Analysis of Contractility and Invasion Potential of Two Canine Mammary Tumor Cell Lines

Peer reviewe

Structure based de novo design of IspD inhibitors as anti-tubercular agents

Tuberculosis is one of the leading contagious diseases, caused by Mycobacterium tuberculosis. Despite improvements in anti-tubercular agents, it remains one of the most prevalent infectious diseases worldwide, responsible for a total of 1.6 million deaths annually. The emergence of multidrug resistant strains highlighted the need of discovering novel drug targets for the development of anti-tubercular agents. 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) is an enzyme involved in MEP pathway for isoprenoid biosynthesis, which is considered an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. In the present study, we have employed structure based drug design approach to develop novel and potent inhibitors for IspD receptor. To explore binding affinity and hydrogen bond interaction between the ligand and active site of IspD receptor, docking studies were performed. ADMET and synthetic accessibility filters were used to screen designed molecules. Finally, ten compounds were selected and subsequently submitted for the synthesis and in vitro studies as IspD inhibitors

Molecular Rationale behind the Differential Substrate Specificity of Bacterial RND Multi-Drug Transporters

Resistance-Nodulation-cell Division (RND) transporters AcrB and AcrD of Escherichia coli expel a wide range of substrates out of the cell in conjunction with AcrA and TolC, contributing to the onset of bacterial multidrug resistance. Despite sharing an overall sequence identity of ~66% (similarity ~80%), these RND transporters feature distinct substrate specificity patterns whose underlying basis remains elusive. We performed exhaustive comparative analyses of the putative substrate binding pockets considering crystal structures, homology models and conformations extracted from multi-copy μs-long molecular dynamics simulations of both AcrB and AcrD. The impact of physicochemical and topographical properties (volume, shape, lipophilicity, electrostatic potential, hydration and distribution of multi-functional sites) within the pockets on their substrate specificities was quantitatively assessed. Differences in the lipophilic and electrostatic potentials among the pockets were identified. In particular, the deep pocket of AcrB showed the largest lipophilicity convincingly pointing out its possible role as a lipophilicity-based selectivity filter. Furthermore, we identified dynamic features (not inferable from sequence analysis or static structures) such as different flexibilities of specific protein loops that could potentially influence the substrate recognition and transport profile. Our findings can be valuable for drawing structure (dynamics)-activity relationship to be employed in drug design

Dynamics of Cell Area and Force during Spreading

AbstractExperiments on human pulmonary artery endothelial cells are presented to show that cell area and the force exerted on a substrate increase simultaneously, but with different rates during spreading; rapid-force increase systematically occurred several minutes past initial spreading. We examine this theoretically and present three complementary mechanisms that may accompany the development of lamellar stress during spreading and underlie the observed behavior. These include: 1), the dynamics of cytoskeleton assembly at the cell basis; 2), the strengthening of acto-myosin forces in response to the generated lamellar stresses; and 3), the passive strain-stiffening of the cytoskeleton

Computer simulations of the activity of RND efflux pumps

Abstract The putative mechanism by which bacterial RND-type multidrug efflux pumps recognize and transport their substrates is a complex and fascinating enigma of structural biology. How a single protein can recognize a huge number of unrelated compounds and transport them through one or just a few mechanisms is an amazing feature not yet completely unveiled. The appearance of cooperativity further complicates the understanding of structure-dynamics-activity relationships in these complex machineries. Experimental techniques may have limited access to the molecular determinants and to the energetics of key processes regulating the activity of these pumps. Computer simulations are a complementary approach that can help unveil these features and inspire new experiments. Here we review recent computational studies that addressed the various molecular processes regulating the activity of RND efflux pumps

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump

Background Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically. Methods A computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process. Results We provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile. Conclusions Our findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions. General significance We provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps

Vimentin intermediate filaments control actin stress fiber assembly through GEF-H1 and RhoA

The actin and intermediate filament cytoskeletons contribute to numerous cellular processes, including morphogenesis, cytokinesis and migration. These two cytoskeletal systems associate with each other, but the underlying mechanisms of this interaction are incompletely understood. Here, we show that inactivation of vimentin leads to increased actin stress fiber assembly and contractility, and consequent elevation of myosin light chain phosphorylation and stabilization of tropomyosin-4.2 (see Geeves et al., 2015). The vimentin-knockout phenotypes can be rescued by re-expression of wild-type vimentin, but not by the non-filamentous ` unit length form' vimentin, demonstrating that intact vimentin intermediate filaments are required to facilitate the effects on the actin cytoskeleton. Finally, we provide evidence that the effects of vimentin on stress fibers are mediated by activation of RhoA through its guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2). Vimentin depletion induces phosphorylation of the microtubule-associated GEF-H1 on Ser886, and thereby promotes RhoA activity and actin stress fiber assembly. Taken together, these data reveal a new mechanism by which intermediate filaments regulate contractile actomyosin bundles, and may explain why elevated vimentin expression levels correlate with increased migration and invasion of cancer cells.Peer reviewe

Assembly of Peripheral Actomyosin Bundles in Epithelial Cells Is Dependent on the CaMKK2/AMPK Pathway

Summary Defects in the maintenance of intercellular junctions are associated with loss of epithelial barrier function and consequent pathological conditions, including invasive cancers. Epithelial integrity is dependent on actomyosin bundles at adherens junctions, but the origin of these junctional bundles is incompletely understood. Here we show that peripheral actomyosin bundles can be generated from a specific actin stress fiber subtype, transverse arcs, through their lateral fusion at cell-cell contacts. Importantly, we find that assembly and maintenance of peripheral actomyosin bundles are dependent on the mechanosensitive CaMKK2/AMPK signaling pathway and that inhibition of this route leads to disruption of tension-maintaining actomyosin bundles and re-growth of stress fiber precursors. This results in redistribution of cellular forces, defects in monolayer integrity, and loss of epithelial identity. These data provide evidence that the mechanosensitive CaMKK2/AMPK pathway is critical for the maintenance of peripheral actomyosin bundles and thus dictates cell-cell junctions through cellular force distribution.Peer reviewe

Rehospitalization following percutaneous coronary intervention for commercially insured patients with acute coronary syndrome: a retrospective analysis

BACKGROUND: While prior research has provided important information about readmission rates following percutaneous coronary intervention, reports regarding charges and length of stay for readmission beyond 30 days post-discharge for patients in a large cohort are limited. The objective of this study was to characterize the rehospitalization of patients with acute coronary syndrome receiving percutaneous coronary intervention in a U.S. health benefit plan. METHODS: This study retrospectively analyzed administrative claims data from a large US managed care plan at index hospitalization, 30-days, and 31-days to 15-months rehospitalization. A valid Diagnosis Related Group code (version 24) associated with a PCI claim (codes 00.66, 36.0X, 929.73, 929.75, 929.78–929.82, 929.84, 929.95/6, and G0290/1) was required to be included in the study. Patients were also required to have an ACS diagnosis on the day of admission or within 30 days prior to the index PCI. ACS diagnoses were classified by the International Statistical Classification of Disease 9 (ICD-9-CM) codes 410.xx or 411.11. Patients with a history of transient ischemic attack or stroke were excluded from the study because of the focus only on ACS-PCI patients. A clopidogrel prescription claim was required within 60 days after hospitalization. RESULTS: Of the 6,687 ACS-PCI patients included in the study, 5,174 (77.4%) were male, 5,587 (83.6%) were <65 years old, 4,821 (72.1%) had hypertension, 5,176 (77.4%) had hyperlipidemia, and 1,777 (26.6%) had diabetes. At index hospitalization drug-eluting stents were the most frequently used: 5,534 (82.8%). Of the 4,384 patients who completed the 15-month follow-up, a total of 1,367 (31.2%) patients were rehospitalized for cardiovascular (CV)-related events, of which 811 (59.3%) were revascularization procedures: 13 (1.0%) for coronary artery bypass graft and 798 (58.4%) for PCI. In general, rehospitalizations associated with revascularization procedures cost more than other CV-related rehospitalizations. Patients rehospitalized for revascularization procedures had the shortest median time from post-index PCI to rehospitalization when compared to the patients who were rehospitalized for other CV-related events. CONCLUSIONS: For ACS patients who underwent PCI, revascularization procedures represented a large portion of rehospitalizations. Revascularization procedures appear to be the most frequent, most costly, and earliest cause for rehospitalization after ACS-PCI

Regulation of Mitochondrial Structure and Dynamics by the Cytoskeleton and Mechanical Factors

Mitochondria supply cells with energy in the form of ATP, guide apoptosis, and contribute to calcium buffering and reactive oxygen species production. To support these diverse functions, mitochondria form an extensive network with smaller clusters that are able to move along microtubules aided by motor proteins. Mitochondria are also associated with the actin network, which is involved in cellular responses to various mechanical factors. In this review, we discuss mitochondrial structure and function in relation to the cytoskeleton and various mechanical factors influencing cell functions. We first summarize the morphological features of mitochondria with an emphasis on fission and fusion as well as how network properties govern function. We then review the relationship between the mitochondria and the cytoskeletal structures, including mechanical interactions. We also discuss how stretch and its dynamic pattern affect mitochondrial structure and function. Finally, we present preliminary data on how extracellular matrix stiffness influences mitochondrial morphology and ATP generation. We conclude by discussing the more general role that mitochondria may play in mechanobiology and how the mechanosensitivity of mitochondria may contribute to the development of several diseases and aging