietary supplementation with cholesterol and docosahexaenoic acid affects concentrations of amino acids in tissues of young pigs.

Cholesterol and docosahexaenoic acid (DHA) are important nutrients for neural development of infants. However, little is known about the effect of cholesterol or DHA on concentrations of amino acids (AA) in neonatal tissues. This study was conducted with the piglet (an established model for studying human infant nutrition) to test the hypothesis that dietary supplementation with the lipids may modulate AA availability in tissues. Sixteen newborn pigs were nursed by sows for 24 h and then assigned to one of four treatment groups, representing supplementation with 0.0% (control), 0.2% cholesterol, 0.2% DHA, or cholesterol plus DHA to the basal milk-formula. All piglets were euthanized at 49 days of age. In brain, cholesterol supplementation reduced (P < 0.05) concentrations of glutamate, serine, glutamine, threonine, beta-alanine, alanine, methionine, isoleucine, leucine, and gamma-aminobutyrate but increased (P < 0.05) concentrations of glycine and lysine, whereas DHA supplementation similarly affected (P < 0.05) concentrations of the same AA (except for isoleucine and lysine) and taurine. In addition, concentrations of most AA in liver, muscle and plasma were substantially altered by dietary supplementation of cholesterol and DHA in a tissue-dependent manner. Further, DHA reduced concentrations of carnosine in skeletal muscle, as well as ammonia in both plasma and brain. The results reveal that cholesterol and DHA can regulate AA metabolism and availability in various tissues of piglets. These novel findings have important implications for designing the next generation of infant formula to optimize neonatal growth and development.

Dietary supplementation with cholesterol and docosahexaenoic acid increases the activity of the arginine-nitric oxide pathway in tissues of young pigs.

Nitric oxide (NO), synthesized from l-arginine by tetrahydrobiopterin (BH4)-dependent NO synthase (NOS), is critical for neurological and muscular development and function. This study was designed to test the hypothesis that cholesterol and docosahexaenoic acid (DHA) may modulate the arginine-NO pathway in tissues of the young pig. Sixteen newborn pigs were nursed by sows for 24h and then assigned to one of four treatment groups, representing supplementation with 0.0%, 0.2% cholesterol, 0.2% DHA, or cholesterol plus DHA to the basal milk-formula. All piglets were euthanized at 49 days of age. Brain, liver and gastrocnemius muscle were analyzed for BH4, NADPH and arginine, GTP cyclohydrolase-I (GTP-CH) and NOS activities, and NOS protein isoforms. Hepatic NOS activity was below the detection limit in all pigs. DHA supplementation (P<0.01) increased GTP-CH activities, as well as BH4 and NADPH concentrations in brain, liver, and muscle by 24-46%, while enhancing (P<0.05) NOS activities by 45-48% in brain and muscle. Dietary cholesterol supplementation increased (P<0.05) NOS and GTP-CH activities by 17-26% in brain but had no effect in liver or muscle. The enhanced NOS activity in the brain or muscle of cholesterol- or DHA-supplemented piglets was attributable to the combined effects of increased eNOS and nNOS activation (changes in phosphorylation levels) and total iNOS protein. Additionally, DHA and cholesterol enhanced (P<0.05) arginine concentrations in brain (35-42%), but not in liver or muscle. These tissue-specific effects of cholesterol and DHA on NO synthesis may play an important role in postnatal growth and development.