Osmotic pressure modulates single cell cycle dynamics inducing reversible growth arrest and reactivation of human metastatic cells

Biophysical cues such as osmotic pressure modulate proliferation and growth arrest of bacteria, yeast cells and seeds. In tissues, osmotic regulation takes place through blood and lymphatic capillaries and, at a single cell level, water and osmoregulation play a critical role. However, the effect of osmotic pressure on single cell cycle dynamics remains poorly understood. Here, we investigate the effect of osmotic pressure on single cell cycle dynamics, nuclear growth, proliferation, migration and protein expression, by quantitative time-lapse imaging of single cells genetically modified with fluorescent ubiquitination-based cell cycle indicator 2 (FUCCI2). Single cell data reveals that under hyperosmotic stress, distinct cell subpopulations emerge with impaired nuclear growth, delayed or growth arrested cell cycle and reduced migration. This state is reversible for mild hyperosmotic stress, where cells return to regular cell cycle dynamics, proliferation and migration. Thus, osmotic pressure can modulate the reversible growth arrest and reactivation of human metastatic cells

Variability of Optical Counterparts in the Chandra Galactic Bulge Survey

We present optical lightcurves of variable stars consistent with the positions of X-ray sources identified with the Chandra X-ray Observatory for the Chandra Galactic Bulge Survey. Using data from the Mosaic-II instrument on the Blanco 4m Telescope at CTIO, we gathered time-resolved photometric data on timescales from $\sim2$ hr to 8 days over the $\frac{3}{4}$ of the X-ray survey containing sources from the initial GBS catalog. Among the lightcurve morphologies we identify are flickering in interacting binaries, eclipsing sources, dwarf nova outbursts, ellipsoidal variations, long period variables, spotted stars, and flare stars. $87\%$ of X-ray sources have at least one potential optical counterpart. $24\%$ of these candidate counterparts are detectably variable; a much greater fraction than expected for randomly selected field stars, which suggests that most of these variables are real counterparts. We discuss individual sources of interest, provide variability information on candidate counterparts, and discuss the characteristics of the variable population.Comment: Accepted for publication in the Astrophysical Journal Supplement

Gateway vectors for efficient artificial gene assembly in vitro and expression in yeast Saccharomyces cerevisiae

Peer reviewedPublisher PD

Abrogated cryptic activation of lentiviral transfer vectors

Despite significant improvements in lentivirus (LV) vector-based gene therapy there are still several safety risks using LV vectors including the potential formation of replication-competent LV particles. To address this shortcoming, we constructed a novel and safer gene transfer system using modified SIN-based LV gene transfer vectors. Central to our approach is a conditional deletion of the Ψ packaging signal after integration in the target genome. Here we demonstrate that after transduction of target cells, conventional SIN-based LV transfer vectors can still be mobilized. However mobilization is rendered undetectable if transductions are followed by a Cre/loxP-mediated excision of Ψ. Thus conditional elimination of the packaging signal may represent another advance in increasing the safety of LV vectors for gene therapeutic treatment of chronic diseases

Functional Fluorescent Ca(2+) Indicator Proteins in Transgenic Mice under TET Control

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2

Transplantation of canine olfactory ensheathing cells producing chondroitinase ABC promotes chondroitin sulphate proteoglycan digestion and axonal sprouting following spinal cord injury

Olfactory ensheathing cell (OEC) transplantation is a promising strategy for treating spinal cord injury (SCI), as has been demonstrated in experimental SCI models and naturally occurring SCI in dogs. However, the presence of chondroitin sulphate proteoglycans within the extracellular matrix of the glial scar can inhibit efficient axonal repair and limit the therapeutic potential of OECs. Here we have used lentiviral vectors to genetically modify canine OECs to continuously deliver mammalian chondroitinase ABC at the lesion site in order to degrade the inhibitory chondroitin sulphate proteoglycans in a rodent model of spinal cord injury. We demonstrate that these chondroitinase producing canine OECs survived at 4 weeks following transplantation into the spinal cord lesion and effectively digested chondroitin sulphate proteoglycans at the site of injury. There was evidence of sprouting within the corticospinal tract rostral to the lesion and an increase in the number of corticospinal axons caudal to the lesion, suggestive of axonal regeneration. Our results indicate that delivery of the chondroitinase enzyme can be achieved with the genetically modified OECs to increase axon growth following SCI. The combination of these two promising approaches is a potential strategy for promoting neural regeneration following SCI in veterinary practice and human patients

Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (Ptets). We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility

Separating Lentiviral Vector Injection and Induction of Gene Expression in Time, Does Not Prevent an Immune Response to rtTA in Rats

BACKGROUND: Lentiviral gene transfer can provide long-term expression of therapeutic genes such as erythropoietin. Because overexpression of erythropoietin can be toxic, regulated expression is needed. Doxycycline inducible vectors can regulate expression of therapeutic transgenes efficiently. However, because they express an immunogenic transactivator (rtTA), their utility for gene therapy is limited. In addition to immunogenic proteins that are expressed from inducible vectors, injection of the vector itself is likely to elicit an immune response because viral capsid proteins will induce "danger signals" that trigger an innate response and recruit inflammatory cells. METHODOLOGY AND PRINCIPAL FINDINGS: We have developed an autoregulatory lentiviral vector in which basal expression of rtTA is very low. This enabled us to temporally separate the injection of virus and the expression of the therapeutic gene and rtTA. Wistar rats were injected with an autoregulatory rat erythropoietin expression vector. Two or six weeks after injection, erythropoietin expression was induced by doxycycline. This resulted in an increase of the hematocrit, irrespective of the timing of the induction. However, most rats only responded once to doxycycline administration. Antibodies against rtTA were detected in the early and late induction groups. CONCLUSIONS: Our results suggest that, even when viral vector capsid proteins have disappeared, expression of foreign proteins in muscle will lead to an immune respons

Construction and Modelling of an Inducible Positive Feedback Loop Stably Integrated in a Mammalian Cell-Line

Understanding the relationship between topology and dynamics of transcriptional regulatory networks in mammalian cells is essential to elucidate the biology of complex regulatory and signaling pathways. Here, we characterised, via a synthetic biology approach, a transcriptional positive feedback loop (PFL) by generating a clonal population of mammalian cells (CHO) carrying a stable integration of the construct. The PFL network consists of the Tetracycline-controlled transactivator (tTA), whose expression is regulated by a tTA responsive promoter (CMV-TET), thus giving rise to a positive feedback. The same CMV-TET promoter drives also the expression of a destabilised yellow fluorescent protein (d2EYFP), thus the dynamic behaviour can be followed by time-lapse microscopy. The PFL network was compared to an engineered version of the network lacking the positive feedback loop (NOPFL), by expressing the tTA mRNA from a constitutive promoter. Doxycycline was used to repress tTA activation (switch off), and the resulting changes in fluorescence intensity for both the PFL and NOPFL networks were followed for up to 43 h. We observed a striking difference in the dynamics of the PFL and NOPFL networks. Using non-linear dynamical models, able to recapitulate experimental observations, we demonstrated a link between network topology and network dynamics. Namely, transcriptional positive autoregulation can significantly slow down the “switch off” times, as comparared to the nonautoregulatated system. Doxycycline concentration can modulate the response times of the PFL, whereas the NOPFL always switches off with the same dynamics. Moreover, the PFL can exhibit bistability for a range of Doxycycline concentrations. Since the PFL motif is often found in naturally occurring transcriptional and signaling pathways, we believe our work can be instrumental to characterise their behaviour