SARS-CoV-2 main protease (Mpro) is one of the most extensively exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of ...their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.
FlipGFP and protease-Glo luciferase assays, coupled with the FRET and thermal shift binding assays, were applied to validate/invalidate the reported SARS-CoV-2 Mpro inhibitors. Display omitted
A deficit of GABA (γ-aminobutyric acid) transmission will lead to epilepsy and other cognitive disorders. Recent evidence has shown that neuronal miRNAs affect various synapses, including GABAergic ...synapses. However, the miRNAs that control GABAergic synapses remain not fully understood. Here, we identified miR-51, a member of Caenorhabditis elegans miR-99/100 family, as a key regulator of GABAergic synapses. Loss of mir-51 increased PTZ (Pentylenetetrazole) and aldicarb hypersensitivities, and decreased the number of GABAergic synapses and abundance of GABAA receptors. A Rab guaninenucleotide exchange factor (GEF) GLO-4, a well-known component in lysosomal trafficking-related GLO-4/GLO-1/AP-3 (GLO/AP-3) pathway, was discovered to be the direct target of miR-51. Rescue experiments showed that GLO-4 expressed in GABAergic motor neurons functioned as a suppressor of miR-51. Disruption of glo-1 or AP-3 gene apm-3 attenuated the defects of GABAergic synapse in mir-51 mutants, suggesting miR-51 regulated GABAergic synapses through GLO/AP-3 pathway. The present study implies the essential roles of miRNAs on the nervous pathologies characterized by mis-regulated GABA signaling, such as epilepsy.
•miR-51 is a key regulator of GABAergic synapses in C. elegans miR-51 family.•miR-51 increases GABAergic synapse number and GABAA receptor abundance.•A Rab GEF GLO-4 is a direct target of miR-51.•miR-51 regulates GABAergic synapses by inhibiting GLO-4 in GABAergic motor neurons.•GLO/AP-3 pathway is required for miR-51 modulating GABAergic synapses.
The Quark Archipelago, in the northernmost part of the Baltic Sea, provides valuable reproduction areas for coastal fish such as pike (Esox lucius L.). Here, pike stocks are still in good condition ...compared to the southern Baltic Sea, where pike stocks have declined. Hence, this area offers a unique opportunity to study the underlying causes of successful pike reproduction and to identify a productive pike reproduction habitat. This study suggests that the key factor defining suitable reproduction habitats for pike in the Quark Archipelago was the level of shelter. The more sheltered a habitat, in terms of morphology and level of isolation of the site and the site's exposure to wave action and shoreline density, the better the reproduction habitat was for pike. Gloes, which are the most isolated successional stages of coastal lagoons, represented the most sheltered habitat type and showed the highest pike larvae abundance. Additionally, sheltered bays and flads, less isolated successional stages of coastal lagoons compared to gloes, were also used as reproduction habitats by pike but with a lower abundance of larvae compared to the gloes. Gradually increasing salinity and the occurrence of sticklebacks (Gasterosteidae) reflected the morphology of the site. Pike larvae were observed in locations without sticklebacks and with low salinity. Pike larvae were found mostly amongst fallen reed (Phragmites australis) and in areas with moss (Fontinalis sp.) growth, which supports earlier findings. This study provides valuable new knowledge on larval pike habitats and highlights the significance of sheltered bays and gloes in the northern Baltic Sea. The information can be utilised for planning conservation and the restoration of pike reproduction habitats to protect and restore pike populations on both a regional and larger scale.
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-The increase of shelter provided by a habitat improves the reproduction habitat for common coastal predatory fish species such as pike.-Shelterness of the habitat was best described by the morphology and level of isolation of the site and the site's exposure to wave action and shoreline density.-Salinity and occurrence of sticklebacks reflect the morphology of the studied habitats, i.e. sheltered bays, pre-flads, flads and gloes.-Pike prefer fallen reed (Phragmites australis) mats and areas with moss (Fontinalis sp.) growth for spawning.
Neisseria meningitidis is a Gram-negative bacterium that causes meningitis. N. meningitidis serogroup W (NmW) capsule polymerase synthesizes capsular polysaccharide of this serogroup. This enzyme ...could be a tool for meningococcal glycoconjugate vaccine development. Our long-term goal is to control activity of the NmW capsule polymerase for production of defined carbohydrates for vaccines. The enzyme lacks a simple, high-throughput activity assay. Here, we describe the use of high-throughput bioluminescence assays (CMP-Glo and UDP-Glo by Promega) to investigate NmW capsule polymerase activity. These assays detect free nucleotides produced during transfer of sugar from UDP-Galactose and CMP-Sialic Acid to an acceptor. Kinetic studies using NmW hydrolyzed polysaccharide (PS) acceptor are described as well as preliminary work with a sialic acid trimer (DP3) acceptor.
In CMP-Glo kinetic studies, with constant donor (80 µM) and varied NmW hydrolyzed polysaccharide (0-2000 µg/mL), a K
of 629.2 ± 101.4 µg/mL and a V
of 0.8965 ± 0.05823 µM/min was obtained. Using UDP-Glo, K
and V
values of 13.84 ± 9.675 µM and 0.6205 ± 0.1331 µM/min were obtained with varied CMP-NeuNAc (0-80 µM) and constant acceptor (400 µg/mL) and UDP-Gal (80 µM). This is the first report of using bioluminescence assays for NmW kinetics.
Heated tobacco products (HTPs) are non-combustible, inhaled tobacco products that generate an aerosol with fewer and lower levels of toxicants, with a potential to reduce risk relative to cigarette ...smoking. Here, we assessed in vitro toxicological effects of three menthol (glo neo neoCLICK, neo Smooth Menthol and Fresh Menthol) and one non-menthol (neo Smooth Tobacco) variants of glo HTP, along with market comparators for cigarettes and HTPs. Limited chemical characterization of the study products revealed significantly lower levels of acetaldehyde, acrolein, crotanaldehyde and formaldehyde in test samples from HTPs than those from cigarettes. The glo HTPs were non-mutagenic in the bacterial reverse mutagenesis assay. Although, the whole aerosol exposures of glo HTPs were classified as genotoxic in the in vitro micronucleus assay, and cytotoxic in the NRU (monolayer) and MTT (3 dimensional EpiAirway™ tissues) assays, the cigarette comparators were the most toxic study products in each of these assessments. Further, glo HTPs elicited oxidative stress responses only at the highest dose tested, whereas the cigarette comparators were potent inducers of oxidative stress at substantially lower doses in the EpiAirway tissues. The comparator (non-glo) HTP results were similar to the glo HTPs in these assays. Thus, the glo HTPs exhibit substantially lower toxicity compared to cigarettes.
•Low-severity fires were frequent and widespread within the Great Lakes Region.•GLO data described sites (e.g., composition, density), reasonably well, but not scale or frequency of fires.•Tree-ring ...based fire rotation intervals were orders of magnitude shorter than estimates based on GLO data.•Regionally significant fire years occurred ca. every 10 years.
Fire regimes in Eastern North America are often determined from historical data because land-use change and natural resource policy have confounded natural fire processes. It is good practice to combine multiple historical data sources, which can serve to both corroborate findings and fill knowledge gaps that might exist when trying to gain a full picture of historical ecological processes. In the Great Lakes Region (GLR) fire rotation, or the number of years it takes to burn an area equivalent to the area of interest, is an extensively used metric that has been calculated based on the Euro-American settlement era General Land Office Public Land Survey (GLO) records. However, fire rotation methods and GLO records are best suited for understanding high-severity fire, and low- to moderate-severity fires have received less attention in forested ecosystems in this region. We used dendrochronological (tree-ring) data to evaluate GLO data and fire rotation methods in relation to low-severity fires. Tree-ring and GLO data were well-aligned in some ways, with high concurrence of tree species, tree density, and common fire dates. However, GLO data did not identify fires for survey points closest to any of our sites (n = 26), though 71% of sites burned within one year and all sites burned within 8 years of surveys. Mean fire return intervals for our sites ranged from 2 to 9 years for all fires and 6–20 years for fires recorded on ≥25% of samples within sites (1602–2018) with relatively minor effects of filtering on return intervals. Thus, fires were historically frequent and widespread within sites. We estimate that fires burned on average 858 km2 to 2564 km2 per year within five ecological landscapes with rotation intervals ranging between 11 (Northeast Sands) and 34 years (Northern Highlands; µ = 22 years across all five landscapes). We found 25 regional fire years that were synchronous among multiple (2–5) ecological landscapes over a 218-yr period with evidence that drought plays a role in regionally widespread fire years. High-severity fire was likely limited in the GLR; however, low- to moderate-severity fires were abundant, large-scale, widespread, and an important forcing mechanism shaping forests of the GLR over millennia.
Daylight fluorescent pigments are complex artists' colorants made of multiple admixed dyes and additives infused in a polymer resin. Their unique photochemistry creates interesting optical effects ...that make them appealing for a range of applications. The wavelength dependence of the dyes' emissive properties has also led to their use in works meant to be displayed under nontraditional high energy light sources, such as blacklights. The intended display methods and the photochemistry of the constituent dyes can lead to chemical instability and also pose unique challenges for conservation and exhibition. In Part 1 of this research, we reported on the chemical constituents of colorants from two major manufacturers. In this paper, we provide a comprehensive report of the spectral properties and lightfastness of paints prepared with the pigments. The optical properties and chemical stability of these colorants are correlated with the composition. Variation in composition can lead to unique markers that could be useful for conservation treatment and exhibition considerations. Changes in the dyes used in the pigment formulation over time are considered in the comparison of two objects: a Stephen Sprouse silvered leather motorcycle jacket featuring fluorescent art by Stefano Castronovo and selections from the Day-Glo Designer's Guide from 1969.
Heated tobacco products (HTP) are a form of nicotine delivery intended to be an alternative to traditional cigarettes. HTP tobacco products are sold to consumers as a less harmful alternative to ...traditional cigarettes, both for users and bystanders. The actual impact of HTP on the health of users and its overall impact on public health is still not fully known. A systematic search of the literature was carried out to identify relevant studies published in English from 2015 to February 2021. The following databases were used: PubMed, Scopus, Elsevier and ClinicalKey. 25 studies (independent and sponsored by the tobacco industry) were considered. The analysis of exposure biomarkers and cardiovascular and respiratory biomarkers showed differences between smokers and people using heated tobacco products. Improvements in clinically relevant risk markers, especially cholesterol, sICAM-1, 8-epi-PGF2α, 11-DTX-B2, HDL and FEV1, were observed compared to persistent cigarette smokers. On the other hand, exposure to IQOS has been reported to alter mitochondrial function, which may further exaggerate airway inflammation, airway remodeling and lung cancer. These products have the potential to increase oxidative stress and increase respiratory tract infections by increasing microbial adherence to the respiratory tract. Our review suggests that HTP products may be products with a reduced risk of chronic diseases, including respiratory and cardiovascular diseases and cancer compared to traditional smoking, although in the case of non-smokers so far, they may pose a risk of their occurrence. Research seems to be necessary to assess the frequency of HTP use and its potential negative health effects.
Vitamin C (ascorbic acid) plays important roles as an anti-oxidant and in collagen synthesis. These important roles, and the relatively large amounts of vitamin C required daily, likely explain why ...most vertebrate species are able to synthesize this compound. Surprisingly, many species, such as teleost fishes, anthropoid primates, guinea pigs, as well as some bat and Passeriformes bird species, have lost the capacity to synthesize it. Here, we review the genetic bases behind the repeated losses in the ability to synthesize vitamin C as well as their implications. In all cases so far studied, the inability to synthesize vitamin C is due to mutations in the L-gulono-γ-lactone oxidase (GLO) gene which codes for the enzyme responsible for catalyzing the last step of vitamin C biosynthesis. The bias for mutations in this particular gene is likely due to the fact that losing it only affects vitamin C production. Whereas the GLO gene mutations in fish, anthropoid primates and guinea pigs are irreversible, some of the GLO pseudogenes found in bat species have been shown to be reactivated during evolution. The same phenomenon is thought to have occurred in some Passeriformes bird species. Interestingly, these GLO gene losses and reactivations are unrelated to the diet of the species involved. This suggests that losing the ability to make vitamin C is a neutral trait.
The objective of the article was to evaluate the relationship between the Covid-19 pandemic and globalisation (GLO) and de-globalisation (de-GLO) tendencies. Based on a theoretical evaluation of this ...relationship as well as historical development, it concludes that Covid-19 is not the mother of de-GLO but its child, born in a completely new global context facilitating the pandemic. The roots of the pandemic's conception are more deeply embedded in the capitalist system, in its principal elements (market system, consumerist development model...). GLO as a global division of labour is not over; the factors enhancing GLO are winning over those slowing it down, provided that GLO becomes more egalitarian and more human. A fully-fledged de-GLO would be inefficient and painful. While the pandemic is not a black swan, it could have been predicted. Such an unprecedented crisis impersonated by the pandemic also offers an opportunity to fundamentally rethink of our theories, way of life and development paradigm and, not the least, the whole system to be better prepared for future similar crises.
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