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

Clubroot resistance gene Rcr6 in Brassica nigra resides in a genomic region homologous to chromosome A08 in B. rapa

Background: Clubroot, caused by Plasmodiophora brassicae Woronin, is a very important disease of Brassica species. Management of clubroot relies heavily on genetic resistance. In a cross of Brassica nigra lines PI 219576 (highly resistant, R) × CR2748 (highly susceptible, S) to clubroot, all F1 plants were resistant to clubroot. There was a 1:1 ratio of R:S in the BC1 and 3R:1S in the F2, which indicated that a single dominant gene controlled clubroot resistance in PI 219576. This gene was designated Rcr6. Mapping of Rcr6 was performed using genome sequencing information from A-genome of B. rapa and B-genome of B. nigra though bulked segregant RNA sequencing (BSR-Seq) and further mapping with Kompetitive Allele Specific PCR (KASP) analysis. Results: Reads of R and S bulks from BSR-Seq were initially aligned onto B. rapa (A-genome; B. nigra has the B-genome) where Rcr6 was associated with chromosome A08. KASP analysis showed that Rcr6 was flanked by SNP markers homologous to the region of 14.8-15.4 Mb of chromosome A08. There were 190 genes annotated in this region, with five genes (Bra010552, Bra010588, Bra010589, Bra010590 and Bra010663) identified as encoding the toll-interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat (TIR-NBS-LRR; TNL) class of proteins. The reads from BSR-Seq were then aligned into a draft B-genome of B. nigra, where Rcr6 was mapped on chromosome B3. KASP analysis indicated that Rcr6 was located on chromosome B3 in a 0.5 Mb region from 6.1-6.6 Mb. Only one TNL gene homologous to the B. rapa gene Bra010663 was identified in the target region. This gene is a likely candidate for Rcr6. Subsequent analysis of the Rcr6 equivalent region based on a published B. nigra genome was performed. This gene is located into chromosome B7 of the published B-genome, homologous to BniB015819. Conclusion: Rcr6 was the first gene identified and mapped in the B-genome of Brassica species. It resides in a genomic region homologous to chromosome A08 of A-genome. Based on this finding, it could possibly integrate into A08 of B. napus using marker assisted selection with SNP markers tightly linked to Rcr6 developed in this study

Use of cultivar resistance and crop rotation with Bacillus subtilis for clubroot control in canola

Non-Peer ReviewedThis study was conducted to assess additional strategies potentially complimentary to cultivar resistance or biocontrol in control of clubroot. New granular Bacillus subtilis formulations and a seed dressing method were developed to facilitate biofungicide delivery in field trials. The granular formulations were applied in furrow during seeding at 50 kg/ha to a clubroot resistant (CR) and susceptible (CS) canola cultivar, respectively, in three field trials. The seed dressing applied approximately 1×105 to 5×106 cfu/seed doses of the biocontrol agent, and was evaluated on the CS cultivar seeded to different crop-rotation scenarios where the plots had a 1-year, 3-year, or 11-year break from last canola crop. Clubroot disease pressure was high at all trial sites with disease severity indexes (DSI) ranging from 69% to 98% on the CS cultivar. None of the granular formulations reduced clubroot substantially, whereas the CR cultivar showed a high effect, reducing DSI to below 15% and doubling the yield over that of CS cultivar. Plots of varying rotation showed a pattern of clubroot pathogen pressure, with those of 1-year break from canola being the highest. The DSI for all rotational scenarios was high, reaching 100% in short-rotation plots. Biofungicide seed dressing did not reduce DSI, but longer crop rotation often reduced gall size slightly, showed much milder above-ground damage, and increased the yield significantly relative to short rotation in two separate trials. Even a 3-year break from canola was highly beneficial, with the yield doubled as opposed to that with only 1-year break from canola

ОЦЕНКА СОДЕРЖАНИЯ ГУМУСА В ПОЧВАХ РЕКУЛЬТИВИРОВАННЫХ ОТВАЛОВ УГОЛЬНЫХ РАЗРЕЗОВ КУЗБАССА

On the basis of the approach suggested by the authors and resting on functional characteristics of pedogenic organic substance the content of humus is determined in soil heaps of coal-mining sections reclaimed following different technologies. It is shown that the distribution of nitrogen-containing organic substances across the profile reflects the specificity of organic substances transformation systems in the soils examined. This index is the most reliable in estimating the content of humus in initial embryoearths where nitrogen consumption by plants is minimum and in humus-accumulative ones where pedogenic organic substance is deposited. The humus content increase is revealed in the evolutionary series of soils, on average, from 2.4% in the initial soils to 3.6 and 4.2% in organ-accumulative and cespitose soils, respectively, the 4.7% increase being marked in humus-accumulative embryoearths. The content of humus in embryoearths is identified to depend much more on reclamation technology applied and soil formation stage, the same content depends much less on organic substances system status inherited from soil formation rocks.На основе предложенного авторами подхода, опирающегося на функциональные особенности педогенного органического вещества, определено содержание гумуса в почвах отвалов каменноугольных разрезов, рекультивированных по различным технологиям. Показано, что специфику трансформации систем органических веществ в исследуемых почвах отражает распределение по профилю азотсодержащих органических веществ. Этот показатель наиболее достоверен при оценке содержания гумуса в инициальных эмбриоземах, где потребление азота растениями минимально, и в гумусово-аккумулятивных, где происходит депонирование педогенного органического вещества. Выявлено увеличение содержания гумуса в эволюционном ряду почв в среднем от 2,4% в инициальных до 3,6 и 4,2% соответственно в органо-аккумулятивных и дерновых и более 4,7% в гумусово-аккумулятивных эмбриоземах. Установлено, что содержание гумуса в эмбриоземах в большей степени зависит от применяемой технологии рекультивации и стадии почвообразования и в меньшей степени – от состояния системы органических веществ, унаследованной от почвообразующих пород

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

An engineered mammalian band-pass network

Gene expression circuitries, which enable cells to detect precise levels within a morphogen concentration gradient, have a pivotal impact on biological processes such as embryonic pattern formation, paracrine and autocrine signalling, and cellular migration. We present the rational synthesis of a synthetic genetic circuit exhibiting band-pass detection characteristics. The components, involving multiply linked mammalian trans-activator and -repressor control systems, were selected and fine-tuned to enable the detection of ‘low-threshold’ morphogen (tetracycline) concentrations, in which target gene expression was triggered, and a ‘high-threshold’ concentration, in which expression was muted. In silico predictions and supporting experimental findings indicated that the key criterion for functional band-pass detection was the matching of componentry that enabled sufficient separation of the low and high threshold points. Using the circuitry together with a fluorescence-encoded target gene, mammalian cells were genetically engineered to be capable of forming a band-like pattern of differentiation in response to a tetracycline chemical gradient. Synthetic gene networks designed to emulate naturally occurring gene behaviours provide not only insight into biological processes, but may also foster progress in future tissue engineering, gene therapy and biosensing applications

Toxin–antitoxin based transgene expression in mammalian cells

Long-term, recombinant gene expression in mammalian cells depends on the nature of the transgene integration site and its inherent properties to modulate transcription (epigenetic effects). Here we describe a method by which high transgene expression is achieved and stabilized in extensively proliferating cultures. The method is based on strict co-expression of the transgene with an antitoxin in cells that express the respective toxin. Since the strength of antitoxin expression correlates with an advantage for cell growth, the cells with strong antitoxin expression are enriched over time in cultures of heterogeneous cells. This principle was applied to CHO cell lines that conditionally express the toxin kid and that are transduced to co-express the antitoxin kis together with different transgenes of interest. Cultivation of pools of transfectants that express the toxin steadily increase their transgene expression within several weeks to reach a maximum that is up to 120-fold over the initial status. In contrast, average transgene expression drops in the absence of toxin expression. Together, we show that cells conditionally expressing kid can be employed to create overexpressing cells by a simple coupling of kis to the transgene of interest, without further manipulation and in absence of selectable drugs

An Inducible and Reversible Mouse Genetic Rescue System

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications

Conditional mouse models demonstrate oncogene-dependent differences in tumor maintenance and recurrence

Diversity in the pathophysiology of breast cancer frustrates therapeutic progress. We need to understand how mechanisms activated by specific combinations of oncogenes, tumor suppressors, and hormonal signaling pathways govern response to therapy and prognosis. A recent series of investigations conducted by Chodosh and colleagues offers new insights into the similarities and differences between specific oncogenic pathways. Expression of three oncogenes relevant to pathways activated in human breast cancers (c-myc, activated neu and Wnt1) were targeted to murine mammary epithelial cells using the same transgenic tetracycline-responsive conditional gene expression system. While the individual transgenic lines demonstrate similarly high rates of tumor penetrance, rates of oncogene-independent tumor maintenance and recurrence following initial regression are significantly different, and are modifiable by mutations in specific cooperating oncogenes or loss of tumor suppressor gene expression. The experiments make three notable contributions. First, they illustrate that rates of tumor regression and recurrence following initial regression are dependent upon the pathways activated by the initiating oncogene. The experiments also demonstrate that altered expression or mutation of specific cooperating oncogenes or tumor suppressor genes results in different rates of tumor regression and recurrence. Finally, they exemplify the power of conditional mouse models for elucidating how specific molecular mechanisms give rise to the complexity of human cancer