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Ubiquitin and ubiquitin-like proteins (UBLs) function as critical post-translational modifiers in the maintenance of genome stability. Ubiquitin/UBL-conjugating enzymes (E2s) are responsible, as part of a wider enzymatic cascade, for transferring single moieties or polychains of ubiquitin/UBLs to one or multiple residues on substrate proteins. Recent advances in structural and mechanistic understanding of how ubiquitin/UBL substrate attachment is orchestrated indicate that E2s can exert control over chain topology, substrate-site specificity, and downstream physiological effects to help maintain genome stability. Drug discovery efforts have typically focussed on modulating other members of the ubiquitin/UBL cascades or the ubiquitin-proteasome system. Here, we review the current standing of E2s in genome stability and revisit their potential as pharmacological targets for developing novel anti-cancer therapies.

DNA double-strand breaks (DSBs) represent the most cytotoxic DNA lesions, as—if mis- or unrepaired—they can cause cell death or lead to genome instability, which in turn can cause cancer. DSBs are repaired by two major pathways termed homologous recombination and non-homologous end-joining (NHEJ). NHEJ is responsible for repairing the vast majority of DSBs arising in human cells. Defects in NHEJ factors are also associated with microcephaly, primordial dwarfism and immune deficiencies. One of the key proteins important for mediating NHEJ is XRCC4. XRCC4 is a dimer, with the dimer interface mediated by an extended coiled-coil. The N-terminal head domain forms a mixed alpha–beta globular structure. Numerous factors interact with the C-terminus of the coiled-coil domain, which is also associated with significant self-association between XRCC4 dimers. A range of construct lengths of human XRCC4 were expressed and purified, and the 1–164 variant had the best NMR properties, as judged by consistent linewidths, and chemical shift dispersion. In this work we report the 1H, 15 N and 13C backbone resonance assignments of human XRCC4 in the solution form of the 1–164 construct. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 156 of 161 assignable residues of XRCC4 were assigned to resonances in the TROSY spectrum, with an additional 11 resonances assigned to His-Tag residues. Prediction of solution secondary structure from a chemical shift analysis using the TALOS + webserver is in good agreement with the published X-ray crystal structures of this protein. Supplementary Information The online version contains supplementary material available at 10.1007/s12104-021-10035-6.

The organotypic human air‐liquid‐interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high‐throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error‐corrected next‐generation sequencing method capable of detecting a single mutation per 107 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 μg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time‐ and concentration‐dependent increases in DNA damage and a concentration‐dependent increase in mutant frequency. The mutations observed in the EMS‐treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti‐p63‐positive basal cell frequency, but produced concentration‐responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration‐responsive decreases in culture viability, goblet cell and anti‐Ki67‐positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28‐day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue‐specific general toxicity.

[Image: see text] This report describes the development of an orally active analgesic that resolves inflammation and neuropathic pain without the addictive potential of opioids. EC5026 acts on the cytochrome P450 branch of the arachidonate cascade to stabilize epoxides of polyunsaturated fatty acids (EpFA), which are natural mediators that reduce pain, resolve inflammation, and maintain normal blood pressure. EC5026 is a slow-tight binding transition-state mimic that inhibits the soluble epoxide hydrolase (sEH) at picomolar concentrations. The sEH rapidly degrades EpFA; thus, inhibiting sEH increases EpFA in vivo and confers beneficial effects. This mechanism addresses disease states by shifting endoplasmic reticulum stress from promoting cellular senescence and inflammation toward cell survival and homeostasis. We describe the synthesis and optimization of EC5026 and its development through human Phase 1a trials with no drug-related adverse events. Additionally, we outline fundamental work leading to discovery of the analgesic and inflammation-resolving CYP450 branch of the arachidonate cascade.

The paper reports on multicomponent (Ziegler) catalytic systems based on acetylacetonate complexes of cobalt, nickel or palladium and various aluminum-containing cocatalysts of general formula M(acac)2–Red (where M = Co, Ni, Pd; Red = AlEt3, LiAlH4, AlEt2(OEt), LiAlH(tert-BuO)3), which are active in alkenes hydrogenation under mild conditions (T = 30 °C, P = 2 atm). The key features (size, composition, nature of the ligand shell) of metal-containing nanoscale particles formed during the interaction of the initial components under inert medium and hydrogenation catalysis are considered. Also, the dynamics of the reaction solution changes in the course of catalytic processes as well as the kinetic parameters (activity, productivity, selectivity) of alkenes hydrogenation are described. The models of nanoparticles formed under the hydrogenation catalysis conditions during the interaction of M(acac)2–Red (where M = Co, Ni, Pd; Red = AlEt3, LiAlH4) are discussed. The role of proton-donor compounds (contained in the initial complexes of transition metals or specially introduced into the reaction systems) in the formation of catalytically active sites is explained. In particular, it is shown that the activating effect of these compounds is associated with alcoholysis of the cocatalyst and products of its catalytic decomposition, which are located on the surface of transition metal nanoparticles. A general scheme of activation of the [M]n particles formed by the interaction of M(acac)2 with AlEt3 (M = Ni and Co) is presented. The concept for the directed design of Ziegler catalytic systems is proposed. In order to evaluate the application scope of this concept, the reasons of different catalytic activity and productivity of bimetallic nickel–copper catalysts of the same composition, obtained by sequential and simultaneous reduction of Ni(acac)2 and Cu(acac)2 by LiAlH4, are revealed. In addition, a number of priority issues that should be addressed are discussed to highlight the prospects for the further development of this field of catalysis.

The effect of the composition of the catalytic system and reaction conditions on the properties of phosphorus-modified palladium catalysts in hydrogenations of alkynols was studied. Modification with phosphorus increased the activity and turnover number of palladium catalysts in the hydrogenation of the model compound 2-methyl-3-butyn-2-ol (MBY) without any reduction in the selectivity to 2-methyl-3-butene-2-ol at 95–98% MBY conversion. The promoting effect of phosphorus on the properties of the palladium catalyst is caused not only by an increase in the particle size, but also, probably, by a change in the energy of interaction of reagents with the active sites. Hypotheses on the nature of the carriers of catalytic activity in Pd–P particles were discriminated using kinetic methods with the differential selectivity of catalytic systems as the main measured parameter under the conditions of competition between two alkynols. The hydrogenation of acetylenic alcohols involves only one of the two potentially active forms in Pd–P nanoparticles—Pd(0) clusters, whereas the hydrogenation of the resulting allyl alcohols involves both Pd(0) clusters and palladium phosphides.

Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal “CaaX” sequence in their protein substrates, but recent studies in yeast- and mammalian-based systems have demonstrated FTase can also accept sequences that diverge in length from the canonical four-amino acid motif, such as the recently reported five-amino acid C(x)(3)X motif. In this work, we further expand the substrate scope of FTase by demonstrating sequence-dependent farnesylation of shorter three-amino acid “Cxx” C-terminal sequences using both genetic and biochemical assays. Strikingly, biochemical assays utilizing purified mammalian FTase and Cxx substrates reveal prenyl donor promiscuity leading to both farnesylation and geranylgeranylation of these sequences. These findings expand the substrate pool of sequences that can be potentially prenylated, further refine our understanding of substrate recognition by FTase and GGTase-I, and suggest the possibility of a new class of prenylated proteins within proteomes.

Cryoconite is a mixture of mineral and organic material covering glacial ice, playing important roles in biogeochemical cycles and lowering the albedo of a glacier's surface. Understanding the differences in structure of cryoconite across the globe can be important in recognizing past and future changes in supraglacial environments and ice-organisms-dust interactions. Despite the worldwide distribution and over a century of studies, the basic characteristics of cryoconite, including its forms and geochemistry, remain poorly studied. The major purpose of our study is the presentation and description of morphological diversity, chemical and photoautotrophs composition, and organic matter content of cryoconite sampled from 33 polar and mountain glaciers around the globe. Observations revealed that cryoconite included various morphologies including loose and granular forms. Granular cryoconite includes smooth, rounded, or irregularly shaped forms; with some having their surfaces covered by cyanobacteria filaments. The occurrence of granules increased with the organic matter content in cryoconite. Moreover, a major driver of cryoconite colouring was the concentration of organic matter and its interplay with minerals. The structure of cyanobacteria and algae communities in cryoconite differs between glaciers, but representatives of cyanobacteria families Pseudanabaenaceae and Phormidiaceae, and algae families Mesotaeniaceae and Ulotrichaceae were the most common. The most of detected cyanobacterial taxa are known to produce polymeric substances (EPS) that may cementing matter and form granules. Organic matter content in cryoconite varied between glaciers, ranging from 1% to >40%. The geochemistry of all the investigated samples reflected local sediment sources, except of highly concentrated Pb and Hg in cryoconite collected from European glaciers near industrialized regions, corroborating cryoconite as element-specific collectors and potential environmental indicator of anthropogenic activity. Our work supports a notion that cryoconite may be more than just simple sediment and instead exhibits complex structure with relevance for biodiversity and the functioning of glacial ecosystems.

The nature and size of particles, formed under conditions of the ethylene oligomerization in the systems based on nickel bis(acetylacetonate) with Et2AlCl or Et2AlCl·Cl2AlEt (Al: Ni = 50: 1), have been determined using ex situ HRTEM, electron diffraction and EDAX in combination with EPR spectroscopy and kinetic studies. It was revealed that the average sizes of Ni-containing particles depend on the nature of cocatalyst and are ∼3.5 and ∼6 nm for the systems derived from Et2AlCl and Et2AlCl·Cl2AlEt, respectively. It was shown that these nanoparticles act as the carriers of catalytically active sites during the C2H4 conversion.

A key challenge to understanding the effects of climate change and nutrient deposition on ecosystem functioning is our lack of knowledge about nutrient limitations of heterotrophic and phototrophic microbial communities. This is especially true in high elevation ecosystems where it has been shown that earlier melt-out of snow beds and glacial retreat is allowing photosynthetic microbes and plants to move into previously unvegetated areas. We used landscape-level analyses of microbial enzyme stoichiometries combined with soil microcosm fertilization studies to determine which nutrients are limiting to microbes in plant-free or sparsely vegetated, snow bed areas of the Colorado Front Range. Both of these independent approaches indicated that the ultimate limiting nutrient in unvegetated and sparsely vegetated soils is phosphorus (P) for phototrophic microbes, with co-limitation by carbon (C) for the entire microbial community. In contrast, vegetated soils in the same watersheds showed more balanced nitrogen (N), P and C co-limitation similar to patterns seen in other plant-dominated ecosystems. In microcosm experiments, P additions resulted in increased growth rates and percent cover by phototrophs, whereas N additions decreased the relative abundances of phototrophs. Taken together, our findings indicate that the colonization of high elevation ecosystems being impacted by N deposition and climate warming will likely be constrained by P limitation of both heterotrophic and phototrophic microbes and by negative impacts of N on microbial phototrophs. These effects may in turn limit the ability of these fragile ecosystems to immobilize inputs of atmospheric N causing increased runoff of excess N to downstream ecosystems.

Objective Although mu-opioid receptor agonists have been the mainstay of analgesic regimens for moderate to severe pain, they are associated with serious side effects, risks, and limitations. We evaluate the most serious risks associated with conventional opioids and compare these with the pharmacology of CYT-1010, a prototypical endomorphin and mu-opioid receptor agonist. Results Addiction and respiratory depression are serious risks of traditional mu-opioid analgesics. Mitigation strategies have been inadequate at addressing the opioid crisis and may interfere with the effective treatment of pain. Improved understanding of mu-opioid receptor biology and the discovery in 1997 of an additional and unique family of endogenous opioid peptides (endomorphins) have provided a pathway for dissociating analgesia from opioid-related adverse events and developing new classes of mu-opioid receptor agonists that use biased signaling and/or target novel sites to produce analgesia with reduced side effect liability. Endomorphin-1 and -2 are endogenous opioid peptides highly selective for mu-opioid receptors that exhibit potent analgesia with reduced side effects. CYT-1010 is a cyclized, D-lysine-containing analog of endomorphin-1 with a novel mechanism of action targeting traditional mu- and exon 11/truncated mu-opioid receptor 6TM variants. CYT-1010 preclinical data have demonstrated reduced abuse potential and analgesic potency exceeding that of morphine. In an initial phase 1 clinical study, CYT-1010 demonstrated significant analgesia vs baseline and no respiratory depression at the dose levels tested. Conclusions CYT-1010 and other novel mu-opioid receptor agonists in clinical development are promising alternatives to conventional opioids that may offer the possibility of safer treatment of moderate to severe pain.

Simple Summary The research field of 3D cell cultivation in hydrogels is continuously growing. To be able to analyze the reaction of melanoma cells to 3D cultivation in alginate hydrogel on a molecular level, whole transcriptome sequencing was performed. Intriguingly, we could not only unravel differences between the gene regulation in 2D and 3D cultures but could also correlate the culture switch to the physiological process of tumor plasticity based on the observed patterns. Thereby, the role of EGR1 in controlling tumor plasticity and progression in melanoma was revealed. We conclude that the combination of cell culture models using biomaterials and whole transcriptome analysis leads to a deeper molecular understanding of cancer cells, herewith defining new therapeutic targets. Abstract Alginate hydrogels have been used as a biomaterial for 3D culturing for several years. Here, gene expression patterns in melanoma cells cultivated in 3D alginate are compared to 2D cultures. It is well-known that 2D cell culture is not resembling the complex in vivo situation well. However, the use of very intricate 3D models does not allow performing high-throughput screening and analysis is highly complex. 3D cell culture strategies in hydrogels will better mimic the in vivo situation while they maintain feasibility for large-scale analysis. As alginate is an easy-to-use material and due to its favorable properties, it is commonly applied as a bioink component in the growing field of cell encapsulation and biofabrication. Yet, only a little information about the transcriptome in 3D cultures in hydrogels like alginate is available. In this study, changes in the transcriptome based on RNA-Seq data by cultivating melanoma cells in 3D alginate are analyzed and reveal marked changes compared to cells cultured on usual 2D tissue culture plastic. Deregulated genes represent valuable cues to signaling pathways and molecules affected by the culture method. Using this as a model system for tumor cell plasticity and heterogeneity, EGR1 is determined to play an important role in melanoma progression.

Rheumatoid arthritis (RA) belongs to the most often occurring autoimmune diseases in the world. For serological diagnosis, IgM auto-antibodies directed against the Fc portion of IgG referred to as rheumatoid factor are used as biomarkers. The autoantibody detection is usually done by ELISA. Such assays are reliable but are not suitable for point-of-care testing in contrast to lateral flow assays. Here, we report the development of a lateral flow assay based on carboxylated fluorescence-encoded poly(methyl methacrylate) nanoparticles. Poly(methyl methacrylate) is a non-toxic plastic with an excellent biocompatibility and high optical transparency which promises especially high sensitive fluorescence detection thereby leading to very sensitive assays. We could detect a positive signal in samples with a nephelometric reading down to 0.4 U/mL. By analyzing 30 sera of patients with a RA diagnosis and 34 sera of healthy test subjects we could confirm positive ELISA results in 72% of all cases and negative ELISA results in 97% of all cases.

SUMOylation is critical for a plethora of cellular signalling pathways including the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Based on systematic proteome microarray screening combined with widely applicable carbene footprinting and high-resolution structural profiling, we define two non-conventional SUMO2-binding modules on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, interaction of SUMO2 with XRCC4 is incompatible with XRCC4 binding to at least two other NHEJ proteins – XLF and DNA ligase 4 (LIG4). These findings are consistent with SUMO2 interactions of XRCC4 acting as backup pathways at different stages of NHEJ, in the absence of these factors or their dysfunctioning. Such scenarios are not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. This work reveals insights into topology-specific SUMO recognition and its potential for modulating DSB repair by NHEJ. Moreover, it provides a rich resource on binary SUMO receptors that can be exploited for uncovering regulatory layers in a wide array of cellular processes.

A recent work by Hartwig et al. (Science, 2020) details an efficient method to borylate strong primary C(sp3)–H bonds via iridium-catalysed C–H activation. The conditions confer counterintuitive selectivity over more labile C–H bonds, while providing an avenue for a gamut of synthetically valuable late-stage functionalisations through versatile borylate intermediates.

DNA suffers constant insult from a variety of endogenous and exogenous sources. To deal with the arising lesions, cells have evolved complex and coordinated pathways, collectively termed the DNA damage response (DDR). Importantly, an improper DDR can lead to genome instability, premature ageing and human diseases, including cancer as well as neurodegenerative disorders. As a crucial process for cell survival, regulation of the DDR is multi-layered and includes several post-translational modifications. Since the discovery of ubiquitin in 1975 and the ubiquitylation cascade in the early 1980s, a number of ubiquitin-like proteins (UBLs) have been identified as post-translational modifiers. However, while the importance of ubiquitin and the UBLs SUMO and NEDD8 in DNA damage repair and signalling is well established, the roles of the remaining UBLs in the DDR are only starting to be uncovered. Herein, we revise the current status of the UBLs ISG15, UBL5, FAT10 and UFM1 as emerging co-regulators of DDR processes. In fact, it is becoming clear that these post-translational modifiers play important pleiotropic roles in DNA damage and/or associated stress-related cellular responses. Expanding our understanding of the molecular mechanisms underlying these emerging UBL functions will be fundamental for enhancing our knowledge of the DDR and potentially provide new therapeutic strategies for various human diseases including cancer.

Arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) are two fungal groups that colonize plant roots and can benefit plant growth, but little is known about their landscape distributions. We performed sequencing and microscopy on a variety of plants across a high-elevation landscape featuring plant density, snowpack, and nutrient gradients. Percent colonization by both AMF and DSE varied significantly among plant species, and DSE colonized forbs and grasses more than sedges. AMF were more abundant in roots at lower elevation areas with lower snowpack and lower phosphorus and nitrogen content, suggesting increased hyphal recruitment by plants to aid in nutrient uptake. DSE colonization was highest in areas with less snowpack and higher inorganic nitrogen levels, suggesting an important role for these fungi in mineralizing organic nitrogen. Both of these groups of fungi are likely to be important for plant fitness and establishment in areas limited by phosphorus and nitrogen.

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.

The effect of the composition of the catalytic system and the size of particles on the properties of palladium catalysts in 2-ethyl-9,10-anthraquinone hydrogenation was studied. It was shown that, depending on the nature of a reducing agent (H2, AlEt3), palladium species formed in the absence of a modifying agent catalyze various side processes to a substantial extent together with 2-ethyl-9,10-anthraquinone hydrogenation: mostly hydrogenolysis (in the case of H2 as a reducing agent) and hydrogenation of aromatic rings in 2-ethyl-9,10-anthrahydroquinone (in the case of AlEt3 as a reducing agent). Elementary phosphorus was found to have a promoting effect on the selectivity of palladium catalysts in the synthesis of hydrogen peroxide by the anthraquinone method. The main factors that make it possible to control the selectivity of palladium catalysts were discussed.

The turnover frequencies of catalytic systems based on Со(acac)2–AlEt3 or AlEt2(OEt) in the hydrogenation of benzene have been determined at temperatures of 80–150 °С, initial РН2 = 15 bar, and Al/Со ratios = 1–8. It has been shown that the benzene hydrogenation rate is directly proportional to the Со(acac)2 concentration and hydrogen pressure, while it does not depend on the concentration of benzene at СС6Н6 > 1 × 10−2 mol/L. The parameters of the Arrhenius equation in the hydrogenation of benzene for the systems based on Со(асас)2–AlEt3/AlEt2(OEt) were determined. It has been found that nanosized particles containing cobalt in the zero oxidation state exhibit catalytic activity in the benzene hydrogenation.

Protein prenylation is a post-translational modification that has been most commonly associated with enabling protein trafficking to and interaction with cellular membranes. In this process, an isoprenoid group is attached to a cysteine near the C terminus of a substrate protein by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I or II (GGTase-I and GGTase-II). FTase and GGTase-I have long been proposed to specifically recognize a four-amino acid CAAX C-terminal sequence within their substrates. Surprisingly, genetic screening reveals that yeast FTase can modify sequences longer than the canonical CAAX sequence, specifically C(x)(3)X sequences with four amino acids downstream of the cysteine. Biochemical and cell-based studies using both peptide and protein substrates reveal that mammalian FTase orthologs can also prenylate C(x)(3)X sequences. As the search to identify physiologically relevant C(x)(3)X proteins begins, this new prenylation motif nearly doubles the number of proteins within the yeast and human proteomes that can be explored as potential FTase substrates. This work expands our understanding of prenylation's impact within the proteome, establishes the biologically relevant reactivity possible with this new motif, and opens new frontiers in determining the impact of non-canonically prenylated proteins on cell function.

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathways. In the canonical understanding of FTase, the isoprenoids are attached to the cysteine residue of a four-amino-acid CaaX box sequence. However, recent work has shown that five-amino-acid sequences can be recognized, including the pentapeptide CMIIM. This paper describes a new systematic approach to discover novel peptide substrates for FTase by combining the combinatorial power of solid-phase peptide synthesis (SPPS) with the ease of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The workflow consists of synthesizing focused libraries containing 10–20 sequences obtained by randomizing a synthetic peptide at a single position. Incubation of the library with FTase and farnesyl pyrophosphate (FPP) followed by mass spectrometric analysis allows the enzymatic products to be clearly resolved from starting peptides due to the increase in mass that occurs upon farnesylation. Using this method, 30 hits were obtained from a series of libraries containing a total of 80 members. Eight of the above peptides were selected for further evaluation, reflecting a mixture that represented a sampling of diverse substrate space. Six of these sequences were found to be bona fide substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Experiments in yeast demonstrated that proteins bearing these sequences can be efficiently farnesylated within live cells. Additionally, a bioinformatics search showed that a variety of pentapeptide CaaaX sequences can be found in the mammalian genome, and several of these sequences display excellent farnesylation in vitro and in yeast cells, suggesting that the number of farnesylated proteins within mammalian cells may be larger than previously thought.

Eicosanoids are biologically active lipid signaling molecules derived from polyunsaturated fatty acids. Many of the actions of eicosanoid metabolites formed by cyclooxygenase and lipoxygenase enzymes have been characterized, however, the epoxy-fatty acids (EpFAs) formed by cytochrome P450 enzymes are newly described by comparison. The EpFA metabolites modulate a diverse set of physiologic functions that include inflammation and nociception among others. Regulation of EpFAs occurs primarily via release, biosynthesis and enzymatic transformation by the soluble epoxide hydrolase (sEH). Targeting sEH with small molecule inhibitors has enabled observation of the biological activity of the EpFAs in vivo in animal models, greatly contributing to the overall understanding of their role in the inflammatory response. Their role in modulating inflammation has been demonstrated in disease models including cardiovascular pathology and inflammatory pain, but extends to neuroinflammation and neuroinflammatory disease. Moreover, while the EpFA demonstrate activity against inflammatory pain, interestingly, this action extends to blocking chronic neuropathic pain as well. This review outlines the role of modulating sEH and the biological action of EpFA in models of pain and inflammatory diseases.

A dominant paradigm in ecology is that plants are limited by nitrogen (N) during primary succession. Whether generalizable patterns of nutrient limitation are also applicable to metabolically and phylogenetically diverse soil microbial communities, however, is not well understood. We investigated if measures of N and phosphorus (P) pools inform our understanding of the nutrient(s) most limiting to soil microbial community activities during primary succession. We evaluated soil biogeochemical properties and microbial processes using two complementary methodological approaches-a nutrient addition microcosm experiment and extracellular enzyme assays-to assess microbial nutrient limitation across three actively retreating glacial chronosequences. Microbial respiratory responses in the microcosm experiment provided evidence for N, P and N/P co-limitation at Easton Glacier, Washington, USA, Puca Glacier, Peru, and Mendenhall Glacier, Alaska, USA, respectively, and patterns of nutrient limitation generally reflected site-level differences in soil nutrient availability. The activities of three key extracellular enzymes known to vary with soil N and P availability developed in broadly similar ways among sites, increasing with succession and consistently correlating with changes in soil total N pools. Together, our findings demonstrate that during the earliest stages of soil development, microbial nutrient limitation and activity generally reflect soil nutrient supply, a result that is broadly consistent with biogeochemical theory.

The turnover frequencies of catalytic systems based on Ni(acac)2–AlEt3 or AlEt2(OEt) in the hydrogenation of benzene and its methyl-substituted homologs (toluene, three isomers of xylene, and 1,3,5-trimethylbenzene) have been determined at temperatures of 80–120 °C, initial PH2 = 15 bar, and different ratios of Al/Ni. The size and nature of the nanoparticles forming in the systems based on Ni(acac)2–AlEt3 or AlEt2(OEt) under the benzene hydrogenation condition shave been resolved by high-resolution electron microscopy and X-ray microanalysis. This study included the performance of competitive hydrogenation reactions of benzene with toluene or three xylene isomers. The relative adsorption constants of toluene and three xylene isomers have been determined and the stereochemistry of the hydrogen addition to the arene ring has also been elucidated.

Pd-P nanoparticles were obtained by low-temperature reduction of Pd(acac)2 with hydrogen in the presence of elemental phosphorus. Using the EDX analysis, XRD, and HRTEM, the composition, size, and morphology of the particles were determined for different P:Pd ratios. The results of testing Pd-P nanoparticles showed that they are 8–9 times higher in activity in the hydrogenation of mono- and di-substituted acetylenic compounds than Pd-black and phase-pure Pd3P phosphide, while maintaining high selectivity to alkenes at 95–98% conversion of alkynes. Colloidal solutions of Pd-P nanoparticles can act as long-lived supported catalysts (TON = 27,444 mol PA·(mol Pd total)−1). The effect of the P:Pd ratio on the change in the rates of hydrogenation of alkyne (r1) and the resulting alkene (r2) under the action of Pd-P nanoparticles has been found and experimentally substantiated. Electron-deficient small clusters of palladium contained along with palladium phosphides in the Pd-P nanoparticles are responsible for the decrease in the ratio r1:r2 in the ratio range P:Pd = 1–1.5.

The precursor nature effect on the state of the Pd–P surface layer in palladium catalysts and on their properties in the liquid-phase hydrogenation of chloronitrobenzenes under mild conditions has been investigated. A general feature of the Pd–P-containing nanoparticles obtained from different precursors and white phosphorus at P/Pd = 0.3 (PdCl2 precursor) and 0.7 (Pd(acac)2 precursor) is that their surface contains palladium in phosphide form (BE(Pd3d 5/2) = 336.2 eV and BE(Р2р) = 128.9 eV) and Pd(0) clusters (BE(Pd3d5/2) = 335.7 eV). Factors having an effect on the chemoselectivity of the palladium catalysts in chloronitrobenzenes hydrogenation are considered, including the formation of small palladium clusters responsible for hydrogenation under mild conditions.

For the enantiopure synthesis of novel chiral GABA uptake inhibitors, nipecotic acid (1) is an important key precursor. To characterize accurately the pharmacological activity of these interesting target compounds, the determination of the correct enantiomeric purity of nipecotic acid as the starting material is indispensable. In this report, a sensitive high-performance liquid chromatography (HPLC) based method for the separation and quantitation of both enantiomers of nipecotic acid as 1-(7-nitrobenzo[c][1,2,5] oxadiazol-4-yl) derivatives (5) on a Chiralpak ID-3 column (Daicel, Illkirch, France) was established. UV/Vis-detection at 490 nm was chosen to ensure a selective determination of even highly enantioenriched samples. Reliability was demonstrated by validation of specificity, linearity, lower limit of quantification (LLOQ), accuracy, and precision. By spiking highly enantiopure samples with small amounts of racemic rac-5, it was proven that the established HPLC method is able to detect even slight changes in enantiomeric excess (ee) values. Thus, accurate determination of ee values up to 99.87% ee for (R)-5 and 99.86% ee for (S)-5 over a linear concentration range of 1-1500 mu M for (R)-5 and of 1-1455 mu M for (S)-5 could be demonstrated.

Phase composition and surface layer state of the Pd–P hydrogenation catalyst formed at various P/Pd ratios from Pd(acac)2 and white phosphorus in a hydrogen atmosphere were determined. Palladium on the catalyst surface is mainly in two chemical states: as Pd(0) clusters and as palladium phosphides. As the P/Pd ratio increases, the fraction and size of palladium clusters decrease, and also the phase composition of formed palladium phosphides changes: Pd3P0.8 → Pd5P2 → PdP2. The causes of the modifying action of phosphorus on the properties of palladium catalysts for hydrogenation of unsaturated compounds were considered.

A sequence of possible reactions occurring during evolution of deep-lying fluids, components of which could be sources of biogenous oil substances, is studied within the confines of biogenetic concept of formation of oil. Oxidative condensation transformation of methane and its immediate homologs under the action of sulfur present in some fluids might have facilitated formation of various types of hydrocarbons and organosulfur compounds. The possibility of such transformations is supported by the stable direct correlations between sulfur concentration in oils, their density, viscosity, content of asphaltic-resinous components and heavy fractions, and the scale of oil deposits. The proportion of gas in the oil-gas systems decreases with increase of sulfur content. Metals, like V and Ni, in the deep-lying fluids affect the geochemical type of oil. Vanadium oils are heavier (V > Ni), have high sulfur contents, and occur closer to the surface, while Ni oils are lighter (Ni < V), have low sulfur contents, and occur in deeper strata. These characteristics apparently stem from the different nature of catalytic properties of the metals and the structures containing them. Transformations of the oil fluid with the involvement of heterocomponents thus affect the fractional and chemical composition of oil-gas systems.

The preparation of a series of planar chiral, ferrocenyl-substituted hydrosilanes as precursors of ferrocene-stabilized silicon cations is described. These molecules also feature stereogenicity at the silicon atom. The generation and (29)Si NMR spectroscopic characterization of the corresponding silicon cations is reported, and problems arising from interactions of the electron-deficient silicon atom and adjacent C(sp(3))-H bonds or aromatic π donors are discussed. These issues are overcome by tethering another substituent at the silicon atom to the ferrocene backbone. The resulting annulation also imparts conformational rigidity and steric hindrance in such a way that the central chirality at the silicon atom is set with complete diastereocontrol. These chiral Lewis acid catalysts were then tested in difficult Diels-Alder reactions, but no enantioinduction was seen.

The turnover frequencies of multicomponent ternary catalytic systems formed on the basis of Ni(acac)2 and Cu(acac)2 with LiAlH4 at Т = 30 оС and РН2 = 2 atm in styrene hydrogenation were determined. It was shown that, at other things being equal, catalytic activity is defined by the order of mixing of the components at the catalyst formation stage. Namely, three particular cases are considered: sequential reduction of Ni(acac)2 and Cu(acac)2 by LiAlH4 (first Ni(acac)2, then Cu(acac)2 and vice versa) and simultaneous reduction of Ni(acac)2 and Cu(acac)2 with LiAlH4. It has been established that nano-sized structures of the core@shell type act as carriers of catalytic activity. The nano-sized structures are stabilized by the ligand shell, which contain Li(acac) as well as AlH4– and AlH63–-anions. The model of the catalytically active (in styrene hydrogenation) nanoparticle formed by sequential reduction of Cu(acac)2 with LiAlH4 and Ni(acac)2 is presented.

The catalytic properties of palladium crystallites obtained from various precursors were studied in the hydrogenation of 2-ethyl-9,10-anthraquinone. The highest yield of H2O2 (88%) is achieved on unmodified Pd crystallites obtained from Pd(dba)2 at 50°C. A sigmoidal relationship between the coherent domain size (CDS) of Pd particles and the content of anthrones, which are by-products of the eAQ transformation, is established. Small Pd clusters, along with the reduction of eAQ carbonyl groups, accelerate predominantly the hydrogenation of the eAQH2 aromatic rings. The smallest contribution of the side processes is observed for a palladium catalyst with a CDS of 3–5 nm.

Aristolochic acids (AA) are a potent class of mutagen that occur in plants of the genera Aristolochia and Asarum. Exposures to AA are caused both by intentional ingestion of traditional herbal medicines containing Aristolochia and through inadvertent consumption from contaminated crops. The carcinogenic potential and epidemiologic connection of AA exposure with upper-tract urothelial cancers (UTUC), liver cancer and kidney failure have been known for years; however, the extent of AA exposure and its influence on cancer risk and mortality have yet to be fully explored in many world populations. Using whole exome sequencing of tumor samples, a recent study determined that 78% of HCCs from Taiwan exhibit distinct mutation signatures of AA exposure (Ng, Poon, Huang, et al., 2017). Exposure to AA is geographically widespread and there is a major opportunity for primary and secondary prevention of AA-associated diseases if it were possible to detect the exposure through non-invasively sampled body fluids such as blood or urine sediment. Although AA adducts can be measured in body fluids, this measurement only reflects recent ingestion, not the life-integrated exposure that better approximates disease risk. A mutation burden-based assay could circumvent this problem; however, a major challenge is that AA-mediated mutations are extremely rare in bulk tissue or fluid samples from non-tumor sources. Duplex Sequencing (DS) is a tag-based NGS error-correction method that enables the detection of ultra-rare mutations that exist at the mutation frequency of normal tissues (~10e-8). DS can be applied to DNA from any tissue of any organism without the need for tumor formation or ex vivo cloning. We developed a DS-based assay that measures mutations across a 50 kb genome-representative panel and applied it to the tissues and liquid biopsies of cancer patients of known AA-exposure status. We sampled the blood, urine, kidney, normal-adjacent urothelium and ureteral tumors from 12 UTUC patients where 6 of the patients were exposed to AA, and 6 were not. Under blinded conditions, we positively identified AA mutation signatures in the normal adjacent urothelium tissues of 5 AA-exposed patients. Remarkably, we also detected AA signatures in all available blood and urine samples from the same patients. The proportion of mutations attributed to AA-exposure ranged from 2.5% in blood to 67.8% in kidney. These mutations, on the order of one-in-a-million, were exposure-related, not tumor derived. We did not detect AA mutation signatures in any samples from the non-AA-exposed patients. To our knowledge, this is the first time an AA mutation signature has been detected in non-invasively sampled body fluids. DS is a promising approach for the detection of mutagenic signatures caused by environmental carcinogens, and we foresee it being a powerful metric that can be used to measure life-integrated carcinogenic processes and cancer risk. Citation Format: Fang Yin Lo, Charles Valentine, Elizabeth Schmidt, Lindsey Williams, Arnoud Boot, Steve Rozen, Jesse Salk. Non-invasive detection of aristolochic acid exposure using ultra-sensitive duplex sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3147.

Osteoarthritis (OA) is a degenerative joint disease that causes pain and bone deterioration driven by an increase in prostaglandins (PGs) and inflammatory cytokines. Current treatments focus on inhibiting prostaglandin production, a pro-inflammatory lipid metabolite, with NSAID drugs; however, other lipid signaling targets could provide safer and more effective treatment strategies. Epoxides of polyunsaturated fatty acids (PUFAs) are anti-inflammatory lipid mediators that are rapidly metabolized by the soluble epoxide hydrolase (sEH) into corresponding vicinal diols. Interestingly, diol levels are increased in the synovial fluid of humans with OA, warranting further research on the biological role of this lipid pathway in the progression of OA. sEH inhibitors (sEHI) stabilize these biologically active, anti-inflammatory lipid epoxides, resulting in analgesia in both neuropathic, and inflammatory pain conditions. Most experimental studies testing the analgesic effects of sEH inhibitors have used experimental rodent models, which do not completely represent the complex etiology of painful diseases. Here, we tested the efficacy of sEHI in aged dogs with natural arthritis to provide a better representation of the clinical manifestations of pain. Two sEHI were administered orally, once daily for 5 days to dogs with naturally occurring arthritis to assess efficacy and pharmacokinetics. Blinded technicians recorded the behavior of the arthritic dogs based on pre-determined criteria to assess pain and function. After 5 days, EC1728 significantly reduced pain at a dose of 5 mg/kg compared to vehicle controls. Pharmacokinetic evaluation showed concentrations exceeding the enzyme potency in both plasma and synovial fluid. In vitro data showed that epoxyeicosatrienoic acid (EETs), epoxide metabolites of arachidonic acid, decreased inflammatory cytokines, IL-6 and TNF-α, and reduced cytotoxicity in canine chondrocytes challenged with IL1β to simulate an arthritic environment. These results provide the first example of altering lipid epoxides as a therapeutic target for OA potentially acting by protecting chondrocytes from inflammatory induced cytotoxicity. Considering the challenges and high variability of naturally occurring disease in aged dogs, these data provide initial proof of concept justification that inhibiting the sEH is a non-NSAID, non-opioid, disease altering strategy for treating OA, and warrants further investigation.

[EN] Guard cells integrate various hormone signals and environmental cues to balance plant gas exchange and transpiration. The wounding-associated hormone jasmonic acid (JA) and the drought hormone abscisic acid (ABA) both trigger stomatal closure. In contrast to ABA however, the molecular mechanisms of JA-induced stomatal closure have remained largely elusive. Here, we identify a fast signaling pathway for JA targeting the K+ efflux channel GORK. Wounding triggers both local and systemic stomatal closure by activation of the JA signaling cascade followed by GORK phosphorylation and activation through CBL1-CIPK5 Ca2+ sensor-kinase complexes. GORK activation strictly depends on plasma membrane targeting and Ca2+ binding of CBL1-CIPK5 complexes. Accordingly, in gork, cbl1, and cipk5 mutants, JA-induced stomatal closure is specifically abolished. The ABA-coreceptor ABI2 counteracts CBL1-CIPK5-dependent GORK activation. Hence, JA-induced Ca2+ signaling in response to biotic stress converges with the ABA-mediated drought stress pathway to facilitate GORK-mediated stomatal closure upon wounding., The authors would like to thank Stefanie Schultke and Linda Beckmann for their contribution of early interaction studies and Jan Niklas Offenborn for providing reagents and constructs. We thank Rainer Hedrich, Tracey Ann Cuin, Elzbieta Krol, and Dietmar Geiger for helpful discussions and critical comments on the manuscript and Irene Marten for supporting the patch-clamp experiments. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) within the GK1342 "lipid signaling" to D.B. and within the DFG research group FOR964 to D.B., E.G., and J.K.

The DNA damage response (DDR) is a designation for a number of pathways that protects our DNA from various damaging agents. In normal cells, the DDR is extremely important for maintaining genome integrity, but in cancer cells these mechanisms counteract therapy-induced DNA damage. Inhibition of the DDR could therefore be used to increase the efficacy of anti-cancer treatments. Hyperthermia is an example of such a treatment&mdash, it inhibits a sub-pathway of the DDR, called homologous recombination (HR). It does so by inducing proteasomal degradation of BRCA2 &mdash, one of the key HR factors. Understanding the precise mechanism that mediates this degradation is important for our understanding of how hyperthermia affects therapy and how homologous recombination and BRCA2 itself function. In addition, mechanistic insight into the process of hyperthermia-induced BRCA2 degradation can yield new therapeutic strategies to enhance the effects of local hyperthermia or to inhibit HR. Here, we investigate the mechanisms driving hyperthermia-induced BRCA2 degradation. We find that BRCA2 degradation is evolutionarily conserved, that BRCA2 stability is dependent on HSP90, that ubiquitin might not be involved in directly targeting BRCA2 for protein degradation via the proteasome, and that BRCA2 degradation might be modulated by oxidative stress and radical scavengers.

We theoretically investigate a new class of silicon waveguides for achieving Stimulated Brillouin Scattering (SBS) in the mid-infrared (MIR). The waveguide consists of a rectangular core supporting a low-loss optical mode, suspended in air by a series of transverse ribs. The ribs are patterned to form a finite quasi-one-dimensional phononic crystal, with the complete stopband suppressing the transverse leakage of acoustic waves, confining them to the core of the waveguide. We derive a theoretical formalism that can be used to compute the opto-acoustic interaction in such periodic structures, and find forward intramodal-SBS gains up to 1750 m-1 W-1 , which compares favorably with the proposed MIR SBS designs based on buried germanium waveguides. This large gain is achieved thanks to the nearly complete suppression of acoustic radiative losses.

Background The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise question regarding the unclarified heterogeneity of disease pathomechanisms. While biomarkers for the differentiation of Crohn’s disease (CD) vs. ulcerative colitis (UC) have been suggested, specific markers for a subclassification of CD phenotypes are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease, we sought to characterise potential biomarkers focusing on the downstream metabolites of kynurenine metabolism. The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise question regarding the unclarified heterogeneity of disease pathomechanisms. While biomarkers for the differentiation of Crohn’s disease (CD) vs. ulcerative colitis (UC) have been suggested, specific markers for a subclassification of CD phenotypes are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease, we sought to characterise potential biomarkers focusing on the downstream metabolites of kynurenine metabolism. Methods Using immunohistochemical staining, we analysed and compared the mucosal tryptophan immune metabolism in biotic samples from patients with UC (n = 11), CD (n = 11) and healthy control (n = 12). Localisation-specific quantification of immune cell infiltration, tryptophan-metabolizing enzyme expression and mucosal tryptophan downstream metabolite levels was performed. Results As expected, we found generally increased immune cell infiltrates in the tissue of all patients with IBD. However, in patients with CD, significant differences were found between regulatory T-cell markers in the ileum compared with the colon. In line with this finding, we identified kynureninase as a modulator of immunosuppressive kynurenine levels specifically in the ileum of patients with CD. Correspondingly, significantly elevated levels of the kynurenine metabolite 3-hydroxyanthranilic acid were detected in CD ileum samples. Conclusion Highlighting the heterogeneity of the different phenotypes of CD, we identified 3-hydroxyanthranilic acid as a potential mucosal biomarker allowing the localisation-specific differentiation of ileum or colon inflammation in patients with CD. Moreover, we characterised the kynurenine-degrading enzyme kynureninase as a modulator of immunosuppression and chronic inflammation with potential therapeutic relevance.

Many alpine areas are experiencing deglaciation, biogeochemical changes driven by temperature rise, and changes in atmospheric deposition. There is mounting evidence that the water quality of alpine streams may be related to these changes, including rising atmospheric deposition of carbon (C) and nutrients. Given that barren alpine soils can be severely C limited, atmospheric deposition sources may be an important source of C and nutrients for these environments. We evaluated the magnitude of atmospheric deposition of C and nutrients to an alpine site, the Green Lake 4 catchment in the Colorado Rocky Mountains. Using a long-term dataset (2002–2010) of weekly atmospheric wet deposition and snowpack chemistry, we found that volume weighted mean dissolved organic carbon (DOC) concentrations were 1.12 ± 0.19 mg l−1, and weekly concentrations reached peaks as high at 6–10 mg l−1 every summer. Total dissolved nitrogen concentration also peaked in the summer, whereas total dissolved phosphorus and calcium concentrations were highest in the spring. To investigate potential sources of C in atmospheric deposition, we evaluated the chemical quality of dissolved organic matter (DOM) and relationships between DOM and other solutes in wet deposition. Relationships between DOC concentration, fluorescence, and nitrate and sulfate concentrations suggest that pollutants from nearby urban and agricultural sources and organic aerosols derived from sub-alpine vegetation may influence high summer DOC wet deposition concentrations. Interestingly, high DOC concentrations were also recorded during "dust-in-snow" events in the spring, which may reflect an association of DOM with dust. Detailed chemical and spectroscopic analyses conducted for samples collected in 2010 revealed that the DOM in many late spring and summer samples was less aromatic and polydisperse and of lower molecular weight than that of winter and fall samples. Our C budget estimates for the Green Lake 4 catchment illustrated that wet deposition (9.9 kg C ha−1 yr−1) and dry deposition (6.9 kg C ha−1 yr−1) were a combined input of approximately 17 kg C ha−1 yr−1, which could be as high as 24 kg C ha−1 yr−1 in high dust years. This atmospheric C input approached the C input from microbial autotrophic production in barren soils. Atmospheric wet and dry deposition also contributed 4.3 kg N ha−1 yr−1, 0.15 kg P ha−1 yr−1, and 2.7 kg Ca2+ ha−1 yr−1 to this alpine catchment.

Size and phase composition of the Pd-P catalyst, which is highly active in the hydrogenation of unsaturated compounds have been established using X-ray powder diffraction (XRD) analysis, high resolution transmission electron microscopy (HRTEM) technique in combination with energy-dispersive X-ray (EDX) analysis and selected area electron diffraction (SAED) analysis. The reduction of Pd(acac)₂ with hydrogen in the presence of white phosphorus (P/Pd = 0.3) in N,N-dimethylformamide (DMF) at 80 °C results in formation of Pd-enriched polycrystalline nanoparticles with a predominant diameter of 5.7 nm (C(Pd) = 1 mmol·L⁻¹) and 5.1 nm (C( Pd) = 5 mmol · L⁻¹). The polycrystallites consist of palladium phosphides (Pd5P2, Pd3P0.8 and palladium crystallites. It is found that phase composition of the Pd-P catalyst is changed at the stage of Pd-P sample preparation for XRD analysis. It is established that palladium phosphide Pd6P detected by XRD technique is a product of the solid-phase reaction between Pd crystallites and Pd₅P₂, Pd₃P(0.8) palladium phosphides at the stage of high-temperature sample preparation (400 °C). The mechanism of low-temperature synthesis of palladium phosphides was proposed.