FISH mapping and molecular organization of the major repetitive sequences of tomato

This paper presents a bird's-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA](4), a microsatellite that also forms part of the pericentromeres, together with [GA](8), [GATA](4) and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer regio

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene NDC80 during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in other yeast strains and growth conditions

Trick or Heat? Manipulating Critical Temperature-Based Control Systems Using Rectification Attacks

Temperature sensing and control systems are widely used in the closed-loop control of critical processes such as maintaining the thermal stability of patients, or in alarm systems for detecting temperature-related hazards. However, the security of these systems has yet to be completely explored, leaving potential attack surfaces that can be exploited to take control over critical systems. In this paper we investigate the reliability of temperature-based control systems from a security and safety perspective. We show how unexpected consequences and safety risks can be induced by physical-level attacks on analog temperature sensing components. For instance, we demonstrate that an adversary could remotely manipulate the temperature sensor measurements of an infant incubator to cause potential safety issues, without tampering with the victim system or triggering automatic temperature alarms. This attack exploits the unintended rectification effect that can be induced in operational and instrumentation amplifiers to control the sensor output, tricking the internal control loop of the victim system to heat up or cool down. Furthermore, we show how the exploit of this hardware-level vulnerability could affect different classes of analog sensors that share similar signal conditioning processes. Our experimental results indicate that conventional defenses commonly deployed in these systems are not sufficient to mitigate the threat, so we propose a prototype design of a low-cost anomaly detector for critical applications to ensure the integrity of temperature sensor signals.Comment: Accepted at the ACM Conference on Computer and Communications Security (CCS), 201

Molecular cytogenetic mapping of Cucumis sativus and C. melo using highly repetitive DNA sequences

Chromosomes often serve as one of the most important molecular aspects of studying the evolution of species. Indeed, most of the crucial mutations that led to differentiation of species during the evolution have occurred at the chromosomal level. Furthermore, the analysis of pachytene chromosomes appears to be an invaluable tool for the study of evolution due to its effectiveness in chromosome identification and precise physical gene mapping. By applying fluorescence in situ hybridization of 45S rDNA and CsCent1 probes to cucumber pachytene chromosomes, here, we demonstrate that cucumber chromosomes 1 and 2 may have evolved from fusions of ancestral karyotype with chromosome number n= 12. This conclusion is further supported by the centromeric sequence similarity between cucumber and melon, which suggests that these sequences evolved from a common ancestor. It may be after or during speciation that these sequences were specifically amplified, after which they diverged and specific sequence variants were homogenized. Additionally, a structural change on the centromeric region of cucumber chromosome 4 was revealed by fiber-FISH using the mitochondrial-related repetitive sequences, BAC-E38 and CsCent1. These showed the former sequences being integrated into the latter in multiple regions. The data presented here are useful resources for comparative genomics and cytogenetics of Cucumis and, in particular, the ongoing genome sequencing project of cucumbe

Applying watershed algorithms to the segmentation of clustered nuclei

Cluster division is a critical issue in fluor escence micr oscopy-based analytical cytology when preparation protocols do not provide appropriate separation of objects. Overlooking cluster ed nuclei and analyzing only isolated nuclei may dramatically incr ease analysis time or af fect the statistical validation of the r esults. Automatic segmentation of cluster ed nuclei r equir es the implementation of specific image segmentation tools. Most algorithms are inspired by one of the two following strategies: 1) cluster division by the detection of inter nuclei gradients; or 2) division by definition of domains of influence (geometrical approach). Both strategies lead to completely different implementations, and usually algorithms based on a single view strategy fail to corr ectly segment most cluster ed nuclei, or per for m well just for a specific type of sample. An algorithm based on morphological watersheds has been implemented and tested on the segmentation of micr oscopic nuclei clusters. This algorithm pr ovides a tool that can be used for the implementation of both gradient- and domain-based algorithms, and, mor e importantly, for the implementation of mixed (gradient- and shape-based) algorithms. Using this algorithm, almost 90% of the test clusters wer e corr ectly segmented in peripheral blood and bone marr ow pr eparations. The algorithm was valid for both types of samples, using the appr opriate markers and transfor mations.Contract grant sponsor: ARCADIM Project; Contract grant number: CICYT TIC92-0922-C02-01 (Comisión Interministerial de Ciencia y Tecnología); Contract grant sponsor: European Concerted Action CA-AMCA; Contract grant number: BMH1-CT92-1307; Contract grant sponsor: Comunidad Autónoma de Madrid (CAM); Contract grant sponsor: Universidad Politécnica de Madrid (UPM).Publicad

Stomagen positively regulates stomatal density in Arabidopsis.

葉の気孔の数を増加させる因子の発見～CO2削減や食糧増産へ向けて～. 京都大学プレスリリース. 2009-12-10.Stomata in the epidermal tissues of leaves are valves through which passes CO(2), and as such they influence the global carbon cycle. The two-dimensional pattern and density of stomata in the leaf epidermis are genetically and environmentally regulated to optimize gas exchange. Two putative intercellular signalling factors, EPF1 and EPF2, function as negative regulators of stomatal development in Arabidopsis, possibly by interacting with the receptor-like protein TMM. One or more positive intercellular signalling factors are assumed to be involved in stomatal development, but their identities are unknown. Here we show that a novel secretory peptide, which we designate as stomagen, is a positive intercellular signalling factor that is conserved among vascular plants. Stomagen is a 45-amino--rich peptide that is generated from a 102-amino-acid precursor protein designated as STOMAGEN. Both an in planta analysis and a semi-in-vitro analysis with recombinant and chemically synthesized stomagen peptides showed that stomagen has stomata-inducing activity in a dose-dependent manner. A genetic analysis showed that TMM is epistatic to STOMAGEN (At4g12970), suggesting that stomatal development is finely regulated by competitive binding of positive and negative regulators to the same receptor. Notably, STOMAGEN is expressed in inner tissues (the mesophyll) of immature leaves but not in the epidermal tissues where stomata develop. This study provides evidence of a mesophyll-derived positive regulator of stomatal density. Our findings provide a conceptual advancement in understanding stomatal development: inner photosynthetic tissues optimize their function by regulating stomatal density in the epidermis for efficient uptake of CO(2)

Progranulin contributes to endogenous mechanisms of pain defense after nerve injury in mice

Progranulin haploinsufficiency is associated with frontotemporal dementia in humans. Deficiency of progranulin led to exaggerated inflammation and premature aging in mice. The role of progranulin in adaptations to nerve injury and neuropathic pain are still unknown. Here we found that progranulin is up-regulated after injury of the sciatic nerve in the mouse ipsilateral dorsal root ganglia and spinal cord, most prominently in the microglia surrounding injured motor neurons. Progranulin knockdown by continuous intrathecal spinal delivery of small interfering RNA after sciatic nerve injury intensified neuropathic pain-like behaviour and delayed the recovery of motor functions. Compared to wild-type mice, progranulin-deficient mice developed more intense nociceptive hypersensitivity after nerve injury. The differences escalated with aging. Knockdown of progranulin reduced the survival of dissociated primary neurons and neurite outgrowth, whereas addition of recombinant progranulin rescued primary dorsal root ganglia neurons from cell death induced by nerve growth factor withdrawal. Thus, up-regulation of progranulin after neuronal injury may reduce neuropathic pain and help motor function recovery, at least in part, by promoting survival of injured neurons and supporting regrowth. A deficiency in this mechanism may increase the risk for injury-associated chronic pain

Expansion microscopy of C. elegans.

Funder: John DoerrFunder: The Open Philanthropy ProjectFunder: Lisa YangWe recently developed expansion microscopy (ExM), which achieves nanoscale-precise imaging of specimens at ~70 nm resolution (with ~4.5x linear expansion) by isotropic swelling of chemically processed, hydrogel-embedded tissue. ExM of C. elegans is challenged by its cuticle, which is stiff and impermeable to antibodies. Here we present a strategy, expansion of C. elegans (ExCel), to expand fixed, intact C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical structures at resolutions of ~65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins after 20x linear expansion. We demonstrate the utility of the ExCel toolbox for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal