SUSIG: an on-line signature database, associated protocols and benchmark results

We present a new online signature database (SUSIG). The database consists of two parts that are collected using different pressure-sensitive tablets ( one with and the other without an LCD display). A total of 100 people contributed to each part, resulting in a database of more than 3,000 genuine signatures and 2,000 skilled forgeries. The genuine signatures in the database are real signatures of the contributors. In collecting skilled forgeries, forgers were shown the signing process on the monitor and were given a chance to practice. Furthermore, for a subset of the forgeries ( highly skilled forgeries), this animation was mapped onto the LCD screen of the tablet so that the forgers could trace over the mapped signature. Forgers in this group were also informed of how close they were to the reference signature, so that they could improve their forgery quality. We describe the signature acquisition process and several verification protocols for this database. We also report the performance of a state-of-the-art signature verification system using the associated protocols. The results show that the highly skilled forgery set is significantly more difficult compared to the skilled forgery set, providing researchers with challenging forgeries. The database is available through http://icproxy.sabanciuniv.edu:215

Salivary Glucose Concentration and Excretion in Normal and Diabetic Subjects

The present report aims mainly at a reevaluation of salivary glucose concentration and excretion in unstimulated and mechanically stimulated saliva in both normal and diabetic subjects. In normal subjects, a decrease in saliva glucose concentration, an increase in salivary flow, but an unchanged glucose excretion rate were recorded when comparing stimulated saliva to unstimulated saliva. In diabetic patients, an increase in salivary flow with unchanged salivary glucose concentration and glucose excretion rate were observed under the same experimental conditions. Salivary glucose concentration and excretion were much higher in diabetic patients than in control subjects, whether in unstimulated or stimulated saliva. No significant correlation between glycemia and either glucose concentration or glucose excretion rate was found in the diabetic patients, whether in unstimulated or stimulated saliva. In the latter patients, as compared to control subjects, the relative magnitude of the increase in saliva glucose concentration was comparable, however, to that of blood glucose concentration. The relationship between these two variables was also documented in normal subjects and diabetic patients undergoing an oral glucose tolerance test

Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases

Activity-Based Protein Profiling Reveals Dynamic Substrate-Specific Cellulase Secretion by Saprotrophic Basidiomycetes

BACKGROUND: Fungal saccharification of lignocellulosic biomass occurs concurrently with the secretion of a diverse collection of proteins, together functioning as a catalytic system to liberate soluble sugars from insoluble composite biomaterials. How different fungi respond to different substrates is of fundamental interest to the developing biomass saccharification industry. Among the cornerstones of fungal enzyme systems are the highly expressed cellulases (endo-β-glucanases and cellobiohydrolases). Recently, a cyclophellitol-derived activity-based probe (ABP-Cel) was shown to be a highly sensitive tool for the detection and identification of cellulases. RESULTS: Here we show that ABP-Cel enables endo-β-glucanase profiling in diverse fungal secretomes. In combination with established ABPs for β-xylanases and β-d-glucosidases, we collected multiplexed in-gel fluorescence activity-based protein profiles of 240 secretomes collected over ten days from biological replicates of ten different basidiomycete fungi grown on maltose, wheat straw, or aspen pulp. Our results reveal the remarkable dynamics and unique enzyme fingerprints associated with each species substrate combination. Chemical proteomic analysis identifies significant arsenals of cellulases secreted by each fungal species during growth on lignocellulosic biomass. Recombinant production and characterization of a collection of probe-reactive enzymes from GH5, GH10, and GH12 confirm that ABP-Cel shows broad selectivity towards enzymes with endo-β-glucanase activity. CONCLUSION: Using small-volume samples with minimal sample preparation, the results presented here demonstrate the ready accessibility of sensitive direct evidence for fungal enzyme secretion during early stages of growth on complex lignocellulosic substrates. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13068-022-02107-z

A fungal lytic polysaccharide monooxygenase is required for cell wall integrity, thermotolerance, and virulence of the fungal human pathogen Cryptococcus neoformans

Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment

A relational model of perceived overqualification : the moderating role of interpersonal influence on social acceptance.

Theories of perceived overqualification have tended to focus on employees’ job-related responses to account for effects on performance. We offer an alternative perspective and theorize that perceived overqualification could influence work performance through a relational mechanism. We propose that relational skills, in the form of interpersonal influence of overqualified employees, determine their tendency to experience social acceptance and, thus, engage in positive work-related behaviors. We tested this relational model across two studies using time-lagged, multisource data. In Study 1, the results indicated that for employees high on interpersonal influence, perceived overqualification was positively related to self-reported social acceptance, whereas for employees low on interpersonal influence, the relationship was negative. Social acceptance, in turn, was positively related to in-role job performance, interpersonal altruism, and team member proactivity evaluated by supervisors. In Study 2, we focused on peer-reported social acceptance and found that the indirect relationships between perceived overqualification and supervisor-reported behavioral outcomes via social acceptance were negative when interpersonal influence was low and nonsignificant when interpersonal influence was high. The implications of the general findings are discussed

Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family, display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class Agaricomycetes, associated with wood-decay, but no function has been demonstrated for any Arg-AA9. Through bioinformatics, transcriptomic and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, LsAA9B, a naturally occurring protein from Lentinus similis. The LsAA9B structure reveals gross changes in the region equivalent to the canonical LPMO copper binding site, whilst features implicated in carbohydrate binding in AA9 LPMOs have been maintained. We obtained a structure of LsAA9B with xylotetraose bound on the surface of the protein although with considerably different binding mode compared to other AA9 complex structures. In addition, we have found indications of protein phosphorylation near the N-terminal Arg and the carbohydrate binding site, for which the potential function is currently unknown. Our results are strong evidence that Arg-AA9s function markedly different from canonical AA9 LPMO, but nonetheless may play a role in fungal conversion of lignocellulosic biomass

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.Peer reviewe

Comparison of CT and integrated PET-CT based radiation therapy planning in patients with malignant pleural mesothelioma

<p>Abstract</p> <p>Background</p> <p>When combined with adequate tumoricidal doses, accurate target volume delineation remains to be the one of the most important predictive factors for radiotherapy (RT) success in locally advanced or medically inoperable malignant pleural mesothelioma (MPM) patients. Recently, 18-fluorodeoxyglucose positron emission tomography (PET) has demonstrated significant improvements in diagnosis and accurate staging of MPM. However, role of additional PET data has not been studied in RT planning (RTP) of patients with inoperable MPM or in those who refuse surgery. Therefore, we planned to compare CT with co-registered PET-CT as the basis for delineating target volumes in these patients group.</p> <p>Methods</p> <p>Retrospectively, the CT and co-registered PET-CT data of 13 patients with histologically proven MPM were utilized to delineate target volumes separately. For each patient, target volumes (gross tumor volume [GTV], clinical target volume [CTV], and planning target volume [PTV]) were defined using the CT and PET-CT fusion data sets. The PTV was measured in two ways: PTV1 was CTV plus a 1-cm margin, and PTV2 was GTV plus a 1-cm margin. We analyzed differences in target volumes.</p> <p>Results</p> <p>In 12 of 13 patients, compared to CT-based delineation, PET-CT-based delineation resulted in a statistically significant decrease in the mean GTV, CTV, PTV1, and PTV2. In these 12 patients, mean GTV decreased by 47.1% ± 28.4%, mean CTV decreased by 38.7% ± 24.7%, mean PTV1 decreased by 31.1% ± 23.1%, and mean PTV2 decreased by 40.0% ± 24.0%. In 4 of 13 patients, hilar lymph nodes were identified by PET-CT that was not identified by CT alone, changing the nodal status of tumor staging in those patients.</p> <p>Conclusion</p> <p>This study demonstrated the usefulness of PET-CT-based target volume delineation in patients with MPM. Co-registration of PET and CT information reduces the likelihood of geographic misses, and additionally, significant reductions observed in target volumes may potentially allow escalation of RT dose beyond conventional limits potential clinical benefits in tumor control rates, which needs to be tested in future studies.</p

SNi from SN2: a front-face mechanism ‘synthase’ engineered from a retaining hydrolase

SNi or SNi-like mechanisms, in which leaving group departure and nucleophile approach occur on the same ‘front’ face, have been observed previously experimentally and computationally in both the chemical and enzymatic (glycosyltransferase) substitution reactions of α-glycosyl electrophiles. Given the availability of often energetically comparable competing pathways for substitution (SNi vs SN1 vs SN2) the precise modulation of this archetypal reaction type should be feasible. Here, we show that the drastic engineering of a protein that catalyzes substitution, a retaining β-glycosidase (from Sulfolobus solfataricus SSβG), apparently changes the mode of reaction from “SN2” to “SNi”. Destruction of the nucleophilic Glu387 of SSβG-WT through Glu387Tyr mutation (E387Y) created a catalyst (SSβG-E387Y) with lowered but clear transglycosylation substitution activity with activated substrates, altered substrate and reaction preferences and hence useful synthetic (‘synthase’) utility by virtue of its low hydrolytic activity with unactivated substrates. Strikingly, the catalyst still displayed retaining β stereoselectivity, despite lacking a suitable nucleophile; pH-activity profile, mechanism-based inactivators and mutational analyses suggest that SSβG-E387Y operates without either the use of nucleophile or general acid/base residues, consistent with a SNi or SNi-like mechanism. An x-ray structure of SSβG-E387Y and subsequent metadynamics simulation suggest recruitment of substrates aided by a π-sugar interaction with the introduced Tyr387 and reveal a QM/MM free energy landscape for the substitution reaction catalyzed by this unnatural enzyme similar to those of known natural, SNi-like glycosyltransferase (GT) enzymes. Proton flight from the putative hydroxyl nucleophile to the developing p-nitrophenoxide leaving group of the substituted molecule in the reactant complex creates a hydrogen bond that appears to crucially facilitate the mechanism, mimicking the natural mechanism of SNi-GTs. An oxocarbenium ion-pair minimum along the reaction pathway suggests a step-wise SNi-like DN*ANss rather than a concerted SNi DNAN mechanism. This first observation of a front face mechanism in a β-retaining glycosyl transfer enzyme highlights, not only that unusual SNi reaction pathways may be accessed through direct engineering of catalysts with suitable environments, but also suggests that ‘β-SNi’ reactions are also feasible for glycosyl transfer enzymes and the more widespread existence of SNi or SNi-like mechanism in nature