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This study aimed to expand our understanding of myelin basic protein (MBP), a key component of central nervous system myelin, by developing a protocol to track and quantifying individual MBP particles during oligodendrocyte (OL) differentiation. MBP particle directionality, confinement, and diffusion were tracked by rapid TIRF and HILO imaging of Dendra2 tagged MBP in three stages of mouse oligodendroglia: OL precursors, early myelinating OLs, and mature myelinating OLs. The directionality and confinement of MBP particles increased at each stage consistent with progressive transport toward, and recruitment into, emerging myelin structures. Unexpectedly, diffusion data presented a more complex pattern with subpopulations of the most diffusive particles disappearing at the transition between the precursor and early myelinating stage, before reemerging in the membrane sheets of mature OLs. This diversity of particle behaviors, which would be undetectable by conventional ensemble-averaged methods, are consistent with a multifunctional view of MBP involving roles in myelin expansion and compaction., Competing Interests: No competing interests, either financial or nonfinancial, are declared by the authors., (© The Author(s) 2023.)

Traditional methods for the assembly of functionalised DNA structures, involving enzyme restriction and modification, present difficulties when working with small DNA fragments (<100 bp), in part due to a lack of control over enzymatic action during the DNA modification process. This limits the design flexibility and range of accessible DNA structures. Here, we show that these limitations can be overcome by introducing chemical modifications into the DNA that spatially restrict enzymatic activity. This approach, sterically controlled nuclease enhanced (SCoNE) DNA assembly, thereby circumvents the size limitations of conventional Gibson assembly (GA) and allows the preparation of well-defined, functionalised DNA structures with multiple probes for specific analytes, such as IL-6, procalcitonin (PCT), and a biotin reporter group. Notably, when using the same starting materials, conventional GA under typical conditions fails. We demonstrate successful analyte capture based on standard and modified sandwich ELISA and also show how the inclusion of biotin probes provides additional functionality for product isolation., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, limiting the numbers of three-dimensional (3D) nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy (ExM) solves many of these limitations, but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here, we demonstrate the conditions for isotropic expansion of the nucleus at a resolution equal to or better than 120-130 nm (pre-expansion). Using the DNA damage response proteins BRCA1, 53BP1 (also known as TP53BP1) and RAD51 as exemplars, we quantitatively describe the 3D nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin-regulated events and show the simultaneous assessment of four elements. This study thus demonstrates how ExM can contribute to the investigation of nanoscale nuclear processes., Competing Interests: Competing interests R.K.N. is founder of Chrometra, a company that sells probes for expansion microscopy., (© 2022. Published by The Company of Biologists Ltd.)

We present a measurement of ν&#95;{e} interactions from the Fermilab Booster Neutrino Beam using the MicroBooNE liquid argon time projection chamber to address the nature of the excess of low energy interactions observed by the MiniBooNE Collaboration. Three independent ν&#95;{e} searches are performed across multiple single electron final states, including an exclusive search for two-body scattering events with a single proton, a semi-inclusive search for pionless events, and a fully inclusive search for events containing all hadronic final states. With differing signal topologies, statistics, backgrounds, reconstruction algorithms, and analysis approaches, the results are found to be either consistent with or modestly lower than the nominal ν&#95;{e} rate expectations from the Booster Neutrino Beam and no excess of ν&#95;{e} events is observed.

We report a measurement of the energy-dependent total charged-current cross section σ(E&#95;{ν}) for inclusive muon neutrinos scattering on argon, as well as measurements of flux-averaged differential cross sections as a function of muon energy and hadronic energy transfer (ν). Data corresponding to 5.3×10^{19} protons on target of exposure were collected using the MicroBooNE liquid argon time projection chamber located in the Fermilab booster neutrino beam with a mean neutrino energy of approximately 0.8 GeV. The mapping between the true neutrino energy E&#95;{ν} and reconstructed neutrino energy E&#95;{ν}^{rec} and between the energy transfer ν and reconstructed hadronic energy E&#95;{had}^{rec} are validated by comparing the data and Monte Carlo (MC) predictions. In particular, the modeling of the missing hadronic energy and its associated uncertainties are verified by a new method that compares the E&#95;{had}^{rec} distributions between data and a MC prediction after constraining the reconstructed muon kinematic distributions, energy, and polar angle to those of data. The success of this validation gives confidence that the missing energy in the MicroBooNE detector is well modeled and underpins first-time measurements of both the total cross section σ(E&#95;{ν}) and the differential cross section dσ/dν on argon.

We report results from a search for neutrino-induced neutral current (NC) resonant Δ(1232) baryon production followed by Δ radiative decay, with a ⟨0.8⟩  GeV neutrino beam. Data corresponding to MicroBooNE's first three years of operations (6.80×10^{20} protons on target) are used to select single-photon events with one or zero protons and without charged leptons in the final state (1γ1p and 1γ0p, respectively). The background is constrained via an in situ high-purity measurement of NC π^{0} events, made possible via dedicated 2γ1p and 2γ0p selections. A total of 16 and 153 events are observed for the 1γ1p and 1γ0p selections, respectively, compared to a constrained background prediction of 20.5±3.65(syst) and 145.1±13.8(syst) events. The data lead to a bound on an anomalous enhancement of the normalization of NC Δ radiative decay of less than 2.3 times the predicted nominal rate for this process at the 90% confidence level (C.L.). The measurement disfavors a candidate photon interpretation of the MiniBooNE low-energy excess as a factor of 3.18 times the nominal NC Δ radiative decay rate at the 94.8% C.L., in favor of the nominal prediction, and represents a greater than 50-fold improvement over the world's best limit on single-photon production in NC interactions in the sub-GeV neutrino energy range.

We present a search for the decays of a neutral scalar boson produced by kaons decaying at rest, in the context of the Higgs portal model, using the MicroBooNE detector. We analyze data triggered in time with the Fermilab NuMI neutrino beam spill, with an exposure of 1.93×10^{20} protons on target. We look for monoenergetic scalars that come from the direction of the NuMI hadron absorber, at a distance of 100 m from the detector, and decay to electron-positron pairs. We observe one candidate event, with a standard model background prediction of 1.9±0.8. We set an upper limit on the scalar-Higgs mixing angle of θ<(3.3-4.6)×10^{-4} at the 95% confidence level for scalar boson masses in the range (100-200)  MeV/c^{2}. We exclude, at the 95% confidence level, the remaining model parameters required to explain the central value of a possible excess of K&#95;{L}^{0}→π^{0}νν[over ¯] decays reported by the KOTO collaboration. We also provide a model-independent limit on a new boson X produced in K→πX decays and decaying to e^{+}e^{-}.

DNA methyltransferase activity is associated with a host of diseases, including cancers, where global hypomethylation of the genome, as well as marked changes in local DNA methylation patterns, can be both diagnostic and prognostic for the disease. Despite this, we currently lack a method for directly measuring the activity of the DNA methyltransferases, which would support the development of DNA methyltransferase-targeted therapies. Here, we demonstrate an assay for the direct measurement of methyltransferase activity, in real time. We employ a fluorescent methyltransferase cofactor analogue, which when bound by the enzyme to a labeled target DNA sequence results in fluorescence resonance energy transfer (FRET) between the donor dye (DNA) and the acceptor dye (cofactor). We demonstrate that the method can be used to monitor the activity of DNA MTases in real time and can be applied to screen inhibitors of the DNA methyltransferases. We show this in both bulk phase and single molecule imaging experiments, highlighting the potential application of the assay in screening and biophysical studies of methyltransferase function.

We report on the first measurement of flux-integrated single differential cross sections for charged-current (CC) muon neutrino (ν&#95;{μ}) scattering on argon with a muon and a proton in the final state, ^{40}Ar (ν&#95;{μ},μp)X. The measurement was carried out using the Booster Neutrino Beam at Fermi National Accelerator Laboratory and the MicroBooNE liquid argon time projection chamber detector with an exposure of 4.59×10^{19} protons on target. Events are selected to enhance the contribution of CC quasielastic (CCQE) interactions. The data are reported in terms of a total cross section as well as single differential cross sections in final state muon and proton kinematics. We measure the integrated per-nucleus CCQE-like cross section (i.e., for interactions leading to a muon, one proton, and no pions above detection threshold) of (4.93±0.76&#95;{stat}±1.29&#95;{sys})×10^{-38}  cm^{2}, in good agreement with theoretical calculations. The single differential cross sections are also in overall good agreement with theoretical predictions, except at very forward muon scattering angles that correspond to low-momentum-transfer events.

Super-resolution fluorescence microscopy is a key tool in the elucidation of biological fine structures, providing insights into the distribution and interactions of biomolecular complexes down to the nanometer scale. Expansion microscopy is a recently developed approach for achieving nanoscale resolution on a conventional microscope. Here, biological samples are embedded in an isotropically swollen hydrogel. This physical expansion of the sample allows imaging with resolutions down to the tens-of-nanometers. However, because of the requirement that fluorescent labels are covalently bound to the hydrogel, standard, small-molecule targeting of fluorophores has proven incompatible with expansion microscopy. Here, we show a chemical linking approach that enables direct, covalent grafting of a targeting molecule and fluorophore to the hydrogel in expansion microscopy. We show application of this series of molecules in the antibody-free targeting of the cell cytoskeleton and in an example of lipid membrane staining for expansion microscopy. Furthermore, using this trivalent linker strategy, we demonstrate the benefit of introducing fluorescent labels post-expansion by visualizing an immunostaining through fluorescent oligonucleotide hybridization after expanding the polymer. Our probes allow different labeling approaches that are compatible with expansion microscopy.

Expansion microscopy is a novel, fluorescence imaging technique, which allows three-dimensional nanoscale imaging of specimens on a conventional fluorescence microscope. This is achieved through an innovative sample treatment, which culminates in approximately 4.5-fold expansion of specimens in each dimension. This allows 70 nm lateral and 200 nm axial resolution. To further develop application of the technique, there has been considerable focus on improving the methodology by i) extending the efficacy of labelling, ii) enabling multi-colour labelling of different biomolecules simultaneously, iii) further improving resolving power through alterations to sample preparation and iv) by combination of expansion microscopy with other well-established super resolution techniques. This review will highlight some of these recent advances and suggest ways that the technique could be developed further in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. RKN is founder of Chrometra, which sells trifunctional labels for expansion microscopy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Current methods for bioconjugation rely on the introduction of stable linkers that lack the required versatility to perform sequential functionalizations. However, sequential manipulations are an increasing requirement in chemical biology because they can underpin multiple analyses of the same sample to provide a wider understanding of cell behavior. Here, we present a new method to site-selectively write , remove , and rewrite chemical functionality to a biomolecule, DNA in this case. Our method combines the precision and robustness of methyltransferase-directed labeling with the reversibility of acyl hydrazones and the efficiency of click chemistry. Underpinning the method is a new S -adenosyl-l-methionine derivative to site-selectively label DNA with a bifunctional chemical handle containing an acyl hydrazone-linker and a terminal azide. Functional tags are conjugated via the azide and can be removed (i.e., untagged) when needed at the acyl hydrazone via exchange with hydroxyl amine. The formed hydrazide-labeled DNA is a versatile intermediate that can be either rewritten to reset the original chemical handle or covalently reacted with a permanent tag . This ability to write , tag , untag , and permanently tag DNA is exploited to sequentially introduce two fluorescent dyes on DNA. Finally, we demonstrate the potential of the method by developing a protocol to sort labeled DNA using magnetic beads, with subsequent amplification of the sorted DNA sample for further analysis. The presented method opens new avenues for site-selective bioconjugation and should underpin integrative approaches in chemical biology where sequential functionalizations of the same sample are required., Competing Interests: The authors declare the following competing financial interest(s): A.W., E.J., R.K.N., and P.F.-T. are named on a patent application (GB1913598.7) related to this work., (Copyright © 2020 American Chemical Society.)

We report an approach for visualizing DNA sequence and using these 'DNA barcodes' to search complex mixtures of genomic material for DNA molecules of interest. We demonstrate three applications of this methodology; identifying specific molecules of interest from a dataset containing gigabasepairs of genome; identification of a bacterium from such a dataset and, finally, by locating infecting virus molecules in a background of human genomic material. As a result of the dense fluorescent labelling of the DNA, individual barcodes of the order 40 kb pairs in length can be reliably identified. This means DNA can be prepared for imaging using standard handling and purification techniques. The recorded dataset provides stable physical and electronic records of the total genomic content of a sample that can be readily searched for a molecule or region of interest., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The methyltransferase enzymes can be applied to deliver a range of modifications to pre-determined sites on large DNA molecules with exceptional specificity and efficiency. To date, however, a limited number of modifications have been delivered in this way because of the complex chemical synthesis that is needed to produce a cofactor analogue carrying a specific function, such as a fluorophore. Here, we describe a method for the direct transfer of a series of functional compounds (seven fluorescent dyes, biotin and polyethylene glycol) to the DNA duplex. Our approach uses a functional cofactor analogue, whose final preparative step is performed alongiside the DNA modification reaction in a single pot, with no purification needed. We show that fluorophore conjugation efficiency in these mixtures is significantly improved compared to two-step labeling approaches. Our experiments highlight the remarkable malleability and selectivity of the methyltransferases tested. Additional analysis using high resolution localization of the fluorophore distribution indicates that target sites for the methyltransferase are predominantly labeled on a single strand of their palindromic site and that a small and randomly-distributed probability of off-site labeling exists.

Methyltransferases (MTases) form a large family of enzymes that methylate a diverse set of targets, ranging from the three major biopolymers to small molecules. Most of these MTases use the cofactor S-adenosyl-l-Methionine (AdoMet) as a methyl source. In recent years, there have been significant efforts toward the development of AdoMet analogues with the aim of transferring moieties other than simple methyl groups. Two major classes of AdoMet analogues currently exist: doubly-activated molecules and aziridine based molecules, each of which employs a different approach to achieve transalkylation rather than transmethylation. In this review, we discuss the various strategies for labelling and functionalizing biomolecules using AdoMet-dependent MTases and AdoMet analogues. We cover the synthetic routes to AdoMet analogues, their stability in biological environments and their application in transalkylation reactions. Finally, some perspectives are presented for the potential use of AdoMet analogues in biology research, (epi)genetics and nanotechnology., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report an assay for determining the number of fluorophores conjugated to single plasmid DNA molecules and apply this to compare the efficiency of fluorophore coupling strategies for covalent DNA labelling. We compare a copper-catalyzed azide-alkyne cycloaddition reaction, amine to N -hydroxysuccinimidyl ester coupling reaction and strain-promoted azide-alkyne cycloaddition reaction for fluorescent DNA labelling. We found increased labelling efficiency going from the amine to N -hydroxysuccinimidyl ester coupling reaction to the copper-catalyzed azide-alkyne cycloaddition and found the highest degree of DNA labelling with the strain-promoted azide-alkyne cycloaddition reaction. We also examined the effect of labelling on the DNA structure using atomic force microscopy. We observe no distortions or damage to the DNA that was labeled using the amine to N -hydroxysuccinimidyl ester and strain-promoted azide-alkyne cycloaddition coupling reactions. This was in contrast to the copper-catalyzed azide-alkyne cycloaddition reaction, which, despite the use of copper-coordinating ligands in the labelling mixture, leads to some structural DNA damage (single-stranded DNA breaks).

The formation and repair of myelin involves alterations in the molecular and physical properties of oligodendrocytes, and highly coordinated interactions with their target axons. Characterising the nature and timing of these events at the molecular and cellular levels illuminates the fundamental events underlying myelin formation, and provides opportunities for the development of therapies to replace myelin lost through traumatic injury and inflammation. The dynamic nature of these events requires that live-imaging methods be used to capture this information accurately and completely. Developments in imaging technologies, and model systems suitable for their application to myelination, have advanced the study of myelin formation, injury and repair. Similarly, new techniques for single molecule imaging, and novel imaging probes, are providing opportunities to resolve the dynamics of myelin proteins during myelination. Here, we explore these developments in the context of myelin formation and injury, identify unmet needs within the field where progress can be advanced through live-imaging approaches, identify technical challenges that are limiting this progress, and highlight practical applications for these approaches that could lead to therapies for the protection of oligodendrocytes and myelin from injury, and restore myelin lost through injury and disease. This article is part of the Special Issue entitled 'Oligodendrocytes in Health and Disease'., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nearly 50 years since its potential as a fluorescent base analogue was first recognized, 2-aminopurine (2AP) continues to be the most widely used fluorescent probe of DNA structure and the perturbation of that structure by interaction with enzymes and other molecules. In this review, we begin by considering the origin of the dramatic and intriguing difference in photophysical properties between 2AP and its structural isomer, adenine; although 2AP differs from the natural base only in the position of the exocyclic amine group, its fluorescence intensity is one thousand times greater. We then discuss the mechanism of interbase quenching of 2AP fluorescence in DNA, which is the basis of its use as a conformational probe but remains imperfectly understood. There are hundreds of examples in the literature of the use of changes in the fluorescence intensity of 2AP as the basis of assays of conformational change; however, in this review we will consider in detail only a few intensity-based studies. Our primary aim is to highlight the use of time-resolved fluorescence measurements, and the interpretation of fluorescence decay parameters, to explore the structure and dynamics of DNA. We discuss the salient features of the fluorescence decay of 2AP when incorporated in DNA and review the use of decay measurements in studying duplexes, single strands and other structures. We survey the use of 2AP as a probe of DNA-enzyme interaction and enzyme-induced distortion, focusing particularly on its use to study base flipping and the enhanced mechanistic insights that can be gained by a detailed analysis of the decay parameters, rather than merely monitoring changes in fluorescence intensity. Finally we reflect on the merits and shortcomings of 2AP and the prospects for its wider adoption as a fluorescence-decay-based probe.

Deposition of linear DNA molecules is a critical step in many single-molecule genomic approaches including DNA mapping, fiber-FISH, and several emerging sequencing technologies. In the ideal situation, the DNA that is deposited for these experiments is absolutely linear and uniformly stretched, thereby enabling accurate distance measurements. However, this is rarely the case, and furthermore, current approaches for the capture and linearization of DNA on a surface tend to require complex surface preparation and large amounts of starting material to achieve genomic-scale mapping. This makes them technically demanding and prevents their application in emerging fields of genomics, such as single-cell based analyses. Here we describe a simple and extremely efficient approach to the deposition and linearization of genomic DNA molecules. We employ droplets containing as little as tens of picograms of material and simply drag them, using a pipet tip, over a polymer-coated coverslip. In this report we highlight one particular polymer, Zeonex, which is remarkably efficient at capturing DNA. We characterize the method of DNA capture on the Zeonex surface and find that the use of droplets greatly facilitates the efficient deposition of DNA. This is the result of a circulating flow in the droplet that maintains a high DNA concentration at the interface of the surface/solution. Overall, our approach provides an accessible route to the study of genomic structural variation from samples containing no more than a handful of cells.

Magnetite nanoparticles have size- and shape-dependent magnetic properties. In addition, assemblies of magnetite nanoparticles forming one-dimensional nanostructures have magnetic properties distinct from zero-dimensional or non-organized materials due to strong uniaxial shape anisotropy. However, assemblies of free-standing magnetic nanoparticles tend to collapse and form closed-ring structures rather than chains in order to minimize their energy. Magnetotactic bacteria, ubiquitous microorganisms, have the capability to mineralize magnetite nanoparticles, the so-called magnetosomes, and to direct their assembly in stable chains via biological macromolecules. In this contribution, the synthesis and assembly of biological magnetite to obtain functional magnetic dipoles in magnetotactic bacteria are presented, with a focus on the assembly. We present tomographic reconstructions based on cryo-FIB sectioning and SEM imaging of a magnetotactic bacterium to exemplify that the magnetosome chain is indeed a paradigm of a 1D magnetic nanostructure, based on the assembly of several individual particles. We show that the biological forces are a major player in the formation of the magnetosome chain. Finally, we demonstrate by super resolution fluorescence microscopy that MamK, a protein of the actin family necessary to form the chain backbone in the bacteria, forms a bundle of filaments that are not only found in the vicinity of the magnetosome chain but are widespread within the cytoplasm, illustrating the dynamic localization of the protein within the cells. These very simple microorganisms have thus much to teach us with regards to controlling the design of functional 1D magnetic nanoassembly.

We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes transalkylate DNA with the cofactor we tested (a readily prepared s-adenosyl-l-methionine analogue).

Microbial communities associated with submerged detritus in aquatic ecosystems often comprise a diverse mixture of autotrophic and heterotrophic microbes, including algae, bacteria, protozoa, and fungi. Recent studies have documented increased rates of plant litter mass loss when periphytic algae are present. We conducted laboratory and field experiments to assess potential metabolic interactions between natural autotrophic and heterotrophic microbial communities inhabiting submerged decaying plant litter of Typha angustifolia and Schoenoplectus acutus. In the field, submerged plant litter was either exposed to natural sunlight or placed under experimental canopies that manipulated light availability and growth of periphytic algae. Litter was collected and returned to the laboratory, where algal photosynthesis was manipulated (light/dark incubation), while rates of bacterial and fungal growth and productivity were simultaneously quantified. Bacteria and fungi were rapidly stimulated by exposure to light, thus establishing the potential for algal priming of microbial heterotrophic decay activities. Experimental incubations of decaying litter with 14C- and 13C-bicarbonate established that inorganic C fixed by algal photosynthesis was rapidly transferred to and assimilated by heterotrophic microbial decomposers. Periphytic algal stimulation of microbial heterotrophs, especially fungal decomposers, is an important and largely unrecognized interaction within the detrital microbial landscape, which may transform our current conceptual understanding of microbial secondary production and organic matter decomposition in aquatic ecosystems.

Structural and temporal inhomogeneities can have a marked influence on the performance of inorganic and biocatalytic systems alike. While these subtle variations are hardly ever accessible through bulk or ensemble averaged activity screening, insights into the molecular mechanisms underlying these diverse phenomena are absolutely critical for the development of optimized or novel catalytic systems and processes. Fortunately, state-of-the-art fluorescence microscopy methods have allowed experimental access to this intriguing world at the nanoscale. In this tutorial review we will first provide a broad overview of key concepts and developments in the application of single molecule fluorescence spectroscopy to (bio)catalysis research. In the second part topics specific to both bio and heterogeneous catalysis will be reviewed in more detail.

A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme-substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.

We present Localizer, a freely available and open source software package that implements the computational data processing inherent to several types of superresolution fluorescence imaging, such as localization (PALM/STORM/GSDIM) and fluctuation imaging (SOFI/pcSOFI). Localizer delivers high accuracy and performance and comes with a fully featured and easy-to-use graphical user interface but is also designed to be integrated in higher-level analysis environments. Due to its modular design, Localizer can be readily extended with new algorithms as they become available, while maintaining the same interface and performance. We provide front-ends for running Localizer from Igor Pro, Matlab, or as a stand-alone program. We show that Localizer performs favorably when compared with two existing superresolution packages, and to our knowledge is the only freely available implementation of SOFI/pcSOFI microscopy. By dramatically improving the analysis performance and ensuring the easy addition of current and future enhancements, Localizer strongly improves the usability of superresolution imaging in a variety of biomedical studies.

Structural knowledge of interactions amongst the ~ 40 proteins of the human complement system, which is central to immune surveillance and homeostasis, is expanding due primarily to X-ray diffraction of co-crystallized proteins. Orthogonal evidence, in solution, for the physiological relevance of such co-crystal structures is valuable since intermolecular affinities are generally weak-to-medium and inter-domain mobility may be important. In this current work, Förster resonance energy transfer (FRET) was used to investigate the 10 μM K(D) (210 kD) complex between the N-terminal region of the soluble complement regulator, factor H (FH1-4), and the key activation-specific complement fragment, C3b. Using site-directed mutagenesis, seven cysteines were introduced individually at potentially informative positions within the four CCP modules comprising FH1-4, then used for fluorophore attachment. C3b possesses a thioester domain featuring an internal cycloglutamyl cysteine thioester; upon hydrolysis this yields a free thiol (Cys988) that was also fluorescently tagged. Labeled proteins were functionally active as cofactors for cleavage of C3b to iC3b except for FH1-4(Q40C) where conjugation with the fluorophore likely abrogated interaction with the protease, factor I. Time-resolved FRET measurements were undertaken to explore interactions between FH1-4 and C3b in fluid phase and under near-physiological conditions. These experiments confirmed that, as in the cocrystal structure, FH1-4 binds to C3b with CCP module 1 furthest from, and CCP module 4 closest to, the thioester domain, placing subsequent modules of FH near to any surface to which C3b is attached. The data do not rule out flexibility of the thioester domain relative to the remainder of the complex., (Copyright © 2011 The Protein Society.)

The technologies associated with DNA sequencing are rapidly evolving. Indeed, single-molecule DNA sequencing strategies are cheaper and faster than ever before. Despite this progress, every sequencing platform to date relies on reading the genome in small, abstract fragments, typically of less than 1000 bases in length. The overarching aim of the optical map is to complement the information derived from DNA sequencing by providing long-range context on which these short sequence reads can be built. This is typically done using an enzyme to target and modify at short DNA sequences of, say, six bases in length throughout the genome. By accurately placing these short pieces of sequence on long genomic DNA fragments, up to several millions of bases in length, a scaffold for sequence assembly can be obtained. This review focuses on three enzymatic approaches to optical mapping. Optical mapping was first developed using restriction enzymes to sequence-specifically cleave DNA that is immobilized on a surface. More recently, nicking enzymes have found application in the sequence-specific fluorescent labeling of DNA for optical mapping. Such covalent modification allows the DNA to be imaged in solution, and this, in combination with developing nanofluidic technologies, is enabling new high-throughput approaches to mapping. And, finally, this review will discuss the recent development of mapping with subdiffraction-limit precision using methyltransferase enzymes to label the DNA with an ultrahigh density., (Copyright © 2011 Wiley Periodicals, Inc.)

Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.

Background: Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC., Results: The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360), cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases., Conclusion: We have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.

We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state: base flipping is accompanied by the loss of the very short ( approximately 50 ps) lifetime component associated with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by pi-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the proposed key residue for base flipping by M.TaqI, the target base partner thymine, is substituted by an abasic site analog. The corresponding cocrystal structure shows 2-aminopurine in the active site of M.TaqI, demonstrating that the partner thymine is not essential for base flipping. However, in this structure, a shift of the 3' neighbor of the target base into the vacancy left after base flipping is observed, apparently replicating a stabilizing role of the missing partner thymine. Time-resolved fluorescence and acrylamide quenching measurements of M.TaqI complexes in solution provide evidence for an alternative binding site for the extra-helical target base within M.TaqI and suggest that the partner thymine assists in delivering the target base into the active site.

To explore the effect of intermolecular interactions on the photophysics of 2-aminopurine (2AP) in a well-defined environment, we have investigated the fluorescence properties of single 2AP crystals, having determined their X-ray structure. In the crystal, 2AP is subject to base-stacking and hydrogen-bonding interactions similar to those found in DNA. The crystal shows dual fluorescence: pi-stacked molecules in the bulk of the lattice have redshifted excitation and emission spectra, while molecules at defect sites have spectra similar to those of 2AP in solution or in DNA. Heterogeneous intermolecular interactions in the crystal give rise to multiexponential fluorescence decay characteristics similar to those observed for 2AP-labelled DNA. The presence of about 13 % of the 7H tautomer in the crystal confirms that 9H-7H tautomerisation of 2AP occurs in the ground state. Long-wavelength excitation of a 2AP-labelled oligonucleotide duplex produced redshifted emission similar to that observed in the crystal, indicating that pi-stacking interaction of 2AP with nucleobases gives rise to a low energy excited state.

We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements.

Time-resolved fluorescence of 2-aminopurine-labeled DNA duplexes at 77 K reveals the relationship between base dynamics and the conformational heterogeneity that results in the well-known multiexponential fluorescence decay at room temperature. The conformation that exhibits rapid interbase charge transfer at room temperature is not populated in the frozen duplex at 77 K; this geometry is accessed by thermal motion of the bases, it is not a minimum energy structure of the duplex. Three photophysically distinct conformational states persist in the frozen duplex; these are minimum energy structures and do not interconvert at room temperature on the time scale of the 2-aminopurine excited-state lifetime.

We examined the effect of light on extracellular enzyme activities of periphytic/endogenous microbial assemblages associated with decomposing litter of an emergent macrophyte Typha angustifolia within a small inland wetland in southeastern Michigan. Standing-dead Typha leaf litter was collected, placed into floating wire mesh litter baskets, and submerged in a wetland pool. Enzyme saturation assays were conducted on three occasions following litter submergence (days 9, 28, and 44) to generate saturation curves for the individual enzymes tested and to examine potential differences in enzyme saturation kinetics during microbial colonization and development. Experimental light manipulations were conducted on two occasions during microbial development (days 10 and 29). Short-term (30 min) light exposure significantly increased extracellular beta-glucosidase activity of litter-associated microbial communities. Activities of beta-xylosidase and leucine-aminopeptidase were not stimulated, and stimulation of phosphatase activity was variable. The exact mechanism for increased enzyme activity remains unknown, but it may have been increased pH arising from periphytic algal photosynthesis. These results suggest that extracellular enzyme activity in microbial communities colonizing natural organic substrata may be influenced by light/photosynthesis, as has previously been demonstrated for periphyton communities grown on artificial, inert substrata. Thus, light/photosynthetic mediated stimulation of extracellular enzyme activities may be a common occurrence in microbial communities associated with natural decaying plant litter in wetlands and might engender diurnal patterns in other microbial decay processes (e.g., production, organic matter decomposition, and mineralization).

The radiolabeled leucine incorporation technique for quantifying rates of bacterial production has increased in popularity since its original description for bacterioplankton communities. Prior studies addressing incorporation conditions (e.g., substrate saturation) for bacterial communities in other habitats, such as decaying plant litter, have reported a wide range of final leucine concentrations (400 nM to 50 microM) required to achieve saturation-level uptake. We assessed the application of the [(3)H]leucine incorporation procedure for measuring bacterial production on decaying wetland plant litter. Substrate saturation experiments (nine concentrations, 10 nM to 50 microM final leucine concentration) were conducted on three dates for microbial communities colonizing the submerged litter of three emergent plant species (Typha angustifolia, Schoenoplectus validus, and Phragmites australis). A modified [(3)H]leucine protocol was developed by coupling previously described incubation and alkaline extraction protocols with microdialysis (500 molecular weight cutoff membrane) of the final radiolabeled protein extract. The incorporation of [(3)H]leucine into protein exhibited a biphasic saturation curve, with lower apparent K(m) values ranging from 400 nM to 4.2 microM depending on the plant species studied. Upper apparent K(m) values ranged from 1.3 to 59 microM. These results suggest differential uptake by litter-associated microbial assemblages, with the lower apparent K(m) values possibly representing bacterial uptake and higher apparent K(m) values representing a combination of both bacterial and nonbacterial (e.g., eukaryotic) uptake.

DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 A resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (approximately 100 ps) decay component and the large increase in the amplitude of the long (approximately 10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA-enzyme complexes that cannot be discerned from the present X-ray structures.

A novel method of detecting extracellular protease activity at biofilm-substratum interfaces was developed. This method utilizes fluorescent molecules bound to cellulose substrata with a lectin. Extracellular proteases degrade the lectin and release the fluorochrome into solution. This new technique and a standard dissolved-substrate assay detected similar responses of biofilm extracellular protease activity to experimental manipulation of N supply. Combination of this technique with confocal scanning laser microscopy allowed direct visualization of microspatial patterns of bacterial distribution and extracellular protease activity at the biofilm-substratum interface.

Students' organization of the knowledge that they acquire is an important factor in determining the degree to which it is retained and used. In the past we have used the "fill-in-the-structure" (FITS) task as a direct method of inferring students' cognitive structures of course content (Naveh-Benjamin, Lin & McKeachie, 1995). This study goes one step further by using the FITS task to assess the flexibility of students' cognitive structures of the material learned; that is, whether students are able to relate the same concepts in different ways when the concepts are embedded in two different conceptual frameworks. We assessed the flexibility of students' cognitive structures in three studies by asking students in an ecology course to complete two different structures, each based on a different major dimension in the course. Results of the first study showed that the FITS technique could be used to assess students' ability to use concepts learned in the course appropriately in two different frameworks. The flexibility measures obtained were positively related to academic performance. The second study demonstrated the usefulness of the technique in measuring the development of conceptual flexibility during the course. Finally, the third study employed the technique to show that students' flexible use of concepts can be enhanced by appropriate instruction. Copyright 1998 Academic Press.

A method of simultaneously quantifying photoautotrophic (algae and cyanobacteria) and bacterial production in periphyton communities by (14)C-bicarbonate and (3)H-leucine incorporation was investigated and applied to communities subjected to specific intensities of photosynthetically active radiation (400-700 nm). Maximum photosynthetic output (2.23 ± 0.29 (SE) μg C cm(-2) h(-1)) and bacterial production (0.07 ± 0.006 μg C cm(-2) h(-1)) occurred at the highest photon flux density (400 μmol m(-2) s(-1)). Over a photon flux density range of 20-400 μmol m(-2) s(-1), bacterial and autotroph productivity were significantly and positively correlated (r = 0.89). Furthermore, application of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, a photosystem 11 inhibitor, to periphyton films reduced bacterial production by 46%, but it had no such effect on bacteria-only cultures. Therefore, the magnitude of bacterial production in periphyton was coupled to the photosynthesis/metabolism of algae and/or cyanobacteria.