Challenges Of Small Dance Companies Meeting Artistic And Administrative Demands

M.S., Arts Administration -- Drexel University, 200

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Lethality of PAK3 and SGK2 shRNAs to Human Papillomavirus Positive Cervical Cancer Cells Is Independent of PAK3 and SGK2 Knockdown

<div><p>The p21-activated kinase 3 (PAK3) and the serum and glucocorticoid-induced kinase 2 (SGK2) have been previously proposed as essential kinases for human papillomavirus positive (HPV+) cervical cancer cell survival. This was established using a shRNA knockdown approach. To validate PAK3 and SGK2 as potential targets for HPV+ cervical cancer therapy, the relationship between shRNA-induced phenotypes in HPV+ cervical cancer cells and PAK3 or SGK2 knockdown was carefully examined. We observed that the phenotypes of HPV+ cervical cancer cells induced by various PAK3 and SGK2 shRNAs could not be rescued by complement expression of respective cDNA constructs. A knockdown-deficient PAK3 shRNA with a single mismatch was sufficient to inhibit HeLa cell growth to a similar extent as wild-type PAK3 shRNA. The HPV+ cervical cancer cells were also susceptible to several non-human target shRNAs. The discrepancy between PAK3 and SGK2 shRNA-induced apoptosis and gene expression knockdown, as well as cell death stimulation, suggested that these shRNAs killed HeLa cells through different pathways that may not be target-specific. These data demonstrated that HPV+ cervical cancer cell death was not associated with RNAi-induced PAK3 and SGK2 knockdown but likely through off-target effects.</p></div

Loss of cell viability induced by PAK3 or SGK2 shRNAs.

<p>(A) HeLa cells were infected with PAK3 shRNA at a 1:10 dilution. Cell apoptosis was determined using a caspase 3/7 glo luciferase assay 72 hours after infection. The bar graph presents fold changes of caspase 3/7 activity (luminescence) induced by various shRNAs compared with a no-shRNA control. Data represent the average ± standard deviation of 4 replicates from one of two separate experiments with similar results; (B) Inhibition of proliferation/viability of HeLa cells by PAK3 shRNAs was assessed using a CellTiter blue assay 5 days after infection. The bar graph presents percent viability (fluorescence) of lentiviral shRNA-infected cells compared with a control lentivirus without shRNA expression. Data represent the mean ± standard deviation of three independent experiments; (C) SGK2 lentiviral shRNAs induced HeLa cell apoptosis. Data represent 4 replicates from one of two separate experiments with similar results; (D) SGK2 shRNAs inhibited proliferation/viability of HeLa cells. Data represent the average ± standard deviation of two independent experiments; (E) SGK2 lentiviral shRNAs induced autophagy of HeLa cells. Cells were infected with SGK2 lentiviral shRNAs at 1:16 and 1:32 dilution, respectively. 1 μM Rapamycin was included as an autophagy control on each plate. Cell plates were fixed 72 hour after infection and immunostained for induction of autophagy with a LC3B primary antibody, followed with an Alexa 488-conjugated secondary antibody. Images were captured using the confocal Opera High Content Imager. Images of SGK2 lentiviral shRNA-infected HeLa cells (~1200 cells/well) were captured and cell numbers counted. The average intensity of LC3B staining per cell was measured and calculated using a modified Capella (Perkin Elmer) algorithm. The bar graph presents the average ± standard deviation of LC3B staining intensity derived from two wells.</p

Rescue of SGK2 shRNA-induced phenotypes.

<p>(A) HeLa cells were transfected with serially diluted SGK2 expressing plasmids harboring silent mutations at the shRNA 2111 annealing site. Six hours after transfection, the cells were infected with SGK2 lentiviral shRNA 2111 (1:15 dilution). SGK2 mRNA expression levels were determined 72 hours after infection. The bar graph presents quantities of SGK2 mRNA normalized with GAPDH mRNA. Data represent the average ± standard deviation of 4 replicates from one of two experiments with similar results; (B) HeLa cell apoptosis induced by SGK2 shRNA 2111 was quantified with a caspase 3/7 glo assay 72 hours after infection. Two variants of SGK2 (alpha and beta) were used for the phenotype rescue analysis. Data represent the average ± standard deviation of two independent experiments; (C) HeLa cell proliferation/ viability inhibition induced by SGK2 shRNA 2111 was determined using the CellTiter Blue assay 5 days after infection. Data represents the average ± standard deviation of two independent experiments.</p

Inhibition of HPV+ cervical cancer cells by lentiviral shRNAs that do not target human genes.

<p>Cell viability was assessed using a CellTiter Blue assay 5 days after infection. Each of the viruses was titrated for infection to determine its potency of inducing HeLa cell proliferation/viability inhibition. Percent cell viability (fluorescence) in the presence of lentiviral shRNAs was calculated by comparing with a control lentivirus without shRNA expression. Data represent the average ± standard deviation of 4 replicates from one of three independent experiments with similar results; (A) Inhibition of HeLa cell proliferation/viability; (B) Inhibition of proliferation/viability of CaSki cells.</p

Study shRNA lentivirus vectors.

<p>^a NA—not available</p><p>Study shRNA lentivirus vectors.</p

Knockdown of PAK3 and SGK2 by shRNAs.

<p>mRNA levels were determined by quantitative reverse transcription PCR analysis at 72 hours after infection with the respective shRNA vectors; control denotes infection with a vector encoding non-target shRNA. Each bar on the bar graph represents the average ± standard deviation of 4 replicates from one of three independent experiments with similar results. mRNA levels were normalized with GAPDH expression; PAK3 and SGK2 protein expression were determined 72 hours after infection using a Western immunoblot. GAPDH protein acted as a protein loading control for each sample. (A) PAK3 shRNA decreased PAK3 mRNA levels in HeLa cells; (B) PAK3 shRNAs reduced PAK3 mRNA expression in DMS-79 cells; (C) PAK3 shRNAs reduced PAK3 protein expression in DMS-79 cells. DMS-79 cell lysates were analyzed with PAK3 monoclonal antibody N-19; (D) SGK2 shRNAs reduced SGK2 mRNA levels in HeLa cells; (E) SGK2 shRNAs reduced SGK2 mRNA levels in GTL16 cells; (F) SGK2 shRNAs reduced SGK2 protein levels in GTL16 cells. GTL16 cell lysates were analyzed with SGK2 monoclonal antibody 3Q-2.</p

Longitudinal Changes and Associations Between Quantitative Sensory Testing and Psychological Factors in Whiplash-associated Disorders:A Systematic Review and Meta-analyses-based Data Synthesis

Whiplash-associated disorders (WAD) represent a multifactorial condition often accompanied by altered nociceptive processing and psychological factors. This systematic review on acute and chronic WAD aimed to investigate the relationship between quantitative sensory testing (QST) and psychological factors and quantify whether their trajectories over time follow a similar pattern to disability levels. Eight databases were searched until October 2022. When 2 prospective studies examined the same QST or psychological variable, data synthesis was performed with random-effects meta-analysis by pooling within-group standardized mean differences from baseline to 3-, 6-, and 12-month follow-ups. From 5,754 studies, 49 comprising 3,825 WAD participants were eligible for the review and 14 for the data synthesis. Altered nociceptive processing in acute and chronic WAD, alongside worse scores on psychological factors, were identified. However, correlations between QST and psychological factors were heterogeneous and inconsistent. Furthermore, disability levels, some QST measures, and psychological factors followed general positive improvement over time, although there were differences in magnitude and temporal changes. These results may indicate that altered psychological factors and increased local pain sensitivity could play an important role in both acute and chronic WAD, although this does not exclude the potential influence of factors not explored in this review