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- Beal, Jacob, et al.
(author)
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Robust estimation of bacterial cell count from optical density
- 2020
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Journal article (peer-reviewed)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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- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)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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- You, Xiaohu, et al.
(author)
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Towards 6G wireless communication networks: vision, enabling technologies, and new paradigm shifts
- 2021
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In: Science China Information Sciences. - : Science Press. - 1674-733X .- 1869-1919. ; 64:1
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Research review (peer-reviewed)abstract
- The fifth generation (5G) wireless communication networks are being deployed worldwide from 2020 and more capabilities are in the process of being standardized, such as mass connectivity, ultra-reliability, and guaranteed low latency. However, 5G will not meet all requirements of the future in 2030 and beyond, and sixth generation (6G) wireless communication networks are expected to provide global coverage, enhanced spectral/energy/cost efficiency, better intelligence level and security, etc. To meet these requirements, 6G networks will rely on new enabling technologies, i.e., air interface and transmission technologies and novel network architecture, such as waveform design, multiple access, channel coding schemes, multi-antenna technologies, network slicing, cell-free architecture, and cloud/fog/edge computing. Our vision on 6G is that it will have four new paradigm shifts. First, to satisfy the requirement of global coverage, 6G will not be limited to terrestrial communication networks, which will need to be complemented with non-terrestrial networks such as satellite and unmanned aerial vehicle (UAV) communication networks, thus achieving a space-air-ground-sea integrated communication network. Second, all spectra will be fully explored to further increase data rates and connection density, including the sub-6 GHz, millimeter wave (mmWave), terahertz (THz), and optical frequency bands. Third, facing the big datasets generated by the use of extremely heterogeneous networks, diverse communication scenarios, large numbers of antennas, wide bandwidths, and new service requirements, 6G networks will enable a new range of smart applications with the aid of artificial intelligence (AI) and big data technologies. Fourth, network security will have to be strengthened when developing 6G networks. This article provides a comprehensive survey of recent advances and future trends in these four aspects. Clearly, 6G with additional technical requirements beyond those of 5G will enable faster and further communications to the extent that the boundary between physical and cyber worlds disappears.
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| 7. |
- Bu, Junling, et al.
(author)
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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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In: Horticulture Research. - : Oxford University Press (OUP). - 2662-6810 .- 2052-7276. ; 9
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Journal article (peer-reviewed)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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- Chen, Shao-Chun, et al.
(author)
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First nationwide study regarding ceftriaxone resistance and molecular epidemiology of Neisseria gonorrhoeae in China
- 2016
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In: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 71:1, s. 92-99
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Journal article (peer-reviewed)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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| 9. |
- Chen, Shao-Chun, et al.
(author)
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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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In: Sexually Transmitted Diseases. - 0148-5717 .- 1537-4521. ; 40:11, s. 872-876
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Journal article (peer-reviewed)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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