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- 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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| 2. |
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| 3. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 4. |
- 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. |
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| 6. |
- Albert, Benjamin B., et al.
(författare)
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Supplementation with milk enriched with complex lipids during pregnancy : A double-blind randomized controlled trial
- 2021
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 16:2
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Tidskriftsartikel (refereegranskat)abstract
- Background Gangliosides are a class of sphingolipids that are present in the cell membranes of vertebrates. Gangliosides influence a broad range of cellular processes through effects on signal transduction, being found abundantly in the brain, and having a role in neurodevelopment.Objective We aimed to assess the effects of maternal daily consumption of ganglioside-enriched milk vs non-enriched milk and a non-supplemented group of pregnant women on maternal ganglioside levels and pregnancy outcomes.Design Double-blind parallel randomized controlled trial. Methods 1,500 women aged 20-40 years were recruited in Chongqing (China) between 11 and 14 weeks of a singleton pregnancy, and randomized into three groups: Control-received standard powdered milk formulation (>= 4 mg gangliosides/day); Complex milk lipid-enhanced (CML-E) group-same formulation enriched with complex milk lipids (>= 8 mg gangliosides/day) from milk fat globule membrane; Reference-received no milk. Serum ganglioside levels were measured in a randomly selected subsample of 250 women per group.Results CML-E milk was associated with marginally greater total gangliosides levels in maternal serum compared to Control (13.02 vs 12.69 mu g/ml; p = 0.034) but not to Reference group. CML-E milk did not affect cord blood ganglioside levels. Among the 1500 women, CML-E milk consumption was associated with a lower rate of gestational diabetes mellitus than control milk [relative risk 0.80 (95% CI 0.64, 0.99)], but which was not different to the Reference group. CML-E milk supplementation had no other effects on maternal or newborn health.Conclusions Maternal supplementation with milk fat globule membrane, as a source of gangliosides, was not associated with any adverse health outcomes, and did not increase serum gangliosides compared with the non-supplemented reference group.
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| 7. |
- Sun, Hui-Min, et al.
(författare)
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SERPINA4 is a novel independent prognostic indicator and a potential therapeutic target for colorectal cancer
- 2016
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Ingår i: American Journal of Cancer Research. - Madison : E-CENTURY PUBLISHING CORP. - 2156-6976. ; 6:8, s. 1636-1649
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Tidskriftsartikel (refereegranskat)abstract
- Serpina family A member 4 (SERPINA4), also known as kallistatin, exerts important effects in inhibiting tumor growth and angiogenesis in many malignancies. However, the precise role of SERPINA4 in CRC has not been fully elucidated. The present study aimed to investigate the expression of SERPINA4 and its clinical significance in CRC. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses showed that the mRNA and protein expression of SERPINA4 in colorectal cancer (CRC) specimens was significantly decreased than that in adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to characterize the expression pattern of SERPINA4 by using a tissue microarray (TMA) containing 327 archived paraffin-embedded CRC specimens. Statistical analyses revealed that decreased SERPINA4 expression was significantly associated with invasion depth, nodal involvement, distant metastasis, American Joint Committee on Cancer (AJCC) stage, and tumor differentiation. SERPINA4 was also an independent prognostic indicator of disease-free survival and overall survival in patients with CRC. Furthermore, the impact of altered SERPINA4 expression on CRC cells was analyzed with a series of in vitro and in vivo assays. The results demonstrated that SERPINA4 significantly inhibits malignant tumor progression and serves as a novel prognostic indicator and a potential therapeutic target for CRC.
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| 9. |
- Zhang, Xiao-Jie, et al.
(författare)
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Auto-suppression of Tet dioxygenases protects the mouse oocyte genome from oxidative demethylation
- 2024
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Ingår i: Nature Structural & Molecular Biology. - : NATURE PORTFOLIO. - 1545-9993 .- 1545-9985. ; 31:1
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Tidskriftsartikel (refereegranskat)abstract
- DNA cytosine methylation plays a vital role in repressing retrotransposons, and such derepression is linked with developmental failure, tumorigenesis and aging. DNA methylation patterns are formed by precisely regulated actions of DNA methylation writers (DNA methyltransferases) and erasers (TET, ten-eleven translocation dioxygenases). However, the mechanisms underlying target-specific oxidation of 5mC by TET dioxygenases remain largely unexplored. Here we show that a large low-complexity domain (LCD), located in the catalytic part of Tet enzymes, negatively regulates the dioxygenase activity. Recombinant Tet3 lacking LCD is shown to be hyperactive in converting 5mC into oxidized species in vitro. Endogenous expression of the hyperactive Tet3 mutant in mouse oocytes results in genome-wide 5mC oxidation. Notably, the occurrence of aberrant 5mC oxidation correlates with a consequent loss of the repressive histone mark H3K9me3 at ERVK retrotransposons. The erosion of both 5mC and H3K9me3 causes ERVK derepression along with upregulation of their neighboring genes, potentially leading to the impairment of oocyte development. These findings suggest that Tet dioxygenases use an intrinsic auto-regulatory mechanism to tightly regulate their enzymatic activity, thus achieving spatiotemporal specificity of methylome reprogramming, and highlight the importance of methylome integrity for development. Here the authors show that TET dioxygenases, the erasers of DNA methylation, use a self-limiting mechanism via their LCD domain to ensure adaptable methylome status and protect the genome from excessive oxidative methylation.
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| 10. |
- Zhao, Ying, et al.
(författare)
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Intramyocardial Dissecting Hematoma after Acute Myocardial Infarction : Echocardiographic Features and Clinical Outcome
- 2016
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Ingår i: Echocardiography. - : Wiley-Blackwell. - 0742-2822 .- 1540-8175. ; 33:7, s. 962-969
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Intramyocardial dissecting hematoma (IDH) after acute myocardial infarction (MI) is a rare form of subacute cardiac rupture and hence management uncertainties. The objective of this study was to describe the clinical course of a small series of IDH patients and to review the available evidence for managing similar cases. Methods: Eight IDH patients from our center had echocardiographic, coronary angiographic and clinical outcome data reviewed. PubMed was also searched for IDH following MI. Cases were divided into three groups and compared according to the dissection location. Results: In our 8 patients, 3 had septal, 1 right ventricular (RV), and 4 left ventricular (LV) dissection. Five were medically treated and 3 surgically repaired. Reviewing the literature revealed 68 IDH patients, of mean age 66 +/- 10 years, 43 males. The percentage of IDH involving the LV free wall, septal, and RV free wall were 47%, 26.5%, and 26.5%, respectively. In the cohort as a whole, mortality was not different between surgically and medically treated patients (33.3% vs. 54.3%, P = 0.08), neither based on the IDH location (P = 0.49). While surgical and medical treatment of the LV free wall (20.0% vs. 40.9%, P = 0.25) and septal (46.2% vs. 60.0%, P = 0.60) were not different, surgical repair of RV free wall had significantly better survival (30.0% vs. 87.5%, P = 0.015). The LVEF (P = 0.82), mitral regurgitation (P = 0.49) failed to predict mortality. Conclusion: While survival following medical and surgical treatment of LV IDH is not different, patients with RV free wall dissection benefit significantly from surgical repair.
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