Glycosaminoglycan remodeling during chondrogenic differentiation of human bone marrow−/synovial-derived mesenchymal stem/stromal cells under normoxia and hypoxia

Glycosaminoglycans (GAGs) are major components of cartilage extracellular matrix (ECM), which play an important role in tissue homeostasis not only by providing mechanical load resistance, but also as signaling mediators of key cellular processes such as adhesion, migration, proliferation and differentiation. Specific GAG types as well as their disaccharide sulfation patterns can be predictive of the tissue maturation level but also of disease states such as osteoarthritis. In this work, we used a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to perform a comparative study in terms of temporal changes in GAG and disaccharide composition between tissues generated from human bone marrow- and synovial-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) after chondrogenic differentiation under normoxic (21% O₂) and hypoxic (5% O₂) micromass cultures. The chondrogenic differentiation of hBMSC/hSMSC cultured under different oxygen tensions was assessed through aggregate size measurement, chondrogenic gene expression analysis and histological/immunofluorescence staining in comparison to human chondrocytes. For all the studied conditions, the compositional analysis demonstrated a notable increase in the average relative percentage of chondroitin sulfate (CS), the main GAG in cartilage composition, throughout MSC chondrogenic differentiation. Additionally, hypoxic culture conditions resulted in significantly different average GAG and CS disaccharide percentage compositions compared to the normoxic ones. However, such effect was considerably more evident for hBMSC-derived chondrogenic aggregates. In summary, the GAG profiles described here may provide new insights for the prediction of cartilage tissue differentiation/disease states and to characterize the quality of MSC-generated chondrocytes obtained under different oxygen tension culture conditions.

     

Epidemiological,Clinical and Serological Characteristics of Children with Coronavirus Disease 2019 in Wuhan: A Single-centered,Retrospective Study

In December 2019, SARS-CoV-2 was first detected in the samples obtained from three adult patients who suffered from an unknown viral pneumonia in Wuhan (Li et al. 2020). This unknown viral pneumonia is further named as coronavirus disease 2019 (COVID-19) by the World Health Organization. To date, the number of new COVID-19 cases has continued to skyrocket and the impact of SARS-CoV-2 on humans is far greater than any pathogen of this century in both breadth and depth. Previous studies have shown that adults with COVID-19 have symptoms of fever, dry cough, dyspnea, fatigue and lymphocytopenia. Moreover, COVID-19 is more likely to cause death in the elderly, especially those with chronic comorbidities (Huang et al. 2020). In Wuhan, more than 50, 000 COVID-19 cases have been confirmed, including over 780 pediatric patients, and only one child death case (Lu et al. 2020). Although the number of children cases was far fewer than that of adults, COVID-19 might endanger children's health and the information on children remains limited, especially in serological study. In the retrospective study, the investigators analyzed the epidemiological, clinical and serological characteristics of children with COVID-19 in Wuhan in the early stages of the outbreak, which might provide theoretical and practical help in controlling COVID-19 and similar emerging infectious diseases in the future.     

Quantitative analysis of severe acute respiratory syndrome (SARS)-associated coronavirus-infected cells using proteomic approaches: implications for cellular responses to virus infection

We present the first proteomic analysis on the cellular response to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. The differential proteomes of Vero E6 cells with and without infection of the SARS-CoV were resolved and quantitated with two-dimensional differential gel electrophoresis followed by ESI-MS/MS identification. Moreover isotope-coded affinity tag technology coupled with two-dimensional LC-MS/MS were also applied to the differential proteins of infected cells. By combining these two complementary strategies, 355 unique proteins were identified and quantitated with 186 of them differentially expressed (at least 1.5-fold quantitative alteration) between infected and uninfected Vero E6 cells. The implication for cellular responses to virus infection was analyzed in depth according to the proteomic results. Thus, the present work provides large scale protein-related information to investigate the mechanism of SARS-CoV infection and pathogenesis.     

The cytosolic loop of the gamma-secretase component presenilin enhancer 2 protects zebrafish embryos from apoptosis

The gamma-secretase complex, composed of presenilin, presenilin enhancer 2 (Pen-2), nicastrin, and Aph-1, catalyzes the final cleavage of amyloid precursor protein to generate the toxic amyloid beta protein, the major component of plaques in the brains of Alzheimer disease patients. To understand the in vivo function of Pen-2, we used morphant technology available in zebrafish and transiently knocked down the expression of endogenous Pen-2 by injecting the morpholino (MO) against Pen-2. Two truncated Pen-2 proteins lacking either the cytosolic or the C-terminal domain were expressed in MO-injected embryos. This deletion analysis demonstrated that the Pen-2 cytosolic loop is essential for protecting developing embryos from caspase-dependent apoptosis caused by the reduction of Pen-2. Twelve amino acids in the C terminus of Pen-2 were dispensable and could not rescue the Pen-2 knockdown-induced apoptotic phenotype. Surprisingly, double knockdown of Pen-2 and nuclear factor kappaB component p65 abrogated the single Pen-2 MO-induced caspase activation, indicating that a previously reported pro-apoptotic role of NF-kappaB in some cell types could be manifested in a whole animal and that knockdown of Pen-2 may trigger pro-apoptotic activation of NF-kappaB.     

Unique Insights in the Cervicovaginal Lactobacillus iners and L. crispatus Proteomes and Their Associations with Microbiota Dysbiosis

Background

A Lactobacillus-dominated cervicovaginal microbiota (VMB) protects women from adverse reproductive health outcomes, but the role of L. iners in the VMB is poorly understood. Our aim was to explore the association between the cervicovaginal L. iners and L. crispatus proteomes and VMB composition.

Methods

The vaginal proteomes of 50 Rwandan women at high HIV risk, grouped into four VMB groups (based on 16S rDNA microarray results), were investigated by mass spectrometry using cervicovaginal lavage (CVL) samples. Only samples with positive 16S results for L. iners and/or L. crispatus within each group were included in subsequent comparative protein analyses: Lactobacillus crispatus-dominated VMB cluster (with 16S-proven L. iners (n_i) = 0, and with 16S-proven L. crispatus (n_c) = 5), L. iners-dominated VMB cluster (n_i = 11, n_c = 4), moderate dysbiosis (n_i = 12, n_c = 2); and severe dysbiosis (n_i = 8, n_c = 2). The relative abundances of proteins that were considered specific for L. iners and L. crispatus were compared among VMB groups.

Results

Forty Lactobacillus proteins were identified of which 7 were specific for L. iners and 11 for L. crispatus. The relative abundances of L. iners DNA starvation/stationary phase protection protein (DPS), and the glycolysis enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate isomerase (GPI), were significantly decreased in women with L. iners-containing dysbiosis compared to women with a L. iners-dominated VMB, independent of vaginal pH and L. iners abundance. Furthermore, L. iners DPS, GAPDH, GPI, and fructose-bisphosphate aldolase (ALDO) were significantly negatively associated with vaginal pH. Glycolysis enzymes of L. crispatus showed a similar negative, but nonsignificant, trend related to dysbiosis.

Conclusions

Most identified Lactobacillus proteins had conserved intracellular functions, but their high abundance in CVL supernatant might imply an additional extracellular (moonlighting) role. Our findings suggest that these proteins can be important in maintaining a Lactobacillus-dominated VMB. Functional studies are needed to investigate their roles in vaginal bacterial communities and whether they can be used to prevent vaginal dysbiosis.     

Spatial phylogenetics of the Chinese angiosperm flora provides insights into endemism and conservation

The flora of China is well known for its high diversity and endemism. Identifying centers of endemism and designating conservation priorities are essential goals for biodiversity studies.However, there is no comprehensive study from a rigorous phylogenetic perspective to understand patterns of diversity and endemism and to guide biodiversity conservation in China. We conducted a spatial phylogenetic analysis of the Chinese angiosperm flora at the generic level to identify centers of neo-and pale...     

核酸适配体及其在病原微生物学中的应用

核酸适配体指利用指数富集配体系统进化技术筛选出的寡聚核苷酸片段,它可以特异性地识别靶标并与之结合,已经广泛应用于基础研究、临床诊断、纳米技术等。以下综述了适配体在微生物学方面的应用。     

A pseudo-receiver domain in Atg32 is required for mitophagy

Mitochondria are targeted for degradation by mitophagy, a selective form of autophagy. In Saccharomyces cerevisiae, mitophagy is dependent on the autophagy receptor, Atg32, an outer mitochondrial membrane protein. Once activated, Atg32 recruits the autophagy machinery to mitochondria, facilitating mitochondrial capture in phagophores, the precursors to autophagosomes. However, the mechanism of Atg32 activation remains poorly understood. To investigate this crucial step in mitophagy regulation, we examined the structure of Atg32. We have identified a structured domain in Atg32 that is essential for the initiation of mitophagy, as it is required for the proteolysis of the C-terminal domain of Atg32 and the subsequent recruitment of Atg11. The solution structure of this domain was determined by NMR spectroscopy, revealing that Atg32 contains a previously undescribed pseudo-receiver (PsR) domain. Our data suggests that the PsR domain of Atg32 regulates Atg32 activation and the initiation of mitophagy.

Abbreviations:AIM: Atg8-interacting motif; GFP: green fluorescent protein; LIR: LC3-interacting region; NMR: nuclear magnetic resonance; NOESY: nuclear Overhauser effect spectroscopy; PDB: protein data bank; PsR: pseudo-receiver; RMSD: root-mean-square deviation     

毛木耳子实体中活性多糖APPⅡA的分离纯化与结构初探

毛木耳Auricularia polytricha子实体500g经热水浸提、乙醇沉淀、Sevage法脱蛋白后,得到粗多糖3.94g,得率为0.79%。经小鼠S180载体实验证实,50mg/kg该粗多糖对肿瘤的抑制率达47.89%,呈极显著差异。利用离子交换柱对其进行分离,共得到三个部分,分别命名为APPⅠ、APPⅡ、APPⅢ。其中,APPⅡ对小鼠S180的抑制率达到56.36%,明显优于其他两部分。通过SephacrylS300凝胶过滤层析柱后,APPⅡ被进一步分离为A和B两个部分,APPⅡA的抑瘤率为53.61%,远高于APPⅡB,呈极显著差异。通过紫外光谱、红外光谱等方法对该单一多糖组分进行结构分析,表明APPⅡA为一相对分子质量约为110,000Da、可能同时具有α和β构象的杂多糖,主要有木糖和甘露糖组成,且纯度较高,含有硫酸基。