Time-resolved secretome analysis of three Colletotrichum species identifies copper radical alcohol oxidases for the production of fatty aldehydes

Copper Radical Alcohol Oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens are attractive for the production of fragrant fatty aldehydes. With the initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time-course analyses of the secretomes of three Colletotrichum species ( C. graminicola , C. tabacum and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture in absence of any plant-biomass mimicking compounds to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29-52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11-AA13, AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were thus expressed in Pichia pastoris and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their corresponding fragrant aldehydes. IMPORTANCE In the context of the industrial shift toward greener processes, the biocatalytic production of aldehydes is of utmost interest owing to their importance for their use as flavors and fragrances ingredients. CRO-AlcOx have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored, while the use of crude fungal secretomes is an appealing approach for industrial application to alleviate various costs pertaining to biocatalysts production. While investigating this primary objective, the secretomics studies revealed unexpected results showing that under the oxidative-stressful conditions we probed, Colletotrichum species can secrete a broad diversity of copper-containing enzymes (laccases, sugar oxidoreductases, LPMOs) usually assigned to “plant-cell wall degradation”, despite the absence of any plant-biomass mimicking compound, and only little amount of CRO-AlcOx were secreted, pointing out at recombinant expression as the most promising path for their biocatalytic application.

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Time course of chemical and structural events in protein crystals measured by microspectrophotometry

Philosophical Transactions of the Royal Society of London Series A Physical and Engineering Sciences ◽

10.1098/rsta.1992.0060 ◽

1992 ◽

Vol 340 (1657) ◽

pp. 191-207 ◽

Cited By ~ 19

Keyword(s):

Time Course ◽

Equilibrium Distribution ◽

Synergistic Effects ◽

Conformational Transitions ◽

Structural Studies ◽

Time Resolved ◽

The Past ◽

Catalytic Intermediates ◽

Single Form ◽

Half Site

The functional properties of proteins in the crystalline state have been investigated over the past 30 years by a variety of methods, including single crystal polarized absorption spectroscopy. This technique has provided information on the accumulation and equilibrium distribution of protein-ligand complexes in the crystal and, in a few cases, on the rates of interconversion of catalytic intermediates. It has been possible to detect synergistic effects in the binding of different ligands, cooperativity and half-site reactivity and even formation of active multiprotein complexes, obtained by diffusion of one small protein in the pre-formed crystals of the other. Lattice interactions restrain the conformational transitions of some proteins existing in multiple states in solution. The crystal offers the unique opportunity to analyse not only the structure but also the function of a single form of the protein. The relevance of these data to the planning and interpretation of structural studies, especially in the perspectives of time-resolved crystallography, will be discussed with reference to well-characterized systems.

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Two Quenchers Formed During Photodamage of Phostosystem II and The Role of One Quencher in Preemptive Photoprotection

Scientific Reports ◽

10.1038/s41598-019-53030-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Alonso Zavafer ◽

Ievgeniia Iermak ◽

Mun Hon Cheah ◽

Wah Soon Chow

Keyword(s):

Chlorophyll Fluorescence ◽

Photosystem Ii ◽

Time Course ◽

Physiological Role ◽

Reaction Centre ◽

Time Resolved ◽

Fluorescence Quencher

AbstractThe quenching of chlorophyll fluorescence caused by photodamage of Photosystem II (qI) is a well recognized phenomenon, where the nature and physiological role of which are still debatable. Paradoxically, photodamage to the reaction centre of Photosystem II is supposed to be alleviated by excitation quenching mechanisms which manifest as fluorescence quenchers. Here we investigated the time course of PSII photodamage in vivo and in vitro and that of picosecond time-resolved chlorophyll fluorescence (quencher formation). Two long-lived fluorescence quenching processes during photodamage were observed and were formed at different speeds. The slow-developing quenching process exhibited a time course similar to that of the accumulation of photodamaged PSII, while the fast-developing process took place faster than the light-induced PSII damage. We attribute the slow process to the accumulation of photodamaged PSII and the fast process to an independent quenching mechanism that precedes PSII photodamage and that alleviates the inactivation of the PSII reaction centre.

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Time course and local distribution of skin exposure of hand and fingers from [68Ga]Ga-DOTA-NOC synthesis using a self-shielded module

Nuklearmedizin ◽

10.1055/a-1134-4374 ◽

2020 ◽

Vol 59 (04) ◽

pp. 308-315

Author(s):

Oliver Stephan Grosser ◽

Heiko Wissel ◽

Maurice Klopfleisch ◽

Dennis Kupitz ◽

Nadine Paetzold ◽

...

Keyword(s):

Dose Distribution ◽

Time Course ◽

Index Finger ◽

Product Yield ◽

Skin Dose ◽

Exposure Level ◽

Time Resolved ◽

Left Hand ◽

Skin Exposure ◽

Local Exposure

Abstract Aim The study examined the local dose distribution as well as the time course of skin exposure of hand and fingers from [68Ga]Ga-DOTA-NOC synthesis using a self-shielded synthesis module. Methods A compact calibrated electronic dosimeter (ED) with a miniaturized probe was used for real-time measurements of skin dose equivalent Hp (0.07) (reference point: left and right index finger). A time resolved assessment of exposure during radiotracer production was performed. Additionally, thermoluminescence dosimeters (TLD) were used to determine local dose distribution for five different positions (e. g. fingertips). Cumulated Hp (0.07) estimated by ED was analysed and correlated with the measurements obtained by a TLD positioned close to the ED. Results The cumulative skin exposure from the production process measured by ED, was 74.7 ± 32.7 µSv/GBq and 40.1 ± 14.3 µSv/GBq for the right and left hand, respectively. The exposure recorded by the ED was in the average 19.4 % ± 40.0 % (median = 21.3 %) lower compared to the results from TLD. Highest exposure was recorded during synthesis (guided hand: 24.5 ± 12.2 µSv/GBq) and measuring of product yield including preparation of probes for quality control (guided hand: 36.1 ± 12.7 µSv/GBq). The highest local exposure was measured by a TLD close to the tip of the index finger of the guiding hand (range: 773–1257 µS/GBq). Conclusion The chosen methodology using ED, proved to be a good concept for identifying procedure steps with an increased exposure level and to determine the time course of skin exposure and to identify procedure steps for further optimization of handling. Furthermore, miniaturized electronic dosimeters may be used for online surveillance of local exposure rates at hands and fingers.

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THE ROLE OF THE NUCLEOLUS

The Journal of Cell Biology ◽

10.1083/jcb.23.1.39 ◽

1964 ◽

Vol 23 (1) ◽

pp. 39-51 ◽

Cited By ~ 28

Author(s):

Sasha Koulish ◽

Ruth G. Kleinfeld

Keyword(s):

Time Course ◽

Specific Activity ◽

Unit Area ◽

Freeze Substitution ◽

Male Rats ◽

Ethanol Mixture ◽

Cytoplasmic Rna ◽

Total Structure ◽

Parenchymal Cells

Male rats of the Sherman strain were fed for 2 weeks a diet of ground purina rat chow containing 0.04 per cent thioacetamide. Animals were injected intraperitoneally with tritiated cytidine, 200µc/100 gm body weight, and sacrificed in pairs, a control and a thioacetamide-treated rat, at prescribed intervals. Liver tissues were preserved with the freeze-substitution method and postfixed in anhydrous OsO4. Other samples were fixed directly with an acetic acid-ethanol mixture (1:3). AR-10 stripping film was applied to 2- and 4-µ sections and exposed for appropriate lengths of time. Nuclear and nucleolar volumes were obtained by direct measurement. Cytoplasmic volumes were obtained with the aid of Chalkley ratios. Nucleolar and cytoplasmic RNA concentrations were calculated from cytophotometric extinction (E540 mµ) measurements. Data were expressed as grains/unit area, grains/unit area/concentration (or specific activity) and grains/total structure. In the liver parenchymal cells of thioacetamide-treated rats, the nucleolus shows vast increases in volume, RNA content, and grain count/total structure, 14-fold, 25-fold, and over 30-fold, respectively. The nucleus increases 2-fold in volume and about 3-fold in total grain count. Cytoplasmic volume increases only 20 per cent and displays a total grain count about equal to that in the control. The time course of incorporation curves for nucleolus and non-nucleolar nucleus (NNN) contain 2 distinct turnover fractions, rapid and slow. Both fractions were increased after thioacetamide treatment but remained proportional to those of controls. The unique stimulated RNA turnover in the nucleus and nucleolus, coupled to a "normal" turnover in the cytoplasm, suggests that this nuclear-nucleolar loss of label does not represent an exclusive passage of formed nuclear RNA to the cytoplasm.

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Frontal and Parietal Cortices Show Different Spatiotemporal Dynamics across Problem-solving Stages

Journal of Cognitive Neuroscience ◽

10.1162/jocn_a_00960 ◽

2016 ◽

Vol 28 (8) ◽

pp. 1098-1110 ◽

Cited By ~ 12

Author(s):

Nadja Tschentscher ◽

Olaf Hauk

Keyword(s):

Problem Solving ◽

Time Course ◽

Strategy Selection ◽

Arithmetic Task ◽

Time Resolved ◽

Source Power ◽

Time Frequency ◽

Problem Solving Process ◽

The Right ◽

Parietal Cortices

Arithmetic problem-solving can be conceptualized as a multistage process ranging from task encoding over rule and strategy selection to step-wise task execution. Previous fMRI research suggested a frontal–parietal network involved in the execution of complex numerical and nonnumerical tasks, but evidence is lacking on the particular contributions of frontal and parietal cortices across time. In an arithmetic task paradigm, we evaluated individual participants' “retrieval” and “multistep procedural” strategies on a trial-by-trial basis and contrasted those in time-resolved analyses using combined EEG and MEG. Retrieval strategies relied on direct retrieval of arithmetic facts (e.g., 2 + 3 = 5). Procedural strategies required multiple solution steps (e.g., 12 + 23 = 12 + 20 + 3 or 23 + 10 + 2). Evoked source analyses revealed independent activation dynamics within the first second of problem-solving in brain areas previously described as one network, such as the frontal–parietal cognitive control network: The right frontal cortex showed earliest effects of strategy selection for multistep procedural strategies around 300 msec, before parietal cortex activated around 700 msec. In time–frequency source power analyses, memory retrieval and multistep procedural strategies were differentially reflected in theta, alpha, and beta frequencies: Stronger beta and alpha desynchronizations emerged for procedural strategies in right frontal, parietal, and temporal regions as function of executive demands. Arithmetic fact retrieval was reflected in right prefrontal increases in theta power. Our results demonstrate differential brain dynamics within frontal–parietal networks across the time course of a problem-solving process, and analyses of different frequency bands allowed us to disentangle cortical regions supporting the underlying memory and executive functions.

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Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis thaliana

Genome ◽

10.1139/g93-082 ◽

1993 ◽

Vol 36 (3) ◽

pp. 610-618 ◽

Cited By ~ 54

Author(s):

John P. McNevin ◽

Wendy Woodward ◽

Abdelali Hannoufa ◽

Kenneth A. Feldmann ◽

Bertrand Lemieux

Keyword(s):

Arabidopsis Thaliana ◽

Length Distribution ◽

Transformation Method ◽

Epicuticular Wax ◽

Primary Alcohols ◽

Chain Length Distribution ◽

Isolation And Characterization ◽

Mutant Phenotypes ◽

Mutant Lines ◽

Fatty Aldehydes

Thirteen Arabidopsis thaliana mutants with deviating epicuticular wax layers (i.e., cer mutants) were isolated by screening 13 000 transformed lines produced by the seed transformation method. After crossing the 13 mutants to some of the previously known cer mutant lines, 12 of our mutants mapped to 6 of the 21 known complementation groups (cer1 through cer4 as well as cer6 and cer10), while the other mutant corresponded to a previously unknown locus, cer21. Mutant phenotypes of 6 of the 13 mutant lines were caused by T-DNA insertions within cer genes. We also analyzed the chemical composition of the epicuticular wax layers of the cer mutants isolated in this study relative to that of Arabidopsis wild-type plants. Our results suggest that the five genes we tagged regulate different steps in wax biosynthesis, i.e., the decarbonylation of fatty aldehydes to alkanes, the elongation of hexacosanoic acid to octacosanoic acid, the reduction of fatty aldehydes to primary alcohols and the production of free aldehydes, while an insertion in the fifth gene causes an alteration in the chain length distribution of the different classes of wax compounds.Key words: epicuticular wax, glossy mutants, gas chromotography – mass spectroscopy.

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Time-resolved multivariate pattern analysis of infant EEG data

10.1101/2021.06.16.448720 ◽

2021 ◽

Author(s):

Kira Ashton ◽

Benjamin Zinszer ◽

Radoslaw Cichy ◽

Charles Nelson ◽

Richard Aslin ◽

...

Keyword(s):

Time Course ◽

Pattern Analysis ◽

Multivariate Pattern Analysis ◽

Time Resolved ◽

Promising Tool ◽

Eeg Data ◽

Neuroimaging Data ◽

Linear Svm ◽

Multivariate Pattern ◽

The Impact

Time-resolved multivariate pattern analysis (MVPA), a popular technique for analyzing magneto- and electro-encephalography (M/EEG) neuroimaging data, quantifies the extent and time-course by which neural representations support the discrimination of relevant stimuli dimensions. As EEG is widely used for infant neuroimaging, time-resolved MVPA of infant EEG data is a particularly promising tool for infant cognitive neuroscience. MVPA methods have recently been applied to common infant imaging methods such as EEG and fNIRS. In this tutorial, we provide and describe code to implement time-resolved, within-subject MVPA with infant EEG data. A pipeline for time-resolved MVPA based on linear SVM classification is described and implemented with accompanying code in both Matlab and Python. Results from a test dataset indicated that in both infants and adults this method reliably produced above chance classification accuracy. Extensions of the core pipeline are presented including both geometric- and accuracy-based representational similarity analysis, implemented in Python. Common choices of implementation are presented and discussed. As the amount of artifact-free EEG data contributed by each participant is lower in studies of infants than in studies of children and adults, we also explore and discuss the impact of varying participant-level inclusion thresholds on resulting MVPA findings in these datasets.

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Comparison of transient and successful fusion pores connecting influenza hemagglutinin expressing cells to planar membranes.

The Journal of General Physiology ◽

10.1085/jgp.106.5.803 ◽

1995 ◽

Vol 106 (5) ◽

pp. 803-819 ◽

Cited By ~ 28

Author(s):

G B Melikyan ◽

W D Niles ◽

V A Ratinov ◽

M Karhanek ◽

J Zimmerberg ◽

...

Keyword(s):

Time Course ◽

Cell Surfaces ◽

Bilayer Membranes ◽

Time Resolved ◽

Influenza Hemagglutinin ◽

Influenza Virus Hemagglutinin ◽

Time Courses ◽

Fusion Pores ◽

Planar Membranes ◽

Pore Enlargement

Time-resolved admittance measurements were used to investigate the evolution of fusion pores formed between cells expressing influenza virus hemagglutinin (HA) and planar bilayer membranes. The majority of fusion pores opened in a stepwise fashion to semistable conductance levels of several nS. About 20% of the pores had measurable rise times to nS conductances; some of these opened to conductances of approximately 500 pS where they briefly lingered before opening further to semistable conductances. The fall times of closing were statistically similar to the rise times of opening. All fusion pores exhibited semistable values of conductance, varying from approximately 2-20 nS; they would then either close or fully open to conductances on the order of 1 microS. The majority of pores closed; approximately 10% fully opened. Once within the semistable stage, all fusion pores, even those that eventually closed, tended to grow. Statistically, however, before closing, transient fusion pores ceased to grow and reversed their conductance pattern: conductances decreased with a measurable time course until a final drop to closure. In contrast, pore enlargement to the fully open state tended to occur from the largest conductance values attained during a pore's semistable stage. This final enlargement was characterized by a stepwise increase in conductance. The density of HA on the cell surface did not strongly affect pore dynamics. But increased proteolytic treatment of cell surfaces did lead to faster growth within the semistable range. Transient pores and pores that fully opened had indistinguishable initial conductances and statistically identical time courses of early growth, suggesting they were the same upon formation. We suggest that transient and fully open pores evolved from common structures with stochastic factors determining their fate.

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Time-Resolved fMRI of Activation Patterns in M1 and SMA During Complex Voluntary Movement

Journal of Neurophysiology ◽

10.1152/jn.2001.85.5.1858 ◽

2001 ◽

Vol 85 (5) ◽

pp. 1858-1863 ◽

Cited By ~ 68

Author(s):

Florian Weilke ◽

Sabine Spiegel ◽

Henning Boecker ◽

Helga Gräfin von Einsiedel ◽

Bastian Conrad ◽

...

Keyword(s):

Time Course ◽

Primary Motor Cortex ◽

Planar Imaging ◽

Hemodynamic Response Function ◽

Time Resolved ◽

Activation Patterns ◽

Signal Change ◽

Repetition Time ◽

Report Data ◽

Echo Planar

The aim of this study was to use time-resolved functional magnetic resonance imaging (fMRI) to investigate temporal differences in the activation of the supplementary motor area (SMA) and the primary motor cortex (M1). We report data from eight human volunteers who underwent fMRI examinations in a 1.5T Philips Gyroscan ACS-NT MRI scanner. While wearing a contact glove, subjects executed a complex automated sequence of finger movements either spontaneously or in response to external auditory cues. Based on the result of a functional scout scan, a single slice that included the M1 and the SMA was selected for image acquisition (echo planar imaging, repetition time 100 ms, echo time 50 ms, 64 × 64 matrix, 1,000 images). Data were analyzed with a shifting cross-correlation approach using the STIMULATE program and in-house programs written in Interactive Data Language (IDL™). Time-course data were generated for regions of interest in the M1 as well as in the rostral and caudal SMA. Mean time between onset of the finger movement sequence and half-maximum of the signal change in M1 was 3.6 s for the externally cued execution (SD 0.5) and 3.5 s for the spontaneous execution (SD 0.6). Activation in the rostral section of the SMA occurred 0.7 s earlier than it did in the M1 during the externally cued execution and 2.0 s earlier during the spontaneous execution, a difference significant at the P < 0.01 level. Our results indicate that rostral SMA activation precedes M1 activation by varying time intervals in the sub-second range that are determined by the mode of movement initialization. By applying a paradigm that exerts a differential influence on temporal activation, we could ensure that the observed timing differences were not the result of differences in hemodynamic response function.

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