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Cotoneaster integerrimus represents a multiploid and facultative apomictic system of widely distributed mountain populations. We used flow cytometry to determine genome size, ploidy level, and reproduction mode variation of the Balkan populations, supplemented by analysis of nuclear microsatellites in order to address: (i) geographic distribution and variation of cytotypes among the populations; (ii) variation of reproduction mode and the frequency of sexuality; (iii) pathways of endosperm formation among the sampled polyploids and their endosperm balance requirements; (iv) genotypic diversity and geographic distribution of clonal lineages of polyploids. The prevalence of apomictic tetraploid cytotype followed by sexual diploids and extremely rare triploids was demonstrated. This prevalence of tetraploids affected the populations’ structure composed from clonal genotypes with varying proportions. The co-occurrence of diploids and tetraploids generated higher cytotype, reproductive mode, and genotypic diversity, but mixed-ploidy sites were extremely rare. The endosperm imbalance facilitates the development and the occurrence of intermediate triploids in mixed-ploidy populations, but also different tetraploid lineages elsewhere with unbalanced endosperm. All these results showed that the South European populations of C. integerrimus have higher levels of cytotype and reproductive diversity compared to the Central European ones. Therefore, the South European populations can be considered as a potential reservoir of regional and global diversity for this species.

Rhabdomyosarcoma (RMS) and rhabdoid tumors (RT) are rare soft-tissue malignancies with the highest incidence in infants, children, and adolescents. Advanced, recurrent, and/or metastatic RMS and RT exhibit poor response to treatment. One of the main mechanisms behind resistance to treatment is believed to be intratumoral heterogeneity. In this study, we investigated the myogenic determination factor 1 (MYOD1) and Noggin (NOG) markers in an embryonal RMS (ERMS) cell line and an RT cell line and the differential response of the MYOD1 and NOG expressing subpopulations to chemotherapy. Importantly, we found that these markers together identify a subpopulation of cells (MYOD1+ NOG+ cells) with primary resistance to Vincristine and Doxorubicin, two commonly used chemotherapies for ERMS and RT. The chemoresistant MYOD1+ NOG+ cells express markers of undifferentiated cells such as myogenin and ID1. Combination of Vincristine with TPA/GSK126, a drug combination shown to induce differentiation of RMS cell lines, is able to partially overcome MYOD1/NOG cells chemoresistance.

Flow cytometry has become the method of choice to measure the DNA content (genome size) in plants. Ease of sample preparation, fast acquisition, and accurate measurements have made the method popular in the domains of plant cell biology, systematics, evolution, genetics and biotechnology. Although the cell wall is a problem when isolating plant cells, cytometry remains a powerful tool in plant sciences. Based on our 30-years’ experience in this field, this review will focus at first on genome size measurement using simply isolated nuclei: the good practice for acquisition, nuclei isolation, appropriate buffers, kind of tissues to use. The second part will briefly review what kind of measurements it is possible to make in plant cytometry, and for what purpose: base composition, ploidy level, cell cycle, endoreplication, seed screening, and nuclei/chromosomes sorting. We will address troubleshooting. The commonly-used mathematic tools will be discussed.

To test how cerebellar crus I/II Purkinje cells and their targets in the lateral cerebellar nuclei (CbN) integrate sensory and motor-related inputs and contribute to reflexive movements, we recorded extracellularly in awake, head-fixed mice during non-contact whisking. Ipsilateral or contralateral air puffs elicited changes in population Purkinje simple spike rates that matched whisking kinematics (∼1 Hz/1° protraction). Responses remained relatively unaffected when ipsilateral sensory feedback was removed by lidocaine but were reduced by optogenetically inhibiting the reticular nuclei. Optogenetically silencing cerebellar output suppressed movements. During puff-evoked whisks, both Purkinje and CbN cells generated well-timed spikes in sequential 2- to 4-ms windows at response onset, such that they alternately elevated their firing rates just before protraction. With spontaneous whisks, which were smaller than puff-evoked whisks, well-timed spikes were absent and CbN cells were inhibited. Thus, sensory input can facilitate millisecond-scale, well-timed spiking in Purkinje and CbN cells and amplify reflexive whisker movements.

International audience; As part of normal development most eukaryotic organisms, ranging from insects and mammals to plants, display variations in nuclear ploidy levels resulting from somatic endopolyploidy. Endoreduplication is the major source of endopolyploidy in higher plants. Endoreduplication is a remarkable characteristic of the fleshy pericarp tissue of developing tomato fruits, where it establishes a highly integrated cellular system that acts as a morphogenetic factor supporting cell growth. However, the functional significance of endoreduplication is not fully understood. Although endoreduplication is thought to increase metabolic activity due to a global increase in transcription, the issue of gene-specific ploidy-regulated transcription remains open. To investigate the influence of endoreduplication on transcription in tomato fruit, we tested the feasibility of a RNA sequencing (RNA-Seq) approach using total nuclear RNA extracted from purified populations of flow cytometry-sorted nuclei based on their DNA content. Here we show that cell-based approaches to the study of RNA-Seq profiles need to take into account the putative global shift in expression between samples for correct analysis and interpretation of the data. From ploidy-specific expression profiles we found that the activity of cells inside the pericarp is related both to the ploidy level and their tissue location.

International audience; Plant response to their environment stresses is a complex mechanism involving secondary metabolites. Stilbene phytoalexins, namely resveratrol, pterostilbene, piceids and viniferins play a key role in grapevine (Vitis vinifera) leaf defense. Despite their well-established qualities, conventional analyses such as HPLC-DAD or LC-MS lose valuable information on metabolite localization during the extraction process. To overcome this issue, a correlative analysis combining mass spectroscopy imaging (MSI) and fluorescence imaging was developed to localize in situ stilbenes on the same stressed grapevine leaves. High-resolution images of the stilbene fluorescence provided by macroscopy were supplemented by specific distributions and structural information concerning resveratrol, pterostilbene, and piceids obtained by MSI. The two imaging techniques led to consistent and complementary data on the stilbene spatial distribution for the two stresses addressed: UV-C irradiation and infection by Plasmopara viticola. Results emphasize that grapevine leaves react differently depending on the stress. A rather uniform synthesis of stilbenes is induced after UV-C irradiation, whereas a more localized synthesis of stilbenes in stomata guard cells and cell walls is induced by P. viticola infection. Finally, this combined imaging approach could be extended to map phytoalexins of various plant tissues with resolution approaching the cellular level.

The report from Reeds et al represents data from a US Food and Drug Administration (FDA)–approved protocol that examined both safety and efficacy of injecting a combination of phosphatidylcholine (PC) and deoxycholate (DC) into abdominal fat. Mesotherapy, the use of multiple injections of chemical agents, pharmaceutical agents, or other ingredients into subdermal adipose tissue, is designed to ultimately generate necrosis that will lead to contour changes. Mesotherapy treatments have a long history, but with a lack of regulatory oversight and rigorous clinical protocols, the clinical outcomes have been suspect until recently.1,2 The PC-DC protocol from Reeds et al was designed to specifically test the premise that injections of PC-DC may be safe and efficacious. The article describes 7 women who received 4 treatment sessions spaced 8 weeks apart. Tissue biopsies were obtained at the control site and treated sites at 1 week after the first treatment and at the end of the study. A number of tissue inflammatory markers (interleukin-6, tumor necrosis factor–α, monocyte chemotactic protein–1) and adipocyte-related enzymatic biomarkers associated with adipose tissue metabolism (leptin, adiponectin) were measured. In general, at 1 week posttreatment, a number of biomarkers in treated sites were noted to have significantly different levels from the respective control sites; many returned to their normal levels at the end of the study. The Discussion section of the main article states that the enzymatic or protein biomarker results were associated with an implied favorable adipose tissue loss, for which there were no results shown. It is this point that requires clarification within the context of this study. The major hypothesis to be tested was the potential fat volume decrease in the treated versus control sites. I was asked to review these data on behalf of the American Society for Aesthetic Plastic Surgery (ASAPS) and presented my interpretation of the results at the annual conference. Dr Young, a senior author on the current study, had promptly provided all the data requested for this report. This FDA-approved study involved 6 women who completed the entire protocol (although 20 subjects were initially approved). Each subject received a different dose of PC-DC according to the various surface sizes of the abdominal areas that were labeled and treated. It is important to note that there were no clinical safety issues, and only anticipated adverse events were noted. At the end of the 4 injection sessions for each subject, the following summary results were noted: There were no significant differences in body mass index, skin fold thickness, fat mass, fat-free mass, percentage of body fat, visceral fat tissue, subcutaneous fat area, or adipose volume, size, diameter, or lipid content. There was 1 subset of magnetic resonance imaging data—subcutaneous fat site thicknesses—that did change over time in the treated sites (vs no change in control sites over the treatment intervals). The mean decrease in anterior fat width was 4.1 mm, and a 2.3-mm decrease was observed in the side fat width. In summary, the null hypothesis was met; there was no clinically distinguishable fat volume change in the patients who underwent hundreds of injections over a long period compared with controls. The study was challenging on many different levels. Initially, FDA approval was acquired for PC-DC injections with an approved clinical protocol. However, many of the treatment parameters (dosing levels, surface area to be treated, timing of doses, number of doses, subject selection, adipose target site selection, etc) were not well documented in terms of safety, which is the first criterion for the FDA. A more conservative treatment protocol was ultimately approved. The methodology and techniques used in this study were impressive to document adipose volume changes and adipose-related metabolic consequences at 1 week for safety indications and at the end of the study for both safety and efficacy outcome measures. The scientific rigor presented in the protocol by Reeds and his coauthors met the criteria of ASAPS and its research arm, the Aesthetic Surgery Education and Research Foundation (ASERF), for examining the safety and possible efficacy of mesotherapy using PC-DC solutions. Under these experimental conditions, the injection of PC-DC did not produce anticipated aesthetic results. This narrative does not imply that under different circumstances, PC-DC will not work. In fact, Kythera Biopharmaceuticals, Inc (Calabasas, California) has produced a first-in-class injectable drug that has undergone 4 successful clinical studies: 2 randomized, double-blind, placebo-controlled, Phase 2 studies using the injectable drug in the reduction of submental fat and 2 Phase 1 pharmacokinetic and histology studies.3 The company reports that the injectable is safe and shows efficacy in reducing localized fat deposits. The advancement of clinically translatable therapies carries risks and requires the fully informed consent of participating patients, but clinicians and patients ultimately benefit from the culmination of many studies in achieving evidence-based research milestones.

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-X L plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-X L homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal β-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.

In the lecanoid chromosomal system, as exemplified by the mealy bug, Planococcus citri (Risso), one chromosome set becomes heterochromatic during embryogeny of the male and is maintained as such during development. At spermatogenesis, the first division is equational for both the euchromatic and heterochromatic chromosomes which are segregated from each other in the second; only the euchromatic derivatives form sperm. After paternal irradiation, the induced aberrations appear in the heterochromatic set of the male embryos while they occur in the euchromatic set after maternal treatment. After paternal irradiation of doses up to 30000 rep, dominant lethality is induced in daughters but not in sons. There is thus a clear picture of the failure of damage to the heterochromatic set to produce a detectable genetic effect. These results do not rule out a secondary genetic or physiological function of the heterochromatic set. No breakage-fusion-bridge cycles, which would complicate the results whether the heterochromatic set were inert or not, were observed in the cytological studies after low-dosage treatments. After maternal treatment, both sexes diminish with increasing dosage but the results are complicated by the effect of maternal age on the sequence in which the two sexes of offspring occur during oviposition (sexualmore » dichronism) as well as on total progeny. At high-dosage paternal treatments, 60, 90, and 120,000 rep, the number of sons is drastically reduced while the number of daughters is about 40% of the controls. (P.C.H.)« less

SUMMARY In the diaspine scale, Pseudaulacaspis pentagona, the males are haploid, with eight chromosomes, and the females are diploid, with 16. Meiosis is normal in the female and replaced by a simple mitosis in the male. Mating is required for the production of any offspring, either male or female. Egg production affords an example of combined sexual dimorphism and dichronism; the mothers first produce a series of eggs containing coral-colored female embryos and then, without interruption, lay a series containing pinkish-white male embryos. Cytological study of early embryogeny shows that a chromosome set is eliminated to produce the haploid condition of the male embryos. Chromosomes broken by X-rays were used as genetic markers to demonstrate that the eliminated set is of paternal origin. Symbionts, yeast-like in appearance, are present at the distal end of both male and female embryos. The polyploid sector of the embryo stems from the fusion of a cleavage nucleus with both polar bodies and is pentaploid with 40 chromosomes. As would be expected on a simple genetic basis, irradiation of fathers preferentially reduces the number of daughters.

Purpose To describe a pilot study for a novel preclinical model used to test human tissue-based therapies in the setting of cutaneous radiation injury. Methods A protocol was designed to irradiate the skin of athymic rats while sparing the body and internal organs by utilizing a non-occlusive skin clamp along with an x-ray image guided stereotactic irradiator. Each rat was irradiated both on the right and the left flank with a circular field at a 20 cm source-to-surface distance (SSD). Single fractions of 30.4 Gy, 41.5 Gy, 52.6 Gy, 65.5 Gy, and 76.5 Gy were applied in a dose-finding trial. Eight additional wounds were created using the 41.5 Gy dose level. Each wound was photographed and the percentage of the irradiated area ulcerated at given time points was analyzed using ImageJ software. Results No systemic or lethal sequelae occurred in any animals, and all irradiated skin areas in the multi-dose trial underwent ulceration. Greater than 60% of skin within each irradiated zone underwent ulceration within ten days, with peak ulceration ranging from 62.1% to 79.8%. Peak ulceration showed a weak correlation with radiation dose (r = 0.664). Mean ulceration rate over the study period is more closely correlated to dose (r = 0.753). With the highest dose excluded due to contraction-related distortions, correlation between dose and average ulceration showed a stronger relationship (r = 0.895). Eight additional wounds created using 41.5 Gy all reached peak ulceration above 50%, with all healing significantly but incompletely by the 65-day endpoint. Conclusions We developed a functional preclinical model which is currently used to evaluate human tissue-based therapies in the setting of cutaneous radiation injury. Similar models may be widely applicable and useful the development of novel therapies which may improve radiotherapy management over a broad clinical spectrum.