Genetic connection of carotid atherosclerosis with coat color and body weight in an intercross between hyperlipidemic mouse strains.

Atherosclerosis in the carotid artery is a major cause of ischemic stroke and has a strong genetic component. The aim of this study was to identify genetic factors contributing to carotid atherosclerosis. One hundred fifty-four female F2 mice were generated from an intercross between LP/J and BALB/cJ Apoe-null (Apoe-/-) mice and fed 12 wk of Western diet. Atherosclerotic lesions, body weight, and coat color were measured and genotyping was performed using miniMUGA genotyping arrays. A significant quantitative trait locus (QTL) on chromosome (Chr) 7, named Cath20, and five suggestive QTL on Chr 6, 12, 13, 15, and X were identified for carotid lesions. Three significant QTL, Bwfq2, Bw1n, Bwtq6, on Chr 2, 7, and 15 were identified for body weight. Two significant QTL, Chop2 and Albc2, on Chr 4 and 7 were identified for coat color, with Tyr, encoding tyrosinase, being the causal gene of Albc2. Cath20 overlapped with or was close to QTL Bw1n and Albc2 on Chr7. Carotid lesion sizes were significantly correlated with body weight and graded coat color in F2 mice. Cath20 on Chr7 disappeared after adjustment for coat color but remained after adjustment for body weight. Tyr was abundantly expressed in atherosclerotic lesions. These results demonstrate genetic connections of carotid atherosclerosis with body weight and coat color in hyperlipidemic mice and suggest a potential role for Tyr in carotid atherosclerosis.

RNA-sequencing analysis of differential gene expression associated with arterial stiffness.

OBJECTIVES: Arterial stiffness is recognized as an important predictor of cardiovascular disease morbidity and mortality, independent of traditional cardiovascular disease risk factors. Given that arterial tissue is not easily accessible, most gene expression studies on arterial stiffness have been conducted on animals or on patients who have undergone by-pass surgeries. In order to obtain a deeper understanding of early changes of arterial stiffness, this study compared transcriptome profiles between healthy adults with higher and lower arterial stiffness. METHODS: The sample included 20 healthy female adults without cardiovascular disease. Arterial stiffness was measured by carotid-femoral pulse wave velocity, the "gold-standard" measure of central arterial stiffness. Peripheral blood samples collected to PAXgene™ RNA tubes were used for RNA sequencing (RNA-seq). The potential confounding effects of age, body mass index, and mean arterial pressure were controlled for in RNA-seq analysis. To validate RNA-seq results, quantitative real-time PCR (qRT-PCR) was performed for six selected genes. RESULTS: The findings demonstrated that genes including CAPN9, IL32, ERAP2, RAB6B, MYBPH, and miRNA626 were down-regulated, and that MOCS1 gene was up-regulated among the people with higher arterial stiffness. Real-time PCR showed that the changes of CAPN9, IL32, ERAP2, and RAB6B were in concordance with RNA-seq data, and confirmed the validity of the gene expression profiles obtained by RNA-seq analysis. CONCLUSIONS: Previous studies have suggested the potential roles of CAPN9, IL32, and ERAP2 in structural changes of the arterial wall through up-regulation of metalloproteinases. However, the current study showed that CAPN9, IL32, and ERAP2 were down-regulated in the individuals with higher arterial stiffness, compared with those with lower arterial stiffness. The unexpected directions of expression of these genes may indicate an effort to maintain vascular homeostasis during increased arterial stiffness among healthy individuals. Further studies are guaranteed to investigate the roles of CAPN9, IL32, and ERAP2 in regulating arterial stiffness in people with and without cardiovascular disease.

TLR2/6 agonists and interferon-gamma induce human melanoma cells to produce CXCL10.

Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor-reactive T-cell infiltration of metastases. However, most tumors lack significant immune infiltration prior to therapy. Selected chemokines promote T-cell migration into tumors; thus, agents that induce these chemokines in the tumor microenvironment (TME) may improve responses to systemic immune therapy. CXCL10 has been implicated as a critical chemokine supporting T-cell infiltration into the TME. Here, we show that toll-like receptor (TLR) agonists can induce chemokine production directly from melanoma cells when combined with IFNγ treatment. We find that TLR2 and TLR6 are widely expressed on human melanoma cells, and that TLR2/6 agonists (MALP-2 or FSL-1) synergize with interferon-gamma (IFNγ) to induce production of CXCL10 from melanoma cells. Furthermore, melanoma cells and immune cells from surgical specimens also respond to TLR2/6 agonists and IFNγ by upregulating CXCL10 production, compared to treatment with either agent alone. Collectively, these data identify a novel mechanism for inducing CXCL10 production directly from melanoma cells, with TLR2/6 agonists +IFNγ and raise the possibility that intratumoral administration of these agents may improve immune signatures in melanoma and have value in combination with other immune therapies, by supporting T-cell migration into melanoma metastases.

Effects of n-3 fish oil on metabolic and histological parameters in NASH: a double-blind, randomized, placebo-controlled trial.

BACKGROUND & AIMS: This study's aim was to assess the histological and metabolic effects of n-3 polyunsaturated fatty acids (PUFAs) vs. placebo while adjusting for the impact of age and weight change in NASH patients. (ClinicalTrials.gov: NCT00681408). METHODS: Forty-one subjects with non-cirrhotic NASH were enrolled, and 34 completed the study. 17 received n-3 fish oil 3000 mg/day and 17 received placebo daily for 1 year with typical counselling on caloric intake and physical activity for all subjects. RESULTS: N-3- and placebo-treated groups showed no significant difference for the primary end point of NASH activity score (NAS) reduction ⩾ 2 points without fibrosis progression after adjustment for known covariates (n-3, 4/17 (23.5%); placebo, 3/17, (17.6%), p = 0.99). Among subjects with increased or stable weight, n-3 subjects showed a larger decrease in liver fat content by MRI than placebo-treated subjects (p = 0.014 for 2nd quartile, p = 0.003 for 3rd quartile of weight change). N-3 treatment showed significant fat reduction on the paired analysis of image-assisted fat morphometry regardless of weight loss or gain. Exercise capacity remained markedly reduced in all subjects. No independent effects on markers of hepatocyte injury or insulin sensitivity indices were observed. CONCLUSION: N-3 PUFAs at 3000 mg/day for one year did not lead to an improvement in the primary outcome of histological activity in NASH patients (⩾ 2 point NAS reduction). N-3 led to reduced liver fat by multiple measures. Other metabolic effects were not seen, although no detrimental effects were apparent. Whether longer duration, higher dose, or different composition of n-3 therapy would lead to additional benefits is uncertain.

Genes determining the severity of cerebral palsy: the role of single nucleotide polymorphisms on the amount and structure of apolipoprotein E.

AIM: Apolipoprotein E (apoE) influences repair and other processes in the brain, and the apoE4 variant is a risk factor for Alzheimer's disease and for prolonged recovery following traumatic brain injury. We previously reported that specific single nucleotide polymorphisms in the APOE or TOMM40 genes affecting the structure and production of apoE were associated with epilepsy, more impaired hand function and gastrostomy tube feeding in children with cerebral palsy (CP). This study explored how various combinations of the same polymorphisms may affect these clinical manifestations. METHODS: Successful DNA analyses of APOE and TOMM40 were carried out on 227 children. The CP Register of Norway provided details of gross and fine motor function, epilepsy and gastrostomy tube feeding. Possible associations between these clinical manifestations and various combinations of the APOEε2, ε3 or ε4 alleles and of the rs59007384 polymorphism in the TOMM40 gene were explored. RESULTS: Epilepsy, impaired fine motor function and gastrostomy tube feeding were less common in children carrying the combination of rs59007384 GG and APOEε2 or ε3 than in children with other combinations. CONCLUSION: Our findings suggest that specific combinations of genes influence the structure and production of apoE differently and affect the clinical manifestations of CP.

Nanosphere pharmacodynamics improves safety of immunostimulatory cytokine therapy.

Systemic administration of interleukin (IL)-12 induces potent anti-tumor immune responses in preclinical cancer models through the systemic activation of effector immune cells and release of proinflammatory cytokines. IL-12-loaded PLGA nanospheres (IL12ns) are hypothesized to improve therapeutic efficacy and thwart unwanted side effects observed in previous human clinical trials. Through the investigation of peripheral blood and local tissue immune responses in healthy BALB/c mice, the immune-protective pharmacodynamics of IL12ns were suggested. Nanospheres increased pro-inflammatory plasma cytokines/chemokines (IFN-γ, IL-6, TNF-α, and CXCL10) without inducing maladaptive transcriptomic signatures in circulating peripheral immune cells. Gene expression profiling revealed activation of pro-inflammatory signaling pathways in systemic tissues, the likely source of these effector cytokines. These data support that nanosphere pharmacodynamics, including shielding IL-12 from circulating immune cells, depositing peripherally in systemic immune tissues, and then slowly eluting bioactive cytokine, thereafter, are essential to safe immunostimulatory therapy.

Apolipoprotein E polymorphisms and severity of cerebral palsy: a cross-sectional study in 255 children in Norway.

AIM: The aim of this study was to examine whether the presence of the apolipoprotein E (ApoE) allele APOEε4 is associated with less severe manifestations of cerebral palsy (CP), consistent with the suggested beneficial effect of this allele on neurodevelopment in children. METHOD: ApoE genotyping was performed on buccal epithelial cells from 255 children (141 males 114 females; mean age 12y, SD 2y 3mo, range 9-17y) recorded in the Cerebral Palsy Register of Norway. The main outcome measure of CP severity was the Gross Motor Function Classification System (GMFCS). Secondary outcome measures were fine motor function, epilepsy, and the need for gastrostomy tube feeding (GTF). RESULTS: There was no association between the APOEε4 genotype and GMFCS levels (odds ratio [OR] 1.15; 95% confidence interval [CI] 0.66-1.99). However, the APOEε4 genotype was more often present among children with epilepsy (OR 2.2; 95% CI 1.1-4.2) and/or receiving GTF (OR 2.7; 95% CI 1.1-6.6). Among children with unilateral CP, the presence of APOEε4 was associated with more severe fine motor impairment (OR 2.6; 95% CI 1.3-6.9). INTERPRETATION: Our main hypothesis that APOEε4 would have a protective effect on neurodevelopment was not supported. Instead, subgroup analyses suggested an adverse effect of the APOEε4 genotype on the developing brain after injury.

Characterization of Bglu3, a mouse fasting glucose locus, and identification of Apcs as an underlying candidate gene.

Bglu3 is a quantitative trait locus for fasting glucose on distal chromosome 1 identified in an intercross between C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE(-/-)) mice. This locus was subsequently replicated in two separate mouse intercrosses. The objective of this study was to characterize Bglu3 through construction and analysis of a congenic strain and identify underlying candidate genes. Congenic mice were constructed by introgressing a genomic region harboring Bglu3 from C3H.apoE(-/-) into B6.apoE(-/-) mice. Mice were started with a Western diet at 6 wk of age and maintained on the diet for 12 wk. Gene expression in the liver was analyzed by microarrays. Congenic mice had significantly higher fasting glucose levels and developed more significant glucose intolerance compared with B6.apoE(-/-) mice on the Western diet. Microarray analysis revealed 336 genes to be differentially expressed in the liver of congenic mice. Further pathway analysis suggested a role for acute phase response signaling in regulating glucose intolerance. Apcs, encoding an acute phase response protein serum amyloid P (SAP), is located underneath the linkage peak of Bglu3. Multiple single nucleotide polymorphisms between B6 and C3H mice were detected within and surrounding Apcs. Apcs expression in the liver was significantly higher in congenic and C3H mice compared with B6 mice. The Western diet consumption led to a gradual rise in plasma SAP levels, which was accompanied by rising fasting glucose in both B6 and C3H apoE(-/-) mice. Expression of C3H Apcs in B6.apoE(-/-) mice aggravated glucose intolerance. Bglu3 is confirmed to be a locus affecting diabetes susceptibility, and Apcs is a probable candidate gene.

Genome-Wide mRNA Expression Analysis of Acute Psychological Stress Responses.

INTRODUCTION: Most previous studies have examined the effects of acute psychological stress in humans based on select gene panels. The genomic approach may help identify novel genes that underline biological mechanisms of acute psychological stress responses. OBJECTIVE: This exploratory study aimed to investigate genome-wide transcriptional activity changes in response to acute psychological stress. METHODS: The sample included 40 healthy women (mean age 31.4 ± 11.6 years). Twenty-two participants had a stress experience induced by the Trier Social Stress Test (experimental group) and 18 did not (control group). Psychological stress levels and hemodynamic changes were assessed before and after the Trier Social Stress Test. Peripheral blood samples obtained before and after the Trier Social Stress Test were processed for mRNA sequencing. RESULTS: Psychological and hemodynamic stress parameters indicated that the Trier Social Stress Test induced moderate levels of stress in the experimental group. Six genes (HCG26, HCP5, HLA-F, HLA-F-AS1, LOC1019287, and SLC22A16) were up-regulated, and fi ve genes (CA1, FBXO9, SNCA, STRADB, and TRMT12) were down-regulated among those who experienced stress induction, compared with the control group. Nine genes of eleven were linked to endocrine system disorders, neurological disease, and organismal injury and abnormalities. CONCLUSIONS: Of the genes identifi ed in this study, HCP5, SLC22A16, and SNCA genes have previously been proposed as therapeutic targets for cancer and Parkinson disease. Further studies are needed to examine pathological mechanisms through which these genes mediate eff ects of psychological stress on adverse health outcomes. Such studies may ultimately identify therapeutic targets that enhance biological resilience to adverse eff ects of psychological stress.

Genetic Connection between Hyperglycemia and Carotid Atherosclerosis in Hyperlipidemic Mice.

Type 2 diabetes (T2D) is a major risk for atherosclerosis and its complications. Apoe-null (Apoe-/-) mouse strains exhibit a wide range of variations in susceptibility to T2D and carotid atherosclerosis, with the latter being a major cause of ischemic stroke. To identify genetic connections between T2D and carotid atherosclerosis, 145 male F2 mice were generated from LP/J and BALB/cJ Apoe-/- mice and fed 12 weeks of a Western diet. Atherosclerotic lesions in the carotid arteries, fasting, and non-fasting plasma glucose levels were measured, and genotyping was performed using miniMUGA arrays. Two significant QTL (quantitative trait loci) on chromosomes (Chr) 6 and 15 were identified for carotid lesions. The Chr15 QTL coincided precisely with QTL Bglu20 for fasting and non-fasting glucose levels. Carotid lesion sizes showed a trend toward correlation with fasting and non-fasting glucose levels in F2 mice. The Chr15 QTL for carotid lesions was suppressed after excluding the influence from fasting or non-fasting glucose. Likely candidate genes for the causal association were Tnfrsf11b, Deptor, and Gsdmc2. These results demonstrate a causative role for hyperglycemia in the development of carotid atherosclerosis in hyperlipidemic mice.

A germline SNP in BRMS1 predisposes patients with lung adenocarcinoma to metastasis and can be ameliorated by targeting c-fos.

About 50% of patients with early-stage, surgically resected lung cancer will develop distant metastasis. There remains an unmet need to identify patients likely to develop recurrence and to design innovative therapies to decrease this risk. Two primary isoforms of BRMS1, v1 and v2, are present in humans. Using next-generation sequencing of BRMS1 on matched human noncancerous lung tissue and non-small cell lung cancer (NSCLC) specimens, we identified single-nucleotide polymorphism (SNP) rs1052566 that results in an A273V mutation of BRMS1v2. This SNP is homozygous (BRMS1v2A273V/A273V) in 8% of the population and correlates with aggressive biology in lung adenocarcinoma (LUAD). Mechanistically, we show that BRMS1v2 A273V abolishes the metastasis suppressor function of BRMS1v2 and promotes robust cell invasion and metastases by activation of c-fos-mediated gene-specific transcriptional regulation. BRMS1v2 A273V increases cell invasion in vitro and increases metastases in both tail-vein injection xenografts and LUAD patient-derived organoid (PDO) intracardiac injection metastasis in vivo models. Moreover, we show that BRMS1v2 A273V fails to interact with nuclear Src, thereby activating intratumoral c-fos in vitro. Higher c-fos results in up-regulation of CEACAM6, which drives metastases in vitro and in vivo. Using both xenograft and PDO metastasis models, we repurposed T5224 for treatment, a c-fos pharmacologic inhibitor investigated in clinical trials for arthritis, and observed suppression of metastases in BRMS1v2A273V/A273V LUAD in mice. Collectively, we elucidate the mechanism of BRMS1v2A273V/A273V-induced metastases and offer a putative therapeutic strategy for patients with LUAD who have this germline alteration.

Cell density-dependent transcriptional activation of endocrine-related genes in human adipose tissue-derived stem cells.

Adipose tissue is recognized as an endocrine organ that plays an important role in human diseases such as type II diabetes and cancer. Human adipose tissue-derived stem cells (ASCs), a distinct cell population in adipose tissue, are capable of differentiating into multiple lineages including adipogenesis. When cultured in vitro under a confluent condition, ASCs reach a commitment stage for adipogenesis, which can be further induced into terminally differentiated adipocytes by a cocktail of adipogenic factors. Here we report that the confluent state of ASCs triggers transcriptional activation cascades for genes that are responsible for the endocrine function of adipose tissue. These include insulin-like growth factor 1 (IGF-1) and aromatase (Cyp19), a key enzyme in estrogen biosynthesis. Despite similar adipogenic potentials, ASCs from different individuals display huge variations in activation of these endocrine-related genes. Bioinformatics and experimental data suggest that transcription factor Foxo1 controls a large number of "early" confluency-response genes, which subsequently induce "late" response genes. Furthermore, siRNA-mediated knockdown of Foxo1 substantially compromises the ability of committed ASCs to stimulate tumor cell migration in vitro. Thus, our work suggests that cell density is an important determinant of the endocrine potential of ASCs.

Estrogen signals via an extra-nuclear pathway involving IGF-1R and EGFR in tamoxifen-sensitive and -resistant breast cancer cells.

Activation of IGF-1R can activate metalloproteinases which release heparin-binding EGF (Hb-EGF) and lead to EGFR-dependent MAPK activation in certain tissues. We postulated that this pathway is operative in E(2)-induced MAPK activation in breast cancer tissues. As evidence, we showed that E(2) rapidly induced the phosphorylation of both IGF-1R and EGFR and that siRNA knockdown or selective inhibitors against either growth factor receptor inhibited E(2)-induced MAPK activation. The selective inhibitors or knockdown of either IGF-1R or EGFR significantly inhibited cell growth and reversed cell death protection induced by E(2) in MCF-7 cells. Our data support the conclusion that the IGF-1R acts upstream of EGFR in a linear pathway which mediates E(2) action on MAPK activation, cell growth stimulation and anti-apoptosis in breast cancer cells. During the process of development of tamoxifen resistance this pathway is up-regulated with increased sensitivity to activate EGFR for cell growth and protection against apoptosis. Surprisingly, translocation of ERalpha out of the nucleus into the cytoplasm, mediated by c-Src, occurs during development of resistance. This effect can be abrogated by administration of the c-Src inhibitor, PP2 which also restores sensitivity to tamoxifen.

Dickkopf homolog 1 mediates endothelin-1-stimulated new bone formation.

Tumor-produced endothelin-1 (ET-1) stimulates osteoblasts to form new bone and is an important mediator of osteoblastic bone metastasis. The anabolic actions of ET-1 in osteoblasts were investigated by gene microarray analyses of murine neonatal calvarial organ cultures. Targets of ET-1 action were validated by real-time RT-PCR in murine primary osteoblast cultures. IL-6, IL-11, the CCN (CYR61, CTGF, NOV) family members cysteine-rich protein 61 and connective tissue growth factor, inhibin beta-A, serum/glucocorticoid regulated kinase, receptor activator of nuclear factor kappaB ligand, snail homolog 1, tissue inhibitor of metalloproteinase 3, and TG-interacting factor transcripts were increased by ET-1. ET-1 decreased the transcript for the Wnt signaling pathway inhibitor, dickkopf homolog 1 (Dkk1). Calvarial organ cultures treated with ET-1 had lower concentrations of DKK1 protein in conditioned media than control cultures. High DKK1 concentrations in bone marrow suppress bone formation in multiple myeloma. We hypothesized that the converse occurs in osteoblastic bone metastasis, where ET-1 stimulates osteoblast activity by reducing autocrine production of DKK1. Recombinant DKK1 blocked ET-1-mediated osteoblast proliferation and new bone formation in calvarial organ cultures, whereas a DKK1-neutralizing antibody increased osteoblast numbers and new bone formation. ET-1 directed nuclear translocation of beta-catenin in osteoblasts, indicating activation of the Wnt signaling pathway. The data suggest that ET-1 increases osteoblast proliferation and new bone formation by activating the Wnt signaling pathway through suppression of the Wnt pathway inhibitor DKK1.

Revisiting the Therapeutic Potential of Bothrops jararaca Venom: Screening for Novel Activities Using Connectivity Mapping.

Snake venoms are sources of molecules with proven and potential therapeutic applications. However, most activities assayed in venoms (or their components) are of hemorrhagic, hypotensive, edematogenic, neurotoxic or myotoxic natures. Thus, other relevant activities might remain unknown. Using functional genomics coupled to the connectivity map (C-map) approach, we undertook a wide range indirect search for biological activities within the venom of the South American pit viper Bothrops jararaca. For that effect, venom was incubated with human breast adenocarcinoma cell line (MCF7) followed by RNA extraction and gene expression analysis. A list of 90 differentially expressed genes was submitted to biosimilar drug discovery based on pattern recognition. Among the 100 highest-ranked positively correlated drugs, only the antihypertensive, antimicrobial (both antibiotic and antiparasitic), and antitumor classes had been previously reported for B. jararaca venom. The majority of drug classes identified were related to (1) antimicrobial activity; (2) treatment of neuropsychiatric illnesses (Parkinson's disease, schizophrenia, depression, and epilepsy); (3) treatment of cardiovascular diseases, and (4) anti-inflammatory action. The C-map results also indicated that B. jararaca venom may have components that target G-protein-coupled receptors (muscarinic, serotonergic, histaminergic, dopaminergic, GABA, and adrenergic) and ion channels. Although validation experiments are still necessary, the C-map correlation to drugs with activities previously linked to snake venoms supports the efficacy of this strategy as a broad-spectrum approach for biological activity screening, and rekindles the snake venom-based search for new therapeutic agents.

Estrogen signaling via a linear pathway involving insulin-like growth factor I receptor, matrix metalloproteinases, and epidermal growth factor receptor to activate mitogen-activated protein kinase in MCF-7 breast cancer cells.

We present an integrated model of an extranuclear, estrogen receptor-alpha (ERalpha)-mediated, rapid MAPK activation pathway in breast cancer cells. In noncancer cells, IGF-I initiates a linear process involving activation of the IGF-I receptor (IGF-IR) and matrix metalloproteinases (MMP), release of heparin-binding epidermal growth factor (HB-EGF), and activation of EGF receptor (EGFR)-dependent MAPK. 17beta-Estradiol (E2) rapidly activates IGF-IR in breast cancer cells. We hypothesize that E2 induces a similar linear pathway involving IGF-IR, MMP, HB-EGF, EGFR, and MAPK. Using MCF-7 breast cancer cells, we for the first time demonstrated that a sequential activation of IGF-IR, MMP, and EGFR existed in E2 and IGF-I actions, which was supported by evidence that the selective inhibitors of IGF-IR and MMP or knockdown of IGF-IR all inhibited E2- or IGF-I-induced EGFR phosphorylation. Using the inhibitors and small inhibitory RNA strategies, we also demonstrated that the same sequential activation of the receptors occurred in E2-, IGF-I-, but not EGF-induced MAPK phosphorylation. Additionally, a HB-EGF neutralizing antibody significantly blocked E2-induced MAPK activation, further supporting our hypothesis. The biological effects of sequential activation of IGF-IR and EGFR on E2 stimulation of cell proliferation were also investigated. Knockdown or blockade of IGF-IR significantly inhibited E2- or IGF-I-stimulated but not EGF-induced cell growth. Knockdown or blockade of EGFR abrogated cell growth induced by E2, IGF-I, and EGF, indicating that EGFR is a downstream molecule of IGF-IR in E2 and IGF-I action. Together, our data support the novel view that E2 can activate a linear pathway involving the sequential activation of IGF-IR, MMP, HB-EGF, EGFR, and MAPK.

Gene expression profiling reveals cross-talk between melanoma and fibroblasts: implications for host-tumor interactions in metastasis.

Host-tumor interaction is considered critical in carcinogenesis, tumor invasion, and metastasis. To explore the reciprocal effects of host-tumor interaction, we developed a system to assess the gene expression patterns of A2058 human melanoma cells cocultured in fibrillar collagen with HS-68 primary human fibroblasts. The gene expression pattern of the cocultured A2058 cells was only modestly affected, whereas the HS-68 fibroblast gene expression pattern was significantly altered. Interleukin-11 and inhibitor of DNA-binding domain-1 gene expression in the cocultured A2058 cells was down-regulated, indicative of a proinflammatory response and resistance to apoptosis, respectively. The overall pattern of up-regulated genes indicated triggering of the proinflammatory process. In addition, the melanoma growth and migration stimulatory chemokines CXCL1 and CXCL2 were significantly up-regulated in the cocultured fibroblasts. These results were corroborated by additional coculture experiments with the melanoma cell lines WM-164, BLM, and SK-Mel-28 and immunohistochemistry on invasive human melanoma sections. Taken together, these results indicate that tumor cells cause a proinflammatory and melanoma growth-promoting response in stromal fibroblasts. The role of inflammation in carcinogenesis, tumor promotion, invasion, and metastasis is viewed as being increasingly important and the results of these studies underscore this as well as identify certain key proteins that are expressed as a result of the complex interactive processes in the host-tumor microenvironment.

Prevalence and extent of heteroresistance by next generation sequencing of multidrug-resistant tuberculosis.

Amplicon-based Next Generation Sequencing (NGS) is an emerging method for Mycobacterium tuberculosis drug susceptibility testing (DST) but has not been well described. We examined 158 clinical multidrug-resistant M. tuberculosis isolates via NGS of 11 resistance-associated gene regions covering 3519 nucleotides. Across these gene regions, complete resistance or heteroresistance (defined as 1%-99% mutation) was present in at least one isolate in 6.3% of loci. The number of isolates with heteroresistance was highest for gyrA codon 94, rpoB codons 526 and 531, and embB codons 306, 372 and 406 (range 11-26% of isolates exhibited heteroresistance). 57% of MDR strains had heteroresistance of one or more recognized resistance-associated mutation. Heteroresistant loci generally exhibited high or low degrees of mutation (>90% or <10%). The deep sensitivity of NGS for detecting low level pncA heteroresistance appeared to improve genotypic-phenotypic PZA susceptibility correlations over that of Sanger. NGS demonstrates that heteroresistance in TB in the regions of key genes is common and will need to be bioinformatically managed. The clinical significance of such heteroresistance is unclear, and further study of pncA should be pursued.

Correction: Prevalence and extent of heteroresistance by next generation sequencing of multidrug-resistant tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0176522.].