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AC susceptibility measurements of [FeIII(L5)(NCSe)] reveal a field supported slow magnetic relaxation. On cooling, the relaxation time of the high-frequency fraction decreases which is a sign of reciprocating thermal behaviour. The relaxation time for the low-frequency mode at T = 2.0 K is as high as τ(LF) = 2.0 s.
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Two-dimensional (2D) wide bandgap materials are gaining significant interest for next-generation optoelectronic devices. However, fabricating electronic-grade 2D nanosheets from non-van der Waals (n-vdW) oxide semiconductors poses a great challenge due to their stronger interlayer coupling compared with vdW crystals. This strong coupling typically introduces defects during exfoliation, impairing the optoelectronic properties. Herein, we report the liquid-phase exfoliation of few-atomic-layer thin, defect-free, free-standing ZnO nanosheets. These micron-sized, ultrathin ZnO structures exhibit three different orientations aligned along both the polar c-plane as well as the nonpolar a- and m-planes. The superior crystalline quality of the ZnO nanosheets is validated through comprehensive characterization techniques. This result is supported by density functional theory (DFT) calculations, which reveals that the formation of oxygen vacancies is energetically less favorable in 2D ZnO and that the c-plane loses its polarity upon exfoliation. Unlike bulk ZnO, which is typically dominated by defect-induced emission, the exfoliated nanosheets exhibit a strong, ambient-stable excitonic UV emission. We further demonstrate the utility of solution processing of ZnO nanosheets by their hybrid integration with organic components to produce stable light emitting diodes (LEDs) for display applications.
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PURPOSE: Eye plaque brachytherapy (EPBT) is the most common treatment for uveal melanoma with high local control rates of 95-100%. When local recurrences occur following EPBT, salvage options include enucleation, transpupillary thermotherapy (TTT), external beam radiation, or re-irradiation with EPBT. The purpose of this study is to report our institution's experience with EPBT re-irradiation for locally recurrent uveal melanoma. METHODS AND MATERIALS: Patients were included if they were previously treated for uveal melanoma with EPBT, experienced local recurrence, and were subsequently treated at our institution with EPBT from 2016- 2020. RESULTS: A total of 5 patients with median age 68 years were included. All patients were initially treated at an outside institution (OSI) with Iodine-125 or Ruthenium-106 EPBT. Mean time between EPBT at the OSI and EPBT at our facility was 130 months (range 28-231 months). Patients were re-irradiated with Iodine-125 EPBT prescribed to 85 Gy over 168 hours. Median follow up after re-treatment at our center was 24 months. Local control among this cohort was 100%. Metastasis occurred in two patients after re-treatment, at 8 months and 7 months. At last follow up, all treated lesions were decreased in size. Four patients experienced worsening visual acuity. Four patients developed cataracts, while two patients developed radiation retinopathy with cystoid macular edema requiring anti-VEGF injections. One patient developed radiation retinopathy but did not require injections. No patients required enucleation. CONCLUSIONS: Re-treatment of locally recurrent uveal melanomas with EPBT is a feasible alternative to enucleation with a high local control rate. Ocular toxicities have not been significant enough to require enucleation.
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Harmful algal bloom (HAB) formation leads to the eutrophication of water ecosystems and may render recreational lakes unsuitable for human use. We evaluated the applicability and comparison of metabarcoding, metagenomics, qPCR, and ELISA-based methods for cyanobacteria/cyanotoxin detection in bloom and non-bloom sites for the Great Lakes region. DNA sequencing-based methods robustly identified differences between bloom and non-bloom samples (e.g., the relative prominence of Anabaena and Planktothrix). Shotgun sequencing strategies also identified the enrichment of metabolic genes typical of cyanobacteria in bloom samples, though toxin genes were not detected, suggesting deeper sequencing or PCR methods may be needed to detect low-abundance toxin genes. PCR and ELISA indicated microcystin levels and microcystin gene copies were significantly more abundant in bloom sites. However, not all bloom samples were positive for microcystin, possibly due to bloom development by non-toxin-producing species. Additionally, microcystin levels were significantly correlated (positively) with microcystin gene copy number but not with total cyanobacterial 16S gene copies. In summary, next-generation sequencing-based methods can identify specific taxonomic and functional targets, which can be used for absolute quantification methods (qPCR and ELISA) to augment conventional water monitoring strategies.
Subject(s)
Cyanobacteria , Environmental Monitoring , Harmful Algal Bloom , Lakes , Microcystins , Microcystins/genetics , Microcystins/analysis , Lakes/microbiology , Cyanobacteria/genetics , Cyanobacteria/growth & development , Cyanobacteria/classification , Environmental Monitoring/methods , Ecosystem , Metagenomics , Recreation , Water Microbiology , Enzyme-Linked Immunosorbent AssayABSTRACT
Coordination compounds, like iron(II) triazole complexes, exhibit spin crossover (SCO) behavior at around room temperature. Therefore, they are interesting for a variety of possible applications, and it is convenient to integrate them into polymers. Due to a reduction of the iron content and thus also 57Fe content in the sample through integration in polymers, Mössbauer measurements are only possible with greater difficulty or very long measurement times without expensive enrichment of the samples with 57Fe. So, other ways of improving the Mössbauer signal for these composite materials are necessary. Therefore, we pressed these composite materials to improve the Mössbauer spectra. In this study, we synthesized an iron(II) triazole spin crossover complex and an electrospun polymer complex composite nanofiber material including the same complex. For both products, Mössbauer measurements were performed at room temperature before and after using a press to show that the complex composite is not harmed through pressing. We investigate the influence of the pressing impact on the Mössbauer measurements in the context of measurement statistics and the measured signals. We show that pressing is not connected to any changes in the sample regarding the spin and oxidation state. We present that pressing improves the statistics of the Mössbauer measurements significantly. Furthermore, we use SEM measurements and PXRD to investigate whether or not the obtained fiber mats are destroyed in the pressing process.
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INTRODUCTION: Tropomyosin related kinase B (TrkB) and C (TrkC) receptor signaling promotes synaptic plasticity and interacts with pathways affected by amyloid beta (Aß) toxicity. Upregulating TrkB/C signaling could reduce Alzheimer's disease (AD)-related degenerative signaling, memory loss, and synaptic dysfunction. METHODS: PTX-BD10-2 (BD10-2), a small molecule TrkB/C receptor partial agonist, was orally administered to aged London/Swedish-APP mutant mice (APPL/S) and wild-type controls. Effects on memory and hippocampal long-term potentiation (LTP) were assessed using electrophysiology, behavioral studies, immunoblotting, immunofluorescence staining, and RNA sequencing. RESULTS: In APPL/S mice, BD10-2 treatment improved memory and LTP deficits. This was accompanied by normalized phosphorylation of protein kinase B (Akt), calcium-calmodulin-dependent kinase II (CaMKII), and AMPA-type glutamate receptors containing the subunit GluA1; enhanced activity-dependent recruitment of synaptic proteins; and increased excitatory synapse number. BD10-2 also had potentially favorable effects on LTP-dependent complement pathway and synaptic gene transcription. DISCUSSION: BD10-2 prevented APPL/S/Aß-associated memory and LTP deficits, reduced abnormalities in synapse-related signaling and activity-dependent transcription of synaptic genes, and bolstered transcriptional changes associated with microglial immune response. HIGHLIGHTS: Small molecule modulation of tropomyosin related kinase B (TrkB) and C (TrkC) restores long-term potentiation (LTP) and behavior in an Alzheimer's disease (AD) model. Modulation of TrkB and TrkC regulates synaptic activity-dependent transcription. TrkB and TrkC receptors are candidate targets for translational therapeutics. Electrophysiology combined with transcriptomics elucidates synaptic restoration. LTP identifies neuron and microglia AD-relevant human-mouse co-expression modules.
Subject(s)
Alzheimer Disease , Microglia , Receptor, trkB , Synapses , Animals , Male , Mice , Alzheimer Disease/drug therapy , Disease Models, Animal , Hippocampus/drug effects , Hippocampus/metabolism , Long-Term Potentiation/drug effects , Mice, Transgenic , Microglia/drug effects , Microglia/metabolism , Neuronal Plasticity/drug effects , Receptor, trkB/metabolism , Receptor, trkC/metabolism , Receptor, trkC/genetics , Synapses/drug effects , Transcriptome/drug effectsABSTRACT
BACKGROUND: Lung cancer, the second most common cancer, presents persistently dismal prognoses. Radiomics, a promising field, aims to provide novel imaging biomarkers to improve outcomes. However, clinical translation faces reproducibility challenges, despite efforts to address them with quality scoring tools. OBJECTIVE: This study had two objectives: 1) identify radiomics biomarkers in post-radiotherapy stage III/IV nonsmall cell lung cancer (NSCLC) patients, 2) evaluate research quality using the CLEAR (CheckList_for_EvaluAtion_of_Radiomics_research), RQS (Radiomics_Quality_Score) frameworks, and formulate an amalgamated CLEAR-RQS tool to enhance scientific rigor. MATERIALS AND METHODS: A systematic literature review (Jun-Aug 2023, MEDLINE/PubMed/SCOPUS) was conducted concerning stage III/IV NSCLC, radiotherapy, and radiomic features (RF). Extracted data included study design particulars, such as sample size, radiotherapy/CT technique, selected RFs, and endpoints. CLEAR and RQS were merged into a CLEAR-RQS checklist. Three readers appraised articles utilizing CLEAR, RQS, and CLEAR-RQS metrics. RESULTS: Out of 871 articles, 11 met the inclusion/exclusion criteria. The Median cohort size was 91 (range: 10-337) with 9 studies being single-center. No common RF were identified. The merged CLEAR-RQS checklist comprised 61 items. Most unreported items were within CLEAR's "methods" and "open-source," and within RQS's "phantom-calibration," "registry-enrolled prospective-trial-design," and "cost-effective-analysis" sections. No study scored above 50% on RQS. Median CLEAR scores were 55.74% (32.33/58 points), and for RQS, 17.59% (6.3/36 points). CLEAR-RQS article ranking fell between CLEAR and RQS and aligned with CLEAR. CONCLUSION: Radiomics research in post-radiotherapy stage III/IV NSCLC exhibits variability and frequently low-quality reporting. The formulated CLEAR-RQS checklist may facilitate education and holds promise for enhancing radiomics research quality. CLINICAL RELEVANCE STATEMENT: Current radiomics research in the field of stage III/IV postradiotherapy NSCLC is heterogenous, lacking reproducibility, with no identified imaging biomarker. Radiomics research quality assessment tools may enhance scientific rigor and thereby facilitate radiomics translation into clinical practice. KEY POINTS: There is heterogenous and low radiomics research quality in postradiotherapy stage III/IV nonsmall cell lung cancer. Barriers to reproducibility are small cohort size, nonvalidated studies, missing technical parameters, and lack of data, code, and model sharing. CLEAR (CheckList_for_EvaluAtion_of_Radiomics_research), RQS (Radiomics_Quality_Score), and the amalgamated CLEAR-RQS tool are useful frameworks for assessing radiomics research quality and may provide a valuable resource for educational purposes in the field of radiomics.
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Escherichia coli serves as a proxy indicator of fecal contamination in aquatic ecosystems. However, its identification using traditional culturing methods can take up to 24 h. The application of DNA markers, such as conserved signature proteins (CSPs) genes (unique to all species/strains of a specific taxon), can form the foundation for novel polymerase chain reaction (PCR) tests that unambiguously identify and detect targeted bacterial taxa of interest. This paper reports the identification of three new highly-conserved CSPs (genes), namely YahL, YdjO, and YjfZ, which are exclusive to E. coli/Shigella. Using PCR primers based on highly conserved regions within these CSPs, we have developed quantitative PCR (qPCR) assays for the evaluation of E. coli/Shigella species in water ecosystems. Both in-silico and experimental PCR testing confirmed the absence of sequence match when tested against other bacteria, thereby confirming 100% specificity of the tested CSPs for E. coli/Shigella. The qPCR assays for each of the three CSPs provided reliable quantification for all tested enterohaemorrhagic and environmental E. coli strains, a requirement for water testing. For recreational water samples, CSP-based quantification showed a high correlation (r > 7, p < 0.01) with conventional viable E. coli enumeration. This indicates that novel CSP-based qPCR assays for E. coli can serve as robust tools for monitoring water ecosystems and other critical areas, including food monitoring.
Subject(s)
Escherichia coli , Water Microbiology , Water Quality , Escherichia coli/genetics , Escherichia coli/classification , Escherichia coli Proteins/genetics , Real-Time Polymerase Chain Reaction/methods , Shigella/genetics , Shigella/classification , Shigella/isolation & purification , Conserved Sequence , Environmental Monitoring/methods , Polymerase Chain Reaction/methods , Feces/microbiologyABSTRACT
It has long been recognized that the environment experienced by parents can influence the traits of offspring (i.e. 'parental effects'). Much research has explored whether mothers respond to predictable shifts in environmental signals by modifying offspring phenotypes to best match future conditions. Many organisms experience conditions that theory predicts should favor the evolution of such 'anticipatory parental effects', but such predictions have received limited empirical support. 'Condition transfer effects' are an alternative to anticipatory effects that occur when the environment experienced by parents during development influences offspring fitness. Condition transfer effects occur when parents that experience high-quality conditions produce offspring that exhibit higher fitness irrespective of the environmental conditions in the offspring generation. Condition transfer effects are not driven by external signals but are instead a byproduct of past environmental quality. They are also likely adaptive but have received far less attention than anticipatory effects. Here, we review the generality of condition transfer effects and show that they are much more widespread than is currently appreciated. Condition transfer effects are observed across taxa and are commonly associated with experimental manipulations of resource conditions experienced by parents. Our Review calls for increased research into condition transfer effects when considering the role of parental effects in ecology and evolution.
Subject(s)
Ecology , Exercise , Female , Humans , Mothers , PhenotypeABSTRACT
With the advent of the first laser sources and suitable detectors, optical sensor applications immediately also came into focus. During the last decades, a huge variety of optical sensor concepts were developed, yet the forecast for the future application potential appears even larger. In this context, the development of new sensor probes at different scales down to the atomic or molecular level open new avenues for research and development. We investigated an iron based triazole molecular spin-crossover complex changing its absorption characteristics significantly by varying environmental parameters such as humidity, temperature, magnetic or electric field, respectively, with respect to its suitability for a new class of versatile molecular sensor probes. Hereby, besides the investigation of synthesized pure bulk material using different analyzing methods, we also studied amorphous micro particles which were applied in or onto optical waveguide structures. We found that significant changes of the reflection spectra can also be obtained after combining the particles with different types of optical waveguides.The obtained results demonstrate the suitability of the material complex for a broad field of future sensor applications.
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The morpholine heterocycle is a structural unit found in many bioactive compounds and FDA-approved drugs, but the generation of more complex C-functionalized morpholine derivatives remains considerably underexplored. Using systematic chemical diversity (SCD), a concept that guides the expansion of saturated drug-like scaffolds through regiochemical and stereochemical variation, we describe the synthesis of a collection of methyl-substituted morpholine acetic acid esters starting from enantiomerically pure amino acids and amino alcohols. In total, 24 diverse substituted morpholines were produced that vary systematically in regiochemistry and stereochemistry (relative and absolute). These diverse C-substituted morpholines can be directly applied in fragment screening or incorporated as building blocks in medicinal chemistry and library synthesis.
Subject(s)
Morpholines , Morpholines/chemistry , Molecular Structure , Stereoisomerism , Esters/chemistry , Amino Acids/chemistry , Amino Acids/chemical synthesis , Chemistry, PharmaceuticalABSTRACT
BACKGROUND: Fecal bacterial densities are proxy indicators of beach water quality, and beach posting decisions are made based on Beach Action Value (BAV) exceedances for a beach. However, these traditional beach monitoring methods do not reflect the full extent of microbial water quality changes associated with BAV exceedances at recreational beaches (including harmful cyanobacteria). This proof of concept study evaluates the potential of metagenomics for comprehensively assessing bacterial community changes associated with BAV exceedances compared to non-exceedances for two urban beaches and their adjacent river water sources. RESULTS: Compared to non-exceedance samples, BAV exceedance samples exhibited higher alpha diversity (diversity within the sample) that could be further differentiated into separate clusters (Beta-diversity). For Beach A, Cyanobacterial sequences (resolved as Microcystis and Pseudanabaena at genus level) were significantly more abundant in BAV non-exceedance samples. qPCR validation supported the Cyanobacterial abundance results from metagenomic analysis and also identified saxitoxin genes in 50% of the non-exceedance samples. Microcystis sp and saxitoxin gene sequences were more abundant on non-exceedance beach days (when fecal indicator data indicated the beach should be open for water recreational purposes). For BAV exceedance days, Fibrobacteres, Pseudomonas, Acinetobacter, and Clostridium sequences were significantly more abundant (and positively correlated with fecal indicator densities) for Beach A. For Beach B, Spirochaetes (resolved as Leptospira on genus level) Burkholderia and Vibrio sequences were significantly more abundant in BAV exceedance samples. Similar bacterial diversity and abundance trends were observed for river water sources compared to their associated beaches. Antibiotic Resistance Genes (ARGs) were also consistently detected at both beaches. However, we did not observe a significant difference or correlation in ARGs abundance between BAV exceedance and non-exceedance samples. CONCLUSION: This study provides a more comprehensive analysis of bacterial community changes associated with BAV exceedances for recreational freshwater beaches. While there were increases in bacterial diversity and some taxa of potential human health concern associated with increased fecal indicator densities and BAV exceedances (e.g. Pseudomonas), metagenomics analyses also identified other taxa of potential human health concern (e.g. Microcystis) associated with lower fecal indicator densities and BAV non-exceedances days. This study can help develop more targeted beach monitoring strategies and beach-specific risk management approaches.
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PURPOSE: To associate clinical factors and radiation doses delivered by iodine-125 plaque brachytherapy to visual outcomes and development of radiation-induced ocular complications in patients with uveal melanoma in the era of anti-vascular endothelial growth factor (anti-VEGF) injections. DESIGN: Retrospective cohort study. METHODS: A retrospective chart review was performed for 225 patients treated with iodine-125 brachytherapy for uveal melanoma. The effects of radiation doses (focal doses, average dose to the entire eye, and integral dose) on visual outcomes and development of radiation complications (radiation retinopathy, radiation optic neuropathy, vitreous hemorrhage, and neovascular glaucoma) were analyzed using multivariate Cox regression snalysis. RESULTS: Median follow-up was 33.6 months (range, 12-105.6 months). Radiation retinopathy was associated with younger age, tumor distance to optic nerve <6 mm, and maximum radiation dose to fovea. Radiation optic neuropathy was associated with White race, tumor distance to optic nerve <6 mm, and integral radiation dose. Vitreous hemorrhage was associated with White race and integral radiation dose. Incidence of neovascular glaucoma was low in our study, with 2 patients (0.9%) developing the complication. Of the 123 patients who developed radiation retinopathy, 82 patients (66.7% of radiation retinopathy patients, 37.3% of total patients) received anti-VEGF injections. CONCLUSIONS: Our study found multiple associations between radiation doses and complications as well as visual outcomes on multivariate analysis. Given that the majority of our patients who developed radiation retinopathy received anti-VEGF injections, our study helps to illustrate the course and progression of radiation-induced complications in the new era of anti-VEGF.
Subject(s)
Brachytherapy , Eye Injuries , Glaucoma, Neovascular , Iodine Radioisotopes , Melanoma , Optic Nerve Diseases , Retinal Diseases , Uveal Neoplasms , Humans , Brachytherapy/adverse effects , Retrospective Studies , Vitreous Hemorrhage , Glaucoma, Neovascular/drug therapy , Glaucoma, Neovascular/etiology , Retinal Diseases/etiology , Uveal Neoplasms/radiotherapy , Optic Nerve Diseases/etiology , Eye Injuries/etiologyABSTRACT
Brown adipose tissue (BAT) dissipates energy as heat, contributing to temperature control, energy expenditure, and systemic homeostasis. In adult humans, BAT mainly exists in supraclavicular areas and its prevalence is associated with cardiometabolic health. However, the developmental origin of supraclavicular BAT remains unknown. Here, using genetic cell marking in mice, we demonstrate that supraclavicular brown adipocytes do not develop from the Pax3+/Myf5+ epaxial dermomyotome that gives rise to interscapular BAT (iBAT). Instead, the Tbx1+ lineage that specifies the pharyngeal mesoderm marks the majority of supraclavicular brown adipocytes. Tbx1Cre-mediated ablation of peroxisome proliferator-activated receptor gamma (PPARγ) or PR/SET Domain 16 (PRDM16), components of the transcriptional complex for brown fat determination, leads to supraclavicular BAT paucity or dysfunction, thus rendering mice more sensitive to cold exposure. Moreover, human deep neck BAT expresses higher levels of the TBX1 gene than subcutaneous neck white adipocytes. Taken together, our observations reveal location-specific developmental origins of BAT depots and call attention to Tbx1+ lineage cells when investigating human relevant supraclavicular BAT.
Subject(s)
Adipocytes, Brown , Adipose Tissue, White , Adult , Humans , Mice , Animals , Transcription Factors , Adipose Tissue, Brown/physiology , Adipocytes, White , T-Box Domain Proteins/geneticsABSTRACT
BACKGROUND: In several eukaryotes, DNA methylation occurs within the coding regions of many genes, termed gene body methylation (GbM). Whereas the role of DNA methylation on the silencing of transposons and repetitive DNA is well understood, gene body methylation is not associated with transcriptional repression, and its biological importance remains unclear. RESULTS: We report a newly discovered type of GbM in plants, which is under constitutive addition and removal by dynamic methylation modifiers in all cells, including the germline. Methylation at Dynamic GbM genes is removed by the DRDD demethylation pathway and added by an unknown source of de novo methylation, most likely the maintenance methyltransferase MET1. We show that the Dynamic GbM state is present at homologous genes across divergent lineages spanning over 100 million years, indicating evolutionary conservation. We demonstrate that Dynamic GbM is tightly associated with the presence of a promoter or regulatory chromatin state within the gene body, in contrast to other gene body methylated genes. We find Dynamic GbM is associated with enhanced gene expression plasticity across development and diverse physiological conditions, whereas stably methylated GbM genes exhibit reduced plasticity. Dynamic GbM genes exhibit reduced dynamic range in drdd mutants, indicating a causal link between DNA demethylation and enhanced gene expression plasticity. CONCLUSIONS: We propose a new model for GbM in regulating gene expression plasticity, including a novel type of GbM in which increased gene expression plasticity is associated with the activity of DNA methylation writers and erasers and the enrichment of a regulatory chromatin state.
Subject(s)
DNA Methylation , Plants , Plants/genetics , Biological Evolution , Gene Expression , ChromatinABSTRACT
Introduction: TrkB and TrkC receptor signaling promotes synaptic plasticity and interacts with pathways affected by amyloid-ß (Aß)-toxicity. Upregulating TrkB/C signaling could reduce Alzheimer's disease (AD)-related degenerative signaling, memory loss, and synaptic dysfunction. Methods: PTX-BD10-2 (BD10-2), a small molecule TrkB/C receptor partial agonist, was orally administered to aged London/Swedish-APP mutant mice (APP L/S ) and wild-type controls (WT). Effects on memory and hippocampal long-term potentiation (LTP) were assessed using electrophysiology, behavioral studies, immunoblotting, immunofluorescence staining, and RNA-sequencing. Results: Memory and LTP deficits in APP L/S mice were attenuated by treatment with BD10-2. BD10-2 prevented aberrant AKT, CaMKII, and GLUA1 phosphorylation, and enhanced activity-dependent recruitment of synaptic proteins. BD10-2 also had potentially favorable effects on LTP-dependent complement pathway and synaptic gene transcription. Conclusions: BD10-2 prevented APP L/S /Aß-associated memory and LTP deficits, reduced abnormalities in synapse-related signaling and activity-dependent transcription of synaptic genes, and bolstered transcriptional changes associated with microglial immune response.
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OBJECTIVES: To investigate the pre-analytics of the molecular testing of cytology specimens, we studied the effects of time in refrigerator storage (4°C) of malignant effusions on RNA sequencing (RNAseq) results. METHODS: Ten effusion specimens were stored in a refrigerator (4°C) for different durations (day 0, 1, 4, and 7). All specimens were prepared as cytospins fixed in either Carnoy's solution or 95% ethanol (EtOH) and in an RNA preservative for a fresh frozen (FF) high-quality reference. Whole transcriptome (wt) and targeted (t)RNAseq of two multigene expression signatures were performed. We then compared transcript expression levels (including mutant allele fraction) according to pre-analytical variables using a concordance correlation coefficient (CCC) and a mixed effect model. RESULTS: Sequencing results were mostly stable over increasing time in storage. Cytospins fixed in Carnoy's solution were more concordant with FF samples than cytospins fixed in 95% EtOH at all timepoints. This finding was consistent for both wtRNAseq (averages: day 0 CCC = 0.98 vs 0.91; day 7 CCC = 0.88 vs 0.78) and tRNAseq methods (averages: day 0 CCC = 0.98 vs 0.81; day 7 CCC = 0.98 vs 0.90). Cytospins fixed in Carnoy's solution did not show significant changes in expression over timepoints or between expression signatures, whereas 95% EtOH did. CONCLUSION: RNAseq can be accurately performed on effusion specimens after prolonged refrigerator storage. RNA extracted from scraped cytospin slides fixed in Carnoy's solution was marginally superior to 95% EtOH fixation, but either method had comparable analytic performance to high-quality FF RNA samples.
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Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. SIGNIFICANCE: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109.
Subject(s)
Interferons , Methyltransferases , Animals , Mice , Interferons/genetics , Methyltransferases/genetics , Methyltransferases/metabolism , RNA, Double-StrandedABSTRACT
We report a heterocyclic merging approach to construct novel indazolo-piperazines and indazolo-morpholines. Starting from chiral diamines and amino alcohols, novel regiochemically (1,3 and 1,4) and stereochemically diverse (relative and absolute) cohorts of indazolo-piperazines and indazolo-morpholines were obtained within six or seven steps. The key transformations involved are a Smiles rearrangement to generate the indazole core structure and a late-stage Michael addition to build the piperazine and morpholine heterocycles. We further explored additional vector diversity by incorporating substitutions on the indazole aromatic ring, generating a total of 20 unique, enantiomerically pure heterocyclic scaffolds.
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Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.