Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells

Summary: As cerebellar granule cells (GCs) coordinate the formation of regular cerebellar networks during postnatal development, molecules in GCs are expected to be involved. Here, we test the effects of the knockdown (KD) of multiple epidermal growth factor-like domains protein 11 (MEGF11), which is a homolog of proteins mediating astrocytic phagocytosis but is substantially increased at the later developmental stages of GCs on cerebellar development. MEGF11-KD in GCs of developing mice results in abnormal cerebellar structures, including extensively ectopic Purkinje cell (PC) somas, and in impaired motor functions. MEGF11-KD also causes abnormally asynchronous synaptic release from GC axons, parallel fibers, before the appearance of abnormal cerebellar structures. Interestingly, blockade of this abnormal synaptic release restores most of the cerebellar structures. Thus, apart from phagocytic functions of its related homologs in astrocytes, MEGF11 in GCs promotes proper PC development and cerebellar network formation by regulating immature synaptic transmission

Transcriptome and Proteome Co-Profiling Offers an Understanding of Pre-Harvest Sprouting (PHS) Molecular Mechanisms in Wheat (Triticum aestivum)

While wheat (Triticum aestivum L.) is a widely grown and enjoyed crop, the diverse and complex global situation and climate are exacerbating the instability of its supply. In particular, pre-harvest sprouting (PHS) is one of the major abiotic stresses that frequently occurs due to irregular climate conditions, causing serious damage to wheat and its quality. In this study, transcriptomic analysis with RNA-seq and proteomic analysis with LC-MS/MS were performed in PHS-treated spikes from two wheat cultivars presenting PHS sensitivity and tolerance, respectively. A total of 13,154 differentially expressed genes (DEGs) and 706 differentially expressed proteins (DEPs) were identified in four comparison groups between the susceptible/tolerant cultivars. Gene function and correlation analysis were performed to determine the co-profiled genes and proteins affected by PHS treatment. In the functional annotation of each comparative group, similar functions were confirmed in each cultivar under PHS treatment; however, in Keumgang PHS+7 (K7) vs. Woori PHS+7 (W7), functional annotations presented clear differences in the ”spliceosome” and ”proteasome” pathways. In addition, our results indicate that alternative splicing and ubiquitin–proteasome support the regulation of germination and seed dormancy. This study provides an advanced understanding of the functions involved in transcription and translation related to PHS mechanisms, thus enabling specific proposals for the further analysis of germination and seed dormancy mechanisms and pathways in wheat

Comprehensive Profiling of Surface Gangliosides Extracted from Various Cell Lines by LC-MS/MS

Gangliosides act as a surface marker at the outer cellular membrane and play key roles in cancer cell invasion and metastasis. Despite the biological importance of gangliosides, they have been still poorly characterized due to the lack of effective analytical tools. Herein, we performed molecular profiling and structural elucidation of intact gangliosides in various cell lines including CFPAC1, A549, NCI-H358, MCF7, and Caski. We identified and quantified a total of 76 gangliosides on cell membrane using C18 LC-MS/MS. Gangliosides found in each cell line exhibited high complexity and diversity both qualitatively and quantitatively. The most abundant species was GM3(d34:1) in CFPAC1, NCI-H358, and MCF7, while GM2(d34:1) and GM1(d34:1) were major components in A549 and Caski, respectively. Notably, glycan moieties showed more diversity between cancer cell lines than ceramide moieties. In addition, noncancerous pancreatic cell line (hTERT/HPNE) could be distinguished by gangliosides containing different levels of sialic acid compared with cancerous pancreatic cell line (CFPAC1). These results clearly demonstrated the feasibility of our analytical platform to comprehensive profile of cell surface gangliosides for identifying cell types and subgrouping cancer cell types

Image2_Advanced assessment through intact glycopeptide analysis of Infliximab’s biologics and biosimilar.pdf

Characterization of therapeutic monoclonal antibodies (mAbs) represents a major challenge for analytical sciences due to their heterogeneity associated with post-translational modifications (PTMs). The protein glycosylation requires comprehensive identification, which could influence on the mAbs’ structure and their function. Here, we demonstrated high-resolution tandem mass spectrometry with an ultra-high-performance liquid chromatography for characterization and comparison between biologics and biosimilar of infliximab at an advanced level. Comparing the N- and O-glycopeptides profiles, a total of 49 and 54 glycopeptides was identified for each product of the biologics and biosimilar, respectively. We also discovered one novel N-glycosylation site at the light chain from both biopharmaceuticals and one novel O-glycopeptide at the heavy chain from only biosimilar. Site-specific glycopeptide analysis process will be a robust and useful technique for evaluating therapeutic mAbs and complex glycoprotein products.</p

Image1_Advanced assessment through intact glycopeptide analysis of Infliximab’s biologics and biosimilar.pdf

Characterization of therapeutic monoclonal antibodies (mAbs) represents a major challenge for analytical sciences due to their heterogeneity associated with post-translational modifications (PTMs). The protein glycosylation requires comprehensive identification, which could influence on the mAbs’ structure and their function. Here, we demonstrated high-resolution tandem mass spectrometry with an ultra-high-performance liquid chromatography for characterization and comparison between biologics and biosimilar of infliximab at an advanced level. Comparing the N- and O-glycopeptides profiles, a total of 49 and 54 glycopeptides was identified for each product of the biologics and biosimilar, respectively. We also discovered one novel N-glycosylation site at the light chain from both biopharmaceuticals and one novel O-glycopeptide at the heavy chain from only biosimilar. Site-specific glycopeptide analysis process will be a robust and useful technique for evaluating therapeutic mAbs and complex glycoprotein products.</p

Cultured meat with enriched organoleptic properties by regulating cell differentiation

Abstract Research on cultured meat has primarily focused on the mass proliferation or differentiation of muscle cells; thus, the food characteristics of cultured meat remain relatively underexplored. As the quality of meat is determined by its organoleptic properties, cultured meat with similar sensory characteristics to animal-derived meat is highly desirable. In this study, we control the organoleptic and nutritional properties of cultured meat by tailoring the 2D differentiation of primary bovine myoblasts and primary bovine adipose-derived mesenchymal stem cells on gelatin/alginate scaffolds with varying stiffness. We assess the effect of muscle and adipose differentiation quality on the sensory properties of cultured meat. Thereafter, we fabricate cultured meat with similar sensory profiles to that of conventional beef by assembling the muscle and adipose constructs composed of highly differentiated cells. We introduce a strategy to produce cultured meat with enriched food characteristics by regulating cell differentiation with scaffold engineering

Image3_Advanced assessment through intact glycopeptide analysis of Infliximab’s biologics and biosimilar.pdf

Characterization of therapeutic monoclonal antibodies (mAbs) represents a major challenge for analytical sciences due to their heterogeneity associated with post-translational modifications (PTMs). The protein glycosylation requires comprehensive identification, which could influence on the mAbs’ structure and their function. Here, we demonstrated high-resolution tandem mass spectrometry with an ultra-high-performance liquid chromatography for characterization and comparison between biologics and biosimilar of infliximab at an advanced level. Comparing the N- and O-glycopeptides profiles, a total of 49 and 54 glycopeptides was identified for each product of the biologics and biosimilar, respectively. We also discovered one novel N-glycosylation site at the light chain from both biopharmaceuticals and one novel O-glycopeptide at the heavy chain from only biosimilar. Site-specific glycopeptide analysis process will be a robust and useful technique for evaluating therapeutic mAbs and complex glycoprotein products.</p

gFinder: A Web-Based Bioinformatics Tool for the Analysis of <i>N</i>‑Glycopeptides

Glycoproteins influence numerous indispensable biological functions, and changes in protein glycosylation have been observed in various diseases. The identification and characterization of glycoprotein and glycosylation sites by mass spectrometry (MS) remain challenging tasks, and great efforts have been devoted to the development of proteome informatics tools that facilitate the MS analysis of glycans and glycopeptides. Here we report on the development of gFinder, a web-based bioinformatics tool that analyzes mixtures of native <i>N</i>-glycopeptides that have been profiled by tandem MS. gFinder not only enables the simultaneous integration of collision-induced dissociation (CID) and high-energy collisional dissociation (HCD) fragmentation but also merges the spectra for high-throughput analysis. These merged spectra expedite the identification of both glycans and <i>N</i>-glycopeptide backbones in tandem MS data using the glycan database and a proteomic search tool (e.g., Mascot). These data can be used to simultaneously characterize peptide backbone sequences and possible <i>N</i>-glycan structures using assigned scores. gFinder also provides many convenient functions that make it easy to perform manual calculations while viewing the spectrum on-screen. We used gFinder to detect an additional protein (Q8N9B8) that was missed from the previously published data set containing N-linked glycosylation. For <i>N</i>-glycan analysis, we used the GlycomeDB glycan structure database, which integrates the structural and taxonomic data from all of the major carbohydrate databases available in the public domain. Thus, gFinder is a convenient, high-throughput analytical tool for interpreting the tandem mass spectra of <i>N</i>-glycopeptides, which can then be used for identification of potential missing proteins having glycans. gFinder is available publicly at http://gFinder.proteomix.org/

Table1_Advanced assessment through intact glycopeptide analysis of Infliximab’s biologics and biosimilar.XLSX

Characterization of therapeutic monoclonal antibodies (mAbs) represents a major challenge for analytical sciences due to their heterogeneity associated with post-translational modifications (PTMs). The protein glycosylation requires comprehensive identification, which could influence on the mAbs’ structure and their function. Here, we demonstrated high-resolution tandem mass spectrometry with an ultra-high-performance liquid chromatography for characterization and comparison between biologics and biosimilar of infliximab at an advanced level. Comparing the N- and O-glycopeptides profiles, a total of 49 and 54 glycopeptides was identified for each product of the biologics and biosimilar, respectively. We also discovered one novel N-glycosylation site at the light chain from both biopharmaceuticals and one novel O-glycopeptide at the heavy chain from only biosimilar. Site-specific glycopeptide analysis process will be a robust and useful technique for evaluating therapeutic mAbs and complex glycoprotein products.</p