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The hybrid network of Si3N4 whiskers and conducting carbon fiber has great potential for microwave absoprtion applications. The high electrical conductivity of the carbon fiber helps to transform the microwave transparent Si3N4 into microwave absorbing materials. Herein, the microwave absorption performance of 5–20 vol % of carbon fiber reinforced reaction bonded Si3N4 (Cf-RBSN) composites have been discussed in detail. The Cf reinforcement tuned the X-band dielectric properties of the RBSN composites. The 5 vol % Cf-RBSN composite exhibit a minimum reflection loss (RLmin) of −36.16 dB (99.998% microwave absorption) at 11.89 GHz and a high specific reflection loss of 920 dB. g−1 for 5.9 mm thickness, while 20 vol % Cf-RBSN composites resulted in RLmin of −22.86 dB at 11.56 GHz with a low thickness of 1.5 mm. Thus, the superior microwave absorption performance of the prepared lightweight composites results from the multiple interfacial polarization, dipole polarization, and conduction loss due to the 3D network of interconnected Si3N4 whiskers and Cf.

Janus Kinases (JAKs) have emerged as an important drug target for the treatment of a number of immune disorders due to the central role that they play in cytokine signalling. 4 isoforms of JAKs exist in mammalian cells and the ideal isoform profile of a JAK inhibitor has been the subject of much debate. JAK3 has been proposed as an ideal target due to its expression being largely restricted to the immune system and its requirement for signalling by cytokine receptors using the common γ-chain. Unlike other JAKs, JAK3 possesses a cysteine in its ATP binding pocket and this has allowed the design of isoform selective covalent JAK3 inhibitors targeting this residue. We report here that mutating this cysteine to serine does not prevent JAK3 catalytic activity but does greatly increase the IC50 for covalent JAK3 inhibitors. Mice with a Cys905Ser knockin mutation in the endogenous JAK3 gene are viable and show no apparent welfare issues. Cells from these mice show normal STAT phosphorylation in response to JAK3 dependent cytokines but are resistant to the effects of covalent JAK3 inhibitors. These mice therefore provide a chemical-genetic model to study JAK3 function.

The COVID-19 pandemic has triggered outbreaks of unanticipated toxicities, including methanol toxicity. Multiple methanol outbreaks have been described, including contaminated hand sanitizer in the southwest USA. In this case, we describe a fatal case of methanol toxicity from hand sanitizer ingestion, geographically separated from the outbreak in the southwest USA and prior to the announcement of nationwide warnings by the Food and Drug Administration (FDA). The product was identified as one later recalled by the FDA for methanol contamination. Additionally, the consumption in this case was related to a desire to conceal alcohol consumption from family members. This case of methanol toxicity should increase awareness of the ease of which contaminated products can be widely distributed and of the use of alternative ethanol-containing products to obscure relapse in alcohol use disorder.

A novel lead-free Ba(1-x)(La,Na)xTiO3 (BLNT), (0 ≤ x ≤ 0.08) ferroelectric ceramic with enhanced energy storage density was fabricated by the conventional solid-state method. With an increase in La...

Surface functionalisation of diamond can produce negative electron affinity (NEA), where the conduction band lies higher in energy than the vacuum level. Hydrogen termination is well-known to produce NEA on diamond but can be removed by temperatures above ∼700 °C, making it unsuitable for diamond-based thermionic applications, where high temperatures are converted into electricity via an electron emission process. In this review article we give an overview of thermionic emission, describe the potential applications of diamond-based thermionic energy converters, then review the different surface termination schemes that can provide a diamond surface with NEA. We discuss the relative merits of the different NEA surfaces that have been developed both computationally and experimentally as alternative cathode materials for thermionic devices.

SrBi4Ti4O15 is a bismuth layered perovskite structure material, its thin film form gained much interest due to their geometrical flexibility and thickness. In the present study, SrBi4Ti4O15 thin films are deposited on amorphous substrate using radio frequency sputtering technique. SrBi4Ti4O15 thin films found to be single phase orthorhombic structure by using X-ray diffraction technique. Surface morphology studies such as grain size, shape and roughness were explored through atomic force microscopy. EDX analysis identified the individual elements and its percentage in the compound. Optical properties (refractive index and bandgap) were determined with varying the oxygen mixing percentage (OMP). The best optical properties (refractive index ~ 2.35 and bandgap ~ 3.4 eV) were obtained for films deposited at 50% OMP. These results shows the potential of using SrBi4Ti4O15 thin films in electro-optic devices.

Background: Local anesthetics are widely used to decrease sensitivity to pain in specific regions of the body while performing medical tasks. Many studies have probed the mechanism of action of local anesthetics but still many questions remain. (2R - (-) 2 - (2, 6-dimethylphenylaminocarbonyl) - 1 – methyl piperidinium chloride (DAMP), is an extensively used amide-type local anesthetic. Objective: This study aims at revealing the various electrophysical and chemical properties of the title compound. This study will be useful for future research by pharmacologists. Method: Density Functional Theory (DFT) computations were executed using Gaussian’09 program package and were optimized with the B3LYP /6-311+G (d, p) basis set. Natural bond orbital (NBO) analysis was carried out with version 3.1. Normal Coordinate Analysis (NCA) was used to systematically calculate the harmonic vibrational wavenumbers. Molecular docking simulations were carried out to understand the pharmacokinetic behavior of the drug. Results: The presence of strong N-H…Cl intra molecular hydrogen bonding was evidently revealed from the FT-IR spectrum due to the shifting of NH stretching wavenumber. Stability of the molecule arising from hyper conjugative interactions exhibits the bioactivity of the molecule by natural bond orbital analysis. The title molecule binds to the inner pore and blocks voltage - gated sodium channels in peripheral neurons. Conclusion: A detailed molecular picture of DAMP and its interactions were obtained by modeling analysis, IR, Raman, and UV-Vis spectroscopy. The geometrical parameters agree well with the XRD data. NBO analysis indicates the bioactivity of the molecule. The HOMO-LUMO energy gap indicates the possibility of intramolecular charge transfer of the molecule. From the ligand docking studies it is concluded that the title molecule binds to the inner pore and blocks voltage - gated sodium channels in peripheral neurons.

Deubiquitylating enzymes (DUBs) play a vital role in the ubiquitin pathway by editing or removing ubiquitin from their substrate. As breakthroughs within the ubiquitin field continue to highlight the potential of deubiquitylating enzymes as drug targets, there is increasing demand for versatile high-throughput (HT) tools for the identification of potent and selective DUB modulators. Here we present the HT adaptation of the previously published MALDI-TOF–based DUB assay method. In a MALDI-TOF DUB assay, we quantitate the amount of mono-ubiquitin generated by the in vitro cleavage of ubiquitin chains by DUBs. The method has been specifically developed for use with nanoliter-dispensing robotics to meet drug discovery requirements for the screening of large and diverse compound libraries. Contrary to the most common DUB screening technologies currently available, the MALDI-TOF DUB assay combines the use of physiological substrates with the sensitivity and reliability of the mass spectrometry–based readout. The authors describe a protocol for a high-throughput version of the MALDI-TOF–based deubiquitylating enzyme assay method.

The influence of Nickel and Cobalt co-substitution on the structure and dielectric properties of barium titanate (BaTiO3) ceramics has been investigated. Nickel and Cobalt co-substituted BaTiO3 ceramics with general formula BaTi1−x(Ni,Co)xO3 has been synthesized for x = 0.0, 0.02, 0.04 and 0.08 through conventional solid state reaction route. The structural analysis carried out by X-ray diffraction reveals the successful incorporation of Ni2+ and Co2+ ions in the Ti4+ site of BaTiO3 with all samples under study having tetragonal structure. However the tetragonality (c/a ratio) seems to be decreasing with increase of co-substitution. The dielectric measurements are done at different frequencies and temperatures. The room temperature dielectric constant decreased considerably for the Ni and Co co-substituted samples. The phase transition of the BaTi1−x(Ni,Co)xO3 has been investigated by studying the temperature dependence of dielectric constant. Nickel and Cobalt co-substituted samples showed diffused nature of phase transition with a notable reduction in the Curie temperature with the increase of co-substitution. The Curie temperature is reduced to 104 °C for x = 0.08 composition whereas for x = 0.0 the Curie temperature is 123 °C. Enhancement in conductivity is observed with addition of Nickel and Cobalt addition. Nearest-neighbour hopping model is used to explain the conduction in the samples under study and energy of activation for conduction is calculated from Arrhenius plot.

In quest of finding an alternative for lead-based materials, ferroelectric materials with Bi-layered structure have attracted great attention due to their potential applications in non-volatile memory, electro-optic devices, and microelectromechanical systems. In this work, samarium (Sm)-substituted SrBi4Ti4O15 (SBTi) ceramics have been prepared by solid-state reaction method and sintered using microwave, as well as conventional heating. XRD phase analysis reveals the formation of single phase compound, and there is no effect of Sm-substitution on basic crystal structure. SEM micrographs showed the plate-like structure of grains both in microwave and conventional furnace. Temperature-dependent dielectric properties were investigated in the temperature range 40–600 °C and frequency range 10 kHz–1 MHz. Sm-substitution increases dielectric constant upto Sm ~ 0.75 and also introduces small relaxor behavior. Hardness and Young’s modulus of SrBi4-xSmxTi4O15 ceramics were measured by nanoindentation. The structural, morphology, dielectric, and mechanical properties of pure and substituted SrBi4Ti4O15 ceramics along with sintering methods have been investigated in detail.

Myocardial ischemia leads to conduction slowing, cell-to-cell uncoupling, and arrhythmias. We previously demonstrated that varying perfusate sodium (Na(+)) and calcium (Ca(2+)) attenuates conduction slowing and arrhythmias during simulated ischemia with continuous perfusion. Cardioprotection was selectively associated with widening of the perinexus, a gap junction adjacent nanodomain important to ephaptic coupling. It is unknown whether perfusate composition affects the perinexus or ischemic conduction during nonsimulated ischemia, when coronary flow is reduced or halted. We hypothesized that altering preischemic perfusate composition could facilitate perinexal expansion and attenuate conduction slowing during global ischemia. To test this hypothesis, ex vivo guinea pig hearts (n = 49) were Langendorff perfused with 145 or 153 mM Na(+) and 1.25 or 2.0 mM Ca(2+) and optically mapped during 30 min of no-flow ischemia. Altering Na(+) and Ca(2+) did not substantially affect baseline conduction. Increasing Na(+) and decreasing Ca(2+) both lowered pacing thresholds, whereas increasing Ca(2+) narrowed perinexal width (W(p)). A least squares mean estimate revealed that reduced perfusate Na(+) and Ca(2+) resulted in the most severe conduction slowing during ischemia. Increasing Na(+) alone modestly attenuated conduction slowing, yet significantly delayed the median time to conduction block (10 to 16 min). Increasing both Na(+) and Ca(2+) selectively widened W(p) during ischemia (22.7 vs. 15.7 nm) and attenuated conduction slowing to the greatest extent. Neither repolarization nor levels of total or phosphorylated connexin43 correlated with conduction slowing or block. Thus, perfusate-dependent widening of the perinexus preserved ischemic conduction and may be an adaptive response to ischemic stress. NEW & NOTEWORTHY Conduction slowing during acute ischemia creates an arrhythmogenic substrate. We have shown that extracellular ionic concentrations can alter conduction by modulating ephaptic coupling. Here, we demonstrate increased extracellular sodium and calcium significantly attenuate conduction slowing during no-flow ischemia. This effect was associated with selective widening of the perinexus, an intercalated disc nanodomain and putative cardiac ephapse. These findings suggest that acute changes in ephaptic coupling may serve as an adaptive response to ischemic stress.

Narrowing the bandgap energy of perovskite ferroelectrics is a potential task for improving PV applications. This study examined the effect of Ni2+/Na+ co-substitution on the structural, optical, electrical, and magnetic properties of Ba1-x(Nix/2,Nax)TiO3 (BTNaNi-x) ceramics, where (0 ≤ x ≤ 0.08), prepared by solid-state reaction method. X-ray diffraction along with structure refinement verifies the existence of perovskite structure with a tetragonal phase in all samples. The morphological study reveals a non-uniform grain size distribution with a decrease in grain size with an increase in co-substitution concentration. XPS study gives direct evidence of oxygen vacancies in the investigated sample. The oxygen vacancies and octahedral distortion accompanying the Ni2+/Na+ co-substitution significantly reduce the optical bandgap of BaTiO3 ceramics from 3.14 to 2.688 eV. The temperature dependence of the dielectric constant is measured in the range of 30–250 °C at the frequency of 1 kHz. The results reveal that the transition temperature gradually decreased with an increase in Ni2+ and Na+ concentrations. This phenomenon was interpreted based on reducing grain size and tetragonality with co-substitution. The ferroelectric and magnetic properties are determined by (P–E) and (M–H) hysteresis loops at room temperature. Magnetic properties are significantly enhanced with an increase in the Ni2+, Na+ contents in the system. The correlation between the ESR width parameter (ΔH) and asymmetry parameter (Pays) with concentrations (x) reveals that the sample with x = 0.08 has a strong magnetic nature compared to other samples.

Summary Background The ICON8 study reported no significant improvement in progression-free survival (a primary endpoint) with weekly chemotherapy compared with standard 3-weekly treatment among patients with epithelial ovarian cancer. All ICON8 patients were eligible to take part in the accompanying health-related quality-of-life study, which measured the effect of treatment on self-reported wellbeing, reported here. Methods In this open-label, randomised, controlled, phase 3, three-arm, Gynecologic Cancer Intergroup (GCIG) trial done at 117 hospital sites in the UK, Australia, New Zealand, Mexico, South Korea, and Republic of Ireland, women (aged at least 18 years) with newly diagnosed, histologically confirmed International Federation of Gynecology and Obstetrics stage IC–IV ovarian cancer and an Eastern Cooperative Oncology Group performance status of 0–2 were randomly assigned (1:1:1) centrally using minimisation to group 1 (intravenous carboplatin area under the curve [AUC]5 or AUC6 and 175 mg/m2 intravenous paclitaxel every 3 weeks), group 2 (carboplatin AUC5 or AUC6 every 3 weeks and 80 mg/m2 paclitaxel weekly), or group 3 (carboplatin AUC2 weekly and 80 mg/m2 paclitaxel weekly). Randomisation was stratified by GCIG group, disease stage, and outcome and timing of surgery. Patients and clinicians were not masked to treatment assignment. Patients underwent immediate or delayed primary surgery according to clinicians' choice. Patients were asked to complete European Organisation for Research and Treatment of Cancer QLQ-C30 and QLQ-OV28 questionnaires at enrolment, before each chemotherapy cycle, then 6-weekly up to 9 months, 3-monthly up to 2 years, and 6-monthly up to 5 years. Quality of life was a prespecified secondary outcome of the ICON8 study. Within the quality-of-life study, the co-primary endpoints were QLQ-C30 global health score at 9 months (cross-sectional analysis) and mean QLQ-C30 global health score from randomisation to 9 months (longitudinal analysis). Data analyses were done on an intention-to-treat basis. The trial is registered on ClinicalTrials.gov , NCT01654146 and ISRCTN Registry, ISRCTN10356387, and is currently in long-term follow up. Findings Between June 6, 2011, and Nov 28, 2014, 1566 patients were recruited into ICON8 (522 were included in group 1, 523 in group 2, and 521 in group 3). Baseline quality-of-life questionnaires were completed by 1438 (92%) of 1566 patients and 9-month questionnaires by 882 (69%) of 1280 patients. We observed no significant difference in global health score at 9 months (cross-sectional analysis) between study groups (group 2 vs group 1, difference in mean score 2·3, 95% CI −0·4 to 4·9, p=0·095; group 3 vs group 1, −0·8, −3·8 to 2·2, p=0·61). Using longitudinal analysis, we found lower global health scores for those receiving weekly paclitaxel than for those receiving 3-weekly chemotherapy (group 2 vs group 1, mean difference −1·8, 95% CI −3·6 to −0·1, p=0·043; group 3 vs group 1, −2·9, −4·7 to −1·1, p=0·0018). Interpretation We found no evidence of a difference in global quality of life between treatment groups at 9 months; however, patients receiving weekly treatment reported lower mean quality of life across the 9-month period after randomisation. Taken together with the lack of progression-free survival benefit, these findings do not support routine use of weekly paclitaxel-containing regimens in the management of newly diagnosed ovarian cancer. Funding Cancer Research UK, Medical Research Council, Health Research Board Ireland, Irish Cancer Society, and Cancer Australia.

The food we consume and its interactions with the host and their gut microbiota affect normal gut function and health. Functional gut disorders (FGDs), including irritable bowel syndrome (IBS), can result from negative effects of these interactions, leading to a reduced quality of life. Certain foods exacerbate or reduce the severity and prevalence of FGD symptoms. IBS can be used as a model of perturbation from normal gut function with which to study the impact of foods and diets on the severity and symptoms of FGDs and understand how critical processes and biochemical mechanisms contribute to this impact. Analyzing the complex interactions between food, host, and microbial metabolites gives insights into the pathways and processes occurring in the gut which contribute to FGDs. The following review is a critical discussion of the literature regarding metabolic pathways and dietary interventions relevant to FGDs. Many metabolites, for example bile acids, SCFAs, vitamins, amino acids, and neurotransmitters, can be altered by dietary intake, and could be valuable for identifying perturbations in metabolic pathways that distinguish a “normal, healthy” gut from a “dysfunctional, unhealthy” gut. Dietary interventions for reducing symptoms of FGDs are becoming more prevalent, but studies investigating the underlying mechanisms linked to host, microbiome, and metabolite interactions are less common. Therefore, we aim to evaluate the recent literature to assist with further progression of research in this field.

Several different biomaterials are being introduced for clinical applications. However, no current material-specific systematic studies define parameters for evaluating these materials. The aim of this retrospective animal study is to classify biomaterials according to the in vivo induced cellular reaction and outline the clinical consequence of the biomaterial-specific cellular reaction for the regeneration process. A retrospective histologic analysis was performed for 13 polymeric biomaterials and 19 bone substitute materials (BSMs) (of various compositions and origins) that were previously implanted in a standardized subcutaneous model. Semiquantitative analyses were performed at days 3, 15, and 30 after implantation according to a standardized score for the induction of multinucleated giant cells (MNGCs) and vascularization rate. The induced cellular reaction in response to different polymeric materials allowed their classification according to the MNGC score in the following groups: class I induced no MNGCs at any time point, class II induced and maintained a constant number of MNGCs over 30 days, and class III induced MNGCs and provided an increasing number over 30 days. All BSMs induced MNGCs to varying extents. Therefore, the resultant BSM classifications are as follows: class I induced MNGCs with a decreasing number, class II induced and maintained constant MNGCs over 30 days, and class III induced MNGCs with increasing number over 30 days. These observations were mostly related to the biomaterial physicochemical properties and were independent of the biomaterial origin. Consequently, the induction of MNGCs and their increase over 30 days resulted in disintegration of the biomaterial. By contrast, the absence of MNGCs resulted in an integration of the biomaterial within the host tissue. This novel classification provides clinicians a tool to assess the capacity and suitability of biomaterials in the intended clinical indication for bone and soft tissue implantations.

Background:\ud Carboplatin and paclitaxel administered every 3 weeks is standard-of-care first-line chemotherapy for epithelial ovarian cancer. The Japanese JGOG3016 trial showed a significant improvement in progression-free and overall survival with dose-dense weekly paclitaxel and 3-weekly carboplatin. In this study, we aimed to compare efficacy and safety of two dose-dense weekly regimens to standard 3-weekly chemotherapy in a predominantly European population with epithelial ovarian cancer.\ud Methods:\ud In this phase 3 trial, women with newly diagnosed International Federation of Gynecology and Obstetrics stage IC–IV epithelial ovarian cancer were randomly assigned to group 1 (carboplatin area under the curve [AUC]5 or AUC6 and 175 mg/m2 paclitaxel every 3 weeks), group 2 (carboplatin AUC5 or AUC6 every 3 weeks and 80 mg/m2 paclitaxel weekly), or group 3 (carboplatin AUC2 and 80 mg/m2 paclitaxel weekly). Written informed consent was provided by all women who entered the trial. The protocol had the appropriate national research ethics committee approval for the countries where the study was conducted. Patients entered the trial after immediate primary surgery, or before neoadjuvant chemotherapy with subsequent planned delayed primary surgery. The trial coprimary outcomes were progression-free survival and overall survival. Data analyses were done on an intention-to-treat basis, and were powered to detect a hazard ratio of 0·75 in progression-free survival. The main comparisons were between the control group (group 1) and each of the weekly research groups (groups 2 and 3).\ud Findings:\ud Between June 6, 2011, and Nov 28, 2014, 1566 women were randomly assigned to treatment. 72% (365), completed six protocol-defined treatment cycles in group 1, 60% (305) in group 2, and 63% (322) in group 3, although 90% (454), 89% (454), and 85% (437) completed six platinum-based chemotherapy cycles, respectively. Paclitaxel dose intensification was achieved with weekly treatment (median total paclitaxel dose 1010 mg/m2 in group 1; 1233 mg/m2 in group 2; 1274 mg/m2 in group 3). By February, 2017, 1018 (65%) patients had experienced disease progression. No significant progression-free survival increase was observed with either weekly regimen (restricted mean survival time 24·4 months [97·5% CI 23·0–26·0] in group 1, 24·9 months [24·0–25·9] in group 2, 25·3 months [23·9–26·9] in group 3; median progression-free survival 17·7 months [IQR 10·6–not reached] in group 1, 20·8 months [11·9–59·0] in group 2, 21·0 months [12·0–54·0] in group 3; log-rank p=0·35 for group 2 vs group 1; group 3 vs 1 p=0·51). Although grade 3 or 4 toxic effects increased with weekly treatment, these effects were predominantly uncomplicated. Febrile neutropenia and sensory neuropathy incidences were similar across groups.\ud Interpretation:\ud Weekly dose-dense chemotherapy can be delivered successfully as first-line treatment for epithelial ovarian cancer but does not significantly improve progression-free survival compared with standard 3-weekly chemotherapy in predominantly European populations.\ud Funding:\ud Cancer Research UK, Medical Research Council, Health Research Board in Ireland, Irish Cancer Society, Cancer Australia.

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain, and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. Here we report a proteomic screen for cellular factors that interact with the cytoplasmic tail of S. We confirm interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding site promotes exit from the endoplasmic reticulum, and although binding to COPI should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.

The COVID‐19 pandemic, caused by the SARS‐CoV‐2 coronavirus, has triggered a worldwide health emergency. Here, we show that ferritin‐like Dps from hyperthermophilic Sulfolobus islandicus, covalently coupled with SARS‐CoV‐2 antigens via the SpyCatcher system, forms stable multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS‐CoV‐2 receptor binding domain (RBD) coupled to Dps (RBD‐S‐Dps) elicited a higher antibody titre and an enhanced neutralising antibody response compared to monomeric RBD. A single immunisation with RBD‐S‐Dps completely protected hACE2‐expressing mice from serious illness and led to viral clearance from the lungs upon SARS‐CoV‐2 infection. Our data highlight that multimerised SARS‐CoV‐2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra‐stable scaffold., Preparation and quality control of coupled antigen–Dps complexes (Ag‐S‐Dps). (A) SDS/PAGE of the three expressed and purified antigens as introduced in Fig. 1C, Coomassie stained. Glycosylation of Spike leads to a fuzzy appearance of its band. RBD‐SpyT2 and Spike‐SpyT2 were expressed in mammalian cells, and SpyT2‐NP was expressed in bacteria, as was the SpyC‐Dps scaffold. (B) Size exclusion chromatography to separate excess antigens after the SpyCatcher/Spytag2 coupling reactions; Superose 6 Increase in PBS. (C) SDS/PAGE of the coupled and purified Ag‐S‐Dps complexes. ‘RT’, no heating; ‘99’, heated to 99 °C. The SpyC‐Dps scaffold alone, as well as all the three coupled complexes show high‐molecular weight complexes, presumably dodecameric, that disappear only after heating of the samples in SDS loading buffer (Coomassie stained). (D) Negative‐stain electron microscopy analyses of the three multimeric Ag‐S‐Dps complexes, showing that all samples form defined and monodisperse spheres that display the antigens on their surface, leading to particles of different sizes for the three differently sized antigens.

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins. Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.

Accumulating evidence supports a prion-like mechanism in the spread of assembled tau in neurodegenerative diseases. Prion-like spread is proposed to require the transit of tau assemblies to the interior of neurons in order to seed aggregation of native, cytosolic tau. This process is poorly understood and remains largely hypothetical. Here, we develop sensitive techniques to quantify the cytosolic entry of tau in real-time. We find that tau does not promote its own entry but, rather, is wholly dependent on cellular machinery. We find that entry to the widely used reporter cell line HEK293 requires clathrin whereas entry to neurons does not. Cholesterol depletion or knockdown of cholesterol transport protein Niemann-Pick type C1 in neurons renders cells highly vulnerable to cytosolic entry and seeded aggregation. Our findings establish entry as the rate-limiting step in seeded aggregation and demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation. Graphical Abstract

Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of cancers, neurodegenerative diseases and autoimmune disorders. Herein a novel series of pyrrolo[2,3-d]pyrimidine-based HDAC inhibitors were designed, synthesized and biologically evaluated, among which compounds 7a, 12a1, and 16a1 exhibited potent inhibitory activities and selectivities against HDAC6. Notably, compared with the well-known HDAC6 inhibitor Tubastatin A, our pyrrolo[2,3-d]pyrimidine-based HDAC6 inhibitors showed superior in vitro antiproliferative activity against human multiple myeloma cell lines RPMI 8226, U266 and MM.1S, while maintaining the low cytotoxicity against human breast cancer cell line MDA-MB-231 and two normal cell lines. The HDAC6 selective inhibition of one representative compound 12a1 in RPMI 8226 cells was confirmed by western blot analysis. Although pyrrolo[2,3-d]pyrimidine is a privileged structure in many kinase inhibitors, compound 12a1 showed negligible inhibition against several kinases including JAK family members and Akt1, indicating its acceptable off-target profile. Besides, compound 12a1 exhibited desirable metabolic stability in mouse liver microsome. The in vivo anti-multiple myeloma potency of 12a1, alone and in combination with bortezomib, was demonstrated in a RPMI 8226 xenograft model.

Coronaviruses such as SARS-CoV-2, which is responsible for COVID-19, depend on virus spike protein binding to host cell receptors to cause infection. The SARS-CoV-2 spike protein binds primarily to ACE2 on target cells and is then processed by membrane proteases, including TMPRSS2, leading to viral internalisation or fusion with the plasma membrane. It has been suggested, however, that receptors other than ACE2 may be involved in virus binding. We have investigated the interactions of recombinant versions of the spike protein with human epithelial cell lines that express low/very low levels of ACE2 and TMPRSS2 in a proxy assay for interaction with host cells. A tagged form of the spike protein containing the S1 and S2 regions bound in a temperature-dependent manner to all cell lines, whereas the S1 region alone and the receptor-binding domain (RBD) interacted only weakly. Spike protein associated with cells independently of ACE2 and TMPRSS2, while RBD required the presence of high levels of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we tested the effects of various heparins on ACE2-independent spike protein interaction with cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of

Different amounts of graphene-wrapped magnesium oxide (G@MgO) powders are uniformly dispersed in poly(vinyl alcohol) (PVA) solution in different experiments to obtain solutions which are coagulated to obtain solid materials, which are then hot pressed at 413 K and 3 t of pressure to finally obtain 1 mm thick freestanding G@MgO/PVA composite sheets in which the constituents, namely, graphene and MgO (in the form of G@MgO), are the nanofillers in PVA matrix. During synthesis of G@MgO powder, MgO nanoparticles are in situ wrapped by the graphene nanosheets as revealed by electron microscopy. Uniformity of G@MgO dispersion in PVA was confirmed by secondary electron micrographs and the consistency in X-ray diffraction and Raman scattering data collected from different locations of the samples. Temperature (303-393 K) dependent complex permittivity of G@MgO/PVA composite sheets (including those prepared by casting) in low frequency (20 Hz to 2 MHz) and high frequency (i.e., X-band, 8.2-12.4 GHz) ranges are measured. In both frequency ranges, G@MgO/PVA composite sheets prepared by coagulation exhibited dielectric properties superior to those of PVA and G@MgO/PVA composite sheets prepared by casting. A strong interfacial polarization is observed in coagulated and as-cast G@MgO/PVA composite sheets. It is noticed from the calculated activation energies that conduction is the dominating mechanism for energy transfer in both composite sheets' cases, while it is predominating in coagulated composite sheets due to the better network formation of the fillers in the coagulated samples than in the cast composite samples. The electromagnetic interference shielding effectiveness (EMI SE) values in the X-band frequency range (i.e., 8.2-12.4 GHz) of the G@MgO/PVA composite sheets prepared by coagulation are more than those prepared by casting for a particular weight fraction of G@MgO. At 393 K, for a particular G@MgO/PVA composite sheet prepared by coagulation, an excellent EMI SE of ∼27.5 dB is measured. It is also experimentally elucidated that the absorption is the dominating mechanism for EMI SE in the prepared composite sheets.

Concurrent inhibition of Janus kinase (JAK) and histone deacetylase (HDAC) could potentially improve the efficacy of the HDAC inhibitors in the treatment of cancers and resolve the problem of HDAC inhibitor resistance in some tumors. Here, a novel series of pyrimidin-2-amino-pyrazol hydroxamate derivatives as JAK and HDAC dual inhibitors was designed, synthesized, and evaluated, among which 8m possessed potent and balanced activities against both JAK2 and HDAC6 with half-maximal inhibitory concentration at the nanomolar level. 8m exhibited improved antiproliferative and proapoptotic activities over SAHA and ruxolitinib in several hematological cell lines. Remarkably, 8m exhibited more potent antiproliferation effect than the combination of SAHA and ruxolitinib in HEL cells bearing JAK2V617F mutation. Pharmacokinetic studies in mice showed that 8m possessed good bioavailability after intraperitoneal administration. Finally, 8m showed antitumor efficacy with no significant toxicity in a HEL xenograft model....

Volcanic eruptions in otherwise clean environments are “natural experiments” wherein the effects of aerosol emissions on clouds and climate can be partitioned from meteorological variability and anthropogenic activities. In this work, we combined satellite retrievals, reanalysis products, and atmospheric modeling to analyze the mechanisms of aerosol–cloud interactions during two degassing events at the Kilauea volcano in 2008 and 2018. The eruptive nature of the 2008 and 2018 degassing events was distinct from long-term volcanic activity for Kilauea. Although previous studies assessed the modulation of cloud properties from the 2008 event, this is the first time such an analysis has been reported for the 2018 event and that multiple degassing events have been analyzed and compared at this location. Both events resulted in significant changes in cloud effective radius and cloud droplet number concentration that were decoupled from local meteorology and in line with an enhanced cloud albedo. However, it is likely that the effects of volcanic emissions on liquid water path and cloud fraction were largely offset by meteorological variability. Comparison of cloud anomalies between the two events suggested a threshold response of aerosol–cloud interactions to overcome meteorological effects, largely controlled by aerosol loading. In both events, the ingestion of aerosols within convective parcels enhanced the detrainment of condensate in the upper troposphere, resulting in deeper clouds than observed under pristine conditions. Accounting for ice nucleation on ash particles led to enhanced ice crystal concentrations at cirrus levels and a slight decrease in ice water content, improving the correlation of the model results with the satellite retrievals. Overall, aerosol loading, plume characteristics, and meteorology contributed to changes in cloud properties during the Kilauea degassing events.

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. So far, several different types of vaccines have shown strong efficacy. However, both the emergence of new SARS-CoV-2 variants and the need to vaccinate a large fraction of the world’s population necessitate the development of alternative vaccines, especially those that are simple and easy to store, transport and administer. Here, we showed that ferritin-like Dps protein from hyperthermophilic Sulfolobus islandicus can be covalently coupled with different SARS-CoV-2 antigens via the SpyCatcher system, to form extremely stable and defined multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) shows particular promise as it elicited a higher antibody titre and an enhanced neutralising antibody response compared to the monomeric RBD. Furthermore, we showed that a single immunisation with the multivalent RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to efficient viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.

Graphical abstract, Highlights • N—H⋯O intermolecular interactions elucidates the effect of hyperconjugation. • C—H⋯O intermolecular interactions elucidates the effect of rehybridization. • FT-IR and FT-Raman spectral analysis substantiates the red shift and blue shift in stretching frequencies. • Drug likeness and ADMET analysis reveals pharmacokinetic properties. • Molecular docking shows the interaction of MCMH with SARS-CoV-2 protease., Prospective antiviral molecule (2E)-N-methyl-2-[(4-oxo-4H-chromen-3-yl)methylidene]-hydrazinecarbothioamide has been probed using Fourier transform infrared (FTIR), FT-Raman and quantum chemical computations. The geometry equilibrium and natural bond orbital analysis have been carried out with density functional theory employing Becke, 3-parameter, Lee-Yang-Parr method with the 6-311G++(d,p) basis set. The vibrational assignments pertaining to different modes of vibrations have been augmented by normal coordinate analysis, force constant and potential energy distributions. Drug likeness and oral activity have been carried out based on Lipinski's rule of five. The inhibiting potency of 2(2E)-methyl-2-[(4-oxo-4H-chromen-3-yl)methylidene]-hydrazinecarbothioamide has been investigated by docking simulation against SARS-CoV-2 protein. The optimized geometry shows a planar structure between the chromone and the side chain. Differences in the geometries due to the substitution of the electronegative atom and intermolecular contacts due to the chromone and hydrazinecarbothioamide were analyzed. NBO analysis confirms the presence of two strong stable hydrogen bonded N—H⋯O intermolecular interactions and two weak hydrogen bonded C—H⋯O interactions. The red shift in N—H stretching frequency exposed from IR substantiates the formation of N—H⋯O intermolecular hydrogen bond and the blue shift in C—H stretching frequency substantiates the formation of C—H⋯O intermolecular hydrogen bond. Drug likeness, absorption, distribution, metabolism, excretion and toxicity property gives an idea about the pharmacokinetic properties of the title molecule. The binding energy of the nonbonding interaction with Histidine 41 and Cysteine 145, present a clear view that 2(2E)-methyl-2-[(4-oxo-4H-chromen-3-yl)methylidene]-hydrazinecarbothioamide can irreversibly interact with SARS-CoV-2 protease.

Complex I is the largest and most intricate of the protein complexes of mitochondrial electron transport chain (ETC). This L-shaped enzyme consists of a peripheral hydrophilic matrix domain and a membrane-bound orthogonal hydrophobic domain. The interfacial region between these two arms is known to be critical for binding of ubiquinone moieties and has also been shown to be the binding site of Complex I inhibitors. Knowledge on specific roles of the ETC interfacial region proteins is scarce due to lack of knockout cell lines and animal models. Here we mutated nuclear encoded NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2), one of three protein subunits of the interfacial region, in a human embryonic kidney cell line 293 using a CRISPR/Cas9 procedure. Disruption of NDUFS2 significantly decreased cell growth in medium, Complex I specific respiration, glycolytic capacity, ATP pool and cell-membrane integrity, but significantly increased Complex II respiration, ROS generation, apoptosis, and necrosis. Treatment with idebenone, a clinical benzoquinone currently being investigated in other indications, partially restored growth, ATP pool, and oxygen consumption of the mutant. Overall, our results suggest that NDUFS2 is vital for growth and metabolism of mammalian cells, and respiratory defects of NDUFS2 dysfunction can be partially corrected with treatment of an established mitochondrial therapeutic candidate. This is the first report to use CRISPR/Cas9 approach to construct a knockout NDUFS2 cell line and use the constructed mutant to evaluate the efficacy of a known mitochondrial therapeutic to enhance bioenergetic capacity.

A fundamental property of infectious agents is their particulate nature: infectivity arises from independently-acting particles rather than as a result of collective action. Assemblies of the protein tau can exhibit seeding behaviour, potentially underlying the apparent spread of tau aggregation in many neurodegenerative diseases. Here we ask whether tau assemblies share with classical pathogens the characteristic of particulate behaviour. We used organotypic hippocampal slice cultures from P301S tau transgenic mice in order to precisely control the concentration of extracellular tau assemblies in neural tissue. Whilst untreated slices displayed no overt signs of pathology, exposure to recombinant tau assemblies could result in the formation of intraneuronal, hyperphosphorylated tau structures. However, seeding ability of tau assemblies did not titrate in a one-hit manner in neural tissue. The results suggest that seeding behaviour of tau arises at high concentrations, with implications for the interpretation of high-dose intracranial challenge experiments and the possible contribution of seeded aggregation to human disease. Introduction Neurodegenerative diseases are typified by the accumulation of specific proteins into fibrillar assemblies. In around twenty distinct neurodegenerative diseases, including the most common, Alzheimer’s disease, the protein tau forms hyperphosphorylated, filamentous inclusions within the cytoplasm of neurons. Tau pathology can be a direct cause of neurodegeneration. For instance, human genetic studies reveal that around 50 distinct mutations in MAPT, the gene that encodes tau, cause inherited forms of dementia with evidence of tau filaments [1]. The origin of tau assemblies in the human brain remains uncertain. Cell-autonomous processes may lead to the spontaneous nucleation of oligomeric forms of tau within the cytoplasm of neurons. Some of these assemblies adopt filamentous conformations that are able to undergo extension by the addition of tau monomers to the filament ends. Over the past decade it has been postulated that, in addition to these cell-autonomous mechanisms, tau pathology may occur through a spreading or ‘prion-like’ mechanism [2]. Several lines of evidence demonstrate that assemblies of tau can be taken up into cells, whereupon they seed the conversion of native tau to the assembled state. Addition of tau assemblies to the exterior of cells, or the injection of tau assemblies into the brains of tau-transgenic and wildtype mice, can induce intracellular tau assembly in the recipient [3,4,5,6]. Population cross-sectional studies demonstrate that the appearance of tau pathology follows a predictable pattern over time and space in the human brain, potentially indicating spreading via a prion-like mechanism. Immunoreactivity to antibodies such as AT8, which detects tau that is phosphorylated at positions S202 and T205 [7], progresses in a manner that can be systematically categorised into stages according to anatomical distribution (Braak stages 0–VI) [8, 9]. In young adults, some AT8 immunoreactivity is observed in the vast majority of brains by the third decade of life. However, it is generally confined to neurons within the locus coeruleus (LC) in the brainstem (Braak pretangle stages 0 a–c and 1a,b). Subsequently, AT8 staining is observed in the entorhinal cortex (EC) and hippocampus (HC) (Braak stages I–II). Later stages are characterised by progressive dissemination and increasing density of staining in neocortical regions (Braak stages III–VI). These late stages are associated with severe disease and the overall burden of tau pathology negatively correlates with cognitive function [10]. Though intracranial challenge experiments demonstrate that seeded aggregation can in principle occur, they provide little insight as to whether physiological concentrations of extracellular tau species might support prion-like activity. The concentration of tau in wildtype mouse interstitial fluid (ISF) is around 50 ng/ml total tau (equivalent to ~ 1 nM tau monomer). Wildtype mouse ISF levels typically exceed cerebrospinal fluid (CSF) tau levels by around tenfold [11]. In humans between ages 21–50 years, CSF total tau is below 300 pg/ml increasing to 500 pg/ml over age 70 [12]—approximately 7–12 pM if considering the average mass of full length tau isoforms. Levels are increased 2–threefold in Alzheimer’s disease [13]. If a similar relationship between ISF and CSF tau concentration exists in humans as in mice, ISF tau levels are likely in the order of 100 pM, rising to 300 pM in Alzheimer’s disease. Intracranial injection experiments typically supply tau in the high micromolar range. Even if this were distributed broadly across the brain, micromolar concentrations would be exceeded and local concentration at the injection site may plausibly be 100-fold greater. Thus, intracranial injection experiments likely exceed physiological concentrations of extracellular tau by two to seven orders of magnitude. For classical infectious agents, infectivity is related to dose by a “one-hit” relationship wherein the amount of infectivity decreases linearly upon dilution until end-point [14]. This property is also evident in PrPSc prions, though it is complicated by the presence of multiple aggregation states and the size distribution of particles [15]. The relationship between dose and prion-like activity for tau has not been established. It is therefore currently not possible to reconcile high-dose challenge experiments with the low concentrations of tau observed in the extracellular spaces of the brain. To address this, we developed a model of seeded tau aggregation in mouse organotypic hippocampal slice cultures, allowing direct control of the concentration of tau neurons were exposed to. Brain slice cultures have been used for over 40 years [16], though developments in recent years have rendered them increasingly relevant for the study of neurodegenerative diseases [17,18,19,20,21]. We prepared slices from transgenic mice expressing human tau bearing the P301S mutation [22], which is causative of familial fronto-temporal dementia and displays accelerated fibrilisation compared to wildtype tau [23]. Using our system, we show that neurons within CA1 are preferentially susceptible to seeded aggregation, displaying intracellular hyperphosphorylated tau tangles. We find that seeding activity cannot be titrated down and only occurs at high concentrations of tau assemblies. The concentrations of tau assemblies required to initiate seeding in this model exceed reported physiological ranges of ISF and CSF tau. Our results imply that a model of tau spread via seeded aggregation requires these concentrations to be locally exceeded or requires other mechanisms not captured here to facilitate seeded aggregation. Results Sagittal hippocampal slices of ~ 300 µm thickness were prepared from homozygous P301S tau-transgenic mice at age 7 d, and cultured at the air–liquid interface on culture support membranes (Fig. 1a). Hippocampal structures are well developed at this age, yet the tissue exhibits plasticity that aids recovery from the slicing procedure [24]. We stained OHSCs with antibodies against markers of the major cell types of the brain: neurons (Map2), microglia (Iba1) and astrocytes (Gfap) (Fig. 1b). Similar to previous studies [20], neurons were found to maintain extensive arborisation with evidence of intact neuronal tracts. Microglia were observed with normal morphology with extensive processes, similar to quiescent cells in whole brains [25]. Astrocytes were also well represented throughout the cultures. Immunostaining for P301S tau revealed widespread expression in all regions of the hippocampus (Fig. 1c). After 5 weeks in culture no overt signs of tau pathology were apparent, as visualised by staining with AT8 (Fig. 1d). These results demonstrate that hippocampal architecture and cell types from P301S tau transgenic mice are maintained through the slicing and culture process. Importantly, they demonstrate that OHSCs from P301S tau transgenic mice do not undergo detectable spontaneous aggregation over this time period. Fig. 1 figure1 Organotypic hippocampal slice cultures (OHSCs) maintain cellular diversity and display no spontaneous tau pathology. a Schematic of the production of OHSCs. 300 µm thick sagittal slices are cut from brain hemispheres. The hippocampus is then dissected and cultured at the air–liquid interface on semi-permeable membranes with media supplied to the basal side of the membrane. b OHSCs from mice transgenic for P301S tau were fixed after 2 weeks in culture and stained for nuclei (DAPI), the neuronal marker Map2, the astrocyte marker Gfap, and the microglial marker Iba1. Scale bars, 50 µm. c OHSCs from P301S tau transgenic mice stain positive for human tau-specific antibody HT7 whereas OHSCs from WT mice do not. Scale bars, 250 µm and 25 µm. d OHSCs from P301S tau transgenic mice after 5 weeks in culture display only background levels of staining with the phospho-tau specific antibody AT8. Slices were stained with DAPI and Map2 as above and with pan-tau. Scale bars, 250 µm Full size image To investigate the response of slice cultures to challenge with tau assemblies, we prepared tau from two independent sources. First, we expressed the 0N4R isoform of tau bearing the P301S mutation in E. coli. Recombinant protein was incubated with heparin and, following a lag period, was found to give a fluorescent signal in the presence of thioflavin T, a dye whose fluorescence increases upon binding to β-sheet rich amyloid structures (Fig. 2a). Negative stain transmission electron microscopy revealed the presence of abundant filamentous structures (Fig. 2b). Second, we prepared the sarkosyl-insoluble (SI) fraction from aged P301S tau transgenic mice, a procedure that enriches insoluble tau species. Brain-derived assemblies were subjected to western blot, confirming the presence of hyperphosphorylated, insoluble tau (Fig. 2c). The samples were quantified using a dot-blot method using recombinant fibrillar tau as a standard (Additional file 1: Supplementary Fig. 1). Tau assemblies were added to HEK293 cells stably expressing 0N4R P301S tau-venus, a reporter cell line for seeded aggregation [26]. In this assay, transfection reagents are used to deliver tau assemblies into cells, whereupon tau-venus is observed to form puncta over 1–2 d. This aggregation was previously found to result in the accumulation of tau-venus in the sarkosyl-insoluble pellet [26]. In the present study, abundant venus-positive puncta were detected following challenge with recombinant fibrils or mouse brain derived tau (Fig. 2d). To investigate whether these seeded assemblies bore markers of tau hyperphosphorylation, we stained with the monoclonal antibody AT8 and AT100, which recognises tau phosphorylated at pT212 and pS214. These epitopes occur on tau filaments extracted from post-mortem tauopathy brains, including those from Alzheimer’s disease patients. We observed that challenge with recombinant tau assemblies resulted in colocalization between tau-venus puncta and AT8 or AT100 (Fig. 2e, f). We therefore concluded that our tau preparations contained species able to induce bona fide seeded aggregation in recipient cells. Fig. 2 figure2 Characterisation of seed competent tau assemblies. a Time course of recombinant P301S tau assembly, monitored by Thioflavin T fluorescence. b Representative transmission electron microscopy image of heparin assembled P301S tau. c Aged P301S tau transgenic mouse brain homogenate was immunoblotted for human tau (HT7 antibody) to detect transgenic tau and for cyclophilin B which served as a loading control. Presence of sarkosyl insoluble (SI) tau was confirmed with the HT7 antibody and AT100 (tau phosphorylated at pT212, pT214). Lanes represent preparations from different mice which were subsequently pooled. d Representative images from HEK293 cells expressing P301S tau-venus 48 h after challenge with either recombinant P301S tau assemblies or mouse SI tau in the presence of LF2000. e, f Tau-venus aggregates observed following challenge with tau assemblies stain with AT8 and AT100 demonstrating that the induced tau aggregates are phosphorylated. Scale bars, 20 µm Full size image To test whether OHSCs support seeded aggregation, we treated slices at DIV7 with recombinant tau assemblies or mouse brain derived sarkosyl insoluble (SI) tau. After three days a complete media change was performed to remove the tau, and the OHSCs were incubated for a further three weeks (Fig. 3a). Following treatment with tau assemblies, we observed pronounced AT8 staining, suggesting the presence of intracellular hyperphosphorylated tau structures (Fig. 3b, c). The addition of monomeric tau did not induce these same structures, indicating that the misfolded state of tau was responsible for seeded aggregation (Fig. 3b, d). Furthermore, addition of tau assemblies to OHSCs prepared from wildtype mice did not induce seeded aggregation suggesting that the transgenic P301S tau construct is responsible for the phenotype (Fig. 3b, c). The hyperphosphorylation of tau was accompanied by the accumulation of sarkosyl-insoluble species following seeding in P301S transgenic OHSCs but not in wildtype OHSCs (Fig. 3e, f) (Additional file 1: Supplementary Fig. 2). Whilst the human transgenic tau is therefore required for seeding to occur, we cannot exclude the possibility that endogenous mouse tau also contributes to aggregates in the transgenic slices since the AT8 epitope is identical between human and mouse tau. To test whether exogenously applied tau could be found within the slices, recombinant tau assemblies were supplied to the culture media of wildtype slices, beneath the membrane, for a period of three days. After this time, we observed human tau present in the slices by western blot, indicating the transfer of tau assemblies from the media to the slice (Fig. 3g) (Additional file 1: Supplementary Fig. 2). Taken together, these data demonstrate that exogenously supplied tau assemblies are taken up into OHSCs and can induce the formation of hyperphosphorylated, insoluble tau species. Fig. 3 figure3 Challenging OHSCs with exogenous tau assemblies induces seeded tau aggregation. a Timeline of organotypic hippocampal slice culture preparation and treatment. OHSCs were prepared from P7 pups. After 7 days in vitro (DIV), tau assemblies were added to the media and incubated for 3 days. A complete media change was carried out at the end of the seeding period (pink). At other times (green) 50% media changes were performed twice weekly until fixation at 28 days in vitro (DIV). b OHSCs derived from P301S tau transgenic mice were challenged with either 100 nM recombinant tau assemblies, 100 nM monomeric tau, 5 µL (~ 300 nM) of mouse brain origin sarkosyl insoluble (SI) tau or buffer only. WT OHSCs were challenged with 100 nM recombinant tau assemblies or buffer only. Scale bars, 50 µm. c Quantification of seeding levels in WT and P301S tau transgenic OHSCs, following addition of 100 nM recombinant tau assemblies or buffer only. d Levels of AT8 immunostaining following challenge of OHSCs with monomeric tau (Mono) or heparin assembled tau (Seed). e, f Levels of sarkosyl insoluble tau present in transgenic P301S (e) or WT (f) OHSCs with and without the addition of 100 nM recombinant tau assemblies. Data derived from Western blot, normalised to input levels of tau and loading control. g Levels of human tau (HT7) present in WT OHSCs after 3 days of exposure to 300 nM recombinant tau assemblies applied beneath the membrane. c and d, statistical significance determined by Kruskal–Wallis test by ranks and Dunn’s multiple comparisons test. e–g statistical significance determined by unpaired t-test. For panels c–f, data points represent individual fields of view from multiple slices derived from N = 3 mice per condition. ****P

Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the RING E3 ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when repurposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce virus neutralization or drive Trim-Away. We harness this mechanism for selective degradation of disease-causing huntingtin protein containing long polyglutamine tracts and expand the Trim-Away toolbox with highly active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has implications for targeted protein degradation technologies.

Funder: Boehringer Ingelheim Fonds (Stiftung für medizinische Grundlagenforschung); doi: https://doi.org/10.13039/501100001645, Attachment of ubiquitin (Ub) to proteins is one of the most abundant and versatile of all posttranslational modifications and affects outcomes in essentially all physiological processes. RING E3 ligases target E2 Ub-conjugating enzymes to the substrate, resulting in its ubiquitination. However, the mechanism by which a ubiquitin chain is formed on the substrate remains elusive. Here we demonstrate how substrate binding can induce a specific RING topology that enables self-ubiquitination. By analyzing a catalytically trapped structure showing the initiation of TRIM21 RING-anchored ubiquitin chain elongation, and in combination with a kinetic study, we illuminate the chemical mechanism of ubiquitin conjugation. Moreover, biochemical and cellular experiments show that the topology found in the structure can be induced by substrate binding. Our results provide insights into ubiquitin chain formation on a structural, biochemical and cellular level with broad implications for targeted protein degradation.

The HIV capsid self-assembles a protective conical shell that simultaneously prevents host sensing whilst permitting the import of nucleotides to drive DNA synthesis. This is accomplished through the construction of dynamic, highly charged pores at the centre of each capsid multimer. The clustering of charges required for dNTP import is strongly destabilising and it is proposed that HIV uses the metabolite IP6 to coordinate the pore during assembly. Here we have investigated the role of inositol phosphates in coordinating a ring of positively charged lysine residues (K25) that forms at the base of the capsid pore. We show that whilst IP5, which can functionally replace IP6, engages an arginine ring (R18) at the top of the pore, the lysine ring simultaneously binds a second IP5 molecule. Dose dependent removal of K25 from the pore severely inhibits HIV infection and concomitantly prevents DNA synthesis. Cryo-tomography reveals that K25A virions have a severe assembly defect that inhibits the formation of mature capsid cones. Monitoring both the kinetics and morphology of capsids assembled in vitro reveals that while mutation K25A can still form tubes, the ability of IP6 to drive assembly of capsid cones has been lost. Finally, in single molecule TIRF microscopy experiments, capsid lattices in permeabilised K25 mutant virions are rapidly lost and cannot be stabilised by IP6. These results suggest that the coordination of IP6 by a second charged ring in mature hexamers drives the assembly of conical capsids capable of reverse transcription and infection., Author summary HIV protects its RNA genome while copying it into DNA by carrying out reverse transcription inside its capsid. This is accomplished by importing nucleotides through highly charged pores at the centre of each capsid multimer. These pores contain two rings of positively charged residues–R18 and K25 –but assembling capsids with these features is challenging because they are intrinsically destabilising. Here we show that the metabolite IP6 coordinates both residues within the pore to drive the assembly of stable capsids capable of nucleotide import. R18 or K25 mutants lose infectivity and the ability to synthesise DNA but have differing assembly phenotypes. Mutant K25A is unable to undergo efficient capsid assembly, while replacing K25 with a neutral polar residue partially restores assembly but not infectivity. We propose that IP6-driven assembly is conserved by HIV not because it is the only way to build a capsid, but because it allows the construction of a capsid with a charged pore that can import nucleotides.

The majority of SARS-CoV-2 vaccines in use or in advanced clinical development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described that act against both open and closed conformations. The long-term success of vaccination strategies will depend upon inducing antibodies that provide long-lasting broad immunity against evolving, circulating SARS-CoV-2 strains, while avoiding the risk of antibody dependent enhancement as observed with other Coronavirus vaccines. Here we have assessed the results of immunization in a mouse model using an S protein trimer that is arrested in the closed state to prevent exposure of the receptor binding site and therefore interaction with the receptor. We compared this with a range of other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induce a long-lived, strongly neutralizing antibody response as well as T-cell responses. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralising responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, virus-inhibiting immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines.

Three different procedures were used to deposit aluminium onto O-terminated (100) and (111) boron-doped diamond, with the aim of producing a thermally stable surface with low work function and negative electron affinity. The methods were: (i) deposition of a >20 nm film Al by high-vacuum evaporation, followed by HCl acid wash to remove excess metallic Al, (ii) deposition of

HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.

Aria is a plant hosting a$${350}\,\hbox {m}$$350mcryogenic isotopic distillation column, the tallest ever built, which is being installed in a mine shaft at Carbosulcis S.p.A., Nuraxi-Figus (SU), Italy. Aria is one of the pillars of the argon dark-matter search experimental program, lead by the Global Argon Dark Matter Collaboration. It was designed to reduce the isotopic abundance of$${^{39}\hbox {Ar}}$$39Arin argon extracted from underground sources, called Underground Argon (UAr), which is used for dark-matter searches. Indeed,$${^{39}\hbox {Ar}}$$39Aris a$$\beta $$β-emitter of cosmogenic origin, whose activity poses background and pile-up concerns in the detectors. In this paper, we discuss the requirements, design, construction, tests, and projected performance of the plant for the isotopic cryogenic distillation of argon. We also present the successful results of the isotopic cryogenic distillation of nitrogen with a prototype plant.

The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6) inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant effects without losing affinity and selectivity at HDAC6. Structure-activity relationships led to compound 10b as a hybrid molecule showing pronounced and selective inhibition of HDAC6 (IC50 = 30.7 nM, > 25-fold selectivity over other subtypes). This compound shows comparable DPPH radical scavenging ability to ferulic acid, comparable ORAC value to melatonin and comparable Cu2+ chelating ability to EDTA. It also lacks neurotoxicity on HT-22 cells, exhibits a pronounced immunomodulatory effect, and is active in vivo showing significantly higher efficacy in an AD mouse model to prevent both Aβ25-35-induced spatial working and long-term memory dysfunction at lower dose (0.3 mg/kg) compared to positive control HDAC6 inhibitor ACY1215 and an equimolar mixture of the three entities ACY1215, melatonin and ferulic acid, suggesting potentially disease-modifying properties.

Novel antiviral active molecule 2- [(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluoro- phenyl)acetamide has been synthesised and characterized by FT-IR and FT-Raman spectra. The equilibrium geometry, natural bond orbital calculations and vibrational assignments have been carried out using density functional B3LYP method with the 6-311G++(d,p) basis set. The complete vibrational assignments for all the vibrational modes have been supported by normal coordinate analysis, force constants and potential energy distributions. A detailed analysis of the intermolecular interactions has been performed based on the Hirshfeld surfaces. Drug likeness has been carried out based on Lipinski's rule and the absorption, distribution, metabolism, excretion and toxicity of the title molecule has been calculated. Antiviral potency of 2- [(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluoro-phenyl) acetamide has been investigated by docking against SARS-CoV-2 protein. The optimized geometry shows near-planarity between the phenyl ring and the pyrimidine ring. Differences in the geometries due to the substitution of the most electronegative fluorine atom and intermolecular contacts due to amino pyrimidine were analyzed. NBO analysis reveals the formation of two strong stable hydrogen bonded N – H···N intermolecular interactions and weak intramolecular interactions C – H···O and N – H···O. The Hirshfeld surfaces and consequently the 2D-fingerprint confirm the nature of intermolecular interactions and their quantitative contributions towards the crystal packing. The red shift in N – H stretching frequency exposed from IR substantiate the formation of N – H···N intermolecular hydrogen bond. Drug likeness and absorption, distribution, metabolism, excretion and toxicity properties analysis gives an idea about the pharmacokinetic properties of the title molecule. The binding energy -8.7 kcal/mol of the nonbonding interaction present a clear view that 2- [(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluoro- phenyl) acetamide can irreversibly interact with SARS-CoV-2 protease., Graphical abstract Unlabelled Image, Highlights • NBO analysis elucidates the effect of hyperconjugation and rehybridization. • FT-IR and FT-Raman Spectral analysis substantiates the formation of intermolecular interactions • Hirshfeld surface analysis reveals mutiple hydrogen bonded interactions • Drug likeness and ADMET analysis reveals pharmacokinetic properties • Molecular docking shows the interaction of DAPF with SARS-CoV-2 protease.

Replication forks encounter numerous challenges as they move through eu- and hetero-chromatin during S phase in mammalian cells. These include a variety of impediments to the unwinding of DNA by the replicative helicase such as alternate DNA structures, transcription complexes and R-loops, DNA–protein complexes, and DNA chemical adducts. Much of our knowledge of these events is based on analysis of markers of the replication stress and DNA Damage Response that follow stalling of replisomes. To examine consequences for the replisomes more directly, we developed an approach for imaging collisions of replication forks with the potent block presented by an interstrand crosslink (ICL). The strategy is based on the visualization on DNA fibers of the encounter of replication tracts and an antigen tagged ICL. Our studies revealed an unexpected restart of DNA synthesis past an intact ICL. In addition, and also unexpected, we found two distinct versions of the replisome, one biased toward euchromatin and the other more prominent in heterochromatin. Here, we present details of our experimental procedures that led to these observations.

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain (TMD) and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. We have applied proteomics to identify cellular factors that interact with the cytoplasmic tail of S. We confirmed interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding-site promotes exit from the endoplasmic reticulum (ER), and although COPI-binding should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.

With five histone deacetylase (HDAC) inhibitors approved for cancer treatment, proteolysis-targeting chimeras (PROTACs) for degradation of HDAC are emerging as an alternative strategy for HDAC-targeted therapeutic intervention. Herein, three bestatin-based hydroxamic acids (P1, P2 and P3) were designed, synthesized and biologically evaluated to see if they could work as HDAC degrader by recruiting cellular inhibitor of apoptosis protein 1 (cIAP1) E3 ubiquitin ligase. Among the three compounds, the bestatin-SAHA hybrid P1 exhibited comparable even more potent inhibitory activity against HDAC1, HDAC6 and HDAC8 relative to the approved HDAC inhibitor SAHA. It is worth noting that although P1 could not lead to intracellular HDAC degradation after 6 h of treatment, it could dramatically decrease the intracellular levels of HDAC1, HDAC6 and HDAC8 after 24 h of treatment. Intriguingly, the similar phenomenon was also observed in the HDAC inhibitor SAHA. Cotreatment with proteasome inhibitor bortezomib could not reverse the HDAC decreasing effects of P1 and SAHA, confirming that their HDAC decreasing effects were not due to protein degradation. Moreover, all three bestatin-based hydroxamic acids P1, P2 and P3 exhibited more potent aminopeptidase N (APN, CD13) inhibitory activities than the approved APN inhibitor bestatin, which translated to their superior anti-angiogenic activities. Taken together, a novel bestatin-SAHA hybrid was developed, which worked as a potent APN and HDAC dual inhibitor instead of a PROTAC.

We describe scalable and cost-efficient production of full length, His-tagged SARS-CoV-2 spike glycoprotein trimer by CHO cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both HEK and CHO cells mediated by PEI was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Nichelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in ELISA format to detect IgG antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by PCR, including those who had displayed COVID-19 symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of “stressed” replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late., Eukaryotic replisomes are multiprotein complexes. Here the authors reveal two distinct stressed replisomes, associated with DONSON and FANCM, displaying a bias in replication timing and chromatin domain.

We describe scalable and cost‐efficient production of full length, His‐tagged severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS‐CoV‐2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9‐fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS‐CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9‐fold to 53 mg/L. Purification of recombinant spike by Ni‐chelate affinity chromatography initially yielded a variety of co‐eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme‐linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS‐CoV‐2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID‐19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS‐CoV‐2 trimer which can be used as antigen for mass serological testing.

SUMMARYTrim-Away is a powerful new technology that exploits off-the-shelf antibodies and the E3 RING ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically-activated upon substrate engagement during either its normal immune function or when re-purposed for targeted protein degradation is unknown. Here we show that a mechanism of substrate-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce an antiviral response or drive Trim-Away. We harness this mechanism to expand the Trim-Away toolbox with highly-active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has important implications for targeted protein degradation technologies.

Cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. Various techniques can provide a qualitative survey of which components are found in a given complex. However, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. Here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. We have devised a system in which a given biomolecule is differentially labelled with spectrally distinct fluorescent dyes (label A or B), which are then mixed such that B-labelled molecules are vastly outnumbered by those with label A. Complexes, containing this component, are then simply scored as either being positive or negative for label B. The frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. We demonstrate this method using complexes of Adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. Beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology.The statistical models used in our analysis are available here: https://github.com/sophiamersmann/molecular-counting, the raw data used for molecular counting can be found here: 10.5281/zenodo.3955142.