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Following a waste-To-wealth approach, humic acid (HA) was exploited as a flame retardant additive. The effect of its addition alone and in combination with urea (UR) and ammonium polyphosphate (APP) on the thermal, fire, and mechanical performances of a bisphenol A diglycidyl ether (DGEBA)-based epoxy resin modified with (3-Aminopropyl)-Triethoxysilane (AP) and cured with aliphatic isophoronediamine (IDA) has been investigated. Unlike in previous studies, a UL 94-V-0 classification was achieved for epoxy resin containing HA at 6 wt % and APP at only 1 wt % phosphorus (P) loading. The presence of silicon-modified epoxy chains ameliorated the distribution of the biowaste within the resin, and the addition of HA alone avoided melt dripping. Besides, APP and UR promoted a remarkable reduction (up to 52%) of the peak heat release rate (pHRR) values and a significant delay (up to 21%) of the time to ignition in cone calorimetry tests, and hence an increase (up to 1.8 min) of the time to flashover, without any detrimental effect on the overall mechanical behavior. The evolved gas, thermal, and fire analysis was used to propose the combined mode of action of HA, UR, APP, and silicon in the fire performance improvement of the hybrid epoxy system.

The infection of the floral tissues of snap bean and other crops by Sclerotinia sclerotiorum, the causative agent of white mold, is by ascospores. Irrespective of the fungicide mode of action being evaluated, in vitro fungicide sensitivity tests are conducted almost exclusively using mycelial growth assays. This is likely because of difficulties and time involved in sclerotial conditioning required to produce apothecia and ascospores. The objective of this research was to compare estimates of fungicide sensitivity between mycelial growth and ascospore germination assays for S. sclerotiorum. Sensitivity assays were conducted using serial doses of three fungicides commonly used to control white mold: boscalid, fluazinam, and thiophanate-methyl. A total of 27 isolates were evaluated in replicated trials conducted for each fungicide and assay type. The effective concentration to reduce mycelial growth or ascospore germination by 50% (EC50) was estimated for each isolate, fungicide, and assay type. The median EC50 values obtained from ascospore germination assays were 52.7, 10.0, and 2.7 times higher than those estimated from the mycelial growth for boscalid, fluazinam, and thiophanate-methyl, respectively. No significant correlation was found between EC50 values estimated by the two methods. These findings highlight differences that may be important in evaluating the sensitivity of S. sclerotiorum given the fungicide mode of action and how they will be used in the field.

The effective delivery of drugs to the inner ear is still an unmet medical need. Local controlled drug delivery to this sensory organ is challenging due to its location in the petrous bone, small volume, tight barriers, and high vulnerability. Local intracochlear delivery of drugs would overcome the limitations of intratympanic (extracochlear) and systemic drug application. The requirements for such a delivery system include small size, appropriate flexibility, and biodegradability. We have developed biodegradable PLGA-based implants for controlled intracochlear drug release that can also be used in combination with cochlear implants (CIs), which are implantable neurosensory prosthesis for hearing rehabilitation. The drug carrier system was tested for implantation in the human inner ear in 11 human temporal bones. In five of the temporal bones, CI arrays from different manufacturers were implanted before insertion of the biodegradable PLGA implants. The drug carrier system and CI arrays were implanted into the scala tympani through the round window. Implanted temporal bones were evaluated by ultra-high-resolution computed tomography (µ-CT) to illustrate the position of implanted electrode carriers and the drug carrier system. The µ-CT measurements revealed the feasibility of implanting the PLGA implants into the scala tympani of the human inner ear and co-administration of the biodegradable PLGA implant with a CI array. Graphical abstract

The numerical modelling of chloride diffusion in concrete structures requires an appropriate description of input parameters. The main inputs for the model are the diffusion coefficient of concrete and derived aging factor. The model itself can be susceptible to the values of these parameters because of the size of the finite elements and size of the time step. Due to the potential use in probabilistic calculations, which requires millions of simulations, it is desirable to create a highly optimizedmodel. It is important to pay attention to the accuracy of the calculation, but also to its calculation time. The presented results show the possible limits of the finite element modelof the diffusion in a concrete structure. This is demonstrated on the reference ordinary Portland cement mixture and 32 various binary and ternary concrete mixtures, which show a significant effect of different diffusion coefficients and aging factors on the overall convergence. This study provides guidance on which extreme material parameters, out of thepotential range, may adversely affect model results.

A case of feline intoxication and fatality with the illicit drug heroin is described. A 5-year-old castrated male domestic shorthair cat was recently diagnosed with an active pneumonitis and left at home for a couple of days under the care of another resident. Upon return, the owner found his cat dead with strong suspicion of foul play. The cat was necropsied by a local veterinary clinic to retrieve the liver for diagnostic toxicology. The postmortem liver sample screened positive for 6-acetylmorphine and 6-acetylcodeine by gas chromatography mass spectrometry. Deconvolution techniques were applied to chromatograms, which revealed the additional presence of morphine and mirtazapine. Subsequent quantitation of mirtazapine, heroin, morphine, 6-acetylmorphine and 6-acetylcodeine was performed by gas chromatography--tandem quadrupole mass spectrometry. Although companion animal fatalities arising from toxicities are a likely consequence of drug abuse in a home, this is the first reported case of a malicious feline fatality resulting from heroin with quantitation of heroin metabolites.

As the prevalence of plastic micro- and nanoparticles in the environment, foods, and beverages continues to increase, the risk of human exposure to and uptake of such particles, notably via ingestion or inhalation, is also elevated. Despite this development, relatively little is known about the potential adverse effects of plastic particles on humans. The lack of relevant plastic nanoparticles for use in studies investigating their behavior and effect on human cells is a key hurdle that must be overcome prior to generating hazard data. We herein demonstrate the creation of fluorescent nanoparticles of the thermoplastic polymers poly(ethylene terephthalate), polypropylene, and polystyrene. The particles were produced by melt-processing and milling. The analysis of scanning electron microscopy images showed core diameters of less than 75 nm. Furthermore, the images revealed that the milled particles had highly heterogeneous shapes, as is often seen in environmental samples. The particles were exposed to relevant cell lines (i.e. Caco-2 intestinal epithelial cells and J774A.1 macrophages) to determine their uptake, assessed by confocal laser microscopy, and biocompatibility, assessed by measuring the release of lactate dehydrogenase. Exposure data showed no cytotoxicity at the concentrations utilized in this study. Interaction of the particles was found to be cell type dependent, with agglomeration on the apical surface and few intracellular particles in the intestinal epithelial cells in comparison to numerous internalized particles in the macrophages. In conclusion, the herein presented melt-processing and milling methods resulted in heterogeneously shaped plastic nanoparticles with a fluorescence label allowing their behavior within a complex biological environment to be studied.

Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export., Lysosomes play an important role in cellular LDL-cholesterol uptake. Here, the authors perform a genome-wide genetic screen for cholesterol regulators and identify C18orf8 as a conserved subunit of a trimeric Rab7 GEF that controls LDL trafficking and NPC1-dependent lysosomal cholesterol export.

Objective The objectives were to quantify collagen content in nonlactating uninfected and Staphylococcus aureus–infected mammary glands and determine the relative abundance of collagen in different gland regions. Materials and Methods Mammary tissues that were previously collected from a mastitis-challenge trial were examined. Mammary tissues were collected along a vertical midline through each gland and included parenchymal tissues that were proximal to the gland cistern (cisternal parenchyma) and tissues near the abdominal wall (deep parenchyma) from each saline-infused (n = 18) and Staph. aureus–infused (n = 18) gland. Collagen was detected via picro-sirus red staining and visualized using polarized light for image acquisition and subsequent analysis. Results and Discussion Most interlobular collagen was associated with larger ducts and stromal tissues. Some collagen was intralobular and was mostly associated with these larger ducts but was also present in epithelial basement membranes. Staphylococcus aureus–infused glands contained more tissue area occupied by collagen than saline-infused glands (19.78 vs. 17.26 ± 1.19%). Additionally, parenchyma near the gland cistern contained more collagen than deep parenchyma (19.95 vs. 17.09 ± 1.19%). Results indicate that mastitis elicits the deposition of collagen in affected glands and that collagen abundance is affected by gland region. Implications and Applications Nonlactating mammary glands affected by Staph. aureus mastitis may have a greater amount of collagen, believed to be resulting scar tissue. We speculate that it may have long-term, and possibly irreversible, effects on glandular structure and, consequently, lifetime productivity.

Cholesterol is an essential component of mammalian membranes, and its homeostasis is strictly regulated, with imbalances causing atherosclerosis, Niemann Pick disease, and familial hypercholesterolemia. Cellular cholesterol supply is mediated by LDL-cholesterol import and de novo cholesterol biosynthesis, and both pathways are adjusted to cellular demand by the cholesterol-sensitive SREBP2 transcription factor. Cholesterol homeostasis is modulated by a wide variety of metabolic pathways and the ubiquitination machinery, in particular E3 ubiquitin ligases. In this article, we review recent progress in understanding the role of E3 ubiquitin ligases in the metabolic control of cellular sterol homeostasis.

In this study a robust, whole organism screening based on Caenorhabditis elegans is presented for the discovery of natural products (NP) with beneficial effects against obesity and age-related diseases. Several parameters of the elaborated workflow were optimized to be adapted for probing multicomponent mixtures combining knowledge from traditional medicine and NP chemistry by generating optimized small-scale extracts considering scarcity of the natural source, solubility issues, and potential assay interferences. The established miniaturized assay protocol allows for in vivo probing of small amounts of even complex samples (~ 1 mg) to test their ability to increase the nematodes’ survival time and the suppression of fat accumulation assessed by Nile red staining as hall marks of “healthy aging”. The workflow was applied on 24 herbal and fungal materials traditionally used against symptoms of the metabolic syndrome and revealed promising results for the extracts of Gardenia jasminoides fruits and the sclerotia from Inonotus obliquus. Tested at 100 µg/mL they were able to significantly reduce the Nile red fluorescence and extend the 50% survival rate (DT50) compared to the control groups. This phenotype-directed in vivo approach opens up new horizons for the selection of natural starting materials and the investigation of their active principles as fast drug discovery tool with predictive value for human diseases.

Significance Molecular ON-switches are important tools in chemical biology, enabling protein–protein interactions to be regulated by small molecules. However, currently available ON-switches that induce conditional heterodimerization are suboptimal for therapeutic applications. In this study, we present an ON-switch system based on human retinol binding protein 4 (hRBP4) and the orally available small molecule A1120. Two distinct protein scaffolds, FN3 and rcSso7d, were successfully engineered to bind to hRBP4 in a small molecule-dependent manner, demonstrating the flexibility of the system. The binders specifically associated with the drug-induced conformation of hRBP4. Our study demonstrates that lipocalin-based ON-switches can enable specific regulation of protein heterodimerization and provides proof of concept for potential applications in controlling the activity of human CAR T cells., Molecular ON-switches in which a chemical compound induces protein–protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

This chapter sets the scene for the potential role of water electrolysis in the global future energy system. It introduces hydrogen, water electrolysis, and other hydrogen production routes and discusses historic drivers for hydrogen. It then identifies climate change mitigation as the key motivation for contemporary hydrogen developments. This chapter also provides a review of the potential future role of hydrogen and water electrolysis in publicly available global energy system studies. It shows that in order to reach net-zero CO2 emissions, between 1.7 and 13 Terawatt of water electrolysis capacity could be needed by 2050 worldwide. This would require the water electrolyzer industry to scale up manufacturing to a hundred or more gigawatts per year by 2040. This chapter closes with an outlook of water electrolysis deployments planned at the beginning of the 2020s.

Publisher Copyright: © 2022 Northern forest soils are a major carbon (C) reservoir of global importance. To estimate how the C balance in these soils will change, the roles of tree roots and soil microbes in C balance should first be decoupled. This study determined how the activity of heterotrophs and tree roots together with root-associated microbes in the rhizosphere varies in coniferous forest soils in boreal, hemiboreal, and temperate climates along a latitudinal gradient using a trenching approach. We created experimental plots without living tree roots, measured soil respiration (CO2 efflux) from these and from unmanipulated plots using the chamber technique, and partitioned the efflux into root-rhizosphere (RR) and heterotrophic (RH) respiration. The share of RR in ecosystem gross primary production (GPP) decreased from north to south in the Scots pine (Pinus sylvestris L.) and the Norway spruce (Picea abies (L.) Karst.) forests, with the exception of a mixed site, where the share of RR in GPP varied strongly between the years. RR per ground area and per root biomass were mainly independent of climate within the gradient. RH per ground area increased from north to south with temperature, while RH per soil C did not change with temperature. Soil moisture did not significantly affect the respiration components in the northernmost site, whereas soil moisture was positively connected with RH and negatively with RR in other Scots pine sites and positively connected with RR in pure Norway spruce stands. The dynamic ecosystem model LPJ-GUESS was able to capture the seasonal dynamics of RH and RR at the sites, but overall accuracy varied markedly between the sites, as the model underestimated RH in the southern site and RR elsewhere. Our study provides knowledge about the nature of soil respiration components. The valuable insights can be used in more accurate land-ecosystem modelling of forest ecosystems.

The recycling rates, especially those from plastic packaging waste, have to be increased according to the European Union directive in the next years. Besides many other technologies, the pyrolysis of plastic wastes seems to be an efficient supplementary opportunity to treat mixed and unpurified plastic streams. For this reason, a pyrolysis process was developed for the chemical recycling of hydrocarbons from waste polyolefins. The obtained products can be further processed and upgraded in crude oil refineries, so that also monomers can be recovered, which are used for the plastic polymerization again. However, to achieve a scale up to a demo plant, a kinetic model for predicting the yields of the plastic pyrolysis in a tubular reactor is needed. For this reason, a pilot plant was built, in which different plastics and carrier fluids can be tested. Based on the data generated at the pilot plant, a very practical and suitable model was found to describe the plastic co-pyrolysis of the carrier fluid with polypropylene (PP) and low density and high density polyethylene (HDPE and LDPE), respectively. The physical and chemical mechanisms of the co-pyrolysis in the tubular reactor are successfully investigated.

To date, it is widely accepted by the scientific community that many agricultural regions will experience more extreme temperature fluctuations. These stresses will undoubtedly impact crop production, particularly fruit and seed yields. In fact, pollination is considered as one of the most temperature-sensitive phases of plant development and until now, except for the time-consuming and costly processes of genetic breeding, there is no immediate alternative to address this issue. In this work, we used a multidisciplinary approach using physiological, biochemical, and molecular techniques for studying the effects of two carbohydrate-based natural activators on in vitro tomato pollen germination and pollen tube growth cultured in vitro under cold conditions. Under mild and strong cold temperatures, these two carbohydrate-based compounds significantly enhanced pollen germination and pollen tube growth. The two biostimulants did not induce significant changes in the classical molecular markers implicated in pollen tube growth. Neither the number of callose plugs nor the CALLOSE SYNTHASE genes expression were significantly different between the control and the biostimulated pollen tubes when pollens were cultivated under cold conditions. PECTIN METHYLESTERASE (PME) activities were also similar but a basic PME isoform was not produced or inactive in pollen grown at 8°C. Nevertheless, NADPH oxidase (RBOH) gene expression was correlated with a higher number of viable pollen tubes in biostimulated pollen tubes compared to the control. Our results showed that the two carbohydrate-based products were able to reduce in vitro the effect of cold temperatures on tomato pollen tube growth and at least for one of them to modulate reactive oxygen species production.

Treatment options for COVID-19 are currently limited. Drugs reducing both viral loads and SARS-CoV-2-induced inflammatory responses would be ideal candidates for COVID-19 therapeutics. Previous in vitro and clinical studies suggest that the proprietary Pelargonium sidoides DC. root extract EPs 7630 has antiviral and immunomodulatory properties, limiting symptom severity and disease duration of infections with several upper respiratory viruses. Here we assessed if EPs 7630 affects SARS-CoV-2 propagation and the innate immune response in the human lung cell line Calu-3. In direct comparison to other highly pathogenic CoV (SARS-CoV, MERS-CoV), SARS-CoV-2 growth was most efficiently inhibited at a non-toxic concentration with an IC50 of 1.61 μg/ml. Particularly, the cellular entry step of SARS-CoV-2 was significantly reduced by EPs 7630 pretreatment (10–100 μg/ml) as shown by spike protein-carrying pseudovirus particles and infectious SARS-CoV-2. Using sequential ultrafiltration, EPs 7630 was separated into fractions containing either prodelphinidins of different oligomerization degrees or small molecule constituents like benzopyranones and purine derivatives. Prodelphinidins with a low oligomerization degree and small molecule constituents were most efficient in inhibiting SARS-CoV-2 entry already at 10 μg/ml and had comparable effects on immune gene regulation as EPs 7630. Downregulation of multiple pro-inflammatory genes (CCL5, IL6, IL1B) was accompanied by upregulation of anti-inflammatory TNFAIP3 at 48 h post-infection. At high concentrations (100 μg/ml) moderately oligomerized prodelphinidins reduced SARS-CoV-2 propagation most efficiently and exhibited pronounced immune gene modulation. Assessment of cytokine secretion in EPs 7630-treated and SARS-CoV-2-coinfected Calu-3 cells showed that pro-inflammatory cytokines IL-1β and IL-6 were elevated whereas multiple other COVID-19-associated cytokines (IL-8, IL-13, TNF-α), chemokines (CXCL9, CXCL10), and growth factors (PDGF, VEGF-A, CD40L) were significantly reduced by EPs 7630. SARS-CoV-2 entry inhibition and the differential immunomodulatory functions of EPs 7630 against SARS-CoV-2 encourage further in vivo studies.

Allosteric communication between distant sites in proteins is central to nearly all biological regulation but still poorly characterised for most proteins, limiting conceptual understanding, biological engineering and allosteric drug development. Typically only a few allosteric sites are known in model proteins, but theoretical, evolutionary and some experimental studies suggest they may be much more widely distributed. An important reason why allostery remains poorly characterised is the lack of methods to systematically quantify long-range communication in diverse proteins. Here we address this shortcoming by developing a method that uses deep mutational scanning to comprehensively map the allosteric landscapes of protein interaction domains. The key concept of the approach is the use of ‘multidimensional mutagenesis’: mutational effects are quantified for multiple molecular phenotypes—here binding and protein abundance—and in multiple genetic backgrounds. This is an efficient experimental design that allows the underlying causal biophysical effects of mutations to be accurately inferred en masse by fitting thermodynamic models using neural networks. We apply the approach to two of the most common human protein interaction domains, an SH3 domain and a PDZ domain, to produce the first global atlases of allosteric mutations for any proteins. Allosteric mutations are widely dispersed with extensive long-range tuning of binding affinity and a large mutational target space of network-altering ‘edgetic’ variants. Mutations are more likely to be allosteric closer to binding interfaces, at Glycines in secondary structure elements and at particular sites including a chain of residues connecting to an opposite surface in the PDZ domain. This general approach of quantifying mutational effects for multiple molecular phenotypes and in multiple genetic backgrounds should allow the energetic and allosteric landscapes of many proteins to be rapidly and comprehensively mapped.

The seminal description of cellular restriction factor APOBEC3G and its antagonism by HIV-1 Vif has underpinned two decades of research on the host-virus interaction. As well as APOBEC3G and its homologues, however, we have recently discovered that Vif is also able to degrade the PPP2R5 family of regulatory subunits of key cellular phosphatase PP2A (PPP2R5A-E) (Greenwood et al., 2016; Naamati et al., 2019). We now identify amino acid polymorphisms at positions 31 and 128 of HIV-1 Vif which selectively regulate the degradation of PPP2R5 family proteins. These residues covary across HIV-1 virusesin vivo, favouring depletion of PPP2R5A-E. Through analysis of point mutants and naturally occurring Vif variants, we further show that degradation of PPP2R5 family subunits is both necessary and sufficient for Vif-dependent G2/M cell cycle arrest. Antagonism of PP2A by HIV-1 Vif is therefore independent of APOBEC3 family proteins, and regulates cell cycle progression in HIV-infected cells.

Overcoming drug resistance remains a key challenge to cure patients with acute and chronic B cell malignancies. Here, we describe a stromal cell–autonomous signaling pathway, which contributes to drug resistance of malignant B cells. We show that protein kinase C (PKC)–β–dependent signals from bone marrow–derived stromal cells markedly decrease the efficacy of cytotoxic therapies. Conversely, small-molecule PKC-β inhibitors antagonize prosurvival signals from stromal cells and sensitize tumor cells to targeted and nontargeted chemotherapy, resulting in enhanced cytotoxicity and prolonged survival in vivo. Mechanistically, stromal PKC-β controls the expression of adhesion and matrix proteins, required for activation of phosphoinositide 3-kinases (PI3Ks) and the extracellular signal–regulated kinase (ERK)–mediated stabilization of B cell lymphoma–extra large (BCL-X L ) in tumor cells. Central to the stroma-mediated drug resistance is the PKC-β–dependent activation of transcription factor EB, regulating lysosome biogenesis and plasma membrane integrity. Stroma-directed therapies, enabled by direct inhibition of PKC-β, enhance the effectiveness of many antileukemic therapies.

Silica-epoxy nanocomposites were prepared via an “in-situ” sol-gel synthesis process and a phosphorus (P) flame-retardant i.e. 6H-dibenz[c,e][1,2]oxaphosphorin,6-[(1-oxido-2,6,7-trioxa-1-phosphabicyclo[2.2.2]oct-4-yl)methoxy]-, 6-oxide (DP) and melamine (Mel) were further added to the matrix to improve its fire performance. The main components of epoxy resin were bisphenol A diglycidyl ether (DGEBA) and isophorone diamine (IPDA) hardener. The addition of DP as well as silica alone into the epoxy system stopped the melt dripping phenomena in the vertical fire test (UL 94), however, the addition of melamine was crucial for achieving the highest fire classification (UL 94-V0 rating). The presence of DP and Mel in the silica-epoxy nanocomposite promoted a large reduction (ranging from 53% up to 80%) in the heat release rate (HRR) and a delay (up to 31%) in the ignition time in the cone calorimetry experiments. Improved fire performance of the epoxy system was attributed to i) a condensed phase activity of silica, DP and melamine to form a protective thermal barrier during combustion and ii) a minor gas phase flame inhibition activity of DOPO component of DP. The mechanical characterization of the epoxy nanocomposites through tensile tests showed that the addition of DP increases the stiffness of the epoxy resin, resulting in a strong increase of Young modulus (up to 32%) and in a slight decrease of fracture strength, elongation at break and toughness. An increased glass transition temperature (up to 8%) of the epoxy system possibly due to hydrogen bonds and polar interactions of DP with the matrix was also observed.

Hepatic steatosis is defined by accumulation of neutral lipids in lipid droplets (LDs) including triacylglycerol (TG) and steryl esters. Two distinct diacylglycerol acyltransferases (DGAT1 and DGAT2) catalyze synthesis of TG in hepatocytes. TG formed through either DGAT1 or DGAT2 appears to be preferentially directed to distinct intercellular fates, such as fatty acid production for oxidation or very-low density lipoprotein assembly, respectively. Because of the preferential use of TG generated by DGAT1 and DGAT2, we hypothesized that targeting/association of lipolytic machinery to LDs would differ depending on whether the TG stores were generated through DGAT1 or DGAT2 activities. Inhibition of DGAT1 or DGAT2 in human hepatoma cells (Huh7) incubated with oleic acid resulted in only a small change in TG accretion suggesting that the two DGATs can compensate for each other in fatty acid esterification. This compensation was not accompanied by changes in DGAT1 or DGAT2 mRNA expression. DGAT1 inhibition (TG synthesized by DGAT2) resulted in large LDs, whereas DGAT2 inhibition (TG synthesized by DGAT1) caused the accumulation of numerous small LDs. Oleic acid treatment increased mRNA and protein expression of the LD-associated protein PLIN2 but not PLIN5 or the lipase ATGL and its activator ABHD5/CGI-58. Inactivation of DGAT1 or DGAT2 did not alter expression (mRNA or protein) of ATGL, ABHD5/CGI-58, PLIN2 or PLIN5, but inactivation of both DGATs increased PLIN2 abundance despite a dramatic reduction in the number of LDs. ATGL localized preferentially to DGAT1-made LDs rather than to DGAT2-made LDs, and TG in these LDs was preferentially used for fatty acid (FA) oxidation. A combination of DGAT2 inhibitor and the pan lipase inhibitor E600 resulted in large LDs, suggesting that the small size of DGAT1-made LDs is due to a lipolytic process.

Herein, a series of aryl-substituted derivatives of 3-amino-1-aryl-9-methoxy-1H-benzo[f]chromene-2-carbonitriles (4a-4q) were designed and synthesized via reaction of 7-methoxy-2-naphthol with a mixture of appropriate aromatic aldehydes and malononitrile under microwave conditions. Among the tested benzochromene, the known compound 4e and four novel compounds 4f, 4j, 4k, 4m exhibited the highest cytotoxicity towards a panel of six human cancer cell lines MDA-MB-231, A549, HeLa, MIA PaCa-2, RPMI 7951, and PC-3. Compound 4j with 2,4-dichloro substitution on the pendant phenyl ring exhibited the highest broad-spectrum cytotoxicity towards all tested cancer cell lines. Compounds 4e, 4f, 4j, 4k, 4m were further selected to study the mechanism of cellular toxicity using the triple-negative breast cancer cells MDA-MB-231. Compounds 4e, 4f, 4j, 4k, 4m induced accumulation of the treated MDA-MB-231 cells in the S phase and 4k additionally in the G2/M phase of the cell cycle. Compounds 4e, 4f, 4j, 4k, 4m induced dissipation of mitochondrial transmembrane potential and activation of caspase 3/7 in MDA-MB-231 cells with 4j being one of the most active. In an in vivo model, compound 4j and less efficiently 4e and 4f inhibited growth and proliferation and triggered DNA fragmentation in MDA-MB-231 xenografts grown on chick chorioallantoic membranes. SAR study confirmed that the 2,4-dichloro substitution pattern on the pendant phenyl ring enhanced the cytotoxic activity of benzochromene.

Drug resistance poses a major challenge for targeted cancer therapy. To be able to functionally screen large randomly mutated target gene libraries for drug resistance mutations, we developed a biochemically defined high-throughput assay termed PhosphoFlowSeq. Instead of selecting for proliferation or resistance to apoptosis, PhosphoFlowSeq directly analyzes the enzymatic activities of randomly mutated kinases, thereby reducing the dependency on the signaling network in the host cell. Moreover, simultaneous analysis of expression levels enables compensation for expression-based biases on a single cell level. Using EGFR and its kinase inhibitor erlotinib as a model system, we demonstrate that the clinically most relevant resistance mutation T790M is reproducibly detected at high frequencies after four independent PhosphoFlowSeq selection experiments. Moreover, upon decreasing the selection pressure, also mutations which only confer weak resistance were identified, including T854A and L792H. We expect that PhosphoFlowSeq will be a valuable tool for the prediction and functional screening of drug resistance mutations in kinases.

Pyrolysis of waste plastics to recycle valuable hydrocarbons represents an attractive technology for reducing waste and providing feedstocks for petrochemical products and fuels. Via the simultaneous processing of heavy petroleum residue fractions, synergies can be harnessed by converting bottom-of-the-barrel refining products into lighter fractions with higher value while improving processability of plastic waste materials. To investigate the effect of reactor pressure, a continuous laboratory co-pyrolysis plant was operated. The setup consisted of two consecutive tubular zones to convert a mixture of LDPE and a heavy petroleum residue to a final temperature of 450 °C at different pressures between 2 and 10 bar. The products were evaluated regarding obtained mass yields and their boiling range. Gaseous and liquid products increased with enhanced pressure, resulting in nearly tripled gas and light liquid formation, whereas more unconverted feed was consumed. Because the reactor pressure also affects the residence time by suppressing evaporation, which subsequently varies between 360 and 440 s, further investigations considering the dependence of product yields on the residence time over a range of 280 to 480 s were necessary. The comparison resulted in the conclusion that the enhancing effect of increased reactor pressure is not only caused by a retention time elongation in the hot reactor zone. Other physical effects also play a role, such as promoted heat transmission and a direct intervention of reactor pressure with the chemical reactions. In the tested range, an enhancing effect of higher reactor pressures on the cracking of the reaction mixture was observed. These novel experimental results indicate, that conversion toward lighter cracking products can be increased by pressure adjustments and highlights that the pressure should be included in process optimizations.

Although many attempts have been made since 1930 to explain the production of cancer in experimental animals by oxidized metabolites of polynuclear aromatic hydrocarbons (PAH) with either molecular structure types ArCH2X or ArX, the simplest interpretation is that one type must function in experimental animals to produce cancer, whereas the other structural type prevents the production of cancer by formation of detoxification products. The specific predictions of the Unified Theory of carcinogenesis by PAH or PACs (Polynuclear Aromatic Compounds) depends on the validity of the predictions for the production of cancer by PACs with structural types ArCH2X or ArX. These two structural types have been adopted as crucial models for PAC metabolites by Flesher, Sydnor and colleagues since the 1970s. We were aided by the experiments and strong inferences of Professors Fieser, Fried, Boyland, Sims, and Huggins, and the theoretical studies of a possible structure of the ultimate carcinogen for 7-methylbenz[a]anthracene and closely related hydrocarbons by Brookes and Dipple together with the calculations of Dewar that predict the most reactive centers for substitution reactions in unsubstituted hydrocarbons. Shear in the late 1930s and 1940s was the first to show that meso-aldehydes and meso-methyl-substituted hydrocarbons function as precursors to active compounds with structure type ArCH2X. The discoveries by Flesher, Sydnor and coworkers demonstrated that biological methylation must take place at the most reactive center in non-methylated hydrocarbons catalyzed by cytosolic SAM-dependent methyltransferases, and the methyl group must subsequently be hydroxylated and conjugated with a good leaving group, if the PAC is to be carcinogenic. This is precisely the same site of special reactivity required for methylation by chemical synthesis as calculated by the Dewar molecular orbital approach. Blocking the reactive center in the aromatic nucleus (in the form of products as ArX) with the same groups attached to the meso-methyl group of methyl-substituted hydrocarbons (in the form ArCH2X) which produces cancer typically abolishes the production of cancer. Consequently, no theory relating ring oxidation to the production of cancer can account for the fact that substitution of reactive centers in non-methylated hydrocarbons with hydroxy or acetoxy groups, while simultaneously blocking methylation, also abolish its carcinogenic activity. If alternative hypotheses or theories do not cite the fundamental studies of Fieser and of Shear and Leiter there is little possibility that the alternative theory is correct. It seems obvious that most, if not all, hypotheses or theories that attempt to relate ring oxidized metabolites of PAH to the production of cancer have been disproved.

Diese Arbeit liefert einen Uberblick uber die Verteilung der stabilen Isotope von Wasserstoff (δ2H) und Sauerstoff (δ18O) im Grundwasser Niedersachsens und Bremens anhand von 133 Grundwasserproben, die mit der Cavity-Ring-Down-Spectroscopy untersucht wurden. Die Ergebnisse zeigen das Auftreten eines Kontinentaleffekts in Nordwest-Sudost-Richtung entlang der vorherrschenden Windrichtung mit einer Abreicherung des Isotopensignals im Wasser mit zunehmender Entfernung von der Kuste (δ2H: −48,3 ‰; δ18O: −6,8 ‰) ins Landesinnere (δ2H: −56,8 ‰; δ18O: −7,8 ‰) sowie einen vermeintlichen Hoheneffekt, der allerdings uber eine grose raumliche Entfernung auftritt und daher hochstwahrscheinlich nicht real ist, sondern durch den Kontinentaleffekt bestimmt wird. Des Weiteren wird die Abhangigkeit der δ2H- und δ18O-Werte des Grundwassers von der Evaporationsrate deutlich, die ihrerseits von der Bodenporositat und dem Pflanzenbewuchs abhangt und auf unterschiedliche Landnutzung oder das Vorkommen von verschiedenen hydrogeologischen Raumen zuruckzufuhren ist. Letztgenannte Effekte beeinflussen sich wechselseitig und sind ebenfalls durch den Kontinentaleffekt uberpragt.

It is well recognized that many cancers are addicted to a constant supply of fatty acids (FAs) and exhibit brisk de novo FA synthesis. Upregulation of a key lipogenic enzyme, fatty acid synthase (FASN), is a near-universal feature of human cancers and their precursor lesions, and has been associated with chemoresistance, tumor metastasis, and diminished patient survival. FASN inhibition has been shown to be effective in killing cancer cells, but progress in the field has been hindered by off-target effects and poor pharmaceutical properties of candidate compounds. Our initial hit (compound 1) was identified from a high-throughput screening effort by the Sanford-Burnham Center for Chemical Genomics using purified FASN thioesterase (FASN-TE) domain. Despite being a potent inhibitor of purified FASN-TE, compound 1 proved highly unstable in mouse plasma and only weakly cytotoxic to breast cancer (BC) cells in vitro. An iterative process of synthesis, cytotoxicity testing, and plasma stability assessment was used to identify a new lead (compound 41). This lead is more cytotoxic against multiple BC cell lines than tetrahydro-4-methylene-2S-octyl-5-oxo-3R-furancarboxylic acid (the literature standard for inhibiting FASN), is stable in mouse plasma, and shows negligible cytotoxic effects against nontumorigenic mammary epithelial cells. Compound 41 also has drug-like physical properties based on Lipinski’s rules and is, therefore, a valuable new lead for targeting fatty acid synthesis to exploit the requirement of tumor cells for fatty acids. SIGNIFICANCE STATEMENT: An iterative process of synthesis and biological testing was used to identify a novel thioesterase domain FASN inhibitor that has drug-like properties, is more cytotoxic to breast cancer cells than the widely used tetrahydro-4-methylene-2S-octyl-5-oxo-3R-furancarboxylic acid, and has negligible effects on the growth and proliferation of noncancerous mammary epithelial cells. Our studies have confirmed the value of using potent and selective FASN inhibitors in the treatment of BC cells and have shown that the availability of exogenous lipoproteins may impact both cancer cell FA metabolism and survival.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Triacylglycerol accumulation in the liver is a hallmark of NAFLD. Metabolic studies have confirmed that increased hepatic de novo lipogenesis (DNL) in humans contributes to fat accumulation in the liver and to NAFLD progression. Mice deficient in carboxylesterase (Ces)1d expression are protected from high-fat diet-induced hepatic steatosis. To investigate whether loss of Ces1d can also mitigate steatosis induced by over-activated DNL, WT and Ces1d-deficient mice were fed a lipogenic high-sucrose diet (HSD). We found that Ces1d-deficient mice were protected from HSD-induced hepatic lipid accumulation. Mechanistically, Ces1d deficiency leads to activation of AMP-activated protein kinase and inhibitory phosphorylation of acetyl-CoA carboxylase. Together with our previous demonstration that Ces1d deficiency attenuated high-fat diet-induced steatosis, this study suggests that inhibition of CES1 (the human ortholog of Ces1d) might represent a novel pharmacological target for prevention and treatment of NAFLD. Fil: Lian, Jihong. University of Alberta; Canadá Fil: Watts, Russell. University of Alberta; Canadá Fil: Quiroga, Ariel Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Beggs, Megan R.. University of Alberta; Canadá Fil: Alexander, R. Todd. University of Alberta; Canadá Fil: Lehner, Richard. University of Alberta; Canadá

Plasma osmolalities of marine vertebrates are generally lower than the surrounding medium; therefore, marine organisms must cope with the osmoregulatory challenges of life in a salty environment. The salt glands serve to maintain osmotic and ionic homeostasis in a number of lower marine vertebrates. One marine reptile, the leatherback sea turtle (Dermochelys coriacea), ingests excessive amounts of salts due to their diet of gelatinous zooplankton. Outside of the normal osmoregulatory function of the salt gland, little research has been conducted on contaminant accumulation and excretion in this organ. Here, we established arsenic, cadmium, lead, mercury, and selenium concentrations in red blood cells (RBCs) and salt gland secretions (SGSs) of nesting leatherbacks. We also collected salt glands from different life stage classes of dead stranded leatherbacks from the western Atlantic Ocean to determine if inorganic contaminants accumulate in this organ. Using non-metric multidimensional scaling and regression analyses, we determined that RBC and SGS inorganic contaminant concentrations were not correlated. Additionally, RBCs showed significantly higher concentrations of these contaminants in comparison to SGSs, likely due to the affinity of inorganic contaminants for the heme group of RBCs. Lastly, we found that salt gland cadmium and mercury concentrations tended to increase with increasing curved carapace length (CCL) in stranded leatherbacks. Our results indicate that different physiological mechanisms determine the distribution of inorganic contaminants in blood and SGSs. Increases in salt gland contaminant concentrations with increasing CCL suggest this organ as a potential target for accumulation.

Background/objective Ischemic heart disease is a major cause of mortality worldwide. Myocardial tissue engineering aims to create transplantable units of myocardium for the treatment of myocardial necrosis caused by ischemic heart disease - bioreactors are used to condition these bioartificial tissues before application. Methods Our group developed a multimodal bioreactor consisting of a linear drive motor for pulsatile flow generation (500 ml/min) and an external pacemaker for electrical stimulation (10 mA, 3 V at 60 Hz) using LinMot-Talk Software to synchronize these modes of stimulation. Polyurethane scaffolds were seeded with 0.750 × 106 mesenchymal stem cells from umbilical cord tissue per cm2 and stimulated in our system for 72 h, then evaluated. Results After conditioning histology showed that the patches consisted of a cell multilayer surviving stimulation without major damage by the multimodal stimulation, scanning electron microscopy showed a confluent cell layer with no cell-cell interspaces visible. No cell viability issues could be identified via Syto9-Propidium Iodide staining. Conclusions This bioreactor allows mechanical stimulation via pulsatile flow and electrical stimulation through a pacemaker. Our stem cell-polyurethane constructs displayed survival after conditioning. This system shows feasibility in preliminary tests.

Several biotypes of the Gram-negative bacterium Serratia marcescens produce the tri-pyrole pigment and secondary metabolite prodigiosin. The biological activities of this pigment have therapeutic potential. For over half a century it has been known that biosynthesis of prodi giosin is inhibited when bacteria are grown at elevated temperatures, yet the fundamental mechanism underlying this thermoregulation has not been characterized. In this study, chromosomal and plasmid-borne luxCDABE transcriptional reporters revealed reduced transcription of the prodigiosin biosynthetic operon at 37°C compared to 30°C indicating transcriptional control of pigment production. Moreover, induced expression of the prodigiosin biosynthetic operon at 37°C was able to produce pigmented colonies and cultures demonstrating that physiological conditions at 37°C allow prodigiosin production and indicating that post-transcriptional control is not a major contributor to the thermoregulation of prodigiosin pigmentation. Genetic experiments support the model that the HexS transcription factor is a key contributor to thermoregulation of pigmentation, whereas CRP plays a minor role, and a clear role for EepR and PigP was not observed. Together, these data indicate that thermoregulation of prodigiosin production at elevated temperatures is controlled largely, if not exclusively, at the transcriptional level. Several biotypes of the Gram-negative bacterium Serratia marcescens produce the tri-pyrole pigment and secondary metabolite prodigiosin. The biological activities of this pigment have therapeutic potential. For over half a century it has been known that biosynthesis of prodi giosin is inhibited when bacteria are grown at elevated temperatures, yet the fundamental mechanism underlying this thermoregulation has not been characterized. In this study, chromosomal and plasmid-borne luxCDABE transcriptional reporters revealed reduced transcription of the prodigiosin biosynthetic operon at 37°C compared to 30°C indicating transcriptional control of pigment production. Moreover, induced expression of the prodigiosin biosynthetic operon at 37°C was able to produce pigmented colonies and cultures demonstrating that physiological conditions at 37°C allow prodigiosin production and indicating that post-transcriptional control is not a major contributor to the thermoregulation of prodigiosin pigmentation. Genetic experiments support the model that the HexS transcription factor is a key contributor to thermoregulation of pigmentation, whereas CRP plays a minor role, and a clear role for EepR and PigP was not observed. Together, these data indicate that thermoregulation of prodigiosin production at elevated temperatures is controlled largely, if not exclusively, at the transcriptional level.

Polycystic ovary syndrome (PCOS) is highly associated with cardiometabolic risk and the metabolic syndrome (MetS), predisposing women to increased risk of developing type 2 diabetes and cardiovascular disease. Metformin is commonly used to treat insulin resistance-glucose intolerance, and flutamide, an androgen receptor (AR) antagonist, is used to target hyperandrogenemia and dyslipidemia. Currently, the physiological mechanism of action of these treatments on androgen, lipidogenic, and insulin signaling pathways remains unclear in PCOS. The aim of this study was to investigate the effects and mechanisms of action of metformin and flutamide on plasma lipid-apolipoprotein (Apo)B-lipoprotein and insulin-glucose metabolism, and endocrine-reproductive indices in a PCOS-prone MetS rodent model. PCOS-prone rodents were treated with metformin (300 mg/kg body wt), flutamide (30 mg/kg body wt), or metformin + flutamide combination treatment for 6 wk. Metformin was shown to improve fasting insulin and HOMA-IR, whereas flutamide and combination treatment were shown to reduce plasma triglycerides, ApoB48, and ApoB100, and this was associated with decreased intestinal secretion of ApoB48/triglyceride. Flutamide and metformin were shown to reduce plasma androgen indices and to improve ovarian primary and preovulatory follicle frequency. Metformin treatment increased hepatic estrogen receptor (ER)α, and metformin-flutamide decreased intestinal AR and increased ERα mRNA expression. Metformin-flutamide treatment upregulated hepatic and intestinal insulin signaling, including insulin receptor, MAPK1, and AKT2. In conclusion, cardiometabolic risk factors, in particular ApoB-hypertriglyceridemia, are independently modulated via the AR, and understanding the contribution of AR and insulin-signaling pathways further may facilitate the development of targeted interventions in high-risk women with PCOS and MetS.

RESUMEN: Se ha planteado la hipótesis de que la productividad primaria neta de bosques lluviosos tropicales de baja altitud se encuentra limitada por la concentración de nutrientes del suelo, principalmente por el fósforo edáfico. Para probar dicha hipótesis se evaluó la producción de hojarasca como una medida de la productividad primaria neta, en dos bosques pluviales tropicales ubicados en las localidades de Opogodó y Pacurita en el Chocó biogeográfico. Se instalaron 125 colectores de hojarasca, que se monitorearon mensualmente durante un año. Dichos colectores se distribuyeron en cinco tratamientos de fertilización (control, nitrógeno, fósforo, potasio y NPK), para determinar los efectos del adicionamiento de nutrientes sobre la producción de hojarasca total y sus componentes hojas, ramas, material reproductivo y miscelánea (material en descomposición), y la relación reproductivo/hojas. En Opogodó, la aplicación de nitrógeno o fósforo incrementó la producción de hojarasca total, la fertilización con nitrógeno aumentó la producción de ramas, y el adicionamiento de nitrógeno, fósforo o potasio, por separado, aumentó la producción de miscelánea. Mientras que, con la fertilización se redujo la producción de material reproductivo y la relación reproductivo/hojas. En síntesis, la fertilización cambió la contribución relativa de los diferentes componentes de la hojarasca, y favoreció la construcción de estructuras fotosintéticas (hojas), en comparación con las estructuras reproductivas. Además, se demostró que la caída de hojarasca está limitada de por nitrógeno, fósforo y potasio, que influyen sobre la captura de carbono de estos ecosistemas boscosos. En Pacurita, la fertilización no tuvo efectos significativos sobre la producción de hojarasca.

Chlorpyrifos (CPF) is an organophosphate (OP) pesticide that causes acute toxicity by inhibiting acetylcholinesterase (AChE) in the nervous system. However, endocannabinoid (eCB) metabolizing enzymes in brain of neonatal rats are more sensitive than AChE to inhibition by CPF, leading to increased levels of eCBs. Because eCBs are immunomodulatory molecules, we investigated the association between eCB metabolism, lipid mediators, and immune function in adult and neonatal mice exposed to CPF. We focused on lung effects because epidemiologic studies have linked pesticide exposures to respiratory diseases. CPF was hypothesized to disrupt lung eCB metabolism and alter lung immune responses to lipopolysaccharide (LPS), and these effects would be more pronounced in neonatal mice due to an immature immune system. We first assessed the biochemical effects of CPF in adult mice (≥ 8 weeks old) and neonatal mice after administering CPF (2.5 mg/kg, oral) or vehicle for 7 days. Tissues were harvested 4 h after the last CPF treatment and lung microsomes from both age groups demonstrated CPF-dependent inhibition of carboxylesterases (Ces), a family of xenobiotic and lipid metabolizing enzymes, whereas AChE activity was inhibited in adult lungs only. Activity-based protein profiling (ABPP)-mass spectrometry of lung microsomes identified 31 and 32 individual serine hydrolases in neonatal lung and adult lung, respectively. Of these, Ces1c/Ces1d/Ces1b isoforms were partially inactivated by CPF in neonatal lung, whereas Ces1c/Ces1b and Ces1c/BChE were partially inactivated in adult female and male lungs, respectively, suggesting age- and sex-related differences in their sensitivity to CPF. Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) activities in lung were unaffected by CPF. When LPS (1.25 mg/kg, i.p.) was administered following the 7-day CPF dosing period, little to no differences in lung immune responses (cytokines and immunophenotyping) were noted between the CPF and vehicle groups. However, a CPF-dependent increase in the amounts of dendritic cells and certain lipid mediators in female lung following LPS challenge was observed. Experiments in neonatal and adult Ces1d(−/−) mice yielded similar results as wild type mice (WT) following CPF treatment, except that CPF augmented LPS-induced Tnfa mRNA in adult Ces1d(−/−) mouse lungs. This effect was associated with decreased expression of Ces1c mRNA in Ces1d(−/−) mice versus WT mice in the setting of LPS exposure. We conclude that CPF exposure inactivates several Ces isoforms in mouse lung and, during an inflammatory response, increases certain lipid mediators in a female-dependent manner. However, it did not cause widespread altered lung immune effects in response to an LPS challenge.

Introduction Inflammation induces a procoagulant phenotype of endothelial cells (EC) with the exposure of tissue factor (TF), a potent initiator of the extrinsic coagulation cascade. Although systemic inflammation affects the whole vascular system, thrombotic lesions occur particularly in microcirculation. This raises the question of whether TF-procoagulant activity (TF-PCA) differs between EC from arterial, venous, and microvascular beds. Materials and methods Functional coagulation tests, including TF-PCA, and inflammatory responses were investigated on arterial, venous and microvascular endothelial cells. Interleukin-6 (IL-6) and TF-levels were determined in cohort of 59 septic patients. Results We found that tumor necrosis factor alpha (TNFα), lipopolysaccharide, and interleukin-1β induce a solid, dose-dependent increase in TF-PCA, which is highest in microvascular EC. A positive correlation of interleukin-6 (IL-6) with TF levels was observed in a cohort of 59 septic patients. In contrast, TF-PCA was independent of IL-6 concentrations in vitro. Re-analysis of publicly available gene expression data revealed that among the top 50 genes annotated to coagulation, TF is one of three regulated genes common to the three investigated EC subtypes. The response to inflammatory stimuli in terms of exposure of leukocyte-endothelial- and platelet-endothelial adhesion molecules (E-selectin and PECAM-1), remodeling of adherens junctions, co-exposure of negatively charged surfaces nor breakdown of the glycocalyx was comparable between the EC subtypes and did not explain the higher TF-PCA on microvascular cells. We found that the ratio of TF and TFPI exposure on the endothelial membrane significantly differs between the EC subtypes. Conclusions These findings indicate that the ratio of TF to its inhibitor TFPI is a determinant of endothelial TF-PCA, which is most pronounced on microvascular endothelial cells and might explain why the microvascular system is particularly susceptible to inflammation-induced thrombosis.

Background Metabolic alkalosis is a frequently occurring problem during continuous veno-venous hemofiltration (CVVH) with regional citrate anticoagulation (RCA). This study aimed to evaluate the effectiveness of switching from high to low bicarbonate (HCO3−) replacement fluid in alkalotic critically ill patients with acute kidney injury treated by CVVH and RCA. Methods A retrospective-comparative study design was applied. Patients who underwent CVVH with RCA in the ICU between 09/2016 and 11/2017 were evaluated. Data were available from the clinical routine. A switch of the replacement fluid Phoxilium® (30 mmol/l HCO3−) to Biphozyl® (22 mmol/l HCO3−) was performed as blood HCO3− concentration persisted ≥ 26 mmol/l despite adjustments of citrate dose and blood flow. Data were collected from 72 h before the switch of the replacement solutions until 72 h afterwards. Results Of 153 patients treated with CVVH during that period, 45 patients were switched from Phoxilium® to Biphozyl®. Forty-two patients (42 circuits) were available for statistical analysis. After switching the replacement fluid from Phoxilium® to Biphozyl® the serum HCO3− concentration decreased significantly from 27.7 mmol/l (IQR 26.9–28.9) to 25.8 mmol/l (IQR 24.6–27.7) within 24 h (p 0.001). Base excess (BE) decreased significantly from 4.0 mmol/l (IQR 3.1–5.1) to 1.8 mmol/l (IQR 0.2–3.4) within 24 h (p 0.001). HCO3− and BE concentration remained stable from 24 h till the end of observation at 72 h after the replacement fluid change (p = 0.225). pH and PaCO2 did not change significantly after the switch of the replacement fluid until 72 h. Conclusions This retrospective analysis suggests that for patients developing refractory metabolic alkalosis during CVVH with RCA the use of Biphozyl® reduces external HCO3− load and sustainably corrects intracorporeal HCO3− and BE concentrations. Future studies have to prove whether correcting metabolic alkalosis during CVVH with RCA in critically ill patients is of relevance in terms of clinical outcome.

BackgroundThere are limited effective prophylactic treatments for SARS-CoV-2 infection, and limited early treatment options. Viral cell entry requires spike protein binding to the ACE2 receptor and spike cleavage by TMPRSS2, a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is already in clinical trials in early SARS-CoV-2 infection.MethodsThe likely initial cells of SARS-CoV-2 entry are the ciliated cells of the upper airway. We therefore used differentiated primary human airway epithelial cells maintained at the air-liquid interface (ALI) to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection.ResultsWe first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-Cov-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, camostat is rapidly metabolised in the circulation in vivo, and systemic bioavailability after oral dosing is low. We therefore modelled local airway administration by applying camostat to the apical surface of the differentiated ALI cultures. We demonstrated that a brief exposure to topical camostat is effective at restricting SARS-CoV-2 viral infection.ConclusionThese experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial.

CD19 is among the most relevant targets in cancer immunotherapy. However, its extracellular domain (ECD) is prone to aggregation and misfolding, representing a major obstacle for the development and analysis of CD19-targeted therapeutics. Here, we engineered stabilized CD19-ECD (termed SuperFolder) variants, which also showed improved expression rates and, in contrast to the wild type protein, they could be efficiently purified in their monomeric forms. Despite being considerably more stable, these engineered mutants largely preserved the wild type sequence (>98.8%). We demonstrate that the variant SF05 enabled the determination of the monovalent affinity between CD19 and a clinically approved FMC63-based CAR, as well as monitoring and phenotypic characterization of CD19-directed CAR-T cells in the blood of lymphoma patients. We anticipate that the SuperFolder mutants generated in this study will be highly valuable tools for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.

This report examines the feasibility of determination of Vitamin D3, D2 and their 25-hydroxy metabolites utilizing Gas Chromatography Tandem Mass Spectrometry (GC/MS/MS) as a potential alternative to popular Liquid Chromatography Tandem Mass Spectrometric (LC/MS/MS) methodologies. The GC/MS/MS approach was found to operate reasonably well despite long-standing concerns that gas-liquid chromatography of vitamin D compounds invoke thermal rearrangements owing to the relatively high inlet and capillary column temperatures used. The workup procedure involved incubation of feed samples with concentrated potassium hydroxide for overnight fat saponification, extraction of D Vitamins in n-hexane and reaction with N,O-bis(trimethylsilyl)trifluoroacetamide at 70 °C for 30 mins. In addition to parent compounds, small amounts of pyro-, isopyro-, and iso-vitamin D and isotachysterol3 variants were obtained from each Vitamin D-related compound upon extraction and GC/MS/MS analysis. Mass spectral and chromatographic behavior of these compounds are herein described and interpreted. Multiple Reaction Monitoring settings on GC/MS/MS included m/z 456→351 for Vitamin D3 and m/z 486→363 for Vitamin D2. Trimethylsilylation enabled single predominant peaks for Vitamins D3 and D2, and sample workup in the presence of deuterated Vitamin D analogs enabled accurate and precise sensitivity to 1 ppb (ng/g) in feeds. The method could be extended with reasonable accuracy to 25-hydroxy (25OH) compounds, but accuracies would be significantly improved by inclusion of respective 25OH-specific deuterated internal standards. The method was applied to 27 submissions of suspect dog foods of which 22% were discovered elevated and 44% were discovered to contain toxic levels of Vitamin D3. The described method was thus discovered to provide a suitable mass spectrometric approach for Vitamin D, proving itself here specifically of value in detection of ergocalciferol and cholecalciferol in animal feeds. The specificity and sensitivity of the tandem quadrupole approach can enable suitable applicability to serum determination if desired.

Semi-malignant giant cell tumors of bone (GCTB) are associated with large osteolytic defects and significant bone destructions. Surgical resection remains the standard therapy that is, however, associated with very high recurrence rates. Bioactive glasses (BGs) that are osteogenic but under certain conditions also cytotoxic might be suitable to achieve biological reconstruction with simultaneous reduction of tumor recurrence in GCTB. In this study, a concentration and time dependent cytotoxic effect of five different BG compositions towards neoplastic GCTB cells was identified while bone marrow derived mesenchymal stromal cells were mostly unaffected. Time course and extent of the cytotoxic effect were dependent on the BG composition and were not associated with caspases activation, indicating that apoptotic mechanisms are not involved. Rather, detection of BG-induced disruption of the cell membranes and a rapid drop of intracellular HMG1 (High Mobility Group Box 1 protein) levels suggest a necrotic cell death. Notably, the cytotoxic effects were dependent on a direct contact of cells and BGs and could not be observed using indirect cultivation settings. Our data suggest that BGs might represent promising materials for the treatment of GCTB in order to reduce tumor recurrence with simultaneous enhancement of bone regeneration.

In this paper, an arc was drawn over ten years of research activities from three chairs of the Montanuniversitaet Leoben, as well as industrial partners. The superior objective of this research effort was to develop a wet-mechanical process for the recovery of polyolefin concentrates (90 wt% polyolefins) from mixed waste fraction for use in chemical recycling and to advance this new technology to commercial maturity. As a bridge technology, it would close the gap between state-of-the-art dry processing of mixed plastic waste materials and chemical plastic recycling via thermo-chemical conversion. The methods used were mainly tested in a lab-scale plant with a throughput capacity of 50 to 200 kg/h depending on the bulk density of the used feedstock. Further studies for the treatment and usage of the main products and by-products, as well as chemical analyses of them, were completed during the investigation. Within these series of tests, polyolefin concentrates, which satisfied the requirements for chemical recycling, could be recovered. With these data, a concept for an industrial pilot plant was developed and evaluated from an economic point of view. According to this evaluation, the realization of such an industrial pilot plant can be recommended.

A simple method is reported for the preparation of silver nanoparticle (AgNP) embedded pH-responsive hydrogel microparticle catalyst via Michael addition gelation and in-situ silver nitrate (AgNO3) reduction. The AgNP-hydrogel microsphere exhibited an efficient reduction of pollutants like 4-Nitrophenol (4-NP) and Congo red (CR) under acidic medium with turn over frequency (TOF) of ~170 h–1 and ~124 h–1 respectively. Interestingly, the activity of the catalysts was turned-OFF under a basic medium (≥ pH 12) due to the deswelling pH-responsive matrix surrounding the AgNPs. On the contrary, turning-OFF the hydrogenation of a cationic pollutant like methylene blue (MB) using high pH (≥ 12) was not possible, due to ionic interaction of MB molecules with the negatively charged catalyst at this pH. This feature was used to demonstrate selective hydrogenation of only MB from a mixture of 4-NP and MB. Finally, five recycling steps confirmed the reusability and practical application potential of the catalyst.

Plaques of the amyloid beta (Aß) peptide are a pathological hallmark of Alzheimer’s disease (AD), the most common form of dementia. Mutations in Aß also cause familial forms of AD (fAD). Here, we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of Aß. The resulting genetic landscape reveals mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the empirical nucleation scores accurately identify all known dominant fAD mutations in Aß, genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease. These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in Aß., eLife digest Alzheimer’s disease is the most common form of dementia, affecting more than 50 million people worldwide. Despite more than 400 clinical trials, there are still no effective drugs that can prevent or treat the disease. A common target in Alzheimer’s disease trials is a small protein called amyloid beta. Amyloid beta proteins are ‘sticky’ molecules. In the brains of people with Alzheimer’s disease, they join to form first small aggregates and then long chains called fibrils, a process which is toxic to neurons. Specific mutations in the gene for amyloid beta are known to cause rare, aggressive forms of Alzheimer’s disease that typically affect people in their fifties or sixties. But these are not the only mutations that can occur in amyloid beta. In principle, any part of the protein could undergo mutation. And given the size of the human population, it is likely that each of these mutations exists in someone alive today. Seuma et al. reasoned that studying these mutations could help us understand the process by which amyloid beta forms new aggregates. Using an approach called deep mutational scanning, Seuma et al. mutated each point in the protein, one at a time. This produced more than 14,000 different versions of amyloid beta. Seuma et al. then measured how quickly these mutants were able to form aggregates by introducing them into yeast cells. All the mutations known to cause early-onset Alzheimer’s disease accelerated amyloid beta aggregation in the yeast. But the results also revealed previously unknown properties that control how fast aggregation occurs. In addition, they highlighted a number of positions in the amyloid beta sequence that act as ‘gatekeepers’. In healthy brains, these gatekeepers prevent amyloid beta proteins from sticking together. When mutated, they drive the protein to form aggregates. This comprehensive dataset will help researchers understand how proteins form toxic aggregates, which could in turn help them find ways to prevent this from happening. By providing an ‘atlas’ of all possible amyloid beta mutations, the dataset will also help clinicians interpret any new mutations they encounter in patients. By showing whether or not a mutation speeds up aggregation, the atlas will help clinicians predict whether that mutation increases the risk of Alzheimer’s disease.

Three novel alkyl sulfone bridged phosphorus (P) compounds namely 6,6′-(sulfonylbis(ethane-2,1-diyl))bis(dibenzo[c,e][1,2]oxaphosphinine 6-oxide) (SEDOPO), sulfonylbis(ethane-2,1-diyl))bis(diphenylphosphine oxide) (SEDPPO), and tetraphenyl (sulfonyl bis(ethane-2,1-diyl)) bis(phosphonate) (SEDPP) (i.e. phosphine oxide, phosphinate and phosphonate, respectively) were synthesized via a Michael addition reaction with good yields (≥ 85%) at a 200-g scale. They exhibited thermal stability above 250 °C, which allowed them to be melt-processed with polypropylene (PP) and formed into thin films (~ 0.6 mm). Rheological measurements of the PP blends exhibited a typical shear thinning behavior and provided evidence for the synthesized compound's thermo-oxidative stabilizing effect. This was also confirmed by thermal analysis showing that the thermo-oxidative stability of PP-SEDOPO and PP-SEDPPO blends was higher (~25 °C) than the blank PP; however, PP-SEDPP had a smaller impact. Small scale fire tests of the PP-FR blends confirmed the flame retardant efficacy of the new P-compounds. Cone calorimetry on PP-SEDOPO blends showed a reduction in the heat release rate (HRR) (~48%) compared to blank PP. Further thermal and evolved gas analysis of the PP blends confirmed that the new P-compounds are primarily active in the gas-phase.

Early root growth is critical for plant establishment and survival. We have identified a molecular pathway required for oscillatory root tip movement known as circumnutation. Here we report a multiscale investigation of the regulation and function of this phenomenon. We identify key cell signaling events comprising interaction of the ethylene, cytokinin, and auxin hormone signaling pathways. We identify the gene Oryza sativa Histidine Kinase-1/OsHK1, as well as the auxin influx carrier gene OsAUX1, as essential regulators of this process in rice. Robophysical modelling demonstrated the benefits of tip movement for navigating past obstacles, prompting us to challenge mutant and wild-type plants with different substrates. Consistent with model behavior, root circumnutation facilitated exploration of a solid surface and promoted seedling establishment in rocky soil. Thus, the integration of robotics, physics and biology elucidated the functional importance of root circumnutation and uncovered the molecular mechanisms underlying its regulation.One sentence summaryCircumnutation facilitates root exploration.

The liver is the central organ regulating cholesterol synthesis, storage, transport, and elimination. Mouse carboxylesterase 1d (Ces1d) and its human ortholog CES1 have been described to possess lipase activity and play roles in hepatic triacylglycerol metabolism and VLDL assembly. It has been proposed that Ces1d/CES1 might also catalyze cholesteryl ester (CE) hydrolysis in the liver and thus be responsible for the hydrolysis of HDL-derived CE; this could contribute to the final step in the reverse cholesterol transport (RCT) pathway, wherein cholesterol is secreted from the liver into bile and feces, either directly or after conversion to water-soluble bile salts. However, the proposed function of Ces1d/CES1 as a CE hydrolase is controversial. In this study, we interrogated the role hepatic Ces1d plays in cholesterol homeostasis using liver-specific Ces1d-deficient mice. We rationalized that if Ces1d is a major hepatic CE hydrolase, its absence would (1) reduce in vivo RCT flux and (2) provoke liver CE accumulation after a high-cholesterol diet challenge. We found that liver-specific Ces1d-deficient mice did not show any difference in the flux of in vivo HDL-to-feces RCT nor did it cause additional liver CE accumulation after high-fat, high-cholesterol Western-type diet feeding. These findings challenge the importance of Ces1d as a major hepatic CE hydrolase.

Hyperosmotic stresses represent some of the most serious abiotic factors that adversely affect plants growth, development and fitness. Despite their central role, the early cellular events that lead to plant adaptive responses remain largely unknown. In this study, using Arabidopsis thaliana cultured cells we analyzed early cellular responses to sorbitol-induced hyperosmotic stress. We observed biphasic and dual responses of A. thaliana cultured cells to sorbitol-induced hyperosmotic stress. A first set of events, namely singlet oxygen (1O2) production and cell hyperpolarization due to a decrease in anion channel activity could participate to signaling and osmotic adjustment allowing cell adaptation and survival. A second set of events, namely superoxide anion (O2-) production by RBOHD-NADPH-oxidases and SLAC1 anion channel activation could participate in programmed cell death (PCD) of a part of the cell population. This set of events raises the question of how a survival pathway and a death pathway could be induced by the same hyperosmotic condition and what could be the meaning of the induction of two different behaviors in response to hyperosmotic stress.