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Using a mini-library of 1062 lentiviral shRNAs targeting 40 nuclear hormone receptors and 70 of their co-regulators, we searched for potential therapeutic targets that would be important during in vivo tumor growth using a parallel in vitro and in vivo shRNA screening strategy in the non-small cell lung cancer (NSCLC) line NCI-H1819. We identified 21 genes essential for in vitro growth, and nine genes specifically required for tumor survival in vivo, but not in vitro: NCOR2, FOXA1, HDAC1, RXRA, RORB, RARB, MTA2, ETV4, and NR1H2. We focused on FOXA1, since it lies within the most frequently amplified genomic region in lung adenocarcinomas. We found that 14q-amplification in NSCLC cell lines was a biomarker for FOXA1 dependency for both in vivo xenograft growth and colony formation, but not mass culture growth in vitro. FOXA1 knockdown identified genes involved in electron transport among the most differentially regulated, indicating FOXA1 loss may lead to a decrease in cellular respiration. In support of this, FOXA1 amplification was correlated with increased sensitivity to the complex I inhibitor phenformin. Integrative ChipSeq analyses reveal that FOXA1 functions in this genetic context may be at least partially independent of NKX2-1. Our findings are consistent with a neomorphic function for amplified FOXA1, driving an oncogenic transcriptional program. These data provide new insight into the functional consequences of FOXA1 amplification in lung adenocarcinomas, and identify new transcriptional networks for exploration of therapeutic vulnerabilities in this patient population., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

A 75-year-old man with severe aortic stenosis, severe chronic obstructive pulmonary disease, NYHA class III heart failure and a large abdominal aortic aneurysm underwent concurrent transfemoral transcatheter aortic valve replacement (TF-TAVR) and endovascular aneurysm repair (EVAR). An Edwards Sapien device was implanted with resolution of hemodynamics. EVAR was performed using an Endurant bifurcated stent graft system. We describe the procedure technique, periprocedural management and one year outcome. To the authors' best knowledge, this is the first case of simultaneous TF-TAVR and EVAR published in North America., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Transglutaminase (TG) enzymes and protein crosslinking have long been implicated in the formation of mineralized tissues. The aim of this study was to analyze the expression, activity and function of TGs in differentiating osteoblasts to gain further insight into the role of extracellular matrix protein crosslinking in bone formation. MC3T3-E1 (subclone 14) pre-osteoblast cultures were treated with ascorbic acid and beta-glycerophosphate to induce cell differentiation and matrix mineralization. Expression of TG isoforms was analyzed by RT-PCR. TG activity was assessed during osteoblast differentiation by in vitro biochemical assays and by in situ labeling of live cell cultures. We demonstrate that MC3T3-E1/C14 osteoblasts express two TG isoforms--TG2 and FXIIIA. Abundant TG activity was observed during cell differentiation which increased significantly after thrombin treatment, a result confirming the presence of FXIIIA in the cultures. Ascorbic acid treatment, which stimulated collagen secretion and assembly, also stimulated externalization of TG activity, likely from FXIIIA which was externalized upon this treatment as analyzed by immunofluoresence microscopy. Inhibition of TG activity in the cultures by cystamine resulted in complete abrogation of mineralization, attributable to decreased matrix accumulation and an arrested state of osteoblast differentiation as measured by decreased levels of bone sialoprotein, osteocalcin and alkaline phosphatase. Additional functional studies and substrate characterization showed that TG activity was required for the formation of a fibronectin-collagen network during the early stages of matrix formation and assembly. This network, in turn, appeared to be essential for further matrix production and progression of the osteoblast differentiation program, and ultimately for mineralization.

An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) was developed for a novel protein granulophysin, a constituent of the platelet dense granule (DG) membrane and used to characterize patients with dense granule storage pool deficiency (delta-SPD). The assay uses two monoclonal antibodies against the protein, one of which is conjugated to peroxidase. Purified DGs, an enriched source of the protein, were used for the standard curve. Granulophysin levels were only low in forms of delta-SPD associated with albinism. Granulophysin levels in platelet homogenates of 30 patients with the Hermansky-Pudlak syndrome form of delta-SPD were 1/4 to 1/5 of levels in controls or obligate heterozygotes. Two patients with the Chediak-Higashi form of delta-SPD syndrome also had markedly reduced levels of granulophysin. Patients with other forms of delta-SPD had normal levels of granulophysin. Two sisters with delta-SPD in one family had normal granulophysin present in empty dense granule membrane vesicles. Three members of another family with delta-SPD had low DG counts but normal granulophysin levels, indicating that in this group the level of granulophysin was maintained despite the reduction in granule formation. Thus, granulophysin quantitation facilitates characterization of delta-SPD patients and may provide clues to the nature of defective granules in delta-SPD subtypes.

Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.

A monoclonal immunocytochemical method with 25 monoclonal antibodies was used to study the distribution in human tonsil of determinants expressed on T cells (mature and immature), Langerhans cells, B cells, killer/natural killer cells, macrophages, immature myeloid and lymphoblastic leukemia cells. Many of the respective determinants were found to have a discrete topographic distribution in normal reactive tonsil. The common acute lymphoblastic leukemia antigen and a determinant found on myeloblastic leukemia cells (My10) were not found in the specimens of the tonsil examined.

The immune phenotype of the infiltrating cells in 13 positive patch tests from 8 cases of contact dermatitis and 1 case of poison ivy was studied. An indirect immunoperoxidase technique was used in conjunction with monoclonal antibodies directed against mature T cells (Leu-1, T11), helper T cells (Leu-3A), suppressor/cytotoxic T cells (Leu-2A), killer and natural killer cells (HNK 1), B cells (B1), Langerhans cells (HLA-DR), and the common acute lymphoblastic leukemia antigen (CALLA), (J5). The majority of infiltrating mononuclear cells were Leu-1+, T11+, Leu-3A+, Leu-2A-, HLA-DR+, T9-, T10-, HNK-, B1-, J5-. Occasional T6+ cells were observed in the epidermis (including spongiotic microvesicles) and also isolated in the dermis and within the dermal mononuclear infiltrates. The phenotype was compared with cutaneous T-cell lymphoma, a disease in which contact allergy and antigenic persistence may play a role.