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Short-chain fatty acids (SCFAs), produced by anaerobic fermentation of mainly indigestible dietary carbohydrates by gut microbiota, have a profound influence on intestinal function and host energy metabolism. Antibiotics may seriously disturb the balance of fecal SCFAs. To evaluate the impacts of antibiotics on fecal SCFAs produced by gut microbiota, a simple, reproducible and accurate gas chromatography (GC) method, which can simultaneously analyze seven SCFAs in fecal samples, was developed and validated. The ranges of detection and quantitation of the SCFAs reached 0.0868 ~ 0.393 and 0.261 ~ 1.18 μg·mL-1 respectively, in an optimized protocol for SCFAs extraction and analysis that used 10 mL 75% ethanol aqueous solution containing 1% HCl, without ultrasonication. The technique exhibited excellent intra-day (relative standard deviation (RSD) ≤ 2.54%) and inter-day (RSD ≤ 4.33%) precisions for all the SCFAs. Later, we administered broad-spectrum antibiotics, cefdinir or azithromycin to rats and analyzed the alterations in fecal SCFAs. The total amount, types and distribution of nearly all fecal SCFAs were significantly altered during the administration and even after withdrawal of the antibiotics in rats. The effects of cefdinir on the SCFAs were more pronounced than those of azithromycin. Our findings suggest SCFAs may serve as sensitive indicators to monitor the influences of antibiotics on SCFAs originated by intestinal bacteria. Our improved SCFAs analysis method is a potential platform for a standard clinical test of the effects of new antibiotics on SCFAs.
Citation: Zhao X, Jiang Z, Yang F, Wang Y, Gao X, Wang Y, et al. (2016) Sensitive and Simplified Detection of Antibiotic Influence on the Dynamic and Versatile Changes of Fecal Short-Chain Fatty Acids. PLoS ONE 11(12): e0167032. https://doi.org/10.1371/journal.pone.0167032
Editor: Marie-Joelle Virolle, Universite Paris-Sud, FRANCE
Received: March 17, 2016; Accepted: November 8, 2016; Published: December 1, 2016
Copyright: © 2016 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by grants from the National Science and Technology Major Projects for "Major New Drugs Innovation and Development" (No. 2015ZX09J15102-004-004 and 2013ZX09201020).
Competing interests: The authors have declared that no competing interests exist.
Gut microbiota, a complex microbial community densely inhabits in the intestinal tract, plays essential roles in our health via multiple mechanisms, one of which is to provide functional metabolites for the host. Short-chain fatty acids (SCFAs) are major end-products of anaerobic fermentation by microbiota in the large intestine, including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid and hexanoic acid [1–3]. Acetic acid is produced by most enteric bacteria, such as acetogenic bacteria through the Wood-Ljungdahl pathway . But propionic acid is mainly produced by Bacteroides and Propionibacterium through succinate pathway . Butyrate-producing bacteria mainly include Faecalibacterium, Eubacterium, and Roseburia genera , whereas Roseburia Inulinivorans and Coprococcus Catus are able to produce propionic acid and butyric acid . A recent study showed that most dominant species of the Bacteroides positively associated with fecal concentrations of isovaleric and isobutyric acid, which are negatively correlated with blood levels of triglycerides . SCFAs are important anions in the colonic lumen not only to provide the major source of energy for colonocytes , but also to exert anti-inflammatory  effects, which influence various functions of the intestinal tract [11–13]. In addition, some SCFAs could regulate the growth of known pathogens . Therefore, SCFAs may reflect the metabolic activity of intestinal microbiota , which could be modified by dietary and environmental changes , particularly by antibiotics  and diet [18,19]. Recent studies indicate that SCFAs are produced by gut microbial fermentation of dietary fiber , thus, compared to the plant-based diet, an animal-based diet resulted in significantly lower levels of the products of carbohydrate fermentation .
Antibiotics plays an effective role in controlling infections caused by pathogens. However, antibiotics also cause adverse effects, including the emergence of resistant bacteria  and dysbiosis [22–27]. For example, Young and Schmidt , who investigated change of bacterial populations in fecal samples from patients with antibiotic-associated diarrhea by amoxicillin-clavulanic acid treatment, found a reduction of genus Faecalibacterium and absence of Clostridium on the fourth day of antibiotic treatment. Small sub-unit rRNA sequencing is often employed to determine the community structure of the gut microbiota to monitor microbial community and diversity after antibiotic administration [23,28]. Antibiotics may therefore also affect the intestinal presence and balance of the major metabolites of gut microbiota, including SCFAs, vitamins , and amino acids . Previous studies have used a number of techniques for SCFAs analysis, including gas chromatography (GC) [31,32], gas chromatography-mass spectrometry (GC-MS) , high performance liquid chromatography (HPLC) , liquid chromatography–mass spectrometry (LC-MS) , capillary electrophoresis (CE) , and stable isotope tracer methods . GC is the most common technique to analyze volatile compounds, but it is usual to pretreat feces by derivatization with different reagents before analysis [33,38]. Derivatization is time-consuming and affects the accuracy and reproducibility of the assay. Although several methods have been developed to improve SCFAs determination, a more efficient and simplified approach is still in demand.
Here, we aimed to develop a simple, accurate and robust GC method for the analysis of fecal SCFAs, and to investigate the effects of antibiotics on the fecal SCFAs of rats. Two representative broad-spectrum antibiotics were selected: cefdinir, an advanced-generation cephalosporin, and azithromycin, a macrolide. We found all the SCFAs decreased upon cefdinir or azithromycin treatment and most of the SCFAs did not recover to control levels within 8 days after withdrawal of the antibiotics. The changes of fecal SCFAs may be used as a clinical index to guide antibiotic use.
Materials and Methods
Chemicals and reagents
Acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, hexanoic acid, 2-ethyl butyric acid and 2-ethyl hexanoic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). We used 2-ethyl butyric acid and 2-ethyl hexanoic acid as internal standards (IS) for final determination of the quantity of each compound present in fecal samples. Ethanol was purchased from Tianjin Damao Chemical Reagent Factory (Tianjin, China). Hydrochloric acid was from Tianjin Jindong Tianzheng Precision Chemical Reagent Factory (Tianjin, China). Ultra-pure water was generated using a Milli-Q water purification system (Millipore, USA). Antibiotics employed were cefdinir dispersible tablets (Shenzhen Zhijun Pharmaceutical Co., Ltd., production batch number A150103), and azithromycin dispersible tablets (Harbin Pharmaceutical Group, Sanchine Nuojie Pharmaceutical Co., Ltd., production batch number 1502224), which were provided by first teaching hospital of Tianjin university of TCM (Tianjin, China).
Eighteen male Sprague-Dawley rats weighing 190 ~ 210 g were supplied by Vital River Lab Animal Co., Ltd. (Beijing, China) [Certification number: SCXK (Jing) 2012–0001]. Rats were housed individually in metabolic cages in conditions of controlled temperature (24 ± 1°C), humidity (50−60%) and photoperiod (12 hours light/dark) with free access to drinking water and food pellets (standard chow: 18.0% protein, 4.0% lipids, and 5.0% fiber) during the experiments.
The 18 rats were randomly divided into three groups, two experimental groups and a control group. Dispersible tablets of cefdinir (135 mg·kg-1·d-1) and azithromycin (450 mg·kg-1·d-1) dissolved in saline were respectively gavaged to the two experimental groups of rats every day for 6 days. The control group was treated by gavage with an equivalent volume of saline. In the next 8 days after withdrawal of the antibiotics, the rats accessed water and food freely under the conditions described above. Fecal samples were collected in the following 24h after the first administration, which is labeled as day 1. Subsequently, the rest feces were collected every 2 days at 9 a.m. and labeled as days 3, 5, 7, 9, 11 and 13. Fecal samples were respectively transferred into 4 mL eppendorf tubes and immediately stored at −80°C. Rats were weighed at 9 a.m. during the period of drug administration and recovery.
Individual stock standard solutions of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, hexanoic acid, 2-ethyl butyric acid and 2-ethyl hexanoic acid were prepared by dissolving the analytes in 75% ethanol aqueous solution, the concentration of which were 23.58, 5.34, 0.46, 9.96, 0.70, 1.28, 1.04, 1.43, and 0.19 mg·mL-1. 2-ethyl butyric acid was employed as internal standard for determination of acetic acid, propionic acid and butyric acid. 2-ethyl hexanoic acid was chosen as internal standard for quantitation of isobutyric acid, isovaleric acid, valeric acid and hexanoic acid.
Working standard solutions were prepared and serially diluted with 1% HCl/75% ethanol aqueous solution containing 2-ethyl butyric acid and 2-ethyl hexanoic acid at a final concentration of 44.51 and 2.26 μg·mL-1, respectively. The detail concentrations of 7 analytes were as following: acetic acid (33.16 ~ 1061 μg·mL-1), propionic acid (7.51 ~ 240.3 μg·mL-1), isobutyric acid (0.78 ~ 25.08 μg·mL-1), butyric acid (9.34 ~ 298.7 μg·mL-1), isovaleric acid (0.98 ~ 31.37 μg·mL-1), valeric acid (1.61 ~ 51.36 μg·mL-1), and hexanoic acid (0.98 ~ 31.26 μg·mL-1), respectively. Once prepared, the solutions were stored at −20°C until analysis.
Sample solution preparation
As follows, the sample preparation procedure was optimized in terms of extraction solvent, extraction volume, the percentage concentration of HCl in sample solution, and ultrasonication time.
Ultra-pure water (2.5 mL) was added to each 0.5 g fecal sample in a 15 mL tube, vortexed briefly, and then a total of 7.5 mL 1.33% HCl/ethanol solution containing internal standards were added to give the final concentration of 44.51 μg·mL-1 (2-ethyl butyric acid) and 2.26 μg·mL-1 (2-ethyl hexanoic acid). The samples were homogenized for 2 min, and centrifuged at 17,968 × g for 10 min at ambient temperature. Supernatant was promptly transferred to a 2 mL sample vial.
Analysis was performed using a GC-2010 (Shimadzu, Kyoto, Japan) system equipped with an AOC-20i autosampler, and coupled to flame ionization detector. The chromatographic separation was performed on a DB-FFAP capillary column (30 m × 0.25 mm × 0.25 μm; Agilent Technologies, Palo Alto, CA, USA). A glass liner stoppered with a glass wool plug in the middle of the liner was used to prevent contamination of the GC column with nonvolatile fecal materials. Three microliters of the sample solution was injected in split mode at a ratio of 50:1. The injection temperature and detector temperature were 250°C. The initial temperature of the column was 50°C for 1 min, increased to 120°C at a rate of 15°C·min-1, to 170°C at a rate of 5°C·min-1, and then to 240°C at a rate of 15°C·min-1 for 3 min. The total run time was 23.33 min. The carrier gas was N2 (99.999%) at 1 mL·min-1. The make-up gas (N2) was at 30 mL·min-1. The flow rate of H2 and air were 47 mL·min-1 and 400 mL·min-1, respectively.
The method established in this study was validated with method of internal standard for linearity, precision (intra- and inter-day), stability, repeatability and recovery yield, except for limit of detection (LOD) and limit of quantification (LOQ). Calibration curves were constructed based on the peak area ratios of the analytes to the IS (y) versus the corresponding concentrations (x) of seven standard solutions at different concentrations. The LOD and LOQ were determined at a signal-to-noise ratio (S/N) of about 3 and 10, respectively. The intra- and inter-day precisions of the sample solution were conducted with six replicate injections performed on the same day and on three consecutive days, respectively. The stability of the sample solution stored in the GC autosampler at room temperature was investigated by replicate injection at 0, 2, 4, 6, 8, 10 and 12 h. To confirm the repeatability, six replicates of the same sample were processed and analyzed. A recovery test was used to further evaluate the accuracy of the method.
Area under concentration versus time curve was calculated by DAS 3.0 software (Data Analysis System). Line charts and histograms were analyzed with GraphPad Prism 5.01 (GraphPad Software, Inc., USA) and pie graphs were performed by Microsoft Excel 2010 (Microsoft Corporation, USA). Nonparametric t-test was used on every antibiotic group and control group comparison. Statistical significance was accepted at p < 0.05.
This article does not contain any studies with human participants performed by any of the authors. All procedures performed in studies involving animals were in accordance with the ethical standards of Tianjin International Joint Academy of Biotechnology and Medicine (TJAB-TJU20140006).
Results and Discussion
Optimization of sample preparation
SCFAs, especially acetic, propionic and butyric acid, are vital to maintain the normal function of intestine and human body. Acetate is absorbed and transported to the liver and peripheral tissues, less metabolized in the colon and acts as substrate for cholesterol synthesis and lipogenesis . Propionate is a primary precursor for gluconeogenesis and it reduces the synthesis of hepatic cholesterol . Butyric acid is a preferred energy source for colonocytes and protects from inflammation . Most of butyrate is metabolized by the colonocyte. Butyrate also affects the regulation of cellular proliferation and apoptosis, resulting in reduced risk of colon cancer . Therefore, we focused on these interesting SCFAs in this study.
The single variable method was used to successively inspect the effect on extraction efficiency of SCFAs from the feces of rats from four sample preparation factors, including (1) the ratio of ethanol to water (25%:75%, 50%:50%, 75%:25% and 100%:0%; v/v); (2) the percentage of HCl (0.1%, 0.5%, 1% and 1.5%); (3) ultrasonication time (0, 5, 10 and 15 min); and (4) extraction volume (5, 10, 20 and 40 mL). Duplicate samples were prepared for each condition to ensure the repeatability of the method.
The ratio of ethanol to water (Fig 1A) and the percentage of HCl (Fig 1B) both had obvious influences on the extraction results. The ultrasonication time (Fig 1C) and the extraction volume (Fig 1D) had no significant effect, but when the samples were diluted with an extraction volume of 10 mL, without ultrasonication, the detection level of SCFAs was increased by 4.3% and 8.2%, respectively. The optimum conditions for extracting SCFAs from rat feces were: extraction in 10 mL 75% ethanol aqueous solution containing 1% HCl, without ultrasonication.
(A) ratio of ethanol to water; (B) the percentage of HCl; (C) ultrasonication time; and (D) extraction volume.
Previous studies reported that fecal SCFAs could be extracted by one-step or more step derivatization [31–38]. The development of reported methods was an essential issue for investigation of SCFAs in different biological samples, which provided more options for analysts. Nonetheless, these methods suffered from limits of the complicated and time-consuming procedure for derivatization [33, 38], or long analytical time [33, 35, 38], or unsuitability for analysis of fat soluble SCFAs , or poor sensitivity by UV detection , or focus on the determination of SCFAs in other biological samples beyond feces [31, 34–37]. Thus, we developed a simple, rapid and more suitable method for accurate quantification for SCFAs in feces.
Using the optimized sample extraction and GC methods, we acquired chromatograms for the mixed standards solution (Fig 2A), the control fecal sample (Fig 2B) and the cefdinir-treated sample (Fig 2C) on day 1. A correlation coefficient (r) >0.999 was obtained for all the calibration curves (Table 1). LOD and LOQ of all the SCFAs were 0.0868 ~ 0.393 and 0.261 ~ 1.18 μg·mL-1 respectively, indicating that the method is sufficiently sensitive for analyzing fecal SCFAs. The new method exhibited excellent precision (intra-day and inter-day RSD ≤2.54% and ≤4.33%, respectively) (Table 2). The result showed that the SCFAs were stable over 12 h at room temperature (Table 2), and showed an excellent repeatability with RSD ≤4.40% (Table 2). The average recovery values for each SCFAs varied from 92.81 to 104.1% (n = 6) with RSD ≤3.98% (Table 2).
(A) mixed standards solution; (B) the control fecal sample; (C) the fecal sample from the cefdinir-treated group on the first day. 1. acetic acid; 2. propionic acid; 3. isobutyric acid; 4. butyric acid; 5. isovaleric acid; 6. valeric acid; 7. hexanoic acid; IS-1. 2-ethyl butyric acid; IS-2. 2-ethyl hexanoic acid.
The variation in the weight of the rats
The variation in the weight of rats of the control, cefdinir and azithromycin treatment groups was shown in Fig 3. There were significant increases in body weight for all groups of rats from Day 0 to Day 13. During this experimental period, the weight gain of rats was no significantly different between control and cefdinir-treated group (p >0.05), but was significantly different between control and azithromycin-treated group (p <0.01). Studies have shown that azithromycin cause gastrointestinal side effects , but few study report adverse effects of cefdinir related to loss of appetite. Our observation of decreased weight gain in azithromycin-treated rats in this study is consistent with its reported gastrointestinal side effects .
Antibiotic treatments alter fecal SCFAs
The validated GC method was subsequently applied to analyze fecal SCFAs over time. The feces of 18 rats were collected for three days prior to the administration of antibiotics to carry out the analysis of seven SCFAs. As normal level of SCFAs, the average concentrations were 2768.6 ± 106 μg·g-1 for acetic acid, 758.1 ± 34.4 μg·g-1 for propionic acid, 43.58 ± 0.89 μg·g-1 for isobutyric acid, 951.1 ± 32.3 μg·g-1 for butyric acid, 53.03 ± 1.22 μg·g-1 for isovaleric acid, 75.49 ± 3.18 μg·g-1 for valeric acid, and 62.00 ± 1.52 μg·g-1 for hexanoic acid. The detail results of fecal SCFAs of rats at day 1, 3, 5, 7, 9, 11, 13 during the period of drug administration and post-drug administration were displayed in Tables A-G in S1 File. The data of seven SCFAs was summarized in Table 3. Treated with cefdinir and azithromycin, the pronounced changes of SCFAs were detected compared with the control group. These variations were found to be in line with other studies performed with other antibiotics, such as ampicillin and clindamycin .
Fig 4 shows time courses of the changes in the seven SCFAs levels in fecal samples from control rats and rats treated with cefdinir or azithromycin. During cefdinir administration, the concentrations of all the SCFAs were remarkably decreased relative to the controls. Acetic acid, propionic acid and butyric acid were detected in much lower level than those from control rats, while isobutyric acid, isovaleric acid, valeric acid and hexanoic acid were undetectable, suggesting a pronounced variation in the gut microflora of the rats. After cefdinir administration ended, the concentrations of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid gradually recovered compared with those in the control group, but the levels of butyric acid and valeric acid remained lower than those in the control group. On azithromycin treatment, the concentrations of all SCFAs (except hexanoic acid) declined to below the control levels during the experiment. The fecal SCFAs concentrations during the recovery period (post-drug administration) were higher than those during the period of administration, but still lower than the respective concentrations in the control group, suggesting that the gut microbiota recovered, but did not reach their normal level within 8 days following withdrawal of azithromycin.
Antibiotics (cefdinir or azithromycin) were administered for the first 6 days. (A) acetic acid; (B) propionic acid; (C) isobutyric acid; (D) butyric acid; (E) isovaleric acid; (F) valeric acid; and (G) hexanoic acid. Arrow means administration of antibiotic.
Few studies systematically explored the influences of antibiotics on fecal SCFAs , and none on the influences of cefdinir or azithromycin. A very recent report showed that the microbiota of preterm infant involves a concomitant alteration in the levels of SCFAs . Moreover, it can be further affected by perinatal antibiotic use, for example, a preterm infants’ mother who received penicillin, ampicillin, or ampicillin plus erythromycin . Our results showed that nearly all the SCFAs were impacted by cefdinir or azithromycin, the two antibiotics widely used in clinics. Our data also indicate that SCFAs could be a sensitive indicator of the influences of antibiotics on gut microbiota.
Variations of SCFAs by area under concentration versus time curves (AUC)
To further clarify the impact of antibiotics on SCFAs, an area under concentration versus time curve (AUC, μg·g-1·d) of SCFAs is introduced, as presented in Fig 5A. The shaded areas between days 1 to 5 and days 7 to 13 represent the AUC in the period of antibiotic administration (Table H in S1 File) and the recovery period of post administration (Table I in S1 File), respectively.
The schematic diagram of AUC with a function of time (A), and AUC of SCFAs in fecal samples in the periods of antibiotic-treated and recovery (B). (a) acetic acid; (b) propionic acid; (c) isobutyric acid; (d) butyric acid; (e) isovaleric acid; (f) valeric acid; (g) hexanoic acid.
As displayed in Fig 5B, during antibiotic administration, the AUC of fecal SCFAs, especially the high abundant SCFAs, such as acetic acid, propionic acid and butyric acid, were significantly reduced (p <0.05), except for isobutyric acid and hexanoic acid, in the antibiotic-treated groups compared with the control group. Effects of cefdinir on SCFAs were more pronounced than those of azithromycin. During post administration period, the AUC values for acetic acid, propionic acid, butyric acid and valeric acid in the cefdinir group were remarkably reduced (p <0.01) compared with those in the control group, but were higher than those during the period of drug administration. During the recovery period, the AUC of isobutyric acid was significantly increased (p <0.01) and the AUC of isovaleric acid showed no significant difference (p >0.05) compared with the control group, indicating that the bacteria which produced isobutyric acid and isovaleric acid, the branched-chain fatty acids, rapidly returned to the control level. The AUC of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid in the azithromycin-treated group were obviously decreased (p <0.05) compared with the control group and none of them recovered to the control level within 8 days, except for hexanoic acid. These disparate results could be partially due to different antimicrobial spectrum and activities of cefdinir and azithromycin [46,47].
Overall quantities and relative proportions of seven SCFAs in fecal samples
As illustrated in Fig 6, compared with the control group, the overall quantities and relative proportions of SCFAs fluctuated remarkably throughout the study in the antibiotic-treated groups. Quantities and proportions of SCFAs produced by the gut microbiota remained relative stable in the control rats. During days 1 and 13, the mean quantities of total SCFAs in the control group ranged from 3026.6 μg·g-1 to 5616.4 μg·g-1 with the distribution of proportion: acetic acid 56.33% ± 3.78% (mean ± SD), propionic acid 19.81% ± 4.27%, butyric acid 21.47% ± 3.95%, isobutyric acid 0.50% ± 0.17%, isovaleric acid 0.76% ± 0.30%, valeric acid 1.02% ± 0.19%, and hexanoic acid 0.12% ± 0.08%, respectively.
(The size of the pie charts represents the relative total quantity of SCFAs; “N” represents the control group. “C” represents the cefdinir group. “A” represents the azithromycin group).
During cefdinir treatment (days 1, 3 and 5), the quantities and proportions of the seven SCFAs in fecal samples were changed markedly compared with the control group. In detail, compared with the control group, the total SCFAs decreased significantly (p <0.01) and mostly were undetectable except acetic acid, propionic acid and butyric acid. The ratio of these three remaining SCFAs was also dramatically altered, as follows: acetic acid 77.25% ± 1.51%, propionic acid 7.97% ± 1.41%, and butyric acid 14.78% ± 2.26%. In the recovery period following cefdinir administration (days 7, 9, 11 and 13), the mean quantities of total SCFAs were restored from 642.9 μg·g-1 to 3942.8 μg·g-1, but it did not reach the normal level of the control group (p <0.01) except at day 11. The ratio of the SCFAs gradually returned to control levels eventually with acetic acid at 55.87% ± 8.65%, propionic acid at 28.42% ± 6.61%, butyric acid at 13.10% ± 7.52%, isobutyric acid at 0.56% ± 0.18%, isovaleric acid at 1.26% ± 0.35%, and valeric acid at 0.79% ± 0.66%, respectively. In azithromycin-treated group, the overall quantities and relative proportions of the SCFAs dramatically changed within 24 h after the first administration. In day 3 and 5, the quantities and proportions of SCFAs recovered compared with day 1. The mean quantities of total SCFAs in the azithromycin group were higher than that in the cefdinir group during administration period with a concentration between 590.5 μg·g-1 and 2552.3 μg·g-1, which reduced significantly (p <0.01) compared with the control group. During post-administration recovery (days 7, 9, 11 and 13), the mean quantities of total SCFAs increased gradually from 2600.0 μg·g-1 to 3308.8 μg·g-1, however, did not recover to normal level (p <0.05) except at day 11. The ratios of the seven SCFAs in the azithromycin group gradually returned to the ratios in the control group with 60.55% ± 7.47% for acetic acid, 17.82% ± 3.34% for propionic acid, 20.13% ± 6.91% for butyric acid, 0.33% ± 0.06% for isobutyric acid, 0.78% ± 0.28% for isovaleric acid, 0.39% ± 0.07% for valeric acid at day 13, respectively.
Early studies suggested that SCFAs produced in the intestinal tract might inhibit the survival of harmful or conditional pathogenic bacteria , leading to protection of the intestinal tract against infection and inflammation . Loss of total bacteria, especially loss of beneficial bacteria in the gut may result in decreases in diversity and quantities of SCFAs in the feces of antibiotic-treated rats.
Conventional studies have shown that antibiotics can disrupt the equilibrium of gut microbial communities [22–25]. Traditionally, disruption of the richness and diversity indices of gut microbial communities are often used to interpret the side effects of oral antibiotics , such disruption is one of the probable mechanisms how antibiotics influence gut SCFAs.
Our new method has potential clinical applications if SCFAs can serve as a personalized drug safety and efficacy index. Antibiotic treatments should be guided according to the profile of SCFAs.
A rapid and quantitative GC method was developed for the analysis of fecal SCFAs of rats and applied to investigate the influences of cefdinir and azithromycin in a time–saving manner. Using this new method, we unveiled that a short course of antibiotic treatment resulted in a significant reduction in the sum, diversity and structure of the SCFAs in fecal samples. The fluctuation range and proportional change of SCFAs could provide more insights to evaluate the effects of new antibiotics in future clinical trials
This work was supported by grants from the National Science and Technology Major Projects for "Major New Drugs Innovation and Development" (No. 2015ZX09J15102-004-004 and 2013ZX09201020).
- Conceptualization: XG YW YZ.
- Formal analysis: XZ ZJ YW YZ.
- Funding acquisition: YW.
- Methodology: XZ ZJ YW.
- Project administration: YW.
- Software: XZ FY YW GP.
- Supervision: YW.
- Validation: XZ ZJ YW.
- Writing – original draft: XZ ZJ FY XC.
- Writing – review & editing: YW YZ.
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- 27. Bokulich NA, Chung J, Battaglia T, Henderson N, Jay M, Li H, et al. (2016) Antibiotics, birth mode, and diet shape microbiome maturation during early life. Sci Transl Med 8: 343ra382.
- 28. Yap IK, Li JV, Saric J, Martin FP, Davies H, Wang Y, et al. (2008) Metabonomic and microbiological analysis of the dynamic effect of vancomycin-induced gut microbiota modification in the mouse. J Proteome Res 7: 3718–3728. pmid:18698804
- 29. Zoetendal EG, Rajilic-Stojanovic M, de Vos WM (2008) High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota. Gut 57: 1605–1615. pmid:18941009
- 30. Romick-Rosendale LE, Goodpaster AM, Hanwright PJ, Patel NB, Wheeler ET, Chona DL, et al. (2009) NMR-based metabonomics analysis of mouse urine and fecal extracts following oral treatment with the broad-spectrum antibiotic enrofloxacin (Baytril). Magn Reson Chem 47 Suppl 1: S36–46.
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- 35. van Eijk HM, Bloemen JG, Dejong CH (2009) Application of liquid chromatography-mass spectrometry to measure short chain fatty acids in blood. J Chromatogr B Analyt Technol Biomed Life Sci 877: 719–724. pmid:19230798
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- 38. Gao X, Pujos-Guillot E, Martin JF, Galan P, Juste C, Jia W, et al. (2009) Metabolite analysis of human fecal water by gas chromatography/mass spectrometry with ethyl chloroformate derivatization. Anal Biochem 393: 163–175. pmid:19573517
- 39. Zambell KL, Fitch MD, Fleming SE (2003) Acetate and butyrate are the major substrates for de novo lipogenesis in rat colonic epithelial cells. J Nutr 133: 3509–3515. pmid:14608066
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- 41. Canani RB, Costanzo MD, Leone L, Pedata M, Meli R, Calignano A. (2011) Potential beneficial effects of butyrate in intestinal and extraintestinal diseases. World J Gastroenterol 17: 1519–1528. pmid:21472114
- 42. Scheppach W, Bartram HP, Richter F (1995) Role of short-chain fatty acids in the prevention of colorectal cancer. Eur J Cancer 31A: 1077–1080. pmid:7576995
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By Sophia Whitfield
Dame Stella Rimington joined the Security Service (MI5) in 1968. During her career she worked in all the main fields of the Service: counter-subversion, counter-espionage and counter-terrorism. She was appointed Director General in 1992, the first woman to hold the post. Rimington certainly has the experience to make her thrillers seem credible. Her first Liz Carlyle novel was published in 2004, Breaking Cover is her ninth novel featuring Liz Carlyle.
The astute Liz Carlyle is still recovering from the death of her partner. She is working in a more sedate environment, in a desk job for MI5ís counter-espionage desk. However, a new Cold War is heating up and as a result, Liz finds herself on the hunt for a Russian spy.
With MI5 and MI6 coming under public scrutiny, security is something that is beginning to feel increasingly remote. The public is concerned about new surveillance tactics.
The Secret Service hires young civil rights lawyer Jasminder Kapoor who had previously been outspoken about civil liberties to be their media advisor to stem he publicís discontent. Things donít quite go to plan with the newly appointed position causing even more problems.
By Sophia WhitfieldOn July 9, 2012
Jilly Cooper's scandalous bestselling Rutshire Chronicles novels are to be adapted for the small screen for ITV.On September 14, 2016
Nicole Trope is a former high school teacher with a Masters Degree in Children's Literature. In 2005 she was one of the winners of the Varuna Awards for Manuscript Development....On June 4, 2013
By Sophia WhitfieldOn July 23, 2012
Charlotte grew up with her nose in a book and her head in the clouds. At fourteen, her English teacher told her that the short story she'd submitted was wildly...On August 13, 2013
Jane Austenís Rogues and Romances has taken Facebook by storm. Jane Austen fans worldwide have been logging on to Facebook to rescue beloved heroines Elizabeth and Mr Darcy.On July 12, 2012

domain: literature


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Why is my french bulldog so tall: Most of the time, your long-legged friend will probably be a mixed breed, which should not be a surprise to you. Your Frenchie may be the product of a rare genetic mutation, but chances are they aren’t purebreds. If they were neutered before puberty, that could explain it. It also could be to do with growth plate injuries. Neutering dogs before they are fully grown interferes with the closure of their growth plates.
You may feel slightly confused if you notice that your French Bulldog’s legs are abnormally long. You probably expected your Frenchie to grow up with a small head, broad chest, and short legs, but instead, they ended up with a height more like their English cousins.
There is a direct relationship between height and genetics. In humans, for example, short parents sometimes give birth to tall children. In some cases, height skips a generation, which means that a grandparent (or even a great-grandparent) may have been taller and that gene may have been passed down to each parent. When those parents came together to produce puppies, they probably passed this gene on to your French Bulldog, making her taller.
French Bulldog Growth Plate Injuries?
As puppies grow, their long bones develop growth plates that ossify slowly. Once he is fully grown, the growth plate will have fully ossified, and your dog will have finished growing. There is a possibility that your dog may injure his growth plate acutely or chronically. Trauma such as being hit by a car or falling off a couch is considered an acute injury.
The growth plate may be permanently damaged by a growth plate fracture in some cases, resulting in an abnormal growth in the future. As a result of this abnormal growth, the bone on the injured side may become longer or grow differently, resulting in a deformed or crooked arm or leg.
As puppies grow, their long bones develop growth plates that ossify slowly. Once he is fully grown, the growth plate will have fully ossified, and your dog will have finished growing. There is a possibility that your dog may injure his growth plate acutely or chronically. Trauma such as being hit by a car or falling off a couch is considered an acute injury. Chronic injuries can be caused by repetitive microtraumas, such as jumping off a bed or running on hard surfaces. French Bulldogs are particularly prone to growth plate injuries because of their breed-specific conformations.
If your French Bulldog experiences a growth plate injury, he will likely show signs of lameness and decreased range of motion. You may also notice that his bone is not growing at the same rate as his peers. If your dog has an acute injury, you will need to take him to the veterinarian for x-rays to determine the extent of the damage. Treatment for an acute injury may include surgery, physical rehabilitation, and bracing/casting. You will need to work if your French Bulldog has a chronic injury.
When Do French Bulldogs Stop Growing and How Big Will My Puppy Get?
Dogs’ growth plates usually close between 9 and 11 months of age. Between six and eight months, smaller dogs reach their full growth. However, giant breeds continue to grow until they are about a year old. When your dog reaches that age, you should be able to gauge its ultimate size and height.
The size of your dog’s siblings is a good indicator of its eventual size. A large and medium-breed dog may exhibit a ‘juvenile’ appearance for the first one to two years of their lives, but they are no longer growing. You can get a better idea of your dog’s future size if you check out a previous litter of the sire and dam.
Little French Dog participates in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.
How tall should my French Bulldog be?
Generally speaking, a healthy adult male French Bulldog is 11 to 12 inches tall, and female French Bulldogs are also similar in height. When fully grown, a Frenchie will reach 28 to 30 centimeters when it is 9 to 12 months old.
According to the statistics, there is no hard and fast rule for how tall a French Bulldog should be over what’s considered typical based on averages. Although it is impossible to predict how tall your dog will grow, it is possible to make a prediction. These two metrics can be used to predict the height of a French Bulldog in a very simple way.
|Approximately two-thirds of the size of a Frenchie when he is an adult is what he was when he was 16 weeks old.
|French Bulldogs’ adult measurements are four times larger than they were at eight weeks of age.
You will be able to get an approximation of your Frenchie’s height with those two rules of thumb. The father’s height can also be used to predict how tall your male French Bulldog will grow. In the same way, you can measure the height of female puppies by comparing them to their mothers.
How Big Do French Bulldogs Get?
Across dog breeds, weight and size vary depending on the gender, but the average height of a French bulldog is the same as other bulldog breeds. Typically, male French bulldogs weigh between 20 and 28 pounds, while females weigh between 18 and 26 pounds. Due to their short stature, French dogs are distinctive from other breeds. Their unique walk makes them stand out from other breeds. Known for their low center of gravity and leggy gait, French bulldogs have a low center of gravity.
Are There Different Sizes Of French Bulldogs?
French Bulldogs also come in mini and teacup versions that are considerably smaller than traditional French Bulldogs. Runts from several different litters are bred to create these smaller versions. The American Kennel Club (AKC) does not recognize Micro (or Mini) or Teacup French Bulldogs because they do not meet traditional breed standards.
Breeders of these miniature dogs state that Mini and Teacup refer to the same dog, but the titles depend on its size.
In contrast to regular French Bulldogs, who can weigh up to 28 pounds and stand up to 12″ tall, these little ones are considerably smaller. Most Teacup French Bulldogs weigh less than 18 pounds, and Mini Frenchies weigh anywhere from 7 to 14 pounds, with some weighing as little as 5 pounds.

domain: pet


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- Hi-tech wall lighting with Videprojektoren – the installation of Struct
- Balcony and terrace patio umbrella – a maritime climax
- Parabolic innovative design armchair by Stefan Heiliger for Leolux
- Sofa Muuto
- Mirror cabinet in the bathroom – designs for minimalist interior
- Idea of creative decorating with hand mirrors
- Living room with fireplace
- Designer chandelier made of paper – Architecture meets art
- Brown Retro Wallpaper
- A room glass house design with perfect balance built on a slope
The Garden of Eden play in a modern bedroom design
This interesting project for a room design was inspired by the biblical Garden of Eden. Modern bedroom with en-suite bathroom in the middle was created by Spanish designer Egue there Seta workshop. The Eden Project was presented at Casa Decor Barcelona 2010 and is a modern interpretation of the Garden of Eden idea.
The design of the bedroom is a modern interpretation of the Garden of Eden
The design of the chamber there Egue Seta was inspired by the Garden of Eden and is a modern interpretation dar. Many items in the bedroom have biblical scenes from the Garden of Eden dar. Particularly striking is the neon sign "Eva", which, as a wall decoration used on the bed. The shelves on both sides of the bed with apples as a decoration are also a reference to the original Garden of Eden. The rich decoration plants bedroom can really look like a garden.
Rooms with "Eva" Neon for wall decoration
Original decoration with fresh apples
Room design with integrated bathroom in the middle
The project opens in the Garden of Eden is not only a bedroom but also a modern bathroom. The design of the room was still decorated in the center of a bathroom. The walls were made entirely of glass, and the floor is made of river stones. Again, the plants have been widely used as decoration. In this way, the bathroom looks like a garden of paradise.
Modern design bathroom with glass walls
Abundant plant used as decoration
Vertical garden wall as decoration
wall panels in a more natural look
Architectural plans of the garden of Eden Project

domain: design


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Lulu is a strange dog. She doesn't really like many other dogs, mainly just tolerates them. But she LOVES cats. They just don't love her back. I don't think she'd ever hurt them, just exuberently say hi to them. She doesn't understand that cats have been hard-wired to fear dogs. Case in point - the cat from downstairs, Kitty. (Yes, that's really her name. Those stoner college boys are really original.) Kitty freezes whenever Lulu comes near her. Like she blends into the grass or something. But lately Kitty has been coming onto our balcony to hang out. I have no idea how she gets up here. This morning (the first time I noticed this) I put Lulu in the bedroom, let Kitty in and took her downstairs. A few hours later she was here again! Lulu was beside herself! She was trying so hard to get to Kitty through the door. And barking like crazy! HI! NOTICE ME! PAY ATTENTION TO ME! I wasn't quick enough to get it on video, but I did get a picture.
I was seriously cracking up. I don't think Lulu appreciated my laughter. She stopped barking to stare at me like I hurt her feelers. Funny dog. :)
P.S. Kitty somehow got down on her own this time. Michelle thinks she's a special wall climbing cat.


While more and more people are calling for the electrification of transportation and an energy transition, the mining projects upon which this transition depends are suffering from political instability that keeps putting them in peril.
On average, health spending corresponds to 37% of provinces’ 2022 budgets, and is increasing rapidly according to a Deloitte report commissioned by the Canadian Medical Association and released today.
The City of Montreal has a problem. While it is experiencing an increase in violent events, it is struggling to retain staff.
It’s no great revelation that governments across the world are competing to attract investments, especially in sectors such as green technology.
Supply management is an artificial control of the dairy supply through quotas and controlling imports “as a means of setting stable prices for both farmers and consumers.”
Yesterday morning Ontario announced a plan to provide additional stability to their health care system which is under severe strain, a situation that has been dubbed a crisis.
With all major car makers now on board with electric vehicle production, demand for lithium has sent prices rocketing 750% since 2021.
If universal dental coverage for low-income children under 12 isn’t implemented by the end of the year, the NDP-Liberal Supply and Confidence Agreement is dead in the water according to the leader of the NDP.
This week has been a big one for new parents. There is a shortage of baby formula in the United States that has the potential to be devastating.
Two thirds of Canadians bought organic groceries on a weekly basis in 2020, but with the rising cost of food, families are having to make tough decisions at the cash register.


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|Year of Last Renovation:||2010|
|Corridors:||All Rooms Have Interior Hallways|
|Non Smoking Rooms:||0|
|Room Windows:||Windows Do Open|
|Dist. to Food/Bars:||0.3 Mi|
|Dist. to 24hr Restaurant:||1.0 Mi|
Groups must cancel 30 days prior to the arrival date. Individual must cancel before 6 pm on the expected day of arrival.
|Typical Group Deposit:||25%|
|Total Misc Fees:||$0.00 USD |
(mandatory resort fees/taxes)
The Fayetteville Regional Airport/Grannis Field is seven miles away. Visitors will enjoy shopping at the nearby Cross Creek Mall. Baywood Golf Club, Cypress Lakes Golf Course and King's Grant Golf Club are less than 10 miles from the hotel. Many major businesses and a civic center are also nearby.Airport Shuttle? Sorry, no airport shuttle available.
The Sleep Inns highlights above are subject to change without notice.

domain: travel


Flamenco facts for kids
Flamenco is a style of entertainment with song, music and dance. it is very popular in Spain and is known worldwide. Its foundation is in Andalusia in Spain. In its evolution Andalusian Gypsies played an important part. The term 'flamenco' was first recorded in the late 18th century but many believe the art form is much older.
Flamenco has acoustic guitar music, singing, hand claps, heel stamps, castanets. It is danced by a man and a woman in traditional Spanish costumes. The dance has no set tempo. It may have both fast and slow passages, rising to a climax near the end.
Flamenco performance has evolved during the history of this musical genre. In the beginning (the 18th century at the latest), songs were sung without any guitar accompaniment. During the 19th century, the guitar was used to accompany songs. Since the second half of the 19th century, the solo guitar is played in flamenco concerts.
On November 16, 2010, UNESCO declared Flamenco one of the Masterpieces of the Oral and Intangible Heritage of Humanity.
Images for kids
Flamenco Facts for Kids. Kiddle Encyclopedia.


Hawkwind Hall Of The Mountain Grill chords
Highlighted Show chord diagrams
Hall of the Mountain Grill ========================== Although this instrumental is played on the piano, here's a guitar version. One guitar should strum the chords and a second guitar should play the chords arpeggiated using the notes shown as 'Melody'. The general idea is to play the arpeggiated chords from the root note upwards until you hit the 2nd octave, then come back down again. The final couplet in each phrase should be played 2 octaves above the root note. If this makes no sense, just muck about with it till it sounds right, that's all I did anyway... Chord: Am Melody: A - E - A - D A - E - D B__A (x2) Chord: D#m Melody: D# - Bb - D# - F# - Bb - F# - D# F__D (x2) Chord: Cm Melody: C - G - C - D# - G - D# - C D__C (x2) Chord: F#m Melody: F# - C# - F# - B F# - C# - B Ab__F# (x2) This tab is not my own work and is made by the talented folks at starfarer.net and is from their collection of tabs! I am simply transferring it here for simplicities sake.


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Today we will discuss about Mennonite Recipes. All information about this topic come from the expert. So you can trust our content and keep update on this site to get more information about Mennonite Recipes. So please check the article below and please bookmark our site on your browser to get update.
coleen s recipes maple twists. peppernuts pfeffernusse cookies baker bettie. one days work iv barnraising arched barn summer amish. zwieback recipe food com. watermelon baby fruit basket. dumplings recipes dishmaps. lancaster farm bed and breakfast mennonite amp amish farm stay. amish facts wanda brunstetter. open for business.
Coleen's Recipes: MAPLE TWISTS. Peppernuts (Pfeffernusse Cookies) | Baker Bettie. One Days Work IV barnraising arched barn summer | Amish .... Zwieback Recipe - Food.com. WATERMELON BABY FRUIT BASKET. Dumplings Recipes — Dishmaps. Lancaster Farm Bed and Breakfast | Mennonite & Amish Farm Stay. Amish Facts - Wanda Brunstetter. Open for Business.
How to Make Strawberry Jam!! Homemade Small Batch Preserves Recipe. Million Dollar Deviled Eggs. Best Bean Soup Recipe ...easy and delicious. The best BREAD & BUTTER PICKLES!. POOR MAN'S STEAK AMISH RECIPE | HOW TO MAKE RECIPES | EASY WAY TO MAKE RECIPES. S1E11 Jeselle Hadley-Bacon: Converting into the Amish + A Father Who Groomed Her for Sexual Abuse. How to Make Brooke's Saltine Cracker Pudding. How to make Amish Style Fruit Slush | Refreshing Summer Frozen Dessert!. How Mennonite Girls Can Cook.. Firecracker Sundae Recipe.

domain: food


Saturday, 6 June 2015
Experimental Mechanised Force/Experimental Armoured Force
I have long been interested in British tank development and doctrine between the wars, in particular in the Experimental Mechanised Force/Experimental Armoured Force of the late 1920s, and have been assembling some vehicles to portray it in 1/76 and 1/72. While looking for information on the armoured cars with the force this morning I came across this - Pathe News Super Gazette newsreel of Winston Churchill visiting manoeuvers at Tidworth, listed on the IWM site as 1926 and on the Pathe site as 1927 (which makes more sense).
There are Medium Mk IIs, Carden Lloyd Mk IVs, Rolls Royce armoured cars (1920 pattern?), Birch guns on the move, tank signalling with flags, and Burford Kegresse infantry half tracks.
The clip may be well known, but I have never come across it before.
If anyone can suggest a reasonable proxy for the Burford Kegresse half track in 1/72 or 1/76 I'd be very grateful to hear from you. - the nearest I have come so far is the EWM Citroen P19 for Dragons Portees.
Posted by Vintage Wargaming at 10:17


BALTIMORE — When P.J. Higgins changed positions in 2016, pitch calling came later in the process.
His move to catcher in the Chicago Cubs organization created an opportunity and path to the big leagues that did not appear to exist as an infielder. Higgins’ conversion to the position featured a year-by-year build-up, ultimately earning a major-league call-up last year before a season-ending injury thwarted him.
Higgins has impressed since joining the team May 29 to replace the injured Yan Gomes. Namely, Higgins’ work with the pitching staff and pitch calling has stood out in his 10 starts at catcher.
Manager David Ross, a former catcher, knows what goes into good game calling and pitch sequencing. He considers it one of Higgins’ strength, noting how the 29-year-old has a good feel behind the plate and complementing him on how he retains information.
“That’s something that can be learned but also is a little bit of just innate ability to understand competition and being able to adjust in the moment from going from Plan A to Plan B to Plan C sometimes,” Ross said. “I think he does a really nice job with that.”
For Higgins, the pitch-calling side of the position was “down the road,” instead heavily focusing on the physical and defensive side as he worked up through the minors. Once he became comfortable with that aspect of catching, Higgins transitioned to focusing on reading hitters and their swings. Incorporating that into his game made catching more enjoyable and easier because he had better preparation and a game plan.
“Honestly, you’ve got to trust your gut sometimes,” Higgins told the Tribune. “You can have a scouting report to the T against guys, but you might have the opposing team be absolutely on everything some days. So being able to just watch the game and make sure you’re in tune to the game and be able to adjust on the fly and trust your gut because sometimes we overthink it.”
Higgins believes his time as an infielder has helped him read swings and subsequently adjust his pitch calls and sequencing. Experience and reps, however, can’t be replicated.
Higgins estimates it took until 2019, when he played at Double-A Tennessee for a second straight season, to feel more in control behind the plate. Making the jump to Triple-A Iowa for the first time that year meant more experienced hitters, including some who had been in the majors. Higgins delved deeper into his own scouting reports and equipped himself with as much information as possible.
“I’m not the biggest toolsy guy out there so I feel like I have to think the game through mentally a little more than physically,” Higgins said.
Pitching coach Tommy Hottovy appreciates Higgins’ calmness and ability to read the game. Although Higgins is good at breaking down hitters’ swings, Hottovy doesn’t see him overreact to that element when calling a game.
“He’s really in tune with what is going on,” Hottovy told the Tribune. “He does a great job of doing his own homework and really taking what the pitcher does really well and finding ways to explore that in-game. He’s humble. He really likes information. He loves talking with pitchers and you can’t ask for more from the guy that’s back there to do that at his age and and where he is in the game.”
Chicago Tribune Sports
When Higgins isn’t catching, he typically sits by the starting pitchers in the dugout to discuss the opposing lineup, just in case they play them again, especially division foes. After games he starts behind the plate, Higgins breaks down video of his receiving and sequencing.
Right-hander Kyle Hendricks said he never would have guessed Higgins is a converted infielder. Hendricks, who teamed with Higgins for one start this season, credited Higgins’ ability to simplify things and his understanding of the game.
“He’s so good back there,” Hendricks told the Tribune. “Just his feel for the game, his pitch calling just complements that. He really can feel what the hitters are doing. He’s locked in on the pitching staff and helping you get through the game and navigate the lineup, which there’s so much to be said for that. But how he goes about it, it’s a special kind of way. He’s a ballplayer.”
Left-hander Justin Steele played with Higgins in the minors and remembers how defensive prowess in the infield carried over to catcher, saying, “you want somebody like that touching the ball all the time.” Steele is looking forward to watching Higgins’ continued development.
“All the guys love throwing to him,” Steele said. “He is really good at paying attention and being aware of what’s going on and he calls pitches accordingly. He’s one of those guys that has very good instincts. He cares about the pitchers’ results, and you want a guy like that behind the plate for you.”
With Gomes nearing a return from his oblique injury, Higgins likely will soon be headed back to Triple A until another big-league spot opens, perhaps after the trade deadline in August or sooner if another catcher injury arises. He made the most of his chance, though, and left a strong impression on the pitching staff.
“He’s always locked in,” Hendricks said. “He’s always trying to find something to make him better. He just loves baseball, which translates and flows over to us.”


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Kate Moss beamed with pride as she watched her daughter Lila take to the runway during the Richard Quinn show and cocktail party in the midst of London Fashion Week.
The model, 46, looked effortlessly chic in a blue velvet blazer and flared jeans as she made her way into The Londoner Hotel to support her daughter, 18.
Kate, who over the weekend was reported to be returning to her partying ways, showcased her ageless complexion in the simple look as she took her place in the front row of the fashion show.
Happy: Kate Moss beamed with pride as she watched her daughter Lila take to the runway during the Richard Quinn show and cocktail party in the midst of London Fashion Week
Kate opted for a casual ensemble as she made her way into the fashion show, dressing in a crisp pale blue shirt and royal-shaded velvet blazer.
The fashion icon teamed the look with simple flared jeans and brown boots, with her essentials in a simple silver clutch bag.
Despite claims that she’s embraced her return to partying, Kate showcased her ageless visage as she posed with Edward, and her model protegé Jordan Barrett on the front row of the show.
Here she is! Lila was among a slew of models who took to the catwalk for the fashion show in a stunning red one-piece
Exciting: She was every inch the proud mother as she chatted to the models, including her own daughter, backstage during the fashion show
Stylish: Kate looked effortlessly chic in a blue velvet blazer and flared jeans as she made her way into the venue to watch the show alongside Vogue Editor Edward Enninful
Sophisticated: Kate opted for a casual ensemble as she made her way into the fashion show, dressing in a crisp pale blue shirt and royal-shaded velvet blazer.
Happy: Despite claims that she’s embraced her return to partying, Kate showcased her ageless visage as she posed with Edward, and her model protegé Jordan Barrett on the front row of the show
Say cheese! Kate was seen posing backstage with editor Edward, who looked dapper in a classic black suit
Kate was every inch the proud mother as she arrived to watch her daughter Lila storm the catwalk, with the brunette beauty dressed in a strapless red frilled jumpsuit.
Displaying a bold new look for the show, Lila sported bleached eyebrows as she made her way along the runway while Kate watched on.
Lila is the daughter of both Kate and Dazed founder Jefferson Hack, who dated from 2001 to 2004 and have maintained an amicable relationship and would ensure their daughter spent a regular amount of time with each of them in her childhood.
Back in 2016, Jefferson admitted that despite Lila having two famous parents, she found them both to be ‘deeply uncool’.
Star-studded: Seated on the front row, Kate was joined by (L-R) Edward, Jordan Barrett, Boy George and Bond Girl Olga Kurylenko as they prepared to watch the fashion show
Beautiful: Lila stunned in the red ensemble as she made her way along the catwalk while her mother watched on
Amazing! Her arrival on the catwalk earned a thunderous reception from the stars in attendance, including Kate herself
Legend: Kate made her runway debut at the same age as her daughter in 1992, after being scouted as a 14-year-old in 1988 at JFK Airport in New York
Plotting: It was reported by the Mail On Sunday that fashionistas are suggesting that Moss senior is masterminding her daughter’s style and her fledgling career
Speaking to the Sunday Times Magazine, he explained his daughter keeps himself and her model mother rooted in the ‘real’ world.
Despite her mother’s status as a bonafide British icon, thanks to her status in the fashion industry, and Jefferson’s own achingly cool CV, Lila – wouldn’t label her parents as cool.
‘She thinks me and her mum are deeply uncool,’ he explained. ‘I don’t think her music tastes are cool — Capital radio and Justin Bieber. She keeps me real.’
Flawless: Kate was among a star-studded front row for the fashion event
While Lila still has a good relationship with both parents, she is often seen out with mum Kate.
Kate made her runway debut at the same age as her daughter in 1992, after being scouted as a 14-year-old in 1988 at JFK Airport in New York.
She first walked for Dolce & Gabbana in their spring show, in a blue brocade suit teamed with a matching wide-brimmed hat.
Kate became a stalwart on the runway during the nineties and regularly appeared for big name designers including Prada, Tommy Hilfiger and Stella McCartney.
Ready to go! The eye-catching show saw models dressed in form-fitting jumpsuits with piped detailing in a range of primary colours
Like mother like daughter: Kate and Lila were seen chatting backstage during the fashion show
Friendly: The star couldn’t hide her pride at watching her daughter follow in her fashion footsteps
It was reported by the Mail On Sunday that fashionistas are suggesting that Moss senior is masterminding her daughter’s style and her fledgling career.
Industry sources reported that Kate’s priorities have shifted from her own career to launching her only daughter onto the fashion scene.
Lila is the principal client at the Kate Moss Agency, which also boasts singer Rita Ora and Game Of Thrones actress Gwendoline Christie.
Low-key: Model Jordan, who has been seen on many nights out with Kate, opted for an olive green mohair jumper and corduroy trousers, along with a plaid shirt tied around his waist
Off they go! Following the show, Kate was seen leaving the hotel with Jordan after watching Lila in the catwalk show
Simple: The pair both sported more laid-back looks than some of the other stars in attendance as they made their departure
Lila has ditched her publishing executive father Jefferson Hack’s surname and is now known simply as Lila Moss.
Kate’s work as a momager appears to be paying off, as last month Lila was unveiled as the face of the new Perfect perfume by Marc Jacobs.
In a campaign for the fragrance Lila paid tribute to her mother for introducing her to the world of fashion and beauty.
Ab-tastic: After bleaching her own eyebrowns, Amelia Gray Hamlin was also seen making her way into the fashion show in a black crop top and baggy trousers
Daring: Channelling her inner alien, Amelia still wowed with her bleached eyebrows as she arrived for the fashion show
She said: ‘I remember when I was two, my mum and I were staying in New York. She was getting ready to go out and I saw this super sparkly, deep black eye shadow on her dressing table.
‘I made one of her friends apply it on me and spent the rest of the evening tottering around the room in a pair of my mum’s high heels.’
The teen has been working with Marc Jacobs since 2018 when she became the face of the brand’s beauty range.
Sensational: The Don’t Call Me Up hitmaker was hard to miss in the plunging dress which accentuated her ample bust, which she teamed with matching pointed-toe heels
Say cheese! Inside the fashion show, Mabel was seen posing alongside fellow singer Mahalia, who sported an eye-catching floral bodysuit and matching bomber jacket
Also in attendance was hitmaker Mabel, who made a bold statement in an extravagant floral gown with a dramatic double train and a perilous thigh-high split.
The Don’t Call Me Up hitmaker was hard to miss in the plunging dress which accentuated her ample bust, which she teamed with matching pointed-toe heels.
Sweeping her hair into a high bun, Mabel posed up a storm in her jaw-dropping gown as she headed into the hotel for the fashion event.
Stunning: Bond girl Olga Kurylenko was a vision in an intricate pale pink dress with eye-catching embroidery and a beautifully sequinned trim
Posing up a storm: The actress took her seat on the front row in the beautiful pink dress alongside music legend Boy George
Elsewhere, Bond girl Olga Kurylenko, 41, was a vision in an intricate pale pink dress with eye-catching embroidery and a beautifully sequinned trim that appeared to be inspired by the 1920s.
The actress, pulled her brunette tresses into a sleek ponytail and accessorised her look with nude strapped heels.
Olympics star Dina Asher-Smith proudly showcased her athletic physique as she posed in a thigh-skimming white beaded blazer dress covered with pink roses and silver detailing.
Sensational: Olympics star Dina Asher-Smith proudly showcased her athletic physique as she posed in a thigh-skimming white beaded blazer dress covered with pink roses and silver detailing
Fashion favourite: Dina showed off her incredible pins in the revealing vintage-inspired mini dress with an incredibly detailed print, teamed with black pointed-toe heels
Gorgeous: She pulled her dark tresses into a sleek bun to allow the dress to take centre stagte
On Sunday sources told The Mail On Sunday that after a holiday in Ibiza last month, Kate ‘is back’ and has ‘a new lease of life,’ three years after halting her partying antics following the death of her friend Annabelle Neilson, from a heart attack at the age of just 49.
‘Mossy is back and everyone is talking about it,’ said a source. The pal said that with lockdown over, Kate has been out with make-up queen Charlotte Tilbury, designer Stella McCartney and model Jordan Barrett over the summer.
‘In Ibiza it was all anyone was talking about,’ said the source. ‘And a few famous people who know her mentioned it at a Chiltern Firehouse lunch last week.
‘She has been seen occasionally acting quite wild. She still knows how to gyrate on dance floors when she feels like it, just like the old days.’
Star-studded: Dina also posed with Edward’s assistant Deborah Ababio at the fashion event
Standing out! Erin O’Connor commanded attention in a 70s-inspired floral frilled dress, while Boy George opted for one of his signature bold jackets
Glammed up! Erin and Olga were seen posing together with Pom Klementieff in their colourful ensembles at the fashion show
In July, Kate attended the launch of the autobiography of her close friend DJ Fat Tony, who has been sober for 14 years. At the party he said: ‘Kate’s been clean for over two years. Me and my sober mates now have a better time than we ever did when we used to drink and take drugs.’
Kate has also given interviews saying she likes to ‘hit the sack at 11pm-ish and watch Netflix’, adding that she was trying to cut down on cigarettes and instead dealt with stress by doing yoga, which helped her ‘concentrate on the here and now’.
Yet around a month after she attended the launch of the so-called ‘quit-lit’ book championing sobriety, Kate attended Jordan’s wedding to fellow model Fernando Casablancas at a raucous venue in Ibiza as a ring-bearer… and apparently decided to let her hair down.
Bye! After the show George made a solo exit, documenting the outing on his phone as he was greeted by a throng of fans
Pictures from the island showed her dancing with a topless Jordan at a wild party, crashing out on the floor next to him, and semi-naked as she applied make-up in the bathroom, while he looked on.
Other photos from a reportedly rowdy lunch on the same holiday showed Kate looking tired and dishevelled as she dangled over a balcony to puff on a cigarette.
There were no signs of her boyfriend Nikolai von Bismarck during the trip, and one friend believes they hit a rough patch in their six-year relationship at around that time – although there is no suggestion they have split up.
Bouncng back: On Sunday sources told The Mail On Sunday that after a holiday in Ibiza last month, Kate ‘is back’ and has ‘a new lease of life,’ three years after halting her partying antics

domain: celebrity


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Package this colorful mix in a holiday tin for gift-giving.
* One-half cup semisweet chocolate chips can be substituted for the candy pieces.
Chex® Muddy Buddies®
Peanut Butter Cookie Granola
Original Chex® Party Mix
Kentucky Bourbon Bacon Chex Mix®


“Interview by Sebastiann Hofstede”
Sebastiaan: Chien Wei is an expert in the production of precision jig grinders and coordinate measuring machines. Let’s take a closer look inside to learn more about their products.
Chien Wei General Manager Justin Lee has agreed to answer some of our questions. Thank you. Could you tell us a little bit about your company and your product range?
Justin Lee: Chien Wei Precise Technology started by producing precision grinding tools, and then later after 30 years of evolution, developed precision measuring equipment as well as high precision machine tools. Chien Wei now has three main divisions, one specializes in precision grinding equipment, one in precision measuring equipment and one in the manufacturing of machine tools. Currently our business is focused on promoting each of these three business units.
Sebastiaan: I have seen you have a new precision jig grinder on display, could you tell us a bit about it?
Justin Lee: This machine was completed very recently and the JG-510DC is a jig grinder that can be used for grinding gears, cycloid gears and precision tools. It is also used for the making of gear molds, jigs and a diverse range of other products. This machine has a very high precision with a grinding accuracy of between 1-2mm, and can provide a range of industrial applications which is why we recently developed this new product.
Sebastiaan: Could you tell us a bit about your other products?
Justin Lee: Besides the latest JG-510DC jig grinder, we also offer high precision machine tools, such as vertical grinders and coordinate measuring machines that can be used in a range of industries. This includes the electronics industry and the precision grinding for either the plastic injection industry or the sheet metal working industry. We can provide a wide range of equipment for these industries, to help them with production, measurement and specific applications as we aim to offer a fully integrated service. We therefore hope our products can continue to be well-received and we are very grateful for the positive feedback we have received so far. Thank you.
Sebastiaan: If you would like to know more about Chien Wei and their latest products, click the link at the end of this video.


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hey, just bought your shirt! and i LOVE IT!!!! and i was at the dentist the other day and they were asking me about it and i was able to point them to God! :D
flag as inappropriate
It came in the mail today! I love it! I didn't even notice the doves until it came in today!
I just ordered this one from C28! I can't wait to get it!
this is really cute :)
yes, the cloud would look better if it was metallic silver or something and then a little silver on the hearts. But its still awesome i bought it!
Love the design, just ordered the shirt today can wait to get it!
cool design, & congratulations on it being selected for printing!
I have to say this is probably my favorite design
congrats on the win!
Soooo pretty! I love this!
Thanks everyone for your support
hearts as raindrops? such a cool picture!!!! This better be a girls shirt cuz I want it :)
i love this!!
cool! i love all the colors and the heart are like God's love all over us
this is an awesome shirt i love the colors and the graphics
this is awesome!!
This shirt rocks!!! No doubt in my mind i'd buy this shirt!!
I really like this, I would buy it.
i luv this! be sure to put this on a womens t-shirt! love the colors & hearts!! i like it the way it is, with black clouds... that must represent darkness, then into the LIGHT. i get it!
Ver cute!!! I agree about a different color for the clouds and I also think the hearts should be right side up only because upside down hearts make me think of love gone wrong.
I agree it is great, but would be better w/ different color clouds
Ya sorry it's hard to read in the pic...it's Romans 5:5
I just noticed there's a scripture reference, but I can't tell what it is...oh and the shirt is awesome!
Cool design but I would've liked it better if the clouds were a different color like gold or something, just because black clouds make me think of a bad storm not a good one


Phew, you ready? This is a good one…
Today we’re talking about one of those “secrets” that will help you transition from good to great songs.
This one is all about flow…
You already know what flow is of course: When there’s no distractions in the music and one line takes you smoothly to the next, when everything grooves, when there’s no clutter or lack of elements, when it feels “right” – that’s when you have flow.
Today we will take a look at a technique that can actually help you create this flow, and it is called…
This is one of those techniques every professional instinctively knows, but it’s a hard one to get your head around if you haven’t been writing songs for decades.
Trading Space is all about focussing your audience. Our brain can really only focus on one thing at a time, so whenever we hear a song that splits our attention, we get confused.
For example, a mistake often made is to have a singer sing over a intricate drum fill. This doesn’t work because it splits your audience’s attention.
“Should I listen to the drums or the vocals? I’m confused”
What you want to do instead is to trade space: Imagine your song like a stage in a nice old theater. Every sound, every instrument in your song is standing on that stage.
Now all of them are waiting for their turn to enter center stage. Here is where the magic happens.
Whatever is center stage you focus on. If your singer sings, he becomes the center of attention. All the other instruments accompany him in the background.
If the guitarist has a solo, the singer moves back and the guitarist enters center stage (yeah I know it’s not the 90s anymore, stop asking me that). Now he is the focus of the audience and the singer is not important.
This much shouldn’t come as a big surprise to you. It’s clear that you don’t let the piano and guitar solo at the same time.
What is not so obvious is that this rule applies to many other situations as well.
For example, if you’re mixing a call & response section in your song, don’t put too much delay on the “call”, because it will clash with the response.
As I stated earlier, if you want to use a drum fill while your singer is still singing, make it dead simple. Don’t let that drum fill steal the show of your singer. It should be felt, and nothing more.
Don’t write a drum fill and have the bass play a pick-up phrase at the same time unless they fit together perfectly (btw: If you want to learn how to write fills, here is the best way to learn).
And here’s a biggie I hear all the time: For god’s sake, if you’re using synth leads, don’t play them at the same time as your vocals. They are both competing for center stage. For some reason songwriters still put distracting synths in their verses and choruses
Why is this mental image of trading space so important? Why should you be seeing your song as a stage?
Because it prevents you from overwriting, from getting yet another idea for a part that could also fit this section. Being a great songwriter is NOT writing a lot of good things. It’s trying out a lot of things, finding one that’s great and then ONLY using that one.
It also helps you focus on what’s important. In your arrangement and your mix you will have less problems because you will know what to mix around.
And lastly, when you envision all the instruments playing your song, you will get new ideas for “trades”. In other words, you will notice if your vocalist has taken up all the space the entire time.
If this is the case, then maybe it’s time to let another instrument take over for a short while. Let the guitar play a short riff or interesting rhythm into the gaps of the vocals. Start your next song with just the bass instead of the piano.
Think “fair”. Let every instrument have its little moment. This becomes especially important over the course of an album or live gig.
Even if it’s just a second, make sure you let each instrument remind your listener that it’s there. This excludes soft pads and other very subtle instruments that are used to double or fill the mix, of course.
And one more thing: Trading space quickly can be used as an effect. Remember Sum41’s Fatlip? Listen to the first verse and see how it creates excitement and speed by letting different vocalists quickly trade center stage.