Long-lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to PLIM and beyond

Lifetime imaging microscopy with sub-micron resolution provides essential understanding of living systems by allowing both the visualisation of their structure, and the sensing of bio-relevant analytes in vivo using external probes. Chemistry is pivotal for the development of the next generation of bio-tools, where contrast, sensitivity, and molecular specificity facilitate observation of processes fundamental to life. A fundamental limitation at present is the nanosecond lifetime of conventional fluorescent probes which typically confines the sensitivity to sub-nanosecond changes, whilst nanosecond background autofluorescence compromises the contrast. High-resolution visualization with complete background rejection and simultaneous mapping of bio-relevant analytes including oxygen – with sensitivity orders of magnitude higher than that currently attainable – can be achieved using time-resolved emission imaging microscopy (TREM) in conjunction with probes with microsecond (or longer) lifetimes. Yet the microsecond timescale has so far been incompatible with available multiphoton excitation/detection technologies. Here we realize for the first time microsecond-imaging with multiphoton excitation whilst maintaining the essential sub-micron spatial resolution. The new method is background-free and expands available imaging and sensing timescales 1000-fold. Exploiting the first engineered water-soluble member of a family of remarkably emissive platinum-based, microsecond-lived probes amongst others, we demonstrate (i) the first instance of background-free multiphoton-excited microsecond depth imaging of live cells and histological tissues, (ii) over an order-of-magnitude variation in the probe lifetime in vivo in response to the local microenvironment. The concept of two-photon TREM can be seen as “FLIM + PLIM” as it can be used on any timescale, from ultrafast fluorescence of organic molecules to slower emission of transition metal complexes or lanthanides/actinides, and combinations thereof. It brings together transition metal complexes as versatile emissive probes with the new multiphoton-excitation/microsecond-detection approach to create a transformative framework for multiphoton imaging and sensing across biological, medicinal and material sciences

Multimodal probes : superresolution and transmission electron microscopy imaging of mitochondria, and oxygen mapping of cells, using small-molecule Ir(III) luminescent complexes

We describe an Ir(III)-based small-molecule, multimodal probe for use in both light and electron microscopy. The direct correlation of data between light- and electron-microscopy-based imaging to investigate cellular processes at the ultrastructure level is a current challenge, requiring both dyes that must be brightly emissive for luminescence imaging and scatter electrons to give contrast for electron microscopy, at a single working concentration suitable for both methods. Here we describe the use of Ir(III) complexes as probes that provide excellent image contrast and quality for both luminescence and electron microscopy imaging, at the same working concentration. Significant contrast enhancement of cellular mitochondria was observed in transmission electron microscopy imaging, with and without the use of typical contrast agents. The specificity for cellular mitochondria was also confirmed with MitoTracker using confocal and 3D-structured illumination microscopy. These phosphorescent dyes are part of a very exclusive group of transition-metal complexes that enable imaging beyond the diffraction limit. Triplet excited-state phosphorescence was also utilized to probe the O2 concentration at the mitochondria in vitro, using lifetime mapping techniques

Effect of Mesophase Order on the Dynamics of Side Group Liquid Crystalline Polymers

Rheology and X-ray scattering were employed to probe the viscoelastic properties and structural transitions of model cyano-biphenyl-based side-group liquid−crystalline polymers (SGLCPs) with molecular weights ranging from 91 to 1900 kg/mol. Temperature-dependent rheological data show a rapid change in dynamics over a small temperature range. Small-angle X-ray scattering reveals these changes to be associated with an isotropic to smectic transition with an appreciable biphasic region. The presence of a biphasic region is attributed to inhomogeneity in chain structure resulting from incomplete attachment of mesogens to every monomeric unit in the SGLCP polymer. While isotropic and smectic phase data may be separately time−temperature shifted to create master curves for the individual phases, we argue against attempts to achieve superposition between the two phases in the high-frequency regime, since smectic ordering may not simply slow the dynamics but also increase the modulus of the sample. Molecular weight has a strong influence on rheology in the isotropic phase, where an entanglement plateau emerges; however, the smectic-phase rheology is dominated by the layer structure and is fairly insensitive to molecular weight

Genetic basis of thermal nociceptive sensitivity and brain weight in a BALB/c reduced complexity cross

Thermal nociception involves the transmission of temperature-related noxious information from the periphery to the CNS and is a heritable trait that could predict transition to persistent pain. Rodent forward genetics complement human studies by controlling genetic complexity and environmental factors, analysis of end point tissue, and validation of variants on appropriate genetic backgrounds. Reduced complexity crosses between nearly identical inbred substrains with robust trait differences can greatly facilitate unbiased discovery of novel genes and variants. We found BALB/cByJ mice showed enhanced sensitivity on the 53.5°C hot plate and mechanical stimulation in the von Frey test compared to BALB/cJ mice and replicated decreased gross brain weight in BALB/cByJ versus BALB/cJ. We then identified a quantitative trait locus (QTL) on chromosome 13 for hot plate sensitivity (LOD = 10.7; p < 0.001; peak = 56 Mb) and a QTL for brain weight on chromosome 5 (LOD = 8.7; p < 0.001). Expression QTL mapping of brain tissues identified H2afy (56.07 Mb) as the top transcript with the strongest association at the hot plate locus (FDR = 0.0002) and spliceome analysis identified differential exon usage within H2afy associated with the same locus. Whole brain proteomics further supported decreased H2AFY expression could underlie enhanced hot plate sensitivity, and identified ACADS as a candidate for reduced brain weight. To summarize, a BALB/c reduced complexity cross combined with multiple-omics approaches facilitated identification of candidate genes underlying thermal nociception and brain weight. These substrains provide a powerful, reciprocal platform for future validation of candidate variants

Условия формирования и проблемы функционирования крупных диверсифицированных производственно-корпоративных структур в Украине

У статті розглянуто умови формування та функціонування, а також історія розвитку великих диверсифікованих виробничо-корпоративних структур в Україні. Пропонуються підходи оцінки результативності процесу диверсифікації з використанням різних методик. Визначено, що в даний час оцінка результативності процесу диверсифікації можлива лише непрямими математичними методами.В статье рассмотрены условия формирования и функционирования, а также история развития крупных диверсифицированных производственно-корпоративных структур в Украине. Предлагаются подходы оценки результативности процесса диверсификации с использованием разных методик. Определено, что в настоящее время оценка результативности процесса диверсификации возможна лишь косвенными математическими методами.In the article address the formation and functioning of the conditions, as well as story development of large industrial and corporate structures, becoming diversification in Ukraine. Proposes approaches assessing impact of the process of diversification, using of different methods. Proved that the current performance assessment process of diversification can only be indirect mathematical methods

Multiple factors interact to produce responses resembling spectrum of human disease in Campylobacter jejuni infected C57BL/6 IL-10-/- mice

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter jejuni </it>infection produces a spectrum of clinical presentations in humans – including asymptomatic carriage, watery diarrhea, and bloody diarrhea – and has been epidemiologically associated with subsequent autoimmune neuropathies. This microorganism is genetically variable and possesses genetic mechanisms that may contribute to variability in nature. However, relationships between genetic variation in the pathogen and variation in disease manifestation in the host are not understood. We took a comparative experimental approach to explore differences among different <it>C. jejuni </it>strains and studied the effect of diet on disease manifestation in an interleukin-10 deficient mouse model.</p> <p>Results</p> <p>In the comparative study, C57BL/6 interleukin-10^-/-mice were infected with seven genetically distinct <it>C. jejuni </it>strains. Four strains colonized the mice and caused disease; one colonized with no disease; two did not colonize. A DNA:DNA microarray comparison of the strain that colonized mice without disease to <it>C. jejuni </it>11168 that caused disease revealed that putative virulence determinants, including loci encoding surface structures known to be involved in <it>C. jejuni </it>pathogenesis, differed from or were absent in the strain that did not cause disease. In the experimental study, the five colonizing strains were passaged four times in mice. For three strains, serial passage produced increased incidence and degree of pathology and decreased time to develop pathology; disease shifted from watery to bloody diarrhea. Mice kept on an ~6% fat diet or switched from an ~12% fat diet to an ~6% fat diet just before infection with a non-adapted strain also exhibited increased incidence and severity of disease and decreased time to develop disease, although the effects of diet were only statistically significant in one experiment.</p> <p>Conclusion</p> <p><it>C. jejuni </it>strain genetic background and adaptation of the strain to the host by serial passage contribute to differences in disease manifestations of <it>C. jejuni </it>infection in C57BL/6 IL-10^-/-mice; differences in environmental factors such as diet may also affect disease manifestation. These results in mice reflect the spectrum of clinical presentations of <it>C. jejuni </it>gastroenteritis in humans and contribute to usefulness of the model in studying human disease.</p

An intranasal ASO therapeutic targeting SARS-CoV-2

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5′ leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 “variants of concern” tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics

Association of cannabis, cannabidiol and synthetic cannabinoid use with mental health in UK adolescents

No abstract available

Long-term responders on olaparib maintenance in high-grade serous ovarian cancer: Clinical and molecular characterization

Purpose: Maintenance therapy with olaparib has improved progression-free survival in women with high-grade serous ovarian cancer (HGSOC), particularly those harboring BRCA1/2 mutations. The objective of this study was to characterize long-term (LT) versus short-term (ST) responders to olaparib. Experimental Design: A comparative molecular analysis of Study 19 (NCT00753545), a randomized phase II trial assessing olaparib maintenance after response to platinum-based chemotherapy in HGSOC, was conducted. LT response was defined as response to olaparib/placebo > 2 years, ST as < 3 months. Molecular analyses included germline BRCA1/2 status, three-biomarker homologous recombination deficiency (HRD) score, BRCA1 methylation, and mutational profiling. Another olaparib maintenance study (Study 41; NCT01081951) was used as an additional cohort. Results: Thirty-seven LT (32 olaparib) and 61 ST (21 olaparib) patients were identified. Treatment was significantly associated with outcome (P < 0.0001), with more LT patients on olaparib (60.4%) than placebo (11.1%). LT sensitivity to olaparib correlated with complete response to chemotherapy (P < 0.05). In the olaparib LT group, 244 genetic alterations were detected, with TP53, BRCA1, and BRCA2 mutations being most common (90%, 25%, and 35%, respectively). BRCA2 mutations were enriched among the LT responders. BRCA methylation was not associated with response duration. High myriad HRD score (>42) and/or BRCA1/2 mutation was associated with LT response to olaparib. Study 41 confirmed the correlation of LT response with olaparib and BRCA1/2 mutation. Conclusions: Findings show that LT response to olaparib may be multifactorial and related to homologous recombination repair deficiency, particularly BRCA1/2 defects. The type of BRCA1/2 mutation warrants further investigation. (C) 2017 AACR