Evaluation of graphical models for multi-group metabolomics data.

Gaussian graphical model is a strong tool for identifying interactions from metabolomics data based on conditional correlation. However, data may be collected from different stages or subgroups of subjects with heterogeneity or hierarchical structure. There are different integrating strategies of graphical models for multi-group data proposed by data scientists. It is challenging to select the methods for metabolism data analysis. This study aimed to evaluate the performance of several different integrating graphical models for multi-group data and provide support for the choice of strategy for similar characteristic data. We compared the performance of seven methods in estimating graph structures through simulation study. We also applied all the methods in breast cancer metabolomics data grouped by stages to illustrate the real data application. The method of Shaddox et al. achieved the highest average area under the receiver operating characteristic curve and area under the precision-recall curve across most scenarios, and it was the only approach with all indicators ranked at the top. Nevertheless, it also cost the most time in all settings. Stochastic search structure learning tends to result in estimates that focus on the precision of identified edges, while BEAM, hierarchical Bayesian approach and birth-death Markov chain Monte Carlo may identify more potential edges. In the real metabolomics data analysis from three stages of breast cancer patients, results were in line with that in simulation study.

Roles and mechanisms of exosomal non-coding RNAs in human health and diseases.

Exosomes play a role as mediators of cell-to-cell communication, thus exhibiting pleiotropic activities to homeostasis regulation. Exosomal non-coding RNAs (ncRNAs), mainly microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), are closely related to a variety of biological and functional aspects of human health. When the exosomal ncRNAs undergo tissue-specific changes due to diverse internal or external disorders, they can cause tissue dysfunction, aging, and diseases. In this review, we comprehensively discuss the underlying regulatory mechanisms of exosomes in human diseases. In addition, we explore the current knowledge on the roles of exosomal miRNAs, lncRNAs, and circRNAs in human health and diseases, including cancers, metabolic diseases, neurodegenerative diseases, cardiovascular diseases, autoimmune diseases, and infectious diseases, to determine their potential implication in biomarker identification and therapeutic exploration.

Adiponectin attenuates the premature senescence of vascular smooth muscle cells induced by high glucose through mTOR signaling pathway.

OBJECTIVE: Cardiovascular diseases and vascular aging are common in patients with diabetes. High glucose is a major cause of vascular aging and cardiovascular diseases. Premature senescence of vascular smooth muscle cells (VSMCs) is one of the main contributors to vascular aging. Adiponectin has been demonstrated to have an anti-aging effect. The present study explored the mechanisms by which adiponectin protects VSMCs against high-glucose-induced senescence. METHODS: Senescence-associated ß-galactosidase (SA-ß-gal) staining was used to detect senescence cells. Western blot was used for measuring protein levels. Flow cytometry was carried out to detect the cell cycle and telomeric repeat amplification protocol (TRAP)-polymerase chain reaction (PCR) silver staining was selected to measure the telomerase activity. RESULTS: Premature senescence of VSMCs was induced by high glucose (30 mM) in a time-dependent manner, which was verified by an increased number of senescence cells, p21 and p53 expression, as well as the decreased proliferation index. High glucose reduced telomerase activity of VSMCs via inhibition of the AMPK/TSC2/mTOR/S6K1 pathway and activation of the PI3K/Akt/mTOR/S6K1 pathway, while adiponectin treatment significantly increased telomerase activity of VSMCs through activation of AMPK/TSC2/mTOR/S6K1 signaling and inhibition of PI3K/Akt/mTOR/S6K1 signaling. CONCLUSION: Adiponectin attenuated the high-glucose-induced premature senescence of VSMCs via increasing telomerase activity of VSMCs, which was achieved by activation of AMPK/TSC2/mTOR/S6K1 signaling and inhibition of PI3K/Akt/mTOR/S6K1 signaling.

[Evaluation of key production processes of Schizonepetae Herba formula granules based on characteristic chromatogram and quantitative transfer relationship].

The aim of this paper was to evaluate the key production processes of Schizonepetae Herba formula granules based on the new model of combining characteristic chromatogram with quantitative transfer relationship. The rationality of production process design was evaluated by studying the intermediates in different processes of formula granules, analyzing the loss of index component pulegone in each step, and establishing the characteristic chromatogram. The content of pulegone in 10 batches of standard decoction ranged between 0.067% and 0.124%(70%-130% of the average value), and the transfer rate of pulegone was 44.58%-93.97%. After the improvement of the production process, the content of pulegone in Schizonepetae Herba formula granules was 0.093%, and the transfer rate of pulegone was 68.38%, which was consistent with the parameters range of standard decoction. This study emphasized the integrality of the research process of traditional Chinese medicine(TCM) formula granules, and provided a new idea for the quality control of TCM with content determination as the main evaluation index for a long time.

Exosomal Notch3 from high glucose-stimulated endothelial cells regulates vascular smooth muscle cells calcification/aging.

AIMS: Vascular calcification/aging can cause different kind of serious diabetic vascular complications. High glucose could induce vascular smooth muscle cells (VSMCs) calcification/aging and then lead to diabetes-related vascular calcification/aging. In this study, we investigated how information in the blood is transmitted to VSMCs and the mechanisms of VSMCs calcification/aging under hyperglycaemic conditions. MATERIALS AND METHODS: Transmission electron microscopy and molecular size analysis were used to assess the morphology and size of exosomes. Alizarin Red S staining and senescence-associated ß galactosidase (SA-ß-gal) staining were carried out to detect calcification and senescence in VSMCs, respectively. Proteomics analysis was carried out to detect the different expression of exosomal proteins. Protein levels were measured by western blot analysis. KEY FINDINGS: The results show that exosomes isolated from high glucose stimulated human umbilical vein endothelial cell (HG-HUVEC-Exo) exhibited a bilayer structure morphology with a mean diameter of 63.63 ±â€¯2.96 nm. The presence of exosome markers including CD9, CD63 and TSG101 were also detected in HG-HUVEC-Exo. High glucose could induce VSMCs calcification/aging by increasing the expression of osteocalcin (OC) and p21 as well as the formation of mineralised nodules and SA-ß-gal positive cells. Fluorescence microscopy verified that the exosomes were taken up by VSMCs and Notch3 protein was enriched in HG-HUVEC-Exo. Most importantly, mTOR signalling was closely related to Notch3 protein and was involved in regulating HG-HUVEC-Exo-induced VSMCs calcification/aging. SIGNIFICANCE: The data demonstrate that Notch3 is required for HG-HUVEC-Exo promoted VSMCs calcification/aging and regulates VSMCs calcification/aging through the mTOR signalling pathway.

Exosomes from hyperglycemia-stimulated vascular endothelial cells contain versican that regulate calcification/senescence in vascular smooth muscle cells.

BACKGROUND: To determine whether and how exosomes from human umbilical vein endothelial cells (HUVEC-Exos) regulates vascular smooth muscle cells (VSMCs) calcification/senescence in high glucose condition. METHODS: HUVEC-Exos were isolated from normal glucose (NG) and high glucose (HG) stimulated HUVECs (NG/HG-HUVEC-Exos) by super speed centrifugation. HUVEC-Exos were identified by transmission electron microscopy and Western blot of CD63. Protein profile in HUVEC-Exos was examined to screen the candidate molecules that mediate HUVEC-Exos function. VSMCs were incubated with HUVEC-Exos. A series of functional assays in vitro were performed to assess the effects of HUVEC-Exos on the calcification/senescence of VSMCs. The role of the candidate protein in HUVEC-Exos-induced VSMCs dysfunction was assessed. RESULTS: Exosomes isolated from HG-HUVEC-Exos induced calcification/senescence in VSMCs as assessed by Alizarin Red Staining, senescence-associated ß-galactosidase (SA-ß-gal) staining, and the expression of ALP and p21. HG-HUVEC-Exos significantly increased LDH activity, as well as the product of lipid peroxidation (MDA content), and decreased oxidative stress marker activity, as compared with NG-HUVEC-Exos. Moreover, mechanism studies showed that mitochondrial membrane potential and the expression levels of mitochondrial function related protein HADHA and Cox-4 were significantly decreased in HG-HUVEC-Exos compared to controls. Proteomic analysis showed that HG-HUVEC-Exos consisted of higher level of versican (VCAN), as compared with NG-HUVEC-Exos. Observation under laser confocal microscopy revealed that most green fluorescence of VCAN could overlap with the red fluorescence came from mitochondria, indicating VCAN is mainly localized to the mitochondria of VSMCs. Knockdown of VCAN with siRNA in HUVECs, inhibited HG-HUVEC-Exos-induced mitochondrial dysfunction and calcification/senescence of VSMCs. CONCLUSIONS: Our data indicate an intracellular role for VCAN in VSMCs. VCAN participates in hyperglycemia-induced calcification/senescence via modulation of mitochondrial function in VSMCs.

lncRNA-ES3/miR-34c-5p/BMF axis is involved in regulating high-glucose-induced calcification/senescence of VSMCs.

Vascular calcification/aging is common in diabetes and is associated with increased morbidity and mortality of patients. MiR-34c-5p, not miR-34c-3p, was suppressed significantly in calcification/senescence of human aorta vascular smooth muscle cells (HA-VSMCs) induced by high glucose, which was proven by the formation of mineralized nodules and staining of senescence associated-ß-galactosidase staining (SA ß-gal) positive cells. Overexpression of miR-34c-5p alleviated calcification/senescence of HA-VSMCs, whereas inhibition of miR-34c-5p received the opposite results. Bcl-2 modifying factor (BMF) was a functional target of miR-34c-5p and it was involved in the process of calcification/senescence of HA-VSMCs. Besides, lncRNA-ES3 acted as a competing endogenous RNAs (ceRNA) of miR-34c-5p to enhance BMF expression. Further, lncRNA-ES3 inhibited miR-34c-5p expression by direct interaction and its knockdown suppressed the calcification/senescence of HA-VSMCs. Our results showed for the first time that the calcification/senescence of VSMCs was regulated by lncRNA-ES3 /miR-34c-5p/BMF axis.

Artery calcification, osteoporosis, and plasma adiponectin levels in Chinese elderly.

OBJECTIVES: The purpose of this study is to investigate the plasma adiponectin level and thoracic aortic calcification (TAC) in Chinese elderly. BACKGROUND: The association of serum adiponectin levels and thoracic aortic calcification has not been reported. METHODS: Total plasma adiponectin level was measured by ELISA. TAC was evaluated by posterior-anterior chest radiographs. Osteoporosis was diagnosed using dual energy X-ray absorptiometry. RESULTS: The mean plasma adiponectin level was 6.3 ± 3.7 µg/ml, TAC was detected in 58% of subjects, and comorbidities of TAC and osteoporosis were observed in 33.6% subjects. The percentages of TAC or comorbidities of TAC and osteoporosis were higher in subjects with low adiponectin tertile level than that in patients with intermediate or high adiponectin tertile level. Multivariable logistic regression analysis showed that adiponectin level was negatively associated with TAC prevalence. CONCLUSIONS: Plasma adiponectin level is associated with TAC and lower plasma adiponectin level may be a useful indicator of artery calcification in the elderly.

Sarco-Osteoporosis: Prevalence and Association with Frailty in Chinese Community-Dwelling Older Adults.

The aim was to apply AWGS criteria to estimate the prevalence of sarco-osteoporosis and investigate its relationship with frailty, in a sample of 316 community-dwelling Chinese older people. Regression analysis was performed using frailty as the dependent variable. The results showed that the prevalence rate of sarco-osteoporosis was 10.4% in older men and 15.1% in older women. ≧80 years old (OR 4.8; 95% CI, 3.05-10.76; P = 0.027), women (OR 2.6; 95% CI, 1.18-2.76; P = 0.036), and higher level of comorbidity (OR 3.71; 95% CI, 1.61-10.43; P = 0.021) were independently associated with the likelihood of being sarco-osteoporosis. In the frail group, sarco-osteoporosis occurred in 26.3% of men, in 38.5% of women, and in lower proportion in the prefrail (13.6% of men; 16.2% of women) and nonfrail group (1.6% of men; 1.9% of women) (P < 0.05, resp.). Furthermore, the likelihood of being frail/prefrail was substantially higher in the presence of sarco-osteoporosis (OR 4.16; 95% CI, 2.17-17.65; P = 0.019 in men; and OR 4.67; 95% CI, 2.42-18.86; P = 0.007 in women). The results indicate that patients with sarco-osteoporosis are more likely to be ≧80 yrs with higher burden of comorbidities and to have frailty/prefrailty, especially for women.

Exenatide can inhibit calcification of human VSMCs through the NF-kappaB/RANKL signaling pathway.

BACKGROUND: Arterial calcification is an important pathological change of diabetic vascular complication. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) plays an important cytopathologic role in arterial calcification. The glucagon-like peptide-1 receptor agonists (GLP-1RA), a novel type of antidiabetic drugs, exert cardioprotective effects through the GLP-1 receptor (GLP-1R). However, the question of whether or not GLP-1RA regulates osteoblastic differentiation and calcification of VSMCs has not been answered, and the associated molecular mechanisms have not been examined. METHODS: Calcifying VSMCs (CVSMCs) were isolated from cultured human arterial smooth muscle cells through limiting dilution and cloning. The extent of matrix mineralization was measured by Alizarin Red S staining. Protein expression and phosphorylation were detected by Western blot. Gene expression of receptor activator of nuclear factor-κB ligand (RANKL) was silenced by small interference RNA (siRNA). RESULTS: Exenatide, an agonist of GLP-1 receptor, attenuated ß-glycerol phosphate (ß-GP) induced osteoblastic differentiation and calcification of human CVSMCs in a dose- and time-dependent manner. RANKL siRNA also inhibited osteoblastic differentiation and calcification. Exenatide decreased the expression of RANKL in a dose-dependent manner. 1,25 vitD3 (an activator of RANKL) upregulated, whereas BAY11-7082 (an inhibitor of NF-κB) downregulated RANKL, alkaline phosphatase (ALP), osteocalcin (OC), and core binding factor α1 (Runx2) protein levels and reduced mineralization in human CVSMCs. Exenatide decreased p-NF-κB and increased p-AMPKα levels in human CVSMCs 48 h after treatment. Significant decrease in p-NF-κB (p-Ser(276), p-Ser(536)) level was observed in cells treated with exenatide or exenatide + BAY11-7082. CONCLUSION: GLP-1RA exenatide can inhibit human VSMCs calcification through NF-κB/RANKL signaling.

[XRD, FTIR and thermoluminescence spectra of different types of urinary stones].

Using thermoluminescence spectrometer, X-ray diffraction and Fourier transform infrared spectroscopy, the chemical compositions of four types of kidney stones were investigated. They are calcium oxalate, uric acid, calcium phosphate and magnesium ammonium phosphate calculi (struvite). Experimental results showed that in the 305 cases of stones, calculi oxalate stones were found to account for 63%, uric acid stones 22%, calcium phosphate stones 8%, struvite 5%, and the stones with other compositions 2%. There were significant differences in the thermoluminescence spectra among the 4 types of urinary stones, which can provide an important basis for the clinic diagnosis of urinary stone types.

Measurement of urine crystallites and its influencing factors by nanoparticle size analyzer.

The difference of urine crystallites under 1000 nm in 10 patients with urolithiasis and 10 healthy subjects with no history of urolithiasis was comparatively studied with the nanoparticle size analyzer. By comparing the differences of intensity-autocorrelation curve, polydispersity index (PDI), Zeta potential, and relative error of average diameter of the two kinds of urine crystallites, it was concluded that the urine crystallites of healthy subjects were more stable than those of patients. The urine crystallites of healthy subjects had a narrower size distribution from 100 nm to 350 nm and a better dispersion (PDI < 0.3). However, the urine crystallites of patients with urolithiasis had a wider distribution from dozens of nanometers to 1000 nm and a worse dispersion (PDI > 0.5). The best processing method for urine crystallites detection was found: after antisepticising and protein-coagulating with formaldehyde, the urine was diluted with distilled water of the same volume, then filtrated through a micropore film of 3 microm, and the filtrate was centrifugalized at 4000 rpm for 15 minutes. This method can remove the cell fragments and macromolecular substances in the urine without affecting the detection of the urine crystallites under 1000 nm. The results were consistent with those obtained by transmission electron microscope (TEM).

[Study on nano- and microcrystallites in the urines of calcium oxalate stone formers].

The crystallites in urine are related closely with the formation of urolithiasis. In the present paper the composition, morphology and Zeta potential of crystallites of twenty calcium oxalate stone formers were comparatively studied using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, nanoparticle size analyzer, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results showed that calcium oxalate calculi usually coexisted with a little of uric acid, calcium phosphate, and magnesium ammonium phosphate. By contrast, the compositions of urine crystallites of the patients with calcium oxalate calculi were mainly uric acid, phosphate, calcium oxalate and so on. Most of them had sharp angularity with a particle size distribution ranging from tens of nanometers to tens of microns; and obvious aggregation was observed. The negative value of Zeta potential of urine crystallites in the twenty stone formers (average value -5.92 mV) was less than that in the twenty normal subjects (-12.9 mV). However, there was no obvious difference in the urine pH between stone formers (average pH 6.03) and normal subjects (average pH 5.92). The study on the relationship between urine crystallites and urinary calculi components will be helpful for finding out the causes of urolithiasis and providing an important basis for the scientific prevention methods and reasonable treatments in clinic.

Morphology, particle size distribution, aggregation, and crystal phase of nanocrystallites in the urine of healthy persons and lithogenic patients.

BACKGROUND: The first step in urinary stone formation is the nucleation of urinary mineral from supersaturated urine. The formed nuclei then grow or/and aggregate to a pathological size. Thus, the nanocrystallites in urine may be related to the formation of urinary stones. METHODS: Nanocrystallites with a size of less than 1000 nm in the urine samples of 85 healthy persons and 65 lithogenic patients were comparatively investigated using laser scattering spectroscopy, TEM, and X-ray diffraction. RESULTS: Most of the nanocrystallites in healthy urine samples were spheroidal, less aggregated, well-dispersed, and with a narrow particle size distribution from about 20 to 350 nm. In contrast, most of the particles in lithogenic urines had sharply angled edges and tips, were aggregated, and had a broad particle size distribution from 1.1 to 1000 nm. More calcium oxalate dihydrate (COD) nanocrystallites were found in healthy urine; however, more calcium oxalate monohydrate (COM) nanocrystallites were found in lithogenic urine. CONCLUSIONS: The morphology, particle size, aggregation, and crystal phase of nanocrystallites in the urine of lithogenic patients are pronouncedly different from those of healthy persons. The results suggest, in ascending order of importance, that making nanocrystallites rounded, diminishing their size differentiation, and decreasing their aggregation in urine by physical and chemical methods maybe the means to prevent urinary stone formation. The most crucial among the four differences is the crystal phase differential of calcium oxalate (CaOxa). That is, the formation of COD nanocrystallites in urine can be considered as being relatively more favorable in preventing stone formation than the formation of COM nanocrystallites, which are in accord with those found for larger crystallites.