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| 2. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 3. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 5. |
- Liu, Bingbing, et al.
(författare)
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Effects of silver films with different nano-particle sizes on SERS of single-walled carbon nanotubes
- 2005
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Ingår i: Chemical Journal of the Chinese Universities. - 0251-0790. ; 26:10, s. 1930-1933
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Tidskriftsartikel (refereegranskat)abstract
- Surface-enhanced Raman scattering (SERS) spectra of single-walled carbon nanotubes (SWCNTs) on silver films with different nano-particle sizes from 20 to 100 nm deposited on quartz and glass substrates were studied systematically. The characteristic Raman spectral features of SWCNT G-band and D-band were analyzed. The two bands show a similar tendency with the change of nano-particle size of silver on quartz and glass substrates. The position and the intensity of the G-band are less sensitive to the films' silver particle sizes in the studied range, indicating that hexagonal carbon rings are stable and have a weak interaction with silver films. The shape of the D-band depends on the silver size. The smaller the silver particle size, the more the contribution of high frequency vibrations to the D-band, indicating that disorder carbon has a strong interaction with the active silver films.
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| 6. |
- Wei, Jiang, et al.
(författare)
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17β-estradiol regulates the expression of apolipoprotein M through estrogen receptor α-specific binding motif in its promoter
- 2017
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Ingår i: Lipids in Health and Disease. - : Springer Science and Business Media LLC. - 1476-511X. ; 16:1, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- Background: We have previously demonstrated that estrogen could significantly enhance expression of apolipoprotein M (apoM), whereas the molecular basis of its mechanism is not fully elucidated yet. To further investigate the mechanism behind the estrogen induced up-regulation of apoM expression. Results: Our results demonstrated either free 17β-estradiol (E2) or membrane-impermeable bovine serum albumin-conjugated E2 (E2-BSA) could modulate human apoM gene expression via the estrogen receptor alpha (ER-α) pathway in the HepG2 cells. Moreover, experiments with the luciferase activity analysis of truncated apoM promoters could demonstrate that a regulatory region (from-1580 to −1575 bp (−GGTCA-)) upstream of the transcriptional start site of apoM gene was essential for the basal transcriptional activity that regulated by the ER-α. With the applications of an electrophoresis mobility shift assay and a chromatin immunoprecipitation assay, we could successfully identify a specific ER-α binding element in the apoM promoter region. Conculsion: In summary, the present study indicates that 17β-estradiol induced up-regulation of apoM in HepG2 cells is through an ER-α-dependent pathway involving ER-α binding element in the promoter of the apoM gene.
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| 7. |
- Yu, Miao mei, et al.
(författare)
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Apolipoprotein M increases the expression of Vitamin D receptor mRNA in colorectal cancer cells detected with duplex fluorescence reverse transcription-quantitative polymerase chain reaction
- 2017
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Ingår i: Molecular Medicine Reports. - : Spandidos Publications. - 1791-2997 .- 1791-3004. ; 16:2, s. 1167-1172
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein M (ApoM) and the Vitamin D receptor (VDR) are apolipoproteins predominantly presenting in high-density lipoprotein (HDL) and a karyophilic protein belonging to the steroid-thyroid receptor superfamily, respectively. Previous studies have demonstrated that ApoM and VDR are associated with cholesterol metabolism, immune and colorectal cancer regulation. In order to investigate whether ApoM affected the expression of VDR in colorectal cancer cells, a single-tube duplex fluorescence reverse transcription-quantitative polymerase chain reaction (RT-qPCR) system was developed to simultaneously detect the mRNA levels of VDR and GAPDH in HT-29 cells overexpressing ApoM. The results demonstrated that the amplification products were confirmed as the specific fragment of VDR/GAPDH using the DNA sequencing instrument. The sensitivity, linear range, correlation coefficient, amplification efficiency, intra-assay and inter-assay coefficients of variation were 40 copies/μl, 4.00×101-4.00×105 copies/μl, 0.999, 92.42%, 0.09-0.34% and 0.32-0.65% for VDR, and 40 copies/μl, 400×101-4.00×105 copies/μl, 0.999, 98.07%, 0.19-0.43% and 0.40-0.75% for GAPDH, respectively. The results indicated that the expression of VDR mRNA was significantly higher in HT-29 cells overexpressing ApoM, compared with the negative control group (P<0.05). In conclusion, the current study successfully developed the single-tube duplex RT-qPCR to simultaneously detect VDR and GAPDH expression in colorectal cancer cells. The methodology results demonstrated that the duplex RT-qPCR system with high sensitivity and specificity could ensure the objectivity and credibility of the detection. The present study confirmed that ApoM significantly increased the expression of VDR in HT-29 cells. In addition, it was hypothesized that ApoM may be involved in antineoplastic activity via the upregulation of VDR expression, which may provide novel directions for the investigation of ApoM in cancer.
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| 8. |
- Feng, Yue Hua, et al.
(författare)
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Increased apolipoprotein M induced by lack of scavenger receptor BI is not activated via HDL-mediated cholesterol uptake in hepatocytes
- 2018
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Ingår i: Lipids in Health and Disease. - : Springer Science and Business Media LLC. - 1476-511X. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Scavenger receptor BI (SR-BI) is a classic high-density lipoprotein (HDL) receptor, which mediates selective lipid uptake from HDL cholesterol esters (HDL-C). Apolipoprotein M (ApoM), as a component of HDL particles, could influence preβ-HDL formation and cholesterol efflux. The aim of this study was to determine whether SR-BI deficiency influenced the expression of ApoM. Methods: Blood samples and liver tissues were collected from SR-BI gene knockout mice, and serum lipid parameters, including total cholesterol (TC), triglyceride (TG), high and low-density lipoprotein cholesterol (HDL-C and LDL-C) and ApoM were measured. Hepatic ApoM and ApoAI mRNA levels were also determined. In addition, BLT-1, an inhibitor of SR-BI, was added to HepG2 cells cultured with cholesterol and HDL, under serum or serum-free conditions. The mRNA and protein expression levels of ApoM were detected by RT-PCR and western blot. Results: We found that increased serum ApoM protein levels corresponded with high hepatic ApoM mRNA levels in both male and female SR-BI-/- mice. Besides, serum TC and HDL-C were also significantly increased. Treatment of HepG2 hepatoma cells with SR-BI specific inhibitor, BLT-1, could up-regulate ApoM expression in serum-containing medium but not in serum-free medium, even in the presence of HDL-C and cholesterol. Conclusions: Results suggested that SR-BI deficiency promoted ApoM expression, but the increased ApoM might be independent from HDL-mediated cholesterol uptake in hepatocytes.
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| 9. |
- Zhu, Bin, et al.
(författare)
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Apolipoprotein M Protects Against Lipopolysaccharide-Induced Acute Lung Injury via Sphingosine-1-Phosphate Signaling
- 2018
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Ingår i: Inflammation. - : Springer Science and Business Media LLC. - 0360-3997 .- 1573-2576. ; 41:2, s. 643-653
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: It had been demonstrated that apolipoprotein M (apoM) is an important carrier of sphingosine-1-phosphate (S1P) in blood, and the S1P has critical roles in the pathogenesis of sepsis-induced acute lung injury (ALI). In the present study, we investigated whether apoM has beneficial effects in a mouse model after lipopolysaccharide (LPS)-induced ALI. Forty-eight mice were divided into two groups: male C57BL/6 wild-type (apoM+/+) group (n = 24) and apoM gene-deficient (apoM−/−) group (n = 24) and then randomly subdivided into four subgroups (n = 6 each) according to different intraperitoneal (i.p.) injection: control group, W146 group, LPS group, and LPS + W146 group. Serum levels of interleukin-1 beta (IL-1β) and mRNA levels of IL-1β, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), lung histology, wet/dry weight ratio, and immunohistochemistry were measured at 3 h after the baseline and compared in each group. Our results clearly demonstrated that IL-1β mRNA levels and other inflammatory biomarkers were significantly increased in the lungs of LPS-induced ALI apoM−/− mice compared to those of the apoM+/+ mice. Moreover, when apoM+/+ mice were treated with W146, a S1P receptor (S1PR1) antagonist, these inflammatory biomarkers could be significantly upregulated by LPS-induced ALI. Therefore, it suggests that apoM-S1P-S1PR1 signaling might underlie the pathogenesis of ALI and apoM could have physiological benefits to alleviate LPS-induced ALI.
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