A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa.

Superovulated hamster oocytes were cryopreserved and thawed according to our carefully designed procedures. More than 90% (92 +/- 4%) of oocytes survived freezing and thawing. They were proven to be well conserved, showing excellent performance comparable to freshly ovulated oocytes in the human sperm penetration test (proportion of penetrated ova: 94.7% vs. 93.6%) and human sperm chromosome analysis (proportion of metaphasic ova: 81.8% vs. 83.6%). There was no statistically significant difference in the incidences of sperm chromosome aberrations between assays using fresh and frozen-thawed oocytes. In addition, there was no statistically significant increase of aberrations in female pronuclear (hamster) chromosomes. This freezing-thawing method was found to be reliable, yielding viable hamster oocytes of high quality.     

Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion

Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula which migrate to the final location. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells – postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro-induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were somewhat inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati, employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far of the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled us to distinguish PA and CA glands for microdissection. Using 7.5 × 10⁶ μm³ sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least two of three replicates in either gland type (PA = 455, 40 exclusive; CA = 421, six exclusive; 60 proteins differed significantly in their abundance between the glands). Peptidases of five catalytic types accounted for ca. 8% and 6% of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. While PA glands generally had rich enzymatic equipment, CA glands were conspicuously abundant in venom allergen-like proteins.     

脱血红素细胞色素c插入磷脂单分子层能力的研究

介绍了一种改进的、用于研究膜脂-蛋白相互作用的气-液界面单分子层实验模型及实验装置,并在该实验装置上研究了来自马心和金枪鱼心的线粒体前体蛋白脱血红素细胞色素c与大豆磷脂单分子层的相互作用,实验结果表明这两种前体蛋白对大豆磷脂单分子层都具有较强的亲和性和插膜能力,其临界插膜压力分别为43mN/m、45mN/m.     

Assessment of Potential Sublethal Effects of Various Insecticides on Key Biological Traits of The Tobacco Whitefly,Bemisia tabaci

The tobacco whitefly Bemisia tabaci is one of the most devastating pests worldwide. Current management of B. tabaci relies upon the frequent applications of insecticides. In addition to direct mortality by typical acute toxicity (lethal effect), insecticides may also impair various key biological traits of the exposed insects through physiological and behavioral sublethal effects. Identifying and characterizing such effects could be crucial for understanding the global effects of insecticides on the pest and therefore for optimizing its management in the crops. We assessed the effects of sublethal and low-lethal concentrations of four widely used insecticides on the fecundity, honeydew excretion and feeding behavior of B. tabaci adults. The probing activity of the whiteflies feeding on treated cotton seedlings was recorded by an Electrical Penetration Graph (EPG). The results showed that imidacloprid and bifenthrin caused a reduction in phloem feeding even at sublethal concentrations. In addition, the honeydew excretions and fecundity levels of adults feeding on leaf discs treated with these concentrations were significantly lower than the untreated ones. While, sublethal concentrations of chlorpyrifos and carbosulfan did not affect feeding behavior, honeydew excretion and fecundity of the whitefly. We demonstrated an antifeedant effect of the imidacloprid and bifenthrin on B. tabaci, whereas behavioral changes in adults feeding on leaves treated with chlorpyrifos and carbosulfan were more likely caused by the direct effects of the insecticides on the insects'' nervous system itself. Our results show that aside from the lethal effect, the sublethal concentration of imidacloprid and bifenthrin impairs the phloem feeding, i.e. the most important feeding trait in a plant protection perspective. Indeed, this antifeedant property would give these insecticides potential to control insect pests indirectly. Therefore, the behavioral effects of sublethal concentrations of imidacloprid and bifenthrin may play an important role in the control of whitefly pests by increasing the toxicity persistence in treated crops.     

Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification

The dopaminergic neurons of the basal ganglia play critical roles in CNS function and human disease, but specification of dopamine neuron phenotype is poorly understood in vertebrates. We performed an in vivo screen in zebrafish to identify dopaminergic neuron enhancers, in order to facilitate studies on the specification of neuronal identity, connectivity, and function in the basal ganglia. Based primarily on identification of conserved non-coding elements, we tested 54 DNA elements from four species (zebrafish, pufferfish, mouse, and rat), that included 21 genes with known or putative roles in dopaminergic neuron specification or function. Most elements failed to drive CNS expression or did not express specifically in dopaminergic neurons. However, we did isolate a discrete enhancer from the otpb gene that drove specific expression in diencephalic dopaminergic neurons, although it did not share sequence conservation with regulatory regions of otpa or other dopamine-specific genes. For the otpb enhancer, regulation of expression in dopamine neurons requires multiple elements spread across a large genomic area. In addition, we compared our in vivo testing with in silico analysis of genomic regions for genes involved in dopamine neuron function, but failed to find conserved regions that functioned as enhancers. We conclude that regulation of dopaminergic neuron phenotype in vertebrates is regulated by dispersed regulatory elements.     

When the going gets tough: Responses of Sorghum bicolor L. (cv. Tegemeo) seedlings to soil penetration resistance

Summary

Sorghum bicolor L. (cv. Tegemeo) seedlings were grown for nine days in soil at field capacity packed to give a uniform penetration resistance (PR) of either 0.25, 1.00 or 1.75 MPa. Root biomass was not significantly affected by soil PR treatment. However, as PR increased to 1.75 MPa, the diameter of the seminal root axis increased by 52% whilst its length decreased by 30%. Shoot growth, in terms of oven dry (OD) weight and photo-synthetic area, was reduced in both the 0.25 MPa and 1.75 MPa treatments compared to the 1.00 MPa treatment. A reduced nutrient, water or oxygen supply to the roots were discounted as possible causes of the root and shoot responses to soil PR. It is suggested that the changes in root morphology between treatments were a direct result of the changes in soil PR. For shoot growth, in the 0.25 MPa treatment it is suggested that shoot growth was reduced as a result of an increase in the carbon sink strength of the roots.     

Experimental assessment of ecological restoration options for compacted forest soils

It is ecologically undesirable to solve forest soil compaction due to mechanized harvesting at large spatial scales using agricultural mechanical soil loosening techniques. We therefore examined whether a stimulation of biological activity through litter manipulation, liming and/or inoculation of the anecic earthworm species Lumbricus terrestris could significantly contribute to the ecological restoration of compacted forest soils by comparing the impact of these treatments on the soil within and beside compacted wheel tracks. The replacement of native litter by litter with a better quality resulted in a faster litter decomposition. However, maximal decay rates were obtained only when litter manipulation, earthworm inoculation and liming were combined. Anecic earthworms were initially absent as soils were probably too acid. Liming as well as litter manipulation had a small positive influence on the numbers of retraced L. terrestris, inducing positive feedback mechanisms on soil pH and litter decomposition rates. None of the treatments, however, had a significant effect on the compaction degrees within or beside tracks within the small study period. L. terrestris realized only a small decrease of bulk density beside the tracks. Within the tracks a similar number of L. terrestris was retrieved, but effects on the compaction degree were negligible. Liming decreased penetration resistance, but only in the absence of anecic earthworms. Endogeic earthworms were overall more abundant than the anecics, especially within tracks where soil water contents and pH values were higher. However, endogeics only had a marginal effect on litter decomposition and although they positively influence soil structure, they could not realize a reduction of the compaction degree, quantified by bulk density and penetration resistance, due to different burrowing habits. Our results indicated that a positive impact of anecic earthworms on the structure of compacted forest soils can be obtained in the long-term, at least in case soil conditions (acidity, nutrient availability and moisture content) are favourable. This can be achieved by conversion of forests towards tree species with high quality litter.