Design, Synthesis and Biological Evaluation of Novel MDH Inhibitors Targeting Tumor Microenvironment.

MDH1 and MDH2 enzymes play an important role in the survival of lung cancer. In this study, a novel series of dual MDH1/2 inhibitors for lung cancer was rationally designed and synthesized, and their SAR was carefully investigated. Among the tested compounds, compound 50 containing a piperidine ring displayed an improved growth inhibition of A549 and H460 lung cancer cell lines compared with LW1497. Compound 50 reduced the total ATP content in A549 cells in a dose-dependent manner; it also significantly suppressed the accumulation of hypoxia-inducible factor 1-alpha (HIF-1α) and the expression of HIF-1α target genes such as GLUT1 and pyruvate dehydrogenase kinase 1 (PDK1) in a dose-dependent manner. Furthermore, compound 50 inhibited HIF-1α-regulated CD73 expression under hypoxia in A549 lung cancer cells. Collectively, these results indicate that compound 50 may pave the way for the development of next-generation dual MDH1/2 inhibitors to target lung cancer.

Cancer-specific cytotoxicity of pyridinium-based ionic liquids by regulating hypoxia-inducible factor-1α-centric cancer metabolism.

Owing to their unique properties and biological activities, ionic liquids (ILs) have attracted research interest in pharmaceutics and medicine. Hypoxia-inducible factor (HIF)- 1α is an attractive cancer drug target involved in cancer malignancy in the hypoxic tumor microenvironment. Herein, we report the inhibitory activity of ILs on the HIF-1α pathway and their mechanism of action. Substitution of a dimethylamino group on pyridinium reduced hypoxia-induced HIF-1α activation. It selectively inhibited the viability of the human colon cancer cell line HCT116, compared to that of the normal fibroblast cell line WI-38. These activities were enhanced by increasing the alkyl chain length in the pyridinium. Under hypoxic conditions, dimethylaminopyridinium reduced the accumulation of HIF-1α and its target genes without affecting the HIF1A mRNA level in cancer cells. It suppressed the oxygen consumption rate and ATP production by directly inhibiting electron transfer chain complex I, which led to enhanced intracellular oxygen content and oxygen-dependent degradation of HIF-1α under hypoxia. These results indicate that dimethylaminopyridinium suppresses the mitochondria and HIF-1α-dependent glucose metabolic pathway in hypoxic cancer cells. This study provides insights into the anticancer activity of pyridinium-based ILs through the regulation of cancer metabolism, making them promising candidates for cancer treatment.

Prescription fill rates for acute and chronic medications in claims-EMR linked data.

Nonadherence to prescribed medications poses a significant public health problem. Prescription data in electronic medical records (EMRs) linked with pharmacy claims data provides an opportunity to examine the prescription fill rates and factors associated with it.Using a claims-EMR linked data, patients who had a prescription for either an antibiotic, antihypertensive, or antidiabetic in EMR were identified (index prescription). Prescription fill was defined as a pharmacy claim found within the 90 days following the EMR prescription. For each medication group, patient characteristics and fill rates were examined using descriptive statistics. Multivariate logistic regression was used to evaluate the association between fill rates and factors such as age, race, brand vs generic, and prior treatment during 365 days before the index date.Among 77,996 patients with index antibiotic prescription, 78,462 with index antihypertensive prescription, and 24,013 with index antidiabetic prescription, the prescription fill rate was 73%, 74%, and 76%, respectively. Overall, African American race was negatively associated with fill rates (odds ratio [OR] 0.8 for all 3 groups). Prior treatment history was positively associated with antihypertensives (OR 5.6, 95% confidence interval [CI] 5.4-5.7) or antidiabetics (OR 4.1, CI 3.8-4.4) but negatively with antibiotics (OR 0.6, CI 0.6-0.6). Older age was an additional factor that was negatively associated with first time fill rate among patients without prior treatment.Significant proportions of patients, especially patients with no prior treatment history, did not fill prescriptions for antibiotics, antihypertensives, or antidiabetics. The association between patient factors and medication fill rates varied across different medication groups.

Characteristics of Cricopharyngeal Dysphagia After Ischemic Stroke.

OBJECTIVE: To evaluate the characteristics of cricopharyngeal dysfunction (CPD), the frequency, and correlation with a brain lesion in patients with first-ever ischemic stroke, and to provide basic data for developing a therapeutic protocol for dysphagia management. METHODS: We retrospectively reviewed the medical records of a series of subjects post-stroke who underwent a videofluoroscopic swallowing study (VFSS) from January 2009 to December 2015. VFSS images were recorded on videotape and analyzed. CPD was defined as the retention of more than 25% of residue in the pyriform sinus after swallowing. The location of the brain lesion was assessed using magnetic resonance imaging. RESULTS: Among the 262 dysphagic patients with first-ever ischemic stroke, 15 (5.7%) showed CPD on the VFSS. Patients with an infratentorial lesion had a significantly higher proportion of CPD than those with a supratentorial lesion (p=0.003), and lateral medullary infarction was identified as the single independent predictor of CPD (multivariable analysis: odds ratio=19.417; confidence interval, 5.560-67.804; p<0.0001). Compared to patients without CPD, those with CPD had a significantly prolonged pharyngeal transit time, lower laryngeal elevation, and a higher pharyngeal constriction ratio and functional dysphagia scale score. CONCLUSION: Overall, the results support the notion that an impaired upper esopharyngeal opening is likely related to the specific locations of brain lesions. The association of CPD with lateral medullary infarction can be explained based on the regulation of the pharyngolaryngeal motor system by the motor neurons present in the dorsal nucleus ambiguus. Overall, the results reveal the relation between CPD and the problems in the pharyngeal phase as well as the severity of dysphagia.

Effects of Radiation Therapy on Established Neurogenic Heterotopic Ossification.

Heterotopic ossification (HO) is frequently seen on rehabilitation units after spinal cord injuries, fractures, brain injuries, and limb amputations. Currently, there is no effective treatment for HO other than prophylaxis with anti-inflammatory medications, irradiation, and bisphosphonate administration. These prophylactic treatments are not effective for managing ectopic bone once it has formed. Here we describe three cases of established neurogenic HO treated with radiation therapy (RT). All patients had decreased serum alkaline phosphatase (ALP) and bone-specific ALP levels with decreased pain but increased range of motion immediately after RT. Post-treatment X-rays revealed no further growth of the HO. All patients maintained clinical and laboratory improvements 4 or 6 months after the RT. Our results suggest that RT is safe and effective in decreasing pain and activity of neurogenic HO.

Genetic Analysis of Substrain Divergence in Non-Obese Diabetic (NOD) Mice.

The non-obese diabetic (NOD) mouse is a polygenic model for type 1 diabetes that is characterized by insulitis, a leukocytic infiltration of the pancreatic islets. During ~35 years since the original inbred strain was developed in Japan, NOD substrains have been established at different laboratories around the world. Although environmental differences among NOD colonies capable of impacting diabetes incidence have been recognized, differences arising from genetic divergence have not been analyzed previously. We use both mouse diversity array and whole-exome capture sequencing platforms to identify genetic differences distinguishing five NOD substrains. We describe 64 single-nucleotide polymorphisms, and two short indels that differ in coding regions of the five NOD substrains. A 100-kb deletion on Chromosome 3 distinguishes NOD/ShiLtJ and NOD/ShiLtDvs from three other substrains, whereas a 111-kb deletion in the Icam2 gene on Chromosome 11 is unique to the NOD/ShiLtDvs genome. The extent of genetic divergence for NOD substrains is compared with similar studies for C57BL6 and BALB/c substrains. As mutations are fixed to homozygosity by continued inbreeding, significant differences in substrain phenotypes are to be expected. These results emphasize the importance of using embryo freezing methods to minimize genetic drift within substrains and of applying appropriate genetic nomenclature to permit substrain recognition when one is used.

Enrichment with wood blocks does not affect toxicity assessment in an exploratory toxicology model using Sprague-Dawley rats.

Environmental enrichment in rodents may improve animal well-being but can affect neurologic development, immune system function, and aging. We tested the hypothesis that wood block enrichment affects the interpretation of traditional and transcriptomic endpoints in an exploratory toxicology testing model using a well-characterized reference compound, cyclophosphamide. ANOVA was performed to distinguish effects of wood block enrichment separate from effects of 40 mg/kg cyclophosphamide treatment. Biologically relevant and statistically significant effects of wood block enrichment occurred only for body weight gain. ANOVA demonstrated the expected effects of cyclophosphamide on food consumption, spleen weight, and hematology. According to transcriptomic endpoints, cyclophosphamide induced fewer changes in gene expression in liver than in spleen. Splenic transcriptomic pathways affected by cyclophosphamide included: iron hemostasis; vascular tissue angiotensin system; hepatic stellate cell activation and fibrosis; complement activation; TGFß-induced hypertrophy and fibrosis; monocytes, macrophages, and atherosclerosis; and platelet activation. Changes in these pathways due to cyclophosphamide treatment were consistent with bone marrow toxicity regardless of enrichment. In a second study, neither enrichment nor type of cage flooring altered body weight or food consumption over a 28-d period after the first week. In conclusion, wood block enrichment did not interfere with a typical exploratory toxicology study; the effects of ingested wood on drug level kinetics may require further consideration.

Epigenetic dysregulation of SHANK3 in brain tissues from individuals with autism spectrum disorders.

The molecular basis for the majority of cases of autism spectrum disorders (ASD) remains unknown. We tested the hypothesis that ASD have an epigenetic cause by performing DNA methylation profiling of five CpG islands (CGI-1 to CGI-5) in the SHANK3 gene in postmortem brain tissues from 54 ASD patients and 43 controls. We found significantly increased overall DNA methylation (epimutation) in three intragenic CGIs (CGI-2, CGI-3 and CGI-4). The increased methylation was clustered in the CGI-2 and CGI-4 in ∼15% of ASD brain tissues. SHANK3 has an extensive array of mRNA splice variants resulting from combinations of five intragenic promoters and alternative splicing of coding exons. Altered expression and alternative splicing of SHANK3 isoforms were observed in brain tissues with increased methylation of SHANK3 CGIs in ASD brain tissues. A DNA methylation inhibitor modified the methylation of CGIs and altered the isoform-specific expression of SHANK3 in cultured cells. This study is the first to find altered methylation patterns in SHANK3 in ASD brain samples. Our finding provides evidence to support an alternative approach to investigating the molecular basis of ASD. The ability to alter the epigenetic modification and expression of SHANK3 by environmental factors suggests that SHANK3 may be a valuable biomarker for dissecting the role of gene and environment interaction in the etiology of ASD.

Relationship between methylome and transcriptome in patients with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Cirrhosis and liver cancer are potential outcomes of advanced nonalcoholic fatty liver disease (NAFLD). It is not clear what factors determine whether patients will develop advanced or mild NAFLD, limiting noninvasive diagnosis and treatment before clinical sequelae emerge. We investigated whether DNA methylation profiles can distinguish patients with mild disease from those with advanced NAFLD, and how these patterns are functionally related to hepatic gene expression. METHODS: We collected frozen liver biopsies and clinical data from patients with biopsy-proven NAFLD (56 in the discovery cohort and 34 in the replication cohort). Samples were divided into groups based on histologic severity of fibrosis: F0-1 (mild) and F3-4 (advanced). DNA methylation profiles were determined and coupled with gene expression data from the same biopsies; differential methylation was validated in subsets of the discovery and replication cohorts. We then analyzed interactions between the methylome and transcriptome. RESULTS: Clinical features did not differ between patients known to have mild or advanced fibrosis based on biopsy analysis. There were 69,247 differentially methylated CpG sites (76% hypomethylated, 24% hypermethylated) in patients with advanced vs mild NAFLD (P < .05). Methylation at fibroblast growth factor receptor 2, methionine adenosyl methyltransferase 1A, and caspase 1 was validated by bisulfite pyrosequencing and the findings were reproduced in the replication cohort. Methylation correlated with gene transcript levels for 7% of differentially methylated CpG sites, indicating that differential methylation contributes to differences in expression. In samples with advanced NAFLD, many tissue repair genes were hypomethylated and overexpressed, and genes in certain metabolic pathways, including 1-carbon metabolism, were hypermethylated and underexpressed. CONCLUSIONS: Functionally relevant differences in methylation can distinguish patients with advanced vs mild NAFLD. Altered methylation of genes that regulate processes such as steatohepatitis, fibrosis, and carcinogenesis indicate the role of DNA methylation in progression of NAFLD.

Effect of the myostatin locus on muscle mass and intramuscular fat content in a cross between mouse lines selected for hypermuscularity.

BACKGROUND: This study is aimed at the analysis of genetic and physiological effects of myostatin on economically relevant meat quality traits in a genetic background of high muscularity. For this purpose, we generated G(3) populations of reciprocal crosses between the two hypermuscular mouse lines BMMI866, which carries a myostatin mutation and is lean, and BMMI806, which has high intramuscular and body fat content. To assess the relationship between muscle mass, body composition and muscle quality traits, we also analysed intramuscular fat content (IMF), water holding capacity (WHC), and additional physiological parameters in M. quadriceps and M. longissimus in 308 G(3)-animals. RESULTS: We found that individuals with larger muscles have significantly lower total body fat (r = -0.28) and IMF (r = -0.64), and in females, a lower WHC (r = -0.35). In males, higher muscle mass was also significantly correlated with higher glycogen contents (r = 0.2) and lower carcass pH-values 24 hours after dissection (r = -0.19). Linkage analyses confirmed the influence of the myostatin mutation on higher lean mass (1.35 g), reduced body fat content (-1.15%), and lower IMF in M. longissimus (-0.13%) and M. quadriceps (-0.07%). No effect was found for WHC. A large proportion of variation of intramuscular fat content of the M. longissimus at the myostatin locus could be explained by sex (23%) and direction-of-cross effects (26%). The effects were higher in males (+0.41%). An additional locus with negative over-dominance effects on total fat mass (-0.55 g) was identified on chromosome 16 at 94 Mb (86-94 Mb) which concurs with fat related QTL in syntenic regions on SSC13 in pigs and BTA1 in cattle. CONCLUSION: The data shows QTL effects on mouse muscle that are similar to those previously observed in livestock, supporting the mouse model. New information from the mouse model helps to describe variation in meat quantity and quality, and thus contribute to research in livestock.

Genome-wide association mapping of quantitative traits in outbred mice.

Recent developments in high-density genotyping and statistical analysis methods that have enabled genome-wide association studies in humans can also be applied to outbred mouse populations. Increased recombination in outbred populations is expected to provide greater mapping resolution than traditional inbred line crosses, improving prospects for identifying the causal genes. We carried out genome-wide association mapping by using 288 mice from a commercially available outbred stock; NMRI mice were genotyped with a high-density single-nucleotide polymorphism array to map loci influencing high-density lipoprotein cholesterol, systolic blood pressure, triglyceride levels, glucose, and urinary albumin-to-creatinine ratios. We found significant associations (P < 10(-5)) with high-density lipoprotein cholesterol and identified Apoa2 and Scarb1, both of which have been previously reported, as candidate genes for these associations. Additional suggestive associations (P < 10(-3)) identified in this study were also concordant with published quantitative trait loci, suggesting that we are sampling from a limited pool of genetic diversity that has already been well characterized. These findings dampen our enthusiasm for currently available commercial outbred stocks as genetic mapping resources and highlight the need for new outbred populations with greater genetic diversity. Despite the lack of novel associations in the NMRI population, our analysis strategy illustrates the utility of methods that could be applied to genome-wide association studies in humans.

Discovery of novel variants in genotyping arrays improves genotype retention and reduces ascertainment bias.

BACKGROUND: High-density genotyping arrays that measure hybridization of genomic DNA fragments to allele-specific oligonucleotide probes are widely used to genotype single nucleotide polymorphisms (SNPs) in genetic studies, including human genome-wide association studies. Hybridization intensities are converted to genotype calls by clustering algorithms that assign each sample to a genotype class at each SNP. Data for SNP probes that do not conform to the expected pattern of clustering are often discarded, contributing to ascertainment bias and resulting in lost information - as much as 50% in a recent genome-wide association study in dogs. RESULTS: We identified atypical patterns of hybridization intensities that were highly reproducible and demonstrated that these patterns represent genetic variants that were not accounted for in the design of the array platform. We characterized variable intensity oligonucleotide (VINO) probes that display such patterns and are found in all hybridization-based genotyping platforms, including those developed for human, dog, cattle, and mouse. When recognized and properly interpreted, VINOs recovered a substantial fraction of discarded probes and counteracted SNP ascertainment bias. We developed software (MouseDivGeno) that identifies VINOs and improves the accuracy of genotype calling. MouseDivGeno produced highly concordant genotype calls when compared with other methods but it uniquely identified more than 786000 VINOs in 351 mouse samples. We used whole-genome sequence from 14 mouse strains to confirm the presence of novel variants explaining 28000 VINOs in those strains. We also identified VINOs in human HapMap 3 samples, many of which were specific to an African population. Incorporating VINOs in phylogenetic analyses substantially improved the accuracy of a Mus species tree and local haplotype assignment in laboratory mouse strains. CONCLUSION: The problems of ascertainment bias and missing information due to genotyping errors are widely recognized as limiting factors in genetic studies. We have conducted the first formal analysis of the effect of novel variants on genotyping arrays, and we have shown that these variants account for a large portion of miscalled and uncalled genotypes. Genetic studies will benefit from substantial improvements in the accuracy of their results by incorporating VINOs in their analyses.

Genome-wide association mapping of loci for antipsychotic-induced extrapyramidal symptoms in mice.

Tardive dyskinesia (TD) is a debilitating, unpredictable, and often irreversible side effect resulting from chronic treatment with typical antipsychotic agents such as haloperidol. TD is characterized by repetitive, involuntary, purposeless movements primarily of the orofacial region. In order to investigate genetic susceptibility to TD, we used a validated mouse model for a systems genetics analysis geared toward detecting genetic predictors of TD in human patients. Phenotypic data from 27 inbred strains chronically treated with haloperidol and phenotyped for vacuous chewing movements were subject to a comprehensive genomic analysis involving 426,493 SNPs, 4,047 CNVs, brain gene expression, along with gene network and bioinformatic analysis. Our results identified ~50 genes that we expect to have high prior probabilities for association with haloperidol-induced TD, most of which have never been tested for association with human TD. Among our top candidates were genes regulating the development of brain motor control regions (Zic4 and Nkx6-1), glutamate receptors (Grin1 and Grin2a), and an indirect target of haloperidol (Drd1a) that has not been studied as well as the direct target, Drd2.

Genetic determinants for intramuscular fat content and water-holding capacity in mice selected for high muscle mass.

Intramuscular fat content and water-holding capacity are important traits in livestock as they influence meat quality, nutritive value of the muscle, and animal health. As a model for livestock, two inbred lines of the Berlin Muscle Mouse population, which had been long-term selected for high muscle mass, were used to identify genomic regions affecting intramuscular fat content and water-holding capacity. The intramuscular fat content of the Musculus longissimus was on average 1.4 times higher in BMMI806 than in BMMI816 mice. This was accompanied by a 1.5 times lower water-holding capacity of the Musculus quadriceps in BMMI816 mice. Linkage analyses with 332 G(3) animals of reciprocal crosses between these two lines revealed quantitative trait loci for intramuscular fat content on chromosome 7 and for water-holding capacity on chromosome 2. In part, the identified loci coincide with syntenic regions in pigs in which genetic effects for the same traits were found. Therefore, these muscle-weight-selected mouse lines and the produced intercross populations are valuable genetic resources to identify genes that could also contribute to meat quality in other species.

Subspecific origin and haplotype diversity in the laboratory mouse.

Here we provide a genome-wide, high-resolution map of the phylogenetic origin of the genome of most extant laboratory mouse inbred strains. Our analysis is based on the genotypes of wild-caught mice from three subspecies of Mus musculus. We show that classical laboratory strains are derived from a few fancy mice with limited haplotype diversity. Their genomes are overwhelmingly Mus musculus domesticus in origin, and the remainder is mostly of Japanese origin. We generated genome-wide haplotype maps based on identity by descent from fancy mice and show that classical inbred strains have limited and non-randomly distributed genetic diversity. In contrast, wild-derived laboratory strains represent a broad sampling of diversity within M. musculus. Intersubspecific introgression is pervasive in these strains, and contamination by laboratory stocks has played a role in this process. The subspecific origin, haplotype diversity and identity by descent maps can be visualized using the Mouse Phylogeny Viewer (see URLs).

CGDSNPdb: a database resource for error-checked and imputed mouse SNPs.

The Center for Genome Dynamics Single Nucleotide Polymorphism Database (CGDSNPdb) is an open-source value-added database with more than nine million mouse single nucleotide polymorphisms (SNPs), drawn from multiple sources, with genotypes assigned to multiple inbred strains of laboratory mice. All SNPs are checked for accuracy and annotated for properties specific to the SNP as well as those implied by changes to overlapping protein-coding genes. CGDSNPdb serves as the primary interface to two unique data sets, the 'imputed genotype resource' in which a Hidden Markov Model was used to assess local haplotypes and the most probable base assignment at several million genomic loci in tens of strains of mice, and the Affymetrix Mouse Diversity Genotyping Array, a high density microarray with over 600,000 SNPs and over 900,000 invariant genomic probes. CGDSNPdb is accessible online through either a web-based query tool or a MySQL public login. Database URL: http://cgd.jax.org/cgdsnpdb/

A customized and versatile high-density genotyping array for the mouse.

We designed a high-density mouse genotyping array containing 623,124 single-nucleotide polymorphisms that captures the known genetic variation present in the laboratory mouse. The array also contains 916,269 invariant genomic probes targeted to functional elements and regions known to harbor segmental duplications. The array opens the door to the characterization of genetic diversity, copy-number variation, allele-specific gene expression and DNA methylation, and will extend the successes of human genome-wide association studies to the mouse.

Cancer stem cells are enriched in the side population cells in a mouse model of glioma.

The recent identification of cancer stem cells (CSCs) in multiple human cancers provides a new inroad to understanding tumorigenesis at the cellular level. CSCs are defined by their characteristics of self-renewal, multipotentiality, and tumor initiation upon transplantation. By testing for these defining characteristics, we provide evidence for the existence of CSCs in a transgenic mouse model of glioma, S100beta-verbB;Trp53. In this glioma model, CSCs are enriched in the side population (SP) cells. These SP cells have enhanced tumor-initiating capacity, self-renewal, and multipotentiality compared with non-SP cells from the same tumors. Furthermore, gene expression analysis comparing fluorescence-activated cell sorting-sorted cancer SP cells to non-SP cancer cells and normal neural SP cells identified 45 candidate genes that are differentially expressed in glioma stem cells. We validated the expression of two genes from this list (S100a4 and S100a6) in primary mouse gliomas and human glioma samples. Analyses of xenografted human glioblastoma multiforme cell lines and primary human glioma tissues show that S100A4 and S100A6 are expressed in a small subset of cancer cells and that their abundance is positively correlated to tumor grade. In conclusion, this study shows that CSCs exist in a mouse glioma model, suggesting that this model can be used to study the molecular and cellular characteristics of CSCs in vivo and to further test the CSC hypothesis.

Randomization in laboratory procedure is key to obtaining reproducible microarray results.

The quality of gene expression microarray data has improved dramatically since the first arrays were introduced in the late 1990s. However, the reproducibility of data generated at multiple laboratory sites remains a matter of concern, especially for scientists who are attempting to combine and analyze data from public repositories. We have carried out a study in which a common set of RNA samples was assayed five times in four different laboratories using Affymetrix GeneChip arrays. We observed dramatic differences in the results across laboratories and identified batch effects in array processing as one of the primary causes for these differences. When batch processing of samples is confounded with experimental factors of interest it is not possible to separate their effects, and lists of differentially expressed genes may include many artifacts. This study demonstrates the substantial impact of sample processing on microarray analysis results and underscores the need for randomization in the laboratory as a means to avoid confounding of biological factors with procedural effects.

On the subspecific origin of the laboratory mouse.

The genome of the laboratory mouse is thought to be a mosaic of regions with distinct subspecific origins. We have developed a high-resolution map of the origin of the laboratory mouse by generating 25,400 phylogenetic trees at 100-kb intervals spanning the genome. On average, 92% of the genome is of Mus musculus domesticus origin, and the distribution of diversity is markedly nonrandom among the chromosomes. There are large regions of extremely low diversity, which represent blind spots for studies of natural variation and complex traits, and hot spots of diversity. In contrast with the mosaic model, we found that most of the genome has intermediate levels of variation of intrasubspecific origin. Finally, mouse strains derived from the wild that are supposed to represent different mouse subspecies show substantial intersubspecific introgression, which has strong implications for evolutionary studies that assume these are pure representatives of a given subspecies.