Maciej Kotecki

Haploid Genetic Screens in Human Cells Identify Host Factors Used by Pathogens

“Haploid Human” Genetic screens can provide direct insight into biological processes that are poorly understood. Carette et al. (p. 1231) describe genetic screens using large-scale gene disruption in human cells haploid for all chromosomes except for chromosome 8. One screen was used to identify host factors essential for the activity of cytolethal distending toxin, a toxin found in several pathogenic bacteria. Another screen identified host gene products essential for infection with influenza, and an additional screen revealed genes required for the action of adenosine 5′-diphosphate (ADP)–ribosylating bacterial toxins. This loss-of-function genetic approach in mammalian cells will be widely applicable to study a variety of biological processes and cellular functions. A method identifies human factors required for successful microbial pathogenesis. Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.

Robust relational parsing over biomedical literature: extracting inhibit relations.

We describe the design of a robust parser for identifying and extracting biomolecular relations from the biomedical literature. Separate automata over distinct syntactic domains were developed for extraction of nominal-based relational information versus verbal-based relations. This allowed us to optimize the grammars separately for each module, regardless of any specific relation resulting in significantly better performance. A unique feature of this system is the use of text-based anaphora resolution to enhance the results of argument binding in relational extraction. We demonstrate the performance of our system on inhibition-relations, and present our initial results measured against an annotated text used as a gold standard for evaluation purposes. The results represent a significant improvement over previously published results on extracting such relations from Medline: Precision was 90%, Recall 57%, and Partial Recall 22%. These results demonstrate the effectiveness of a corpus-based linguistic approach to information extraction over Medline.

Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells

BackgroundAstrocytomas are the most common type of primary central nervous system tumors. They are frequently associated with genetic mutations that deregulate cell cycle and render these tumors resistant to apoptosis. STAT3, signal transducer and activator of transcription 3, participates in several human cancers by inducing cell proliferation and inhibiting apoptosis and is frequently activated in astrocytomas.MethodsRNA interference was used to knockdown STAT3 expression in human astrocytes and astrocytoma cell lines. The effect of STAT3 knockdown on apoptosis, cell proliferation, and gene expression was then assessed by standard methods.ResultsWe have found that STAT3 is constitutively activated in several human astrocytoma cell lines. Knockdown of STAT3 expression by siRNA induces morphologic and biochemical changes consistent with apoptosis in several astrocytoma cell lines, but not in primary human astrocytes. Moreover, STAT3 is required for the expression of the antiapoptotic genes survivin and Bcl-xL in the A172 glioblastoma cell line.ConclusionThese results show that STAT3 is required for the survival of some astrocytomas. These studies suggest STAT3 siRNA could be a useful therapeutic agent for the treatment of astrocytomas.

Signal Transducer and Activator of Transcription 3 (STAT3) Regulates Human Telomerase Reverse Transcriptase (hTERT) Expression in Human Cancer and Primary Cells

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that plays a critical role in cytokine and growth factor signaling and is frequently activated in human tumors. Human telomerase reverse transcriptase (hTERT) is also often overexpressed in tumor cells and mediates cellular immortalization. Here we report that STAT3 directly regulates the expression of hTERT in a variety of human cancer cells. Moreover, STAT3 activity is required for the survival of many human tumors, and hTERT expression contributes to the survival of STAT3-dependent tumor cells. In addition, we find that growth factors and cytokines stimulate hTERT expression in primary human cells in a STAT3-dependent manner. Thus, STAT3 is a key regulator of hTERT expression in both normal and tumor cells.

Pericyte actomyosin-mediated contraction at the cell?material interface can modulate the microvascular niche

Pericytes physically surround the capillary endothelium, contacting and communicating with associated vascular endothelial cells via cell-cell and cell-matrix contacts. Pericyte-endothelial cell interactions thus have the potential to modulate growth and function of the microvasculature. Here we employ the experimental finding that pericytes can buckle a freestanding, underlying membrane via actin-mediated contraction. Pericytes were cultured on deformable silicone substrata, and pericyte-generated wrinkles were imaged via both optical and atomic force microscopy (AFM). The local stiffness of subcellular domains both near and far from these wrinkles was investigated by using AFM-enabled nanoindentation to quantify effective elastic moduli. Substratum buckling contraction was quantified by the normalized change in length of initially flat regions of the substrata (corresponding to wrinkle contour lengths), and a model was used to relate local strain energies to pericyte contractile forces. The nature of pericyte-generated wrinkling and contractile protein-generated force transduction was further explored by the addition of pharmacological cytoskeletal inhibitors that affected contractile forces and the effective elastic moduli of pericyte domains. Actin-mediated forces are sufficient for pericytes to exert an average buckling contraction of 38% on the elastomeric substrata employed in these in vitro studies. Actomyosin-mediated contractile forces also act in vivo on the compliant environment of the microvasculature, including the basement membrane and other cells. Pericyte-generated substratum deformation can thus serve as a direct mechanical stimulus to adjacent vascular endothelial cells, and potentially alter the effective mechanical stiffness of nonlinear elastic extracellular matrices, to modulate pericyte-endothelial cell interactions that directly influence both physiologic and pathologic angiogenesis.