Fluorescence lifetime imaging microscopy (FLIM) to demonstrate the nuclear binding of flavanols and (--epigallocatechin gallate

The use of light microscopy and DMACA staining strongly suggested that plant and animal cell nuclei act as sinks for flavanols [1, 2]. Detailed uv-vis spectroscopic titration experiments indicated that histone proteins are the likely binding sites in the nucleus [2]. Here we report the development of a multi-photon excitation microscopy technique combined with fluorescent lifetime measurements of flavanols. Using this technique, (+) catechin, (-) epicatechin and (-) epigallocatechin gallate (EGCG) showed strikingly different excited state lifetimes in solution. Interaction of histone proteins with flavanols was indicated by the appearance of a significant τ2-component of 1.7 to 4.0ns. Tryptophan interference could be circumvented in the in vivo fluorescence lifetime imaging microscopy (FLIM) experiments with 2-photon excitation at 630nm. This enabled visualisation and semi-quantitative measurements that demonstrated unequivocally the absorption of (+)catechin, (-)epicatechin and EGCG by nuclei of onion cells. 3D FLIM revealed for the first time that externally added EGCG penetrated the whole nucleus in onion cells. The relative proportions of EGCG in cytoplasm: nucleus: nucleoli were ca. 1:10:100. FLIM experiments may therefore facilitate probing the health effects of EGCG, which is the major constituent of green tea

Directed Molecular Stacking for Engineered Fluorescent Three-Dimensional Reduced Graphene Oxide and Coronene Frameworks

[EN] Three‐dimensional fluorescent graphene frameworks with controlled porous morphologies are of significant importance for practical applications reliant on controlled structural and electronic properties, such as organic electronics and photochemistry. Here we report a synthetically accessible approach concerning directed aromatic stacking interactions to give rise to new fluorogenic 3D frameworks with tuneable porosities achieved through molecular variations. The binding interactions between the graphene‐like domains present in the in situ‐formed reduced graphene oxide (rGO) with functional porphyrin molecules lead to new hybrids via an unprecedented solvothermal reaction. Functional free‐base porphyrins featuring perfluorinated aryl groups or hexyl chains at their meso‐ and β‐positions were employed in turn to act as directing entities for the assembly of new graphene‐based and foam‐like frameworks and of their corresponding coronene‐based hybrids. Investigations in the dispersed phase and in thin‐film by XPS, SEM and FLIM shed light onto the nature of the aromatic stacking within functional rGO frameworks (denoted rGOFs) which was then modelled semi‐empirically and by DFT calculations. The pore sizes of the new emerging reduced graphene oxide hybrids are tuneable at the molecular level and mediated by the bonding forces with the functional porphyrins acting as the “molecular glue”. Single crystal X‐ray crystallography described the stacking of a perfluorinated porphyrin with coronene, which can be employed as a molecular model for understanding the local aromatic stacking order and charge transfer interactions within these rGOFs for the first time. This opens up a new route to controllable 3D framework morphologies and pore size from the Ångstrom to the micrometre scale. Theoretical modelling showed that the porosity of these materials is mainly due to the controlled inter‐planar distance between the rGO, coronene or graphene sheets. The host‐guest chemistry involves the porphyrins acting as guests held through π‐π stacking, as demonstrated by XPS. The objective of this study is also to shed light into the fundamental localised electronic and energy transfer properties in these new molecularly engineered porous and fluorogenic architectures, aiming in turn to understand how functional porphyrins may exert stacking control over the notoriously disordered local structure present in porous reduced graphene oxide fragments. By tuning the porosity and the distance between the graphene sheets using aromatic stacking with porphyrins, it is also possible to tune the electronic structure of the final nanohybrid material, as indicated by FLIM experiments on thin films. Such nanohybrids with highly controlled pores dimensions and morphologies open the way to new design and assembly of storage devices and applications incorporating π‐conjugated molecules and materials and their π‐stacks may be relevant towards selective separation membranes, water purification and biosensing applications.S.I.P. and S.W.B. thank The Royal Society and STFC for funding. B.Y.M. thanks the University of Bath for a studentship (ORS). D.G.C. thanks the Fundación General CSIC for funding (ComFuturo Program). Dr. Jose A. Ribeiro Martins, Professors Jeremy K. M. Sanders and Paul Raithby are acknowledged for training, helpful discussions and porphyrin supramolecular chemistry. The S.I.P. group thanks the EPSRC for funding to the Centre of Graphene Science (EP/K017160/1) and to the Centre for Doctoral Training in Sustainable Chemical Technologies (EP/L016354/1). The authors thank EPSRC National Service for Mass Spectrometry at Swansea and EPSRC National Service for Crystallography at Southampton for data collection. The authors also acknowledge the ERC for the Consolidator Grant O2SENSE (617107, 2014–2019)

Contribution of advanced fluorescence nano microscopy towards revealing mitotic chromosome structure

The organization of chromatin into higher-order structures and its condensation process represent one of the key challenges in structural biology. This is important for elucidating several disease states. To address this long-standing problem, development of advanced imaging methods has played an essential role in providing understanding into mitotic chromosome structure and compaction. Amongst these are two fast evolving fluorescence imaging technologies, specifically fluorescence lifetime imaging (FLIM) and super-resolution microscopy (SRM). FLIM in particular has been lacking in the application of chromosome research while SRM has been successfully applied although not widely. Both these techniques are capable of providing fluorescence imaging with nanometer information. SRM or nanoscopy is capable of generating images of DNA with less than 50 nm resolution while FLIM when coupled with energy transfer may provide less than 20 nm information. Here, we discuss the advantages and limitations of both methods followed by their contribution to mitotic chromosome studies. Furthermore, we highlight the future prospects of how advancements in new technologies can contribute in the field of chromosome science

High-resolution μCT of a mouse embryo using a compact laser-driven X-ray betatron source

High-resolution microcomputed tomography with benchtop X-ray sources requires long scan times because of the heat load limitation on the anode. We present an alternative, high-brightness plasma-based X-ray source that does not suffer from this restriction. A demonstration of tomography of a centimeter-scale complex organism achieves equivalent quality to a commercial scanner. We will soon be able to record such scans in minutes, rather than the hours required by conventional X-ray tubes

Correction:Microwave gallium-68 radiochemistry for kinetically stable bis(thiosemicarbazone) complexes: Structural investigations and cellular uptake under hypoxia (Dalton Transactions (2016) 45 (144-155))

We report the microwave synthesis of several bis(thiosemicarbazones) and the rapid gallium-68 incorporation to give the corresponding metal complexes. These proved kinetically stable under ‘cold’ and ‘hot’ biological assays and were investigated using laser scanning confocal microscopy, flow cytometry and radioactive cell retention studies under normoxia and hypoxia. (68)Ga complex retention was found to be 34% higher in hypoxic cells than in normoxic cells over 30 min, further increasing to 53% at 120 min. Our data suggests that this class of gallium complexes show hypoxia selectivity suitable for imaging in living cells and in vivo tests by microPET in nude athymic mice showed that they are excreted within 1 h of their administration

An Expanded Multi-scale Monte Carlo Simulation Method for Personalized Radiobiological Effect Estimation in Radiotherapy: a feasibility study

A novel and versatile “bottom-up� approach is developed to estimate the radiobiological effect of clinic radiotherapy. The model consists of multi-scale Monte Carlo simulations from organ to cell levels. At cellular level, accumulated damages are computed using a spectrum-based accumulation algorithm and predefined cellular damage database. The damage repair mechanism is modeled by an expanded reaction-rate two-lesion kinetic model, which were calibrated through replicating a radiobiological experiment. Multi-scale modeling is then performed on a lung cancer patient under conventional fractionated irradiation. The cell killing effects of two representative voxels (isocenter and peripheral voxel of the tumor) are computed and compared. At microscopic level, the nucleus dose and damage yields vary among all nucleuses within the voxels. Slightly larger percentage of cDSB yield is observed for the peripheral voxel (55.0%) compared to the isocenter one (52.5%). For isocenter voxel, survival fraction increase monotonically at reduced oxygen environment. Under an extreme anoxic condition (0.001%), survival fraction is calculated to be 80% and the hypoxia reduction factor reaches a maximum value of 2.24. In conclusion, with biological-related variations, the proposed multi-scale approach is more versatile than the existing approaches for evaluating personalized radiobiological effects in radiotherapy

Hydrothermal conversion of one-photon-fluorescent poly-(4-vinylpyridine) into two-photon-fluorescent carbon nanodots

A novel two-photon-fluorescent N,O-heteroatom-rich carbon nanomaterial has been synthesized and characterized. The new carbon nanoparticles were produced by hydrothermal conversion from a one-photon-fluorescent poly(4-vinylpyridine) precursor (P4VP). The carbonized particles (cP4VP dots) with nonuniform particle diameter (ranging from sub-6 to 20 nm with some aggregates up to 200 nm) exhibit strong fluorescence properties in different solvents and have also been investigated for applications in cell culture media. The cP4VP dots retain their intrinsic fluorescence in a cellular environment and exhibit an average excited-state lifetime of 2.0 ± 0.9 ns in the cell. The cP4VP dots enter HeLa cells and do not cause significant damage to outer cell membranes. They provide one-photon or two-photon fluorescent synthetic scaffolds for imaging applications and/or drug delivery

Induction and processing of the radiation-induced gamma-H2AX signal and Its link to the underlying pattern of DSB: A combined experimental and modelling study

We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy) and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular

The architecture and effect of participation: a systematic review of community participation for communicable disease control and elimination. Implications for malaria elimination

Community engagement and participation has played a critical role in successful disease control and elimination campaigns in many countries. Despite this, its benefits for malaria control and elimination are yet to be fully realized. This may be due to a limited understanding of the influences on participation in developing countries as well as inadequate investment in infrastructure and resources to support sustainable community participation. This paper reports the findings of an atypical systematic review of 60 years of literature in order to arrive at a more comprehensive awareness of the constructs of participation for communicable disease control and elimination and provide guidance for the current malaria elimination campaign.Evidence derived from quantitative research was considered both independently and collectively with qualitative research papers and case reports. All papers included in the review were systematically coded using a pre-determined qualitative coding matrix that identified influences on community participation at the individual, household, community and government/civil society levels. Colour coding was also carried out to reflect the key primary health care period in which community participation programmes originated. These processes allowed exhaustive content analysis and synthesis of data in an attempt to realize conceptual development beyond that able to be achieved by individual empirical studies or case reports.Of the 60 papers meeting the selection criteria, only four studies attempted to determine the effect of community participation on disease transmission. Due to inherent differences in their design, interventions and outcome measures, results could not be compared. However, these studies showed statistically significant reductions in disease incidence or prevalence using various forms of community participation. The use of locally selected volunteers provided with adequate training, supervision and resources are common and important elements of the success of the interventions in these studies. In addition, qualitative synthesis of all 60 papers elucidates the complex architecture of community participation for communicable disease control and elimination which is presented herein.The current global malaria elimination campaign calls for a health systems strengthening approach to provide an enabling environment for programmes in developing countries. In order to realize the benefits of this approach it is vital to provide adequate investment in the 'people' component of health systems and understand the multi-level factors that influence their participation. The challenges of strengthening this component of health systems are discussed, as is the importance of ensuring that current global malaria elimination efforts do not derail renewed momentum towards the comprehensive primary health care approach. It is recommended that the application of the results of this systematic review be considered for other diseases of poverty in order to harmonize efforts at building 'competent communities' for communicable disease control and optimising health system effectiveness