[Determination of the derivatization reactivity between α/ß-dicarbonyl compounds and standard citrullinated peptides based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry].

Protein citrullination is an irreversible post-translational modification process regulated by peptidylarginine deiminases (PADs) in the presence of Ca2+. This process is closely related to the occurrence and development of autoimmune diseases, cancers, neurological disorders, cardiovascular and cerebrovascular diseases, and other major diseases. The analysis of protein citrullination by biomass spectrometry confronts great challenges owing to its low abundance, lack of affinity tags, small mass-to-charge ratio change, and susceptibility to isotopic and deamidation interferences. The methods commonly used to study the protein citrullination mainly involve the chemical derivatization of the urea group of the guanine side chain of the peptide to increase the mass-to-charge ratio difference of the citrullinated peptide. Affinity-enriched labels are then introduced to effectively improve the sensitivity and accuracy of protein citrullination by mass spectrometry. 2,3-Butanedione or phenylglyoxal compounds are often used as derivatization reagents to increase the mass-to-charge ratio difference of the citrullinated peptide, and the resulting derivatives have been observed to contain α-dicarbonyl structures. To date, however, no relevant studies on the reactivity of dicarbonyl compounds with citrullinated peptides have been reported. In this study, we determined whether six α-dicarbonyl and two ß-dicarbonyl compounds undergo derivatization reactions with standard citrullinated peptides using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Among the α-dicarbonyl compounds, 2,3-butanedione and glyoxal reacted efficiently with several standard citrullinated peptides, but yielded a series of by-products. Phenylglyoxal, methylglyoxal, 1,2-cyclohexanedione, and 1,10-phenanthroline-5,6-dione also derivated efficiently with standard citrullinated peptides, generating a single derivative. Thus, a new derivatization method that could yield a single derivative was identified. Among the ß-dicarbonyl compounds, 1,3-cyclohexanedione and 2,4-pentanedione successfully reacted with the standard citrullinated peptides, and generated a single derivative. However, their reaction efficiency was very low, indicating that the ß-dicarbonyl compounds are unsuitable for the chemical derivatization of citrullinated peptides. The above results indicate that the α-dicarbonyl structure is necessary for realizing the efficient and specific chemical derivatization of citrullinated peptides. Moreover, the side chains of the α-dicarbonyl structure determine the structure of the derivatives, derivatization efficiency, and generation (or otherwise) of by-products. Therefore, the specific enrichment and precise identification of citrullinated peptides can be achieved by synthesizing α-dicarbonyl structured compounds containing affinity tags. The proposed method enables the identification of citrullinated proteins and their modified sites by MS, thereby providing a better understanding of the distribution of citrullinated proteins in different tissues. The findings will be beneficial for studies on the mechanism of action of citrullinated proteins in a variety of diseases.

Carnosic acid: an effective phenolic diterpenoid for prevention and management of cancers via targeting multiple signaling pathways.

Cancer is a serious global public health issue, and a great deal of research has been made to treat cancer. Of these, discovery of promising compounds that effectively fight cancer always has been the main point of interest in pharmaceutical research. Carnosic acid (CA) is a phenolic diterpenoid compound widely present in Lamiaceae plants such as Rosemary (Rosmarinus officinalis L.). In recent years, there has been increasing evidence that CA has significant anti-cancer activity, such as leukaemia, colorectal cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, stomach cancer, lymphoma, prostate cancer, oral cancer, etc. The potential mechanisms involved by CA, including inhibiting cell proliferation, inhibiting metastasis, inducing cell apoptosis, stimulating autophagy, regulating the immune system, reducing inflammation, regulating the gut microbiota, and enhancing the effects of other anti-cancer drugs. This article reviews the biosynthesis, pharmacokinetics and metabolism, safety and toxicity, as well as the molecular mechanisms and signaling pathways of the anticancer activity of CA. This will contribute to the development of CA or CA-containing functional foods for the prevention and treatment of cancer, providing important advances in the advancement of cancer treatment strategies.

Extraction, purification, structural characteristics and biological properties of the polysaccharides from Armillaria mellea (Vahl) P. Kumm.: A review.

Armillaria mellea (Vahl) P. Kumm. is a well-known homoeopathic plant with medicinal and culinary uses. Modern phytochemical researchers have successfully extracted and purified over 40 types of A. mellea polysaccharides (AMPs) from the fruiting bodies, hyphae and fermentation broth of A. mellea, and some of them have been analyzed and identified by their chemical structures. The impressive biological activity of these polysaccharides has been recognized by scientists worldwide. Many studies show that AMPs have remarkable antioxidant, anti-diabetic, anti-tumor, anti-inflammatory, immunoregulatory, hypolipidemic, thrombectomy, anti-aging, pulmonary protective, hepatic protective, anti-Alzheimer's properties, etc. However, the current understanding of the relationships between their chemical structure and biological activity, toxicological effects and pharmacokinetics remains limited. This article provides a systematic review of the research conducted over the past decades on the extraction and purification methods, structural characteristics, biological activity and mechanism of action of AMPs. The aim is to provide a research base that will benefit the future application of AMPs as therapeutic drugs and functional foods, and also provide insights for the further development of AMPs.

Robust Glycoproteomics Platform Reveals a Tetra-Antennary Site-Specific Glycan Capping with Sialyl-Lewis Antigen for Early Detection of Gastric Cancer.

The lack of efficient biomarkers for the early detection of gastric cancer (GC) contributes to its high mortality rate, so it is crucial to discover novel diagnostic targets for GC. Recent studies have implicated the potential of site-specific glycans in cancer diagnosis, yet it is challenging to perform highly reproducible and sensitive glycoproteomics analysis on large cohorts of samples. Here, a highly robust N-glycoproteomics (HRN) platform comprising an automated enrichment method, a stable microflow LC-MS/MS system, and a sensitive glycopeptide-spectra-deciphering tool is developed for large-scale quantitative N-glycoproteome analysis. The HRN platform is applied to analyze serum N-glycoproteomes of 278 subjects from three cohorts to investigate glycosylation changes of GC. It identifies over 20 000 unique site-specific glycans from discovery and validation cohorts, and determines four site-specific glycans as biomarker candidates. One candidate has branched tetra-antennary structure capping with sialyl-Lewis antigen, and it significantly outperforms serum CEA with AUC values > 0.89 compared against < 0.67 for diagnosing early-stage GC. The four-marker panel can provide improved diagnostic performances. Besides, discrimination powers of four candidates are also testified with a verification cohort using PRM strategy. This findings highlight the value of this strong tool in analyzing aberrant site-specific glycans for cancer detection.

Stearoyl-CoA Desaturase-1 dependent lipid droplets accumulation in cancer-associated fibroblasts facilitates the progression of lung cancer.

Rationale: Cancer-associated fibroblasts (CAFs) are the main components in the tumor microenvironment (TME) and facilitate lung cancer progression. Studies have reported that metabolic reprogramming can regulate the function of CAFs, especially abnormal lipid metabolism. Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids and have a crucial role in lipid metabolism. However, little is known about the synthesis and functions of LDs in lung CAFs. Methods: TetO-EGFRL858R; CCSP-rtTA transgenic mouse model was used to establish a spontaneous pulmonary tumor model and investigate the accumulation of LDs in CAFs. The effect of LDs accumulation on the phenotype change of fibroblasts was estimated in vitro using mouse fibroblast cell lines. RNA sequencing, Western blotting, RT-PCR, and DNA-pull down were performed to determine the mechanism of LDs synthesis in fibroblasts. Results: We found that LDs were enriched in lung CAFs and induced the pro-tumoral phenotype of CAFs with increased expression of α-smooth muscle actin (α-SMA) and Collagen alpha-2 (I) chain (COL1A2). As the main regulator, hypoxia-inducible factor-1α (HIF-1α) was highly expressed in activated fibroblasts and increased the content of LDs. RNA-sequencing results showed that Stearoyl-CoA Desaturase1 (SCD1) was a downstream gene of HIF-1α, which upregulated the number of LDs in fibroblasts. Importantly, SCD1 inhibition reduced the growth of lung tumors, which was correlated with LDs decrease in CAFs. Analysis of human lung adenocarcinoma tissue chip revealed that CAFs with a high level of SCD1 were positively correlated with the expression of HIF-1α and poor survival in lung cancer patients. Conclusions: The HIF-1α/SCD1 axis regulates the accumulation of LDs in CAFs, which might represent a novel target for lung cancer therapy.

Mass spectrometry-based draft of the mouse proteome.

The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo. Proteogenomic comparison of mouse, human and Arabidopsis reveal common and distinct mechanisms of gene expression regulation and, despite many similarities, numerous differentially abundant orthologs that likely serve species-specific functions. We leverage the mouse proteome by integrating phenotypic drug (n > 400) and radiation response data with the proteomes of 66 pancreatic ductal adenocarcinoma (PDAC) cell lines to reveal molecular markers for sensitivity and resistance. This unique atlas complements other molecular resources for the mouse and can be explored online via ProteomicsDB and PACiFIC.

[Applications of chromatography in giant complex drug-organism system].

Chromatography is an important branch of analytical chemistry that focuses on the separation and analysis of complex structures. Following more than 100 years of development and improvement, chromatography theory and technology have gradually become sophisticated. It has become a coalition of science, technology, and art. Recently, chromatography has been successfully used in combination with mass spectrometry, nuclear magnetic resonance spectroscopy, and atomic emission spectroscopy. Chromatography and the combination with other techniques has significantly improved the analysis of complex systems, such as the environment, food, petrochemicals, biological specimens, and medicine. As one of the oldest healing systems, Traditional Chinese Medicine (TCM) has served to maintain the health of people in China and worldwide for thousands of years. Therefore, it has become a core representative of traditional Chinese culture. In the past two years, TCM has been widely used to treat COVID-19, especially in patients with mild symptoms. Recently, Chinese government emphasized the inheritance and innovation of TCM and stepped up efforts to promote its modernization. TCM includes herbal medicine, acupuncture, moxibustion, massage, food therapy, and physical exercise, such as Tai Chi. In most cases, the patients are administered a mixture of TCM formulas containing more than two herbal medicines, resulting in a highly complicated compound mixture. There is no doubt that long-term clinical practices have demonstrated the safety and therapeutic effect of TCM. However, the compound mixture must be simplified to identify the active compounds. This is mainly because of the existence of carcinogenic compounds, pesticides, and heavy metal residues introduced through plantation and production processes. Moreover, enzymes within the human system generate further new compounds in response to the entry of the TCM containing thousands of components. Consequently, the complex TCM and organism systems interact with each other, constituting a giant complex drug-organism system. The analysis of this giant complex system is acknowledged as a key aspect in the modernization process of TCM. In the last 20 years, many studies have been conducted to screen and identify effective compounds in TCM. These effective compounds can be either the original compounds or new metabolic components generated in vivo. All these efforts are aimed at simplifying the components of TCM and elucidating the therapeutic mechanism. It is well known that chromatography can provide technical support for complex systems owing to its unique advantage of outstanding separation and analysis capabilities. Therefore, chromatography and its combination with other technologies have become mainstream technologies for promoting the compilation of molecular structure, information, digitalization, and modernization of TCM. This paper reviews the research and application of chromatography and combination technologies in a giant complex TCM formula-organism system. Furthermore, the authors briefly introduce and summarize the understanding, research ideas, and activities of the authors' team on the modernization of TCM. "Liang Guanxi" and "He strategy" are proposed as novel strategies for studying the giant complex drug-organism system. A distinguished technology integrated with mathematical model of causal relation, combined receptor chromatography, identification of chemical molecular structure and evaluating of pharmacological activities was established. It was successfully employed to determine the core effector-response substances of "Liang Guanxi" herb pairs in a giant complex drug-organism system. Subsequently, utilizing the proposed technology of Combination of Traditional Chinese Medicine Molecular Chemistry, the author's team designed and developed four series of innovative drugs. Inspired by the hundred years of chromatography history and thousands of years of TCM culture, the future development of chromatographic technology is expected. Furthermore, the mechanisms of TCM in medical healthcare, prevention, and treatment of diseases are likely be explained through chromatography, leading to a new strategy to realize the molecularization and digitalization of TCM, which is beneficial to the development of original new drugs.

Identification of 7 000-9 000 Proteins from Cell Lines and Tissues by Single-Shot Microflow LC-MS/MS.

A current trend in proteomics is to acquire data in a "single-shot" by LC-MS/MS because it simplifies workflows and promises better throughput and quantitative accuracy than schemes that involve extensive sample fractionation. However, single-shot approaches can suffer from limited proteome coverage when performed by data dependent acquisition (ssDDA) on nanoflow LC systems. For applications where sample quantities are not scarce, this study shows that high proteome coverage can be obtained using a microflow LC-MS/MS system operating a 1 mm i.d. × 150 mm column, at a flow-rate of 50 µL/min and coupled to an Orbitrap HF-X mass spectrometer. The results demonstrate the identification of ∼9 000 proteins from 50 µg of protein digest from Arabidopsis roots, 7 500 from mouse thymus, and 7 300 from human breast cancer cells in 3 h of analysis time in a single run. The dynamic range of protein quantification measured by the iBAQ approach spanned 5 orders of magnitude and replicate analysis showed that the median coefficient of variation was below 20%. Together, this study shows that ssDDA by µLC-MS/MS is a robust method for comprehensive and large-scale proteome analysis and which may be further extended to more rapid chromatography and data independent acquisition approaches in the future.̀.

S100A4 enhances protumor macrophage polarization by control of PPAR-γ-dependent induction of fatty acid oxidation.

BACKGROUND: The peroxisome proliferator-activated receptor γ (PPAR-γ)-dependent upregulation of fatty acid oxidation (FAO) mediates protumor (also known as M2-like) polarization of tumor-associated macrophages (TAMs). However, upstream factors determining PPAR-γ upregulation in TAM protumor polarization are not fully identified. S100A4 plays crucial roles in promotion of cancer malignancy and mitochondrial metabolism. The fact that macrophage-derived S100A4 is major source of extracellular S100A4 suggests that macrophages contain a high abundance of intracellular S100A4. However, whether intracellular S100A4 in macrophages also contributes to cancer malignancy by enabling TAMs to acquire M2-like protumor activity remains unknown. METHODS: Growth of tumor cells was evaluated in murine tumor models. TAMs were isolated from the tumor grafts in whole-body S100A4-knockout (KO), macrophage-specific S100A4-KO and transgenic S100A4WT-EGFP mice (expressing enhanced green fluorescent protein (EGFP) under the control of the S100A4 promoter). In vitro induction of macrophage M2 polarization was conducted by interleukin 4 (IL-4) stimulation. RNA-sequencing, real-time quantitative PCR, flow cytometry, western blotting, immunofluorescence staining and mass spectrometry were used to determine macrophage phenotype. Exogenous and endogenous FAO, FA uptake and measurement of lipid content were used to analyze macrophage metabolism. RESULTS: TAMs contain two subsets based on whether they express S100A4 or not and that S100A4+ subsets display protumor phenotypes. S100A4 can be induced by IL-4, an M2 activator of macrophage polarization. Mechanistically, S100A4 controls the upregulation of PPAR-γ, a transcription factor required for FAO induction during TAM protumor polarization. In S100A4+ TAMs, PPAR-γ mainly upregulates CD36, a FA transporter, to enhance FA absorption as well as FAO. In contrast, S100A4-deficient TAMs exhibited decreased protumor activity because of failure in PPAR-γ upregulation-dependent FAO induction. CONCLUSIONS: We find that macrophagic S100A4 enhances protumor macrophage polarization as a determinant of PPAR-γ-dependent FAO induction. Accordingly, our findings provide an insight into the general mechanisms of TAM polarization toward protumor phenotypes. Therefore, our results strongly suggest that targeting macrophagic S100A4 may be a potential strategy to prevent TAMs from re-differentiation toward a protumor phenotype.

HIF-1α is necessary for activation and tumour-promotion effect of cancer-associated fibroblasts in lung cancer.

Cancer-associated fibroblasts (CAFs) activation is crucial for the establishment of a tumour promoting microenvironment, but our understanding of CAFs activation is still limited. In this study, we found that hypoxia-inducible factor-1α (HIF-1α) was highly expressed in CAFs of human lung cancer tissues and mouse spontaneous lung tumour. Accordingly, enhancing the expression of HIF-1α in fibroblasts via hypoxia induced the conversion of normal fibroblasts into CAFs. HIF-1α-specific inhibitor or HIF-1α knockout (KO) significantly attenuated CAFs activation, which was manifested by the decreased expression of COL1A2 and α-SMA. In vivo, during tumour formation, the expression of Ki-67 and proliferating cell nuclear antigen (PCNA) in the tumour tissue with HIF-1α KO fibroblasts was significantly lower than that of normal fibroblasts. Moreover, HIF-1α in fibroblasts could activate the NF-κB signalling pathway and enhance a subsequent secretion of CCL5, thus promoting the tumour growth. In conclusion, our results suggest that HIF-1α is essential for the activation and tumour-promotion function of CAFs in lung cancer (LC). And targeting HIF-1α expression on CAFs may be a promising strategy for LC therapy.

Robust Microflow LC-MS/MS for Proteome Analysis: 38 000 Runs and Counting.

Microflow liquid chromatography tandem mass spectrometry (µLC-MS/MS) is becoming a viable alternative to nanoflow LC-MS/MS for the analysis of proteomes. We have recently demonstrated the potential of such a system operating with a 1 mm i.d. × 150 mm column and at a flow rate of 50 µL/min for high-throughput applications. On the basis of the analysis of ∼38 000 samples measured on two instruments over the past two years, we now show that the approach is extremely robust. Up to 1500 analyses were performed within one month, and >14 000 samples could be analyzed on a single column without loss of chromatographic performance. Samples included proteomes of cell lines, tissues, and human body fluids, which were analyzed with or without prior peptide fractionation or stable isotope labeling. We show that the µLC-MS/MS system is capable of measuring 2600 proteins from undepleted human plasma and ∼5000 proteins from crude human urine in 1 day, demonstrating its potential for in-depth as well as high-throughput clinical application.

Biomimetic and osteogenic 3D silk fibroin composite scaffolds with nano MgO and mineralized hydroxyapatite for bone regeneration.

Artificial bioactive materials have received increasing attention worldwide in clinical orthopedics to repair bone defects that are caused by trauma, infections or tumors, especially dedicated to the multifunctional composite effect of materials. In this study, a weakly alkaline, biomimetic and osteogenic, three-dimensional composite scaffold (3DS) with hydroxyapatite (HAp) and nano magnesium oxide (MgO) embedded in fiber (F) of silkworm cocoon and silk fibroin (SF) is evaluated comprehensively for its bone repair potential in vivo and in vitro experiments, particularly focusing on the combined effect between HAp and MgO. Magnesium ions (Mg2+) has long been proven to promote bone tissue regeneration, and HAp is provided with osteoconductive properties. Interestingly, the weak alkaline microenvironment from MgO may also be crucial to promote Sprague-Dawley (SD) rat bone mesenchymal stem cells (BMSCs) proliferation, osteogenic differentiation and alkaline phosphatase (ALP) activities. This SF/F/HAp/nano MgO (SFFHM) 3DS with superior biocompatibility and biodegradability has better mechanical properties, BMSCs proliferation ability, osteogenic activity and differentiation potential compared with the scaffolds adding HAp or MgO alone or neither. Similarly, corresponding meaningful results are also demonstrated in a model of distal lateral femoral defect in SD rat. Therefore, we provide a promising 3D composite scaffold for promoting bone regeneration applications in bone tissue engineering.

Low SCRIB expression in fibroblasts promotes invasion of lung cancer cells.

AIMS: Cancer associated fibroblasts (CAFs) play a crucial role in lung tumor development, but the underlying mechanism is still not fully understood. MAIN METHODS: SCRIB expression in the CAFs of human lung cancer tissues was examined by immunohistochemistry (IHC). A coculture of mouse Lewis lung cancer cells (LLC) and fibroblasts was used to investigate SCRIB expression in cocultured fibroblasts. Proliferation, scratch wound, and transwell assays were used to examine the proliferation, migration and invasion ability of SCRIB knockdown fibroblasts and their effects on LLC. A 3D-coculture system and co-injection xenograft model were used to examine LLC invasion. RNA sequencing and transwell experiments were used to explore the molecules that may participate in LLC invasion. KEY FINDINGS: Herein, we found that the low expression of SCRIB in CAFs is correlated with advanced tumor stages and poor survival for human lung squamous cell carcinoma. SCRIB expression in fibroblasts is drastically downregulated by LLC cells. SCRIB knockdown fibroblasts not only enhance invasion but also facilitate LLC invasion in a 3D-coculture system and in an in vivo subcutaneous transplantation model. The upregulation of asporin in SCRIB knockdown fibroblasts is involved in LLC invasion in vitro. SIGNIFICANCE: Collectively, the results indicate that fibroblasts with low SCRIB expression promote lung cancer cell invasion, which suggests that the downregulated expression of SCRIB may represent one of the important characteristics of tumor-promoting CAFs in lung squamous cell cancer.

Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC-MS/MS.

Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC-MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC-MS/MS using a 1 × 150 mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.5% CV) using data from >2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. The system identifies >30,000 phosphopeptides in 12 h and protein-protein or protein-drug interaction experiments can be analyzed in 20 min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC-MS/MS is suitable for a broad range of proteomic applications.

Recurrent GNAQ mutation encoding T96S in natural killer/T cell lymphoma.

Natural killer/T cell lymphoma (NKTCL) is a rare and aggressive malignancy with a higher prevalence in Asia and South America. However, the molecular genetic mechanisms underlying NKTCL remain unclear. Here, we identify somatic mutations of GNAQ (encoding the T96S alteration of Gαq protein) in 8.7% (11/127) of NKTCL patients, through whole-exome/targeted deep sequencing. Using conditional knockout mice (Ncr1-Cre-Gnaqfl/fl), we demonstrate that Gαq deficiency leads to enhanced NK cell survival. We also find that Gαq suppresses tumor growth of NKTCL via inhibition of the AKT and MAPK signaling pathways. Moreover, the Gαq T96S mutant may act in a dominant negative manner to promote tumor growth in NKTCL. Clinically, patients with GNAQ T96S mutations have inferior survival. Taken together, we identify recurrent somatic GNAQ T96S mutations that may contribute to the pathogenesis of NKTCL. Our work thus has implications for refining our understanding of the genetic mechanisms of NKTCL and for the development of therapies.

Novel phenotype-genotype correlations of hypertrophic cardiomyopathy caused by mutation in α-actin and myosin-binding protein genes in three unrelated Chinese families.

BACKGROUND: The correlations between genotype and phenotype in hypertrophic cardiomyopathy (HCM) have not been established. Mutation of α-actin gene (ACTC1) is a rare cause of HCM. This study aimed to explore novel genotype-phenotype correlations in HCM patients with the variants in ACTC1 and myosin-binding protein (MYBPC3) genes in three unrelated Chinese families. METHODS: Clinical, electrocardiographic, and echocardiographic examinations were performed in three Han pedigrees. Exon and boarding intron analysis of 96 cardio-disease-related genes was performed using second-generation sequencing on three probands. The candidate variants were validated in 14 available family members and 300 unrelated healthy controls by bi-directional Sanger sequencing. The pathogenicity and conservation were calculated using MutationTaster, PolyPhen-2, SIFT, and Clustal X. Pathogenicity classification of the variants was based on American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS: Nine members fulfilled diagnostic criteria for HCM with clinical characteristics, electrocardiographic, and echocardiographic findings. Two candidate variants in ACTC1 p.Asp26Asn (ACTC1-D26N) and MYBPC3 p.Arg215Cys (MYBPC3-R215C) were identified in patients. Only ACTC1-D26N strongly co-segregated with the HCM phenotype. Seven patients who harbored variant ACTC-D26N only were diagnosed with non-obstructive HCM, and four of these patients exhibited a triphasic left ventricular (LV) filling pattern. Two patients carrying both ACTC1-D26N and MYBPC3-R215C variants showed a higher LV outflow tract pressure gradient. Bioinformatics analysis revealed that the two variants were deleterious and highly conserved across species. According to ACMG guidelines, ACTC1-D26N is classified as a likely pathogenic mutation. The second variation MYBPC3-R215C may function as a genetic modifier, which remains uncertain here. CONCLUSIONS: Novel p.(Asp26Asn) mutation of ACTC1 was associated with HCM phenotype, and the penetrance is extremely high (∼81.8%) in adults. The second variation, MYBPC3-R215C may function as a genetic modifier, which remains uncertain here.

S100A4+ Macrophages Are Necessary for Pulmonary Fibrosis by Activating Lung Fibroblasts.

S100A4, a calcium-binding protein, can promote pulmonary fibrosis via fibroblast activation. Due partly to its various cellular origins, the exact role of S100A4 in the development of lung fibrosis remains elusive. Here, we show that in the bronchoalveolar lavage fluid, numbers of S100A4+ macrophages correlated well with S100A4 protein levels and occurrence of idiopathic pulmonary fibrosis (IPF) in patients. A mouse model of bleomycin-induced pulmonary fibrosis demonstrated S100A4+ macrophages as main source for extracellular S100A4 in the inflammatory phase. In vitro studies revealed that extracellular S100A4 could activate both mouse and human lung fibroblasts by upregulation of α-SMA and type I collagen, during which sphingosine-1-phosphate (S1P) increased. Inhibiting the S1P receptor subtypes S1P1/S1P3 abrogated fibroblast activation. Accordingly, absence or neutralization of S100A4 significantly attenuated bleomycin-induced lung fibrosis in vivo. Importantly, adoptive transfer of S100A4+ but not of S100A4- macrophages installed experimental lung injury in S100A4-/- mice that were otherwise not sensitive to fibrosis induction. Taken together, S100A4 released by macrophages promotes pulmonary fibrosis through activation of lung fibroblasts which is associated with S1P. This suggests that extracellular S100A4 or S100A4+ macrophages within the lung as promising targets for early clinical diagnosis or therapy of IPF.

[Preparation of immobilized ß-glucuronidase enzyme reactor for the analysis of O-glucuronides of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol].

An immobilized ß-glucuronidase enzyme reactor (IMER) based on organic-silica gel hybridized capillary column was prepared by the sol-gel method with acrylamide (AAM) as the organic functional monomer. The effects of the amounts of tetramethyl orthosilicate (TMOS), 3-glycidoxypropyltrimethoxysilane (γ-MAPS), acrylamide (AAM), polyethylene glycol (PEG), and the water bath temperature on the quality of the microscopic structure were studied. After optimization,the monolithic column with good permeability and high mechanical strength was obtained. Then ß-glucuronidase was covalently immobilized, and the hydrolysis efficiency of the ß-glucuronidase IMER was investigated. As the substrate, O-glucuronides of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL-O-Gluc) were hydrolyzed at room temperature, indicating the high hydrolysis efficiency of the ß-glucuronidase IMER, which demonstrated its high potential for the high throughput analysis of tobacco metabolites in the future.

Diazoxide ameliorates severity of experimental osteoarthritis by activating autophagy via modulation of the osteoarthritis-related biomarkers.

Accumulating evidence suggests that autophagy plays a protective role in chondrocytes and prevents cartilage degeneration in osteoarthritis (OA). The objective of this study was to investigate the effect of diazoxide on chondrocyte death and cartilage degeneration and to determine whether these effects are correlated to autophagy in experimental OA. In this study, a cellular OA model was established by stimulating SW1353 cells with interleukin 1ß. A rat OA model was generated by transecting the anterior cruciate ligament combined with the resection of the medial menisci, followed by treatment with diazoxide or diazoxide combination with 3-methyladenine. The percentage of viable cells was evaluated using calcein-acetoxymethyl/propidium iodide double staining. The messenger RNA expression levels of collagen type II alpha 1 chain (COL2A1), matrix metalloproteinase 13 (MMP-13), TIMP metallopeptidase inhibitor 1 (TIMP-1), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were determined using quantitative real-time polymerase chain reaction. The cartilage thickness and joint space were evaluated using ultrasound. SW1353 cell degeneration and autophagosomes were observed using transmission electron microscopy. The expression levels of microtubule-associated protein 1 light chain 3 (LC3), beclin-1, P62, COL2A1, and MMP-13 were evaluated using immunofluorescence staining and Western blot analysis. Diazoxide significantly attenuated articular cartilage degeneration and SW1353 cell death in experimental OA. The restoration of autophagy was observed in the diazoxide-treated group. The beneficial effects of diazoxide were markedly blocked by 3-methyladenine. Diazoxide treatment also modulated the expression levels of OA-related biomarkers. These results demonstrated that diazoxide exerted a chondroprotective effect and attenuated cartilage degeneration by restoring autophagy via modulation of OA-related biomarkers in experimental OA. Diazoxide treatment might be a promising therapeutic approach to prevent the development of OA.

Identifying tumor promoting genomic alterations in tumor-associated fibroblasts via retrovirus-insertional mutagenesis.

Tumor-associated fibroblasts (TAFs) are often essential for solid tumor growth. However, few genetic or epigenetic alterations have been found in TAFs during the progression of solid tumors. Employing a tumor-stromal cell co-injection model, we adapted here retroviral-insertional mutagenesis to stromal cells to identify novel tumor-associated genes in TAFs. We successfully identified 20 gene candidates that might modulate tumor growth if altered in TAFs at genomic level. To validate our finding, the function of one of the candidate genes, tubulin tyrosine ligase (Ttl), was further studied in TAFs from fibrosarcoma, colon, breast and hepatocarcinoma. We demonstrated that down-regulated TTL expression in TAFs indeed promoted tumor growth in mice. Interestingly, decreased expression of TTL in tumor stromal cells also correlated with poor outcome in human colon carcinoma. Thus, the co-injection model of tumor cells with retrovirus-modified fibroblasts proved a valid method to identify tumor-modulating genes in TAFs, allowing for a deeper insight into the role of the stroma for tumor development.