Integrated approach to produce a recombinant, His-tagged human α-galactosidase A in mammalian cells

Successful production of recombinant proteins (r-proteins) by transient gene expression (TGE) depends on several parameters (including producer cells, culture conditions, transfection procedure, or expression vector) that should be optimized when producing any recombinant product. In this work, TGE-based production of human α-galactosidase A (GLA) is described. Producer cells, expression vectors, and parameters influencing cell metabolism after transfection have been tested. The enzyme is secreted, has the right molecular weight, and is enzymatically active. Productivities of up to 30-40 mg/L have been achieved, with a simple, fast procedure. A 6 × His tag allows enzyme purification in a single step, rendering a highly pure product. We propose a TGE-based protocol able to produce up to several milligrams per liter of highly pure, active GLA in a time as short as a few days. By this, enough amounts of engineered versions of the enzyme can be easily produced to be tested in vitro or in preclinical trials.     

Spectroscopic detection of β ‐sheet structure in nascent Aβ oligomers

Deep‐UV resonance Raman (UVRR) spectroscopy and circular dichroism (CD) were employed to study the secondary structure of Aβ(1–42) in fresh samples with increasing fractions of oligomeric peptide. A feature with a minimum at ～217 nm appeared in CD spectra of samples containing oligomeric Aβ(1–42). UVRR spectra more closely resembled those of disordered proteins. The primary difference between UVRR spectra was the ratio of the 1236 cm^–1 to 1260 cm^–1 amide III peak intensities, which shifted in favor of the 1236 cm^–1 band as the fraction of oligomeric peptide increased. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Complete mitochondrial genome of Tubulipora flabellaris (Bryozoa: Stenolaemata): the first representative from the class Stenolaemata with unique gene order

Mitochondrial genomes play a significant role in the reconstruction of phylogenetic relationships within metazoans. There are still many controversies concerning the phylogenetic position of the phylum Bryozoa. In this research, we have finished the complete mitochondrial genome of one bryozoan (Tubulipora flabellaris), which is the first representative from the class Stenolaemata. The complete mitochondrial genome of T. flabellaris is 13,763 bp in length and contains 36 genes, which lacks the atp8 gene in contrast to the typical metazoan mitochondrial genomes. Gene arrangement comparisons indicate that the mitochondrial genome of T. flabellaris has unique gene order when compared with other metazoans. The four known bryozoans complete mitochondrial genomes also have very different gene arrangements, indicates that bryozoan mitochondrial genomes have experienced drastic rearrangements. To investigate the phylogenetic relationship of Bryozoa, phylogenetic analyses based on amino acid sequences of 11 protein coding genes (excluding atp6 and atp8) from 26 metazoan complete mitochondrial genomes were made utilizing Maximum Likelihood (ML) and Bayesian methods, respectively. The results indicate the monopoly of Lophotrochozoa and a close relationship between Chaetognatha and Bryozoa. However, more evidences are needed to clarify the relationship between two groups. Lophophorate appeared to be polyphyletic according to our analyses. Meanwhile, neither analysis supports close relationship between Branchiopod and Phoronida. Four bryozoans form a clade and the relationship among them is T. flabellaris + (F. hispida + (B. neritina + W. subtorquata)), which is in coincidence with traditional classification system.     

Perturbateurs endocriniens et troubles du comportement

Prenatal environmental events that disturb neurodevelopment are suspected to increase the risk of psychiatric disorders. Estrogenic hormones such as diethylstilbestrol (DES) and environmental monomers like Bisphenol A (BPA) have the potential to disturb the development of the foetus and especially its brain. We reviewed the epidemiological studies investigating a possible association between prenatal DES or BPA exposure and risk of psychiatric disorders and discussed the hypothetical biological mechanisms linking this prenatal exposure with psychiatric disorders. The principal methodological issues that could represent confounding factors and may explain conflicting results are discussed. Interestingly, prenatal exposure to DES and BPA has been linked to epigenetic alterations associated with urogenital lesions observed in the exposed offspring, supporting the hypothesis that this environmental factor can indeed alter epigenetic regulations. Following the same line of thinking, these endocrine disruptors may modify the epigenetic mechanisms involved in neurodevelopment and, in turn, increase the occurrence of psychiatric disorders.     

原发性干燥综合征患者肠道菌群特征分析

通过比较原发性干燥综合征(pSS)患者和健康人群肠道菌群的差异, 深入探讨pSS患者肠道菌群与pSS发生发展的关系。

招募2019年1月至2021年2月于大连市中心医院风湿免疫科住院的pSS患者60名, 作为本研究的pSS组; 同时招募大连市中心医院体检中心的健康志愿者50名作为本研究的对照组(Control组)。收集两组粪便标本, 各50例, 提取细菌DNA进行16S rDNA高通量测序后进行菌群多样性和LEfSe分析。

pSS组肠道菌群α-多样性较Control组显著降低(P < 0.05)。与Control组相比, pSS组厚壁菌门/拟杆菌门比值降低; 普雷沃菌科、红蝽菌科、消化链球菌科、Eggerthellaceae, unidentified_Clostridiales丰度降低, 而拟杆菌科升高; 罗姆布茨菌属和Dorea等丰度降低, 拟杆菌属、韦荣球菌属等丰度升高。并且在pSS组肠道菌群中发现大肠埃希菌丰度明显升高。

pSS患者存在肠道菌群失调, 提示菌群失调可能与pSS的发生发展有关。

     

Heterogeneity in viral populations increases the rate of deleterious mutation accumulation

RNA viruses have high mutation rates, with the majority of mutations being deleterious. We examine patterns of deleterious mutation accumulation over multiple rounds of viral replication, with a focus on how cellular coinfection and heterogeneity in viral output affect these patterns. Specifically, using agent-based intercellular simulations we find, in agreement with previous studies, that coinfection of cells by viruses relaxes the strength of purifying selection and thereby increases the rate of deleterious mutation accumulation. We further find that cellular heterogeneity in viral output exacerbates the rate of deleterious mutation accumulation, regardless of whether this heterogeneity in viral output is stochastic or is due to variation in the cellular multiplicity of infection. These results highlight the need to consider the unique life histories of viruses and their population structure to better understand observed patterns of viral evolution.     

Origin and timing of de novo variants implicated in type 2 von Willebrand disease

Very few studies have shown the real origin and timing of de novo variants (DNV) implicated in von Willebrand disease (VWD). We investigated four families with type 2 VWD. First, we conducted linkage analysis using single nucleotide variant genotyping to recognize the possible provenance of DNV. Second, we performed amplification refractory mutation system‐quantitative polymerase chain reaction to confirm the real origin of variant (~0% mutant cells) or presence of a genetic mosaic variant (0%–50% mutant cells) in three embryonic germ layer‐derived tissues and sperm cells. Then, three possible timings of DNV were categorized based on the relative likelihood of occurrence according to the number of cell divisions during embryogenesis. Two each with type 2B VWD (proband 1 p.Arg1308Cys, proband 4 p.Arg1306Trp) and type 2A VWD (proband 2 p.Leu1276Arg, proband 3 p.Ser1506Leu) were identified. Variant origins were identified for families 1, 2 and 3 and confirmed to originate from the mother, father and father, respectively. However, the father of family 4 was confirmed to have isolated germline mosaicism with 2.2% mutant sperm cells. Further investigation confirmed the paternal grandfather to be the origin of variant. Thus, we proposed that DNV originating from the two fathers most likely occurred at the single sperm cell, the one originating from the mother occurred at the zygote during the first few cellular divisions; alternatively, in family 4, the DNV most likely occurred at the early postzygotic development in the father. Our findings are essential for understanding genetic pathogenesis and providing accurate genetic counselling.     

口腔微生物与阿尔茨海默病相关性的研究进展

口腔微生物组是人体五大菌库之一,其微生态失衡不仅在牙周炎和龋齿等口腔疾病中发挥主要作用,还与全身诸多疾病紧密相关。阿尔茨海默病（Alzheimer′s disease,AD）是一种神经系统退行性疾病,随着社会人口老龄化的加剧,其防治已成为严重的公共卫生问题。大量研究表明,AD与某些口腔致病菌及其微生态失衡显著相关。本文就口腔微生物与AD的关系作一综述,总结了口腔微生物影响AD的可能机制,并在此基础上初步推测不同的微生物与AD间的潜在关系。为进一步治疗和预防AD,人们除了关注AD患者大脑的病理改变外,还应关注患者的口腔微生态平衡。

     

Liquid–liquid phase separation of amyloid-β oligomers modulates amyloid fibrils formation

Soluble amyloid-β oligomers (AβOs) are proposed to instigate and mediate the pathology of Alzheimer’s disease, but the mechanisms involved are not clear. In this study, we reported that AβOs can undergo liquid–liquid phase separation (LLPS) to form liquid-like droplets in vitro. We determined that AβOs exhibited an α-helix conformation in a membrane-mimicking environment of SDS. Importantly, SDS is capable of reconfiguring the assembly of different AβOs to induce their LLPS. Moreover, we found that the droplet formation of AβOs was promoted by strong hydrated anions and weak hydrated cations, suggesting that hydrophobic interactions play a key role in mediating phase separation of AβOs. Finally, we observed that LLPS of AβOs can further promote Aβ to form amyloid fibrils, which can be modulated by (−)-epigallocatechin gallate. Our study highlights amyloid oligomers as an important entity involved in protein liquid-to-solid phase transition and reveals the regulatory role of LLPS underlying amyloid protein aggregation, which may be relevant to the pathological process of Alzheimer’s disease.