Photosynthetic regulation under fluctuating light in field-grown Cerasus cerasoides: A comparison of young and mature leaves

Photosystem I (PSI) is a potential target of photoinhibition under fluctuating light. However, photosynthetic regulation under fluctuating light in field-grown plants is little known. Furthermore, it is unclear how young leaves protect PSI against fluctuating light under natural field conditions. In the present study, we examined chlorophyll fluorescence, P700 redox state and the electrochromic shift signal in the young and mature leaves of field-grown Cerasus cerasoides (Rosaceae). Within the first seconds after any increase in light intensity, young leaves showed higher proton gradient (ΔpH) across the thylakoid membranes than the mature leaves, preventing over-reduction of PSI in the young leaves. As a result, PSI was more tolerant to fluctuating light in the young leaves than in the mature leaves. Interestingly, after transition from low to high light, the activity of cyclic electron flow (CEF) in young leaves increased first to a high level and then decreased to a stable value, while this rapid stimulation of CEF was not observed in the mature leaves. Furthermore, the over-reduction of PSI significantly stimulated CEF in the young leaves but not in the mature leaves. Taken together, within the first seconds after any increase in illumination, the stimulation of CEF favors the rapid lumen acidification and optimizes the PSI redox state in the young leaves, protecting PSI against photoinhibition under fluctuating light in field-grown plants.     

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection,Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin—the histone-to-protamine switch.Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.     

Developmental asynchrony caused by steep temperature gradients does not impair pattern formation in the wasp,Pimpla turionellae L.

Generating developmental gradients by temperature gradients established within a developing organism is an easy, non-invasive technique to study physiological interdependencies between locally separated subsystems. A linear temperature gradient of about 10° C/mm was maintained up to 5 h in either direction along the long axis of a long-germ-type hymenopteran egg, which was simultaneously filmed by the 16 mm timelapse technique. The result was a dramatic desynchronization of development, which between the egg poles could reach up to 9.3 h relative to normal development. Within the same egg, up to seven mitotic waves (i.e. eight different nuclear generations) were observed at the same time, and the subsequent cellularization process was extremely asynchronous. The initial regions of the mitotic waves, the fountain flow of the ooplasm, and the gastrulation process were shifted towards the egg pole kept at higher temperatures. Developmental processes occurring successively in normal development now took place simultaneously, with either direction of the temperature gradient. For instance, while gastrulation had started in the warm region, midblastula transition and cellularization were in progress in the middle of the egg, and intravitelline nuclear multiplication occurred at the cold pole, by rapid and still biphasic cell cycles. In some respects, development resembled that of a short-germ-type insect egg. Nevertheless, the developmental processes were resynchronized after the temperature gradient was switched off. Surprisingly, the extreme desynchronization during early development did not affect the segment pattern of the resulting embryos. The technique of inducing well-defined developmental asynchronies might be applied in Drosophila to analyse the subtle interplay between maternal and zygotic gene activities described in this species.     

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.     

HETEROCHRONIC DEVELOPMENTAL PLASTICITY IN LARVAL SEA URCHINS AND ITS IMPLICATIONS FOR EVOLUTION OF NONFEEDING LARVAE

Preexisting developmental plasticity in feeding larvae may contribute to the evolutionary transition from development with a feeding larva to nonfeeding larval development. Differences in timing of development of larval and juvenile structures (heterochronic shifts) and differences in the size of the larval body (shifts in allocation) were produced in sea urchin larvae exposed to different amounts of food in the laboratory and in the field. The changes in larval form in response to food appear to be adaptive, with increased allocation of growth to the larval apparatus for catching food when food is scarce and earlier allocation to juvenile structures when food is abundant. This phenotypic plasticity among full siblings is similar in direction to the heterochronic evolutionary changes in species that have greater nutrient reserves within the ova and do not depend on particulate planktonic food. This similarity suggests that developmental plasticity that is adaptive for feeding larvae also contributes to correlated and adaptive evolutionary changes in the transition to nonfeeding larval development. If endogenous food supplies have the same effect on morphogenesis as exogenous food supplies, then changes in genes that act during oogenesis to affect nutrient stores may be sufficient to produce correlated adaptive changes in larval development.     

股骨短缩截骨全髋置换术治疗青年Crowe IV 型髋关节发育不良

目的：总结股骨短缩截骨结合全髋关节置换术治疗青年Crowe IV 型髋关节发育不良（DDH）的近期疗效。方法：2009 年1 月 至2015 年1 月期间我院收治的22 例（24 髋）青年Crowe IV 型髋关节发育不良患者,其中男4 例（5 髋）,女18 例（19 髋）,年龄 17~27 岁,平均22 岁。记录患者术前、术后髋关节功能Harris评分、髋关节疼痛VAS评分和下肢长度差异。收集患者临床及影像 学资料。结果：22 例患者均获随访,随访时间6～72 个月,平均38 个月。截骨端均骨性愈合,愈合时间3～ 6 个月,平均3.2 个月。 Harris 评分由术前（43.2 ± 2.7）分改善至（95.3 ± 2.8）分,效果显著（P＜0.01）,髋关节疼痛VAS评分由术前(6. 0 ± 1. 0)分改善至 (1.0 ± 0.5) 分,疼痛明显缓解（P＜0.05）,下肢长度差异由术前（55.5± 15.5）mm减少至（16.5± 5.5）mm,显著改善（P＜0.01）。术后2 例合并坐骨神经麻痹,但6 个月内症状完全缓解,6 例遗留轻度跛行。随访期间均无假体翻修,未发现材料与宿主的生物相容性反 应。结论：股骨短缩截骨全髋置换术治疗青年Crowe IV 型髋关节发育不良的近期疗效满意,术中综合考虑软组织松解程度与短缩 截骨方式及长度可有效的解决患者肢体短缩、避免神经血管损伤及改善患者术后髋关节功能。     

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specificationin mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).     

Growth, ageing and death of a photoautotrophic plant cell culture

 Batch cultures of photoautotrophic cell suspensions of Chenopodiumrubrum L., growing in an inorganic medium on CO₂ under a daily balanced light–dark regime of 16 : 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3–4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohomones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. Received: 30 April 1999 / Accepted: 21 August 1999     

Is the number of possible QTL for asymmetry phenotypes dependent on thermal stress?

In bilateral organisms, fluctuating asymmetry (FA) was often used as an index of developmental instability but FA was not always found to be higher in stressful environments. An intercontinental set of recombinant inbred lines (RIL) was used to search for genetic variation in fluctuating asymmetry (FA) of both wing length (WL) and wing width (WW) in Drosophila melanogaster when reared at both benign (25 °C) and stressful (30 °C) temperatures. FA levels did not differ between benign and stressful temperatures. At benign temperature, no QTL was significant for FA. However, at stressful temperature, composite interval mapping revealed some QTL for FA in both WL and WW. QTL-based scans under stressful thermal environments may be an informative approach for either FA or developmental instability analyses, even when FA levels are similar between stressful and benign environments.