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Abstract A metabotropic glutamate receptor coupled to phospholipase D (PLD‐mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch‐evoked firing. We report here the first identification of this PLD‐mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far‐western blotting indicates PLD‐mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD‐mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD‐mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.

Abstract Background Diffuse large B-cell lymphoma (DLBCL) comprises at least two main biologically distinct entities: germinal center B-cell (GCB) and activated B-cell (ABC) subtype. Albeit sharing common lesions, GCB and ABC DLBCL present subtype-specific oncogenic pathway perturbations. ABC DLBCL is typically characterized by a constitutively active NF-kB. However, the latter is seen in also 30% of GCB DLBCL. Another recurrent lesion in DLBCL is an 11q24.3 gain, associated with the overexpression of two ETS transcription factors, ETS1 and FLI1. Here, we showed that FLI1 is more expressed in GCB than ABC DLBCL and we characterized its transcriptional network. Methods Gene expression data were obtained from public datasets GSE98588, phs001444.v2.p1, GSE95013 and GSE10846. ChIP-Seq for FLI1 paired with transcriptome analysis (RNA-Seq) after FLI1 silencing (siRNAs) was performed. Sequencing was carried out using the NextSeq 500 (Illumina). Detection of peaks was done using HOMER (v2.6); differential expressed genes were identified using moderated t-test (limma R-package) and functionally annotated with g:Profiler. ChIP-Seq and RNA-Seq data from GCB DLBCL cell lines after FLI1 downregulation were integrated to identify putative direct targets of FLI1. Results Analysis of clinical DLBCL specimens showed that FLI1 gene was more frequently expressed at higher levels in GCB than in ABC DLBCL and its protein levels were higher in GCB than in ABC DLBCL cell lines. Genes negatively regulated by FLI1 included tumor suppressor genes involved in negative regulation of cell cycle and hypoxia. Among positively regulated targets of FLI1, we found genes annotated for immune response, MYC targets, NF-κB and BCR signaling and NOTCH pathway genes. Of note, direct targets of FLI1 overlapped with genes regulated by ETS1, the other transcription factor gained at the 11q24.3 locus in DLBCL, suggesting a functional convergence within the ETS family. Positive targets of FLI1 included the NF-κB-associated ASB2 , a putative essential gene for DLBCL cell survival. ASB2 gene downregulation was toxic in GCB DLBCL cell lines and induced NF-κB inhibition via downregulation of RelB and increased IκBα. Additionally, downregulation of FLI1, but not ASB2, caused reduction of NF-κB1 and RelA protein levels. Conclusions We conclude that FLI1 directly regulates a network of biologically crucial genes and processes in GCB DLBCL. FLI1 regulates both the classical NF-κB pathway at the transcriptional level, and the alternative NF-κB pathway, via ASB2. FLI1 and ASB2 inhibition represents a potential novel therapeutic approach for GCB DLBCL.

Abstract Lung cancer has the highest mortality rate of any tumour type. The main driver of lung tumour growth and development is uncontrolled cellular proliferation. Poor patient outcomes are partly the result of the limited range of effective anti-cancer therapies available and partly due to the limited accuracy of biomarkers to report on cell proliferation rates in patients. Accordingly, accurate methods of diagnosing, staging and assessing response to therapy are crucial to improve patient outcomes. One effective way of assessing cell proliferation is to employ non-invasive evaluation using 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography [18F]FLT-PET. [18F]FLT, unlike the most commonly used PET tracer [18F]fluorodeoxyglucose ([18F]FDG), can specifically report on cell proliferation and does not accumulate in inflammatory cells. Therefore, this radiotracer could exhibit higher specificity in diagnosis and staging, along with more accurate monitoring of therapy response at early stages in the treatment cycle. This review summarises and evaluates published studies on the clinical use of [18F]FLT to diagnose, stage and assess response to therapy in lung cancer.

Abstract Objective The purpose of the study was to determine the extent and role of systemic hypoxia in the pathogenesis of spinal muscular atrophy (SMA). Methods Hypoxia was assayed in vivo in early‐symptomatic (postnatal day 5) SMA‐model mice by pimonidazole and [18F]‐Fluoroazomycin arabinoside injections, which accumulate in hypoxic cells, followed by immunohistochemistry and tracer biodistribution evaluation. Glucose uptake in hypoxic cells was assayed by [18F]‐Fluorodeoxyglucose labeling. In vitro knockdown of Survival Motor Neuron (SMN) was performed on motor neurons and lactate metabolism measured biochemically, whereas cell cycle progression and cell death were assayed by flow cytometry. Results All assays found significant levels of hypoxia in multiple organ systems in early symptomatic SMA mouse pups, except aerated tissues such as skin and lungs. This was accompanied by significantly increased glucose uptake in many affected organs, consistent with a metabolic hypoxia response. SMN protein levels were shown to vary widely between motor neuron precursors in vitro, and those with lower levels were most susceptible to cell death. In addition, SMA‐model motor neurons were particularly sensitive to hypoxia, with reduced ability to transport lactate out of the cell in hypoxic culture, and a failure in normal cell cycle progression. Interpretation Not only is there widespread tissue hypoxia and multi‐organ cellular hypoxic response in SMA model mice, but SMA‐model motor neurons are especially susceptible to that hypoxia. The data support the hypothesis that vascular defects leading to hypoxia are a significant contributor to disease progression in SMA, and offer a route for combinatorial, non‐SMN related therapy.

A metabotropic glutamate receptor coupled to phospholipase D (PLD‐mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch‐evoked firing. We report here the first identification of this PLD‐mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far‐western blotting indicates PLD‐mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD‐mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD‐mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings. What is the central question of this study?The metabotropic glutamate receptor coupled to phospholipase D (PLD‐mGluR) is a glutamate receptor previously only characterized pharmacologically but essential for maintaining stretch responsiveness in muscle spindle mechanosensory primary endings: what is the PLD‐mGluR protein?What is the main finding and its importance?PLD‐mGluR was identified as a homomeric GluK2 receptor signalling metabotropically. This identifies PLD‐mGluR 30 years after its discovery. This is important because: PLD‐mGluR is essential for muscle spindle stretch sensitivity; it is the first native kainate receptor shown to signal solely metabotropically; and, as it is the only GluR expressed in spindle mechanosensory endings, muscle spindles make a good functional assay of the native receptor. [ABSTRACT FROM AUTHOR]

Abstract The first prospective fluorinated PET tracers for imaging hypoxia obtained via thiazolidine-ligation are reported. Three 1,2-thiol-amine linkers were combined with four different 2-nitroimidazole spacers via amide or urea bond formation. The resulting compounds were submitted to thiazolidine-ring-forming ligation reaction with the fluorinated carbohydrate l-5-fluoro-5-deoxy-ribose (FDR), affording the desired candidate PET tracers in variable yields. The same ligation reactions performed on l-ribose – a by-product of [18F]FDR radiosynthesis – under conditions mimicking a radiochemical production showed that the fluorinated adducts can be efficiently purified and isolated by HPLC. Finally, one of the prospective hypoxia tracers was successfully produced in radiolabelled form in 29.2% radiochemical yield from [18F]FDR.

The discovery of liquid water at several locations in the solar system raises the possibility that microbial life may have evolved outside Earth and as such could be accidently introduced into the Earth’s ecosystem. Unusual sugars or amino acids, like non-proteinogenic isovaline and α-aminoisobutyric acid that are vanishingly rare or absent from life forms on Earth, have been found in high abundance on non-terrestrial carbonaceous meteorites. It is therefore conceivable that exo-microorganisms might contain proteins that include these rare amino acids. We therefore asked whether the mammalian immune system would be able to recognize and induce appropriate immune responses to putative proteinaceous antigens that include these rare amino acids. To address this, we synthesised peptide antigens based on a backbone of ovalbumin and introduced isovaline and α-aminoisobutyric acid residues and demonstrated that these peptides can promote naïve OT-I cell activation and proliferation, but did so less efficiently than the canonical peptides. This is relevant to the biosecurity of missions that may retrieve samples from exoplanets and moons that have conditions that may be permissive for life, suggesting that accidental contamination and exposure to exo-microorganisms with such distinct proteomes might pose an immunological challenge.

Aromatic prenyltransferases from cyanobactin biosynthetic pathways catalyse the chemoselective and regioselective intramolecular transfer of prenyl/geranyl groups from isoprene donors to an electron‐rich position in these macrocyclic and linear peptides. These enzymes often demonstrate relaxed substrate specificity and are considered useful biocatalysts for structural diversification of peptides. Herein, we assess the isoprene donor specificity of the N1‐tryptophan prenyltransferase AcyF from the anacyclamide A8P pathway using a library of 22 synthetic alkyl pyrophosphate analogues, of which many display reactive groups that are amenable to additional functionalization. We further used AcyF to introduce a reactive moiety into a tryptophan‐containing cyclic peptide and subsequently used click chemistry to fluorescently label the enzymatically modified peptide. This chemoenzymatic strategy allows late‐stage modification of peptides and is useful for many applications. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) are neutral mimics of natural DNA and RNA biopolymers that have caught the attention of researchers working on the identification of specific sequences of nucleobases in DNA/RNA strands. For this purpose, specific analytical protocols need to be developed to optimize the nucleic acid recognition ability of PNAs, exploiting both the high intrinsic affinity of PNA/DNA(RNA) couples as well as suitable markers, either linked to the PNA backbone or properly interacting with it in the working medium. In this context, the paper reports on phthalimide and 4‐nitrophthalimide as two cheap, metal‐free electroactive markers covalently bound to the pseudo‐peptide backbone of a PNA decamer. After a preliminary characterization of the markers, as such and in PNA conjugates, attention has been moved toward the optimization of the detectability of the labeled PNA decamers in aqueous solutions. Exploiting the potentiometric stripping analysis on hanging mercury drop electrode it has been possible to reach satisfactory detection limits of ca. 10 nM avoiding the use of expensive transition metal complexes as labels and/or of co‐reagents. [ABSTRACT FROM AUTHOR]

Cyanobactins are linear and cyclic post-translationally modified peptides. Here we show that the prenyl- D -Arg-containing autumnalamide A is a member of the cyanobactin family. Biochemical assays demonstrate that the AutF prenyltransferase targets the guanidinium moiety in arginine and homoarginine and is a useful tool for biotechnological applications. [ABSTRACT FROM AUTHOR]

Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.

Lung cancer has the highest mortality rate of any tumour type. The main driver of lung tumour growth and development is uncontrolled cellular proliferation. Poor patient outcomes are partly the result of the limited range of effective anti-cancer therapies available and partly due to the limited accuracy of biomarkers to report on cell proliferation rates in patients. Accordingly, accurate methods of diagnosing, staging and assessing response to therapy are crucial to improve patient outcomes. One effective way of assessing cell proliferation is to employ non-invasive evaluation using 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography [18F]FLT-PET. [18F]FLT, unlike the most commonly used PET tracer [18F]fluorodeoxyglucose ([18F]FDG), can specifically report on cell proliferation and does not accumulate in inflammatory cells. Therefore, this radiotracer could exhibit higher specificity in diagnosis and staging, along with more accurate monitoring of therapy response at early stages in the treatment cycle. This review summarises and evaluates published studies on the clinical use of [18F]FLT to diagnose, stage and assess response to therapy in lung cancer. [ABSTRACT FROM AUTHOR]

We report the synthesis of five radiotracers incorporating different oxyamine spacers between the hypoxia‐reactive 2‐nitroimidazole moiety and the 5‐[18F]‐fluorodeoxyribose ([18F]FDR, 12) prosthetic group: three linear alkyl chains with 3, 5, 7 carbon atoms (15 a–c), a cyclopropyl ring (15 d) and a 1,4‐disubstituted‐1,2,3‐triazole (15 e). Experiments in hypoxic cells showed that 15 d displays superior uptake kinetics – and similar selectivity for hypoxic cells – relative to the gold standard hypoxia tracers [18F]fluoroazomycin arabinoside ([18F]FAZA) and [18F]fluoromisonidazole ([18F]FMISO). Lipophilicity and structural rigidity have strong influence on the selectivity of tracers 15 towards hypoxic cells: the lead tracer 15 d displays a logP=0.38 and the most rigid spacer. A sixth radiotracer (15 f), with a 2‐H‐imidazole replacing the 2‐nitroimidazole moiety of 15 d, was used to demonstrate that the cyclopropyl group does not play a meaningful role in the sensitivity towards hypoxia. [ABSTRACT FROM AUTHOR]

Objective: The purpose of the study was to determine the extent and role of systemic hypoxia in the pathogenesis of spinal muscular atrophy (SMA). Methods: Hypoxia was assayed in vivo in early‐symptomatic (postnatal day 5) SMA‐model mice by pimonidazole and [18F]‐Fluoroazomycin arabinoside injections, which accumulate in hypoxic cells, followed by immunohistochemistry and tracer biodistribution evaluation. Glucose uptake in hypoxic cells was assayed by [18F]‐Fluorodeoxyglucose labeling. In vitro knockdown of Survival Motor Neuron (SMN) was performed on motor neurons and lactate metabolism measured biochemically, whereas cell cycle progression and cell death were assayed by flow cytometry. Results: All assays found significant levels of hypoxia in multiple organ systems in early symptomatic SMA mouse pups, except aerated tissues such as skin and lungs. This was accompanied by significantly increased glucose uptake in many affected organs, consistent with a metabolic hypoxia response. SMN protein levels were shown to vary widely between motor neuron precursors in vitro, and those with lower levels were most susceptible to cell death. In addition, SMA‐model motor neurons were particularly sensitive to hypoxia, with reduced ability to transport lactate out of the cell in hypoxic culture, and a failure in normal cell cycle progression. Interpretation: Not only is there widespread tissue hypoxia and multi‐organ cellular hypoxic response in SMA model mice, but SMA‐model motor neurons are especially susceptible to that hypoxia. The data support the hypothesis that vascular defects leading to hypoxia are a significant contributor to disease progression in SMA, and offer a route for combinatorial, non‐SMN related therapy. [ABSTRACT FROM AUTHOR]

Purpose: Molecular imaging of αvβ3 integrin has exhibited real potential to guide the appropriate use of anti-angiogenic therapies. However, an incomplete understanding of the factors that influence binding of αvβ3 integrin-specific radiotracers currently limits their use for assessing response to therapy in cancer patients. This study identifies two fundamental factors that modulate uptake of these radiotracers. Procedures Experiments were performed in prostate cancer (PC3) and glioblastoma (U87MG) cells, which differentially express αvβ3 integrin. αvβ3 integrin-specific radiotracers were used to investigate the effect of manipulating αvβ3 integrin expression or activation in cellular binding assays. β3 integrin and αvβ3 integrin expression were measured by western blotting and flow cytometry, respectively. The effect of select pharmacological inhibitors on αvβ3 integrin activation and expression was also determined.Results: Radiotracer binding was proportional to αvβ3 integrin expression when it was decreased (β3 knock-down cells) or increased, either using pharmacological inhibitors of cell signalling or by culturing cells for different times. Studies with both small molecule and arginine-glycine-aspartic acid (RGD)-based radiotracers revealed increased radiotracer binding after activation of αvβ3 integrin with Mn2+ or talin head domain. Moreover, inhibition of fundamental signalling pathways (mitogen-activated protein kinase kinase (MEK), Src and VEGFR2) decreased radiotracer binding, reflecting reduced αvβ3 integrin activity.Conclusion: Binding of small molecule ligands and radiolabelled RGD peptides is modulated by expression and activation status of αvβ3 integrin. αvβ3 integrin-specific radiotracers can provide otherwise inaccessible information of the effect of signalling pathways on αvβ3 integrin. This has significant implications for assessing response to anti-angiogenic therapies in clinical studies. [ABSTRACT FROM AUTHOR]

The bile acid analogue [18F]LCATD (LithoCholic Acid Triazole Derivative) is transported in vitro by hepatic uptake transporters such as OATP1B1 and NTCP and efflux transporter BSEP. In this in vivo “proof of principle” study, we tested if [18F]LCATD may be used to evaluate drug-drug interactions (DDIs) caused by inhibition of liver transporters. Hepatic clearance of [18F]LCATD in rats was significantly modified upon coadministration of rifamycin SV or sodium fusidate, which are known to inhibit clinically relevant uptake transporters (OATP1B1, NTCP) and canalicular hepatic transporters (BSEP) in humans. Treatment with rifamycin SV (total dose 62.5 mg·Kg−1) reduced the maximum radioactivity of [18F]LCATD recorded in the liver from 14.2 ± 0.8% to 10.2 ± 0.9% and delayed t_max by 90 seconds relative to control rats. AUCliver 0–5 min, AUCbile 0–10 min and hepatic uptake clearance CLuptake,in vivo of rifamycin SV treated rats were significantly reduced, whereas AUCliver 0–30 min was higher than in control rats. Administration of sodium fusidate (30 mg·Kg−1) inhibited the liver uptake of [18F]LCATD, although to a lesser extent, reducing the maximum radioactivity in the liver to 11.5 ± 0.3%. These preliminary results indicate that [18F]LCATD may be a good candidate for future applications as an investigational tracer to evaluate altered hepatobiliary excretion as a result of drug-induced inhibition of hepatic transporters. [ABSTRACT FROM AUTHOR]