A Parametric Study on Free Vibration of Multi-perforated Rectangular Plates

AbstractThis work involves study of the first three natural frequencies of the perforated plates. The effect of the parameters which influence them have been studied. The parameters considered are the shape of perforations, pattern of the perforations, aspect ratio of the plate, dimensions of the plate, ligament efficiency and the mass remnant ratio (MRR). The study is focused on the effect of the most influencing parameter on the free vibrations

ARHGEF9 regulates melanoma morphogenesis in environments with diverse geometry and elasticity by promoting filopodial-driven adhesion.

Rho GTP Exchange Factors (RhoGEFs) and Rho GTPase Activating Proteins (RhoGAPs) are large families of molecules that regulate shape determination in all eukaryotes. In pathologies such as melanoma, RhoGEF and RhoGAP activity underpins the ability of cells to invade tissues of varying elasticity. To identify RhoGEFs and RhoGAPs that regulate melanoma cell shape on soft and/or stiff materials, we performed genetic screens, in tandem with single-cell quantitative morphological analysis. We show that ARHGEF9/Collybistin (Cb) is essential for cell shape determination on both soft and stiff materials, and in cells embedded in 3D soft hydrogel. ARHGEF9 is required for melanoma cells to invade 3D matrices. Depletion of ARHGEF9 results in loss of tension at focal adhesions decreased cell-wide contractility, and the inability to stabilize protrusions. Taken together we show that ARHGEF9 promotes the formation of actin-rich filopodia, which serves to establish and stabilize adhesions and determine melanoma cell shape

Nanoneedle-mediated stimulation of cell mechanotransduction machinery

Biomaterial substrates can be engineered to present topographical signals to cells which, through interactions between the material and active components of the cell membrane, regulate key cellular processes and guide cell fate decisions. However, targeting mechanoresponsive elements that reside within the intracellular domain is a concept that has only recently emerged. Here, we show that mesoporous silicon nanoneedle arrays interact simultaneously with the cell membrane, cytoskeleton, and nucleus of primary human cells, generating distinct responses at each of these cellular compartments. Specifically, nanoneedles inhibit focal adhesion maturation at the membrane, reduce tension in the cytoskeleton, and lead to remodeling of the nuclear envelope at sites of impingement. The combined changes in actin cytoskeleton assembly, expression and segregation of the nuclear lamina, and localization of Yes-associated protein (YAP) correlate differently from what is canonically observed upon stimulation at the cell membrane, revealing that biophysical cues directed to the intracellular space can generate heretofore unobserved mechanosensory responses. These findings highlight the ability of nanoneedles to study and direct the phenotype of large cell populations simultaneously, through biophysical interactions with multiple mechanoresponsive components

A whole genome RNAi screen of Drosophila S2 cell spreading performed using automated computational image analysis

An automated, image-based RNAi screen for cell shape reveals roles for membrane secretion factors in cell spreading

Fast fluorescence microscopy for imaging the dynamics of embryonic development

Live imaging has gained a pivotal role in developmental biology since it increasingly allows real-time observation of cell behavior in intact organisms. Microscopes that can capture the dynamics of ever-faster biological events, fluorescent markers optimal for in vivo imaging, and, finally, adapted reconstruction and analysis programs to complete data flow all contribute to this success. Focusing on temporal resolution, we discuss how fast imaging can be achieved with minimal prejudice to spatial resolution, photon count, or to reliably and automatically analyze images. In particular, we show how integrated approaches to imaging that combine bright fluorescent probes, fast microscopes, and custom post-processing techniques can address the kinetics of biological systems at multiple scales. Finally, we discuss remaining challenges and opportunities for further advances in this field

A novel Nav1.5-dependent feedback mechanism driving glycolytic acidification in breast cancer metastasis

The authors wish to acknowledge the roles of the Breast Cancer Now Tissue Bank in collecting and making available the samples and data, and the patients who have generously donated their tissues and shared their data to be used in the generation of this publication. The authors also thank Prof. Miles Whittington (Hull-York Medical School, UK), Dr. John Davey and Dr. Katherine Newling (Technology Facility, University of York, UK), and Prof. Lýdia Vargová (Charles University, Czechia) for providing invaluable advice. For the purpose of open access, a Creative Commons Attribution (CC BY) licence is applied to any author accepted manuscript version arising from this submission.Peer reviewe

Sub-population analysis based on temporal features of high content images

Background: High content screening techniques are increasingly used to understand the regulation and progression of cell motility. The demand of new platforms, coupled with availability of terabytes of data has challenged the traditional technique of identifying cell populations by manual methods and resulted in development of high-dimensional analytical methods. Results: In this paper, we present sub-populations analysis of cells at the tissue level by using dynamic features of the cells. We used active contour without edges for segmentation of cells, which preserves the cell morphology, and autoregressive modeling to model cell trajectories. The sub-populations were obtained by clustering static, dynamic and a combination of both features. We were able to identify three unique sub-populations in combined clustering. Conclusion: We report a novel method to identify sub-populations using kinetic features and demonstrate that these features improve sub-population analysis at the tissue level. These advances will facilitate the application of high content screening data analysis to new and complex biological problems.Computation and Systems Biology Programme of Singapore--Massachusetts Institute of Technology Allianc

Tyrosine Phosphorylation of the UDP-Glucose Dehydrogenase of Escherichia coli Is at the Crossroads of Colanic Acid Synthesis and Polymyxin Resistance

BACKGROUND:In recent years, an idiosyncratic new class of bacterial enzymes, named BY-kinases, has been shown to catalyze protein-tyrosine phosphorylation. These enzymes share no structural and functional similarities with their eukaryotic counterparts and, to date, only few substrates of BY-kinases have been characterized. BY-kinases have been shown to participate in various physiological processes. Nevertheless, we are at a very early stage of defining their importance in the bacterial cell. In Escherichia coli, two BY-kinases, Wzc and Etk, have been characterized biochemically. Wzc has been shown to phosphorylate the UDP-glucose dehydrogenase Ugd in vitro. Not only is Ugd involved in the biosynthesis of extracellular polysaccharides, but also in the production of UDP-4-amino-4-deoxy-L-arabinose, a compound that renders E. coli resistant to cationic antimicrobial peptides. METHODOLOGY/PRINCIPAL FINDINGS:Here, we studied the role of Ugd phosphorylation. We first confirmed in vivo the phosphorylation of Ugd by Wzc and we demonstrated that Ugd is also phosphorylated by Etk, the other BY-kinase identified in E. coli. Tyrosine 71 (Tyr71) was characterized as the Ugd site phosphorylated by both Wzc and Etk. The regulatory role of Tyr71 phosphorylation on Ugd activity was then assessed and Tyr71 mutation was found to prevent Ugd activation by phosphorylation. Further, Ugd phosphorylation by Wzc or Etk was shown to serve distinct physiological purposes. Phosphorylation of Ugd by Wzc was found to participate in the regulation of the amount of the exopolysaccharide colanic acid, whereas Etk-mediated Ugd phosphorylation appeared to participate in the resistance of E. coli to the antibiotic polymyxin. CONCLUSIONS/SIGNIFICANCE:Ugd phosphorylation seems to be at the junction between two distinct biosynthetic pathways, illustrating the regulatory potential of tyrosine phosphorylation in bacterial physiology

A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling

Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling