An in planta haploid induction system in Brassica napus

Doubled haploid technology is widely used to accelerate plant breeding, but its use in the important oilseed crop Brassica napus L. is limited because B. napus haploids could only be obtained through in vitro anther or microspore cultures. Recently, maize (Zea mays) lines containing mutations in Domain of unknown function 679 membrane protein (DMP) were used as haploid inducer lines. This new haploid induction mechanism has been extended to several other plants, including the dicots Arabidopsis thaliana, tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum). Here, we knocked out four BnaDMP genes in the B. napus cultivar Westar using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 vector with an enhanced green fluorescent protein expression cassette. Plants with DMP mutations in B. napus in the T₀, T₁, and T₂ generations exhibited a haploid induction rate up to 2.53%. These results suggest that targeting BnaDMP could be useful for haploid induction in B. napus.     

Early markers of in vitro microspore reprogramming to embryogenesis in olive (Olea europaea L.)

Microspore embryogenesis to form haploid and double-haploid embryos and regenerated plants is an efficient method of producing homozygous lines for crop breeding. In trees, the process is of special interest since classical methods are impractical in many cases, as in Olea europaea L. Recently, a convenient method has been developed for microspore embryogenesis induction by stress in olive isolated microspores in vitro cultures. In the present work, the switch of the microspore developmental pathway and the formation of microspore-derived multicellular proembryos have been achieved and a cytochemical and immunocytochemical analysis was performed in the early stages. The young microspore proembryos displayed defined features different to both, the in vivo gametophytic, and the in vitro non-responsive microspores. Reprogrammed microspores showed an absence of starch, the occurrence of a first symmetrical division and cytokinesis, the presence of an abundant ribosomal population, and changes in cellulosic and pectic cell wall components which constituted early markers of the embryogenic microspore process. They provided new insights on the molecular and cellular events associated with the microspore reprogramming of woody plants, and specifically in olive, providing interesting knowledge which could guide future selection and regeneration strategies in this fruit tree of high economic interest.     

Interaction of glucocorticoid analogues with the human glucocorticoid receptor

Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds. Furthermore, correlation of these data with reported in vivo anti-inflammatory potencies was surprisingly close, with two exceptions. The in vitro data suggest an explanation for the discrepant compounds, consistent with published data on their metabolic fate in vivo. The co-transfection assay has utility as a quantitative predictor of in vivo glucocorticoid pharmacology.     

刺盘孢菌的蛋白互作预测及侵染早期共表达模块分析

刺盘孢菌是一类重要的植物病原真菌,在全球范围内危害众多单双子叶植物。有许多研究对病菌的侵染模式进行了探索,但仍未阐明其确切的分子机制。本研究在预测病菌蛋白互作的基础上,结合表达谱数据对病菌在活体生存环境下的共表达模块进行挖掘和分析,以期为分子机制的研究提供新的线索。通过同源映射法和结构域法,预测得到刺盘孢菌的4 288个蛋白之间存在41 700个潜在互作,其中39 776个互作发生于异源蛋白之间,1 924个互作发生于同一蛋白内。将蛋白互作数据分别与4个表达谱数据进行整合,构建得到离体I、离体II、活体I和活体II 4个共表达互作组。对离体和活体互作组的共有基因的表达水平进行比较分析,结果表明,与离体互作组相比,活体互作组中与翻译、蛋白代谢等有关的基因表达水平下降,与离子转运、糖物质转运等有关的基因表达水平上升,暗示了物质转运在刺盘孢菌侵染早期的重要作用。进一步对活体互作组进行模块化分析,结果表明,活体I和活体II的特异模块分别与胁迫响应、肌动蛋白纤维长度调控有关,其中胁迫响应子网是以热激蛋白Hsp70为核心的互作簇,可能参与病菌对寄主的识别与对抗;肌动蛋白纤维长度调控子网则可能与病菌菌丝在寄主细胞间的延伸有关。     

Novel technologies in doubled haploid line development

haploid inducer line can be transferred (DH) technology can not only shorten the breeding process but also increase genetic gain. Haploid induction and subsequent genome doubling are the two main steps required for DH technology. Haploids have been generated through the culture of immature male and female gametophytes, and through inter‐ and intraspecific via chromosome elimination. Here, we focus on haploidization via chromosome elimination, especially the recent advances in centromere‐mediated haploidization. Once haploids have been induced, genome doubling is needed to produce DH lines. This study has proposed a new strategy to improve haploid genome doubling by combing haploids and minichromosome technology. With the progress in haploid induction and genome doubling methods, DH technology can facilitate reverse breeding, cytoplasmic male sterile (CMS) line production, gene stacking and a variety of other genetic analysis.     

In vivo strain gage implantation in rats

A technique for the fabrication of encapsulated micro-miniature rosette strain gages for in vivo implantation is described. The gage units have an overall area of ten square millimeters (2.5 mm × 4.0 mm), and hence can be installed in very small experimental animals, particularly rodents. Using a rat model, strain data for up to 12 days have been obtained and in vitro studies have validified the in vivo strain recordings.     

Formol-Saline as A Cell Conserving Medium in the Micronucleus Test

It is widely recognized that tests using mammalian cell sytems are essential for assessing mutagenic hazard (Ishidate and Yoshikawa 1980). The micre nucleus test (Von Ledebur and Schmid 1973, Schmid 1973) is a convenient in vivo technique to overcome the shortcomings of in vitro bacterial methods. However, this assay requires high quality smears, for the production of which the solution used to avoid cell damage is critical.     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

Evaluation of a three point pressure method for the determination of arterial transmission characteristics

The mathematical expressions required to analyse wave transmission characteristics (frequency dependent phase velocity, attenuation, and reflection) in arteries, under in vivo conditions, are summarized. In addition, a three point pressure method, which theoretically permits experimental determination of the propagation constant (phase velocity and attenuation), was tested under in vitro conditions. It is found to be a potentially powerful tool for in vivo studies if used with the appropriate constraints.     

In Vivo Esr Studies of Antioxidant Activity on Free Radical Reaction in Living Mice Under Oxidative Stress

In vivo antioxidant activity seems to be quite complicate due to multiple interaction with biomaterials and differs from results by in vitro experiments. In vivo estimation of antioxidant activity is performed by measuring TBA reactive substances in blood or hydrocarbon gases in breath, but these systems do not measure free radical reaction but the final products of oxidative reaction. In the present study, we applied in vivo ESR to evaluate antioxidant activity by monitoring the redox reaction of nitroxide radical and clearly found that the nitroxide is very susceptible to oxidative stress in vivo and quite useful to evaluate antioxidant activity non-invasively.     

Application and prospects of high-throughput screening for in vitro neurogenesis

Over the past few decades, high-throughput screening (HTS) has made great contributions to new drug discovery. HTS technology is equipped with higher throughput, minimized platforms, more automated and computerized operating systems, more efficient and sensitive detection devices, and rapid data processing systems. At the same time, in vitro neurogenesis is gradually becoming important in establishing models to investigate the mechanisms of neural disease or developmental processes. However, challenges remain in generating more mature and functional neurons with specific subtypes and in establishing robust and standardized three-dimensional (3D) in vitro models with neural cells cultured in 3D matrices or organoids representing specific brain regions. Here, we review the applications of HTS technologies on in vitro neurogenesis, especially aiming at identifying the essential genes, chemical small molecules and adaptive microenvironments that hold great prospects for generating functional neurons or more reproductive and homogeneous 3D organoids. We also discuss the developmental tendency of HTS technology, e.g., so-called next-generation screening, which utilizes 3D organoid-based screening combined with microfluidic devices to narrow the gap between in vitro models and in vivo situations both physiologically and pathologically.     

Translating tissue culture results into animal models: the case of Salmonella typhimurium

Investigators use both in vitro and in vivo models to better understand infectious disease processes. Both models are extremely useful in research, but there exists a significant gap in complexity between the highly controlled reductionist in vitro systems and the largely undefined, but relevant variability encompassing in vivo animal models. In an effort to understand how Salmonella initiates disease at the intestinal epithelium, in vitro models have served a useful purpose in allowing investigators to identify molecular mechanisms responsible for Salmonella invasion of host cells and stimulation of host inflammatory responses. Identification of these molecular mechanisms has generated hypotheses that are now being tested using in vivo models. Translating the in vitro findings into the context of an animal model and subsequently to human disease remains a difficult challenge for any disease process.     

油菜双单倍体诱导育种技术研究进展

油菜是食用油、优质饲料蛋白的重要来源,杂种优势利用是油菜培育优势性状最重要的手段,且提高亲本的选育效率对优质品种的培育具有积极的推动作用。现有油菜育种技术存在效率低、周期长、盲目性大、应用范围有限等诸多问题,不适于油菜产业快速发展的需求。双单倍体诱导育种技术是近年来新兴的一种快速选育油菜新品种的技术方法。该技术以操作简便、应用范围广、效率高等优势被广泛应用于油菜新品种的选育过程中。从油菜双单倍体诱导技术创新研究的发现、作用表现、诱导机制、作用价值等方面系统地综述了油菜双单倍体诱导技术的研究进展,展望了油菜双单倍体诱导技术的应用前景,以期为未来油菜双单倍体诱导技术以及其他作物诱导系的研究和利用提供参考。     

基于双碱基编辑系统的植物基因靶向随机突变技术

遗传性变异是表型多样性的基础,靶向饱和突变作物基因可以促进产生具有优异农艺性状的突变体。相较于传统诱变育种和异源物种中的定向进化方法,基于双碱基编辑系统的植物基因靶向随机突变技术可对植物内源基因产生高效突变,从而实现原位定向进化,加快植物育种及功能基因研究进程。该文介绍了使用饱和靶向内源基因突变编辑器(STEME)对植...     

The effect of in vivo heat treatments on blowfly flight muscle mitochondrial function: Effects on partial reactions of the respiratory chain

1. 1. The development of thermotolerance has been shown to protect blowfly flight muscle mitochondrial function from damage resulting from an LD₅₀ in vivo heat dose.

2. 2. The principal sites of the damage have been studied using specific inhibitors of the respiratory chain, rotenone and antimycin A, together with substrates that stimulate respiration through the different complexes.

3. 3. Complex I was identified as the primary site for heat damage. State III respiration was inhibited following the LD₅₀ in vivo heat dose, and uncoupling with FCCP did not restore respiration to control levels, indicating that the respiratory enzymes were inactivated. The development of thermotolerance protected this site from heat damage.

4. 4. In contrast, G3-P stimulated respiration was the same in control, LD₅₀ in vivo treated controls and LD₅₀, in vivo treated thermotolerant mitochondria, and significantly higher than state III respiration of LD₅₀ in vivo treated controls. This suggested that respiration through G3-P dehydrogenase, Co enzyme Q and Complex III is not damaged. However, as G3-P stimulated respiration of coupled mitochondria from LD₅₀ in-vivo treated flies was markedly reduced (El-Wadawi and Bowler, 1995. J. exp. Biol. 198: 2413–2421), phosphorylation at complex III may be inhibited also.

5. 5. Ferrocyanide stimulated respiration through cytochrome c-Complex IV was also inhibited in LD₅₀ in vivo treated flies, as compared with unheated control mitochondria. However, thermotolerance protected this site also from heat damage.

     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

Specific effect of manganese on the allantoinase of Vigna Radiata

Vigna radiata seedlings germinated in the presence of Mn²⁺ show an unusual increase in allantoinase activity which is proportional to Mn²⁺ concentration up to 5 mM. Though Mn²⁺ is not an activator for V. radiata allantoinase, it specifically protects allantoinase against thermal as well as papain-catalysed inactivation. Evidence is presented to show that the primary effect of Mn²⁺ is a protective one, both in vitro and in vivo, and that this is reflected in the observed enhancement of allantoinase activity in Mn²⁺ grown seedlings. That this unusual effect of Mn²⁺ is a specific one is indicated by the lack of a similar effect with Mg²⁺. Cu²⁺ is shown to destabilize V. radiata allantoinase in vitro as well as in vivo.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

Developmental profiles of glial enzymes in the chick embryo: In vivo and in culture

The activities of two glial cell enzymes, glutamine synthetase (a marker for astrocytes) and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (a marker for oligodendrocytes and myelination) were studied in the developing chick embryo brain in vivo and in cultures derived from chick embryos. The in vivo findings showed that the activities of both enzymes parallel the patterns of gliogenesis and myelination. Glutamine synthetase follows similar patterns in culture and in vivo, whereas the developmental profile of 2′,3′-cyclic nucleotide 3′-phosphohydrolase appears to be affected by the culture conditions.