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BackgroundSARS-CoV2 infection causes severe, life-threatening pneumonia. Hyper-inflammation, coagulopathy and lymphopenia are associated with pathology and poor outcomes in these patients. Cell-free (cf) DNA is prominent in COVID-19 patients, amplifies inflammation and promotes coagulopathy and immune dysfunction. We hypothesized that cf-DNA clearance by nebulised dornase alfa may reduce inflammation and improve disease outcomes. Here, we evaluated the efficacy of nebulized dornase alfa in patients hospitalised with severe COVID-19 pneumonia.MethodsIn this randomised controlled single-centre phase 2 proof-of-concept trial, we recruited adult patients admitted to hospital that exhibited stable oxygen saturation (≥94%) on supplementary oxygen and a C-reactive protein (CRP) level ≥30mg/L post dexamethasone treatment. Participants were randomized at a 3:1 ratio to receive twice-daily nebulised dornase alfa in addition to best available care (BAC) or BAC alone for seven days or until hospital discharge. A 2:1 ratio of historical controls to treated individuals (HC, 2:1) were included as the primary endpoint comparators. The primary outcome was a reduction in systemic inflammation measured by blood CRP levels over 7 days post-randomisation, or to discharge if sooner. Secondary and exploratory outcomes included time to discharge, time on oxygen, D-dimer levels, lymphocyte counts and levels of circulating cf-DNA.ResultsWe screened 75 patients and enrolled 39 participants out of which 30 in dornase alfa arm, and 9 in BAC group. We also matched the recruited patients in the treated group (N=30) to historical controls in the BAC group (N=60). For the the primary outcome, 30 patients in the dornase alfa were compared to 69 patients in the BAC group. Dornase alfa treatment reduced CRP by 33% compared to the BAC group at 7-days (P=0.01). The dornase alfa group least squares mean CRP was 23.23 mg/L (95% CI 17.71 to 30.46) and the BAC group 34.82 mg/L (95% CI 28.55 to 42.47). A significant difference was also observed when only randomised participants were compared. Furthermore, compared to the BAC group, the chance of live discharge was increased by 63% in the dornase alfa group (HR 1.63, 95% CI 1.01 to 2.61, P=0.03), lymphocyte counts were improved (least-square mean: 1.08 vs 0.87, P=0.02) and markers of coagulopathy such as D-dimer were diminished (least-square mean: 570.78 vs 1656.96μg/mL, P=0.004). Moreover, the dornase alfa group exhibited lower circulating cf-DNA levels that correlated with CRP changes over the course of treatment. No differences were recorded in the rates and length of stay in the ICU or the time on oxygen between the groups. Dornase alfa was well-tolerated with no serious adverse events reported.ConclusionsIn this proof-of-concept study in patients with severe COVID-19 pneumonia, treatment with nebulised dornase alfa resulted in a significant reduction in inflammation, markers of immune pathology and time to discharge. The effectiveness of dornase alfa in patients with acute respiratory infection and inflammation should be investigated further in larger trials.

RATIONALE: Type 2 innate lymphoid cells (ILC2s) are significant sources of type 2 cytokines, which are implicated in the pathogenesis of asthma and asthma exacerbations. The role of ILC2s in virus-induced asthma exacerbations is not well characterized. OBJECTIVES: To characterize pulmonary ILC responses following experimental rhinovirus challenge in patients with moderate asthma and healthy subjects. METHODS: Patients with moderate asthma and healthy subjects were inoculated with rhinovirus-16 and underwent bronchoscopy at baseline and at Day 3, and Day 8 after inoculation. Pulmonary ILC1s and ILC2s were quantified in bronchoalveolar lavage using flow cytometry. The ratio of bronchoalveolar lavage ILC2:ILC1 was assessed to determine their relative contributions to the clinical and immune response to rhinovirus challenge. MEASUREMENTS AND MAIN RESULTS: At baseline, ILC2s were significantly higher in patients with asthma than in healthy subjects. At Day 8, ILC2s significantly increased from baseline in both groups, which was significantly higher in patients with asthma than in healthy subjects (all comparisons P

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites., Author summary Malaria is a significant global health problem caused by a parasite that invades and multiplies within red blood cells, an invasion cycle resulting in death and disease. Plasmodium falciparum is the parasite that causes most malaria deaths. In most higher organisms, proteins can be marked by addition of a ubiquitin ‘tag’, which determines how quickly they will be broken down or their location in the cell. In this study we found that many parasite proteins are modified with a ubiquitin tag while the parasite matures within red blood cells and that even more proteins seem to be tagged in the extracellular invasive merozoite form. Some small molecules that inhibit the addition of this tag, and in particular one compound that inhibits the activation of the ubiquitin tag, blocked parasite growth. We show that this compound inhibits the parasite enzyme that activates ubiquitin and kills the parasite before it can finish growing and multiplying, and that the same effect is achieved by knocking out the gene for this enzyme. These results point to the importance of ubiquitin tags in the parasite life cycle and highlight the potential of this ubiquitin pathway as a target for the development of new antimalarial drugs.

In this study, we demonstrate that the lack of retinoic acid-related orphan receptor (ROR) γ or α expression in mice significantly reduced the peak expression level of Cry1, Bmal1, E4bp4, Rev-Erbα and Per2 in an ROR isotype- and tissue-selective manner without affecting the phase of their rhythmic expression. Analysis of RORγ/RORα double knockout mice indicated that in certain tissues RORγ and RORα exhibited a certain degree of redundancy in regulating clock gene expression. Reporter gene analysis showed that RORγ was able to induce reporter gene activity through the RORE-containing regulatory regions of Cry1, Bmal1, Rev-Erbα and E4bp4. Co-expression of Rev-Erbα or addition of a novel ROR antagonist repressed this activation. ChIP-Seq and ChIP–Quantitative real-time polymerase chain reaction (QPCR) analysis demonstrated that in vivo RORγ regulate these genes directly and in a Zeitgeber time (ZT)-dependent manner through these ROREs. This transcriptional activation by RORs was associated with changes in histone acetylation and chromatin accessibility. The rhythmic expression of RORγ1 by clock proteins may lead to the rhythmic expression of RORγ1 target genes. The presence of RORγ binding sites and its down-regulation in RORγ−/− liver suggest that the rhythmic expression of Avpr1a depends on RORγ consistent with the concept that RORγ1 provides a link between the clock machinery and its regulation of metabolic genes.

The spirodiaziridines 6 and 9, potential inhibitors of α- and β-glucosidases, were prepared from the validoxylamine A-derived cyclohexanone 5. The trimethylsilyl protecting groups of 5 are crucial for the formation of 6 in good yields. Oxidation of 6 gave 7. The diaziridine 6 (pKHA=2.6) and the diazirine 7 did not inhibit the β-glucosidases from almonds, the β-glucosidase from Caldocellum saccharolyticum, and the α-glucosidase from yeast. The N-benzyl diaziridine 9 is a very weak inhibitor of the α-glucosidase, but did not inhibit the β-glucosidases. To see whether the weak inhibition is due to the low basicity of the diaziridines or to geometric factors, we prepared the spiro-aziridines 21 and 25 and 1-epivalidamine (32). The known cyclohexanone 10 was methylenated and epoxidised to 16 and 17. Azide opening of 16 and 17, mesylation, LiAlH4 reduction, and deprotection gave the aziridines 21 and 25 respectively. 1-Epivalidamine (32) was prepared from the known carba-glucose 29. The aziridine 25 (pKHA=6.8) is a weak irreversible inhibitor of the β-glucosidase from Caldocellum saccharolyticum and a weak reversible inhibitor of the α-glucosidase from yeast, but did not inhibit the β-glucosidases from almonds. The poorly stable aziridine 21 weakly inhibited the three enzymes. Similarly, 1-epivalidamine (pKHA=8.4) proved only a weak inhibitor. The known cyclopentylamine 34 (pKHA=7.9), however, is a micromolar inhibitor of these enzymes. The much stronger inhibition by 34 is related to the pseudoaxial orientation of its amino group.

MAPKAPK5 has been proposed to play a role in regulation of matrix metalloprotease expression and so to be a potential target for intervention in rheumatoid arthritis. We present here the identification of a series of compounds against this target which are effective in both biochemical and cell assays. The expansion of the series is described, along with early SAR and pharmacokinetics for some representative compounds.

A series of 2,3-di-O-benzoyl-4,6-0- [(S)-l-(methoxycarbonyl)ethylidenel-~-glucopyranosyl donors (8phenylthio 3, bromide 4, a-chloride 5, &fluoride 6, and trichloroacetimidate 7) were prepared from the corresponding a-allyl glucoside 1 via the deallylated glucose 2 and were tested in glycosylation reactions with methanol to give the pyruvylated methyl glucosides 8 and 9 and with methyl 2,4,6tri-0-benzoyl-8-D-glucopyranoside 10 to give the disaccharide 11. Best resulta with respect to yield and &selectivity of the coupling were achieved with imidate 7. Thus, the 2-0-benzoyl-4,6-0-[(S)l-(methoxycarbonyl)ethylidenel-3-O-methyl-~-glucopyranosyl trichloroacetimidate 14 was prepared from ita benzyl glucoside 12 and used for the synthesis of fragments related to the Mycobacterium smegma tis lipopentasaccharide. Trimethylsilyl trifluoromethanesulfonate-mediated condensation of 14, 5- [(benzyloxycarbonyl)aminol pentanol, and 10, respectively, followed by deblocking of the producta 15 and 17 gave the pyruvylated aminopentyl glucoside 16 and methyl laminaribioside 18. Treatment of 17 with dichloromethyl methyl ether gave the laminaribiosyl chloride 19, coupling of which with protected 5-aminopentanol gave 20 also obtained from 14 and the monosaccharide nucleophile 26. The trisaccharide 5-aminopentyl glycoside fragment 42 was prepared by first coupling of 14 and benzyl 2-0-benzoyl-4,6-0-[(S)-l-(methoxycarbonyl)ethylidenel-a-~-glucopyranoside 30 that was converted to the bispyruvylated laminaribiose imidate 33, followed by condensation with thioglycoside 38 obtainable in four steps from 1,2,4,6-tetra-O-acetyl-3-O-benzyl-~-~-glucopyranose to give the trisaccharide ethyl thioglycoside 40. Next, NIS-mediated condensation of 40 and 5- [(benzyloxycarbonyl)aminol pentanol followed by deblocking gave 42. Pentasaccharide 45 was similarly prepared from 40 and hepta-0-benzoyltrehalose 43 to give first the blocked saccharide 44, deblocking of which afforded the target pentasaccharide.

Treatment of alkyl D-glycopyranosides and alkyl or phenyl 1-thio-β-D-glycopyranosides 1 with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane gave the corresponding 4,6-0-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl) protected glycosides 2 which were subsequently benzoylated with benzoyl bromide to give the fully blocked glycosides 3. Reaction of the latter with methyl pyruvate and a catalytic amount of trimethylsilyl trifluoromethanesulfonate gave alkyl and phenyl 4,6-0-[1-(methoxycarbonyl)ethylidene]glycopyranosides 4 with high diastereoselectivity

Treatment of alkyl D-glycopyranosides and alkyl or phenyl 1-thio-β-D-glycopyranosides 1 with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane gave the corresponding 4,6-0-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl) protected glycosides 2 which were subsequently benzoylated with benzoyl bromide to give the fully blocked glycosides 3. Reaction of the latter with methyl pyruvate and a catalytic amount of trimethylsilyl trifluoromethanesulfonate gave alkyl and phenyl 4,6-0-[1-(methoxycarbonyl)ethylidene]glycopyranosides 4 with high diastereoselectivity

The study of protein target families, as opposed to single targets, has become a very powerful tool in chemogenomics-led drug discovery. By integrating comprehensive chemoinformatics and bioinformatics databases with customised analytical tools, a 'Toolkit' approach for the target family is possible, thus allowing predictions of the ligand class, affinity, selectivity and likely off-target issues to be made for the guidance of the medicinal chemist. In this review, we highlight the development and application of the Toolkit approach to the protein kinase superfamily, drawing on examples from lead optimisation studies and the design of focused libraries for lead discovery.

Objective. Extracellular microelectrode techniques are the most widely used approach to interrogate neuronal populations. However, regardless of the manufacturing method used, damage to the vasculature and circuit function during probe insertion remains a concern. This issue can be mitigated by minimising the footprint of the probe used. Reducing the size of probes typically requires either a reduction in the number of channels present in the probe, or a reduction in the individual channel area. Both lead to less effective coupling between the probe and extracellular signals of interest. Approach. Here, we show that continuously drawn SiO2-insulated ultra-microelectrode fibres offer an attractive substrate to address these challenges. Individual fibres can be fabricated to >10 m continuous stretches and a selection of diameters below 30 µm with low resistance (−1) continuously conductive metal core of µm and atomically flat smooth shank surfaces. To optimize the properties of the miniaturised electrode-tissue interface, we electrodeposit rough Au structures followed by ∼20 nm IrOx film resulting in the reduction of the interfacial impedance to Main results. We demonstrate that these ultra-low impedance electrodes can record and stimulate both single and multi-unit activity with minimal tissue disturbance and exceptional signal-to-noise ratio in both superficial (∼40 µm) and deep (∼6 mm) structures of the mouse brain. Further, we show that sensor modifications are stable and probe manufacturing is reproducible. Significance. Minimally perturbing bidirectional neural interfacing can reveal circuit function in the mammalian brain in vivo.