CLIPPER 2.0: Peptide-Level Annotation and Data Analysis for Positional Proteomics.

Positional proteomics methodologies have transformed protease research, and have brought mass spectrometry (MS)-based degradomics studies to the forefront of protease characterization and system-wide interrogation of protease signaling. Considerable advancements in both sensitivity and throughput of liquid chromatography (LC)-MS/MS instrumentation enable the generation of enormous positional proteomics datasets of natural and protein termini and neo-termini of cleaved protease substrates. However, concomitant progress has not been observed to the same extent in data analysis and post-processing steps, arguably constituting the largest bottleneck in positional proteomics workflows. Here, we present a computational tool, CLIPPER 2.0, that builds on prior algorithms developed for MS-based protein termini analysis, facilitating peptide-level annotation and data analysis. CLIPPER 2.0 can be used with several sample preparation workflows and proteomics search algorithms and enables fast and automated database information retrieval, statistical and network analysis, as well as visualization of terminomic datasets. We demonstrate the applicability of our tool by analyzing GluC and MMP9 cleavages in HeLa lysates. CLIPPER 2.0 is available at https://github.com/UadKLab/CLIPPER-2.0.

Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30.

The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus "Jettenia caeni" (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus "Scalindua brodae" and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.

Salmonella-driven intestinal edema in mice is characterized by tensed fibronectin fibers.

Intestinal edema is a common manifestation of numerous gastrointestinal diseases and is characterized by the accumulation of fluid in the interstitial space of the intestinal wall. Technical advances in laser capture microdissection and low-biomass proteomics now allow us to specifically characterize the intestinal edema proteome. Using advanced proteomics, we identify peptides derived from antimicrobial factors with high signal intensity, but also highlight major contributions from the blood clotting system, extracellular matrix (ECM) and protease-protease inhibitor networks. The ECM is a complex fibrillar network of macromolecules that provides structural and mechanical support to the intestinal tissue. One abundant component of the ECM observed in Salmonella-driven intestinal edema is the glycoprotein fibronectin, recognized for its structure-function interplay regulated by mechanical forces. Using mechanosensitive staining of fibronectin fibers reveals that they are tensed in the edema, despite the high abundance of proteases able to cleave fibronectin. In contrast, fibronectin fibers increasingly relax in other cecal tissue areas as the infection progresses. Co-staining for fibrin(ogen) indicates the formation of a provisional matrix in the edema, similar to what is observed in response to skin injury, while collagen staining reveals a sparse and disrupted collagen fiber network. These observations plus the absence of low tensional fibronectin fibers and the additional finding of a high number of protease inhibitors in the edema proteome could indicate a critical role of stretched fibronectin fibers in maintaining tissue integrity in the severely inflamed cecum. Understanding these processes may also provide valuable functional diagnostic markers of intestinal disease progression in the future.

Immature Status Epilepticus Alters the Temporal Relationship between Hippocampal Interictal Epileptiform Discharges and High-frequency Oscillations.

The aim was to investigate the long-term effects of a single episode of immature Status Epilepticus (SE) on the excitability of the septal and temporal hippocampus in vitro, by studying the relationship between interictal-like epileptiform discharges (IEDs) and high-frequency oscillations (HFOs; Ripples, Rs and Fast Ripples, FRs). A pentylenetetrazol-induced Status Epilepticus-(SE)-like generalized seizure was induced at postnatal day 20 in 22 male and female juvenile rats, sacrificed >40 days later to prepare hippocampal slices. Spontaneous IEDs induced by Mg2+-free ACSF were recorded from the CA3 area of temporal (T) or septal (S) slices. Recordings were band-pass filtered off-line revealing Rs and FRs and a series of measurements were conducted, with mean values compared with those obtained from age-matched controls (CTRs). In CTR S (vs T) slices, we recorded longer R & FR durations, a longer HFO-IED temporal overlap, higher FR peak power and more frequent FR initiation preceding IEDs (% events). Post-SE, in T slices all types of events duration (IED, R, FR) and the time lag between their onsets (R-IED, FR-IED, R-FR) increased, while FR/R peak power decreased; in S slices, the IED 1st population spike and the FR amplitudes, the R and FR peak power and the (percent) events where Rs or FRs preceded IEDs all decreased. The CA3 IED-HFO relationship offers insights to the septal-to-temporal synchronization patterns; its post-juvenile-SE changes indicate permanent modifications in the septotemporal excitability gradient. Moreover, these findings are in line to region-specific regulation of various currents post-SE, as reported in literature.

Peptidomic analysis of endogenous and bacterial protease activity in human plasma and wound fluids.

Endogenous and bacterial proteases play important roles in wound healing and infection. Analysis of alterations in the low-molecular-weight peptidome by individual enzymes could therefore provide insight into proteolytic events occurring in wounds and may aid in the discovery of biomarkers. Using liquid chromatography with tandem mass spectrometry, we characterized the peptidome of plasma and acute wound fluids digested ex vivo with human (neutrophil elastase and cathepsin G) and bacterial proteases (Pseudomonas aeruginosa LasB and Staphyloccocus aureus V8). We identified over 100 protein targets for each enzyme and characterized enzyme specific peptides and cleavage patterns. Moreover, we found unique peptide regions in V8 digested samples that were also present in dressing extracts from S. aureus infected wounds. Finally, the work indicates that peptidomic analysis of qualitative differences of proteolytic activity of individual enzymes may aid in the discovery of potential diagnostic biomarkers for wound healing status.

A comparative study of protein structure prediction tools for challenging targets: Snake venom toxins.

Protein structure determination is a critical aspect of biological research, enabling us to understand protein function and potential applications. Recent advances in deep learning and artificial intelligence have led to the development of several protein structure prediction tools, such as AlphaFold2 and ColabFold. However, their performance has primarily been evaluated on well-characterised proteins and their ability to predict sturtctures of proteins lacking experimental structures, such as many snake venom toxins, has been less scrutinised. In this study, we evaluated three modelling tools on their prediction of over 1000 snake venom toxin structures for which no experimental structures exist. Our findings show that AlphaFold2 (AF2) performed the best across all assessed parameters. We also observed that ColabFold (CF) only scored slightly worse than AF2, while being computationally less intensive. All tools struggled with regions of intrinsic disorder, such as loops and propeptide regions, and performed well in predicting the structure of functional domains. Overall, our study highlights the importance of exercising caution when working with proteins with no experimental structures available, particularly those that are large and contain flexible regions. Nonetheless, leveraging computational structure prediction tools can provide valuable insights into the modelling of protein interactions with different targets and reveal potential binding sites, active sites, and conformational changes, as well as into the design of potential molecular binders for reagent, diagnostic, or therapeutic purposes.

Sensitive and High-Throughput Exploration of Protein N-Termini by TMT-TAILS N-Terminomics.

Terminal amine isotopic labeling of substrates (TAILS) is a sensitive and robust quantitative mass spectrometry (MS)-based proteomics method used for the characterization of physiological or proteolytically processed protein N-termini, as well as other N-terminal posttranslational modifications (PTMs). TAILS is a well-established, high-throughput, negative enrichment workflow that enables system-wide exploration of N-terminomes independent of sample complexity. TAILS makes use of amine reactivity of free N-termini and a highly efficient aldehyde-functionalized polymer to deplete internal peptides generated after proteolytic digestion during sample preparation. Thereby, it enriches for natural N-termini, allowing for unbiased and complete investigation of differential proteolysis, protease substrate discovery, and analysis of N-terminal PTMs. In this chapter, we provide a state-of-the-art protocol, with detailed steps in all parts of the TAILS sample preparation, MS analysis, and post-processing of acquired data.

Assessment of generalised Bayesian structural equation models for continuous and binary data.

The paper proposes a novel model assessment paradigm aiming to address shortcoming of posterior predictive p -values, which provide the default metric of fit for Bayesian structural equation modelling (BSEM). The model framework presented in the paper focuses on the approximate zero approach (Psychological Methods, 17, 2012, 313), which involves formulating certain parameters (such as factor loadings) to be approximately zero through the use of informative priors, instead of explicitly setting them to zero. The introduced model assessment procedure monitors the out-of-sample predictive performance of the fitted model, and together with a list of guidelines we provide, one can investigate whether the hypothesised model is supported by the data. We incorporate scoring rules and cross-validation to supplement existing model assessment metrics for BSEM. The proposed tools can be applied to models for both continuous and binary data. The modelling of categorical and non-normally distributed continuous data is facilitated with the introduction of an item-individual random effect. We study the performance of the proposed methodology via simulation experiments as well as real data on the 'Big-5' personality scale and the Fagerstrom test for nicotine dependence.

Endodontic management of a double-type IIIB dens invaginatus in a vital maxillary central incisor aided by CBCT: A case report.

Type IIIB dens invaginatus presents with diagnostic and treatment related challenges when in need of endodontic management as a consequence of its complex anatomy, especially when presented in a vital tooth with a periapical lesion. Apical periodontitis associated with two type IIIB invaginations in a central maxillary incisor of a 10-year-old patient was diagnosed. A cone-beam computed tomography (CBCT) scan provided essential diagnostic information and steered the treatment plan. The two invaginations were separate, with no communication between them and the pulp. The pulp appeared vital and non-inflamed. Endodontic treatment of the invaginations was carried out without intervention in the pulp. A 4-month follow-up periapical radiograph showed significant shrinkage of the lesion and a 2-year follow-up CBCT scan confirmed its complete healing. The pulp remains vital, responding normally to sensitivity tests. This outcome indicates that preserving the pulp's vitality is achievable through timely diagnosis.

Longitudinal Evaluation of Biomarkers in Wound Fluids from Venous Leg Ulcers and Split-thickness Skin Graft Donor Site Wounds Treated with a Protease-modulating Wound Dressing.

Venous leg ulcers represent a clinical challenge and impair the quality of life of patients. This study examines impaired wound healing in venous leg ulcers at the molecular level. Protein expression patterns for biomarkers were analysed in venous leg ulcer wound fluids from 57 patients treated with a protease-modulating polyacrylate wound dressing for 12 weeks, and compared with exudates from 10 acute split-thickness wounds. Wound healing improved in the venous leg ulcer wounds: 61.4% of the 57 patients with venous leg ulcer achieved a relative wound area reduction of ≥ 40%, and 50.9% of the total 57 patients achieved a relative wound area reduction of ≥ 60%. Within the first 14 days, abundances of S100A8, S100A9, neutrophil elastase, matrix metalloproteinase-2, and fibronectin in venous leg ulcer exudates decreased significantly and remained stable, yet higher than in acute wounds. Interleukin-1ß, tumour necrosis factor alpha, and matrix metalloproteinase-9 abundance ranges were similar in venous leg ulcers and acute wound fluids. Collagen (I) α1 abundance was higher in venous leg ulcer wound fluids and was not significantly regulated. Overall, significant biomarker changes occurred in the first 14 days before a clinically robust healing response in the venous leg ulcer cohort.

Proteomics and histological assessment of an organotypic model of human skin following exposure to Naja nigricollis venom.

Snakebite envenoming was reintroduced as a Category A Neglected Tropical Disease by the World Health Organization in 2017. Since then, increased attention has been directed towards this affliction and towards the development of a deeper understanding of how snake venoms exert their toxic effects and how antivenoms can counter them. However, most of our in vivo generated knowledge stems from the use of animal models which do not always accurately reflect how the pathogenic effects of snake venoms manifest in humans. Moreover, animal experiments are associated with pain, distress, and eventually animal sacrifice due to the toxic nature of snake venoms. Related to this, the implementation of the 3Rs principle (Replacement, Reduction, and Refinement) in the use of experimental animals in snakebite envenoming research is recommended by the World Health Organization. Therefore, more humane experimental designs and new in vitro/ex vivo alternatives for experimental animals are sought after. Here, we report the use of an organotypic model of human skin to further elucidate the pathophysiology of the dermonecrotic effects caused by the venom of the black-necked spitting cobra, Naja nigricollis, in humans. The goal of this study is to expand the repertoire of available models that can be used to study the local tissue damages induced by cytotoxic venoms.

Timing differences between HFOs and interictal epileptiform discharges generated in vitro by different mechanisms in rat hippocampal slices: A novel approach.

OBJECTIVE: To investigate the effect of generating mechanism on the relationship between interictal-like epileptiform discharges (IEDs) and the underlying High Frequency Oscillations (HFOs; Ripples, R, and Fast Ripples, FR). METHODS: Synchronous spontaneous IEDs were recorded from the CA1 area of hippocampal slices from adult rats, perfused by Mg2+ -free ACSF (n = 41slices/14 animals) or 4-aminopyridine (50 µM, n = 37slices/16 animals); IED filtering revealed Rs and FRs and several metrics were calculated and compared (amplitude, duration, relative onset, time lag, % overlap, peak frequency, peak power, FR/R). RESULTS: Longer IEDs and higher 1st Population Spike (PS) amplitude in Mg2+ -free ACSF (vs 4-AP; P < .001, P < .001) correlated with longer duration and higher amplitude Rs (P < .0001, P = .001) and longer duration FRs (P < .001). In both media, Rs and FRs appeared before IED onset with Rs preceding FRs; R- and FR-IED lag (P = .008, P = .01) as well as R-FR lag (P = .04) were significantly longer in Mg2+ -free ACSF vs in 4-AP. R peak frequency and power were higher in Mg2+ -free ACSF, while no such differences were observed in FRs. Inter-model differences were mostly reflected in Rs, not FRs, suggesting that mechanisms unique to R generation are more active in Mg2+ -free ACSF vs in 4-AP. FRs appeared to contribute equally to IEDs irrespective of generating mechanism. SIGNIFICANCE: Several of the metrics used, particularly those regarding the timing between HFOs and IEDs, appear to correlate with the synchronizing mechanism and we propose that they may be useful when investigating antiepileptic substance effects on neuronal network activity.

The Effect of Cone-Beam Computed Tomography (CBCT) Evaluation on Treatment Planning after Endodontic Instrument Fracture.

Intracanal instrument fracture is a procedural iatrogenic event during endodontic treatment that may affect treatment planning and eventually treatment outcome. Cone Beam Computed Tomography (CBCT) has offered several advantages, especially in endodontic cases in which information from conventional periapical radiograph may not be adequate to allow a precise treatment planning decision and a subsequent appropriate management of the cases. The present study was firstly conducted to assess the effect of CBCT evaluation on the decision-making process after instrument fracture; secondly, to introduce a new clinical approach in cases with fractured instruments located in the mesial roots of mandibular and maxillary molars. The study design was observational. The sample comprised all cases of mandibular and maxillary molars where an instrument fracture had occurred in the mesial roots. Two qualified (National and Kapodistrian University of Athens, Greece) and experienced (more than fifteen years of daily practicing) endodontists evaluated all the cases. The initial treatment plan made by evaluating periapical radiographs of each case was compared to the final plan set after CBCT evaluation. A marginal homogeneity test for paired data was conducted to test the concordance of treatment planning with periapical radiographs versus CBCT. Multivariable logistic regression was structured to identify predictors of modification in treatment planning following CBCT assessment, and to record estimators for decision to remove, bypass or retain the fragment. The level of statistical significance was pre-specified at p < 0.05. Of a total 52 cases evaluated, change in treatment planning with conventional periapical radiograph as a reference, following evaluation of CBCT, was observed in more than half of the teeth. The difference was statistically significant (p < 0.001). Apical location of the fragment was more likely to induce a perceived change in treatment planning after CBCT evaluation (p < 0.01). Canal merging induced 95% lower odds (p = 0.01) for taking a decision to remove or bypass, revealing that retaining the fragment was by far a more likely decision. A significant impact of CBCT preoperative evaluation on treatment planning for the management of such cases was demonstrated. Apical location of the fragment and canal merging seem to influence the decision-making process.

Black-necked spitting cobra (Naja nigricollis) phospholipases A2 may cause Trypanosoma brucei death by blocking endocytosis through the flagellar pocket.

African trypanosomes, such as Trypanosoma brucei, are flagellated protozoa which proliferate in mammals and cause a variety of diseases in people and animals. In a mammalian host, the external face of the African trypanosome plasma membrane is covered by a densely packed coat formed of variant surface glycoprotein (VSG), which counteracts the host's adaptive immune response by antigenic variation. The VSG is attached to the external face of the plasma membrane by covalent attachment of the C-terminus to glycosylphosphatidylinositol. As the trypanosome grows, newly synthesised VSG is added to the plasma membrane by vesicle fusion to the flagellar pocket, the sole location of exo- and endocytosis. Snake venoms contain dozens of components, including proteases and phospholipases A2. Here, we investigated the effect of Naja nigricollis venom on T. brucei with the aim of describing the response of the trypanosome to hydrolytic attack on the VSG. We found no evidence for VSG hydrolysis, however, N. nigricollis venom caused: (i) an enlargement of the flagellar pocket, (ii) the Rab11 positive endosomal compartments to adopt an abnormal dispersed localisation, and (iii) cell cycle arrest prior to cytokinesis. Our results indicate that a single protein family, the phospholipases A2 present in N. nigricollis venom, may be necessary and sufficient for the effects. This study provides new molecular insight into T. brucei biology and possibly describes mechanisms that could be exploited for T. brucei targeting.

The rise of genomics in snake venom research: recent advances and future perspectives.

Snake venoms represent a danger to human health, but also a gold mine of bioactive proteins that can be harnessed for drug discovery purposes. The evolution of snakes and their venom has been studied for decades, particularly via traditional morphological and basic genetic methods alongside venom proteomics. However, while the field of genomics has matured rapidly over the past 2 decades, owing to the development of next-generation sequencing technologies, snake genomics remains in its infancy. Here, we provide an overview of the state of the art in snake genomics and discuss its potential implications for studying venom evolution and toxinology. On the basis of current knowledge, gene duplication and positive selection are key mechanisms in the neofunctionalization of snake venom proteins. This makes snake venoms important evolutionary drivers that explain the remarkable venom diversification and adaptive variation observed in these reptiles. Gene duplication and neofunctionalization have also generated a large number of repeat sequences in snake genomes that pose a significant challenge to DNA sequencing, resulting in the need for substantial computational resources and longer sequencing read length for high-quality genome assembly. Fortunately, owing to constantly improving sequencing technologies and computational tools, we are now able to explore the molecular mechanisms of snake venom evolution in unprecedented detail. Such novel insights have the potential to affect the design and development of antivenoms and possibly other drugs, as well as provide new fundamental knowledge on snake biology and evolution.

Unraveling the structure and function of CdcPDE: A novel phosphodiesterase from Crotalus durissus collilineatus snake venom.

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8-8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities.

Analysis of wound exudates reveals differences in the patterns of tissue damage and inflammation induced by the venoms of Daboia russelii and Bothrops asper in mice.

Clinical manifestations of envenomings by bites of the viperid snakes Bothrops asper and Daboia russelii show marked differences. Both venoms elicit the typical effects induced by viperid venoms (local tissue damage, bleeding, coagulopathies, shock). In addition, envenomings by D. russelii are characterized by a high incidence of acute kidney injury and by systemic capillary leak syndrome. The present investigation aimed to compare the local pathological and inflammatory events induced by the intramuscular injection of these venoms in a mouse model. B. asper venom induced stronger local hemorrhage, whereas D. russelii venom caused a higher extent of myonecrosis, and both venoms induced inflammation. Exudates collected from the site of tissue damage showed higher proteolytic activity in the case of samples from B. asper venom-treated mice. This activity was abrogated by antivenoms, indicating that it is the result of the action of venom proteinases. In addition, an increase in matrix metalloproteinases (MMPs) over time was detected in exudates induced by both venoms. Proteome analysis of exudates revealed higher abundance of extracellular matrix (ECM)-derived protein fragments in samples collected from B. asper venom-injected mice, whereas those from D. russelii venom-injected animals had higher amounts of intracellular proteins. Analysis of the subproteome of inflammatory mediators in exudates showed various patterns of change over time. Some mediators peaked at 180 min and decreased afterwards, whereas others increased and remained elevated during the 360 min observation period. Interestingly, various mediators (MIP-1α, MIP-1ß, KC, MIP-2, GM-CSF, VEGF, and LIX) increased and then decreased in the case of B. asper venom, while they remained elevated at 360 min in the case of D. russelii venom. Our findings show that these venoms induce a different pattern of local tissue damage and suggest that the venom of D. russelii induces a more sustained inflammatory reaction, an observation that may have implications for the pathophysiology of envenomings.

Do Antibiotics Potentiate Proteases in Hemotoxic Snake Venoms?

Antibiotics are often administered with antivenom following snakebite envenomings in order to avoid secondary bacterial infections. However, to this date, no studies have evaluated whether antibiotics may have undesirable potentiating effects on snake venom. Herein, we demonstrate that four commonly used antibiotics affect the enzymatic activities of proteolytic snake venom toxins in two different in vitro assays. Similar findings in vivo could have clinical implications for snakebite management and require further examination.