Purification, identification, and cloning of lysoplasmalogenase, the enzyme that catalyzes hydrolysis of the vinyl ether bond of lysoplasmalogen

Lysoplasmalogenase (EC 3.3.2.2 and EC 3.3.2.5) is an enzyme that catalyzes hydrolytic cleavage of the vinyl ether bond of lysoplasmalogen, forming fatty aldehyde and glycerophosphoethanolamine or glycerophosphocholine and is specific for the sn-2-deacylated form of plasmalogen. Here we report the purification, characterization, identification, and cloning of lysoplasmalogenase. Rat liver microsomal lysoplasmalogenase was solubilized with octyl glucoside and purified 500-fold to near homogeneity using four chromatography steps. The purified enzyme has apparent K(m) values of ～50 μm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent V(m) values of 24.5 and 17.5 μmol/min/mg protein for the two substrates, respectively. The pH optimum was 7.0. Lysoplasmalogenase was competitively inhibited by lysophosphatidic acid (K(i) ～20 μm). The predominant band on a gel at ～19 kDa was subjected to trypsinolysis, and the peptides were identified by mass spectrometry as Tmem86b, a protein of unknown function. Transient transfection of human embryonic kidney (HEK) 293T cells showed that TMEM86b cDNA yielded lysoplasmalogenase activity, and Western blot analyses confirmed the synthesis of TMEM86b protein. The protein was localized in the membrane fractions. The TMEM86b gene was also transformed into Escherichia coli, and its expression was verified by Western blot and activity analyses. Tmem86b is a hydrophobic transmembrane protein of the YhhN family. Northern blot analyses demonstrated that liver expressed the highest level of Tmem86b, which agreed with tissue distribution of activity. Overexpression of TMEM86b in HEK 293T cells resulted in decreased levels of plasmalogens, suggesting that the enzyme may be important in regulating plasmalogen levels in animal cells.     

Quasi-Static and Dynamic Nanoindentation of Some Selected Biomaterials

This study details an investigation of the viscoelastic behavior of some biomaterials （nacre, cattle horn and beetle cuticle） at lamellar length scales using quasi-static and dynamic nanoindentation techniques in the materials＇ Transverse Direction （TD） and Longitudinal Direction （LD）. Our results show that nacre exhibits high fracture toughness moving towards a larger cam- paniForm as the stress frequency varies from 10 Hz to 200 Hz. Elytra cuticle exhibits the least fracture toughness presenting little energy dissipation in TD. It was initially speculated that the fracture toughness of the subject materials would be directly related to energy-dissipating mechanisms （mechanical hysteresis）, but not the maximum value of the loss tangent tan＆ However, it was found that the materials＇ elastic modulus and hardness are similar in both the TD and LD when assessed using the quasi-static nanoindentation method, but not dynamic nanoindentation. It is believed that the reported results can be useful in the design of new crack arrest and damping materials based on biological counterparts.     

Increased number and activation of peripheral basophils in adult‐onset minimal change disease

Nowadays, the pathogenesis of minimal change disease (MCD) is still not well‐known, and the current understanding on MCD is mainly based on data derived from children, and very few adults. Here, we comprehensively analysed the correlation between the changes of peripheral basophils and the incidence rate and relapse of adult‐onset MCD. The results showed that in patients at the onset of MCD, the ratio and activation of basophils were all higher than those of healthy controls (all P < .05). In vitro test results showed that basophils from healthy controls can be activated by the serum taken from patients with MCD. Among 62 patients at the onset of MCD, with complete remission after treatment and 1 year of follow‐up, the relative and absolute basophil counts before treatment were higher in the long‐term remission group (n = 33) than that of the relapse group (n = 29). The basophil counts were significantly higher in the infrequent relapse group (n = 13) than that of the frequent relapse group (n = 16; P < .05). These findings suggested that basophil may play a pathogenic role in adult‐onset MCD, and the increased number and activation of peripheral basophils could predict recurrence in adult MCD.     

SARS-CoV-2 ORF10 impairs cilia by enhancing CUL2ZYG11B activity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal pathogen of the ongoing global pandemic of coronavirus disease 2019 (COVID-19). Loss of smell and taste are symptoms of COVID-19, and may be related to cilia dysfunction. Here, we found that the SARS-CoV-2 ORF10 increases the overall E3 ligase activity of the CUL2^ZYG11B complex by interacting with ZYG11B. Enhanced CUL2^ZYG11B activity by ORF10 causes increased ubiquitination and subsequent proteasome-mediated degradation of an intraflagellar transport (IFT) complex B protein, IFT46, thereby impairing both cilia biogenesis and maintenance. Further, we show that exposure of the respiratory tract of hACE2 mice to SARS-CoV-2 or SARS-CoV-2 ORF10 alone results in cilia-dysfunction-related phenotypes, and the ORF10 expression in primary human nasal epithelial cells (HNECs) also caused a rapid loss of the ciliary layer. Our study demonstrates how SARS-CoV-2 ORF10 hijacks CUL2^ZYG11B to eliminate IFT46 and leads to cilia dysfunction, thereby offering a powerful etiopathological explanation for how SARS-CoV-2 causes multiple cilia-dysfunction-related symptoms specific to COVID-19.     

多胺对荔枝胚性愈伤组织增殖与体胚发生的影响

为探讨外源多胺(PAs)对荔枝胚性愈伤组织(EC)增殖及体胚发生的影响机制,该研究以“妃子笑”荔枝EC为材料,采用单因素法在增殖培养基中添加腐胺(Put)、亚精胺(Spd)及精胺(Spm),分析了不同PAs处理后EC的形态、结构、内源PAs含量及相关酶指标的变化。结果表明:(1)外源Put、Spd和Spm处理均显著提高了EC增殖率,减少了体胚诱导及萌发数量。经外源PAs处理增殖的EC胚性细胞大小较一致,染色深且均匀,多细胞原胚减少,可见已经分化完全的早期子叶胚。(2)外源PAs处理均显著提高了EC中内源PAs含量,其中Put处理的EC中各类内源PAs及总PAs含量最高; 当在含外源PAs培养基上增殖的EC转入不含外源PAs的培养基上增殖时(恢复培养),EC中的Put含量仍然显著高于对照,内源Spd和Spm则显著降低。(3)外源Put处理显著提高了EC中的鸟氨酸脱羧酶(ODC)、精氨酸脱羧酶(ADC)和二胺氧化酶(DAO)活性,而外源Spd、Spm处理显著降低了EC中的ODC及ADC活性,外源Spd显著提高了多胺氧化酶(PAO)活性; 恢复培养后,EC中ADC和DAO活性比恢复培养前显著降低,ODC和PAO无显著性差异。综上认为,外源PAs可以通过调节PAs代谢相关酶活性影响内源PAs含量,进而影响荔枝EC增殖和体胚诱导。该研究结果为进一步研究PAs调节荔枝体胚发生机制及提高荔枝离体再生效率提供了基础。