Mast cells are rare tissue-resident cells of importance to human allergies. To understand the structural basis of principle mast cell functions, we analyzed the proteome of primary human and mouse ...mast cells by quantitative mass spectrometry. We identified a mast-cell-specific proteome signature, indicative of a unique lineage, only distantly related to other immune cell types, including innate immune cells. Proteome comparison between human and mouse suggested evolutionary conservation of core mast cell functions. In addition to specific proteases and proteins associated with degranulation and proteoglycan biosynthesis, mast cells expressed proteins potentially involved in interactions with neurons and neurotransmitter metabolism, including cell adhesion molecules, ion channels, and G protein coupled receptors. Toward targeted cell ablation in severe allergic diseases, we used MRGPRX2 for mast cell depletion in human skin biopsies. These proteome analyses suggest a unique role of mast cells in the immune system, probably intertwined with the nervous system.
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•Mast cell proteome is unique among immune lineages and conserved from mice to men•Mast cells express proteins potentially involved in neuroimmune interactions•Mast cells lack expression of key innate immune sensors•MRGRPX2 is a mast cell surface protein suitable for ablation of skin mast cells
Mast cell functions beyond allergic diseases remain enigmatic. To provide structural information, Plum et al. performed comprehensive proteome analyses on primary human and mouse mast cells, revealing the cells' unique lineage within the immune system, putative roles in neuroimmune interactions, and targets for antibody-mediated mast cell ablation.
The determinants that specify the genomic targets of Polycomb silencing complexes are still unclear. Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC) accelerates flowering and involves a ...cold-dependent epigenetic switch. Here we identify a single point mutation at an intragenci nucleation site within FLC that prevents this epigenetic switch from taking place. The mutation blocks nucleation of plant homeodomain–Polycomb repressive complex 2 (PHD-PRC2) and indicates a role for the transcriptional repressor VAL1 in the silencing mechanism. VAL1 localizes to the nucleation region in vivo, promoting histone deacetylation and FLC transcriptional silencing, and interacts with components of the conserved apoptosis- and splicing-associated protein (ASAP) complex. Sequence-specific targeting of transcriptional repressors thus recruits the machinery for PHD-PRC2 nucleation and epigenetic silencing.
Acquired resistance to chemotherapy remains a major stumbling block in cancer treatment. Chronic inflammation has a crucial role in induction of chemoresistance and results, in part, from the ...induction and expansion of inflammatory cells that include myeloid-derived suppressor cells (MDSCs) and IL-13
Th2 cells. The mechanisms that lead to induction of activated MDSCs and IL-13
Th2 cells have not yet been identified. Here we demonstrated that doxorubicin (DOX) treatment of 4T1 breast tumor-bearing mice led to the induction of IL-13R
miR-126a
MDSCs (DOX-MDSC). DOX-MDSC promote breast tumor lung metastasis through MDSC miR-126a
exosomal-mediated induction of IL-13
Th2 cells and tumor angiogenesis. The induction of DOX-MDSC is regulated in a paracrine manner. DOX treatment not only increases interleukin (IL)-33 released from breast tumor cells, which is crucial for the induction of IL-13
Th2 cells, but it also participates in the induction of IL-13 receptors and miR-126a expressed on/in the MDSCs. IL-13 released from IL-13
Th2 cells then promotes the production of DOX-MDSC and MDSC miR-126a
exosomes via MDSC IL-13R. MDSC miR-126a
exosomes further induce IL13
Th2 cells in a positive feed-back loop manner. We also showed that MDSC miR-126a rescues DOX-induced MDSC death in a S100A8/A9-dependent manner and promotes tumor angiogenesis. Our findings provide insight into the MDSC exosomal-mediated chemoresistance mechanism, which will be useful for the design of inhibitors targeting the blocking of induction of miR-126a
MDSCs.
Background Lysyl oxidase (LOX) is an enzyme expressed by vascular smooth muscle cells (smc) and fibroblasts, that maintains extracellular matrix stability and arterial remodeling. Previously we have ...reported human peripheral vascular disease restenosis showing increased smc and fibroblast proliferation.
Familial hypercholesterolemia (FH), with high LDL (low-density lipoprotein) cholesterol levels, is due to inherited mutations in genes, such as low-density lipoprotein receptor (LDLR). Development of ...therapeutic strategies for FH, which causes atherosclerosis and cardiovascular disease, is urgently needed.
Mice with low-density lipoprotein receptor
deletion (
mice) were used as an FH model.
mRNA was encapsulated into exosomes by forced expression of
in the donor AML12 (alpha mouse liver) cells, and the resultant exosomes were denoted as Exo
.
distribution of exosomes was analyzed by fluorescence labeling and imaging. The delivery efficiency of
mRNA was analyzed by qPCR and Western blotting. Therapeutic effects of Exo
were examined in
mice by blood lipids and Oil Red O staining.
The encapsulated mRNA was stable and could be translated into functional protein in the recipient cells. Following tail vein injection, exosomes were mainly delivered into the liver, producing abundant LDLR protein, resembling the endogenous expression profile in the wild-type mouse. Compared with control exosomes, Exo
treatment significantly decreased lipid deposition in the liver and lowered the serum LDL-cholesterol level. Significantly, the number and size of atherosclerotic plaques and inflammation were reduced in the Exo
-treated mice.
We have shown that exosome-mediated
mRNA delivery effectively restored receptor expression, treating the disorders in the
mouse. Our study provided a new therapeutic approach for the treatment of FH patients and managing atherosclerosis.
Newcastle disease virus (NDV) is the pathogen of a zoonosis that is primarily transmitted by poultry and has severe infectivity and a high fatality rate. Many studies have focused on the role of the ...NDV fusion (F) protein in the cell-cell membrane fusion process. However, little attention has been given to the heptad repeat region, HR4, which is located in the NDV F2 subunit. Here, site-directed mutants were constructed to study the function of the NDV F protein HR4 region and identify the key amino acids in this region. Nine conserved amino acids were substituted with alanine or the corresponding amino acid of other aligned paramyxoviruses. The desired mutants were examined for changes in fusogenic activity through three kinds of membrane fusion assays and expression and proteolysis through IFA, FACS and WB. The results showed that when conserved amino acids (L81, Y84, L88, L91, L92, P94, L95 and I99) were replaced with alanine, the fusogenic activity of the F protein was abolished, possibly because of failed protein expression not only on the cell surface but also inside cells. These data indicated that the conserved amino acids above in NDV F HR4 are critical for normal protein synthesis and expression, possibly for the stability of the F protein monomer, formation of trimer and conformational changes.
•We identified the key amino acids in Newcastle disease virus fusion protein HR4.•Mutations in HR4 decreased the fusogenic activity of fusion protein.•Mutations in HR4 may cause HR4 to form the α-helix unsuccessfully.•The HR4 region is involved in the synthesis and expression of fusion protein.
From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA) annually. In addition, the expiration of blockbuster biologics ...has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX) amplification technology or the glutamine synthetase (GS) system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics.
Immunohistochemistry for mismatch repair protein expression is widely used as a surrogate for microsatellite instability status-an important signature for immunotherapy and germline testing. There ...are no systematic analyses examining the sensitivity of immunohistochemistry for microsatellite instability-high status. Mismatch repair immunohistochemistry and microsatellite instability testing were performed routinely as clinically validated assays. We classified germline/somatic mutation types as truncating (nonsense, frameshift, and in/del) versus missense and predicted pathogenicity of the latter. Discordant cases were compared with concordant groups: microsatellite instability-high/mismatch repair-deficient for mutation comparison and microsatellite stable/mismatch repair-proficient for immunohistochemical comparison. 32 of 443 (7%) microsatellite instability-high cases had immunohistochemistry. Four additional microsatellite instability-high research cases had discordant immunohistochemistry. Of 36 microsatellite instability-high cases with discordant immunohistochemistry, 30 were mismatch repair-proficient, while six (five MLH1 and one MSH2) retained expression of the defective mismatch repair protein and lost its partner. In microsatellite instability-high tumors with discordant immunohistochemistry, we observed an enrichment in deleterious missense mutations over truncating mutations, with 69% (25/36) of cases having pathogenic germline or somatic missense mutations, as opposed to only 19% (7/36) in a matched microsatellite instability-high group with concordant immunohistochemistry (p = 0.0007). In microsatellite instability-high cases with discordant immunohistochemistry and MLH1 or PMS2 abnormalities, less cells showed expression (p = 0.015 and p = 0.00095, respectively) compared with microsatellite stable/mismatch repair-proficient cases. Tumor mutation burden, MSIsensor score, and truncating mismatch repair gene mutations were similar between microsatellite instability-high cases with concordant versus discordant immunohistochemical expression. Approximately 6% of microsatellite instability-high cases have retained mismatch repair protein expression and would be missed by immunohistochemistry-based testing, hindering patient access to immunotherapy. Another 1% of microsatellite instability-high cases show isolated loss of the defective gene's dimerization partner, which may lead to germline testing of the wrong gene. These cases are enriched for pathogenic mismatch repair missense mutations.
Objectives:
Femoroacetabular impingement (FAI) is considered a leading cause of early hip OA, but initial molecular mechanisms that lead FAI patients to OA progression remains to be elucidated. ...Recent articles revealed that epigenetic changes are associated with OA progression, specifically, DNA methyltransferase 3B (DNMT3B) plays a critical role on cartilage homeostasis by attenuating catabolic gene expression. This study aimed to examine the catabolic state of articular chondrocyte (ACs) during OA progression in hip FAI disease, particularly the expressions of DNMT3B and 4-aminobutyrate aminotransferase (ABAT, downstream of DNMT3B), to assess how epigenetic dysregulation contributes to OA progression.
Methods:
Full-thickness cartilage samples were collected from the impingement zone. In total, twelve patients underwent hip preservation surgery for cam FAI (early-FAI) and twelve patients underwent total hip replacement for advanced OA secondary to cam FAI (late FAI-OA) were included in this study. As a non-disease (ND) group, five healthy samples were procured from hip joint cadavers. The explants were cultured in unstimulated conditions or catabolic stimulus (IL1β). Histological analyses was performed with safranin-O/fast-green. Gene expression was analyzed via qPCR for GAPDH, DNMT3B, ABAT, MMP-13, and COL10A1. Protein expression was analyzed by western blotting analysis for DNMT3B and ABAT. Methylation specific PCR was performed to assess the methylation status at ABAT promoter (epigenetic change).
Results:
The early FAI group included younger subjects with lower Tönnis grade than the late FAI OA group; however, there were no significant differences in gender, BMI, and α-angle between groups. Cartilage samples in early-FAI and late FAI-OA showed a histological OA phenotype (Figure 1A). RT-PCR confirmed increased expression of MMP-13 and COL10A1 in early and late FAI-OA (Figure 1B), confirming cartilage degeneration, catabolism, and chondrocyte hypertrophy when compared to ND group. Interestingly, RT-PCR revealed that expression of DNMT3B gradually declined as the disease progressed (ND vs early p<0.001, early vs late p=0.016), while ABAT increased as the disease progressed (ND vs early p<0.001, early vs late p=0.035, Figure 1B). IF staining confirmed that DNMT3B was abundantly expressed in chondrocytes of normal cartilage but the expression gradually decreased with disease progression, contrary ABAT expression gradually increased as the disease progressed (Figure 1C). These results were supported by protein expression in western blotting analysis (Figure 1D). In MSP analysis, the human ABAT promoter is characterized by a typical CpG island (Figure 2A, blue area). Additionally, there was hypomethylation at the ABAT promoter during end stage of disease (ND vs early p=0.022, early vs late p=0.042) (Figure 2B). Catabolic stimulus with IL1β for early FAI and late FAI-OA, accentuated the ABAT promoter hypomethylation (early FAI: nontreatment vs IL1β p=0.056, late FAI-OA: nontreatment vs IL1β p=0.015, Figure 2C) and resulted in further overexpression of ABAT and catabolic markers and decreased expression of DNMT3B (Figure 3A-C). These findings are supported by protein expression in western-blotting analysis (Figure 3D).
Conclusions:
In the present study, histological findings were similar in early and late FAI, but DNMT3B expression levels gradually decreased with OA progression, while ABAT and catabolic marker expression levels increased, suggesting a less catabolic phenotype in the early stage of disease. MSP analysis confirmed ABAT promoter hypo-methylation (epigenetically dysregulation) as hip OA progresses. Notably, in the current study, the inflammatory stimulus with IL1β further reduced DNA methylation at the ABAT promoter and resulted in a concomitant excessive increase of ABAT and catabolic markers in human hip OA ACs. This study suggests, for the first time, that cartilage degeneration in the impingement zone in hip FAI is progressive at the molecular level and may be controlled in part by an epigenetic dysregulation. Understanding the mechanism of OA disease in hip FAI will potentially provide concomitant interventional treatment to hip pre-OA disease.
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