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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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- 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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- Sheng, Xiang, et al.
(författare)
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A Combined Experimental-Theoretical Study of the LigW-Catalyzed Decarboxylation of 5-Carboxyvanillate in the Metabolic Pathway for Lignin Degradation
- 2017
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 7:8, s. 4968-4974
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Tidskriftsartikel (refereegranskat)abstract
- Although it is a member of the amidohydrolase superfamily, LigW catalyzes the nonoxidative decarboxylation of 5-carboxyvanillate to form vanillate in the metabolic pathway for bacterial lignin degradation. We now show that membrane inlet mass spectrometry (MIMS) can be used to measure transient CO2 concentrations in real time, thereby permitting us to establish that C-C bond cleavage proceeds to give CO2 rather than HCO3- as the initial product in the LigW-catalyzed reaction. Thus, incubation of LigW at pH 7.0 with the substrate 5-carboxyvanillate results in an initial burst of CO2 formation that gradually decreases to an equilibrium value as CO2 is nonenzymatically hydrated to HCO3-. The burst of CO2 is completely eliminated with the simultaneous addition of substrate and excess carbonic anhydrase to the enzyme, demonstrating that CO2 is the initial reaction product. This finding is fully consistent with the results of density functional theory calculations, which also provide support for a mechanism in which protonation of the C5 carbon takes place prior to C-C bond cleavage. The calculated barrier of 16.8 kcal/mol for the rate-limiting step, the formation of the C5-protonated intermediate, compares well with the observed kcat value of 27 for Sphingomonas paucimobilis LigW, which corresponds to an energy barrier of 16 kcal/mol. The MIMS-based strategy is superior to alternate methods of establishing whether CO2 or HCO3- is the initial reaction product, such as the use of pH-dependent dyes to monitor very small changes in solution pH. Moreover, the MIMS-based assay is generally applicable to studies of all enzymes that produce and/or consume small-molecule, neutral gases.
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- Yu, Sheng-Xiang, et al.
(författare)
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Phylogeny of Impatiens (Balsaminaceae) : integrating molecular and morphological evidence into a new classification
- 2016
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Ingår i: Cladistics. - : Wiley-Blackwell. - 0748-3007 .- 1096-0031. ; 32:2, s. 179-197
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Tidskriftsartikel (refereegranskat)abstract
- Impatiens L. is one of the largest angiosperm genera, containing over 1000 species, and is notorious for its taxonomic difficulty. Here, we present, to our knowledge, the most comprehensive phylogenetic analysis of the genus to date based on a total evidence approach. Forty-six morphological characters, mainly obtained from our own investigations, are combined with sequence data from three genetic regions, including nuclear ribosomal ITS and plastid atpB-rbcL and trnL-F. We include 150 Impatiens species representing all clades recovered by previous phylogenetic analyses as well as three outgroups. Maximum-parsimony and Bayesian inference methods were used to infer phylogenetic relationships. Our analyses concur with previous studies, but in most cases provide stronger support. Impatiens splits into two major clades. For the first time, we report that species with three-colpate pollen and four carpels form a monophyletic group (clade I). Within clade II, seven well-supported subclades are recognized. Within this phylogenetic framework, character evolution is reconstructed, and diagnostic morphological characters for different clades and subclades are identified and discussed. Based on both morphological and molecular evidence, a new classification outline is presented, in which Impatiens is divided into two subgenera, subgen. Clavicarpa and subgen. Impatiens; the latter is further subdivided into seven sections.
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| 8. |
- Bu, Junling, et al.
(författare)
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Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids
- 2022
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Ingår i: Horticulture Research. - : Oxford University Press (OUP). - 2662-6810 .- 2052-7276. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- O-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquinoline alkaloids (BIAs) in plants. In this study, Corydalis yanhusuo, a plant used in traditional Chinese medicine due to the analgesic effects of its BIA-active compounds, was employed to analyze the catalytic characteristics of O-methyltransferases in the formation of BIA diversity. Seven genes encoding O-methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an O-methyltransferase (CyOMT2) with highly efficient catalytic activity of both 4′- and 6-O-methylations of 1-BIAs was found. CyOMT6 was found to perform two sequential methylations at both 9- and 2-positions of the essential intermediate of tetrahydroprotoberberines, (S)-scoulerine. Two O-methyltransferases (CyOMT5 and CyOMT7) with wide substrate promiscuity were found, with the 2-position of tetrahydroprotoberberines as the preferential catalytic site for CyOMT5 (named scoulerine 2-O-methyltransferase) and the 6-position of 1-BIAs as the preferential site for CyOMT7. In addition, results of integrated phylogenetic molecular docking analysis and site-directed mutation suggested that residues at sites 172, 306, 313, and 314 in CyOMT5 are important for enzyme promiscuity related to O-methylations at the 6- and 7-positions of isoquinoline. Cys at site 253 in CyOMT2 was proved to promote the methylation activity of the 6-position and to expand substrate scopes. This work provides insight into O-methyltransferases in producing BIA diversity in C. yanhusuo and genetic elements for producing BIAs by metabolic engineering and synthetic biology.
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| 9. |
- Chen, Shao-Chun, et al.
(författare)
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First nationwide study regarding ceftriaxone resistance and molecular epidemiology of Neisseria gonorrhoeae in China
- 2016
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 71:1, s. 92-99
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major public health concern worldwide. This is the first nationwide study, performed within the China Gonococcal Antimicrobial Susceptibility Programme (China-GASP), regarding AMR, including ceftriaxone genetic resistance determinants, and molecular epidemiology of gonococci in China.Methods: Gonococcal isolates (naEuroS=aEuroS1257) from consecutive patients were collected at 11 sentinel sites distributed across China during 2012-13. Susceptibility to ceftriaxone, spectinomycin, ciprofloxacin and tetracycline was determined using the agar dilution method. Ceftriaxone resistance determinants penA and penB were examined using sequencing. N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed for molecular epidemiology.Results: Among isolates, 0.2% were resistant to spectinomycin, 4.4% to ceftriaxone, 42.9% to tetracyclines (high-level resistance) and 99.8% to ciprofloxacin. Among 890 sequenced isolates, 16 (1.8%) possessed a penA mosaic allele; 4 of these isolates belonged to the MDR internationally spread NG-MAST genogroup G1407 (first description in China). Non-mosaic penA alleles with an A501T mutation and an A102D alteration in porB1b were statistically associated with decreased susceptibility/resistance to ceftriaxone. NG-MAST G10339, G1424 and G1053 were associated with decreased susceptibility/resistance to ceftriaxone.Conclusions: In China, ceftriaxone and spectinomycin can continue to be recommended for gonorrhoea treatment, with the possible exception of Hainan and Sichuan provinces where ceftriaxone resistance exceeded 5% and AMR surveillance needs to be strengthened. Molecular approaches including genotyping and AMR determinant analysis can be valuable to supplement and enhance conventional surveillance of gonococcal AMR in China.
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| 10. |
- Chen, Shao-Chun, et al.
(författare)
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Prevalence and Molecular Epidemiological Typing of Penicillinase-Producing Neisseria gonorrhoeae and Their bla(TEM-135) Gene Variants in Nanjing, China
- 2013
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Ingår i: Sexually Transmitted Diseases. - 0148-5717 .- 1537-4521. ; 40:11, s. 872-876
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Tidskriftsartikel (refereegranskat)abstract
- Background: This study aimed to investigate the prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG) and their bla(TEM-135) gene variant in 2007 and 2012 in Nanjing, China. In addition, molecular epidemiological typing of all isolates was performed to elucidate the genetic relationships of the PPNG strains. Methods: A total of 199 and 77 N. gonorrhoeae isolates were collected at the National Center for STD Control in 2007 and 2012, respectively. Nitrocefin tests were performed to identify PPNG. Mismatch amplification mutation assay was used to identify bla(TEM-135). All isolates were genotyped using N. gonorrhoeae multiantigen sequence typing (NG-MAST), and additionally, porB-based phylogenetic analysis was performed for the PPNG isolates. Results: The total prevalence of PPNG isolates was 41% (114/276) and 58% (66/114) of these PPNG isolates possessed bla(TEM-135). In 2007, 45% (90/199) produced beta-lactamase, and of those PPNG, 58% (52/90) possessed bla(TEM-135). In 2012, 31% (24/77) were PPNG, and 58% (14/24) of those isolates contained bla(TEM-135). There were 162 NG-MAST STs among the 276 isolates, and 89 of those were novel STs. A strong association between specific NG-MAST STs and bla(TEM-135) was found, and the porB-based phylogenetic analysis showed a distant evolutionary relationship between isolates in 2007 and isolates in 2012. Conclusions: A high prevalence of PPNG and bla(TEM-135) was found in Nanjing, China. bla(TEM-135) might be a precursor in the evolution into an extended-spectrum beta-lactamase that can degrade ceftriaxone, which stresses the need to continuously monitor PPNG, bla(TEM-135), and additional evolving bla(TEM) gene variants.
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