Search

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3-d]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2R)-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S cKi 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18/CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino] derivative 32. Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45, which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.

The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down's syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit. We have used fragment and structure-based discovery methods to identify a highly selective, well-tolerated, brain-penetrant DYRK1A inhibitor which showed in vivo activity in a tumor model. The inhibitor provides a useful tool compound for further exploration of the effect of DYRK1A inhibition in models of disease.

We report the preparation of thermoresponsive poly(N-isopropylacrylamide) (polyNIPAM) hydrogels via the free radical polymerization of deep eutectic monomer solvents (DEMs) for the first time, where NIPAM serves as a polymerizable hydrogen bond donor. DEMs were prepared by a simple heating and stirring protocol using NIPAM and either choline chloride (ChCl) or acetylcholine chloride (AcChCl) (as hydrogen bond acceptor) in various ratios to yield low-melting point liquids (as low as 15 °C). 1D and 2D NMR spectroscopy supported the association of the NIPAM and choline salts present within the DEM structure, in addition to the low self-diffusion coefficients of the species present compared to when dissolved in water. Thermogravimetric analysis demonstrated an enhanced thermal stability of the DEMs compared to NIPAM. Hydrogels prepared by free-radical polymerization of the prepared DEMs in the presence of N,N′-methylenebisacrylamide (BIS) as crosslinker showed a significant increase in reaction rate compared to the equivalent reaction in water, which was attributed in part to the high viscosity of the DEMs. These gels exhibited thermoresponsive swelling behaviour when immersed in water. Specifically, gels prepared via DEMs featured reduced swelling capacity and increased mechanical strength relative to those prepared by aqueous polymerization, attributed to a significant increase in cross-linking density.

1. Procedure (Note 1) A. 3-Ammoniopropylsulfate (1). An oven-dried single-necked 100 mL round-bottomed flask (14/23 joint), equipped with a 2.5-cm Teflon-coated magnetic stirbar, is charged with 3-amino-1-propanol (11.5 mL, 150 mmol, 1 equiv) (Note 2) and anhydrous dichloromethane (35 mL) (Note 3). A 50 mL pressure-equalizing addition funnel (14/23 joint) equipped with a CaCl2 tube is attached to the round-bottomed flask and is then charged with chlorosulfonic acid (10.5 mL, 159 mmol, 1.06 equiv) (Note 4) using a 20 mL glass luer-lock syringe. The flask is immersed in an ice/water bath and the solution is stirred for 5 min. The chlorosulfonic acid is added dropwise over 30 min, allowing the fumes to escape. A white precipitate is formed during the addition. Once the addition is complete, the reaction is stirred at 0 °C for 20 min and left to warm slowly to room temperature over 30 min (Note 5). Once at room temperature, the reaction mixture is stirred for 1 h. The resulting mixture is filtered through a 70 mm diameter Number 3 Glass filter funnel with a Büchner setup. A bent spatula and methanol (25 mL) (Note 6) are used to remove remaining product from the flask walls. The mixture in the filter funnel is triturated with methanol (40 mL, then 2 × 20 mL) (Note 6), using a spatula to break up the lumps each time. The resulting white solid is broken up into a coarse powder and transferred to a 100 mL round-bottomed flask (29/32 joint), where it is placed on a rotary evaporator (40 °C, 12 mmHg pressure) for 1 h. The resulting white solid is ground to a fine white powder using a 10 cm diameterglass mortar and pestle, retransferred to a 100 mL round-bottomedflask and dried on a high vacuum line (25 °C, 0.1 mmHg pressure) for 2 h giving the title compound 1 (20.72 g, 134 mmol, 89% yield) (Note 7) as a fine white powder.

Author(s): Lu, Mei; Bowlus, Christopher L; Lindor, Keith; Rodriguez-Watson, Carla V; Romanelli, Robert J; Haller, Irina V; Anderson, Heather; VanWormer, Jeffrey J; Boscarino, Joseph A; Schmidt, Mark A; Daida, Yihe G; Sahota, Amandeep; Vincent, Jennifer; Li, Jia; Trudeau, Sheri; Rupp, Loralee B; Gordon, Stuart C; FOLD Investigators | Abstract: BackgroundBiopsy remains the gold standard for determining fibrosis stage in patients with primary biliary cholangitis (PBC), but it is unavailable for most patients. We used data from the 11 US health systems in the FibrOtic Liver Disease Consortium to explore a combination of biochemical markers and electronic health record (EHR)-based diagnosis/procedure codes (DPCs) to identify the presence of cirrhosis in PBC patients.MethodsHistological fibrosis staging data were obtained from liver biopsies. Variables considered for the model included demographics (age, gender, race, ethnicity), total bilirubin, alkaline phosphatase, albumin, aspartate aminotransferase (AST) to platelet ratio index (APRI), Fibrosis 4 (FIB4) index, AST to alanine aminotransferase (ALT) ratio, and g100 DPCs associated with cirrhosis/decompensated cirrhosis, categorized into ten clusters. Using least absolute shrinkage and selection operator regression (LASSO), we derived and validated cutoffs for identifying cirrhosis.ResultsAmong 4328 PBC patients, 1350 (32%) had biopsy data; 121 (9%) were staged F4 (cirrhosis). DPC clusters (including codes related to cirrhosis and hepatocellular carcinoma diagnoses/procedures), Hispanic ethnicity, ALP, AST/ALT ratio, and total bilirubin were retained in the final model (AUROC=0.86 and 0.83 on learning and testing data, respectively); this model with two cutoffs divided patients into three categories (no cirrhosis, indeterminate, and cirrhosis) with specificities of 81.8% (for no cirrhosis) and 80.3% (for cirrhosis). A model excluding DPCs retained ALP, AST/ALT ratio, total bilirubin, Hispanic ethnicity, and gender (AUROC=0.81 and 0.78 on learning and testing data, respectively).ConclusionAn algorithm using laboratory results and DPCs can categorize a majority of PBC patients as cirrhotic or noncirrhotic with high accuracy (with a small remaining group of patients' cirrhosis status indeterminate). In the absence of biopsy data, this EHR-based model can be used to identify cirrhosis in cohorts of PBC patients for research and/or clinical follow-up.

The proliferation of successful, cell-free reconstitutions of cytoskeletal networks have prompted measurements of forces between network elements via induced osmotic pressure by the addition of depletants. Indeed, it was through osmotic pressurization that Tau, an intrinsically disordered protein found in neuronal axons, was recently discovered to mediate two distinct microtubule (MT) bundle states, one widely spaced and a second tightly packed, separated by an energy barrier due to polyelectrolyte repulsions between opposing Tau projection domains on neighboring MT surfaces. Here, we compare interfilament force measurements in Tau coated MT bundles using PEO20k (poly(ethylene oxide), Mw = 20000), a commonly used inert depletant, and recently published measurements with PEO102k. While force measurements with either depletant reveals the transition between the two bundled states, measurements with PEO20k cannot recapitulate the correct critical pressure (Pc) at which widely spaced MT bundles transition to ...

Poly ADP-ribose polymerase inhibitors (PARPi) have transformed ovarian cancer (OC) treatment, primarily for tumours deficient in homologous recombination repair. Combining VEGF-signalling inhibitors with PARPi has enhanced clinical benefit in OC. To study drivers of efficacy when combining PARP inhibition and VEGF-signalling, a cohort of patient-derived ovarian cancer xenografts (OC-PDXs), representative of the molecular characteristics and drug sensitivity of patient tumours, were treated with the PARPi olaparib and the VEGFR inhibitor cediranib at clinically relevant doses. The combination showed broad anti-tumour activity, reducing growth of all OC-PDXs, regardless of the homologous recombination repair (HRR) mutational status, with greater additive combination benefit in tumours poorly sensitive to platinum and olaparib. In orthotopic models, the combined treatment reduced tumour dissemination in the peritoneal cavity and prolonged survival. Enhanced combination benefit was independent of tumour cell expression of receptor tyrosine kinases targeted by cediranib, and not associated with change in expression of genes associated with DNA repair machinery. However, the combination of cediranib with olaparib was effective in reducing tumour vasculature in all the OC-PDXs. Collectively our data suggest that olaparib and cediranib act through complementary mechanisms affecting tumour cells and tumour microenvironment, respectively. This detailed analysis of the combined effect of VEGF-signalling and PARP inhibitors in OC-PDXs suggest that despite broad activity, there is no dominant common mechanistic inter-dependency driving therapeutic benefit. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-021-01196-x.

Significance Spatial organization of mitochondria is vital for cellular function. In many specialized cell types, mitochondria are immobilized at specific subcellular loci through interactions with the cytoskeleton. One of the most striking mitochondrial configurations occurs in mammalian sperm, where mitochondria wrap around the flagellum. Malformation of the mitochondrial sheath causes infertility, but the molecular structures underlying this intricate arrangement are unknown. Here, we analyzed the mitochondrial sheath in sperm from three mammalian species. We find that although mitochondrial dimensions and cristae architecture vary across species, molecular assemblies mediating intermitochondria and mitochondria–cytoskeleton interactions are conserved. These findings yield important insight into sperm physiology and evolution and are relevant for other polarized cell types, such as muscles, neurons, photoreceptors, and hair cells., Mitochondria–cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria–cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

The spontaneous zwitterionic copolymerization (SZWIP) of 2-oxazolines and acrylic acid affords biocompatible but low molecular weight linear N-acylated poly(amino ester)s (NPAEs). Here, we present a facile one-step approach to prepare functional higher molar mass cross-linked NPAEs using 2,2′-bis(2-oxazoline)s (BOx). In the absence of solvent, insoluble free-standing gels were formed from BOx with different length n-alkyl bridging units, which when butylene-bridged BOx was used possessed an inherent green fluorescence, a behavior not previously observed for 2-oxazoline-based polymeric materials. We propose that this surprising polymerization-induced emission can be classified as nontraditional intrinsic luminescence. Solution phase and oil-in-oil emulsion approaches were investigated as means to prepare solution processable fluorescent NPAEs, with both resulting in water dispersible network polymers. The emulsion-derived system was investigated further, revealing pH-responsive intensity of emission and excellent photostability. Residual vinyl groups were shown to be available for modifications without affecting the intrinsic fluorescence. Finally, these systems were shown to be cytocompatible and to function as fluorescent bioimaging agents for in vitro imaging.

The occurrence of covalent grafting of polymer chains onto graphene oxide (GO) sheets during aqueous miniemulsion radical polymerizations of common vinyl monomers has been investigated. Styrene, benzyl methacrylate and t-butyl methacrylate were polymerized via miniemulsion polymerization using GO as sole surfactant, and the characterization of GO isolated post-polymerization was investigated by a range of experimental techniques (Fourier-Transform Near Infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), Differential Scanning Calorimetry (DSC), X-Ray Photoelectron spectroscopy (XPS), Raman spectroscopy and X-Ray Diffraction (XRD)). All experimental techniques show consistently that significant grafting occurs for all three monomers under these conditions. Furthermore, Raman spectroscopy indicated that the mechanism of grafting involves the sp2 carbons present in the GO structure, and not the oxygen-containing functional groups (e.g. epoxy, hydroxyl and carboxyl). The extent of grafting was quantified by XPS, revealing that polymer grafting occurred at levels in the approximate range of 20-60 wt% (grafted polymer relative to total mass of GO and grafted polymer). Miniemulsion polymerization using GO as surfactant is an attractive approach for synthesis of polymer/GO nanocomposites, and the present results are important in regards to the specific structures and performances of such materials.

The microtubule (MT)-associated protein tau regulates the critical growing and shortening behaviors of MTs, and its normal activity is essential for neuronal development and maintenance. Accordingly, aberrant tau action is tightly associated with Alzheimer's disease and is genetically linked to several additional neurodegenerative diseases known as tauopathies. Although tau is known to promote net MT growth and stability, the precise mechanistic details governing its regulation of MT dynamics remain unclear. Here, we have used the slowly-hydrolyzable GTP analog, guanylyl-(α,β)-methylene-diphosphonate (GMPCPP), to examine the structural effects of tau at MT ends that may otherwise be too transient to observe. The addition of both four-repeat (4R) and three-repeat (3R) tau isoforms to pre-formed GMPCPP MTs resulted in the formation of extended, multiprotofilament-wide projections at MT ends. Furthermore, at temperatures too low for assembly of bona fide MTs, both tau isoforms promoted the formation of long spiral ribbons from GMPCPP tubulin heterodimers. In addition, GMPCPP MTs undergoing cold-induced disassembly in the presence of 4R tau (and to a much lesser extent 3R tau) also formed spirals. Finally, three pathological tau mutations known to cause neurodegeneration and dementia were differentially compromised in their abilities to stabilize MT disassembly intermediates. Taken together, we propose that tau promotes the formation/stabilization of intermediate states in MT assembly and disassembly by promoting both longitudinal and lateral tubulin–tubulin contacts. We hypothesize that these activities represent fundamental aspects of tau action that normally occur at the GTP-rich ends of GTP/GDP MTs and that may be compromised in neurodegeneration-causing tau variants.

The role of ribavirin (RBV) in the era of direct-acting antivirals (DAA) is not clear, and DAA studies have been largely genotype- and regimen-specific. Using data from the Chronic Hepatitis Cohort Study, we evaluated the role of RBV and increased DAA treatment duration among patients with chronic hepatitis C (HCV) in routine clinical care. We performed multivariable analysis of data from 4133 patients receiving any of the following: sofosbuvir (SOF); daclatasvir + SOF; grazoprevir + elbasvir; paritaprevir/ritonavir + ombitasvir; simeprevir + SOF; and SOF + ledipasvir; SOF + velpatasvir ± voxilaprevir; and glecaprevir + pibrentasvir—all with/ without RBV. Inverse probability treatment weighting was used to adjust for treatment selection bias. Sustained virological response (SVR) was defined by undetectable HCV RNA 12 weeks after end of therapy. The overall SVR rate was 95%. Mean treatment duration was 12 ± 4.5 weeks. The final model included treatment duration and diabetes, as well as the interaction of RBV with previous treatment status (treatment naïve, interferon treatment failure [TF] or previous DAA TF), cirrhosis status, and HCV genotype (GT). Each one-month increment of treatment duration increased odds of SVR by 99% (aOR = 1.99). Diabetes, previous DAA TF, and decompensated cirrhosis significantly reduced odds of SVR. RBV significantly increased the likelihood of SVR among patients with decompensated cirrhosis (aOR = 5.05), previous DAA treatment failure (aOR = 5.43), and GT3 (aOR = 13.28). Among RBV-free regimens, patients with GT3 were less likely to achieve SVR than those with GT1 or 2 (aOR 0.07). Diabetes, decompensated cirrhosis, and prior DAA TF independently reduced the likelihood of SVR. Longer treatment duration increased likelihood of SVR. Conclusion: RBV increased likelihood of SVR among patients with GT3, previous DAA TF, or decompensated cirrhosis.

A photocatalyzed ortho-alkylation of pyridine N-oxide with ynamides and arylacetylenes has been developed, which yields a series of α-(2-pyridinyl) benzyl amides/ketones. Mechanistic studies, including electrochemical studies, radical-trapping experiments, and Stern-Volmer fluorescence quenching studies demonstrate that pyridine N-oxide serves as both a redox auxiliary and radical acceptor to achieve the mild photocatalytic single-electron oxidation of carbon-carbon triple bonds with the generation of a cationic vinyl radical intermediate.

Early development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, much remains to be learned at a molecular level about protective E2-reactive antibodies, since HCV infection persists in some individuals despite early development of broadly neutralizing plasma. To examine B cell repertoire features associated with broad neutralization and viral clearance, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of people with cleared or persistent HCV, including subjects with high or low plasma neutralizing breadth in both clearance and persistence groups. We identified many E2-reactive public BCR clonotypes, which are antibody clones with the same V and J-genes and identical CDR3 sequences, shared among subjects grouped by either clearance or neutralization status. The majority (89) of these public clonotypes were shared by two subjects with broad plasma neutralizing activity and cleared infection, but not found in subjects with high plasma neutralizing breadth and persistent infection. We cloned a potent, cross-reactive neutralizing monoclonal antibody (mAb) by pairing the most abundant public heavy and light chains from these two subjects, providing evidence that broadly E2-reactive public clonotypes arise in a subset of individuals with broadly neutralizing plasma and spontaneous clearance of infection. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.

A direct and asymmetric aldol reaction of N‐acyl thiazinanethiones with aromatic aldehydes catalyzed by chiral nickel(II) complexes is reported. The reaction gives the corresponding O‐TIPS‐protected anti‐aldol adducts in high yields and with remarkable stereocontrol and atom economy. Furthermore, the straightforward removal of the achiral scaffold provides enantiomerically pure intermediates of synthetic interest, which involve precursors for anti‐α‐amino‐β‐hydroxy and α,β‐dihydroxy carboxylic derivatives. Theoretical calculations explain the observed high stereocontrol., Appropriate and simultaneous activation of robust and easy to handle [Tol‐BINAP]NiCl2 and aromatic aldehydes with TIPSOTf orchestrates a direct, asymmetric, and catalytic aldol reaction of a wide array of N‐acyl‐1,3‐thiazinane‐2‐thiones. This method enables the corresponding TIPS‐protected anti aldol adducts to be obtained in a highly efficient and atom economical manner.

Investigation of highly oxidized graphene oxide (GO) by solid-state nuclear magnetic resonance (NMR) spectroscopy has revealed an exceptional level of hitherto undiscovered structural complexity. A number of chemical moieties were observed for the first time, such as terminal esters, furanic carbons, phenolic carbons, and three distinct aromatic and two distinct alkoxy carbon moieties. Quantitative one-dimensional (1D) and two-dimensional (2D) 13C{1H} NMR spectroscopy established the relative populations and connectivity of these different moieties to provide a consistent "local" chemical structure model. An inferred 2 nm GO sheet size from a very large (∼20%) edge carbon fraction by NMR analysis is at odds with the >20 nm sheet size determined from microscopy and dynamic light scattering. A proposed kirigami model where extensive internal cuts/tears in the basal plane provide the necessary edge sites is presented as a resolution to these divergent results. We expect this work to expand the fundamental understanding of this complex material and enable greater control of the GO structure.

1 Mitochondria-cytoskeleton interactions modulate cellular 2 physiology by regulating mitochondrial transport, position-3 ing, and immobilization. However, there is very little struc-4 tural information defining mitochondria-cytoskeleton inter-5 faces in any cell type. Here, we use cryo-focused ion beam 6 milling-enabled cryo-electron tomography to image mam-7 malian sperm, where mitochondria wrap around the ciliary 8 cytoskeleton. We find that mitochondria are tethered to their 9 neighbors through inter-mitochondrial linkers and are an-10 chored to the cytoskeleton through ordered arrays on the 11 outer mitochondrial membrane. We use subtomogram aver-12 aging to resolve in-cell structures of these arrays from three 13 mammalian species, revealing they are conserved across 14 species despite variations in mitochondrial dimensions and 15 cristae organization. We find that the arrays consist of 16 boat-shaped particles anchored on a network of membrane 17 pores whose arrangement and dimensions are consistent 18 with voltage dependent anion channels. Proteomics and in-19 cell cross-linking mass spectrometry suggest that the con-20 served arrays are composed of glycerol kinase-like proteins. 21 Ordered supramolecular assemblies may serve to stabilize 22 similar contact sites in other cell types where mitochondria 23 need to be immobilized in specific subcellular environments, 24 such as in muscles and neurons. 25 sperm | mitochondria-cytoskeleton contact | cryo-electron tomography | cryo-26 FIB milling | cross-linking mass spectrometry | subtomogram averaging 27

Adenosine, a major neuromodulator in the central nervous system (CNS), is involved in a variety of regulatory functions such as the sleep/wake cycle. Because exogenous adenosine displays dark- and night-mimicking effects in the vertebrate retina, we tested the hypothesis that a circadian (24 h) clock in the retina uses adenosine to control neuronal light responses and information processing. Using a variety of techniques in the intact goldfish retina including measurements of adenosine overflow and content, tracer labeling, and electrical recording of the light responses of cone photoreceptor cells and cone horizontal cells (cHCs), which are post-synaptic to cones, we demonstrate that a circadian clock in the retina itself—but not activation of melatonin or dopamine receptors—controls extracellular and intracellular adenosine levels so that they are highest during the subjective night. Moreover, the results show that the clock increases extracellular adenosine at night by enhancing adenosine content so that inward adenosine transport ceases. Also, we report that circadian clock control of endogenous cone adenosine A2A receptor activation increases rod-cone gap junction coupling and rod input to cones and cHCs at night. These results demonstrate that adenosine and A2A receptor activity are controlled by a circadian clock in the retina, and are used by the clock to modulate rod-cone electrical synapses and the sensitivity of cones and cHCs to very dim light stimuli. Moreover, the adenosine system represents a separate circadian-controlled pathway in the retina that is independent of the melatonin/dopamine pathway but which nevertheless acts in concert to enhance the day/night difference in rod-cone coupling.

The vertebrate retina, like most other brain regions, undergoes relatively slow alterations in neural signaling in response to gradual changes in physiological conditions (e.g., activity changes to rest), or in response to gradual changes in environmental conditions (e.g., day changes into night). As occurs elsewhere in the brain, the modulatory processes that mediate slow adaptation in the retina are driven by extrinsic signals (e.g., changes in ambient light level) and/or by intrinsic signals such as those of the circadian (24-h) clock in the retina. This review article describes and discusses the extrinsic and intrinsic modulatory processes that enable neural circuits in the retina to optimize their visual performance throughout day and night as the ambient light level changes by ~10 billion-fold. In the first synaptic layer of the retina, cone photoreceptor cells form gap junctions with rods and signal cone-bipolar and horizontal cells (HCs). Distinct extrinsic and intrinsic modulatory processes in this synaptic layer are mediated by long-range feedback of the neuromodulator dopamine. Dopamine is released by dopaminergic cells, interneurons whose cell bodies are located in the second synaptic layer of the retina. Distinct actions of dopamine modulate chemical and electrical synapses in day and night. The retinal circadian clock increases dopamine release in the day compared to night, activating high-affinity dopamine D4receptors on cones. This clock effect controls electrical synapses between rods and cones so that rod-cone electrical coupling is minimal in the day and robust at night. The increase in rod-cone coupling at night improves the signal-to-noise ratio and the reliability of very dim multi-photon light responses, thereby enhancing detection of large dim objects on moonless nights.Conversely, maintained (30 min) bright illumination in the day compared to maintained darkness releases sufficient dopamine to activate low-affinity dopamine D1receptors on cone-bipolar cell dendrites. This non-circadian light/dark adaptive process regulates the function of GABAAreceptors on ON-cone-bipolar cell dendrites so that the receptive field (RF) surround of the cells is strong following maintained bright illumination but minimal following maintained darkness. The increase in surround strength in the day following maintained bright illumination enhances the detection of edges and fine spatial details.

Key points Although cone and rod photoreceptor cells in the retina have a type of cannabinoid receptor called a CB1 receptor, little is known about how cannabinoids, the active component in marijuana, affect retinal function. Studies have shown that a circadian (24-hour) clock in the retina uses dopamine receptors, which are also on photoreceptors, to regulate gap junctions (a type of cell-to-cell communication) between rods and cones, so that they are functional (open) at night but closed in the day. We show that CB1 receptors have opposite effects on rod-cone gap junctions in day and night, decreasing communication in the day when dopamine receptors are active and increasing communication when dopamine receptors are inactive. CB1 and dopamine receptors thus work together to enhance the day/night difference in rod-cone gap junction communication. The increased rod-cone communication at night due to cannabinoid CB1 receptors may help improve night vision. Abstract Cannabinoid CB1 receptors and dopamine D4 receptors in the brain form receptor complexes that interact but the physiological function of these interactions in intact tissue remains unclear. In vertebrate retina, rods and cones, which are connected by gap junctions, express both CB1 and D4 receptors. Because the retinal circadian clock uses cone D4 receptors to decrease rod-cone gap junction coupling in the day and to increase it at night, we studied whether an interaction between cone CB1 and D4 receptors increases the day/night difference in rod-cone coupling compared to D4 receptors acting alone. Using electrical recording and injections of Neurobiotin tracer into individual cones in intact goldfish retinas, we found that SR141716A (CB1 receptor antagonist) application alone in the day increased both the extent of rod-cone tracer coupling and rod input to cones, which reaches cones via open gap junctions. Conversely, SR141716A application alone at night or SR141716A application in the day following 30-min spiperone (D4 receptor antagonist) application decreased both rod-cone tracer coupling and rod input to cones. These results show that endogenous activation of cone CB1 receptors decreases rod-cone coupling in the day when D4 receptors are activated but increases it at night when D4 receptors are not activated. Therefore, the D4 receptor-dependent day/night switch in the effects of CB1 receptor activation results in an enhancement of the day/night difference in rod-cone coupling. This synergistic interaction increases detection of very dim large objects at night and fine spatial details in the day. This article is protected by copyright. All rights reserved.

SummaryMotile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures rein-forcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

Background TAF demonstrated noninferior efficacy to TDF with a superior bone and renal safety profile, in viremic chronic HBV (CHB) patients through week 96, and in virally suppressed patients switched from TDF to TAF at 48 weeks. The duration of prior TDF therapy may influence the degree and rate of recovery of bone and renal function following switch to TAF. Here, we evaluate the impact of prior TDF treatment duration on the safety profile of TAF in virally suppressed patients. Methods In a double-blind, randomized, multicenter, active-controlled, Phase 3 study, 488 CHB patients who were virologically suppressed on TDF for 1 year, and on TDF monotherapy for 6 months were randomized 1:1 to switch to TAF or continue TDF for 48 weeks. In patients originally randomized to receive TAF, renal, bone, and lipid parameters were categorically evaluated by the duration of prior TDF treatment: Results Of the 243 patients switched to TAF from TDF, 105 (43%) and 138 (57%) had received TDF for 50 years, 74% male, 80% Asian, median eGFRCG91 mL/min, 4% and 12% had osteoporosis at hip and spine, respectively. Similar changes in renal, bone, and lipid parameters following 48 weeks of TAF treatment were observed by TDF treatment duration. A comparable increase in median eGFRCG, decreases in tubular biomarkers (beta-2 microglobulin/creatinine ratio, retinol binding protein/creatinine ratio), increases in mean percent change in hip and spine BMD, and decreases in bone turnover markers (C-type collagen sequence, procollagen type 1 N-terminal propeptide) were observed among patients previously treated with Conclusions In virally suppressed patients on TDF a median of 4 years who switched to TAF treatment, improvements in bone and renal parameters and changes in fasting lipids were not impacted by prior duration of TDF use.

SARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.

Emulsions are mixtures of two immiscible liquids in which droplets of one are dispersed in a continuous phase of the other. The most common emulsions are oil-water systems, which have found widespread use across a number of industries, for example, in the cosmetic and food industries, and are also of advanced scientific interest. In addition, the past decade has seen a significant increase in both the design and application of nonaqueous emulsions. This has been primarily driven by developments in understanding the mechanism of effective stabilization of oil-in-oil (o/o) systems, either using block copolymers (BCPs) or solid (Pickering) particles with appropriate surface functionality. These systems, as highlighted in this review, have enabled emergent applications in areas such as pharmaceutical delivery, energy storage, and materials design (e.g., polymerization, monolith, and porous polymer synthesis). These o/o emulsions complement traditional emulsions that utilize an aqueous phase and allow the use of materials incompatible with water. We assess recent advances in the preparation and stabilization of o/o emulsions, focusing on the identity of the stabilizer (BCP or particle), the interplay between stabilizer and oils, and highlighting applications and opportunities associated with o/o emulsions.

Nanoparticles (NPs) undergo a number of changes in environmental systems which are often influenced by their interaction with natural organic matter (NOM). However, despite their importance and prevalence,these interactions are still not fully understood. To better understand the interaction between NOM and NPs, an affinity capillary electrophoresis (ACE) method is presented, which quantitatively compares the binding between various NPs and NOM by determining the binding or dissociation constant (KD) in addition to the Hill constant (n). The Hill isotherm provided greater agreement with experimental observation than either the Langmuir or Freundlich isotherms. The method was applied to citrate stabilized gold and silver nanoparticles from different suppliers, varying in size, zeta potential and stabilizing agent content. The total amount of stabilizing agent (citrate) was measured by ion chromatography (IC) after dissolution of the NPs and the surface coverage of citrate on the NPs was determined. The surface coverage revealed that the citrate multilayer around a NP is the same regardless of the concentration of citrate in the suspension. The analysis of different NPs demonstrated that when the diameter of the NPs was less than 10 nm (surface area > 10 m2 g−1) the KD was lower than that for the larger sized NPs. Additionally the citrate content inthe suspension was observed to be the main chemical property influencing the binding of NOM to citrate stabilized gold and silver NPs. The proposed method can be applied to other NPs and with different testing conditions to assess how each individual chemical property of the NP and NOM influences their interaction.As such, this method will allow for better fate predictions for NPs in different environmental systems. usc Refereed/Peer-reviewed

Despite widespread use of lactulose and rifaximin for the treatment of hepatic encephalopathy, this complication of advanced liver disease remains a major burden on the health care system in the United States and continues to predispose to high morbidity and mortality. Several agents have surfaced over recent years with promise to treat hepatic encephalopathy and mitigate the cognitive impairment associated with this disease process. The purpose of this article is to highlight the leading emerging therapies in hepatic encephalopathy as well as their therapeutic targets.

The use of amphiphilic triblock copolymers bearing a reactive alkoxysilane middle block as polymeric stabilizers is reported in this work. A series of poly(ethylene glycol) methyl ether methacrylate‐b‐(3‐trimethoxysilyl)propyl methacrylate‐b‐benzyl methacrylate (PEGMA‐b‐MPS‐b‐BzMA) triblock copolymers were prepared by RAFT solution polymerization and polymerization‐induced self‐assembly (PISA), respectively, where the various block lengths and overall composition were varied. The copolymers prepared by solution polymerization were employed as oil‐in‐water stabilizers where upon application of a catalyst, the 3‐(trimethoxysilyl)propyl methacrylate (MPS) block at the droplet interface was crosslinked to yield capsule‐like structures. The effectiveness of interfacial crosslinking was validated by dynamic light scattering and electron microscopy. In situ self‐assembly by the PISA method resulted in spherical nanoparticles of controllable size that were readily crosslinked by addition of base, with significant enhancement of colloidal stability.

The reversibility of chemotherapy-induced peripheral neuropathy (CIPN), a disabling and potentially permanent side effect of microtubule-targeting agents (MTAs), is becoming an increasingly important issue as treatment outcomes improve. The molecular mechanisms regulating the variability in time to onset, severity, and time to recovery from CIPN between the common MTAs paclitaxel and eribulin are unknown. Previously (Benbow et al. in Neurotox Res 29:299-313, 2016), we found that after 2 weeks of a maximum tolerated dose (MTD) in mice, paclitaxel treatment resulted in severe reductions in axon area density, higher frequency of myelin abnormalities, and increased numbers of Schwann cell nuclei in sciatic nerves. Biochemically, eribulin induced greater microtubule-stabilizing effects than paclitaxel. Here, we extended these comparative MTD studies to assess the recovery from these short-term effects of paclitaxel, eribulin, and a third MTA, ixabepilone, over the course of 6 months. Paclitaxel induced a persistent reduction in axon area density over the entire 6-month recovery period, unlike ixabepilone- or eribulin-treated animals. The abundance of myelin abnormalities rapidly declined after cessation of all drugs but recovered most slowly after paclitaxel treatment. Paclitaxel- and ixabepilone- but not eribulin-treated animals exhibited increased Schwann cell numbers during the recovery period. Tubulin composition and biochemistry rapidly returned from MTD-induced levels of α-tubulin, acetylated α-tubulin, and end-binding protein 1 to control levels following cessation of drug treatment. Taken together, sciatic nerve axons recovered more rapidly from morphological effects in eribulin- and ixabepilone-treated animals than in paclitaxel-treated animals and drug-induced increases in protein expression levels following paclitaxel and eribulin treatment were relatively transient.

Chronic Hepatitis Cohort Study (CHeCS) publications using data from "real-world" patients with hepatitis C virus (HCV) have described demographic disparities in access to care; rates of advanced liver disease, morbidity, and mortality (2.5%-3.5% per year during 2006-10, although only 19% of all CHeCS decedents, and just 30% of those with deaths attributed to liver disease, had HCV listed on death certificate); substantial comorbidities, such as diabetes, advanced liver fibrosis (29% prevalence), renal disease, and depression, and partial reversal of all these with successful antiviral therapy; patient risk behaviors; and use of noninvasive markers to assess liver disease.

Treatment of homoallylic N-tosyl amines or allylic N-tosyl hydroxylamines with 1.5 equiv of a malonoyl peroxide provides a stereoselective method to access functionalized pyrrolidines and isoxazolidines. This metal free alkene oxyamination proceeds in 50-85% yield and up to 13:1 trans-selectivity. In addition, the relative stereochemistry of the oxygen and nitrogen substituents can be inverted through an oxidation/reduction sequence or inverting the stereochemistry of the starting alkene. Mechanistic investigations show a higher reactivity for hydroxyl nucleophiles over sulfonamide nucleophiles revealing a preference for dioxygenation over oxyamination.

Copolymer/water interfacial tensions of statistical copolymers of styrene/ n-butyl acrylate were estimated by pendant drop tensiometry using an "inverse" configuration according to which a drop of water was formed in toluene/copolymer solutions. The study first involved the precise measurement of copolymer solutions density using pycnometry. Subsequently, interfacial tensions of copolymer solutions against water were plotted as a function of copolymer concentration in toluene. Several methods were explored to fit the experimental data and obtain estimates of copolymer/water interfacial tensions at 100% copolymer concentration in toluene by extrapolation. The Belton-Evans extrapolation resulted in the best fit with the experimental data. When plotted as a function of the styrene composition of the copolymer, the interfacial tensions estimates followed an additivity relationship. This enabled estimation of the copolymer/water interfacial tensions directly from their respective homopolymer/water interfacial tensions values. These results are particularly useful for the prediction of composite particle morphology involving copolymerization of multiple monomers.

Graphene features an array of extraordinary mechanical, physical and electrical properties, and it is of great interest to impart such properties to polymeric materials by use of graphene and its derivatives as filler materials. The focus of this comprehensive review is on the preparation of graphene-based polymeric nanocomposite materials by use of synthetic approaches involving aqueous emulsions of polymer (nano)particles. These techniques can be broadly categorized as in situ polymerization (where the monomer(s) is/are polymerized in the presence of the composite phase) and solution blending, where an aqueous dispersion of polymer particles is prepared separately and subsequently mixed with an aqueous dispersion of graphene-based material. Many of these techniques involve the use of derivatives of graphene such as graphene oxide and “reduced” graphene oxide. The fundamental challenge is that graphene as well as graphene oxide have low compatibility with most synthetic polymers, and it is consequently difficult to prepare homogeneous polymer nanocomposites without aggregation and restacking. One of the important features of graphene oxide is that it can act as a surfactant in oil-water emulsions, which has been exploited extensively for preparation of a range of composite particles/materials. Polymer/graphene nanocomposite synthesis via emulsion-based approaches is an active field of research with plenty of opportunities for significant future advancement with a range of potential applications.

Methods: MAGELLAN-1 Part 2 was a randomized, open-label, phase 3 study to evaluate the efficacy and safety of ribavirin (RBV)-free glecaprevir and pibrentasvir (G/P; 300 mg/120 mg) in patients with chronic HCV and prior virologic failure on at least one NS3/4A protease and/or NS5A inhibitor-containing therapy. Patients with compensated liver disease, with or without cirrhosis, and HCV genotype (GT) 1, 4, 5, or 6 were randomized 1:1 to receive 12 or 16 weeks of G/P. The primary endpoint was sustained virologic response at 12 weeks post-treatment (SVR12). Results: Among 91 patients treated, 87 had GT1 and four had GT4 infection. SVR12 was achieved by 89% (39/44) and 91% (43/47) of patients who received 12 and 16 weeks of G/P, respectively. Virologic relapse occurred in 9% (4/44) of patients treated with 12 weeks of G/P; there were no relapses with 16 weeks of treatment. Prior treatment history with one class of inhibitor (protease or NS5A) had no impact on SVR12, while prior treatment with both classes of inhibitors was associated with lower SVR12 rate. The most common adverse event was headache (≥10% of patients) and there were no serious adverse events (AEs) assessed as related to study drugs or AEs leading to discontinuation. Conclusions: Sixteen weeks of G/P treatment achieved a high SVR12 rate in patients with HCV GT1 infection and prior failure to regimens containing either NS5A inhibitors or NS3 protease inhibitors. Background: Patients with hepatitis C virus (HCV) who have virologic failure after treatment containing an NS5A inhibitor have limited retreatment options. This article is protected by copyright. All rights reserved.

A series of polymerized high internal phase emulsion (polyHIPE) materials have been prepared by using a water in oil emulsion stabilized by a macro-RAFT agent, 2-(butylthiocarbonothioylthio)-2-poly(styrene)-b-poly(acrylic acid), acting as a polymeric surfactant. The pore structures of the formed polyHIPEs were closed. By removing the RAFT-endgroup of the amphiphilic macro-RAFT agent, the obtained polyHIPEs possessed an open structure with voids. The effect of the RAFT-endgroup of the amphiphilic macro-RAFT agent on the surface chemistry of the polyHIPEs is discussed. The obtained polyHIPEs via this surfactant-assisted functionalization strategies were characterized by FTIR spectroscopy, FTIR mapping, SEM, SEM-EDX, TEM, XPS as well as synchrotron-based scanning transmission X-ray microscopy (STXM). The latter technique revealed the surface chemistry of the obtained polyHIPEs and macro-RAFT agent multicomponents with a surface spatial resolution of the order of 30-100 nm. Refereed/Peer-reviewed

We have studied the electrical and optical characteristics of an air-cooled argon excimer vacuum-ultraviolet lamp ( $\lambda \sim 126$ nm) excited by a dielectric barrier discharge powered by: 1) pulsed or 2) sinusoidal high-voltage drivers from 32 to 100 kHz. Compared to sinusoidal excitation, pulsed excitation gives nearly $\sim 2\times $ higher vacuum-ultraviolet (VUV) output and electrical-to-VUV conversion efficiency at high pressure (800–900 mbar). Visually, the pulse-driven plasma is spatially homogeneous, whereas for sinusoidal excitation the plasma becomes filamentary at higher pressure and/or frequency. Spectral emission is highly monochromatic with most of the output in the desired VUV band ( $\lambda = 115$ –140 nm). With the lamp running at pressure >700 mbar and power loadings >1.6 W/cm3, a sharp spike in VUV output was consistently seen at turn-on. We believe that transient phenomena or favorable initial conditions may be partly responsible for this VUV spike, although the equilibrium VUV output appears to be limited due to thermal dissipation, gas heating, and associated loss of gas from the active region. We propose that we may be observing the same intrinsic VUV spiking phenomena as reported in liquid nitrogen-cooled Xe, Kr, and Ar excimer lamps by Gerasimov et al. More importantly, we believe ours is the first such observation reported for an excimer VUV lamp operating near room temperature. This VUV spiking behavior raises the prospect that designs with improved thermal management may achieve even higher VUV power and efficiency.

Polystyrene/graphene oxide (PSt/GO) nanocomposite latexes have been prepared by Pickering miniemulsion polymerization in the presence of the conventional surfactant sodium dodecyl sulfate (SDS) in order to investigate its influence on the polymerization mechanism. Miniemulsion polymerization of styrene can be conducted successfully using GO as the sole surfactant, but the polymerization rate is very low. In the presence of SDS, a significant rate enhancement is observed. It has been clarified that the presence of SDS leads to additional nucleation pathways besides monomer droplet nucleation, namely nucleation involving free GO sheets in the aqueous phase as well as homogeneous nucleation. These mechanistic pathways are consistent with the rate enhancement and the increase in molecular weight as well as experimentally observed particle distributions in the presence of SDS. The present results represent a significant step forward in terms of our understanding of the fundamental polymerization mechanism of this Pickering miniemulsion system, which will aid in the preparation of advanced nanocomposite materials based on polymers and graphene (oxide).

Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson’s disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LR...

Yeast MTs do not appear to undergo the lattice compaction seen in mammalian MTs upon GTP hydrolysis. Binding of the +TIP Bim1, both between and within αβ-tubulin dimers, causes compaction of yeast MTs and their rapid disassembly., Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. Our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.

The synthesis of composite latex particles possessing core–shell and gradient morphologies, respectively, using seeded starve-fed semibatch emulsion polymerization of styrene (St) and methyl methacrylate (MMA) is presented. The focus is on the effect of the monomer feed order on the particle morphology development. The particle morphology is assessed using a novel approach which entails comparing the experimental surface composition as a function of polymerization time (particle growth) obtained by X-ray photoelectron spectroscopy with the predicted surface composition using a mass balance mathematical model. Both types of composite latexes (core–shell and gradient) feature changes with polymerization time in the oxygen/carbon surface composition which enables one to track the morphology development. Differential scanning calorimetry is also implemented to analyze the extent of phase separation. The monomer feed order is shown to play a crucial role—under the present conditions, gradient and core–shell particles are obtained if the feed order is St/MMA (St fed first), but not if the feed order is reversed. These findings illustrate that thermodynamic factors are important, given that thermodynamically it is more favorable for MMA-rich chains to occupy the oil–water interface to reduce the interfacial tension. Systems where St is the second stage monomer lead to mixed structures rather than the targeted core–shell or gradient morphology with St-rich chains at the particle surface.

A convenient and industrially scalable method for synthesis of homogeneous nanocomposite films comprising poly(styrene-stat-butyl acrylate) and nanodimensional graphene oxide (GO) or reduced GO (rGO) is presented. Importantly, the nanocomposite latex undergoes film formation at ambient temperature, thus alleviating any need for high temperature or high pressure methods such as compression molding. The method entails synthesis of an aqueous nanocomposite latex via miniemulsion copolymerization relying on nanodimensional GO sheets as sole surfactant, followed by ambient temperature film formation resulting in homogeneous film. For comparison, a similar latex obtained by physical mixing of a polymer latex with an aqueous GO dispersion results in severe phase separation, illustrating that the miniemulsion approach using GO as surfactant is key to obtaining homogeneous nanocomposite films. Finally, it is demonstrated that the GO sheets can be readily reduced to rGO in situ by heat treatment of the film.