The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes

Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore’s fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies

Improvement of flexible rotor/active magnetic bearings system performance using pi-d control

Proportional–integral–derivative (PID) control is the most common control approach used to control active magnetic bearings system, especially in the case of supporting rigid rotors. In the case of flexible rotor support, the most common control is again PID control in combination with notch filters. Other control approaches, known as modern control theory, are still in development process and cannot be commonly found in real life industrial application. Right now, they are mostly used in research applications. In comparison to PID control, PI-D control implies that derivate element is in feedback loop instead in main branch of the system. In this paper, performances of flexible rotor/active magnetic bearing system were investigated in the case of PID and PI-D control, both in combination with notch filters. The performances of the system were analysed using an analysis in time domain by observing system response to step input and in frequency domain by observing a frequency response of sensitivity function

Direct imaging of the recruitment and phosphorylation of S6K1 in the mTORC1 pathway in living cells

Knowledge of protein signalling pathways in the working cell is seen as a primary route to identifying and developing targeted medicines. In recent years there has been a growing awareness of the importance of the mTOR pathway, making it an attractive target for therapeutic intervention in several diseases. Within this pathway we have focused on S6 kinase 1 (S6K1), the downstream phosphorylation substrate of mTORC1, and specifically identify its juxtaposition with mTORC1. When S6K1 is co-expressed with raptor we show that S6K1 is translocated from the nucleus to the cytoplasm. By developing a novel biosensor we demonstrate in real-time, that phosphorylation and de-phosphorylation of S6K1 occurs mainly in the cytoplasm of living cells. Furthermore, we show that the scaffold protein raptor, that typically recruits mTOR substrates, is not always involved in S6K1 phosphorylation. Overall, we demonstrate how FRET-FLIM imaging technology can be used to show localisation of S6K1 phosphorylation in living cells and hence a key site of action of inhibitors targeting mTOR phosphorylation

Reticulomics: Protein-protein interaction studies with two plasmodesmata-localised reticulon family proteins identify binding partners enriched at plasmodesmata, ER and the plasma membrane

The ER is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three way junctions, and back to tubules. Plant reticulon (RTNLB) proteins tubulate the ER by dimer- and oligomerization, creating localised ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmal (PD) proteome (Fernandez-Calvino et al., 2011) and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD (Knox et al., 2015). Here we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis (Knox et al., 2015). Co-immunoprecipitation of GFP-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localised proteins as well as plasma-membrane proteins with putative membrane anchoring roles. FRET-FLIM assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modelling, may play important roles in linking the cortical ER to the plasma membrane

Time-resolved fluorescence imaging reveals differential interactions of N-glycan processing enzymes across the Golgi stack in planta

N-glycan processing is one of the most important cellular protein modifications in plants and as such is essential for plant development and defense mechanisms. The accuracy of Golgi-located processing steps is governed by the strict intra-Golgi localization of sequentially acting glycosidases and glycosyltransferases. Their differential distribution goes hand in hand with the compartmentalization of the Golgi stack into cis-, medial and trans-cisternae, which separate early from late processing steps. The mechanisms that direct differential enzyme concentration are still unknown, but formation of multi-enzyme complexes is considered a feasible Golgi protein localization strategy. In this study we used two-photon (2P)-excitation Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) to determine the interaction of N-glycan processing enzymes with differential intra-Golgi locations. Following the coexpression of fluorescent protein-tagged N-terminal Golgi targeting sequences (cytoplasmic-transmembrane-stem region, designated CTS) of enzyme pairs in leaves of tobacco (Nicotiana tabacum or Nicotiana benthamiana), we observed that all tested cis- and medial-Golgi enzymes, namely MNS1, GnTI, GMII and XylT, form homo- and heterodimers, whereas among the late-acting enzymes GALT1, FUT13 and ST (a non-plant Golgi marker) only GALT1 and GMII were found to form a heterodimer. Furthermore, the efficiency of energy transfer indicating the formation of interactions decreased considerably in a cis-to-trans fashion. The comparative 2P-FRET-FLIM analysis of several full-length cis- and medial-Golgi enzymes and their respective catalytic domain-deleted CTS clones further suggested that the formation of protein-protein interactions can occur through their N-terminal CTS region

Fluorescence Lifetime Imaging of Optically Levitated Aerosol: a Technique to Quantitatively Map the Viscosity of Suspended Aerosol Particles

We describe a technique to measure the viscosity of stably levitated single micron-sized aerosol particles. Particle levitation allows the aerosol phase to be probed in the absence of potentially artefact-causing surfaces. To achieve this feat, we combined two laser based techniques: optical trapping for aerosol particle levitation, using a counter-propagating laser beam configuration, and fluorescent lifetime imaging microscopy (FLIM) of molecular rotors for the measurement of viscosity within the particle. Unlike other techniques used to measure aerosol particle viscosity, this allows for the non-destructive probing of viscosity of aerosol particles without interference from surfaces. The well-described viscosity of sucrose aerosol, under a range of relative humidity conditions, is used to validate the technique. Furthermore we investigate a pharmaceutically-relevant mixture of sodium chloride and salbutamol sulphate under humidities representative of in vivo drug inhalation. Finally, we provide a methodology for incorporating molecular rotors into already levitated particles, thereby making the FLIM/optical trapping technique applicable to real world aerosol systems, such as atmospheric aerosols and those generated by pharmaceutical inhalers

The need to address fragmentation and silos in mortality information systems: the case of Ghana and Peru

Objectives: We aimed to understand the information architecture and degree of integration of mortality surveillance systems in Ghana and Peru. Methods: We conducted a cross-sectional study using a combination of document review and unstructured interviews to describe and analyse the sub-systems collecting mortality data. Results: We identified 18 and 16 information subsystems with independent databases capturing death events in Peru and Ghana respectively. The mortality information architecture was highly fragmented with a multiplicity of unconnected data silos and with formal and informal data collection systems. Conclusion: Reliable and timely information about who dies where and from what underlying cause is essential to reporting progress on Sustainable Development Goals, ensuring policies are responding to population health dynamics, and understanding the impact of threats and events like the COVID-19 pandemic. Integrating systems hosted in different parts of government remains a challenge for countries and limits the ability of statistics systems to produce accurate and timely information. Our study exposes multiple opportunities to improve the design of mortality surveillance systems by integrating existing subsystems currently operating in silos

A C-terminal amphipathic helix is necessary for the in vivo tubule-shaping function of a plant reticulon.

Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function

Influence of Topology and Brønsted Acid Site Presence on Methanol Diffusion in Zeolites Beta and MFI

Detailed insight into molecular diffusion in zeolite frameworks is crucial for the analysis of the factors governing their catalytic performance in methanol-to-hydrocarbons (MTH) reactions. In this work, we present a molecular dynamics study of the diffusion of methanol in all-silica and acidic zeolite MFI and Beta frameworks over the range of temperatures 373–473 K. Owing to the difference in pore dimensions, methanol diffusion is more hindered in H-MFI, with diffusion coefficients that do not exceed 10×10−10 m2s−1. In comparison, H-Beta shows diffusivities that are one to two orders of magnitude larger. Consequently, the activation energy of translational diffusion can reach 16 kJ·mol−1 in H-MFI, depending on the molecular loading, against a value for H-Beta that remains between 6 and 8 kJ·mol−1. The analysis of the radial distribution functions and the residence time at the Brønsted acid sites shows a greater probability for methylation of the framework in the MFI structure compared to zeolite Beta, with the latter displaying a higher prevalence for methanol clustering. These results contribute to the understanding of the differences in catalytic performance of zeolites with varying micropore dimensions in MTH reactions

The antibacterial activity of a photoactivatable diarylacetylene against Gram-positive bacteria

The emergence of antibiotic resistance is a growing threat to human health, and therefore, alternatives to existing compounds are urgently needed. In this context, a novel fluorescent photoactivatable diarylacetylene has been identified and characterised for its antibacterial activity, which preferentially eliminates Gram-positive over Gram-negative bacteria. Experiments confirmed that the Gram-negative lipopolysaccharide-rich outer surface is responsible for tolerance, as strains with reduced outer membrane integrity showed increased susceptibility. Additionally, bacteria deficient in oxidative damage repair pathways also displayed enhanced sensitivity, confirming that reactive oxygen species production is the mechanism of antibacterial activity. This new diarylacetylene shows promise as an antibacterial agent against Gram-positive bacteria that can be activated in situ, potentially for the treatment of skin infections