A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists.

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαß structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

Native peptide mapping - A simple method to routinely monitor higher order structure changes and relation to functional activity.

In the biopharmaceutical environment, controlling the Critical Quality Attributes (CQA) of a product is essential to prevent changes that affect its safety or efficacy. Physico-chemical techniques and bioassays are used to screen and monitor these CQAs. The higher order structure (HOS) is a CQA that is typically studied using techniques that are not commonly considered amenable to quality control laboratories. Here, we propose a peptide mapping-based method, named native peptide mapping, which could be considered as straightforward for HOS analysis and applicable for IgG4 and IgG1 antibodies. The method was demonstrated to be fit-for-purpose as a stability-indicating assay by showing differences at the peptide level between stressed and unstressed material. The unfolding pathway induced by a heat stress was also studied via native peptide mapping assay. Furthermore, we demonstrated the structure-activity relationship between HOS and biological activity by analyzing different types of stressed samples with a cell-based assay and the native peptide mapping. The correlation between both sets of results was highlighted by monitoring peptides located in the complementary-determining regions and the relative potency of the biotherapeutic product. This relationship represents a useful approach to interrogate the criticality of HOS as a CQA of a drug.

Proteome of fraction from Tityus serrulatus venom reveals new enzymes and toxins.

BACKGROUND: Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. METHODS: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. RESULTS: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). CONCLUSIONS: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.

Proteome of fraction from Tityus serrulatus venom reveals new enzymes and toxins

Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. Methods: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. Results: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). Conclusions: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.(AU)

The Society of European Robotic Gynaecological Surgery (SERGS) Pilot Curriculum for robot assisted gynecological surgery.

PURPOSE: To set forth experiences in the context of the SERGS Pilot Curriculum-the first standardized educational program for robotic use in gynecological surgery-in terms of feasibility, effectiveness and potential for certification. METHODS: The Society of European Robotic Gynecological Surgery (SERGS) outlined a Pilot Curriculum for standardized education in robot-assisted laparoscopic gynecological surgery. Its feasibility and acceptance were checked in the form of a fellowship pilot program conducted at four European Centers of Excellence for robot-assisted surgery. Results and conclusions derived from this pilot program are presented. RESULTS: The SERGS Pilot Curriculum defines criteria for a standardized training and assessment of performance, boosts the learning curve of the candidate and increases contentment at work. Regarding face validity, it proves valuable as finally all candidates could perform the outlined procedure safely and efficiently without supervision. CONCLUSION: Due to the immense increase of robotic procedures in gynecology standardized training curricula are indispensable. This seems highly necessary to ensure patients' safety and surgical outcome. The SERGS Pilot Curriculum sets standards for a stepwise theoretical and practical training in gynecological robotic procedures. It seems feasible as instrument for accreditation as gynecologic robotic surgeon. Though as a general applicable guideline for systematic training in robot-assisted surgery, a definite curriculum should have a more definite timeline and implementation of a structured assessment of performance.

In-Depth Glyco-Peptidomics Approach Reveals Unexpected Diversity of Glycosylated Peptides and Atypical Post-Translational Modifications in Dendroaspis angusticeps Snake Venom.

Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of Dendroaspis angusticeps (DA), the Eastern Green Mamba, has been intensively studied during recent years. It mainly contains hundreds of large toxins from 6 to 9 kDa, each displaying several disulfide bridges. These toxins are the main target of venom-based studies due to their valuable activities obtained by selectively targeting membrane receptors, such as ion channels or G-protein coupled receptors. This study aims to demonstrate that the knowledge of venom composition is still limited and that animal venoms contain unexpected diversity and surprises. A previous study has shown that Dendroaspis angusticeps venom contains not only a cocktail of classical toxins, but also small glycosylated peptides. Following this work, a deep exploration of DA glycopeptidome by a dual nano liquid chromatography coupled to electrospray ionization mass spectrometry (nanoLC-ESI-MS) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses was initiated. This study reveals unsuspected structural diversity of compounds such as 221 glycopeptides, displaying different glycan structures. Sequence alignments underline structural similarities with natriuretic peptides already characterized in Elapidae venoms. Finally, the presence of an S-cysteinylation and hydroxylation of proline on four glycopeptides, never described to date in snake venoms, is also revealed by proteomics and affined by nuclear magnetic resonance (NMR) experiments.

Diversity in sequences, post-translational modifications and expected pharmacological activities of toxins from four Conus species revealed by the combination of cutting-edge proteomics, transcriptomics and bioinformatics.

Venomous animals have developed a huge arsenal of reticulated peptides for defense and predation. Based on various scaffolds, they represent a colossal pharmacological diversity, making them top candidates for the development of innovative drugs. Instead of relying on the classical, low-throughput bioassay-guided approach to identify innovative bioactive peptides, this work exploits a recent paradigm to access to venom diversity. This strategy bypasses the classical approach by combining high-throughput transcriptomics, proteomics and bioinformatics cutting-edge technologies to generate reliable peptide sequences. The strategy employed to generate hundreds of reliable sequences from Conus venoms is deeply described. The study led to the discovery of (i) conotoxins that belong to known pharmacological families targeting various GPCRs or ion-gated channels, and (ii) new families of conotoxins, never described to date. It also focusses on the diversity of genes, sequences, folds, and PTM's provided by such species.

Ion Mobility-Mass Spectrometry as a Tool for the Structural Characterization of Peptides Bearing Intramolecular Disulfide Bond(s).

Disulfide bonds are post-translationnal modifications that can be crucial for the stability and the biological activities of natural peptides. Considering the importance of these disulfide bond-containing peptides, the development of new techniques in order to characterize these modifications is of great interest. For this purpose, collision cross cections (CCS) of a large data set of 118 peptides (displaying various sequences) bearing zero, one, two, or three disulfide bond(s) have been measured in this study at different charge states using ion mobility-mass spectrometry. From an experimental point of view, CCS differences (ΔCCS) between peptides bearing various numbers of disulfide bonds and peptides having no disulfide bonds have been calculated. The ΔCCS calculations have also been applied to peptides bearing two disulfide bonds but different cysteine connectivities (Cys1-Cys2/Cys3-Cys4; Cys1-Cys3/Cys2-Cys4; Cys1-Cys4/Cys2-Cys3). The effect of the replacement of a proton by a potassium adduct on a peptidic structure has also been investigated. Graphical Abstract ᅟ.

Real-time in-vivo microscopic imaging of the cervix using confocal laser endomicroscopy: preliminary observations and feasibility study.

Confocal laser endomicroscopy (CLE) enables in-vivo, real-time, imaging of tissues with a micron-scale resolution through a fiber optic probe. CLE could be a valuable tool for the detection and characterization of suspicious (dysplastic) areas on the uterine cervix in a minimally invasive manner. This study evaluates the technical feasibility and safety of CLE on the cervix. The study also aims to create a preliminary iconography of normal and dysplastic squamous and columnar cervical epithelium. In-vivo CLE was performed on nine patients scheduled for a cervical loop electric excision procedure for high-grade superficial intraepithelial lesions. The CLE images were compared with standard hematoxylin and eosin analysis of loop electric excision procedure specimens. The histopathological diagnosis on the surgical specimen was established as per standard of care. CLE images were then reviewed by pathologists to point out specific histopathological features. pCLE of the exocervix and the transformation zone was performed successfully on seven out of nine patients. Uninterpretable images were obtained in two other cases: one using the AlveoFlex and one using the GastroFlex UHD after the application of acetic acid 2%. A total of 82.5% of the sequences recorded with the GastroFlex were suitable for interpretation. No adverse event or complications occurred. CLE enables proper in-vivo imaging of healthy and dysplastic cervical tissue. Images correlate well with the histopathological features established through traditional histology. Future blinded prospective analysis will determine the reliability of the real-time diagnosis and its potential use in the assessment and treatment of cervical lesions.

Mass-spectrometry-based method for screening of new peptide ligands for G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. Although implicated in almost all physiological processes in the human body, most of them remain unexploited, mostly because of the lack of specific ligands. The objective of this work is to develop a new mass-spectrometry-based technique capable of identifying new peptide ligands for GPCRs. The strategy is based on the incubation of cellular membranes overexpressing GPCRs with a mixture of peptides that contains potential ligands. Peptide ligands bind to the receptors, whereas other peptides remain in the binding buffer. Bound peptides are eluted from membranes and directly detected, identified, and characterized by MALDI TOF-TOF. The results reveal the efficacy of the procedure for selecting a specific ligand of GPCRs in both simple and complex mixtures of peptides. This new approach may offer direct purification, identification, and characterization of the new ligand in a single workflow. The proposed method is labeling-free and, unlike radio-binding and other techniques, it does not require a previously known labeled ligand of the studied GPCR. All these properties greatly reduce the experimental constraints. Moreover, because it is not based on the principle of a competitive specific binding, this technique constitutes a new tool to discover new ligands not only for known GPCRs, but also for orphan GPCRs.

Peptidomic comparison and characterization of the major components of the venom of the giant ant Dinoponera quadriceps collected in four different areas of Brazil.

Despite the noxious effects inflicted by Dinoponera ant's envenomation, the information about the biological properties and composition of their venom is still very limited. Ants from the genus Dinoponera are believed to be the world's largest living ants with a body length of 3cm. Their occurrence is restricted to tropical areas of South America. In this work, we study the venom of the giant Dinoponera quadriceps ant collected in 4 different regions of Brazil. By using a combination of complementary mass spectrometric approaches, we aim at: (i) characterizing the venom composition of these ants; (ii) establishing a comparative analysis of the venom from four geographically different regions in Brazil. This approach demonstrates that ant venom is a copious source of new compounds. Several peptides were identified and selected for "de novo sequencing". Since most of the new peptides showed similarities with antimicrobial peptides (AMPs), antimicrobial assays were performed with the purpose of evaluating their activity. In regard to the comparative study of the four regions, we observed not only major differences in the venom compositions, but also that the venoms collected in closest areas are more similar than the ones collected in distant regions. These observations seem to highlight an adaption of the ant venoms to the local environment. Concerning the biological assays, the peptides called Dq-3162 and Da-3177 showed a wide-ranging antimicrobial activity. The characterization of new AMPs with a broad spectrum of activity and different scaffolds may aid scientists to design new therapeutic agents and understand the mechanisms of those peptides to interact with microbial membranes. The results obtained betoken the biotechnological potential of ant's venom. BIOLOGICAL SIGNIFICANCE: For the first time this manuscript describes an extensive proteomics characterization of the D. quadriceps venom. In addition this study reports the variation in venom composition of primitive ants from 4 geographically different areas of Brazil. The results reveal the presence of ~335 compounds for each venom/area and inter-colony variations were observed. 16 new peptides were characterized and 2 of them were synthesized and biologically assayed. These findings highlight the considerable and still unexplored diversity of ant's venom which could be used as valuable research tools in different areas of knowledge.

Multi-center experience of robot-assisted laparoscopic para-aortic lymphadenectomy for staging of locally advanced cervical carcinoma.

OBJECTIVES: FIGO classification is commonly used for staging of locally advanced cervical cancer. Laparoscopic para-aortic lymphadenectomy is currently used as a diagnostic tool, since we know that presence of para-aortic lymph node metastases identifies patients with poor prognosis. The application of robotics during this procedure needs to be investigated. DESIGN: Retrospective multi-center study. SETTING: Three centers participated in building one database. POPULATION: Thirty-seven patients with locally advanced cervical cancer underwent a robot-assisted laparoscopic para-aortic lymphadenectomy. METHODS: Patients were prospectively enrolled in one register. Retrospective analysis of the whole database was performed. MAIN OUTCOME MEASURES: Surgical outcomes of the robot-assisted procedure and follow-up data. RESULTS: Median number of lymph nodes collected was 27.5 (1-54) per patient. Five of 37 patients had para-aortic node metastases. The false negative rate for PET-CT diagnosing para-aortic node metastases was 11.4% (4/35). Two major intra-operative complications occurred (5.4%). Postoperative morbidity was low (13.5%). Median follow-up was 27 months [95% confidence interval (95% CI) was 24-30]. Median disease-free survival was 16 months (95% CI 2.4-29.6). Patients with negative nodes had a median disease-free survival of 24 months (not assessable), although patients with positive nodes had a median disease-free survival of 9 months (95% CI 6.9-11.9). CONCLUSIONS: In this series we report that robot-assisted laparoscopic para-aortic lymphadencetomy provided the surgeon with useful information, diagnosing 11.4% of occult para-aortic lymph node metastases in women with locally advanced cervical cancer. Intra-operative and postoperative morbidity were low. The presence of para-aortic lymph node metastases correlated with shorter disease-free survival.

Robotically assisted laparoscopic microsurgical tubal reanastomosis: a retrospective study.

OBJECTIVE: To evaluate the pregnancy and delivery outcome of robot-assisted tubal reanastomosis. DESIGN: Retrospective cohort study. SETTING: University hospital. PATIENT(S): Ninety-seven patients with available follow-up who underwent the reversal of tubal ligation, with a median age of 37 years (range, 24-47 years). INTERVENTION(S): Tubal reanastomosis by robot-assisted laparoscopy. MAIN OUTCOME MEASURE(S): Analysis of the distribution of time to conception and to estimate the crude pregnancy and birth rates at 2 years. RESULT(S): The overall pregnancy and birth rates were 71%, (95% confidence interval [CI], 61%-80%) and 62% (95% CI, 52%-72%). Ninety-one percent (95% CI, 76%-98%) of patients <35 years old became pregnant, and 88% (95% CI, 72%-97%) delivered at least once. The corresponding pregnancy and delivery rates were 75% (95% CI, 57%-89%) and 66% (95% CI, 47%-81%) between 36 and 39 years old, 50% (95% CI, 25%-75%) and 43.8% (95% CI, 20%-70%) between 40 and 42 years old, 33% (95% CI, 10%-65%) and 8.3% (95% CI, <1%-38%) after the age of 43 years. CONCLUSION(S): This study reports satisfactory birth rates after tubal reanastomosis by robot-assisted laparoscopy in patients aged 40 years or less.

Robot assisted laparoscopic transperitoneal para-aortic lymphadenectomy in the management of advanced cervical carcinoma.

OBJECTIVES: Adequate staging of advanced cervical cancer is essential in order to optimally treat the patient. FIGO clinical staging, imaging techniques such as CT scan, MRI and PET sometimes underestimate the extension of tumors. The presence of para-aortic lymph node metastases in advanced cervical cancer identifies patients with poor prognosis who need to be treated aggressively. Laparoscopic para-aortic lymph node dissection is now proposed as a diagnostic tool in many guidelines. We evaluated the feasibility and safety of a robot assisted laparoscopic transperitoneal approach to para-aortic lymph node dissection. STUDY DESIGN: Eight patients with advanced cervical carcinoma who were eligible for primary pelvic radiotherapy combined with concurrent cisplatin chemotherapy or pelvic exenteration underwent a pre-treatment robot assisted transperitoneal laparoscopic para-aortic lymphadenectomy. RESULTS: We isolated from 1 to 38 para-aortic nodes per patient and had one para-aortic node positive patient who was treated with extended doses of pelvic radiotherapy. We did not encounter any major complications and post-operative morbidity was low. CONCLUSIONS: Robot assisted transperitoneal laparoscopic para-aortic lymphadenectomy is feasible and provides the surgeon with greater precision than classical laparoscopy. Larger prospective multicentric trials are needed to validate the generalised usefulness of this technique.