Initial development and testing of an exhaled microRNA detection strategy for lung cancer case-control discrimination.

For detecting field carcinogenesis non-invasively, early technical development and case-control testing of exhaled breath condensate microRNAs was performed. In design, human lung tissue microRNA-seq discovery was reconciled with TCGA and published tumor-discriminant microRNAs, yielding a panel of 24 upregulated microRNAs. The airway origin of exhaled microRNAs was topographically "fingerprinted", using paired EBC, upper and lower airway donor sample sets. A clinic-based case-control study (166 NSCLC cases, 185 controls) was interrogated with the microRNA panel by qualitative RT-PCR. Data were analyzed by logistic regression (LR), and by random-forest (RF) models. Feasibility testing of exhaled microRNA detection, including optimized whole EBC extraction, and RT and qualitative PCR method evaluation, was performed. For sensitivity in this low template setting, intercalating dye-based URT-PCR was superior to fluorescent probe-based PCR (TaqMan). In application, adjusted logistic regression models identified exhaled miR-21, 33b, 212 as overall case-control discriminant. RF analysis of combined clinical + microRNA models showed modest added discrimination capacity (1.1-2.5%) beyond clinical models alone: all subjects 1.1% (p = 8.7e-04)); former smokers 2.5% (p = 3.6e-05); early stage 1.2% (p = 9.0e-03), yielding combined ROC AUC ranging from 0.74 to 0.83. We conclude that exhaled microRNAs are qualitatively measureable, reflect in part lower airway signatures; and when further refined/quantitated, can potentially help to improve lung cancer risk assessment.

Discovery of arylamide-5-anilinoquinazoline-8-nitro derivatives as VEGFR-2 kinase inhibitors: Synthesis, in vitro biological evaluation and molecular docking.

Herein, we embarked on a structural optimization campaign aiming at the discovery of novel anticancer agents with our previously reported XL-6f as a lead compound. A library of 23 compounds has been synthesized based on the highly conserved active site of VEGFR-2. Several title compounds exhibited selective inhibitory activities against VEGFR-2, which also displayed selective anti-proliferation potency against HepG2 cell. All synthesized compounds were evaluated for anti-angiogenesis capability. Compound 7o showed the most potent anti-angiogenesis ability, the efficient cytotoxic activities (in vitro against HUVEC and HepG2 cell lines with IC50 values of 0.58 and 0.23 µM, respectively). The molecular docking analysis revealed 7o is a Type-II inhibitor of VEGFR-2 kinase. In general, these results indicated these arylamide-5-anilinoquinazoline-8-nitro derivatives are promising inhibitors of VEGFR-2 for the potential treatment of anti-angiogenesis.

Design, synthesis and in vitro evaluation of 6-amide-2-aryl benzoxazole/benzimidazole derivatives against tumor cells by inhibiting VEGFR-2 kinase.

Herein, we have carried out a structural optimization campaign to discover the novel anti-tumor agents with our previously screened YQY-26 as the hit compound. A library of thirty-seven 6-amide-2-aryl benzoxazole/benzimidazole derivatives has been designed and synthesized based on the highly conserved active site of VEGFR-2. Several title compounds exhibited selective inhibitory activities against VEGFR-2 than EGFR kinases, which also displayed selective anti-proliferation potency against the HUVEC and HepG2 than the A549 and MDA-MB-231 cancer cell lines. The newly synthesized compounds were evaluated for anti-angiogenesis capability by chick chorioallantoic membrane (CAM) assay. Among them, compounds 9d showed the most potent anti-angiogenesis ability (79% inhibition at 10 nM/eggs), the efficient cytotoxic activities (in vitro against the HUVEC and HepG2 cell lines with IC50 values of 1.47 and 2.57 µM, respectively), and excellent VEGFR-2 kinase inhibition (IC50 = 0.051 µM). The molecular docking analysis revealed that compound 9d is a Type II inhibitor of VEGFR-2 kinase. These results indicated that the 6-amide-2-arylbenzoxazole and 6-amide-2-aryl benzimidazole derivatives are promising inhibitors of VEGFR-2 kinase for the potential treatment of anti-angiogenesis.

Discovery of 6-Arylurea-2-arylbenzoxazole and 6-Arylurea-2-arylbenzimidazole Derivatives as Angiogenesis Inhibitors: Design, Synthesis and in†vitro Biological Evaluation.

We embarked on a structural optimization campaign aimed at the discovery of novel anti-angiogenesis agents with previously reported imidazole kinase inhibitors as a lead compound. A library of 29 compounds was synthesized. Several title compounds exhibited selective inhibitory activities against vascular endothelial growth factor receptor 2 (VEGFR-2) over epidermal growth factor receptor (EGFR) kinase; these compounds also displayed selective and potent antiproliferative activity against three cancer cell lines. The newly synthesized compounds were evaluated for anti-angiogenesis activity by chick chorioallantoic membrane (CAM) assay. Among them, 1-(2-(2-chlorophenyl)benzo[d]oxazol-5-yl)-3-(4-(trifluoromethoxy)phenyl)urea (compound 5 n) showed the most potent anti-angiogenesis capacity, efficient cytotoxic activities (in vitro against human umbilical vein endothelial cells (HUVEC), H1975, A549, and HeLa cell lines, with respective IC50 values of 8.46, 1.40, 7.61, and 0.28 µm), and an acceptable level of VEGFR-2 kinase inhibition (IC50 =0.25 µm). Molecular docking analysis revealed 5 n to be a type II inhibitor of VEGFR-2 kinase. In general, these results indicate that these 6-arylurea-2-arylbenzoxazole/benzimidazole derivatives are promising inhibitors of VEGFR-2 kinase for potential development into anti-angiogenesis drugs.

[Isolation, Identification and Degradation Characteristics of a 17ß-estradiol Degrading Strain Fusarium sp. KY123915].

A 17ß-estradiol (E2) degrading strain (designated as Wu-SP1) was isolated from the activated sludge collected from a wastewater treatment plant (WWTP) in Xi'an. The strain was identified as Fusarium sp. according to 18S rDNA sequence and phylogenetic analysis. The optimal pH and temperature for E2 degradation were 6 and 30℃, respectively. Under these conditions, the E2 biodegradation rate of 2 mg·L-1 E2 amounted to 92.5% within 48 h by this strain. The kinetics of E2 degradation by the strain KY123915 were in good accord with the first-order equation, with the concentration ranged from 10 to 500 mg·L-1. UV spectrum analysis showed the strength of maximum absorption of metabolites became weak compared to E2, indicating that E2 may be degraded via estrone (E1) by Fusarium sp. KY123915.

Myelodysplastic Syndrome and Sweet's Syndrome Are Associated with a Mutation in Isocitrate Dehydrogenase 1.

BACKGROUND: Sweet's syndrome (SS) is a febrile neutrophilic dermatosis that has been clinically linked to hematological malignancies, particularly myelodysplastic syndrome (MDS), in a number of case series. Many epigenetic changes underlying MDS have been identified, such as a mutation in the isocitrate dehydrogenase 1 (IDH1) gene, which causes DNA hypermethylation and alteration of a number of genes that lead to leukemogenesis. However, the pathogenesis of malignancy-associated SS is unknown. CASE REPORT: We present two patients who were diagnosed with SS and concomitant IDH1-mutated MDS. Immunohistochemical staining of their skin lesions showed neutrophils diffusely positive for the IDH1 mutation. CONCLUSION: These cases demonstrate that IDH1 mutation may be implicated in the pathogenesis of malignancy-associated SS. Future investigation to elucidate this pathway is warranted. Establishing this molecular link can provide an earlier identification of patients with SS who are also at increased risk for developing MDS.

Genome Wide Methylome Alterations in Lung Cancer.

Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)-non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p<2.2E-16). Further, when DM was coupled to differential transcriptome (DE) in the same samples, 37,056 differential loci in adenocarcinoma emerged. Approximately 90% of the DM-DE relationships were non-canonical; for example, promoter DM associated with DE in the same direction. Of the canonical changes noted, promoter (PR) DM loci with reciprocal changes in expression in adenocarcinomas included HBEGF, AGER, PTPRM, DPT, CST1, MELK; DM GB loci with concordant changes in expression included FOXM1, FERMT1, SLC7A5, and FAP genes. IPA analyses showed adenocarcinoma-specific promoter DMxDE overlay identified familiar lung cancer nodes [tP53, Akt] as well as less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, PTPN13, DACH1, SMAD6, LAMA3, AR]. The unique findings from this study include the discovery of numerous candidate The unique findings from this study include the discovery of numerous candidate methylation sites in both PR and GB regions not previously identified in NSCLC, and many non-canonical relationships to gene expression. These DNA methylation features could potentially be developed as risk or diagnostic biomarkers, or as candidate targets for newer methylation locus-targeted preventive or therapeutic agents.

RICTOR Amplification Defines a Novel Subset of Patients with Lung Cancer Who May Benefit from Treatment with mTORC1/2 Inhibitors.

UNLABELLED: We identified amplification of RICTOR, a key component of the mTOR complex 2 (mTORC2), as the sole actionable genomic alteration in an 18-year-old never-smoker with lung adenocarcinoma. Amplification of RICTOR occurs in 13% of lung cancers (1,016 cases) in The Cancer Genome Atlas and at a similar frequency in an independent cohort of 1,070 patients identified by genomic profiling. In the latter series, 11% of cases harbored RICTOR amplification as the only relevant genomic alteration. Its oncogenic roles were suggested by decreased lung cancer cell growth both in vitro and in vivo with RICTOR ablation, and the transforming capacity of RICTOR in a Ba/F3-cell system. The mTORC1/2 inhibitors were significantly more active against RICTOR-amplified lung cancer cells as compared with other agents targeting the PI3K-AKT-mTOR pathway. Moreover, an association between RICTOR amplification and sensitivities to mTORC1/2 inhibitors was observed. The index patient has been treated with mTORC1/2 inhibitors that led to tumor stabilization for more than 18 months. SIGNIFICANCE: RICTOR amplification may define a novel and unique molecular subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors.

Transforming growth factor ß1 and extracellular matrix protease expression in the uterosacral ligaments of patients with and without pelvic organ prolapse.

OBJECTIVES: This study was undertaken to evaluate the expression of transforming growth factor ß1 (TGF-ß1) and matrix metalloproteinase 9 (MMP-9), key regulators of the extracellular matrix composition, in the uterosacral ligaments (USLs) of women with pelvic organ prolapse (POP) compared with controls. METHODS: Under an institutional review board approval, USL samples were obtained from women undergoing vaginal hysterectomy for stage 2 or greater POP (cases, n = 21) and from women without POP undergoing vaginal hysterectomy for benign indications (controls, n = 19). Hematoxylin and eosin and trichrome staining were performed on the USL sections, and the distribution of smooth muscle and fibrous tissue were quantified. Immunohistochemical staining was performed using anti-TGF-ß1 and anti-MMP-9 antibodies. The expressions of TGF-ß1 and MMP-9 were evaluated by the pathologist, who was blinded to all clinical data. RESULTS: Transforming growth factor ß1 expression positively correlated with MMP-9 expression (R = 0.4, P = 0.01). The expressions of TGF-ß1 and MMP-9 were similar in subjects with POP versus controls. There was a significant increase in fibrous tissue (P = 0.008) and a corresponding decrease in smooth muscle (P = 0.03), associated with increasing age. The TGF-ß1 expression, but not MMP-9 expression, also significantly increased with age (P = 0.02). DISCUSSION: Although our study uncovered age-related alterations in USL composition and TGF-ß1 expression, there was no difference in the expression of TGF-ß1 or MMP-9 in the subjects with POP versus controls.

Lung cancer transcriptomes refined with laser capture microdissection.

We evaluated the importance of tumor cell selection for generating gene signatures in non-small cell lung cancer. Tumor and nontumor tissue from macroscopically dissected (Macro) surgical specimens (31 pairs from 32 subjects) was homogenized, extracted, amplified, and hybridized to microarrays. Adjacent scout sections were histologically mapped; sets of approximately 1000 tumor cells and nontumor cells (alveolar or bronchial) were procured by laser capture microdissection (LCM). Within histological strata, LCM and Macro specimens exhibited approximately 67% to 80% nonoverlap in differentially expressed (DE) genes. In a representative subset, LCM uniquely identified 300 DE genes in tumor versus nontumor specimens, largely attributable to cell selection; 382 DE genes were common to Macro, Macro with preamplification, and LCM platforms. RT-qPCR validation in a 33-gene subset was confirmatory (ρ = 0.789 to 0.964, P = 0.0013 to 0.0028). Pathway analysis of LCM data suggested alterations in known cancer pathways (cell growth, death, movement, cycle, and signaling components), among others (eg, immune, inflammatory). A unique nine-gene LCM signature had higher tumor-nontumor discriminatory accuracy (100%) than the corresponding Macro signature (87%). Comparison with Cancer Genome Atlas data sets (based on homogenized Macro tissue) revealed both substantial overlap and important differences from LCM specimen results. Thus, cell selection via LCM enhances expression profiling precision, and confirms both known and under-appreciated lung cancer genes and pathways.

Expression of androgen receptor coactivator ARA70/ELE1 in androgenic alopecia.

BACKGROUND: Androgens have been implicated in androgenic alopecia as evidenced by the increased cutaneous expression of androgen receptor (AR), 5alpha-reductase, and decreased aromatase. Abnormalities of the AR-signal transduction pathway probably participate in the development of androgenic alopecia. ARA70/ELE1 is an AR coactivator with two isoforms, one full-length form (ARA70alpha/ELE1alpha), and an internally deleted form (ARA70beta/ELE1beta). We decided to examine the cutaneous expression of both isoforms in male androgenic alopecia. METHODS: Formalin-fixed, paraffin-embedded tissue sections from seven subjects with androgenic alopecia with matched punch biopsies from non-balding and balding areas were examined by in situ hybridization. RESULTS: Expression of at least one of the two probes for ARA70/ELE1 was present in all phases of the hair-growth cycle in all epithelial hair structures except for the inner root sheath. The dermal papilla and hair bulb expressed only the short (beta) but not the long (alpha) form of ARA70/ELE1. In situ labeling for ARA70beta/ELE1beta was weaker in the dermal papilla of balding recipient areas than those from donor ones. CONCLUSIONS: Our data further support that the hair growth is regulated by androgens. The differential expression pattern of ARA70/ELE1 suggests that this key androgen receptor coactivator is involved in androgenic alopecia. Lee P, Zhu C-C, Sadick NS, Diwan AH, Zhang PS, Liu JS, Prieto VG. Expression of androgen receptor coactivator ARA70/ELE1 in androgenic alopecia.

Expression of progesterone receptor is a favorable prognostic marker in ovarian cancer.

OBJECTIVE: Receptors for estrogen (ER), progesterone (PR), or androgen (AR) are predictive and prognostic markers of malignancy of multiple endocrine organs, including endometrial and breast cancer. However, the role of ERs, PRs, or ARs in the carcinogenesis of ovarian cancer, another sex hormone-dependent malignancy, is still controversial despite numerous studies that have attempted to determine their role. The disagreement in the findings may result from the fact that the numbers of tumor samples in studies have been small and that different immunohistochemical methods have been used that can introduce variation in the scoring of the histology. We therefore examined the pattern of expression of ERs, PRs, and ARs in a large number of samples of primary ovarian carcinoma by using a tissue microarray technique. METHODS: We constructed a tissue microarray with 322 samples of primary ovarian carcinoma obtained at surgery performed at The University of Texas M. D. Anderson Cancer Center between 1990 and 2000. Immunohistochemistry studies were performed by using the immunoperoxidase technique against primary antibodies (ER, PR, and AR). RESULTS: ERs, PRs, and ARs were differently expressed in different histotypes of ovarian cancer: ERs were expressed in 77.3% of all cases but more highly expressed in serous and endometrioid types; PRs were expressed in 26.2% of all cases but most highly expressed in the endometrioid type < 64.2%; and ARs were expressed in 43.7% of all cases but were most highly expressed in serous (47.5%) carcinomas. Of particular importance, the expression of PRs, but not ERs or ARs, was associated with better survival (P < 0.0001) in univariate and multivariate analyses. CONCLUSIONS: The PR is an independent marker, with its overexpression associated with a favorable prognosis in women with ovarian cancer.

A simple poly(3,4-ethylene dioxythiophene)/poly(styrene sulfonic acid) transistor for glucose sensing at neutral pH.

We demonstrate a simple transistor based on the conducting polymer poly(3,4-ethylene dioxythiophene)/poly(styrene sulfonic acid), capable of sensing glucose in a neutral pH buffer solution by a mechanism involving sensing of hydrogen peroxide.

Thyrotropin-releasing hormone receptor processing: role of ubiquitination and proteasomal degradation.

These studies were designed to characterize ubiquitination of the G protein-coupled TRH receptor (TRHR). TRHRs and ubiquitin coprecipitated with antibodies to either receptor or ubiquitin in Chinese hamster ovary or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRHRs and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRHR coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRHRs were particularly heavily ubiquitinated. These results show that the proteasome participates in TRHR quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRHRs were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and nonpermissive temperatures, indicating that ligand-dependent ubiquitination of the receptor, or any other protein, is not necessary for TRHR signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.

The concept of bronchioloalveolar cell adenocarcinoma: redefinition, a critique of the 1999 WHO classification, and an ultrastructural analysis of 155 cases.

Bronchioloalveolar cell adenocarcinoma (BACA) is bronchioloalveolar because (1) it arises in bronchioles and alveoli and (2) differentiates into bronchiolar and alveolar cells. Every entity possesses unique characteristics that separate it from other entities. The unique characteristic of BACA is its cell type. Lepidic growth is a clue to the cell type and, even though present in the vast majority, is not unique or absolutely essential. Because of the algebraic nature of concepts, the degree of differentiation, the extent of lepidic growth, and the degree of stromal desmoplasia cannot be used as definitional requirements. Likewise, in malignant tumors, absence of stromal invasion cannot be required. An epistemologically valid definition of BACA is proposed and a study of 155 cases defined this way and examined ultrastructurally is presented.

Dimerization and phosphorylation of thyrotropin-releasing hormone receptors are modulated by agonist stimulation.

Dimerization and phosphorylation of thyrotropin-releasing hormone (TRH) receptors was characterized using HEK293 and pituitary GHFT cells expressing epitope-tagged receptors. TRH receptors tagged with FLAG and hemagglutinin epitopes were co-precipitated only if they were co-expressed, and 10-30% of receptors were isolated as hemagglutinin/FLAG-receptor dimers under basal conditions. The abundance of receptor dimers was increased when cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a dose-dependent manner. TRH increased phosphorylation of both receptor monomers and dimers, documented by incorporation of (32)P and an upshift in receptor mobility reversed by phosphatase treatment. The ability of TRH to increase receptor phosphorylation and dimerization did not depend on signal transduction, because it was not inhibited by the phospholipase C inhibitor. Receptor phosphorylation required an agonist but was not blocked by the casein kinase II inhibitor apigenin, the protein kinase C inhibitor GF109203X, or expression of a dominant negative form of G protein-coupled receptor kinase 2. TRH receptors lacking most of the cytoplasmic carboxyl terminus formed dimers constitutively but failed to undergo agonist-induced dimerization and phosphorylation. TRH also increased phosphorylation and dimerization of TRH receptors expressed in GHFT pre-lactotroph cells.