text
string | predicted_class
string | confidence
float16 |
|---|---|---|
One-step quenching and extraction were applied as previously described to cell samples [14, 15]. Briefly, after the removal of cell culture medium, the cells were washed twice with ice-cold PBS, and then the pellets were quenched by 2 mL −20°C methanol (HPLC grade, Kermel Chemical Reagent, Tianjin, China). Afterwards, the cells were scraped and transferred into a 15-mL centrifugation tube, followed by adding 2 mL −20°C chloroform and ice-cold ddH2O (V/V/V 1:1:0.7). Cell lysates were mixed by vortex for 5mins and left stand for 15 mins, and then centrifuged with 14,000 g for 30 min at 4°C, generating two-phase extraction. The aqueous phase was lyophilized and then dissolved into 450 μL D2O with 50 μL buffer (1.5 M K2HPO4, 0.375 M NaH2PO4, 0.1% TSP, 0.2% NaN3, pH 7.4) . After being mixed with vortex, undissolved substances were removed by 14,000 × g centrifugation for 5 mins at 4°C and the supernatant was transferred into a 5-mm nuclear magnetic tube for NMR analysis.
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study
| 99.94 |
1H NMR analysis was performed by a Bruker AVANCE III 600 MHz NMR spectrometer at 298.15K. The samples were analyzed by pre-saturated pressed water peak pulse sequence noesypr1d ([RD-90°-t1-90°-tM-90°-ACQ]). Relaxation delay was set as 3 s, t1 4 μs, tM 120 ms, sampling time 1.64 s. Two hundred fifty-six free induction decays (FIDs) were collected by TOPSPIN software. The data point at 32 K was kept and adjusted to 64 K with spectral width 10 kHz. Spectral data were acquired from Fourier transform, by a window from all FIDs multiplying exponential function of line width 1 Hz.
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study
| 99.94 |
Phase and baseline of all NMR spectra were adjusted by MestReNova software, and the TMSP (Trimethylsilylpropanoic acid) peak was set as 0 ppm. The text was derived after calculating piecewise integration, with 0.002 ppm as the interval. Data preprocessing was performed with self-prepared MATLAB script. Data points within 0.6–9.5 ppm were kept and the peak water interval (4.5–5.2 ppm) was removed. For each spectrum, 4450 bins were kept. To reduce the difference due to different sample concentration, the dilution factor of each sample was calculated according to a control spectrum after probabilistic quotient normalization (PQN)
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study
| 99.94 |
Pseudo two-dimensional spectra were drawn, based on preprocessing the data of statistical total correlation spectroscopy (STOCSY), which indicated correlation factors among each chemical shift . To reduce the ratio of false positives, the threshold of the correlation factor r was calculated by an adjusted p value and number of varieties by Bonferroni.
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study
| 99.94 |
Dimensionality reduction and pattern recognition analysis was carried out by imputing preprocessed data into the software SIMCA-P+ (Ver. 12.0, Umetrics, Umeå, Sweden). To reduce the difference due to different sample concentrations, data were converted by Pareto .
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study
| 96.7 |
Firstly, principal component analysis (PCA) was applied to perform data-dimensionality reduction and get a data preview . Afterwards, we use partial least squares discriminant analysis (PLS-DA), a supervised pattern recognition method, to determine the modeling that generated the greatest difference between radiated sample and untreated sample. Grouping information used as response variable Y in calculating PLS-DA model. Q2 and R2were generated by software calculation to evaluate the degree of fit and forecasting ability of model. To avoid over-fitting, 7-fold cross-validation was performed for 400 repeats [21–23].
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study
| 100.0 |
The validated model was formally established by orthogonal projections to latent structures discriminant analysis (OPLS-DA), significant components were separated into one predictive component, t1, to describe the differences and one or more orthogonal components to filtering the irrelevant noise, the two datasets may be the most distinguished on score chart . After exporting the model correlation factor p(corr) and variable importance projection (VIP) as well as the load value, the loading diagram was drawn after backtracking transformation, and then differential metabolites were identified.
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study
| 100.0 |
Univariate analysis was performed on those differential metabolites identified by pattern recognition and substances identified by STOCSY. The relative concentration of differential metabolites was acquired by calculating the integration of spectra peaks. According to the p value calculated by the Mann–Whitney test in GraphPad Prism 6 (Ver. 6.01, GraphPad Software, Inc., CA, U.S.), the statistical significance of the differential metabolite was further verified.
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study
| 100.0 |
The names of the metabolites and their KEGG IDs were matched using MetaboAnalyst (http://www.metaboanalyst.ca/) . Pathway enrichment analysis was performed using MBRole . The relevant pathways were acquired by inputting metabolite KEGG ID and running a Hypergeometric Test. The metabolic pathway illustration was generated by Cytoscape and MetScape .
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other
| 99.6 |
To identify the representative metabolites in B16 cells in response to radiation, we firstly compared 1H NMR spectra from X-ray radiated B16 cells and untreated control cells. The representative spectrum of cellular soluble metabolite from X-ray radiated B16 cells and untreated control cells was indicated in Fig 1A and 1B, where the spectrum peak with δ0.5–8.6 ppm chemical shift was kept and water peak was removed. With ChenomxNMR Suite software analysis and statistical total correlation spectroscopy (STOCSY), major metabolites in the spectrum were confirmed. Chemical shifts and corresponding groups were summarized in Table 1.
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study
| 100.0 |
A: NMR spectra of irradiated B16 cells; B: NMR spectra of untreated B16 cells. Spectrum peak with chemical shift δ 0.5–8.6 ppm was kept and water peak as removed. The main metabolites indicated in the spectra were identified by Chenomx NMR Suite software and statistical total correlation spectroscopy (STOCSY).
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study
| 99.94 |
Principal component analysis (PCA) is an unsupervised pattern recognition algorithm, which is able to effectively eliminate the interference of artificial factors and indicates the sample distribution in principal component space. Taking advantage of this, all data were analyzed by PCA. A score plot of the first two principal components are shown in Fig 2A, radiated B16 cells and untreated B16 cells can be distinguished by first two principal components (PC1 and PC2), suggesting a metabolic profiling difference between radiated B16 cells and untreated B16 cells.
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study
| 100.0 |
Differential “metabogram” plotted according to correlation coefficient and VIP value. A: Radiated and untreated B16 cells were distinguished in principal component score chart, which indicated that the metabolic profiling of radiated B16 cells was different with that of untreated cells, t and t are scores on PC1 and PC2, respectively. B: Score chart of partial least squares discriminant analysis model distinguished radiated B16 cells and untreated B16 cells. C: 400 cross validations indicated little over-fitting and a reliable model. D:OPLS-DA model distinguished radiated and untreated B16 cells. E: Differential metabolites were distinguished in radiated and untreated cells according to VIP, relevant index r and loading value.
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study
| 100.0 |
Next, a supervised pattern recognition algorithm was applied using PLS-DA. As shown in Fig 2B, radiated and untreated cells were able to be distinguished in the principal component in the score chart. After 400 cross-validations, little over-fitting was observed and the data presented good predictive feature in the model (Fig 2C). On the basis of a reliable PLS-DA model, orthogonal partial least squares discriminant analysis (OPLS-DA) model was established by rotated principal–component projection to filter irrelevant information, and the predication of principal components in the samples of two individual groups showed the greatest distinction (Fig 2D). Furthermore, differentially expressed metabolites between the two group samples were distinguished by analyzing the VIP of each variety, the correlation coefficient r, and the loading value. According to VIP and the correlation coefficient r, a metabogram was presented that clearly indicated changes in metabolites in different treatments. In summary, in response to radiation, the contents of leucine, lactate, acetate, creatine, creatine phosphate, methanol, ethylene glycol, UDP-glucose, ATP, and formate were decreased in cells, while the contents of alanine, glutamate, taurine, choline, glycerol, and glycine were increased in cells.
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study
| 100.0 |
By calculating the spectra peaks of corresponding metabolites, the relative concentration of metabolites was acquired. Single-variable analysis was performed to validate the statistical difference. For each metabolite, 1 or 2 non-overlapping or little-overlapping spectrum peaks were selected as specific spectrum peaks to calculate the integral area by which the relative concentration of each metabolite was acquired (Table 1). The relative concentration of differentially expressed metabolites was performed in a non-parametric statistical test of single variable to generate a p value by the Mann–Whitney test, and the differentially expressed metabolites with statistical significance were indicated in Fig 3 (* p < 0.05, ** p < 0.01). Consistent with the results of multivariable analysis, in response to radiation, the contents of alanine, glutamate, choline and glycine were increased, while the content of leucine, lactate, acetate, creatine, creatine phosphate, methanol, UDP-glucose, and ATP were decreased.
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study
| 100.0 |
Through pattern recognition analysis and single variable analysis, in total 12 differentially expressed metabolites in B16 cells were identified upon radiation, including 4 content increased metabolites (alanine, glutamate, choline, and glycine) and 8 content-decreased metabolites (leucine, lactate, acetate, creatine, creatine phosphate, methanol, UDP-glucose and ATP). Correspondingly, for those content-changeable metabolites, their KEGG IDs and changes were listed in Table 2.
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study
| 100.0 |
In MBRole, pathway enrichment analysis indicated that differentially expressed metabolites were involved in multiple metabolic pathways, including the metabolism of glycine, taurine, arginine and alanine (Table 3). Using MetScape, metabolic network illustration shows the connection of these metabolites (Fig 4), where hexagons represente metabolites, red frames indicate the decreased metabolites, green frames indicate increased metabolites, squares indicate KEGG ID, rounded rectangles represent enzymes, and blue represents regulative genes.
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study
| 100.0 |
From metabolic pathway and network analysis, we observed that glutamate and pyruvate were converted into alanine and α-ketoglutarate by catalysis of alanine aminotransferase (ALT); Alanine and glyoxalic acid were converted into glycine and pyruvate by catalysis of Alanine-glyoxylate transaminase (AGT) (Fig 5). For those catalytic reactions, key enzymes and the EC number were listed in Table 4.
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study
| 100.0 |
PC, Phosphatidylcholines; PS, Phosphatidylserine; MTHF, 5,10-methylenetetrahydrofolate; THF, Tetrahydrofolate; GAc, Guanidinoacetate; Arg, Arginine; α-KG, α-ketoglutarate, also known as oxoglutarate; Asp, aspartate; OA, Oxaloacetate; TAC, tricarboxylic acid cycle, also known as citrate cycle. Red represents increased metabolites and green represents decreased metabolites in response to radiation.
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other
| 99.9 |
To investigate the impacts of radiation, the response of the metabolome in B16 cells to radiation were analyzed. Though metabolome analysis was performed in many clinic tissues or biofluids [28, 29], using cultured cell samples as a metabolic research model has multiple advantages, as not only can one exclude individual differences in clinic samples and tumor tissue heterogeneity, but also they are stable and have a high controllability; therefore, cell samples are appropriate for preliminary studies .
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study
| 100.0 |
Radiation therapy can generate free radicals and reactive oxygen species (ROS) from X-ray radiation, which induces DNA damage in M-phase and G2-phase cells and subsequently blocks cells in the G2/M phase. By inducing cell cycle arrest–mediated apoptosis, cancer cells are killed with radiation therapy [31–33]. Nevertheless, it has been shown that multiple mechanisms lead to cancer tolerance of radiation therapy (e.g., anti-apoptosis , DNA damage repair , cell cycle regulation ). Previous studies indicated that radiation had little impact on the B16 cell cycle, and the observation of γH2AX foci showed that the ratio of DNA double strand break in radiated B16 cells was higher than that of untreated cells . Impaired DNA double-strand break repair by chemical castration in prostate cancer had an improved response to radiotherapy . In this study, the content of glycine in radiated B16 cells was increased. It is known that glycine can react with 5,10-methylenetetrahydrofolate by catalysis of glycine hydroxymethyltransferase to generate serine and tetrahydrofolic acid, and the latter is an important coenzyme of DNA synthesis process. This conclusion suggested that radiation tolerance in B16 cells may arise from DNA damage repair. Accordingly, by activating specific metabolic pathways to block DNA damage repair, radiation-tolerant cancer cells can be killed, which may possibly be an effective way of radiotherapy sensitization.
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study
| 99.94 |
In response to radiation in B16 cells, the contents of glutamate, alanine, glycine, and choline were increased, which indicated that the activity of aspartate transaminase (AST), alanine transaminase (ALT), and alanine-glyoxylate transaminase (AGT) was elevated. Therefore, we speculate that, with a series of biochemical reactions upon the catalysis of those enzymes, more glycine may be synthesized to participate in DNA damage repair. On the other hand, with the catalysis of phosphatidylcholine (PC) synthase, serine can react with PC to generate choline and phosphatidylserine (PS). Though we did not observe PC and PS in cellular soluble metabolites upon radiation, due to their poor water solubility (no corresponding signal was observed in the hydrogen spectrum), the elevated content of choline in response to radiation indicated that the catalytic reaction occurred.
|
study
| 100.0 |
Though it has been reported that cancer cells, specially cancer stem cells, can synthesize antioxidant substances and reduce the generation of ROS to acquire radiation tolerance [39, 40], in this study, the content of the two antioxidant substances taurine and glutathione did not increase significantly in response to radiation, suggesting that radiation tolerance in B16 cells may have different mechanisms.
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study
| 100.0 |
With NMR analysis, we observed a series of change in metabolites in response to radiation in B16 cells. Based on previous studies, we concluded that these metabolites are involved in radiation tolerance in B16 cells. Taken together, our results suggested that radiation tolerance in B16 cells may result from the repair of radiation induced DNA damage.
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study
| 100.0 |
With intensive bioinformatic analysis, NMR based metabolome analysis can be used to identify the specific metabolic pathways in response to radiation, which may provide potential targets for radiotherapy sensitization and furthermore offers technical support and theoretical evidence for personalized radiation therapy.
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other
| 99.3 |
Non-alcoholic fatty liver disease (NAFLD) constitutes an increasingly prevalent liver disorder and has been suggested to be the hepatic manifestation of the metabolic syndrome . At present, NAFLD is assuming epidemic proportions affecting more than 25% of the world’s population, likely related to a concurrent rise in the prevalence of obesity and type 2 diabetes , although an increasing proportion of normal weight individuals are also affected pointing towards dyslipidemia as an important independent risk factor . NAFLD denotes a broad range of liver pathologies, spanning from simple hepatic steatosis, to advanced non-alcoholic steatohepatitis (NASH), which without intervention may progress to transplant-requiring cirrhosis and hepatocellular carcinoma. Whereas simple hepatic steatosis is mainly characterized by lipid accumulation in > 5% of hepatocytes, NASH is a condition further complicated by lobular inflammatory infiltrations and the presence of ballooning hepatocytes with or without concurrent fibrosis .
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review
| 99.9 |
It has been hypothesized that progression through NAFLD stages involves multiple adverse “hits” where both hepatic inflammation and oxidative stress are key facilitators . Accordingly, serum and hepatic levels of proinflammatory cytokines such as tumor-necrosis factor alpha (TNF-α) and monocyte-chemoattractant protein-1 (MCP-1) have been found to be increased in patients with both simple hepatic steatosis and NASH [6–9]. Also, the glycoproteins haptoglobin and tissue-inhibitor of metalloproteinase 1 (TIMP-1) have been shown to be elevated in plasma of patients with more advanced stages of NAFLD and have therefore recently been suggested as useful clinical plasma biomarkers, indicative of hepatocyte ballooning and liver fibrosis, respectively [10, 11].
|
review
| 99.1 |
Even though the specific etiology of NAFLD remains unclear, dietary fat and cholesterol have been linked to the development of hepatic steatosis and NASH in both humans and animal models [12–14] and more recently, the marked increase in consumption of the simple carbohydrate fructose, has been pointed out as another possible contributory factor [15, 16]. The realization that diet is an important contributor to the pathogenesis of NAFLD has resulted in a considerable variety of diet-induced animal models, where the majority (apart from those based on nutritional deficiencies) is based on a high-fat diet with varying levels of simple carbohydrate and cholesterol [17, 18]. However, the individual roles of fat, carbohydrates and cholesterol in the development of NAFLD are still not entirely clear. Thus, improved insight from animal studies on the individual contribution of fat, simple carbohydrates and cholesterol to the metabolic and inflammatory characteristics of NAFLD is an important prerequisite in understanding the disease complexity in patients. Furthermore, establishment of animal models of NAFLD/NASH that more adequately mimic human pathology will be valuable when testing novel therapeutics for NAFLD/NASH.
|
review
| 99.9 |
The aim of the present study was therefore to compare the effects of diets high in either dietary fat, dietary fructose or a combination diet with added cholesterol on development of NAFLD, dyslipidemia and inflammation in Sprague–Dawley rats. Development of NAFLD was evaluated over time both histologically and biochemically and quantitative computed tomography (qCT) was used as a non-invasive marker of hepatic steatosis, enabling monitoring of NAFLD progression throughout the study period.
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study
| 100.0 |
Forty male Sprague–Dawley rats were purchased from Charles River Laboratories (Sulzfeld, Germany). Animals were acclimatized for 2 weeks upon arrival and were 12 weeks old at the beginning of the experiment, weighing approximately 440–460 g. They were housed two per cage with access to non-chlorinated, non-acidific tap-water and with unrestricted access to standard rodent chow (Altromin 1324, Brogaarden, Denmark) until initiation of the experiment. Temperature in the animal rooms was maintained at 20–25 °C with a light/dark cycle of 12/12 h, a relative humidity of 30–70%, and air change 8–15 times/h.
|
study
| 99.94 |
Immediately prior to study start, rats were block-randomized into four diet groups based on body weight (n = 10/group). Briefly, rats were sorted in order of ascending weight into blocks, and within each block, rats were then randomly assigned to receive either a control diet (Control), a high-fat diet (HFD), a high-fructose diet (HFr) or a high-fat/high-fructose/high-cholesterol-diet (NASH), (Research Diets, NJ, USA) for 16 weeks. The length of the study period was chosen to enhance the probability that NASH with concurrent fibrosis would be established, as has previously been described in Sprague–Dawley rats fed a high-fat diet . The exact nutritional composition of each diet is shown in Table 1. All diets were stored at − 20 °C throughout the study until use, and feed-remains were exchanged with fresh feed twice weekly to prevent purification. To obtain baseline values for all parameters assessed at the 16 week time point, animals were qCT-scanned, quantitative magnetic resonance (qMR)-scanned and blood sampled at study start. After 16 weeks, animals were euthanized by exsanguination under isoflurane anesthesia by incision of the abdominal aorta. Immediately following exsanguination, the liver, a 5 cm section of the proximal jejunum and the two epididymal fat depots were excised, weighed and freeze clamped for further analysis. Food intake and body weight was measured twice weekly throughout the study. Food intake was recorded by calculating the difference between the amount of administered food and remaining food in the cage. These data were used to calculate the accumulated energy intake after the 16 weeks. qCT- and qMR-scans was performed on a bi-monthly basis, and blood samples were collected at baseline and at study termination.Table 1Nutritional composition of dietsNutrient compositionControlNASHHFDHFrFat (%)10406010Carbohydrates (total) (%)70402070 Fructose (%)020060 Sucrose (%)01071 Corn starch (%)55009 Maltodextrin 10 (%)1510130Protein (%)20202020Cholesterol (%)–2––Metabolizable energy (kcal/g)3.854.495.243.85NASH NASH-diet, HFD high-fat diet, HFr high-fructose diet
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study
| 100.0 |
Blood samples were taken from the sublingual vein in conscious non-fasted animals. They were collected in K3-EDTA microvettes and after centrifugation plasma was isolated and kept at − 20 °C until further analysis. Triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), free fatty acids (FFA), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), Haptoglobin and β-hydroxybutyrate were measured using a Cobas 6000 c501 instrument (Roche Diagnostics GmbH, D-68296 Mannheim, Germany), according to manufacturer’s instructions. Plasma levels of MCP-1 and TIMP-1 were analyzed using a multiplex assay, (K15179-C1, Mesoscale Discovery, MD, USA).
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study
| 100.0 |
Additionally, plasma samples were collected from 4-h fasted animals at week 15 (collected 1 week prior to week 16 blood samples, to avoid the fasting interfering with plasma lipid parameters) and assayed for endogenous insulin and glucose. Samples for blood glucose measurements (10 µL) were collected in capillary tubes and transferred to 500 µL system solution. Blood glucose levels were analyzed using the glucose oxidase method at a Biosen apparatus (EKF Diagnostics, Barleben, Germany) according to manufacturer’s instructions. Samples for endogenous insulin measurements were collected in K3-EDTA microvettes and after centrifugation; plasma was isolated and analyzed as previously described . Leptin levels were quantified using a Milliplex assay (RADPKMAG80-K, Merck, Hellerup, DK).
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study
| 100.0 |
To determine total fat mass, all animals underwent qMR-scans after 8 and 16 weeks using an EchoMRI Body Composition Analyser (EchoMRI, Houston, TX, USA). Mass measurements of fat tissue were performed according to manufacturer’s instructions and as previously described .
|
study
| 99.94 |
In order to assess the development and progression of NAFLD, qCT scans were used to quantify liver density as an indirect measure of hepatic fat content. Liver qCT-scans were performed after 8 and 16 weeks in isoflurane-anesthetized rats using a Latheta CT-scanner (LCT-200 series, Aloka co. LTD, Tokyo, Japan). Changes in liver density were calculated by subtracting baseline qCT values from week 8 and 16 time point values.
|
study
| 100.0 |
Levels of hepatic TG, TC and liver glycogen were analyzed on homogenized liver tissue sampled from the left lateral lobe using a Cobas 6000 c501 instrument (Roche Diagnostics GmbH 68206 Mannheim, Germany) according to manufacturer’s instructions and as previously described .
|
study
| 99.94 |
The right medial, the left lateral, and the caudate lobe of the liver were collected for histological examination. Samples were fixed in 10% Neutral Buffered Formalin, processed to paraffin, imbedded and cut in 2–4 µm sections. In addition, a sample from the left medial lobe was snap frozen for cryo-sectioning. Steatosis was evaluated in all three lobes with Mayer’s Haematoxylin and Eosin (H&E) on paraffin section (10 animals/gr) and confirmed by Oil Red O-stain on frozen cryo-sections of the left medial lobe (2–3 animals/group). Inflammation and fibrosis were evaluated based on the morphology on the H&E stain, collagen deposition on a Picro Sirius stain and macrophage infiltration based on a CD68 immunohistochemistry (IHC) stain. For the CD68 IHC stain, antigens were first retrieved by TEG buffer pH 9.0 and subsequently blocked with 0.5% hydrogen peroxide followed by avidin and biotin (Invitrogen, CA, USA). The slides were then incubated with primary antibody (3 µg/mL, MCA341R, AbD serotec, CA, USA) for 60 min, biotinylated secondary antibody for 60 min (1 µg/mL, 715-065-151, Jackson ImmunoSearch, PA, USA) and ABC reagent (Vector Laboratories, CA, USA). Finally, macrophages were visualised with a DAB reagent (Dako, Glostrup, Denmark). All sections were scanned with a Hamamutsu slide scanner and later evaluated using NanoZoomer Digital Pathology Image software (Hamamatsu, Hamamatsu City, Japan). The collagen and macrophage areas were quantified by Visiomorph software (Visiopharm, Hoersholm, Denmark).
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study
| 100.0 |
As described above, livers, epididymal fat depots, and jejunal segments were excised from the animals immediately after sacrifice and stored at − 80 °C until analysis. Tissue protein concentrations of the three tissues were first determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA) method according to manufacturer’s instruction. TNF-α and MCP-1 levels were subsequently determined in tissue homogenates with enzyme-linked immunosorbent assay (ELISA) kits (AB100785 and AB100778; Abcam, Cambridge, UK) according to manufacturer’s instructions. Absorbance was read using a Spectramax 340PC384 microplate reader at 450 nm (Molecular Devices, CA, USA).
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study
| 100.0 |
Statistical analyses were done using GraphPad Prism version 6.05 (GraphPad Software Inc., La Jolla, CA, USA). Data were assumed to be normally distributed and confirmed by visual inspection of qq-plots. In case of severe deviations from normality, statistical analyses were performed on log-transformed data (natural logarithm) or with the use of non-parametric tests. Data are presented as mean ± SEM, except for log-transformed data, which are presented as geometric means with 95% confidence intervals. Differences in means between diet groups for each parameter were analyzed using one-way ANOVA, repeated measures two-way ANOVA or Kruskal–Wallis tests where appropriate, and compared after 16 weeks on the diets. Bonferroni or Dunn corrections, respectively, were used to adjust for multiple comparisons. Outliers in data sets were identified and removed using the ROUT-function in GraphPad Prism. p-values < 0.05 were considered significant.
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study
| 100.0 |
Baseline characteristics for Control, HFD, HFr and NASH-groups are given in Table 2 and show that groups were comparable at study start for all parameters.Table 2Baseline characteristics of rats immediately after randomization to either Control-, NASH-, HFD- or HFr-dietsControlNASHHFDHFrBody weight (g)454.9 ± 6.6457.7 ± 5.7442.7 ± 7.0447.7 ± 8.8Fat mass (g)43.6 ± 2.644.4 ± 2.446.7 ± 2.044.9 ± 2.6Liver density (HU)40.0 ± 1.039.6 ± 0.640.7 ± 1.038.9 ± 0.8Plasma TG (mM)0.9 ± 0.10.9 ± 0.11.0 ± 0.11.3 ± 0.2Plasma FFA (mM)0.3 ± 0.020.3 ± 0.030.4 ± 0.030.3 ± 0.02Plasma cholesterol (mM)1.6 ± 0.11.5 ± 0.11.7 ± 0.11.6 ± 0.1Plasma HDL-C (mM)1.0 ± 0.041.0 ± 0.11.1 ± 0.041.0 ± 0.1ALAT (U/L)33.0 ± 2.131.9 ± 1.735.7 ± 2.833.1 ± 1.9ASAT (U/L)62.4 ± 1.959.9 ± 3.561.5 ± 2.963.2 ± 2.3Results are presented as mean ± SEMHU Hounsfield units, TG triglycerides, FFA free fatty acids, HDL-C high-density-lipoprotein-cholesterol, ALAT alanine aminotransferase, ASAT aspartate aminotransferase. Sample sizes: n = 10/group
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study
| 100.0 |
Figure 1 shows changes in body weight, fat mass and energy intake. Body weight increased throughout the study in all groups, but did not differ from Control after 16 weeks (Fig. 1a). Fat mass was significantly increased in HFD-fed animals compared to both those fed Control and HFr-diets (p < 0.05, Fig. 1b) after 16 weeks. After 8 weeks, accumulated energy intake was significantly higher in NASH-fed rats, compared to both Control- and HFr-fed rats (p < 0.001 and p < 0.05, Fig. 1c) and in HFD-fed rats compared to Control (p < 0.05). This difference remained significant only in NASH-fed compared to Control-fed rats after 16 weeks (p < 0.05).Fig. 1Effects on body weight, fat mass and energy intake of Control-, NASH-, HFD- and HFr-diets. a Body weight increased in all 4 diet groups throughout the study, but did not differ after 16 weeks. b After 8 weeks fat mass was significantly increased in HFD-fed rats compared to Control- and HFr-fed rats. This effect was still present at week 16. c After 8 weeks, accumulated energy intake was significantly higher in NASH-fed rats, compared to both Control- and HFr-fed rats and in HFD-fed rats compared to Control. This remained significant only in NASH-fed compared to Control-fed rats after 16 weeks. Comparisons between groups: *NASH vs. Control; #NASH vs. HFr; †HFD vs. Control. Statistical significance: *p < 0.05; ***p < 0.001; #p < 0.05; ##p < 0.01; †p < 0.05
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study
| 100.0 |
Effects on body weight, fat mass and energy intake of Control-, NASH-, HFD- and HFr-diets. a Body weight increased in all 4 diet groups throughout the study, but did not differ after 16 weeks. b After 8 weeks fat mass was significantly increased in HFD-fed rats compared to Control- and HFr-fed rats. This effect was still present at week 16. c After 8 weeks, accumulated energy intake was significantly higher in NASH-fed rats, compared to both Control- and HFr-fed rats and in HFD-fed rats compared to Control. This remained significant only in NASH-fed compared to Control-fed rats after 16 weeks. Comparisons between groups: *NASH vs. Control; #NASH vs. HFr; †HFD vs. Control. Statistical significance: *p < 0.05; ***p < 0.001; #p < 0.05; ##p < 0.01; †p < 0.05
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study
| 100.0 |
Throughout the study, liver density (used as an indirect measure of liver fat content) continuously declined in all four diet groups as measured by qCT with the effect being most pronounced in the NASH-group (Fig. 2a). At both 8 and 16 weeks, the decrease in liver density was significantly greater in NASH and HFD compared to Control (p < 0.0001 and p < 0.05) and in NASH compared to HFD and HFr (p < 0.0001).Fig. 2Effects on liver in Control-, NASH-, HFD- and HFr-fed rats. a Throughout the study, liver density continuously declined in all four diet groups, with the effect being more pronounced in the NASH-group. Both halfway through the study period and at the week 16 time point the decline in liver density was significantly greater in NASH and HFD compared to Control and in NASH compared to HFD and HFr. b Liver triglyceride content was significantly elevated in NASH- and HFD-fed rats compared to Control and HFr. Additionally, HFD-fed rats had significantly higher hepatic TG levels compared to NASH-fed. Hepatic TG levels in rats fed HFr did not differ from those fed the Control-diet. c Hepatic cholesterol levels were significantly elevated in NASH-fed animals compared to Control, HFD, and HFr. d The level of liver glycogen was significantly lower in both NASH and HFD compared to Control and HFr. a Comparisons between groups: *Control vs. NASH; †Control vs. HFD; ¤NASH vs. HFD; #NASH vs. HFr. Statistical significance: ****p < 0.0001; †p < 0.05; ¤¤¤¤p < 0.0001; ####p < 0.0001. b–d. Statistical significance: **p < 0.01; ****p < 0.0001. TG triglyceride, TC total cholesterol, NASH NASH-diet, HFD high-fat diet, HFr high fructose diet. Results are shown as mean ± SEM
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study
| 100.0 |
Effects on liver in Control-, NASH-, HFD- and HFr-fed rats. a Throughout the study, liver density continuously declined in all four diet groups, with the effect being more pronounced in the NASH-group. Both halfway through the study period and at the week 16 time point the decline in liver density was significantly greater in NASH and HFD compared to Control and in NASH compared to HFD and HFr. b Liver triglyceride content was significantly elevated in NASH- and HFD-fed rats compared to Control and HFr. Additionally, HFD-fed rats had significantly higher hepatic TG levels compared to NASH-fed. Hepatic TG levels in rats fed HFr did not differ from those fed the Control-diet. c Hepatic cholesterol levels were significantly elevated in NASH-fed animals compared to Control, HFD, and HFr. d The level of liver glycogen was significantly lower in both NASH and HFD compared to Control and HFr. a Comparisons between groups: *Control vs. NASH; †Control vs. HFD; ¤NASH vs. HFD; #NASH vs. HFr. Statistical significance: ****p < 0.0001; †p < 0.05; ¤¤¤¤p < 0.0001; ####p < 0.0001. b–d. Statistical significance: **p < 0.01; ****p < 0.0001. TG triglyceride, TC total cholesterol, NASH NASH-diet, HFD high-fat diet, HFr high fructose diet. Results are shown as mean ± SEM
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| 100.0 |
Livers from NASH-fed rats weighed significantly more compared to Control (p < 0.0001), HFD (p < 0.0001) and HFr (p < 0.001, Table 3), as did livers from HFr-fed rats compared to HFD (p < 0.05). Hepatic TG content was higher in NASH- and HFD-fed rats compared to both Control (p < 0.01 and p < 0.0001) and HFr (p < 0.01 and p < 0.0001, Fig. 2b). Additionally, HFD-fed animals had higher hepatic TG levels compared to NASH (p < 0.01). Hepatic TG levels in rats fed HFr did not differ from those fed the Control diet. Hepatic TC levels were significantly elevated in NASH-fed animals compared to Control, HFD, and HFr (p < 0.0001, Fig. 2c). Liver glycogen was significantly lower in both NASH and HFD compared to Control and HFr (p < 0.0001, Fig. 2d). At week 16, ALAT-levels were significantly higher in NASH-fed rats compared to Control- (p < 0.0001, Fig. 3a), HFD- and HFr-fed rats (p < 0.01). Plasma levels of ASAT were higher in the NASH-group compared to both Control- and HFr-groups (p < 0.05, Fig. 3b). Furthermore, plasma levels of the β-oxidation marker β-hydroxybuturate were increased in HFD-fed rats compared to Control-, NASH- and HFr-groups (p < 0.0001, p < 0.01 and p < 0.0001, Table 3).Table 3Metabolic and inflammatory effects in rats after 16 weeks on NASH-, HFD- and HFr-dietControlNASHHFDHFrLiver weight (g)18.9 ± 3.4#32.7 ± 7.9*†‡16.5 ± 2.5# ‡23.1 ± 4.4#†Fasting plasma glucose (mM)b6.4 ± 0.2#†‡7.2 ± 0.2*7.5 ± 0.1*7.16 ± 0.2*Fasting plasma insulin (pM)b560.6 ± 79.5486.7 ± 93.7603.0 ± 43.7695.8 ± 83.7Plasma β-hydroxybutyrate (µmol/L)38.4 ± 4.2†64.0 ± 5.3†115.4 ± 16.8*#‡38.4 ± 2.8†Plasma leptin (ng/mL)3283.3 ± 1035.2†3803.0 ± 1341.1†9366.6 ± 2052.8*#‡2638.0 ± 808.5†Plasma TG (mmol/L)1.6 ± 0.2‡1.7 ± 0.1‡1.5 ± 0.1‡3.9 ± 0.5*#†Plasma FFA (mmol/L)0.3 ± 0.10.4 ± 0.030.4 ± 0.020.3 ± 0.02Plasma Cholesterol (mmol/L)2.1 ± 0.12.5 ± 0.12.1 ± 0.12.3 ± 0.2Plasma HDL-C (mmol/L)1.3 ± 0.11.0 ± 0.11.3 ± 0.11.1 ± 0.1Jejunal MCP-1 (ng/mL)a45.3 [36.3; 55.3]58.1 [48.5; 69.6]49.5 [40.1; 60.5]56.4 [48.1; 65.5]Jejunal TNF-α (ng/mL)a179.9 [129.3; 247.7]87.6 [61.7; 125.5]171.1 [76.1; 384.6]213.2 [112.4; 404.3]Adipose tissue MCP-1 (ng/mL)a14.8 [13.4; 16.5]14.1 [11.9; 16.8]‡14.2 [11.3; 17.7]‡25.6 [15.2; 42.6]#†Adipose tissue TNF-α (ng/mL)a15.4 [14.4; 16.4]16.1 [14.3; 18.2]15.4 [13.6; 18.2]16.2 [14.6; 18.0]TG triglycerides, FFA free fatty acids, HDL-C high-density-lipoprotein-cholesterol, MCP-1 monocyte-chemoattractant-protein-1, TNF-α tissue-necrosis-factor-alphaResults are presented as mean ± SEM. Metabolic parameters denoted with superscript a, were analysed on log-transformed data (due to non-normal distribution), and are consequently presented as geometric means with 95% confidence intervals. Superscripts (*,#,†,‡) indicate significant (p < 0.05) differences in readouts compared to Control-, NASH-, HFD- and HFr-groups respectively. Sample sizes at week 16: n = 6–10/group. b Fasting plasma glucose and fasting plasma insulin levels were assessed after 15 weeks Fig. 3Markers of hepatic function, inflammation and fibrosis. a After 16 weeks ALAT-levels were significantly higher in NASH-fed rats compared to Control-, HFD- and HFr-fed rats. b Plasma levels of ASAT were higher in the NASH-group compared to both Control and HFr-groups. c Rats on NASH, HFD and HFr-diet had increased hepatic levels of MCP-1 compared to Control. d Hepatic TNF-α levels were significantly increased in the HFD fed rats compared to Control. e Haptoglobin was significantly increased in plasma of NASH-fed rats compared to rats fed Control, HFD and HFr. f Plasma TIMP-1 was significantly increased in the NASH-fed rats, compared to all other groups. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Results are shown as mean ± SEM
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Results are presented as mean ± SEM. Metabolic parameters denoted with superscript a, were analysed on log-transformed data (due to non-normal distribution), and are consequently presented as geometric means with 95% confidence intervals. Superscripts (*,#,†,‡) indicate significant (p < 0.05) differences in readouts compared to Control-, NASH-, HFD- and HFr-groups respectively. Sample sizes at week 16: n = 6–10/group. b Fasting plasma glucose and fasting plasma insulin levels were assessed after 15 weeks
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Markers of hepatic function, inflammation and fibrosis. a After 16 weeks ALAT-levels were significantly higher in NASH-fed rats compared to Control-, HFD- and HFr-fed rats. b Plasma levels of ASAT were higher in the NASH-group compared to both Control and HFr-groups. c Rats on NASH, HFD and HFr-diet had increased hepatic levels of MCP-1 compared to Control. d Hepatic TNF-α levels were significantly increased in the HFD fed rats compared to Control. e Haptoglobin was significantly increased in plasma of NASH-fed rats compared to rats fed Control, HFD and HFr. f Plasma TIMP-1 was significantly increased in the NASH-fed rats, compared to all other groups. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Results are shown as mean ± SEM
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The histological evaluation is presented in Fig. 4. After 16 weeks, hepatic steatosis was present in NASH, HFD and HFr-fed rats as confirmed by corresponding Oil Red O stains. No pathological changes were observed in livers of Control-fed rats. Steatosis was phenotypically distinguishable between NASH-, HFD- and HFr-fed rats. While zonal distribution was comparable (originating in zone 1, periportally), steatosis in HFD-rats was almost exclusively found to be of the microvesicular type; in HFr-fed rats almost exclusively macrovesicular; while the NASH-fed rats represented an intermediate between the two, with both macro- and microvesicular steatosis. Furthermore, steatosis in the NASH-group at week 16 was much more pronounced, involving not only zone 1, but occasionally also zones 2 and 3 and in some animals, only few areas displayed normal liver architecture. Inflammatory hepatic infiltration was identified in both NASH-, HFD- and HFr-fed rats. In all three groups, inflammation was characterized by being of primarily mononuclear composition with occasional neutrophilic involvement. However in HFr-fed rats, inflammatory cells appeared to center around lipid-loaded hepatocytes and form structures resembling lipogranulomas, whereas inflammatory infiltrates observed in HFD-fed rats were randomly scattered and not consistently associated with steatosis. As with the steatosis, inflammatory changes in the NASH-group were more pronounced, as exemplified by larger and more disseminated infiltrates. Accordingly, hepatic macrophage numbers were two- to threefold increased in the NASH-group compared to Control- and HFD-fed rats (p < 0.001 and p < 0.01, Fig. 5a, b). Hepatic deposition levels of collagen was higher in NASH-animals than in other groups, albeit only significantly increased compared to HFD (p < 0.001, Fig. 5c, d).Fig. 4Histological evaluation of liver sections from Control-, NASH-, HFD- and HFr-fed rats. Row 1: representative H&E-stains of normal liver from a Control-fed rat, and hepatic steatosis in b NASH-fed rat, c HFD-fed rat and d HFr-fed rat. Hepatic steatosis in HFD-fed rats was almost exclusively found to be microvesicular (c); in HFr-fed rats almost exclusively macrovesicular (d); while steatosis in NASH-fed rats represented an intermediate between the two, with both macro- and microvesicular steatosis (b). Row 2: higher magnification of representative H&E stained liver sections. e Liver morphology appeared normal in Control-fed rats, whereas inflammatory infiltrates were observed in livers from f NASH-fed rats, g HFD-fed rats and h HFr-fed rats Row 3: Oil Red O stains of liver sections from j NASH-fed rats, k HFD-fed rats and l HFr-fed rats confirmed hepatic steatosis observed in b–d. i Oil Red O staining of liver from Control-fed rat Fig. 5Macrophage infiltration and collagen deposition. A significant increase in macrophage infiltration was seen in NASH-fed rats, compared to Control- and HFD.fed rats (a) and collagen deposition in NASH-fed rats trended towards being increased, although only significantly when compared to HFD-rats (b). Representative CD68- (c) and Picro Weigert-stains (d) of liver from NASH-fed rat. Statistical significance: **p < 0.01; ***p < 0.001. Results in a and c are shown as mean ± SEM
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Histological evaluation of liver sections from Control-, NASH-, HFD- and HFr-fed rats. Row 1: representative H&E-stains of normal liver from a Control-fed rat, and hepatic steatosis in b NASH-fed rat, c HFD-fed rat and d HFr-fed rat. Hepatic steatosis in HFD-fed rats was almost exclusively found to be microvesicular (c); in HFr-fed rats almost exclusively macrovesicular (d); while steatosis in NASH-fed rats represented an intermediate between the two, with both macro- and microvesicular steatosis (b). Row 2: higher magnification of representative H&E stained liver sections. e Liver morphology appeared normal in Control-fed rats, whereas inflammatory infiltrates were observed in livers from f NASH-fed rats, g HFD-fed rats and h HFr-fed rats Row 3: Oil Red O stains of liver sections from j NASH-fed rats, k HFD-fed rats and l HFr-fed rats confirmed hepatic steatosis observed in b–d. i Oil Red O staining of liver from Control-fed rat
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Macrophage infiltration and collagen deposition. A significant increase in macrophage infiltration was seen in NASH-fed rats, compared to Control- and HFD.fed rats (a) and collagen deposition in NASH-fed rats trended towards being increased, although only significantly when compared to HFD-rats (b). Representative CD68- (c) and Picro Weigert-stains (d) of liver from NASH-fed rat. Statistical significance: **p < 0.01; ***p < 0.001. Results in a and c are shown as mean ± SEM
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Plasma FFA did not differ between groups (Table 3). HFr-feeding induced a significant increase in circulating TG, compared to Control-, NASH- and HFD (p < 0.0001, Table 3). Plasma HDL-c levels were similar between groups, as were the plasma cholesterol levels (Table 3). Fasting hyperglycemia was present in all groups compared to Control after 16 weeks (NASH and HFr: p < 0.01, HFD: p < 0.0001, Table 3). However, fasting plasma insulin levels did not differ between groups (Table 3). Circulating leptin levels were significantly increased in the HFD-fed rats compared to both Control- (p < 0.05), NASH- (p < 0.05) and HFr-fed rats (p < 0.01, Table 3).
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Hepatic levels of MCP-1 were significantly increased in rats on NASH (p < 0.01), HFD diet (p < 0.0001) and HFr diet (p < 0.01) compared to Control (Fig. 3c). Additionally, hepatic TNF-α levels were higher in HFD fed rats compared to Control- (p < 0.001), NASH (p < 0.0001) and HFr (p < 0.01, Fig. 3d). Haptoglobin was significantly increased in plasma of NASH-fed rats compared to rats fed Control (p < 0.001), HFD (p < 0.0001) and HFr (p < 0.001, Fig. 3e). Concentrations of MCP-1 and TNF-alpha were not elevated in adipose or intestinal tissue in any of the diet-groups compared to Control; however, HFr-fed rats had significantly higher levels of adipose tissue MCP-1 compared to both NASH- and HFD-fed rats (p < 0.05, Table 3). MCP-1 was below detection limit in plasma in all groups. Plasma TIMP-1 levels was significantly increased in the NASH-fed rats, compared to all other groups (vs. Control: p < 0.01; vs. HFD and HFr: p < 0.001, Fig. 3f).
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The present study shows that feeding rats diets high in either dietary fat or dietary fructose results in distinctly different hepatic, metabolic and inflammatory profiles in rats. When comparing the effect of dietary fat and fructose, high-fat feeding more potently induce development of fatty liver and associated hepatic inflammation without affecting the circulating lipid pool. In contrast, high-fructose feeding appear to have the most pronounced effects on the plasma lipid profile, while only subtle effects on the liver are observed. The combination of fat, fructose, and cholesterol exacerbated and intensified overall effects on the liver. The strength of this study is the direct comparison of dietary fat (with very restricted amounts of carbohydrate) and dietary fructose (with very restricted amounts of fat) on parameters associated with NAFLD. This enables a more detailed evaluation of the individual roles of these macronutrients in disease progression. Furthermore, the use of qCT allows the non-invasive assessment of NAFLD-progression throughout the study, a method which to our knowledge has not previously been applied in rat studies comparing NAFLD progression after administration of different diets.
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| 99.94 |
NAFLD in humans is often associated with obesity and insulin resistance, and these metabolic derangements were more closely reflected in the HFD-group. Accordingly, only HFD-fed rats became obese and even though the cumulative energy intake in this group was transiently higher than Control animals at week 8, differences in energy intake did no longer account for the HFD-induced obesity after 16 weeks. Circulating leptin levels were also significantly elevated only in HFD-fed rats, reflecting the increase in adipose tissue depot size within this group. Fasting hyperglycemia was present in all groups at study termination, indicating disturbances in glucose metabolism, even though fasting insulin levels remained similar between groups. These disturbances were further corroborated by the increased levels of hepatic TG and decreased/unaltered hepatic glycogen observed in both NASH-, HFD- and HFr-fed rats, indicative of selective insulin resistance .
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| 100.0 |
Hepatic steatosis was rapidly induced in both NASH- and HFD-fed rats and was shown using qCT to progress in a time-dependent manner. In line with this, higher levels of plasma β-hydroxybutyrate were observed in HFD-fed rats, suggestive of increased β-oxidation due to hepatic lipid overload. The role of hepatic β-oxidation in the pathogenesis of NAFLD remains controversial, but several studies in human NASH-patients have found serum levels of β-hydroxybutyrate to be increased [24, 25]. Although not significant, there was a trend towards an increase of this marker in NASH-fed rats as well. The development of hepatic steatosis in NASH- and HFD-fed rats agrees well with recent studies in other rodent species suggesting fat and cholesterol to be important drivers in the development of experimental NAFLD/NASH [14, 22]. The addition of dietary cholesterol (NASH-diet) caused the most detrimental liver changes, evident by significant accumulation of both hepatic TG and cholesterol, pronounced decreases in liver density, and severe morphological alterations in liver histology. In contrast to what was observed in HFD- and HFr-fed rats, these changes were also accompanied by increased plasma levels of ALAT and ASAT, suggesting impaired liver function. It has previously been observed in both mice [26, 27] and humans that the combined intake of dietary fat and carbohydrates, or of fat and cholesterol can result in accelerated and more severe effects in the liver. The majority of these changes could be driven by the amount of cholesterol in the NASH-diet (2%), as hepatic levels of cholesterol measured in NASH-fed rats were very high. Supporting this, two previous studies in mice have suggested that dietary cholesterol is important in facilitating progression from simple steatosis to NASH and that free cholesterol loading can sensitize the liver to cytokine-mediated hepatocellular death, inflammation and oxidative stress . This could account for the more aggravated inflammatory histological response, supported by the significant increase in infiltrating macrophages observed in livers of NASH-fed rats compared to both HFD and Control groups. Further studies are needed to determine if these changes are caused exclusively by the cholesterol content, by a synergistic effect of fat and fructose, or by a combination of all.
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While fat and cholesterol both seem to contribute to the development of NAFLD, previous studies in rats have shown that also high levels of dietary fructose are capable of inducing hepatic steatosis, inflammation and increase oxidative stress markers within a relatively short time-span of only 2–5 weeks [32–34]. These are in contrast to our findings with the HFr-diet, which only induced subtle changes in the liver. While steatosis was observed histologically in some animals in the HFr-group the condition was not confirmed by increased levels of liver TG at the week 16 time point. The apparent discrepancy between histological and biochemical analyses within the HFr-group could result from sampling variation, a problem also commonly encountered in biopsy-guided diagnosis of human NAFLD/NASH . Biochemical analyses of hepatic TG-content was performed on liver tissue sampled from the left lateral lobe and histologic evaluation of livers in the HFr-group (performed on both left lateral, right medial and the caudate lobe) did in fact, in some animals, show specific lobes to be more severely affected than others, indicating that steatosis, at least in rats fed fructose-enriched diets, may not be homogenously distributed throughout the liver. Further studies, confirming the heterogeneously distributed steatosis would be interesting in terms of evaluating and comparing histology, biochemistry and imaging of the liver in rodent models of NAFLD/NASH.
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| 99.94 |
Dyslipidemia is one of the hallmarks of the metabolic syndrome and has been shown to be strongly associated with NAFLD . Dyslipidemia associated with NAFLD is typically characterized by elevated levels of circulating TG and low-density-lipoprotein cholesterol (LDL-C), as well as decreased HDL-C levels . Only rats fed HFr-diet developed dyslipidemia, as defined by the presence of hypertriglyceridemia. Previous studies have also found that dietary fructose potently increase TG in plasma within a relatively short timeframe in both rats and mice [34, 38]. In rats, this has been suggested to be caused in part by the ability of fructose to both increase hepatic very-low-density-lipoprotein (VLDL)-TG secretion and decrease VLDL-TG clearance from the circulation, even in the absence of hyperinsulinemia [39, 40]. Mechanistically, the increase in VLDL-TG secretion has been proposed to result from a combined effect of fructose-induced hepatic stress responses , and activation of hepatic enzymes involved in the de novo synthesis of fatty acids (de novo lipogenesis) .
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| 99.94 |
In contrast to the HFr-diet, neither the HFD- nor the NASH-diet resulted in increased plasma TG, even though high-fat diets have previously been shown to induce hypertriglyceridemia in rats . However, our findings agree with a recent study in mice investigating the contribution of dietary fat and cholesterol to NAFLD-development, which showed no effect of dietary fat and cholesterol on circulating TG or FFA levels, independently of whether these macronutrients were fed separately or in combination . One explanation could be that accelerated fat accumulation in the liver imposes a significant strain on metabolic pathways responsible for the release of lipids from the liver into plasma. Accordingly, it has been shown both in vitro and in vivo that high levels of fat and cholesterol in the liver result in increased stress in the endoplasmic reticulum, limiting secretion of apolipoprotein B100 (an essential component in the assembly of VLDL-particles) and thereby inhibits hepatic export of TG [29, 43]. The inflammatory profile was clearly distinguishable between groups in the present study. Systemic inflammation was present only in NASH-fed rats with higher circulating levels of haptoglobin. We also found increased levels of the pro-inflammatory cytokines MCP-1 and TNF-alpha in liver tissue homogenates in NASH-, HFD- and HFr- and in HFD-fed rats, respectively, suggesting progression of hepatic steatosis towards NASH with the increased inability of the liver to cope with fat infiltration. The inflammatory infiltrates observed histologically in livers of HFr- and NASH-fed rats at week 16, were reflected only in significantly higher levels of hepatic MCP-1, not TNF-alpha. Compared to the other diet-groups, NASH-fed rats might be expected to have the highest levels of liver inflammatory cytokines, due to the higher macrophage count, increased liver enzymes, and the more severe liver pathology. However, hepatic macrophages are a remarkably heterogeneous population of immune cells, whose effector function depends on origin, underlying pathogenesis and disease stage, which can result in high diversity in inflammatory cytokines released and cell surface markers [44, 45]. This could explain why the NASH-fed group does not have the highest hepatic MCP-1 and TNF-alpha levels despite the higher macrophage count and the more severe liver pathology. Despite increased hepatic MCP-1 levels in all groups compared to Control, MCP-1 plasma levels were below detection limit.
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| 99.94 |
It has been hypothesized that fructose and fat may induce their inflammatory action in the liver not only by dietary overload, but also by stimulation of bacterial overgrowth in the intestine, increasing intestinal permeability and thereby facilitating translocation of endotoxins across the intestinal barrier that are then transported to the liver [46, 47]. In the present study, we were not able to detect increased intestinal levels of TNF-alpha or MCP-1 in any diet group. Moreover, we did not find increased levels of MCP-1 and TNF-alpha in visceral adipose tissue in any of the groups, even though inflammation particularly in the adipose tissue compartment is strongly associated with NAFLD/NASH in humans . Notably, the cytokine analyses in this study were performed on epididymal fat depots, which may not adequately represent visceral adipose tissue depots in humans .
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| 100.0 |
Although fibrosis is not a prerequisite for the NASH diagnosis, it often accompanies the three recognized diagnostic hallmarks (steatosis, lobular inflammation and hepatocyte ballooning), and can be used as a grading tool for evaluation of NASH severity . In the present study, neither cellular ballooning nor fibrosis was present in any of the groups. However, increased levels of plasma haptoglobin and TIMP-1 were observed in NASH-fed rats and are considered predictors of these pathological liver changes, indicating that the NASH diet might be promising in terms of modelling more advanced stages of NASH within a relatively short time-frame. The tendency towards increased collagen deposition in NASH-fed rats further supports this. The inability of the diets used in this study to induce liver fibrosis in rats after only 16 weeks is not entirely surprising. Successful dietary induction of fibrosis in rodent models of NASH has so far mainly been associated with either genetic manipulation, forced overfeeding or diets low in methionine and choline, [51, 52], except for the guinea pig that has been shown to develop NASH after 16 weeks of high fat feeding . However, mild hepatic fibrosis in addition to a NASH-like phenotype has also been induced in C57BL/6 mice on a high-fat diet, but only after 50 weeks . In a recent study, using levels of dietary cholesterol comparable to the level in the NASH-diet in our study, development of fibrosis in Sprague–Dawley rats was observed after only 9 weeks. . However, the diets used by Ichimura and colleagues included relatively high levels of dietary cholate (2%), which is known to potently induce hepatotoxicity and upregulate specific genes related to hepatic fibrosis .
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| 99.94 |
Dietary fat seems to primarily drive NAFLD development in Sprague–Dawley rats with potent effects on hepatic fat accumulation and inflammation, whereas dietary fructose primarily affects circulating lipids with much more subtle effects on the liver. Combining fat, fructose and cholesterol accelerates NAFLD development and increase the overall severity of changes observed in the liver.
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| 99.94 |
Changes in supervisors’ behavior lead to changes in the workplace environment and could reduce aggression, poor manner to job, and physical and emotional strain in workers . Moreover, supervisors’ leadership styles exert an impact on their performance, and leadership style, work environment, and job satisfaction are important factors affecting workers’ performance . Furthermore, support from supervisors increases productivity indirectly by reducing the occurrence of presenteeism , and their communication styles and personality traits affect organizational productivity . In addition, supervisors’ personalities and job stress levels have been associated with workers’ exposure to bullying in the workplace .
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other
| 99.9 |
Supervisors’ behavior could influence workers’ job stress levels, and work-related stress is a serious problem that could exert an adverse effect on employees’ health [6, 7]. In particular, support from supervisors is an important factor affecting workers’ job satisfaction, and supervisors influence workers’ emotion . In addition, supervisors’ behavior influences workers’ well-being, turnover intention, and job satisfaction [8–11]. For example, positive managerial behavior in the workplace could improve employees’ well-being , while destructive managerial leadership could exert an adverse effect . Moreover, support from supervisors and coworkers, the maintenance of a relaxed atmosphere in the workplace, and increased respect for workers could reduce rates of presenteeism .
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other
| 99.8 |
In previous studies, one of the many questions of the job stress questionnaire (e.g., Korean Occupational Stress Scale) used the question, "My boss helps me complete the task.", or there was a study on supervisor support [9, 15, 16]. For this reason, studies in the past have only been able to evaluate some of the various supervisors’ behaviors, and no studies have evaluated the supervisors’ behaviors using more detailed questionnaire.
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| 99.56 |
KWCS is a survey of Korean workers aged 15 and over to investigate the work environment and to identify exposure to risk factors according to job and industry type and risk factors according to employment type. Individual interviews were conducted. The 50,007 subjects included in the KWCS, data for 30,734 wage workers (excluding soldiers and self-employed individuals) and employers were analyzed in the present study. 27,714 subjects who answered the question "How often do you experience stress at work?" And the questions about supervisors’ behaviors were selected. For the other variables, 15,787 subjects were finally selected, excluding those who did not respond or rejected (Fig. 1). After weighting, the weighted frequency was 19,064 and the data were analyzed.Fig. 1Subjects included in the current study
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| 100.0 |
Subjects’ general characteristics, included age, sex, education level, average monthly income, General health condition. The unit of monthly income is 10,000 Korean won. Educational levels were categorized as low for subjects educated to high school level or lower and high for subjects educated to college level or higher. Job stress was set to ‘Low’ for 1 ~ 2 points and ‘High’ for 3 ~ 5 points. General health condition is very good, good, normal is defined as Good, bad, and very bad as Bad.
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| 99.44 |
Occupational characteristics included the current number of employees, sector (public or private), occupation type, employment status, shift work, and working hours. Organizations were classified into 2 groups: those with up to 300 employees and those with more than 300 employees. Privately owned companies were classified as private-sector organizations, while central and local government offices; public institutions; and state-owned schools, hospitals, and universities were classified as public-sector organizations. Occupation types were categorized as white collar (e.g., supervisors, professionals, mechanics, semiprofessionals, and office workers) or service/sales workers (e.g., service workers; sales workers) or blue collar (e.g., skilled workers in agriculture, forestry and fishing; technicians; related technical workers; device or machine operation and assembly workers, and simple laborers) occupations. Employment status was classified as permanent or temporary, and shift pattern (shift work or non-shift work) was included as a variable.
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| 99.94 |
Psychosocial risk factors for job stress were categorized into the following 5 categories: high job demand, insufficient job control, inadequate social support, job insecurity, and lack of reward. The questionnaires used in this study are listed in Appendix. Each item was scored, and scores were classified as high or low based on the median.
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| 99.94 |
Supervisors’ behavior was measured using the following 5 items: my supervisor provides feedback regarding my work (Feedback), my supervisor respects me personally (Respectful behavior), my supervisor is good at resolving conflict (Good conflict abilities), my supervisor is good at planning and organizing work (Good at planning), and my supervisor encourages me to contribute to important decisions (Participation in decisions). The response options were “yes” or “no” for all items.
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| 99.9 |
Chi-square was performed to determine differences in subjects’ general and occupational characteristics according to supervisors’ behavior. Multiple logistic regression analysis was performed to examine subjects’ general characteristics and occupational characteristics and identify the effects of supervisors’ behavior on workers’ job stress levels. All analyses were performed using SPSS ver. 23.0 (Chicago, IL, USA).
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| 99.94 |
The study included 19,064 subjects (9770 men, 9294 women). There were more people in organizations with less than 300 employees, and more people worked in the private sector than in the public sector. Between age and number of employees, age and public/private sector showed significant differences (p < 0.001). In organizations with less than 300 employees, the number of people aged between 41 and 50 was high, while those with more than 300 workers were between 31 and 40. There were many workers in the 41–50 age group in both private and public sectors. The proportion of male and high education level workers was significantly higher in organizations with more than 300 employees than that of organizations with up to 300 employees (p < 0.001). In the private sector, the proportion of men and high education level workers was high (p < 0.001). There was no difference in the general health condition according to the number of workers (p = 0.060), and more people in the public sector answered that they had good general health condition (p < 0.001). Between monthly income and number of employees, monthly income and public/private sector were significantly different (p < 0.001). In organizations with more than 300 employees, the proportion of people with income over 3 million won was high. The proportion of high job stress of the private sector was significantly higher than that of high job stress of the public sector (p < 0.001) (Table 1).Table 1Subjects’ General characteristics according to Occupational characteristicsNumber of employeesSectorTotal<300 (%)≥300 (%)Public (%)Private (%)Total19064a (100%)17436 (91.5)1628 (8.5)2399 (12.6)16665 (87.4)Age ≤ 303302 (17.3)3040 (17.4)262 (16.1)<0.001b 221 (9.2)3081 (18.5)<0.001b 31 ~ 405201 (27.3)4626 (26.5)576 (35.4)580 (24.2)4621 (27.7) 41 ~ 505480 (28.7)5025 (28.8)455 (27.9)750 (31.3)4731 (28.4) 51 ~ 603383 (17.7)3086 (17.7)297 (18.2)448 (18.7)2935 (17.6) ≥ 611698 (8.9)1660 (9.5)38 (2.3)400 (16.7)1297 (7.8)Sex Male9770 (51.2)8582 (49.2)1188 (73.0)<0.001b 1149 (47.9)8622 (51.7)<0.001b Female9294 (48.8)8854 (50.8)440 (27.0)1250 (52.1)8044 (48.3)Educational level High school or lower8928 (46.8)8406 (48.2)523 (32.1)<0.001b 800 (33.3)8129 (48.8)<0.001b College or higher10136 (53.2)9031 (51.8)1105 (67.9)1599 (66.7)8536 (51.2)General health condition Good18525 (97.2)16931 (97.1)1594 (97.9)0.060b 2290 (95.5)16235 (97.4)<0.001b Bad539 (2.8)505 (2.9)34 (2.1)109 (4.5)430 (2.6)Income < 1001906 (10.0)1881 (10.8)25 (1.5)<0.001b 504 (21.0)1402 (8.4)<0.001b 100 ~ 1996475 (34.0)6282 (36.0)192 (11.8)408 (17.0)6066 (36.4) 200 ~ 2995737 (30.1)5289 (30.3)448 (27.5)618 (25.8)5119 (30.7) ≥ 3004946 (25.9)3984 (22.8)962 (59.1)868 (36.2)4078 (24.5)Job stress High14443 (75.8)13174 (75.6)1269 (77.9)0.031b 1711 (71.3)12733 (76.4)<0.001b Low4621 (24.2)4262 (24.4)359 (22.1)688 (28.7)3933 (23.6) aweighted frequency bBased on the chi-square test
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study
| 100.0 |
The proportion of people who answered “yes” to the “my supervisor provides feedback regarding my work,” “my supervisor respects me personally,” “my supervisor is good at resolving conflict,” “my supervisor is good at planning and organizing work,” and “my supervisor encourages me to contribute to important decisions” items, which reflected the quality of supervisors’ behavior, in organizations with more than 300 employees were significantly higher relative to those observed for organizations with up to 300 employees (p < 0.001).
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other
| 99.9 |
The proportion of public-sector organizations who responded in the affirmative for all items pertaining to supervisors’ behavior was significantly greater relative to that observed for private-sector organizations (p < 0.001). However, answer to “my supervisor provides feedback on my work” did not differ significantly between public- and private-sector organizations (p = 0.496) (Table 2).Table 2Supervisors’ behaviors according to Occupational characteristicsNumber of employeesSectorTotal<300 (%)≥300 (%)Public (%)Private (%)Total19064a (100%)17436 (91.5)1628 (8.5)2399 (12.6)16665 (87.4)Feedback Yes14380 (75.4)12991 (74.5)1388 (85.3)*** 1823 (76.0)12557 (75.3) No4684 (24.6)4445 (25.5)240 (14.7)576 (24.0)4108 (24.7)Respectful behavior Yes15880 (83.3)14382 (82.5)1499 (92.1)*** 2120 (88.4)13761 (82.6)*** No3184 (16.7)3055 (17.5)129 (7.9)279 (11.6)2904 (17.4)Good conflict abilities Yes14067 (73.8)12661 (72.6)1406 (86.4)*** 1902 (79.3)12165 (73.0)*** No4997 (26.2)4775 (27.4)221 (13.6)497 (20.7)4500 (27.0)Good at planning Yes14536 (76.2)13111 (75.2)1425 (87.5)*** 1955 (81.5)12581 (75.5)*** No4528 (23.8)4325 (24.8)203 (12.5)444 (18.5)4084 (24.5)Participation in decisions Yes12050 (63.2)10766 (61.7)1285 (78.9)*** 1646 (68.6)10404 (62.4)*** No7014 (36.8)6671 (38.3)343 (21.1)753 (31.4)6261 (37.6) *** p < 0.001 aweighted frequency
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study
| 100.0 |
Organizations with more than 300 employees had high job demands than organizations with less than 300 employees, the private sector has a high proportion of high job demand than the public sector (p < 0.001). Organizations with more than 300 employees and the public sector had high job control (p < 0.001). The high social support proportion was higher in the organizations with more than 300 employees, and the high social support proportion was high in the public sector, showing a significant difference (p < 0.001). Job insecurity and the number of employees showed no significant difference (p = 0.172) (Table 3).Table 3Psychosocial risk factors of study subjects by Occupational characteristicsNumber of employeesSectorTotal<300 (%)≥300 (%)Public (%)Private (%)Total19064a (100%)17436 (91.5)1628 (8.5)2399 (12.6)16665 (87.4)Job demand High8238 (43.2)7398 (42.4)840 (51.6)*** 768 (32.0)7470 (44.8) *** Low10826 (56.8)10038 (57.6)788 (48.4)1630 (68.0)9196 (55.2)Job control High10451 (54.8)9476 (54.3)975 (59.9) *** 1393 (58.1)9059 (54.4) *** Low8613 (45.2)7960 (45.7)653 (40.1)1006 (41.9)7607 (45.6)Social support High10784 (56.6)9557 (54.8)1227 (75.4) *** 1527 (63.7)9257 (55.5) *** Low8280 (43.4)7879 (45.2)401 (24.6)872 (36.3)7408 (44.5)Job insecurity High6072 (31.8)5578 (32.0)494 (30.3)627 (26.1)5445 (32.7)*** Low12992 (68.2)11858 (68.0)1134 (69.7)1772 (73.9)11221 (67.3)Lack of reward High6972 (36.6)6122 (35.1)850 (52.2)*** 1216 (50.7)5757 (34.5)*** Low12092 (63.4)11314 (64.9)778 (47.8)1183 (49.3)10908 (65.5) *** p < 0.001 aweighted frequency
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study
| 100.0 |
The results showed that workers who reported that supervisors provided work-related feedback exhibited high job stress levels (OR = 1.329, 95% CI = 1.203 ~ 1.468). However, those who reported that a supervisor does respectful behaviors (OR = 0.812, 95% CI = 0.722 ~ 0.913) and planning and organizing work exhibited low job stress levels (OR = 0.816, 95% CI = 0.732 ~ 0.910). In addition, job stress did not differ significantly according to conflict solving abilities or participation in decision-making (Table 4).Table 4Multiple logistic regression analysis of factors affecting job stressVariablesUnadjustedAdjustedORb 95% CIc ORb 95% CIc Feedback No1.001.00 Yes1.4041.297 ~ 1.5201.3291.203 ~ 1.468Respectful behavior No1.001.00 Yes1.0740.984 ~ 1.1730.8120.722 ~ 0.913Good conflict abilities No1.001.00 Yes1.1011.022 ~ 1.1860.9770.881 ~ 1.083Good at planning No1.001.00 Yes1.0750.996 ~ 1.1610.8160.732 ~ 0.910Participation in decisions No1.001.00 Yes1.3081.221 ~ 1.4011.1090.999 ~ 1.230adjusted by job demand, job control, social support, job insecurity, lack of reward, sex, shift work, sector, income, age, education level, type of employment, number of employees, working hours, occupation, general health condition bodds ratio, cconfidence interval
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study
| 100.0 |
This study aimed to examine the effects of supervisors’ behavior on job stress in Korean workers. In this study, Feedback, Respectful behaviors, and Good at planning were significant results with job stress. When a supervisor gives feedback, work stress of worker was high. When supervisors respect workers or organize their work well Workers’ stress was low.
|
study
| 99.94 |
First, the results of the current study showed that supervisors’ provision of work-related feedback increased workers’ stress levels. This finding was consistent with those of a previous study indicating that supervisors’ provision of work-related feedback increased job stress in workers . However, another study showed that supervisors’ provision of feedback in an appropriate environment reduced work-related stress in workers . Numerous studies have shown that the environment in which supervisors provided feedback exerted both positive and negative effects on employees’ job stress levels [17–20]. Moreover, supervisors’ feedback was shown to influence both positive factors, such as person-organization fit and organizational commitment, and negative factors, such as role stress or burnout ; in addition, feedback environments exerted either positive or negative effects according to the characteristics of the organization or individual workers . In previous studies, defensive or self-deceitful workers were more affected by bad feedback than good feedback . According to Steelman, “favorable feedback” is expressed as the frequency of receiving positive feedback such as compliments from the perspective of the employee receiving the feedback, and “unfavorable feedback” is the frequency of complaints from managers or colleagues And the frequency of negative feedback such as criticism . Both “favorable feedback” and “unfavorable feedback” are important in reducing the deviance of the worker in terms of the feedback environment .
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study
| 100.0 |
Second, when a supervisor respect workers, the worker’s stress appeared to be low. In previous studies, there have been few studies on the relationship between supervisors’ respectful behaviors and worker’s job stress. According to Cobb, social support consists of three components: information that they are being cared for and loved, information that they are being esteemed, and valued, and information that they belong to one of the group members. In this study, the meaning of ‘Respectful behavior’ means ‘my supervisor respects me personally’ and is one of the components of social support according to the definition of Cobb . There are studies that suggest that social support affects worker’s job stress [23, 24].
|
study
| 99.9 |
Third, when the supervisor’s planning ability was good, the stress level was low. The meaning of “planning” in this study means to organize and plan work well. In previous studies, there have been few study on the relationship between supervisors’ planning ability and job stress. However, there have been studies that have increased job stress when work overload has increased [25, 26]. Supervisors are in an important position to manage role stress. They can influence role overload by determining the scope of work of an employee, and can monitor the worker’s impact on a role. When workers experience quantitative overload, they can help workers by analyzing their work, providing training that helps them work, or introducing workers who do a similar job. Or to help workers who are experiencing quantitative overload by distributing part of the work to another worker or by putting in another worker . With these organizing or planning approaches, managers can reduce psychological strain by preventing worker overload .
|
other
| 99.0 |
The present study was subject to some limitations. For example, although the KWCS, which was a cross-sectional study, demonstrated an association between supervisors’ behavior and workers’ stress, the inference of causality in this relationship was limited. In addition, psychosocial stress, outcome variables, and independent variables were measured via a self-report questionnaire in the current study, which could have led to response biases. Moreover, as work-related stress was not measured using a standardized instrument, such as the Korean Occupational Stress Scale, in the present study, the precision of the measurement of this variable was limited. Despite these limitations, the present study clarified the association between supervisors’ behavior and job stress in workers, using nationally representative data collected via the 2014 KWCS.
|
study
| 100.0 |
According to the present study, supervisors’ behaviors affected on job stress of Korean workers. Therefore, supervisors working in private-sector organizations with fewer than 300 employees should consider the characteristics of workers and be provided appropriate feedback education, receive training in respectful behaviors and organizational skills, which could contribute to the reduction of job stress in workers.
|
other
| 99.9 |
Homologous chromosome recombination in meiotic prophase is required for orderly chromosome segregation. Recombination is preceded by chromosome prealignment and the scheduled formation of DNA double-strand breaks (DSBs), followed by a RAD51-mediated search for homologous DNA sequences and the formation of heteroduplexes involving DNA strands of homologous chromosomes at early stages of meiotic prophase (leptotene and zygotene). Polymerisation of the synaptonemal complex (SC), a meiotic-specific proteinaceous structure, stabilises these connections and completes homologous chromosome synapsis. A small proportion of DSBs (at least one per chromosome pair) is repaired as crossovers (reciprocal exchanges between homologues). The sites of crossing over can be visualised in mid-meiotic prophase (pachytene) as late recombination nodules containing MLH1 (mismatch repair protein), and at diplotene-diakinesis as chiasmata. Sister chromatid cohesion beyond the chiasmata holds homologues together at metaphase-I, ensuring proper orientation and orderly segregation12.
|
study
| 99.94 |
These complex and highly coordinated processes are thoroughly checked by natural selection at each meiosis, and so the genetic unity of the species is preserved. The evolution of geographically isolated populations, however, leads to the fixation of novel chromosomal rearrangements and a divergence of the factors controlling DSB formation and the DNA sequences involved in homology search. In hybrids, karyotypic and genetic divergence can result in meiotic aberrations and variable degrees of infertility due to germ-cell death or the formation of unbalanced gametes.
|
study
| 99.8 |
Although mammalian hybrids have been known for centuries3, studies of the genetic and cellular bases of hybrid sterility in mammals are surprisingly scarce. Several genes causing male sterility in hybrids between karyotypically identical species of the house mouse (Mus musculus x M. domesticus)5678 and felines (Felis catus x Profelis serval, F. catus x Prionailurus bengalensis)9 have been localised and sequenced. One of them, PRDM9 (referred to as a speciation gene), is involved in the control of DSB formation10. Male sterility or reduced fertility accompanied by synaptic aberrations of autosomes and sex chromosomes has been widely reported in hybrids of chromosomally divergent mammalian species, subspecies and local populations3111213141516171819.
|
review
| 98.94 |
Sterility in mammalian hybrids is in a good agreement with Haldane’s rule that “when in the F1 offspring of a cross between two animal species or races one sex is absent, rare, or sterile, that sex is always the heterozygous sex”20. Several hypotheses have been proposed to explain the genetic basis of this rule. Among them, the dominance and “faster male” hypotheses are considered the most plausible2122232425. The dominance model ascribes the predominant inviability/sterility of the heterogametic sex to the alternative fixation of X-linked recessive mutations. The higher the ratio between recessive and dominant mutations of incompatibility, the larger the time lag between hetero- and homogametic sexes in the evolution of hybrid unfitness26. The “faster male” hypothesis suggests that male fertility alleles evolve and diverge faster due to divergent sexual selection. At the same time, strong sperm selection leads to increase of the stringency of male meiotic checkpoints2527. In mammals, male meiosis is very sensitive to genetic and chromosomal aberrations. Meiotic mutants and knockouts affect meiosis earlier and more severely in males than in females28 and this amplifies the effects of genetic and chromosomal incompatibility on the fertility of male hybrids.
|
study
| 99.7 |
Haldane’s rule usually applies only to early stages of speciation. Building reproductive barriers is a snowball process29, or, as Darwin (1866) put it, a series of “graduated steps from very slightly lessened fertility to utter and absolute sterility”30. Therefore, at advanced stages of reproductive isolation, both sexes are sterile. We are currently unclear, however, as to whether males and females proceed to complete sterility by the same or by different routes. Sex differences in meiotic disruption within hybrids, then, can tell us about the genetic and cytological bases of these steps. Unfortunately, due to technical difficulties female meiosis in the hybrids has rarely been analysed. We are aware of only one such study31, which found that female hybrids between Mus musculus and M. domesticus were fertile, even though their oocytes displayed the same pairing abnormalities as male hybrids - with half the frequency, though.
|
study
| 99.94 |
To make insight into the cytological basis of hybrid sterility, we examined chromosome synapsis and recombination in male and female sterile hybrids between two sibling species of the grey vole, Microtus arvalis (dams) and M. levis (sires). These species diverged from 0.5 to 4.3 MYA3233, differ by a series of chromosomal rearrangements3334, yet remain morphologically indistinguishable35. In nature, hybrids occur at the zone of sympatry in the Urals, and can easily be produced in laboratory settings3536373839. Reciprocal hybrids between M. arvalis and M. levis are completely sterile; however, males differ from females in the stage of reproductive collapse38. In males, testis mass is severely reduced and no sperm is found in the epididymis. Male meiosis arrests at prophase I but occasional nuclei reach diakinesis-metaphase I, where they mainly show univalents. More advanced stages of spermatogenesis have not been detected40. In female hybrids, oocyte growth and development was described as normal. However, follicular atresia was detected in hybrids even at the primordial follicle stage, and, at all stages, was more pronounced than in females of the parental species. Hybrid females display abnormal ovulation without follicle wall breakage. Mature oocytes move into Graafian follicles where they undergo the second meiotic division. No corpus luteum was detected in hybrid females41. According to Gileva et al.37 backcross progeny may be produced very rarely, but in our hybridisation experiments none occurred. Thus, these hybrids provide a promising model for studying cellular mechanisms of male and female hybrid sterility.
|
study
| 100.0 |
We analysed key events of chromosome synapsis and recombination. DSBs were detected by immunolocalisation of RAD51, a marker for single-stranded DNA ends42, and γH2A.X, a phosphorylated form of histone H2A.X43. Polymerisation of the lateral elements of SC and the formation of its central element were visualised with antibodies to SYCP3 and SYCP143. The number and distribution of recombination events were estimated by immunolocalisation of MLH144. This analysis allowed us to detect meiotic aberrations leading to hybrid sterility.
|
study
| 100.0 |
Chromosome pairing and recombination in male M. arvalis and M. levis have been described previously454647. Both species showed asynapsis of the X and Y chromosomes (Fig. 1a,b, Supplementary Fig. S1) which underwent meiotic sex chromosome inactivation and were labeled by γH2A.X antibodies (Fig. 1c, Fig. S1). This feature is characteristic of the entire arvalis lineage of the genus Microtus47. Asynapsis of autosomes at pachytene stage, when most chromosomes contained MLH1 foci, was very rare (less than 2% in both sexes) and always affected small chromosomes. M. arvalis and M. levis males did not differ from each other in total autosomal SC length (t212,1 = 2.6, P = 0.09) or MLH1 focus number per cell (t212,1 = 1.1, P = 0.29) (Table 1). The number of MLH1 foci was usually restricted to one per autosomal bivalent in spermatocytes, as was in oocytes (Fig. 1d, Table 1). Assuming at least one focus on the XX bivalent, no significant sex differences in recombination rates can therefore be seen in M. levis.
|
study
| 100.0 |
Leptotene oocytes of hybrids appeared normal with assembling lateral elements of SC labeled by RAD51 (Fig. 2a). At zygotene, lateral elements established contacts with each other while the central elements formed asynchronously. Thus, while some lateral elements were already paired, others displayed extensive asynapsis. Asynaptic regions were often intertwined and labeled by RAD51 and γH2A.X (Fig. 2b,c, Fig. S2). Some regions kept these marks until pachytene when most autosomes were synapsed (Fig. S2).
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study
| 100.0 |
Pachytene oocytes were very variable in their appearance, containing asynaptic and heterosynaptic configurations. However, about 10% of oocytes had 23 “correctly” paired configurations, with four large trivalents, twelve heteromorphic and seven homomorphic bivalents (six acrocentric and one metacentric) (Fig. 3a). The heteromorphic bivalents comprised the X pair with long arms of different length and misaligned centromeres, and one large and ten smaller bivalents with the two centromere signals shifted. This is in a good agreement with pairing expected from comparative metaphase analysis of the parents (Fig. 3b). Parental karyotypes differ by at least four tandem fusions (producing trivalents in F1 hybrids), one paracentric and six pericentric inversions (which may produce inversion loops or straight bivalents with misaligned centromeres), four to seven putative centric transpositions (producing bivalents with misaligned centromeres), and an insertion of a large heterochromatic block in the X of M. levis3334.
|
study
| 100.0 |
Although good correspondence between expected and observed synaptic configurations was found, there were two discordances. One heteromorphic bivalent showed centromere signals on its both ends (Fig. 3b, arrowhead). A trivalent comprising metacentric chromosome 1 of M. arvalis and twin acrocentrics of M. levis contained three misaligned centromeres. Lemskaya et al.34 proposed that chromosome 1 of M. arvalis resulted from tandem fusion of the proximal end of chromosome 3 and the distal end of chromosome 2 of M. levis. However, the synaptic configurations observed in these F1 hybrids indicate that chromosome 1 of M. arvalis was generated by fusion of the distal ends of the acrocentrics followed by inactivation of their centromeres, and re-activation of the centromere at the point of fusion.
|
study
| 100.0 |
More than 90% of pachytene cells in female hybrids contained synaptic aberrations. Supplementary Fig. S2 shows examples of these aberrations. The most common were multivalents containing more than three lateral elements (from four to twenty, 6.5 ± 3.4 on average) (Figs 4 and 5). The number of multivalents varied from one to four per cell (1.7 ± 0.9 on average) and most multivalents were composed of large chromosomes. The probability for large metacentric chromosomes 1 to 4 of M. arvalis and the acrocentric pairs of M. levis to be involved in multivalent formation was 0.60 ± 0.06. This probability was also high for the X chromosomes (0.42 ± 0.11) and submetacentric chromosome 5 of M. arvalis with homologous acrocentric chromosome 1 of M. levis (0.32 ± 0.11). The probability for medium sized and small chromosomes, however, was very low (0.05 ± 0.01).
|
study
| 100.0 |
Univalents were also seen, varying in number from one to nine (3.0 ± 2.4 on average). In many cells odd numbers of univalents were found, indicating that some of their partners were involved in non-homologous synapsis in a multivalent. For the same reason the average number of observed trivalents was lower than the expected four (2.5 ± 1.4 on average). The number of bivalents in oocytes was lower than the expected 19 (15.4 ± 1.5 on average), again due to involvement of one or both partners in non-homologous synapsis in multivalents. Asynapsis was frequently observed around pairing partner switch points and at the ends of multivalent chains. Unpaired regions were labeled with γH2A.X (Fig. 4a,b, Fig. S2) and RAD51 (Fig. 4c,d, Fig. S2), indicating that they contained unrepaired DSBs.
|
study
| 100.0 |
Figure 5 and Fig. S2 show immunolocalisation of MLH1 at the SCs in pachytene oocytes of F1 hybrids between M. аrvalis and M. levis. The mean number of MLH1 foci was significantly lower in hybrid oocytes than in oocytes of M. levis (t96,1 = 9.6, P < 0.001: Table 1), while there was no difference in total SC length (t78,1 = 1.0, P = 0.32: Table 1). The decrease in hybrids was mainly due to a lack of foci in univalents. There was no substantial decrease in recombination between the homologous segments in homomorphic and heteromorphic bivalents, trivalents or multivalents. Most paired arms had at least one MLH1 focus. Even in the cells containing very complex multivalents (Fig. 5a), a series of multivalents (Fig. 5b), or long asynaptic regions (Fig. 5c) we did not observed a substantial decrease in the number of MLH1 foci.
|
study
| 100.0 |
Male meiosis in hybrids was generally arrested at leptotene. The majority of spermatocytes carried only fragments of the axial elements (Fig. 6a, Fig. S2), indicating incomplete assembly. The most advanced spermatocytes contained almost normal axial elements, but even in these cells no initiation of synapsis was seen (Fig. 6b). The halved total length of the lateral elements in the male hybrids at this stage was about 10% longer than the total SC length of the male M. levis at pachytene (t92,1 = 4.4, P < 0.001: Table 1). Hybrid spermatocytes showed heavy labeling with RAD51 antibodies indicating DSB presence.
|
study
| 100.0 |
This study supports breeding records and histological assessments showing the sterility of hybrids between M. arvalis and M. levis353637383941. Hybrids of both sexes are sterile; however, males, in accordance with Haldane’s rule, are “more sterile” than females. Male meiosis is uniformly arrested at leptotene, while female meiosis is affected at pachytene with wide variation between oocytes in the degree of disturbances of chromosome pairing. This might be due to the sex difference in degree of methylation on the meiotic onset. Studies in mice demonstrated that female germ cells enter meiosis in a demethylated state, while the genome of male germ cells is heavily remethylated after mitotic arrest at the beginning of meiosis484950. A faster evolution of male hybrid sterility in the grey voles might also be accelerated by constitutive asynapsis and total lack of recombination between XY in this phylogenetic lineage47.
|
study
| 100.0 |
Several features of meiotic disturbances in female hybrids between M. arvalis and M. levis also characterise male meiosis in many cases of male-only hybrid sterility. The zygotene-pachytene transition is mainly affected, asynapsis and heterosynapsis occur in both heteromorphic and homomorphic chromosomes, while cells vary widely in the degree of disturbances and the number of chromosomes affected1112515253.
|
study
| 99.5 |
Despite the sex differences in the stage and degree of meiotic disturbances, the cause of these disturbances is probably the same. This is failure of a homologous synapsis between homologous chromosome regions, which is complete in males and partial and sporadic in females. The majority of oocytes contains multiple regions of asynapsis and non-homologous synapsis. The establishment and extension of non-homologous synapsis leads to multivalent and univalent formation. Thus, when one homologue is involved in a multivalent, the other either remains univalent or pairs non-homologously with another chromosome region in the same or a different multivalent.
|
study
| 99.9 |
DSB-independent pairing probably depends on associations of centromeres and/or telomeres at early prophase. It restricts the searching area for homologous recognition and alignment2. This process may be impaired in grey vole hybrids, which are heterozygous for at least 15 chromosomal rearrangements3334. Although heterozygosity for a single fusion, pericentric inversion or centromeric shift does not usually lead to pairing abnormalities45545556, a high number of such heterozygosities may impede or delay presynaptic alignment of homologous regions57. It has been shown that multiple heterozygosity for a series of Robertsonian translocations in mice alters the nuclear architecture characteristic of the telocentric karyotype, changes patterns of centromere clustering and other interactions between chromosomal domains and leads to ectopic associations58. Similar problems in centromere clustering should occur in multiple heterozygotes for pericentric inversions and centromeric shifts.
|
study
| 100.0 |
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