Mass spectrometry investigation of glycosylation on the NXS/T sites in recombinant glycoproteins

We used a targeted proteomics approach to investigate whether introduction of new N-linked glycosylation sites in a chimeric protein influence the glycosylation of the existing glycosylation sites. To accomplish our goals, we over-expressed and purified a chimeric construct that contained the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 (IgG-Fc-ZP3E7 protein). Immunoglobulin heavy chain (IgG-HC protein) was used as control. We then analyzed the IgG-HC and IgG-Fc-ZP3E7 proteins by liquid chromatography-tandem mass spectrometry (LC–MS/MS) and by Western blotting (WB). We concluded that in control experiments, the glycosylation site was occupied as expected. However, in the IgG-Fc-ZP3E7 protein, we concluded that only one out of three NXS/T glycosylation sites is occupied by N-linked oligosaccharides. We also concluded that in the IgG-Fc-ZP3E7 protein, upon introduction of additional potential NXS/T glycosylation sites within its sequence, the original NST/S glycosylation site from the Fc region of the IgG-Fc-ZP3E7 protein is no longer glycosylated. The biomedical significance of our findings is discussed.     

Ins and Outs of Interpreting Lipidomic Results

Membrane lipids are essential for life; however, research on how cells regulate cell lipid composition has been falling behind for quite some time. One reason was the difficulty in establishing analytical methods able to cope with the cell lipid repertoire. Development of a diversity of mass spectrometry-based technologies, including imaging mass spectrometry, has helped to demonstrate beyond doubt that the cell lipidome is not only greatly cell type dependent but also highly sensitive to any pathophysiological alteration such as differentiation or tumorigenesis. Interestingly, the current popularization of metabolomic studies among numerous disciplines has led many researchers to rediscover lipids. Hence, it is important to underscore the peculiarities of these metabolites and their metabolism, which are both radically different from protein and nucleic acid metabolism. Once differences in lipid composition have been established, researchers face a rather complex scenario, to investigate the signaling pathways and molecular mechanisms accounting for their results. Thus, a detail often overlooked, but of crucial relevance, is the complex networks of enzymes involved in controlling the level of each one of the lipid species present in the cell. In most cases, these enzymes are redundant and promiscuous, complicating any study on lipid metabolism, since the modification of one particular lipid enzyme impacts simultaneously on many species. Altogether, this review aims to describe the difficulties in delving into the regulatory mechanisms tailoring the lipidome at the activity, genetic, and epigenetic level, while conveying the numerous, stimulating, and sometimes unexpected research opportunities afforded by this type of studies.     

Sources of inocula influence mycorrhizal colonization of plants in restoration projects: a meta‐analysis

Inoculation may influence mycorrhizal colonization and provide benefits to plants in restoration projects. However, it is unclear whether inoculation has consistent effects across ecosystem types, if it has long‐term effects on colonization, and whether sources of inocula differ in their effectiveness. To address these issues, we performed a meta‐analysis of published restoration studies across a variety of ecosystems to examine the effects of mycorrhizal inoculation on mycorrhizal establishment and plant growth under field conditions. Although we included trials from a variety of geographic locations, disturbance types, and ecosystem types, the majority were based in temperate ecosystems in the Northern Hemisphere, and fewer trials were from tropical ecosystems. Across ecosystem types, we found that inoculation consistently increased the abundance of mycorrhizal fungi in degraded ecosystems, and thus improved the establishment of plants. These benefits did not significantly attenuate over time. Moreover, inocula from different sources varied in their effects on mycorrhizal colonization. Inocula sourced from reference ecosystems and inocula with specific fungal species yielded higher increases in mycorrhizal colonization than did inocula from commercial sources. These results suggest that inocula source matters, and that an initial investment into mycorrhizal inoculation could provide lasting benefits for facilitating the establishment of the below‐ and aboveground components of restored ecosystems.     

Which Metric of Relative Weight Best Captures Body Fatness in Children?

Objective: To evaluate the relative merits of BMI (kilograms per meter squared) and age‐ and gender‐adjusted BMI, age‐ and gender‐specific z score of BMI, and age‐ and gender‐specific percentiles of BMI as surrogate measures of body fatness among a sample of youth. Research Methods and Procedures: The sample comprised 596 children and adolescents 5 to 18.7 years old and was 40% male and 55% white. Height and weight were measured by trained research staff. DXA was used to determine body fat mass. BMI, age‐ and gender‐specific percentile of BMI, and age‐ and gender‐specific z scores of BMI were computed, and these metrics were compared with measured body fatness. Results: The BMI values in the sample ranged from 12.9 to 55.0 kg/m², with a mean of 24.9 kg/m². The Spearman correlations with percentage body fat were similar for all of the BMI metrics (r = 0.82 to 0.88). Linear regression models with age‐ and gender‐specific percentiles of BMI explained significantly less of the variance (65%) than models with log‐transformed BMI (81%) or age‐ and gender‐specific z scores of BMI (75% to 79%). z scores were the most accurate at classifying children who were overfat (sensitivity = 0.84, specificity = 0.96 for z score ≥1). However, using a BMI ≥85th percentile or a BMI ≥20 kg/m² was also accurate at classifying youth. Discussion: The BMI metrics had similar correlations with body fatness, but age‐ and gender‐specific percentiles of BMI were the least accurate proxy measure of body fatness. However, a BMI z score ≥1, BMI percentile ≥85, and BMI ≥20 kg/m² are all useful for identifying children who may be overfat.     

Are species–area relationships from entire archipelagos congruent with those of their constituent islands?

Aim To establish the extent to which archipelagos follow the same species–area relationship as their constituent islands and to explore the factors that may explain departures from the relationship. Location Thirty‐eight archipelagos distributed worldwide. Methods We used ninety‐seven published datasets to create island species–area relationships (ISARs) using the Arrhenius logarithmic form of the power model. Observed and predicted species richness of an archipelago and of each of its islands were used to calculate two indices that determined whether the archipelago followed the ISAR. Archipelagic residuals (ArcRes) were calculated as the residual of the prediction provided by the ISAR using the total area of the archipelago, standardized by the total richness observed in the archipelago. We also tested whether any characteristic of the archipelago (geological origin and isolation) and/or taxon accounts for whether an archipelago fits into the ISAR or not. Finally, we explored the relationship between ArcRes and two metrics of nestedness. Results The archipelago was close to the ISAR of its constituent islands in most of the cases analysed. Exceptions arose for archipelagos where (i) the slopes of the ISAR are low, (ii) observed species richness is higher than expected by the ISAR and/or (iii) distance to the mainland is small. The archipelago's geological origin was also important; a higher percentage of oceanic archipelagos fit into their ISAR than continental ones. ArcRes indicated that the ISAR underpredicts archipelagic richness in the least isolated archipelagos. Different types of taxon showed no differences in ArcRes. Nestedness and ArcRes appear to be related, although the form of the relationship varies between metrics. Main conclusions Archipelagos, as a rule, follow the same ISAR as their constituent islands. Therefore, they can be used as distinct units themselves in large‐scale biogeographical and macroecological studies. Departure from the ISAR can be used as a crude indicator of richness‐ordered nestedness, responsive to factors such as isolation, environmental heterogeneity, number and age of islands.     

一种新的肿瘤抑制因子Cylindromatosis

肿瘤抑制因子cylindromatosis(CYLD)是新发现的一种细胞因子,具有去泛素化酶活性。肿瘤坏死因子受体相关因子2(TRAF2)是NF-κB信号转导途径中的一员,TRAF2如果发生Lys48相互连接的多聚泛素化,则导致其自身的降解;如果发生Lys63相互连接的多聚泛素化,则能开启NF—κB的下游信号通路。CYLD很可能是通过把TRAF2上Lys63相互连接的多聚泛素链去掉,而在NF—κB信号转导途径中起负调节物的作用,阻止下游信号途径的转导。因此CYLD功能异常会导致NF—κB过度活化,从而引起多种病理生理反应。NF—κB的抑制剂如水杨酸钠、前列腺素A1等简单的药物试剂,有可能减弱由于CYLD缺失所引起的后果,这为头帕肿瘤综合症和其他癌症在临床治疗中所需药物的研究提供了新的策略。     

Analysis of peptides secreted from cultured mouse brain tissue

Peptides represent a major class of cell–cell signaling molecules. Most peptidomic studies have focused on peptides present in brain or other tissues. For a peptide to function in intercellular signaling, it must be secreted. The present study was undertaken to identify the major peptides secreted from mouse brain slices that were cultured in oxygenated buffer for 3–4 h. Approximately 75% of the peptides identified in extracts of cultured slices matched the previously reported peptide content of heat-inactivated mouse brain tissue, whereas only 2% matched the peptide content of unheated brain tissue; the latter showed a large number of postmortem changes. As found with extracts of heat-inactivated mouse brain, the extracts of cultured brain slices represented secretory pathway peptides as well as peptides derived from intracellular proteins such as those present in the cytosol and mitochondria. A subset of the peptides detected in the extracts of the cultured slices was detected in the culture media. The vast majority of secreted peptides arose from intracellular proteins and not secretory pathway proteins. The peptide RVD-hemopressin, a CB1 cannabinoid receptor agonist, was detected in culture media, which is consistent with a role for RVD-hemopressin as a non-classical neuropeptide. Taken together with previous studies, the present results show that short-term culture of mouse brain slices is an appropriate system to study peptide secretion, especially the non-conventional pathway(s) by which peptides produced from intracellular proteins are secreted. This article is part of a Special Issue entitled: An Updated Secretome.     

Cytodiagnosis through use of a z‐axis video by volunteer observers: a promising tool for external quality assessment

K. Yamashiro, M. Tagami, K. Azuma, A. Nakamura, O. Kato, K. Taira, M. Azuma, H. Takeda and H. Suzuki Cytodiagnosis through use of a z‐axis video by volunteer observers: a promising tool for external quality assessment Objective: This study examined whether cytological diagnosis through the use of a video, which shows the changing depth of focus in the microscopic field, described as a z‐axis video, is useful compared with a still image. Methods: From 17 cytology preparations of fine needle aspiration of the breast, we made six z‐axis videos per case. A frame exhibiting the characteristic features was then extracted from each video and saved as a representative still image. One hundred and twenty‐eight volunteer cytotechnologists were randomly divided into two groups of video observers and still image observers. The participants were asked to make a diagnosis of benign, indeterminate, suspicious or malignant without having any clinical information other than the age of the patient. Diagnoses were categorized as ‘recommended’ or ‘unacceptable’ according to degree of correlation with histology. Results: The number of definitive diagnoses of ‘benign’ or ‘malignant’ were increased in video observers, and indeterminate or suspicious categories were decreased (P = 0.013). The distribution of diagnostic categories in three of the 17 cases was significantly different; the distribution in the remaining cases was similar between the two groups. The z‐axis video observers may have selected the definite diagnoses with confidence because they observed valuable microscopic findings by ‘focusing through observation’. The average number of ‘recommended’ diagnoses by individual observers was significantly higher in the video observer group than in the still image observer group (P = 0.016). In contrast, the average number of ‘unacceptable’ diagnoses was significantly lower (P = 0.019). Conclusions: A z‐axis video is easy to obtain and is therefore expected to become a powerful diagnostic modality for the external quality assessment of clinical cytology and even in the field of primary cytodiagnosis.