The vaccinia virus expression system is known for the efficient production of recombinant proteins with "appropriate" posttranslational modification using desired mammalian cell lines. However, being a replication competent virus, vaccinia virus poses a health threat to immunocompromised individuals and requires biosafety level 2 (BSL2) laboratory precautions, thereby restricting its use by the scientific community. Development of the host range restricted modified vaccinia Ankara (MVA) system has allowed researchers to work with a safer virus even at BSL1. Here, we report on the use of an improved second generation MVA viral system incorporating two selective markers and fluorescent proteins for easier recombinant virus identification. Notably, we demonstrate that this novel system is capable of producing secreted recombinant proteins, a finding not previously reported. Through purification and characterization of wild type and mutant platelet-derived growth factor D (PDGF D) dimer species, we demonstrate this system is capable of producing the latent full-length PDGF D dimer, partially processed intermediate dimer (hemidimer), as well as fully processed growth factor domain dimer that show chemical integrity and biological activity. Importantly, this system is amenable to scaling up for the mass production of recombinant PDGF D (rPDGF D) dimer species.

To assess the clinical feasibility of a virtual mirror therapy system in a pilot sample of patients with phantom pain.

The PB2 protein of the influenza virus RNA polymerase is a major virulence determinant of influenza viruses. It binds to the cap structure at the 5' end of host mRNA to generate short capped RNA fragments that are used as primers for viral transcription named cap-snatching. A large number of the compounds were shown to bind the minimal cap-binding domain of PB2 to inhibit the cap-snatching machinery. However, their binding in the context of an extended form of the PB2 protein has remained elusive. A previous study reported some promising compounds including azaindole and hydroxymethyl azaindole, which were analyzed here to predict binding affinity to PB2 protein using the steered molecular dynamics (SMD) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods. The results show that the rupture force (Fmax) value of three complexes is in agreement with the binding free energy value (ΔGbind) estimated by the MM-PBSA method, whereas for the non-equilibrium pulling work (Wpull) value a small difference between A_PB2-4 and A_PB2-12 was observed. The binding affinity results indicate the A_PB2-12 complex is more favorable than the A_PB2-4 and A_PB2-16 complexes, which means the inhibitor (12) has the potential to be further developed as anti-influenza agents in the treatment of influenza A.

The mitochondrial Bit1 protein exerts tumor-suppressive function in NSCLC through induction of anoikis and inhibition of EMT. Having this dual tumor suppressive effect, its downregulation in the established human lung adenocarcinoma A549 cell line resulted in potentiation of tumorigenicity and metastasis in vivo. However, the exact role of Bit1 in regulating malignant growth and transformation of human lung epithelial cells, which are origin of most forms of human lung cancers, has not been examined. To this end, we have downregulated the endogenous Bit1 expression in the immortalized non-tumorigenic human bronchial epithelial BEAS-2B cells. Knockdown of Bit1 enhanced the growth and anoikis insensitivity of BEAS-2B cells. In line with their acquired anoikis resistance, the Bit1 knockdown BEAS-2B cells exhibited enhanced anchorage-independent growth in vitro but failed to form tumors in vivo. The loss of Bit1-induced transformed phenotypes was in part attributable to the repression of E-cadherin expression since forced exogenous E-cadherin expression attenuated the malignant phenotypes of the Bit1 knockdown cells. Importantly, we show that the loss of Bit1 expression in BEAS-2B cells resulted in increased Erk activation, which functions upstream to promote TLE1-mediated transcriptional repression of E-cadherin. These collective findings indicate that loss of Bit1 expression contributes to the acquisition of malignant phenotype of human lung epithelial cells via Erk activation-induced suppression of E-cadherin expression.

The mitochondrial Bcl-2 inhibitor of transcription 1 (Bit1) protein is part of an anoikis-regulating pathway that is selectively dependent on integrins. We previously demonstrated that the caspase-independent apoptotic effector Bit1 exerts tumor suppressive function in lung cancer in part by inhibiting anoikis resistance and anchorage-independent growth in vitro and tumorigenicity in vivo. Herein we show a novel function of Bit1 as an inhibitor cell migration and epithelial-mesenchymal transition (EMT) in the human lung adenocarcinoma A549 cell line. Suppression of endogenous Bit1 expression via siRNA and shRNA strategies promoted mesenchymal phenotypes, including enhanced fibroblastoid morphology and cell migratory potential with concomitant downregulation of the epithelial marker E-cadherin expression. Conversely, ectopic Bit1 expression in A549 cells promoted epithelial transition characterized by cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Specific downregulation of E-cadherin in Bit1-transfected cells was sufficient to block Bit1-mediated inhibition of cell motility while forced expression of E-cadherin alone attenuated the enhanced migration of Bit1 knockdown cells, indicating that E-cadherin is a downstream target of Bit1 in regulating cell motility. Furthermore, quantitative real-time PCR and reporter analyses revealed that Bit1 upregulates E-cadherin expression at the transcriptional level through the transcriptional regulator Amino-terminal Enhancer of Split (AES) protein. Importantly, the Bit1/AES pathway induction of E-cadherin expression involves inhibition of the TLE1-mediated repression of E-cadherin, by decreasing TLE1 corepressor occupancy at the E-cadherin promoter as revealed by chromatin immunoprecipitation assays. Consistent with its EMT inhibitory function, exogenous Bit1 expression significantly suppressed the formation of lung metastases of A549 cells in an in vivo experimental metastasis model. Taken together, our studies indicate Bit1 is an inhibitor of EMT and metastasis in lung cancer and hence can serve as a molecular target in curbing lung cancer aggressiveness.

The autophagy-lysosome pathway and apoptosis constitute vital determinants of cell fate and engage in a complex interplay in both physiological and pathological conditions. Central to this interplay is the archetypal autophagic cargo adaptor p62/SQSTM1/Sequestosome-1 which mediates both cell survival and endoplasmic reticulum stress-induced apoptosis via aggregation of ubiquitinated caspase-8. Here, we investigated the role of p62-mediated apoptosis in head and neck squamous cell carcinoma (HNSCC), which can be divided into two groups based on human papillomavirus (HPV) infection status. We show that increased autophagic flux and defective apoptosis are associated with radioresistance in HPV(-) HNSCC, whereas HPV(+) HNSCC fail to induce autophagic flux and readily undergo apoptotic cell death upon radiation treatments. The degree of radioresistance and tumor progression of HPV(-) HNSCC respectively correlated with autophagic activity and cytosolic levels of p62. Pharmacological activation of the p62-ZZ domain using small molecule ligands sensitized radioresistant HPV(-) HNSCC cells to ionizing radiation by facilitating p62 self-polymerization and sequestration of cargoes leading to apoptosis. The self-polymerizing activity of p62 was identified as the essential mechanism by which ubiquitinated caspase-8 is sequestered into aggresome-like structures, without which irradiation fails to induce apoptosis in HNSCC. Our results suggest that harnessing p62-dependent sequestration of ubiquitinated caspase-8 provides a novel therapeutic avenue in patients with radioresistant tumors.

The mitochondrial Bit1 (Bcl-2 inhibitor of transcription 1) protein is a part of an apoptotic pathway that is uniquely regulated by integrin-mediated attachment. As an anoikis effector, Bit1 is released into the cytoplasm following loss of cell attachment and induces a caspase-independent form of apoptosis. Considering that anoikis resistance is a critical determinant of transformation, we hypothesized that cancer cells may circumvent the Bit1 apoptotic pathway to attain anchorage-independence and tumorigenic potential. Here, we provide the first evidence of the tumor suppressive effect of Bit1 through a mechanism involving anoikis induction in human lung adenocarcinoma derived A549 cells. Restitution of Bit1 in anoikis resistant A549 cells is sufficient to induce detachment induced-apoptosis despite defect in caspase activation and impairs their anchorage-independent growth. Conversely, stable downregulation of Bit1 in these cells significantly enhances their anoikis resistance and anchorage-independent growth. The Bit1 knockdown cells exhibit significantly enhanced tumorigenecity in vivo. It has been previously shown that the nuclear TLE1 corepressor is a putative oncogene in lung cancer, and we show here that TLE1 blocks Bit1 mediated anoikis in part by sequestering the pro-apoptotic partner of Bit1, the Amino-terminal Enhancer of Split (AES) protein, in the nucleus. Taken together, these findings suggest a tumor suppressive role of the caspase-independent anoikis effector Bit1 in lung cancer. Consistent with its role as a tumor suppressor, we have found that Bit1 is downregulated in human non-small cell lung cancer (NSCLC) tissues.

Mesenchymal stem cells (MSCs) are adult stem cell populations and exhibit great potential in regenerative medicine and oncology. Platelet-derived growth factors (PDGFs) are well known to regulate MSC biology through their chemotactic and mitogenic properties. However, their direct roles in the regulation of MSC lineage commitment are unclear. Here, we show that PDGF D promotes the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into osteoblasts and inhibits hBMSC differentiation into adipocytes. We demonstrate that PDGF D-induced β-actin expression and polymerization are essential for mediating this differential regulation of osteoblastogenesis and adipogenesis. Interestingly, we found that PDGF D induces massive upward molecular weight shifts of its cognate receptor, PDGF receptor beta (β-PDGFR) in hBMSCs, which was not observed in fibroblasts. Proteomic analysis indicated that the E3 ubiquitin ligase HECT, UBA, and WWE domain-containing protein 1 (HUWE1) associates with the PDGF D-activated β-PDGFR signaling complex in hBMSCs, resulting in β-PDGFR polyubiquitination. In contrast to the well-known role of ubiquitin in protein degradation, we provide evidence that HUWE1-mediated β-PDGFR polyubiquitination delays β-PDGFR internalization and degradation, thereby prolonging AKT signaling. Finally, we demonstrate that HUWE1-regulated β-PDGFR signaling is essential for osteoblastic differentiation of hBMSCs, while being dispensable for PDGF D-induced hBMSC migration and proliferation as well as PDGF D-mediated inhibition of hBMSC differentiation into adipocytes. Taken together, our findings provide novel insights into the molecular mechanism by which PDGF D regulates the commitment of hBMSCs into the osteoblastic lineage.

Purpose The COVID-19 pandemic has disproportionately affected the lives of people with disabilities (PWD). How the pandemic affects the employment of PWD and employers has yet to be determined. We aimed to investigate the employment consequences of the pandemic as experienced by PWD and employers. The research questions were: (1) What employment effects do PWD experience, and what business changes do employers encounter as a result of the COVID-19 pandemic? (2) What challenges have PWD encountered during the pandemic? Methods Cross-sectional online surveys of 733 PWD and 67 employers in the Midwestern United States. Results Compared to non-disabled peers, PWD encountered more challenges in employment during the pandemic. We found high percentages of both employers and PWD experiencing employment changes and business shutdown during the pandemic. For PWD whose employment was not affected, 14.6% of the participants (n = 107) expected a loss of income and worried about the economic uncertainty of the pandemic. Unemployment for PWD is high due to illness or disability, being laid-off or furloughed, business reductions, and not feeling safe to work. However, only about 18.6% of unemployed PWD (n = 16) received pay or benefits for the time they were not working even though more than half filed for unemployment benefits. Conclusions The pandemic adversely affected employment of PWD as reported by workers and employers. Findings parallel the experience of the non-disabled workforce, but reveal vulnerabilities that reflect disability consequences and the need for job accommodations. Results reveal emergent needs for policy supports to reduce the disparities experienced by PWD in the workplace.

The Transducin-like enhancer of split 1 (TLE1) corepressor protein is overexpressed in human lung tumors and is a putative lung-specific oncogene. However, the molecular mechanism underlying its oncogenic function remains to be delineated. Here, we report an important role of TLE1 in promoting lung tumorigenesis by a mechanism involving induction of anoikis resistance. Using the human lung adenocarcinoma A549 and immortalized bronchial epithelial BEAS-2B cell lines, we observed that TLE1 inhibits anoikis through transcriptional repression of E-cadherin gene. In support of E-cadherin as a downstream target of TLE1 to block anoikis, forced expression of E-cadherin attenuated TLE1-induced anoikis resistance while E-cadherin downregulation decreased the anoikis sensitivity of TLE1 knockdown cells. Furthermore, we determined that E-cadherin expression is transcriptionally induced upon loss of cell attachment and functions as an effector of anoikis. Loss of E-cadherin via the siRNA strategy or exogenous TLE1 expression was sufficient to attenuate anoikis in A549 and BEAS-2B cells. Importantly, we demonstrated that the ZEB1 transcriptional factor is required for TLE1-mediated E-cadherin repression and anoikis resistance. ZEB1 interacted with and recruited the TLE1 to the E-cadherin promoter to impose histone deacetylation and gene silencing. In vivo, TLE1 strongly promoted tumorigenicity of A549 cells in a ZEB1-dependent manner. Underscoring its role in anoikis insensitivity of lung cancer cells, the TLE1-mediated E-cadherin repression was negatively regulated by the tumor suppressor Bcl-2 inhibitor of transcription 1 (Bit1) to effect anoikis. These findings identify the ZEB1/TLE1/E-cadherin transcriptional mechanism as a novel pathway that promotes anoikis resistance and oncogenicity of lung cancer cells.