The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.     

A method for in situ monitoring of the isotope composition of tree xylem water using laser spectroscopy

Field studies analyzing the stable isotope composition of xylem water are providing important information on ecosystem water relations. However, the capacity of stable isotopes to characterize the functioning of plants in their environment has not been fully explored because of methodological constraints on the extent and resolution at which samples could be collected and analysed. Here, we introduce an in situ method offering the potential to continuously monitor the stable isotope composition of tree xylem water via its vapour phase using a commercial laser‐based isotope analyser and compact microporous probes installed into the xylem. Our technique enables efficient high‐frequency measurement with intervals of only a few minutes per sample while eliminating the need for costly and cumbersome destructive collection of plant material and laboratory‐based processing. We present field observations of xylem water hydrogen and oxygen isotope compositions obtained over several days including a labelled irrigation event and compare them against results from concurrent destructive sampling with cryogenic distillation and mass spectrometric analysis. The data demonstrate that temporal changes as well as spatial patterns of integration in xylem water isotope composition can be resolved through direct measurement. The new technique can therefore present a valuable tool to study the hydraulic architecture and water utilization of trees.     

ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking

COPI‐coated vesicles mediate retrograde membrane traffic from the cis‐Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER‐resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V‐dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI?Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites.     

Identification and molecular characterization of the nicotianamine synthase gene family in bread wheat

Nicotianamine (NA) is a non‐protein amino acid involved in fundamental aspects of metal uptake, transport and homeostasis in all plants and constitutes the biosynthetic precursor of mugineic acid family phytosiderophores (MAs) in graminaceous plant species. Nicotianamine synthase (NAS) genes, which encode enzymes that synthesize NA from S‐adenosyl‐L‐methionine (SAM), are differentially regulated by iron (Fe) status in most plant species and plant genomes have been found to contain anywhere from 1 to 9 NAS genes. This study describes the identification of 21 NAS genes in the hexaploid bread wheat (Triticum aestivum L.) genome and their phylogenetic classification into two distinct clades. The TaNAS genes are highly expressed during germination, seedling growth and reproductive development. Fourteen of the clade I NAS genes were up‐regulated in root tissues under conditions of Fe deficiency. Protein sequence analyses revealed the presence of endocytosis motifs in all of the wheat NAS proteins as well as chloroplast, mitochondrial and secretory transit peptide signals in four proteins. These results greatly expand our knowledge of NAS gene families in graminaceous plant species as well as the genetics underlying Fe nutrition in bread wheat.     

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Plant‐based platforms are extensively used for the expression of recombinant proteins, including monoclonal antibodies. However, to harness the approach effectively and leverage it to its full potential, a better understanding of intracellular processes that affect protein properties is required. In this work, we examined vacuolar (vac) targeting and deposition of the monoclonal antibody (Ab) 14D9 in Nicotiana benthamiana leaves. Two distinct vacuolar targeting signals (KISIA and NIFRGF) were C‐terminal fused to the heavy chain of 14D9 (vac‐Abs) and compared with secreted and ER‐retained variants (sec‐Ab, ER‐Ab, respectively). Accumulation of ER‐ and vac‐Abs was 10‐ to 15‐fold higher than sec‐Ab. N‐glycan profiling revealed the predominant presence of plant typical complex fucosylated and xylosylated GnGnXF structures on sec‐Ab while vac‐Abs carried mainly oligomannosidic (Man 7‐9) next to GnGnXF forms. Paucimannosidic glycans (commonly assigned as typical vacuolar) were not detected. Confocal microscopy analysis using RFP fusions showed that sec‐Ab‐RFP localized in the apoplast while vac‐Abs‐RFP were exclusively detected in the central vacuole. The data suggest that vac‐Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab molecules containing Man structures) and trafficking through the Golgi (for Ab molecules containing complex N‐glycans). Importantly, vac‐Abs were correctly assembled and functionally active. Collectively, we show that the central vacuole is an appropriate compartment for the efficient production of Abs with appropriate post‐translational modifications, but also point to a reconsideration of current concepts in plant glycan processing.     

Dynamic assembly of the exomer secretory vesicle cargo adaptor subunits

The trans‐Golgi network (TGN) is the main secretory pathway sorting station, where cargoes are packed into appropriate transport vesicles targeted to specific destinations. Exomer is a cargo adaptor necessary for direct transport of a subset of cargoes from the TGN to the plasma membrane in yeast. Here, we show that unlike classical adaptor complexes, exomer is not recruited en bloc to the TGN, but rather assembles through a stepwise pathway, in which first the scaffold protein Chs5 and then the cargo‐binding units, the ChAPs, are recruited. Although all ChAPs are able to assemble functional exomer complexes, they do so with different efficiencies. The mutual relationship between ChAPs varies from cooperation to competition depending on their expression levels and affinities to Chs5 allowing an optimized and efficient cargo transport. The multifactorial assembly pathway results in an exquisitely fine‐tuned adaptor complex, enabling the cell to quickly respond and adapt to changes such as stress.     

低分子质量魔芋甘露寡糖的长期毒性与遗传毒性研究

魔芋甘露寡糖是一种具有肠道菌群调节作用的新型食品添加剂．本研究首次通过酶解与有机溶剂沉淀法制备了低分子质量的甘露寡糖(聚合度2～7),并对这类寡糖进行了长期毒性与遗传毒性评价．在长期毒性试验中,以大鼠为实验对象,分低、中、高(2.25,5.25,7.50 g/kg)药物剂量组和阴性对照组,连续灌胃给药90天．一般状况观察、生化指标、血液学指标、病理学等与对照组比较均无显著性差异,而大体解剖观察发现,部分大鼠的肝脏与肾脏形态发生变化,但这些变化均在正常范围内,且其他各项指标差异均无统计学意义．此外,一系列实验包括小鼠骨髓微核实验、Ames试验、小鼠精子畸变试验均未发现低分子质量甘露寡糖有明显的遗传毒性．试验结果提示,本研究方法获得的低分子质量甘露寡糖在本实验条件下未发现长期毒性与遗传毒性．     

裂解多糖单加氧酶高效催化的研究进展

裂解多糖单加氧酶(lytic polysaccharide monooxygenases,LPMOs)是一类新发现的铜离子依赖性的氧化酶,常具有多种模块化组合,能够高效氧化降解生物质多糖.LPMOs的催化结构域为β三明治结构,活性中心含有一个铜离子.该酶的催化反应过程相对于糖苷水解酶类更加复杂,LPMOs结合底物后,首先要接受电子供体提供的电子,通过电子传递链传递给活性中心的Cu[Ⅱ],将其还原为Cu[Ⅰ],Cu[Ⅰ]结合并活化分子氧后,再氧化降解多糖链的糖苷键,生成氧化产物和非氧化产物.近年来的研究表明,在木质纤维素降解酶系中加入LPMOs能显著提高其对结晶纤维素的转化效率,因此LPMOs相关研究的深入开展可以拓展人们对其高效降解机制的认识,从而为高效降解酶系的复配以降低工业规模的生产成本等提供理论指导.本文综述了该领域相关研究的最新进展,分析了LPMOs潜在的研究方向与工业化应用的前景.