Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation

Aim:

Seipin is a protein that resides in endoplasmic reticulum, and involved in both lipid metabolic disorders and motor neuropathy. The aim of this study was to investigate the effects of mutant seipin on autophagy system and the morphology of lipid droplets in vitro.

Methods:

HEK-293, H1299 and MES23.5 cells were transfected with the plasmids of mutated seipin at glycosylation sites (N88S or S90L) and GFP-LC3 plasmids. The cells were subjected to immunofluorescence and flow cytometry assays, and the cell lysates were subjected to immunoblot analysis. Nile Red was used to stain the lipid droplets in the cells.

Results:

Overexpression of the mutated seipin proteins N88S or S90L activated autophagy in the 3 cell lines, and substantially altered the sub-cellular distribution of the autophagosome marker GFP-LC3, leading to a number of large vacuoles appearing in the cytoplasm. The sub-cellular location of GFP-LC3 and mutated seipin proteins highly overlapped. Moreover, and the mutated seipin proteins caused diffuse small lipid droplets to fuse into larger lipid droplets. Treatment of mutated seipin-transfected cells with the autophagy inhibitor 3-MA (5 mmol/L) facilitated the fusion of mutated seipin-induced large vacuoles. The protein glycosylation inhibitor tunicamycin could mimic the mutated seipin-induced effects, and treatment of the wild-type seipin-transfected cells with tunicamycin (2.5 μg/mL) produced similar morphological and biochemical properties as in the mutated seipin-transfected cells.

Conclusion:

The mutation of seipin at glycosylation sites disrupt its function in regulating lipid droplet metabolism, and the autophagy acts as an adaptive response to break down abnormal lipid droplets. The interruption of autophagy would accelerate the fusion of abnormal lipid droplets.     

Interaction of oxalic acid with methylamine and its atmospheric implications

Oxalic acid, which is one of the most common dicarboxylic acids, is expected to be an important component of atmospheric aerosols. However, the contribution of oxalic acid to the generation of new particles is still poorly understood. In this study, the structural characteristics and thermodynamics of (C₂H₂O₄)(CH₃NH₂)_n (n = 1–4) were investigated at the PW91PW91/6-311++G(3df,3pd) level of theory. We found that clusters formed by oxalic acid and methylamine are relatively stable, and the more the atoms participating in the formation of a ring-like structure, the more stable is the cluster. In addition, via the analysis of atmospheric relevance, it can be revealed that clusters of (C₂H₂O₄)(CH₃NH₂)_n (n = 1–4) have a noteworthy concentration in the atmosphere, which indicates that these clusters could be participating in new particle formation. Moreover, by comparison with (H₂C₂O₄)(NH₃)_n (n = 1–6) species, it can be seen that oxalic acid is more readily bound to methylamine than to ammonia, which promotes nucleation or new particle formation. Finally, the Rayleigh scattering properties of clusters of (C₂H₂O₄)(CH₃NH₂)_n (n = 1–4) were investigated for the first time to determine their atmospheric implications.

Oxalic acid, which is one of the most common dicarboxylic acids, is expected to be an important component of atmospheric aerosols.     

Hydration of the methanesulfonate–ammonia/amine complex and its atmospheric implications

Methanesulfonate (MSA⁻), found in substantial concentrations in the atmosphere, is expected to enhance aerosol nucleation and the growth of nanoparticles, but the details of methanesulfonate clusters are poorly understood. In this study, MSA⁻ was chosen along with ammonia (NH₃) or three common amines and water (H₂O) to discuss the roles of ternary homogeneous nucleation and ion-induced nucleation in aerosol formation. We studied the structural characteristics and thermodynamics of the clusters using density functional theory at the PW91PW91/6-311++G(3df,3pd) level. The analysis of noncovalent interactions predicts that the amines can form more stable clusters with MSA⁻ than NH₃, in agreement with the results from structures and thermodynamics; however, the enhancement in stability for amines is not large enough to overcome the difference in the concentrations of NH₃ and amines under typical atmospheric conditions. In addition, the favorable free energies of formation for the (MSA⁻)(NH₃/amines)(H₂O)_n (n = 0–3) clusters at 298.15 K show that MSA⁻ could contribute to the aerosol nucleation process with binding NH₃/amines and H₂O up to n = 3. There are strong temperature and humidity dependences for the formation of complexes; higher humidity and temperature promote the formation of larger hydrates. Finally, for the (MSA⁻)(NH₃/amines)(H₂O)_n clusters, the evaporation rates were determined to further investigate the atmospheric implications.

Methanesulfonate (MSA⁻), found in substantial concentrations in the atmosphere, is expected to enhance aerosol nucleation and the growth of nanoparticles, but the details of methanesulfonate clusters are poorly understood.     

The Application of Biotechnology in Medicinal Plants Breeding Research in China

Breeding is not only an important area of medicinal plants research but also the foundation for the superior varieties acquirement of medicinal plants. The rise of modern biotechnology provides good opportunities and new means for medicinal plants breeding research in China. Biotechnology shows its technical advantages and new development prospects in breeding of new medicinal plants varieties with high and stable yield, good quality, as well as stress-resistance. In this paper, we describe recent advances, problems, and development prospects about the application of modern biotechnology in medicinal plants breeding research in China.     

Applicability of the one-step DVS method for the determination of amorphous amounts for further different hydrophilic and hydrophobic drugs

In a former publication the authors showed that low amounts of amorphous content (LOQ of 0.5%) in a hydrophobic model API (Ciclesonide) can be measured with an individually adjusted one-step dynamic organic vapor sorption (DVS). In this investigation the applicability is tested on various APIs which differ in lipophilicity (poor water solubility) and hygroscopicity (absorption of water).The vapor sorption method proved to be applicable in almost all cases. Moisture sorption isotherms were determined for all five investigated crystalline and amorphous APIs. However, it was necessary to select the parameters individually for each API. The used solvents (water, methanol, isopropanol and methylene chloride) and the humidity-levels (0.05 p/p₀ until 0.5 p/p₀) were chosen carefully because otherwise the amorphous amounts switch to their crystalline counterparts and are not detectable. The production of fully amorphous samples (absence of crystalline material measured by DSC, mDSC and XRPD) was optimized over several trials. As successfully methods proved ball-milling, freeze-drying, spray-drying and/or quench cooling. In the next step these fully amorphous amounts were blended with crystalline starting material to calibration curves (Turbula blender, influence of electrostatic charge to homogeneity) for the calculation of amorphous content.In summary, the following presented methods were used to determine and quantify low amorphous amounts (between 1.5% and 17.0%) in jet-milled powders (grinding pressure of 8 bar, 1–3 grinding cycles), respectively.     

徐经世国医大师扶正祛邪治疗肝癌经验总结

国医大师徐经世教授在60余年的临床实践中总结提出肝癌的病机为正虚为本,邪实为标;早期多表现为正虚邪实,木旺土虚;病延日久累及下元,则出现水不涵木的病理表现。正虚是该病的病机关键,治疗应遵循“扶正祛邪,分期论治”的治疗原则,病初应“调和中州,培土达木”为主,病至后期则累及下元,治疗则应“滋水涵木,濡养下元”;同时强调饮食生活起居和情志调护,做到精神内守,从而使邪去正安。     

Development and validation of a custom panel including 256 Y-SNPs for Chinese Y-chromosomal haplogroups dissection

Y-chromosomal haplogroups determined by Y-chromosomal single nucleotide polymorphisms (Y-SNPs) allow paternal lineage identification and paternal biogeographic ancestry inference, which has attracted a lot of interest in the forensic community. Recently, a comprehensive Y-SNP tool with dominant markers targeting haplogroups in R, E and I branches has been reported, which allows the inference of 640 Y haplogroups. It had a very good performance and could provide a high level of Y haplogroup resolution in most populations. However, the predominant haplogroups in the Chinese populations are O, C and N, suggesting that more Y-SNPs under these clades are needed to achieve the population-specific high resolution. Herein, aiming at the Chinese population, we presented a largely improved custom Y-SNP MPS panel that contains 256 carefully ascertained Y-SNPs based on our previous studies, and evaluated this panel via a series of tests, including the tests for concordance, repeatability, sensitivity, specificity, and stability, as well as the mixture, degraded and case-type sample analysis. The preliminary developmental validation demonstrated that this panel was highly reliable, sensitive, specific, and robust. In the sensitivity test, even when the DNA input was reduced to as low as 0.5 ng, the sample could still be assigned to the correct Y haplogroup. For mixture analysis, even the 1:99 (Male: Female) mixtures had no effects on the assignation of the Y haplogroup of the male contributor. In summary, this assay has provided a high-resolution Y-chromosomal haplogrouping workflow to determine a male’s paternal lineage and/or paternal biogeographic ancestry and could be widely used for Chinese Y-chromosomal haplogroups dissection.     

微创手术与传统手术治疗气胸的对比

目的探讨自发性气胸的最佳治疗方法。方法回顾分析100例自发性气胸患者,其中50例进行微创手术治疗（电视胸腔镜手术35例;腋下小切口手术15例）,与同期进行传统开胸手术治疗50例进行对比分析。结果所有患者均全部治愈。在平均住院天数、术中出血量、术后引流量、带管时间两组差异有统计学意义（P〈0.01）;并发症无差异。结论微创手术是自发性气胸极佳治疗方法。     

不同剂量凯西莱治疗肝豆状核变性模型鼠的实验研究

目的探讨不同剂量凯西莱对肝豆状核变性模型鼠铜代谢变化的影响。方法用铜负荷饮食法复制肝豆状核变性疾病动物模型,将模型大鼠分为3个治疗组与1个模型组,3个治疗组分别给予50mg/（kg·d）、100mg/（kg·d）、200mg/（kg·d）凯西莱（静脉输注）。观察各组大鼠谷丙转氨酶（ALT）、天冬氨酸转氨酶（AST）、总蛋白、白蛋白、肝铜、24h尿铜变化情况。并观察病死率及生长发育情况。结果各治疗组病死率明显降低,100mg/kg组、200mg/（kg·d）组肝铜含量较模型组明显降低（P〈0.05或P〈0.01）,24h尿铜明显升高。结论凯西莱能降低铜负荷大鼠肝铜含量,降低其病死率。     

安徽省汉族人群HLA-A、-B、-C、-DRB1、-DQB1等位基因分布及单体型多态性研究

目的研究安徽省汉族人群HLA-A、-B、-C、-DRB1、-DQB1等位基因和单体型频率分布特征。方法 PCR-测序分型技术(SBT)对3 169例随机无血缘关系的干细胞捐献者进行HLA-A、-B、-C、-DRB1、-DQB1基因分型,利用计数法、最大期望算法和PyPop软件计算等位基因频率、单体型频率和连锁不平衡参数。结果人群共观察到411个HLA等位基因,其中HLA-A、-B、-C、-DRB1、-DQB1分别检出等位基因数量为67、143、65、75和64个。频率>0.1的等位基因有HLA-A*11∶01、A*11∶01、A*24∶02、A*02∶01、C*01∶02、C*07∶02、C*06∶02、DRB1*09∶01、DRB1*15∶01、DRB1*07∶01、DQB1* 03∶01、DQB1* 03∶03、DQB1*02∶01。发现1 426条HLA-A~HLA-B、1 772条HLA-B~HLA-DRB1和798条HLA-B~HLA-C、446条HLA-DRB1~HLA-DQB1单体型,单体型表现有连锁不平衡,其中19条表现为强连锁不平衡(RLD>0.80)。结论获得安徽省汉族人群HLA-A、-B、-C、-DRB1、-DQB1等位基因频率和单体型分布数据,其等位基因和单体型分布特征有其自身的特点。