Cytosolic Crowding Drives the Dynamics of Both Genome and Cytosol in Escherichia coli Challenged with Sub-lethal Antibiotic Treatments.

In contrast to their molecular mode of action, the system-level effect of antibiotics on cells is only beginning to be quantified. Molecular crowding is expected to be a relevant global regulator, which we explore here through the dynamic response phenotypes in Escherichia coli, at single-cell resolution, under sub-lethal regimes of different classes of clinically relevant antibiotics, acting at very different levels in the cell. We measure chromosomal mobility through tracking of fast (<15 s timescale) fluctuations of fluorescently tagged chromosomal loci, and we probe the fluidity of the cytoplasm by tracking cytosolic aggregates. Measuring cellular density, we show how the overall levels of macromolecular crowding affect both quantities, regardless of antibiotic-specific effects. The dominant trend is a strong correlation between the effects in different parts of the chromosome and between the chromosome and cytosol, supporting the concept of an overall global role of molecular crowding in cellular physiology.UKRI grant EP/T002778/

Advancing the Development of the Magneto-Active Slosh Control (MaSC) System for Spacecraft and Launch Vehicles

The Magneto-Active Propellant Management Device (MAPMD) system is designed to address safety hazards in liquid-propellant spaceflight caused by sloshing. This innovative system of Magneto-Active Slosh Control surpasses traditional passive slosh baffles by reducing mass, improving surface wave suppression, and minimizing volumetric intrusion (Santhanam 2012). In prior fight experiments conducted in collaboration between Embry-Riddle Aeronautical University and Carthage College, remnant slosh suppression was observed, however the effective slosh damping did not meet our expectations due to inadequate control forces. We are redesigning the magnetic membrane with multiple layers of ultrahigh-permeability metallic glass film and are developing an optimized configuration of current-carrying coils to increase magnetic force and field performance. These advancements are expected to elevate the MAPMD system\u27s Technology Readiness Level (TRL) from 3 to 4 in order to pave the way for microgravity flight testing. The MAPMD system promises to enhance the safety and performance of liquid-propellant spaceflight by actively managing slosh dynamics

Dynamics of E. coli genome and cytosol under antibiotics

In light of an urgent need for improved antimicrobial diagnostics and therapeutics, understanding bacterial behaviour, and bacterial responses to treatments in particular, is one of the key objectives of modern medical research. While the molecular mode of action of antibiotics is usually well known, their effect on the cell at a "systems" level (on the regulatory networks, metabolism, etc.) is only beginning to be quantitatively understood. We address some of these response phenotypes in Escherichia coli testing different antibiotic classes and growth conditions. We study the short (<15 s) time-scale fluctuation dynamics of fluorescently-tagged chromosomal loci and cytosolic aggregates, which report for the state of locus ”compaction” and the levels of macromolecular crowding of the cytosol, respectively. We improve the precision of those measurements developing a novel data treatment procedure and discover that sub-lethal doses of ciprofloxacin, rifampicin, and vancomycin as well as hyperosmotic shock conditions cause small but consistent changes (unique to each treatment agent) to the physical organisation of chromosomal Ori2 and Ter3 loci and the cytosol. We reveal, among other findings, strong correlations between the effects in different parts of the chromosome and between the chromosome and cytosol. In addition, we complement the marker dynamics work with single-cell level gene expression measurements during sub-lethal translation inhibition. Specifically, we compare responses to tetracycline and chloramphenicol from constitutive and ribosomal promoters in Ori3 and Ter3 chromosomal positions over long (7 h) treatment times in exponentially growing bacteria. We reveal, for the first time, the kinetics of cellular resource allocation and provide novel insights on globally regulated transcription, relevant to the three-component proteome partitioning model, gene-length dependent effects of the processivity of translation, and ”reversibility” of ribosome-binding antibiotics. In addition, we discover a strong correlation between the timing of responses from promoters in the Ori3 and Ter3 positions, and a small but consistent difference in the response magnitude between the two positions.BBSR

Mantle cell lymphoma cells express predominantly cyclin D1a isoform and are highly sensitive to selective inhibition of CDK4 kinase activity

The prognosis for patients with mantle cell lymphoma (MCL) is poor, and at present there is no truly effective therapy. Gene translocation-mediated constitutive expression of cyclin D1 seems to play the key role in the pathogenesis of MCL. Here we report that although 3 of 4 MCL cell lines expressed the recently identified, highly oncogenic cyclin D1b isoform, as well as the canonical cyclin D1a, 8 MCL patient samples expressed only the cyclin D1a protein despite expressing detectable cyclin D1b mRNA. Cell lines and tissue samples displayed constitutive activation of the cyclin D1 signaling cascade, as evidenced by strong expression of CDK4, Rb phosphorylation, and cyclin D1/CDK4 coassociation. All MCL cell lines and tissues examined displayed nondetectable to diminished expression of the cyclin D1 inhibitor p16. Novel small molecule CDK4/CDK6 inhibitor PD0332991 profoundly suppressed—at low nanomolar concentrations—Rb phosphorylation, proliferation, and cell cycle progression at the G0/G1 phase of MCL cells. These findings provide evidence that MCL should be very sensitive to targeted therapy aimed at functional inhibition of the cyclin D1/CDK4 complex

Expression and Oncogenic Role of Brk (PTK6/Sik) Protein Tyrosine Kinase in Lymphocytes

Tyrosine kinases play a fundamental role in cell proliferation, survival, adhesion, and motility and have also been shown to mediate malignant cell transformation. Here we describe constitutive expression of the protein tyrosine kinase Brk in a large proportion of cutaneous T-cell lymphomas and other transformed T- and B-cell populations. The kinase is expressed in the nuclear localization and activated state. Brk expression was also induced in normal T cells on their activation. Introduced expression of the Brk gene resulted in markedly diminished cytokine and growth factor dependence of transfected BaF3 lymphocytes in regard to their in vitro proliferation and survival. Brk also conferred in vivo oncogenicity on the BaF3 cells. siRNA-mediated inhibition of the endogenous Brk in malignant T cells diminished their growth and survival capacity. These findings document inducible expression of Brk in normal T lymphocytes and persistent expression of the activated kinase in malignant T and B cells. Furthermore, our results indicate that Brk may play a key role in lymphomagenesis, hence identifying the kinase as a potential therapeutic target in lymphomas

Self-Terminating Confinement Approach for Large-Area Uniform Monolayer Graphene Directly over Si/SiO_x by Chemical Vapor Deposition

To synthesize graphene by chemical vapor deposition (CVD) both in large area and with uniform layer number directly over Si/SiO_x has proven challenging. The use of catalytically active metal substrates, in particular Cu, has shown far greater success and therefore is popular. That said, for electronics applications it requires a transfer procedure, which tends to damage and contaminate the graphene. Thus, the direct fabrication of uniform graphene on Si/SiO_x remains attractive. Here we show a facile confinement CVD approach in which we simply “sandwich” two Si wafers with their oxide faces in contact to form uniform monolayer graphene. A thorough examination of the material reveals it comprises faceted grains despite initially nucleating as round islands. Upon clustering, they facet to minimize their energy. This behavior leads to faceting in polygons, as the system aims to ideally form hexagons, the lowest energy form, much like the hexagonal cells in a beehive, which requires the minimum wax. This process also leads to a near minimal total grain boundary length per unit area. This fact, along with the high graphene quality, is reflected in its electrical performance, which is highly comparable with graphene formed over other substrates, including Cu. In addition, the graphene growth is self-terminating. Our CVD approach is easily scalable and will make graphene formation directly on Si wafers competitive against that from metal substrates, which suffer from transfer. Moreover, this CVD route should be applicable for the direct synthesis of other 2D materials and their van der Waals heterostructures