To investigate the role of microRNA-145, that regulates gene expression of genes related to differentiation, proliferation and the phenotype of smooth muscle cells (SMCs), in the differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) to SMCs.
Results
Real-time PCR analysis indicated significant upregulation of SMC markers, including SM-α-actin, calponin, caldesmon and SMMHC, in SMCs compared to hBM-MSCs. Conversely, Krüppel-like factor 4, the direct target of microRNA-145 and the suppressor of smooth muscle differentiation, was suppressed in hBM-MSC-derived SMCs. Western blot analysis and immunocytochemistry also confirmed that the introduction of microRNA-145 into hBM-MSCs induced mature contractile SMCs. The functionality of hBM-MSC-derived SMCs was assessed by proliferation assay using PDGF-BB and contractility assay using carbachol. The results showed that the produced SMCs contracted in response to carbachol stimulation.
Conclusion
Overexpression of microRNA-145 in undifferentiated hBM-MSCs results in functionally mature contractile SMCs that can be used in drug discovery and cell therapy in SMC disorders such as vascular disease.
The role of the cytoskeleton in regulating mitochondrial distribution in dividing mammalian cells is poorly understood. We previously demonstrated that mitochondria are transported to the cleavage furrow during cytokinesis in a microtubule-dependent manner. However, the exact subset of spindle microtubules and molecular machinery involved remains unknown.
Methods
We employed quantitative imaging techniques and structured illumination microscopy to analyse the spatial and temporal relationship of mitochondria with microtubules and actin of the contractile ring during cytokinesis in HeLa cells.
Results
Superresolution microscopy revealed that mitochondria were associated with astral microtubules of the mitotic spindle in cytokinetic cells. Dominant-negative mutants of KIF5B, the heavy chain of kinesin-1 motor, and of Miro-1 disrupted mitochondrial transport to the furrow. Live imaging revealed that mitochondrial enrichment at the cell equator occurred simultaneously with the appearance of the contractile ring in cytokinesis. Inhibiting RhoA activity and contractile ring assembly with C3 transferase, caused mitochondrial mislocalisation during division.
Conclusions
Taken together, the data suggest a model in which mitochondria are transported by a microtubule-mediated mechanism involving equatorial astral microtubules, Miro-1, and KIF5B to the nascent actomyosin contractile ring in cytokinesis.
Vascular smooth muscle cells (VSMCs) are mature cells that play critical roles in both normal and aberrant cardiovascular conditions. In response to various environmental cues, VSMCs can dedifferentiate from a contractile state to a highly proliferative synthetic state through the so-called ‘phenotypic switching’ process. Changes in VSMC phenotype contribute to numerous vascular-related diseases, including atherosclerosis, calcification, and restenosis following angioplasty. Adventitial VSMC progenitor cells also contribute to formation of the neointima.
Methods/Results
Herein, we review both, the roles of VSMC differentiation in vascular diseases, and the in vitro models used to investigate the molecular mechanisms involved in the regulation of VSMC differentiation and phenotype modulation.
Conclusion
A comprehensive understanding of VSMC behavior in vascular diseases is essential to identify new therapeutic targets for the prevention and treatment of cardiovascular diseases.
Asthma is characterized by chronic airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling. While exposure of house dust mites (HDM) is a common cause of asthma, the pathogenesis of the HDM-induced asthma is not fully understood. Bronchopulmonary C-fibers (PCFs) contribute to the neurogenic inflammation, viral infection induced-persistent AHR, and ovalbumin induced collagen deposition largely via releasing neuropeptides, such as substance P (SP). However, PCF roles in the pathogenesis of the HDM-induced asthma remain unexplored. The goal of this study was to determine what role PCFs played in generating these characteristics.
Methods
We compared the following variables among the PCF-intact and -degenerated BALB/c mice with and without chronic HDM exposure (four groups): 1) AHR and pulmonary SP; 2) airway smooth muscle (ASM) mass; 3) pulmonary inflammatory cells; and 4) epithelium thickening and mucus secretion.
Results
We found that HDM evoked AHR associated with upregulation of pulmonary SP and inflammation, ASM mass increase, epithelium thickenings, and mucus hypersecretion. PCF degeneration decreased the HDM-induced changes in AHR, pulmonary SP and inflammation, and ASM mass, but failed to significantly affect the epithelium thickening and mucus hypersecretion.
Conclusion
Our data suggest an involvement of PCFs in the mechanisms by which HDM induces allergic asthma via airway inflammation, AHR, and airway remodeling.
Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed.
Objectives
To study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions.
Methods
We present a double-labelling LC–MS approach with a 13C-labeled yeast cellular extract as complex surrogate matrix, and 13C15N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates.
Results
While nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions.
Conclusions
Extraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study.
Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.
Objectives
In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.
Methods
The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.
Results
A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.
Conclusion
The workflow generated repeatable and informative fingerprints for robust metabolome characterization.
The relationship between genetic variation in gene expression and phenotypic variation observable in nature is not well understood. Identifying how many phenotypes are associated with differences in gene expression and how many gene-expression differences are associated with a phenotype is important to understanding the molecular basis and evolution of complex traits.
Results
We compared levels of gene expression among nine natural isolates of Saccharomyces cerevisiae grown either in the presence or absence of copper sulfate. Of the nine strains, two show a reduced growth rate and two others are rust colored in the presence of copper sulfate. We identified 633 genes that show significant differences in expression among strains. Of these genes, 20 were correlated with resistance to copper sulfate and 24 were correlated with rust coloration. The function of these genes in combination with their expression pattern suggests the presence of both correlative and causative expression differences. But the majority of differentially expressed genes were not correlated with either phenotype and showed the same expression pattern both in the presence and absence of copper sulfate. To determine whether these expression differences may contribute to phenotypic variation under other environmental conditions, we examined one phenotype, freeze tolerance, predicted by the differential expression of the aquaporin gene AQY2. We found freeze tolerance is associated with the expression of AQY2.
Conclusions
Gene expression differences provide substantial insight into the molecular basis of naturally occurring traits and can be used to predict environment dependent phenotypic variation.
Stable isotopic labeling experiments are powerful tools to study metabolic pathways, to follow tracers and fluxes in biotic and abiotic transformations and to elucidate molecules involved in metal complexing.
Objective
To introduce a software tool for the identification of isotopologues from mass spectrometry data.
Methods
DeltaMS relies on XCMS peak detection and X13CMS isotopologue grouping and then analyses data for specific isotope ratios and the relative error of these ratios. It provides pipelines for recognition of isotope patterns in three experiment types commonly used in isotopic labeling studies: (1) search for isotope signatures with a specific mass shift and intensity ratio in one sample set, (2) analyze two sample sets for a specific mass shift and, optionally, the isotope ratio, whereby one sample set is isotope-labeled, and one is not, (3) analyze isotope-guided perturbation experiments with a setup described in X13CMS.
Results
To illustrate the versatility of DeltaMS, we analyze data sets from case-studies that commonly pose challenges in evaluation of natural isotopes or isotopic signatures in labeling experiment. In these examples, the untargeted detection of sulfur, bromine and artificial metal isotopic patterns is enabled by the automated search for specific isotopes or isotope signatures.
Conclusion
DeltaMS provides a platform for the identification of (pre-defined) isotopologues in MS data from single samples or comparative metabolomics data sets.
Metastasis is the primary cause of mortality in cancer patients. Therefore, elucidating the genetics and epigenetics of metastatic tumor cells and the mechanisms by which tumor cells acquire metastatic properties constitute significant challenges in cancer research.
Objective
To summarize the current understandings of the specific genotype and phenotype of the metastatic tumor cells.
Method and Result
In-depth genetic analysis of tumor cells, especially with advances in the next-generation sequencing, have revealed insights of the genotypes of metastatic tumor cells. Also, studies have shown that the cancer stem cell (CSC) and epithelial to mesenchymal transition (EMT) phenotypes are associated with the metastatic cascade.
Conclusion
In this review, we will discuss recent advances in the field by focusing on the genomic instability and phenotypic dynamics of metastatic tumor cells.
Recently, measuring phenotype similarity began to play an important role in disease diagnosis. Researchers have begun to pay attention to develop phenotype similarity measurement. However, existing methods ignore the interactions between phenotype-associated proteins, which may lead to inaccurate phenotype similarity.
Results
We proposed a network-based method PhenoNet to calculate the similarity between phenotypes. We localized phenotypes in the network and calculated the similarity between phenotype-associated modules by modeling both the inter- and intra-similarity.
Conclusions
PhenoNet was evaluated on two independent evaluation datasets: gene ontology and gene expression data. The result shows that PhenoNet performs better than the state-of-art methods on all evaluation tests.
Data sharing is being increasingly required by journals and has been heralded as a solution to the ‘replication crisis’.
Objectives
(i) Review data sharing policies of journals publishing the most metabolomics papers associated with open data and (ii) compare these journals’ policies to those that publish the most metabolomics papers.
Methods
A PubMed search was used to identify metabolomics papers. Metabolomics data repositories were manually searched for linked publications.
Results
Journals that support data sharing are not necessarily those with the most papers associated to open metabolomics data.
Conclusion
Further efforts are required to improve data sharing in metabolomics.
Thus far, a handful of genes have been shown to be related to the wing maturation process in insects. A novel heme peroxidase enzyme known as curly suppressor (Cysu)(formerly CG5873), have been characterized in this report because it is involved in wing morphogenesis. Using bioinformatics tools we found that Cysu is remarkably conserved in the genus Drosophila (>95%) as well as in invertebrates (>70%), although its vertebrate orthologs show poor homology. Time-lapse imaging and histochemical analyses have confirmed that the defective wing phenotype of Cysu is not a result of any underlying cellular alterations; instead, its wings fail to expand in mature adults.
Results
The precise requirement of Cysu in wings was established by identifying a bona fide mutant of Cysu from the Bloomington Drosophila Stock Centre collection. Its requirement in the wing has also been shown by RNA knockdown of the gene. Subsequent transgenic rescue of the mutant wing phenotype with the wild-type gene confirmed the phenotype resulting from Cysu mutant. With appropriate GAL4 driver like engrailed-GAL4, the Cysu phenotype was compartmentalized, which raises a strong possibility that Cysu is not localized in the extracellular matrix (ECM); hence, Cysu is not engaged in bonding the dorsal and ventral cuticular layers. Finally, shortened lifespan of the Cysu mutant suggests it is functionally essential for other biological processes as well.
Conclusion
Cysu, a peroxinectin-like gene, is required during the wing maturation process in Drosophila because as a heme peroxidase, Cysu is capable of utilizing H2O2, which plays an essential role in post-eclosion wing morphogenesis.
To investigate the effects of ultrasmall superparamagnetic iron oxide (USPIO) labeling on the maturity or immune tolerance of immature dendritic cells (imDCs) as the success of immunotherapy with immature dendritic cells is highly dependent on immune tolerance.
Results
The feasibility of tracking implanted USPIO-labeled imDCs in vivo by magnetic resonance imaging (MRI) was explored. The effects of USPIO labeling on the immune tolerance of imDCs was examined. USPIO when higher than 200 μg/ml caused considerable damage to imDCs, induced imDC maturation, and impacted the immune tolerance of imDCs. USPIO labeling caused a dose-dependent increase in autophagosome formation in imDCs, and autophagy inhibitors prevented the maturation of imDCs while stimulating their immune tolerance.
Conclusions
We speculate that high concentrations of USPIO can be used to induce imDC maturation, and that this process is likely mediated through an autophagy-related pathway.
In recent years the visualization of biomagnetic measurement data by so-called pseudo current density maps or Hosaka-Cohen (HC) transformations became popular.
Methods
The physical basis of these intuitive maps is clarified by means of analytically solvable problems.
Results
Examples in magnetocardiography, magnetoencephalography and magnetoneurography demonstrate the usefulness of this method.
Conclusion
Hardware realizations of the HC-transformation and some similar transformations are discussed which could advantageously support cross-platform comparability of biomagnetic measurements.
The chemical sensitivity of urine metabolomics analysis is greatly compromised due to the large amounts of inorganic salts in urine (NaCl, KCl), which are detrimental to analytical instrumentation, e.g. chromatographic columns or mass spectrometers. Traditional desalting approaches applied to urine pretreatment suffer from the chemical losses, which reduce the information depth of analysis.
Objectives
We aimed to test a simple approach for the simultaneous preconcentration and desalting of organic solutes in urine based on the collection of induced bursting bubble aerosols above the surface of urine samples.
Method
Bursting bubbles were generated at ambient conditions by feeding gas through an air diffuser at the bottom of diluted (200 times in ultrapure water) urine solution (50–500 mL). Collected aerosols were analyzed by the direct-infusion electrospray ionization mass spectrometry (ESI–MS).
Results
The simultaneous preconcentration (ca. 6–12 fold) and desalting (ca. six–tenfold) of organic solutes in urine was achieved by the bursting bubble sample pretreatment, which allowed ca. three-times higher number of identified urine metabolites by high-resolution MS analysis. No chemical losses due to bubbling were observed. The increased degree of MS data clustering was demonstrated on the principal component analysis of data sets from the urine of healthy people and from the urine people with renal insufficiency. At least ten times higher sensitivity of trace drug detection in urine was demonstrated for clenbuterol and salbutamol.
Conclusion
Our results indicate the high versatility of bubble bursting as a simple pretreatment approach to enhance the chemical depth and sensitivity of urine analysis. The approach could be attractive for personalized medicine as well as for the diagnostics of renal disorders of different etiology (diabetic nephropathy, chronic renal failure, transplant-associated complications, oncological disorders).
Graphical Abstract
Urine desalting and preconcentration in bursting bubbles.
Untargeted metabolomics is a powerful tool for biological discoveries. To analyze the complex raw data, significant advances in computational approaches have been made, yet it is not clear how exhaustive and reliable the data analysis results are.
Objectives
Assessment of the quality of raw data processing in untargeted metabolomics.
Methods
Five published untargeted metabolomics studies, were reanalyzed.
Results
Omissions of at least 50 relevant compounds from the original results as well as examples of representative mistakes were reported for each study.
Conclusion
Incomplete raw data processing shows unexplored potential of current and legacy data.
We aimed to identify new high-yield dextranase strains and study the catalytic potential of dextranase from the strain in industrial applications.
Results
Dextranase-producing strains were screened from soil samples, and a potential strain was identified as Chaetomium globosum according to its phenotype, biochemical characteristics, and rDNA analysis. Crude dextranase was purified to reach 10.97-fold specific activity and 18.7% recovery. The molecular weight of the enzyme was 53 kDa with an optimum temperature and pH of 60 °C and 5.5, respectively. Enzyme activity was stable at pH 4.0–7.0 and displayed sufficient thermal stability at temperatures?<?50 °C. Mn2+ (10 mM) enhanced dextranase activity by 134.44%. The enzyme was identified as an endodextranase. It displayed very high hydrolytic affinity toward high-molecular weight dextran T2000, reaching 97.9% hydrolysis within 15 min at 2 U/mL.
Conclusion
Collectively, these results suggest that Chaetomium globosum shows higher production and specificity of dextranase than that from other reported strains. These findings may offer new insights into the potential of dextranase in the sugar, medical, and food industries.
In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However, undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study, we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients.
Methods
Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated, mucus-secreting and basal cells, and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally, epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay.
Results
Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also, immunofluorescence analysis revealed the presence of ciliated, mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight.
Conclusion
In summary, primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated, mucus-producing and basal cells, which adequately reflect the in vivo properties of the human respiratory epithelium.
Plasma fatty acids are derived from preformed sources in the diet and de novo synthesis through the action of desaturase and elongase enzymes.
Objective
This study was designed to examine the elongation of gamma-linolenic acid (GLA, 18:3n6) into dihomo-gamma-linolenic acid (DGLA, 20:3n6) over an 8-h period using both targeted gas chromatography–flame ionization detection and untargeted liquid chromatography–mass spectrometry-based lipidomics utilizing the sequential window acquisition of all theoretical fragment-ion spectra (SWATH).
Methods
In a single blind, placebo-controlled, crossover design, seven healthy subjects consumed a test meal that consisted of GLA fat (borage oil) or a control fat (a mixture of corn, safflower, sunflower and extra-virgin light olive oils) on three separate test days for each test meal.
Results
Total plasma fatty acid concentrations and 366 unique lipid species were measured at 0, 2, 4, 6 and 8 h in response to the test meals. Mean plasma 18:3n6 was 7-fold higher to the GLA challenge compared with baseline and the control meal. By 8 h, mean plasma 20:3n6 was significantly higher in response to the GLA test meal than baseline and the control group. Five of the seven subjects were “responders” in converting GLA into DGLA, but two subjects did not show this conversion. The conversion was independent of physical activity level.
Conclusion
Using polyunsaturated fatty acid metabolism as an example, this study demonstrates inter-individual differences in enzymatic capacities to inform exact nutritional and metabolic phenotyping that could be used for precision medicine.
To determine the role of microRNA-15b (miR-15b) in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation in the nucleus pulposus (NP).
Results
MiR-15b was up-regulated in degenerative NP tissues and in IL-1β-stimulated NP cells, as compared to the levels in normal controls (normal tissue specimens from patients with idiopathic scoliosis). Bioinformatics and luciferase activity analyses showed that mothers against decapentaplegic homolog 3 (SMAD3), a key mediator of the transforming growth factor-β signaling pathway, was directly targeted by miR-15b. Functional analysis demonstrated that miR-15b overexpression aggravated IL-1β-induced ECM degradation in NP cells, while miR-15b inhibition had the opposite effects. Prevention of IL-1β-induced NP ECM degeneration by the miR-15b inhibitor was attenuated by small-interfering-RNA-mediated knockdown of SMAD3. In addition, activation of MAP kinase and nuclear factor-κB up-regulated miR-15b expression and down-regulated SMAD3 expression in IL-1β-stimulated NP cells.
Conclusions
MiR-15b contributes to ECM degradation in intervertebral disc degeneration (IDD) via targeting of SMAD3, thus providing a novel therapeutic target for IDD treatment.
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