General autophagy is an evolutionarily conserved process in eukaryotes, by which intracellular materials are transported into and degraded inside lysosomes or vacuoles, with the main goal of ...recycling those materials during periods of starvation. The molecular bases of autophagy have been widely described in Saccharomyces cerevisiae, and the specific roles of Atg proteins in the process were first characterized in this model system. Important contributions have been made in Schizosaccharomyces pombe highlighting the evolutionary similarity and, at the same time, diversity of Atg components in autophagy. However, little is known regarding signals, pathways and role of autophagy in this distant yeast. Here, we undertake a global approach to investigate the signals, the pathways and the consequences of autophagy activation. We demonstrate that not only nitrogen but several nutritional deprivations including lack of carbon, sulfur, phosphorus or leucine sources, trigger autophagy, and that the TORC1, TORC2 and MAP kinase Sty1 pathways control the onset of autophagy. Furthermore, we identify an unexpected phenotype of autophagy-defective mutants, namely their inability to survive in the absence of leucine when biosynthesis of this amino acid is impaired.
Abbreviations: ATG: autophagy-related; cAMP: cyclic adenosine monophosphate; cDNA: complementary deoxyribonucleic acid; GFP: green fluorescence protein; Gluc: glucose; Leu: leucine; MAP: mitogen-activated protein; MM: minimal medium; PI: propidium iodine; PKA: protein kinase A; RNA: ribonucleic acid; RT-qPCR: real time quantitative polymerase chain reaction; S. cerevisiae: Saccharomyces cerevisiae; S. pombe: Schizosaccharomyces pombe; TCA: trichloroacetic acid; TOR: target of rapamycin; TORC1: target of rapamycin complex 1; TORC2: target of rapamycin complex 2; YE5S: yeast extract 5 amino acid supplemented.
A strategy for the allylic oxidation of cyclic alkenes with a copper–aluminum mixed oxide as catalyst is presented. The reaction involves the treatment of an alkene with a carboxylic acid employing ...tert-butyl hydroperoxide as the oxidant. In all cases, the corresponding allylic esters are obtained. When l-proline is employed, the allylic alcohol or ketone is obtained. The oxidation of cyclohexene and valencene has been optimized by design of experiments (DoE) statistical methodology.
Many neurodegenerative disorders display protein aggregation as a hallmark, Huntingtin and TDP-43 aggregates being characteristic of Huntington disease and amyotrophic lateral sclerosis, ...respectively. However, whether these aggregates cause the diseases, are secondary by-products, or even have protective effects, is a matter of debate. Mutations in both human proteins can modulate the structure, number and type of aggregates, as well as their toxicity. To study the role of protein aggregates in cellular fitness, we have expressed in a highly tractable unicellular model different variants of Huntingtin and TDP-43. They each display specific patterns of aggregation and toxicity, even though in both cases proteins have to be very highly expressed to affect cell fitness. The aggregation properties of Huntingtin, but not of TDP-43, are affected by chaperones such as Hsp104 and the Hsp40 couple Mas5, suggesting that the TDP-43, but not Huntingtin, derivatives have intrinsic aggregation propensity. Importantly, expression of the aggregating form of Huntingtin causes a significant extension of fission yeast lifespan, probably as a consequence of kidnapping chaperones required for maintaining stress responses off. Our study demonstrates that in general these prion-like proteins do not cause toxicity under normal conditions, and in fact they can protect cells through indirect mechanisms which up-regulate cellular defense pathways.
In this study, we identified specific carbapenemase-producing isolates applying an easy and rapid protocol for the detection of mature KPC-2 β-lactamase by MALDI-TOF MS from colony and positive blood ...culture bottles. In addition, we evaluated the correlation of the ~11,109 Da signal as a biomarker associated with KPC-2 production. A collection of 126 well-characterized clinical isolates were evaluated (including 60 KPC-2-producing strains). Presence of KPC-2 was assessed by MALDI-TOF MS on protein extracts. Samples were prepared using the double layer sinapinic acid technique. In order to identify mature KPC-2, raw spectra were analyzed focusing on the range between m/z 25,000–30,000 Da. A single distinctive peak, at approximately m/z 28,544 Da was found in all clinical and control KPC-2-producing strains, and consistently absent in the control groups (ESBL producers and susceptible strains). This peak was detected in all species independently of where the gene blaKPC-2 was embedded. Statistical results showed 100% sensitivity, CI95%: 94.0%; 100% and 100% specificity, CI95%: 94.6%; 100%, indicating a promising test with a high discriminative power. KPC-2 β-lactamase could be directly detected from both colonies and blood culture bottles. On the other hand, the m/z 11,109 Da signal determinant was only associated with 32% of Klebsiella pneumoniae and Escherichia coli KPC positive isolates. This MALDI-TOF MS methodology has the potential to detect directly the widespread and clinically relevant carbapenemase, KPC-2, in Enterobacterales with a straightforward, low cost process, assuming MALDI-TOF MS is already adopted as the main identification tool, with clear clinical implications on antibiotic stewardship for early infection treatment.
•KPC-2-enzyme was detected by MALDI-TOF MS in different Gram-negative bacilli.•A distinctive peak around 28,544 Da was found in all KPC-2 producers.•Direct KPC-2 detection on positive blood culture bottles was also achieved.•The proposed accompanying 11,109 Da peak was a bad predictor for KPC-2 presence.
The allylic hydroxylation of enones using dioxygen as the oxidant has been studied. The reaction was first examined in the absence of any catalyst, using β‐ionone as a model substrate. Then a new ...copper–aluminium mixed oxide, Cu–Al Ox, was prepared and characterized in order to be used as a catalyst. This oxide showed good activity, and provided the corresponding γ‐ or ϵ‐hydroxylated enones, starting from different α,β‐ or α,β,γ,δ‐unsaturated ketones. In all cases, the yields were significantly improved compared to experiments run in the absence of the catalyst. The reaction was selective, and the formation of epoxides or other overoxidation products was detected only to a minor extent. The described procedure is a technically straightforward synthetic alternative to those methods described to date involving many reaction steps or toxic reagents. The reactions were optimized using design of experiments techniques (DoE).
The direct γ‐hydroxylation of enones using oxygen from the air and potassium tert‐butoxide in the presence of a copper–aluminium mixed oxide as catalyst is reported. This catalyst is inexpensive and easy to prepare. The reactions were optimized using design of experiments techniques.
Abstract
Chromatin remodeling is essential to allow full development of alternative gene expression programs in response to environmental changes. In fission yeast, oxidative stress triggers massive ...transcriptional changes including the activation of hundreds of genes, with the participation of histone modifying complexes and chromatin remodelers. DNA transcription is associated to alterations in DNA topology, and DNA topoisomerases facilitate elongation along gene bodies. Here, we test whether the DNA topoisomerase Top1 participates in the RNA polymerase II-dependent activation of the cellular response to oxidative stress. Cells lacking Top1 are resistant to H2O2 stress. The transcriptome of Δtop1 strain was not greatly affected in the absence of stress, but activation of the anti-stress gene expression program was more sustained than in wild-type cells. Top1 associated to stress open reading frames. While the nucleosomes of stress genes are partially and transiently evicted during stress, the chromatin configuration remains open for longer times in cells lacking Top1, facilitating RNA polymerase II progression. We propose that, by removing DNA tension arising from transcription, Top1 facilitates nucleosome reassembly and works in synergy with the chromatin remodeler Hrp1 as opposing forces to transcription and to Snf22 / Hrp3 opening remodelers.
Graphical Abstract
Graphical Abstract
Mass spectrometry has revolutionized the clinical microbiology field in America's and Europe's industrialized countries, for being a fast, reliable and inexpensive technique. Our study is based on ...the comparison of the performance of two commercial platforms, Microflex LT (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMérieux, Marcy l´Etoile, France) for the identification of unusual and hard-to-diagnose microorganisms in a Reference Laboratory in Argentina. During a four-month period (February-May 2018) the diagnostic efficiency and the concordance between both systems were assessed, and the results were compared with the polyphasic taxonomic identification of all isolates. The study included 265 isolates: 77 Gram-Negative Bacilli, 33 Gram-Positive Cocci, 40 Anaerobes, 35 Actinomycetales, 19 Fastidious Microorganisms and 61 Gram-Positive Bacilli. All procedures were practiced according to the manufacturer's recommendations in each case by duplicate, and strictly in parallel. Other relevant factors, such as the utility of the recommended extraction protocols, reagent stability and connectivity were also evaluated. Both systems correctly identified the majority of the isolates to species and complex level (82%, 217/265). Vitex MS achieved a higher number of correct species-level identifications between the gram-positive microorganisms; however, it presented greater difficulty in the identification of non-fermenting bacilli and a higher number of incorrect identifications when the profile of the microorganism was not represented in the commercial database. Both platforms showed an excellent performance on the identification of anaerobic bacteria and fastidious species. Both systems enabled the fast and reliable identification of most of the tested isolates and were shown to be very practical for the user.
Many experimental studies are carried out to compare biological effectiveness of high dose rate (HDR) with that of low dose rate (LDR). The rational for this is the uncertainty regarding the value of ...the dose rate effectiveness factor (DREF) used in radiological protection. While a LDR is defined as 0.1 mGy/min or lower, anything above that is seen as HDR. In cell and animal experiments, a dose rate around 1 Gy/min is usually used as representative for HDR. However, atomic bomb survivors, the reference cohort for radiological protection, were exposed to tens of Gy/min. The important question is whether gamma radiation delivered at very high dose rate (VHDR—several Gy/min) is more effective in inducing DNA damage than that delivered at HDR. The aim of this investigation was to compare the biological effectiveness of gamma radiation delivered at VHDR (8.25 Gy/min) with that of HDR (0.38 Gy/min or 0.79 Gy/min). Experiments were carried out with human peripheral mononuclear cells (PBMC) and the human osteosarcoma cell line U2OS. Endpoints related to DNA damage response were analysed. The results show that in PBMC, VHDR is more effective than HDR in inducing gene expression and micronuclei. In U2OS cells, the repair of 53BP1 foci was delayed after VHDR indicating a higher level of damage complexity, but no VHDR effect was observed at the level of micronuclei and clonogenic cell survival. We suggest that the DREF value may be underestimated when the biological effectiveness of HDR and LDR is compared.
Different MALDI-TOF MS databases were evaluated for the identification of Achromobacter species. The in-house and extended database generated in this study rendered more accurate identification ...(58/64 and 57/64 isolates, respectively) in comparison with the Bruker commercial database (42/64 isolates), especially in those infrequent species that are not available or poorly represented.
•MALDI-TOF MS proved to be a reliable identification tool at the genus level in Achromobacter•Expansion of MALDI-TOF MS databases rendered more accurate identification at species level•Discriminatory peaks were selected in order to differentiate among highly related species
Nonafluorobutanesulfonyl azide is an efficient, shelf‐stable and cost‐effective diazo transfer reagent for the synthesis of azides from primary amines. The reagent can also be successfully applied to ...the one‐pot regioselective synthesis of 1,2,3‐triazoles from primary amines by a sequential diazo transfer and azide–alkyne 1,3‐dipolar cycloaddition process catalyzed by copper. The cycloaddition step can be conducted in an inter‐ or intramolecular way to afford 1,4‐ or 1,5‐disubstituted triazoles, respectively. The atypical 1,5‐regioselectivity under copper catalysis is a consequence of geometrical constraints of the amino‐alkyne substrates used in the intramolecular version. Nonafluorobutanesulfonyl azide offers an advantageous alternative to the better known and most commonly used trifluoromethanesulfonyl azide.
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