Parasite Mitogen-Activated Protein Kinases as Drug Discovery Targets to Treat Human Protozoan Pathogens

Protozoan pathogens are a highly diverse group of unicellular organisms, several of which are significant human pathogens. One group of protozoan pathogens includes obligate intracellular parasites such as agents of malaria, leishmaniasis, babesiosis, and toxoplasmosis. The other group includes extracellular pathogens such as agents of giardiasis and amebiasis. An unfortunate unifying theme for most human protozoan pathogens is that highly effective treatments for them are generally lacking. We will review targeting protozoan mitogen-activated protein kinases (MAPKs) as a novel drug discovery approach towards developing better therapies, focusing on Plasmodia, Leishmania, and Toxoplasma, about which the most is known

Efficient Foreign Gene Expression in Epstein-Barr Virus-Transformed Human B-Cells

Epstein-Barr virus (EBV) is a herpesvirus that transforms B-cells (B-LCL) and has undergone intense scrutiny owing to its association with Burkitt's lymphoma, nasopharyngeal carcinoma, and immunoblastic lymphomas. B-LCL have also proven useful in the study of human immunology. We describe a novel system for inducing efficient foreign gene expression in B-LCL using biotinylated adenovirus as an endosome-disrupting agent. Plasmid DNA is coupled to the exterior of viral particles by streptavidin-polylysine chimeric proteins. Up to 67% of B-LCL may be induced to express foreign genes in vitro in transient expression systems, and gene expression lasts for at least 17 days. We have expressed firefly luciferase, β-galactosidase (β-gal), chloramphenicol acetyltransferase, HIV gag, and env genes, as well as infectious HIV, and the EBV-specific BZLF gene in B-LCL with this system. In vivo delivery of a β-gal reporter gene to B-LCL was documented in a SCID mouse model. Potential applications include study of genetic regulation of EBV infection and transformation events, study of potential gene therapies for EBV-related B-cell tumors, and production of antigen-presenting cells for use in immunologic assays. Because of the high percentage of cells transformed and the length of foreign gene expression, the possibility of examining foreign gene expression in transient assays, without selection for clonal populations, exists

The Spitzer c2d Survey of Nearby Dense Cores: VI. The Protostars of Lynds Dark Nebula 1221

Observations of Lynds Dark Nebula 1221 from the Spitzer Space Telescope are presented. These data show three candidate protostars towards L1221, only two of which were previously known. The infrared observations also show signatures of outflowing material, an interpretation which is also supported by radio observations with the Very Large Array. In addition, molecular line maps from the Five College Radio Astronomy Observatory are shown. One-dimensional dust continuum modelling of two of these protostars, IRS1 and IRS3, is described. These models show two distinctly different protostars forming in very similar environments. IRS1 shows a higher luminosity and larger inner radius of the envelope than IRS3. The disparity could be caused by a difference in age or mass, orientation of outflow cavities, or the impact of a binary in the IRS1 core.Comment: accepted for publication in Ap

The Detection and Characterization of cm Radio Continuum Emission from the Low-mass Protostar L1014-IRS

Observations by the Cores to Disk Legacy Team with the Spitzer Space Telescope have identified a low luminosity, mid-infrared source within the dense core, Lynds 1014, which was previously thought to harbor no internal source. Followup near-infrared and submillimeter interferometric observations have confirmed the protostellar nature of this source by detecting scattered light from an outflow cavity and a weak molecular outflow. In this paper, we report the detection of cm continuum emission with the VLA. The emission is characterized by a quiescent, unresolved 90 uJy 6 cm source within 0.2" of the Spitzer source. The spectral index of the quiescent component is $\alpha = 0.37\pm 0.34$ between 6 cm and 3.6 cm. A factor of two increase in 6 cm emission was detected during one epoch and circular polarization was marginally detected at the $5\sigma$ level with Stokes {V/I} $= 48 \pm 16$% . We have searched for 22 GHz H2O maser emission toward L1014-IRS, but no masers were detected during 7 epochs of observations between June 2004 and December 2006. L1014-IRS appears to be a low-mass, accreting protostar which exhibits cm emission from a thermal jet or a wind, with a variable non-thermal emission component. The quiescent cm radio emission is noticeably above the correlation of 3.6 cm and 6 cm luminosity versus bolometric luminosity, indicating more radio emission than expected. We characterize the cm continuum emission in terms of observations of other low-mass protostars, including updated correlations of centimeter continuum emission with bolometric luminosity and outflow force, and discuss the implications of recent larger distance estimates on the physical attributes of the protostar and dense molecular core.Comment: 14 pages. Accepted for publication in Ap

Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis.

Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5' end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development

Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis

Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 50 end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development

Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis and autophagy in ovarian cancer and melanoma

PD-L1 antibodies produce efficacious clinical responses in diverse human cancers, but the basis for their effects remains unclear, leaving a gap in understanding of how to rationally leverage the therapeutic activity. PD-L1 is widely expressed in tumor cells but its contributions to tumor pathogenicity are incompletely understood. In this study, we evaluated the hypothesis that PD-L1 exerts tumor cell-intrinsic signals that are critical for pathogenesis. Using RNAi methodology, we attenuated PD-L1 in the murine ovarian cell line ID8agg and the melanoma cell line B16 (termed PD-L1lo cells), which express basal PD-L1. We observed that PD-L1lo cells proliferated more weakly than control cells in vitro. As expected, PD-L1lo cells formed tumors in immunocompetent mice relatively more slowly, but unexpectedly, they also formed tumors more slowly in immunodeficient NSG mice. A comparative microarray analysis identified a number of genes involved in autophagy and mTOR signaling that were affected by PD-L1 expression. In support of a functional role, PD-L1 attenuation augmented autophagy and blunted the ability of autophagy inhibitors to limit proliferation in vitro and in vivo in NSG mice. PD-L1 attenuation also elevated mTORC1 activity and augmented the anti-proliferative effects of the mTORC1 inhibitor rapamycin. PD-L1 cells were also relatively deficient in metastasis to the lung and we found that anti-PD-L1 administration could block tumor cell growth and metastasis in NSG mice. This therapeutic effect was observed with B16 cells but not ID8agg cells, illustrating tumor- or tissue-specific effects in the therapeutic setting. Overall, our findings extend understanding of PD-L1 functions, illustrate non-immune effects of anti-PD-L1 immunotherapy and suggest broader uses for PD-L1 as a biomarker for assessing cancer therapeutic responses