Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow ...for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound. We applied it to identify proteome-wide targets of covalent inhibitors to mutant Kirsten rat sarcoma (KRAS)
and Bruton's tyrosine kinase (BTK). In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines, which includes >20,000 cysteines from >6,000 proteins per line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.
Huntington's disease (HD) is a progressive neurodegenerative disorder caused by expansion of an unstable CAG repeat in the coding sequence of the Huntingtin (HTT) gene. Instability affects both ...germline and somatic cells. Somatic instability increases with age and is tissue-specific. In particular, the CAG repeat sequence in the striatum, the brain region that preferentially degenerates in HD, is highly unstable, whereas it is rather stable in the disease-spared cerebellum. The mechanisms underlying the age-dependence and tissue-specificity of somatic CAG instability remain obscure. Recent studies have suggested that DNA oxidation and OGG1, a glycosylase involved in the repair of 8-oxoguanine lesions, contribute to this process. We show that in HD mice oxidative DNA damage abnormally accumulates at CAG repeats in a length-dependent, but age- and tissue-independent manner, indicating that oxidative DNA damage alone is not sufficient to trigger somatic instability. Protein levels and activities of major base excision repair (BER) enzymes were compared between striatum and cerebellum of HD mice. Strikingly, 5'-flap endonuclease activity was much lower in the striatum than in the cerebellum of HD mice. Accordingly, Flap Endonuclease-1 (FEN1), the main enzyme responsible for 5'-flap endonuclease activity, and the BER cofactor HMGB1, both of which participate in long-patch BER (LP-BER), were also significantly lower in the striatum compared to the cerebellum. Finally, chromatin immunoprecipitation experiments revealed that POLbeta was specifically enriched at CAG expansions in the striatum, but not in the cerebellum of HD mice. These in vivo data fit a model in which POLbeta strand displacement activity during LP-BER promotes the formation of stable 5'-flap structures at CAG repeats representing pre-expanded intermediate structures, which are not efficiently removed when FEN1 activity is constitutively low. We propose that the stoichiometry of BER enzymes is one critical factor underlying the tissue selectivity of somatic CAG expansion.
Compared to tris(2‐phenylpyridine)iridium(III) (Ir(ppy)3), iridium(III) complexes containing difluorophenylpyridine (df‐ppy) and/or an ancillary triazolylpyridine ligand ...3‐phenyl‐1,2,4‐triazol‐5‐ylpyridinato (ptp) or 1‐benzyl‐1,2,3‐triazol‐4‐ylpyridine (ptb) exhibit considerable hypsochromic shifts (ca. 25–60 nm), due to the significant stabilising effect of these ligands on the HOMO energy, whilst having relatively little effect on the LUMO. Despite their lower photoluminescence quantum yields compared with Ir(ppy)3 and Ir(df‐ppy)3, the iridium(III) complexes containing triazolylpyridine ligands gave greater electrogenerated chemiluminescence (ECL) intensities (using tri‐n‐propylamine (TPA) as a co‐reactant), which can in part be ascribed to the more energetically favourable reactions of the oxidised complex (M+) with both TPA and its neutral radical oxidation product. The calculated iridium(III) complex LUMO energies were shown to be a good predictor of the corresponding M+ LUMO energies, and both HOMO and LUMO levels are related to ECL efficiency. The theoretical and experimental data together show that the best strategy for the design of efficient new blue‐shifted electrochemiluminophores is to aim to stabilise the HOMO, while only moderately stabilising the LUMO, thereby increasing the energy gap but ensuring favourable thermodynamics and kinetics for the ECL reaction. Of the iridium(III) complexes examined, Ir(df‐ppy)2(ptb)+ was most attractive as a blue‐emitter for ECL detection, featuring a large hypsochromic shift (λmax=454 and 484 nm), superior co‐reactant ECL intensity than the archetypal homoleptic green and blue emitters: Ir(ppy)3 and Ir(df‐ppy)3 (by over 16‐fold and threefold, respectively), and greater solubility in polar solvents.
Into the blue: Theoretical and experimental studies reveal the most effective strategies for the design of blue‐shifted iridium(III) complexes for efficient electrogenerated chemiluminescence. Stabilisation of the HOMO while only moderately stabilising the LUMO increases the energy gap, thus ensuring favourable thermodynamics and kinetics for the reaction leading to the excited state (see figure).
Longstanding social and economic inequities elevate health risks and vulnerabilities for Black, Indigenous and People of Color (BIPOC) communities. Engagement of BIPOC communities in infectious ...disease research is a critical component in efforts to increase vaccine confidence, acceptability, and uptake of future approved products. Recent data highlight the relative absence of BIPOC communities in vaccine clinical trials. Intentional and effective community engagement methods are needed to improve BIPOC inclusion. We describe the methods utilized for the successful enrollment of BIPOC participants in the U.S. Government (USG)-funded COVID-19 Prevention Network (CoVPN)-sponsored vaccine efficacy trials and analyze the demographic and enrollment data across the efficacy trials to inform future efforts to ensure inclusive participation. Across the four USG-funded COVID-19 vaccine clinical trials for which data are available, 47% of participants enrolled at CoVPN sites in the US were BIPOC. White enrollment outpaced enrollment of BIPOC participants throughout the accrual period, requiring the implementation of strategies to increase diverse and inclusive enrollment. Trials opening later benefitted considerably from strengthened community engagement efforts, and greater and more diverse volunteer registry records. Despite robust fiscal resources and a longstanding collaborative and collective effort, enrollment of White persons outpaced that of BIPOC communities. With appropriate resources, commitment and community engagement expertise, the equitable enrollment of BIPOC individuals can be achieved. To ensure this goal, intentional efforts are needed, including an emphasis on diversity of enrollment in clinical trials, establishment of enrollment goals, ongoing robust community engagement, conducting population-specific trials, and research to inform best practices.
The synthesis and reactivity study of the first isolable boraphosphaketene, cyclic(alkyl)(amino) carbene (CAAC)‐borafluorene‐P=C=O (2), is described. Photolysis of compound 2 results in the formation ...of CAAC‐stabilized BP‐doped phenanthryne (3) through tandem decarbonylation, monoatomic phosphide insertion, and ring‐expansion. Notably, while BN‐doped phenanthryne was previously discussed as a reactive intermediate which could not be isolated, the heavier BP‐doped analogue exhibits remarkable solution and solid‐state stability. The reactivity of 2 with stable carbenes was also explored. Addition of CAAC to 2 led to migration of the original CAAC ligand from boron to phosphorus and coordination of the added CAAC to carbon, affording compound 4. Reaction of 1,3‐diisopropyl‐4,5‐dimethylimidazol‐2‐ylidene (NHC) with 2 resulted in N−C bond activation to give the unusual spiro‐heterocyclic compound (5).
P brings stability: The first BP‐doped phenanthryne has been synthesized via photolysis of the elusive boraphosphaketene. While BN‐doped phenanthryne was previously discussed as a reactive intermediate that could not be isolated, the heavier BP‐doped analogue exhibits remarkable solution and solid‐state stability.
Reactions of 9-carbene-9-borafluorene monoanion (1) with elemental selenium and selenium-containing reagents are reported. When compound 1 is reacted with grey selenium in THF, various ...boryl-substituted selenides and diselenides are produced (2–6), including molecules resulting from migration of the carbene ligand Dipp group (Dipp = 2,6-diisopropylphenyl). However, when a similar reaction between 1 and grey selenium is performed in toluene in the presence of 18-crown-6, boryl-substituted selenide 7 is obtained as the sole boron-containing product. As compound 7 is the monomeric variant of organoselenide 3, 18-crown-6 promotes both product selectivity and solubility in a nonpolar solvent. Diselenide 5, which features a trans-bent B–Se–Se–B core, was directly isolated via reaction of 1 with Se2Cl2 in THF. Computational modeling suggests that the formation of 5 proceeds via a radical mechanism. This was supported by an experiment demonstrating that the CAAC-borafluorene radical also reacts with SeCl2 to yield 5 CAAC = (2,6-diisopropylphenyl)-4,4-diethyl-2,2-dimethyl-pyrrolidin-5-ylidene. Energy decomposition analysis of 5 indicates a covalent borafluorene–diselenide bond (ΔE int, −168.9 kcal mol–1). All of the new compounds were fully characterized via single-crystal X-ray diffraction and multinuclear nuclear magnetic resonance (1H, 13C, 11B, and 77Se).
Protecting the mitochondrial powerhouse Scheibye-Knudsen, Morten; Fang, Evandro F; Croteau, Deborah L ...
Trends in cell biology,
03/2015, Letnik:
25, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Highlights • Mitochondrial maintenance is essential for cellular and organismal function. • Maintenance includes reactive oxygen species (ROS) regulation, DNA repair, fusion–fission, and mitophagy. • ...Loss of function of these pathways leads to disease.
Cellulolytic bacteria and fungi have been shown to use two different approaches to degrade cellulose. Most aerobic microbes secrete sets of individual cellulases, many of which contain a carbohydrate ...binding molecule (CBM), which act synergistically on native cellulose. Most anaerobic microorganisms produce large multienzyme complexes called cellulosomes, which are usually attached to the outer surface of the microorganism. Most of the cellulosomal enzymes lack a CBM, but the cohesin subunit, to which they are bound, does contain a CBM. The cellulases present in each class show considerable overlap in their catalytic domains, and processive cellulases (exocellulases and processive endocellulases) are the most abundant components of both the sets of free enzymes and of the cellulosomal cellulases. Analysis of the genomic sequences of two cellulolytic bacteria, Cytophaga hutchinsonii, an aerobe, and Fibrobacter succinogenes, an anaerobe, suggest that these organisms must use a third mechanism. This is because neither of these organisms, encodes processive cellulases and most of their many endocellulase genes do not encode CBMs. Furthermore, neither organism appears to encode the dockerin and cohesin domains that are key components of cellulosomes.
Summary Background Detention of people who use drugs into compulsory drug detention centres (CDDCs) is common throughout East and Southeast Asia. Evidence-based pharmacological therapies for treating ...substance use disorders, such as opioid agonist treatments with methadone, are generally unavailable in these settings. We used a unique opportunity where CDDCs coexisted with voluntary drug treatment centres (VTCs) providing methadone in Malaysia to compare the timing and occurrence of opioid relapse (measured using urine drug testing) in individuals transitioning from CDDCs versus methadone maintenance in VTCs. Methods We did a parallel, two-arm, prospective observational study of opioid-dependent individuals aged 18 years and older who were treated in Malaysia in the Klang Valley in two settings: CDDCs and VTCs. We used sequential sampling to recruit individuals. Assessed individuals in CDDCs were required to participate in services such as counselling sessions and manual labour. Assessed individuals in VTCs could voluntarily access many of the components available in CDDCs, in addition to methadone therapy. We undertook urinary drug tests and behavioural interviews to assess individuals at baseline and at 1, 3, 6, 9, and 12 months post-release. The primary outcome was time to opioid relapse post-release in the community confirmed by urinary drug testing in individuals who had undergone baseline interviewing and at least one urine drug test (our analytic sample). Relapse rates between the groups were compared using time-to-event methods. This study is registered at ClinicalTrials.gov ( NCT02698098 ). Findings Between July 17, 2012, and August 21, 2014, we screened 168 CDDC attendees and 113 VTC inpatients; of these, 89 from CDDCs and 95 from VTCs were included in our analytic sample. The baseline characteristics of the two groups were similar. In unadjusted analyses, CDDC participants had significantly more rapid relapse to opioid use post-release compared with VTC participants (median time to relapse 31 days IQR 26–32 vs 352 days 256–unestimable, log rank test, p<0·0001). VTC participants had an 84% (95% CI 75–90) decreased risk of opioid relapse after adjustment for control variables and inverse propensity of treatment weights. Time-varying effect modelling revealed the largest hazard ratio reduction, at 91% (95% CI 83–96), occurs during the first 50 days in the community. Interpretation Opioid-dependent individuals in CDDCs are significantly more likely to relapse to opioid use after release, and sooner, than those treated with evidence-based treatments such as methadone, suggesting that CDDCs have no role in the treatment of opioid-use disorders. Funding The World Bank Group, Doris Duke Charitable Foundation, National Institute on Drug Abuse, Australian National Health & Medical Research Council, National Institute of Mental Health, and the University of Malaya-Malaysian Ministry of Higher Education High Impact Research Grant.
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