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Article ; Online: Quantitative Analysis of Intracellular Fluorescent Foci in Live Bacteria.

Moolman, M Charl / Kerssemakers, Jacob W J / Dekker, Nynke H

2015 Volume 109, Issue 5, Page(s) 883–891

Abstract: Fluorescence microscopy has revolutionized in vivo cellular biology. Through the specific labeling of a protein of interest with a fluorescent protein, one is able to study movement and colocalization, and even count individual proteins in a live cell. ... ...

Abstract	Fluorescence microscopy has revolutionized in vivo cellular biology. Through the specific labeling of a protein of interest with a fluorescent protein, one is able to study movement and colocalization, and even count individual proteins in a live cell. Different algorithms exist to quantify the total intensity and position of a fluorescent focus. Although these algorithms have been rigorously studied for in vitro conditions, which are greatly different than the in-homogenous and variable cellular environments, their exact limits and applicability in the context of a live cell have not been thoroughly and systematically evaluated. In this study, we quantitatively characterize the influence of different background subtraction algorithms on several focus analysis algorithms. We use, to our knowledge, a novel approach to assess the sensitivity of the focus analysis algorithms to background removal, in which simulated and experimental data are combined to maintain full control over the sensitivity of a focus within a realistic background of cellular fluorescence. We demonstrate that the choice of algorithm and the corresponding error are dependent on both the brightness of the focus, and the cellular context. Expectedly, focus intensity estimation and localization accuracy suffer in all algorithms at low focus to background ratios, with the bacteroidal background subtraction in combination with the median excess algorithm, and the region of interest background subtraction in combination with a two-dimensional Gaussian fit algorithm, performing the best. We furthermore show that the choice of background subtraction algorithm is dependent on the expression level of the protein under investigation, and that the localization error is dependent on the distance of a focus from the bacterial edge and pole. Our results establish a set of guidelines for what signals can be analyzed to give a targeted spatial and intensity accuracy within a bacterial cell.
MeSH term(s)	Escherichia coli K12/cytology ; Intracellular Space/metabolism ; Microscopy, Fluorescence
Language	English
Publishing date	2015-09-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2015.07.013
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Article ; Online: Measuring In Vivo Protein Dynamics Throughout the Cell Cycle Using Microfluidics.

de Leeuw, Roy / Brazda, Peter / Charl Moolman, M / Kerssemakers, J W J / Solano, Belen / Dekker, Nynke H

Methods in molecular biology (Clifton, N.J.)

2017 Volume 1624, Page(s) 237–252

Abstract: Studying the dynamics of intracellular processes and investigating the interaction of individual macromolecules in live cells is one of the main objectives of cell biology. These macromolecules move, assemble, disassemble, and reorganize themselves in ... ...

Abstract	Studying the dynamics of intracellular processes and investigating the interaction of individual macromolecules in live cells is one of the main objectives of cell biology. These macromolecules move, assemble, disassemble, and reorganize themselves in distinct manners under specific physiological conditions throughout the cell cycle. Therefore, in vivo experimental methods that enable the study of individual molecules inside cells at controlled culturing conditions have proved to be powerful tools to obtain insights into the molecular roles of these macromolecules and how their individual behavior influence cell physiology. The importance of controlled experimental conditions is enhanced when the investigated phenomenon covers long time periods, or perhaps multiple cell cycles. An example is the detection and quantification of proteins during bacterial DNA replication. Wide-field microscopy combined with microfluidics is a suitable technique for this. During fluorescence experiments, microfluidics offer well-defined cellular orientation and immobilization, flow and medium interchangeability, and high-throughput long-term experimentation of cells. Here we present a protocol for the combined use of wide-field microscopy and microfluidics for the study of proteins of the Escherichia coli DNA replication process. We discuss the preparation and application of a microfluidic device, data acquisition steps, and image analysis procedures to determine the stoichiometry and dynamics of a replisome component throughout the cell cycle of live bacterial cells.
MeSH term(s)	Cell Cycle ; DNA Replication ; DNA, Bacterial/genetics ; Escherichia coli/genetics ; Escherichia coli/growth & development ; Escherichia coli/metabolism ; Escherichia coli Proteins/metabolism ; Microfluidics/methods ; Optical Imaging ; Single Molecule Imaging
Chemical Substances	DNA, Bacterial ; Escherichia coli Proteins
Language	English
Publishing date	2017
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-7098-8_18
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Article ; Online: Stochasticity and homeostasis in the E. coli replication and division cycle.

Adiciptaningrum, Aileen / Osella, Matteo / Moolman, M Charl / Cosentino Lagomarsino, Marco / Tans, Sander J

Scientific reports

2015 Volume 5, Page(s) 18261

Abstract: How cells correct for stochasticity to coordinate the chromosome replication and cellular division cycle is poorly understood. We used time-lapse microscopy and fluorescently labelled SeqA to determine the timing of birth, initiation, termination, and ... ...

Abstract	How cells correct for stochasticity to coordinate the chromosome replication and cellular division cycle is poorly understood. We used time-lapse microscopy and fluorescently labelled SeqA to determine the timing of birth, initiation, termination, and division, as well as cell size throughout the cell cycle. We found that the time between birth and initiation (B-period) compensates for stochastic variability in birth size and growth rate. The time between termination and division (D-period) also compensates for size and growth variability, invalidating the notion that replication initiation is the principal trigger for cell division. In contrast, the time between initiation and termination (C-period) did not display such compensations. Interestingly, the C-period did show small but systematic decreases for cells that spontaneously grew faster, which suggests a coupling between metabolic fluctuations and replication. An auto-regressive theoretical framework was employed to compare different possible models of sub-period control.
MeSH term(s)	Cell Cycle ; Cell Division ; DNA Replication ; Escherichia coli/physiology ; Homeostasis ; Microbial Viability ; Models, Biological
Language	English
Publishing date	2015-12-16
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/srep18261
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Article ; Online: Studying genomic processes at the single-molecule level: introducing the tools and applications.

Dulin, David / Lipfert, Jan / Moolman, M Charl / Dekker, Nynke H

Nature reviews. Genetics

2012 Volume 14, Issue 1, Page(s) 9–22

Abstract: To understand genomic processes such as transcription, translation or splicing, we need to be able to study their spatial and temporal organization at the molecular level. Single-molecule approaches provide this opportunity, allowing researchers to ... ...

Abstract	To understand genomic processes such as transcription, translation or splicing, we need to be able to study their spatial and temporal organization at the molecular level. Single-molecule approaches provide this opportunity, allowing researchers to monitor molecular conformations, interactions or diffusion quantitatively and in real time in purified systems and in the context of the living cell. This Review introduces the types of application of single-molecule approaches that can enhance our understanding of genome function.
MeSH term(s)	DNA Replication ; Genetic Phenomena ; Genomics/methods ; Genomics/trends ; Protein Biosynthesis ; Transcription, Genetic
Language	English
Publishing date	2012-10-30
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2035157-4
ISSN	1471-0064 ; 1471-0056
ISSN (online)	1471-0064
ISSN	1471-0056
DOI	10.1038/nrg3316
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Article: Essential validation methods for E. coli strains created by chromosome engineering.

Tiruvadi Krishnan, Sriram / Moolman, M Charl / van Laar, Theo / Meyer, Anne S / Dekker, Nynke H

Journal of biological engineering

2015 Volume 9, Page(s) 11

Abstract: Background: Chromosome engineering encompasses a collection of homologous recombination-based techniques that are employed to modify the genome of a model organism in a controlled fashion. Such techniques are widely used in both fundamental and ... ...

Abstract	Background: Chromosome engineering encompasses a collection of homologous recombination-based techniques that are employed to modify the genome of a model organism in a controlled fashion. Such techniques are widely used in both fundamental and industrial research to introduce multiple insertions in the same Escherichia coli strain. To date, λ-Red recombination (also known as recombineering) and P1 phage transduction are the most successfully implemented chromosome engineering techniques in E. coli. However, due to errors that can occur during the strain creation process, reliable validation methods are essential upon alteration of a strain's chromosome. Results and discussion: Polymerase chain reaction (PCR)-based methods and DNA sequence analysis are rapid and powerful methods to verify successful integration of DNA sequences into a chromosome. Even though these verification methods are necessary, they may not be sufficient in detecting all errors, imposing the requirement of additional validation methods. For example, as extraneous insertions may occur during recombineering, we highlight the use of Southern blotting to detect their presence. These unwanted mutations can be removed via transducing the region of interest into the wild type chromosome using P1 phages. However, in doing so one must verify that both the P1 lysate and the strains utilized are free from contamination with temperate phages, as these can lysogenize inside a cell as a large plasmid. Thus, we illustrate various methods to probe for temperate phage contamination, including cross-streak agar and Evans Blue-Uranine (EBU) plate assays, whereby the latter is a newly reported technique for this purpose in E. coli. Lastly, we discuss methodologies for detecting defects in cell growth and shape characteristics, which should be employed as an additional check. Conclusion: The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non-specific insertions in the chromosome, temperate phage contamination, and defects in growth and cell shape. While techniques such as PCR and DNA sequence verification should standardly be performed, we illustrate the necessity of performing these additional assays. The discussed techniques are highly generic and can be easily applied to any type of chromosome engineering.
Language	English
Publishing date	2015-07-01
Publishing country	England
Document type	Journal Article
ZDB-ID	2391582-1
ISSN	1754-1611
ISSN	1754-1611
DOI	10.1186/s13036-015-0008-x
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Article ; Online: Sustainability elements of companies that are affected by pandemics

Anneke M. Moolman / Jaco P. Fouché / Verona Leendertz

Journal of Economic and Financial Sciences, Vol 16, Iss 1, Pp e1-e

2023 Volume 12

Abstract: Orientation: The havoc created by COVID-19 reaffirmed the pervasive effects of pandemics on companies’ sustainability, which has become an increasingly important consideration for stakeholders. Research purpose: This study determined the sustainability ... ...

Abstract	Orientation: The havoc created by COVID-19 reaffirmed the pervasive effects of pandemics on companies’ sustainability, which has become an increasingly important consideration for stakeholders. Research purpose: This study determined the sustainability elements of companies that are affected by pandemics. Motivation for the study: Pandemics’ recurring nature is evidenced by history. Knowledge of pandemics’ effects on sustainability may assist companies in preparing for and reporting on pandemics, while such information to stakeholders may be important when considering a company’s sustainability. Research design and method: The study followed a systematic review. The final sample constituted 30 records, which were thematically analysed. Main findings: A list of sustainability elements of companies that are affected by pandemics is provided. Government-imposed restrictions led to supply and demand shocks, severely threatening companies’ financial performance and socio-economic targets. Pandemics also present opportunities to improve business models by increasing focus on relationships, nature and digitalisation. Practical implications: This study may assist companies to minimise the effects of future pandemics on sustainability by urging them to recognise the interplay between sustainability’s components. Companies should have some financial leeway and consider the composition of its product/service range (essential versus non-essential) and the delivery thereof (traditional vs. e-commerce), consider and reduce its impact on nature, become more human-centric and finally, revisit their strategy through strong governance. Contribution: Current literature describes some effects of a single pandemic on companies within a specific industry, whereas this study’s scope is broadened to consider all pandemics and industries to derive an extensive list of affected sustainability elements. Current sustainability frameworks do not specify pandemic-related disclosure requirements, making the list useful as a reporting ...
Keywords	covid-19 ; pandemic ; systematic review ; sustainability ; companies ; Economics as a science ; HB71-74
Subject code	650 ; 360
Language	English
Publishing date	2023-02-01T00:00:00Z
Publisher	AOSIS
Document type	Article ; Online
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Article ; Online: The progression of replication forks at natural replication barriers in live bacteria.

Moolman, M Charl / Tiruvadi Krishnan, Sriram / Kerssemakers, Jacob W J / de Leeuw, Roy / Lorent, Vincent / Sherratt, David J / Dekker, Nynke H

Nucleic acids research

2016 Volume 44, Issue 13, Page(s) 6262–6273

Abstract: Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of ... ...

Abstract	Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these Tus-ter barriers in the cell are poorly understood. By performing quantitative fluorescence microscopy with microfuidics, we investigate the effect on the replisome when encountering these barriers in live Escherichia coli cells. We make use of an E. coli variant that includes only an ectopic origin of replication that is positioned such that one of the two replisomes encounters a Tus-ter barrier before the other replisome. This enables us to single out the effect of encountering a Tus-ter roadblock on an individual replisome. We demonstrate that the replisome remains stably bound after encountering a Tus-ter complex from the non-permissive direction. Furthermore, the replisome is only transiently blocked, and continues replication beyond the barrier. Additionally, we demonstrate that these barriers affect sister chromosome segregation by visualizing specific chromosomal loci in the presence and absence of the Tus protein. These observations demonstrate the resilience of the replication fork to natural barriers and the sensitivity of chromosome alignment to fork progression.
MeSH term(s)	Chromosome Segregation/genetics ; Chromosomes, Bacterial/genetics ; DNA Helicases/genetics ; DNA Replication/genetics ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Escherichia coli/genetics ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Macromolecular Substances/metabolism
Chemical Substances	DNA replication terminus site-binding protein, E coli ; DNA-Binding Proteins ; Escherichia coli Proteins ; Macromolecular Substances ; tus protein, E coli ; DNA Helicases (EC 3.6.4.-)
Language	English
Publishing date	2016-05-10
Publishing country	England
Document type	Journal Article
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkw397
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Article ; Online: Electron beam fabrication of a microfluidic device for studying submicron-scale bacteria.

Moolman, M Charl / Huang, Zhuangxiong / Krishnan, Sriram Tiruvadi / Kerssemakers, Jacob W J / Dekker, Nynke H

Journal of nanobiotechnology

2013 Volume 11, Page(s) 12

Abstract: Background: Controlled restriction of cellular movement using microfluidics allows one to study individual cells to gain insight into aspects of their physiology and behaviour. For example, the use of micron-sized growth channels that confine individual ...

Abstract	Background: Controlled restriction of cellular movement using microfluidics allows one to study individual cells to gain insight into aspects of their physiology and behaviour. For example, the use of micron-sized growth channels that confine individual Escherichia coli has yielded novel insights into cell growth and death. To extend this approach to other species of bacteria, many of whom have dimensions in the sub-micron range, or to a larger range of growth conditions, a readily-fabricated device containing sub-micron features is required. Results: Here we detail the fabrication of a versatile device with growth channels whose widths range from 0.3 μm to 0.8 μm. The device is fabricated using electron beam lithography, which provides excellent control over the shape and size of different growth channels and facilitates the rapid-prototyping of new designs. Features are successfully transferred first into silicon, and subsequently into the polydimethylsiloxane that forms the basis of the working microfluidic device. We demonstrate that the growth of sub-micron scale bacteria such as Lactococcus lactis or Escherichia coli cultured in minimal medium can be followed in such a device over several generations. Conclusions: We have presented a detailed protocol based on electron beam fabrication together with specific dry etching procedures for the fabrication of a microfluidic device suited to study submicron-sized bacteria. We have demonstrated that both Gram-positive and Gram-negative bacteria can be successfully loaded and imaged over a number of generations in this device. Similar devices could potentially be used to study other submicron-sized organisms under conditions in which the height and shape of the growth channels are crucial to the experimental design.
MeSH term(s)	Dimethylpolysiloxanes ; Electrons ; Escherichia coli/cytology ; Escherichia coli/growth & development ; Fluorescent Dyes/metabolism ; Gold ; Kymography ; Lactococcus lactis/cytology ; Lactococcus lactis/growth & development ; Microfluidic Analytical Techniques/instrumentation ; Microscopy, Electron, Scanning ; Microtechnology/instrumentation ; Silicon ; Time Factors
Chemical Substances	Dimethylpolysiloxanes ; Fluorescent Dyes ; Gold (7440-57-5) ; Silicon (Z4152N8IUI)
Language	English
Publishing date	2013-04-10
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2100022-0
ISSN	1477-3155 ; 1477-3155
ISSN (online)	1477-3155
ISSN	1477-3155
DOI	10.1186/1477-3155-11-12
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Article ; Online: An Update on Development of Small-Molecule Plasmodial Kinase Inhibitors.

Moolman, Chantalle / Sluis, Rencia van der / Beteck, Richard M / Legoabe, Lesetja J

Molecules (Basel, Switzerland)

2020 Volume 25, Issue 21

Abstract: Malaria control relies heavily on the small number of existing antimalarial drugs. However, recurring antimalarial drug resistance necessitates the continual generation of new antimalarial drugs with novel modes of action. In order to shift the focus ... ...

Abstract	Malaria control relies heavily on the small number of existing antimalarial drugs. However, recurring antimalarial drug resistance necessitates the continual generation of new antimalarial drugs with novel modes of action. In order to shift the focus from only controlling this disease towards elimination and eradication, next-generation antimalarial agents need to address the gaps in the malaria drug arsenal. This includes developing drugs for chemoprotection, treating severe malaria and blocking transmission. Plasmodial kinases are promising targets for next-generation antimalarial drug development as they mediate critical cellular processes and some are active across multiple stages of the parasite's life cycle. This review gives an update on the progress made thus far with regards to plasmodial kinase small-molecule inhibitor development.
MeSH term(s)	Animals ; Antimalarials/pharmacology ; Calcium/metabolism ; Casein Kinase I/metabolism ; Culicidae ; Drug Design ; Drug Discovery/trends ; Drug Resistance ; Glycogen Synthase Kinase 3/metabolism ; Humans ; Imidazoles/pharmacology ; Inhibitory Concentration 50 ; Life Cycle Stages/drug effects ; MAP Kinase Signaling System ; Malaria/drug therapy ; Phosphotransferases/chemistry ; Plasmodium/drug effects ; Plasmodium/enzymology ; Protein Kinase Inhibitors/pharmacology ; Pyridines/pharmacology
Chemical Substances	Antimalarials ; Imidazoles ; Protein Kinase Inhibitors ; Pyridines ; imidazopyridine ; Phosphotransferases (EC 2.7.-) ; Casein Kinase I (EC 2.7.11.1) ; Glycogen Synthase Kinase 3 (EC 2.7.11.26) ; Calcium (SY7Q814VUP)
Keywords	covid19
Language	English
Publishing date	2020-11-07
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	1413402-0
ISSN	1420-3049 ; 1431-5165 ; 1420-3049
ISSN (online)	1420-3049
ISSN	1431-5165 ; 1420-3049
DOI	10.3390/molecules25215182
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Article ; Online: Exploration of benzofuran-based compounds as potent and selective Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3) inhibitors.

Moolman, Chantalle / van der Sluis, Rencia / Beteck, Richard M / Legoabe, Lesetja J

Bioorganic chemistry

2021 Volume 112, Page(s) 104839

Abstract: Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3) has been identified as a potential target for the development of novel drugs against multi-drug resistant malaria. A series of benzofuran-based compounds was synthesised and evaluated as ... ...

Abstract	Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3) has been identified as a potential target for the development of novel drugs against multi-drug resistant malaria. A series of benzofuran-based compounds was synthesised and evaluated as inhibitors of recombinantly expressed and purified PfGSK-3 and human glycogen synthase kinase-3 beta (HsGSK-3β). Of this series, five compounds (5k, 5m, 5p, 5r, 5s) preferentially inhibited PfGSK-3, with four of these compounds exhibiting IC
MeSH term(s)	Benzofurans/chemical synthesis ; Benzofurans/chemistry ; Benzofurans/pharmacology ; Dose-Response Relationship, Drug ; Glycogen Synthase Kinase 3/antagonists & inhibitors ; Glycogen Synthase Kinase 3/metabolism ; Humans ; Molecular Structure ; Plasmodium falciparum/enzymology ; Protein Kinases/chemical synthesis ; Protein Kinases/chemistry ; Protein Kinases/pharmacology ; Structure-Activity Relationship
Chemical Substances	Benzofurans ; Protein Kinases (EC 2.7.-) ; Glycogen Synthase Kinase 3 (EC 2.7.11.26) ; benzofuran (LK6946W774)
Language	English
Publishing date	2021-03-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	120080-x
ISSN	1090-2120 ; 0045-2068
ISSN (online)	1090-2120
ISSN	0045-2068
DOI	10.1016/j.bioorg.2021.104839
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