Membrane Protein Binding Interactions Studied in Live Cells via Diethylpyrocarbonate Covalent Labeling Mass Spectrometry.

Membrane proteins are vital in the human proteome for their cellular functions and make up a majority of drug targets in the U.S. However, characterizing their higher-order structures and interactions remains challenging. Most often membrane proteins are studied in artificial membranes, but such artificial systems do not fully account for the diversity of components present in cell membranes. In this study, we demonstrate that diethylpyrocarbonate (DEPC) covalent labeling mass spectrometry can provide binding site information for membrane proteins in living cells using membrane-bound tumor necrosis factor α (mTNFα) as a model system. Using three therapeutic monoclonal antibodies that bind TNFα, our results show that residues that are buried in the epitope upon antibody binding generally decrease in DEPC labeling extent. Additionally, serine, threonine, and tyrosine residues on the periphery of the epitope increase in labeling upon antibody binding because of a more hydrophobic microenvironment that is created. We also observe changes in labeling away from the epitope, indicating changes to the packing of the mTNFα homotrimer, compaction of the mTNFα trimer against the cell membrane, and/or previously uncharacterized allosteric changes upon antibody binding. Overall, DEPC-based covalent labeling mass spectrometry offers an effective means of characterizing structure and interactions of membrane proteins in living cells.

Physical and Functional Clinical Profile of Older Adults in Specialized Geriatric Rehabilitation Care Services in Saguenay-Québec: A Retrospective Study at La Baie Hospital.

Musculoskeletal disorders, cardiovascular and neurological diseases were the most commonly debilitating conditions and risk factors associated with pain, mobility limitations, increased risk of falls and disability. Studies barely address the profile of older adults in care within a specialized geriatric rehabilitation service (SGRS) to provide subsidies for new actions within the public healthcare to reduce falls and improve management in health investments. This study aimed to establish a clinical physical and functional profile of the patients with neuromusculoskeletal and cognitive disorders and fallers in interventions within SGRS. From a retrospective study design, 127 medical records were compiled and analyzed to determine the physical and functional profile of older adults and differences according to sex, age groups and the benefits for local physical therapy intervention. The users were between 76 and 85 years of age, with diverse clinical diagnoses and debilitating conditions and impairments. A higher proportion presented gait and balance impairments and had two or more falls in 12 months. A significant effect for advanced age was observed. Overall, real benefits were reported with intervention for functional improvement, although the absence of a control group. These results have direct implications for a better understanding of a local SGRS and provide subsidies for developing new approaches for the assessment and treatment of older adults with high a risk of falls in order to reduce costs for the public health system.

Complementary Structural Information for Antibody-Antigen Complexes from Hydrogen-Deuterium Exchange and Covalent Labeling Mass Spectrometry.

Characterizing antibody-antigen interactions is necessary for properly developing therapeutic antibodies, understanding their mechanisms of action, and patenting new drug molecules. Here, we demonstrate that hydrogen-deuterium exchange (HDX) mass spectrometry (MS) measurements together with diethylpyrocarbonate (DEPC) covalent labeling (CL) MS measurements provide higher order structural information about antibody-antigen interactions that is not available from either technique alone. Using the well-characterized model system of tumor necrosis factor α (TNFα) in complex with three different monoclonal antibodies (mAbs), we show that two techniques offer a more complete overall picture of TNFα's structural changes upon binding different mAbs, sometimes providing synergistic information about binding sites and changes in protein dynamics upon binding. Labeling decreases in CL generally occur near the TNFα epitope, whereas decreases in HDX can span the entire protein due to substantial stabilization that occurs when mAbs bind TNFα. Considering both data sets together clarifies the TNFα regions that undergo a decrease in solvent exposure due to mAb binding and that undergo a change in dynamics due to mAb binding. Moreover, the single-residue level resolution of DEPC-CL/MS can clarify HDX/MS data for long peptides. We feel that the two techniques should be used together when studying the mAb-antigen interactions because of the complementary information they provide.

Epitope Mapping with Diethylpyrocarbonate Covalent Labeling-Mass Spectrometry.

Antigen-antibody epitope mapping is essential for understanding binding mechanisms and developing new protein therapeutics. In this study, we investigate diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry as a means of analyzing antigen-antibody interactions using the well-characterized model system of TNFα in complex with three different antibodies. Results show that residues buried in the epitope undergo substantial decreases in labeling, as expected. Interestingly, serine, threonine, and tyrosine residues at the edges of the epitope undergo unexpected increases in labeling. The increased labeling of these weakly nucleophilic residues is caused by the formation of hydrophobic pockets upon antibody binding that presumably increase local DEPC concentrations. Residues that are distant from the epitope generally do not undergo changes in labeling extent; however, some that do change experience variations in their local microenvironment due to side-chain reorganization or stabilization of the TNFα trimer that occurs upon binding. Overall, DEPC labeling of antigen-antibody complexes is found to depend on both changes in solvent exposure and changes to the residue microenvironment.

Evaluation of the Morbidity of Routine Cystoscopy Performed Intraoperatively During Total Laparoscopic Hysterectomies.

STUDY OBJECTIVES: The primary objective is to determine the rate of morbid events (urinary tract infection, hematuria, urinary retention, false positive, incidental finding) associated with routine cystoscopies performed intraoperatively during total laparoscopic hysterectomies (TLH). The secondary objectives are 1) to determine the rate of urinary complications during TLHs in our centers and 2) to determine the detection rate of urinary complications using cystoscopy during TLHs. METHOD: Descriptive retrospective multicenter study. The study took place in Obstetrics & Gynecology departments of 2 university centers in Montreal. Patients underwent a routine cystoscopy during their TLH for a benign reason in our centers. Five hundred thirty-one charts from January 1, 2012 to January 31, 2018 were reviewed. RESULTS: The morbidity rate of routine cystoscopies during TLHs is 4.19% (22/524 cases) in our centers. Our urinary complication rate is 2.45% (13/531 cases). Of these 13 complications, 4 were detected by cystoscopy. CONCLUSION: The usefulness of routine cystoscopies performed intraoperatively during TLHs is questionable due to the number of morbid events and the low rate of urinary trauma in our centers. However, it is hard to establish a direct causality link between certain morbid events and cystoscopy. More studies should be conducted on this subject.

Complementary Structural Information for Stressed Antibodies from Hydrogen-Deuterium Exchange and Covalent Labeling Mass Spectrometry.

Identifying changes in the higher-order structure (HOS) of therapeutic monoclonal antibodies upon storage, stress, or mishandling is important for ensuring efficacy and avoiding adverse effects. Here, we demonstrate diethylpyrocarbonate (DEPC)-based covalent labeling (CL) mass spectrometry (MS) and hydrogen-deuterium exchange (HDX)/MS can be used together to provide site-specific information about subtle conformational changes that are undetectable by traditional techniques. Using heat-stressed rituximab as a model protein, we demonstrate that CL/MS is more sensitive than HDX/MS to subtle HOS structural changes under low stress conditions (e.g., 45 and 55 °C for 4 h). At higher heat stress (65 °C for 4 h), we find CL/MS and HDX/MS provide complementary information, as CL/MS reports on changes in side chain orientation while HDX/MS reveals changes in backbone dynamics. More interestingly, we demonstrate that the two techniques work synergistically to identify likely aggregation sites in the heat-stressed protein. In particular, the CH3 and CL domains experience decreases in deuterium uptake after heat stress, while only the CH3 domain experiences decreases in DEPC labeling extent as well, suggesting the CH3 domain is a likely site of aggregation and the CL domain only undergoes a decrease in backbone dynamics. The combination of DEPC-CL/MS and HDX/MS provides valuable structural information, and the two techniques should be employed together when investigating the HOS of protein therapeutics.

Establishing a peripartum perineal trauma clinic: a narrative review.

INTRODUCTION AND HYPOTHESIS: Obstetric anal sphincter injury (OASI) is not rare, and its consequences are multiple and potentially severe, especially for young women. Some dedicated perineal clinics have been established to improve the management of OASI. Despite their obvious importance, these specific clinics are underrepresented and underdeveloped. The objectives of this review are to explore various options for developing a peripartum perineal clinic and to compare the different practices regarding the mode of delivery for subsequent pregnancies after an OASI. METHODS: This narrative review covers information from patients' questionnaires specific to anal incontinence, anal physiology assessment, pelvic floor and anal sphincter imaging, and the arguments for choosing the mode of delivery after an OASI. RESULTS: This review highlights the extensive range of practices regarding the delivery mode after an OASI throughout national professional organizations and experienced perineal clinics. CONCLUSION: This review summarizes the different choices in developing a perineal clinic to facilitate their development in promoting health care and education specific for peripartum women concerning the perineal consequences of delivery for obstetrician-gynaecologists, family doctors, and residents.

Short- and Long-Term Complications of Intraoperative Benign Ovarian Cyst Spillage: A Systematic Review and Meta-analysis.

OBJECTIVE: To review short- and long-term complications associated with intraoperative rupture of benign ovarian cysts. DATA SOURCES: The Cochrane Central Register of Controlled Trials, BIOSIS, Medline (Ovid), Web of Science, ClinicalTrials.gov, and Google Scholar were searched using the following terms and their combinations: "spillage," "rupture," "leakage," "ovarian cyst," "teratoma," "dermoid," "operative," "surgery," "outcome." METHODS OF STUDY SELECTION: Randomized controlled and observational studies evaluating the operative outcomes of surgical treatment of ovarian cysts with intraoperative spillage compared with those of surgical treatment of ovarian cysts without spillage were included. A systematic review and meta-analysis following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines was performed. TABULATION, INTEGRATION, AND RESULTS: A total of 28 studies were included in the qualitative analysis and 12 in the quantitative analysis. Ovarian cyst diameter was not found to be associated with the risk for spillage (relative risk [RR] 0.75; 95% confidence interval [CI], -0.33 to 1.82). Intraoperative benign ovarian cyst rupture was not associated with adverse short- and long-term outcomes such as reoperation (RR 1.16; 95% CI, 0.39-3.48), infertility (RR 0.73; 95% CI, 0.15-3.63), transient fever (RR 3.22; 95% CI, 0.83-12.51), and readmission (RR 1.00; 95% CI, 0.33-2.98). However, intraoperative spillage was found to be associated with increased risk for benign recurrence (RR 3.1; 95% CI, 1.05-9.14). A subgroup analysis of the studies that included only dermoid cysts showed an association between intraoperative cyst rupture and postoperative chemical peritonitis (RR 9.36; 95% CI, 1.20-73.28). CONCLUSION: Intraoperative ovarian cyst spillage of a benign cyst is associated with limited adverse clinical outcomes. Although the surgical approach (minimally invasive vs open) should not be affected by the concern regarding an intraoperative cyst rupture, maximal efforts should be made to prevent intra-abdominal spillage.

The Cleavage Profile of Protein Substrates by ClpXP Reveals Deliberate Starts and Pauses.

Cells rely on protein degradation by AAA+ proteases. A well-known example is the hexameric ClpX unfoldase, which captures ATP hydrolysis to feed substrates into the oligomeric ClpP peptidase. Recent studies show that an asymmetric ClpX spiral cycles protein translocation upon ATP hydrolysis. However, how this cycle affects peptide products is less explored in part because ClpP cleavage is thought to be solely defined by sequence constraints. Here, we comprehensively characterize peptides from Caulobacter crescentus ClpXP degradation of three different substrates using high-resolution mass spectrometry and find that cleavage of translocated substrates is driven by factors other than sequence. We report that defined locations in a translocated protein are especially sensitive to cleavage spaced on average every 10-13 residues. These sites are not exclusively controlled by sequence and are independent of bulk changes in catalytic peptidase sites, ATP hydrolysis, or the efficiency of initial recognition. These results fit a model in which processive translocation through ClpX starts at a specific location in a polypeptide and pauses during reset of the ClpX hexamer after a cycle of translocation. Our work suggests that defined peptides, which could be used as signaling molecules, can be generated from a given substrate by a nonspecific peptidase.

Endometrial Biopsy in an Outpatient Gynaecological Setting: Overinvestigation.

OBJECTIVE: We reviewed the indications for endometrial biopsy at the general gynaecology outpatient clinic of the Université de Montréal Hospital Center and measured their compliance with the Society of Obstetricians and Gynaecologists of Canada and other international guidelines. METHODS: Three hundred and seventy-one files of patients who had an endometrial biopsy between January and October 2015 were reviewed. Indication for endometrial biopsy and pathology results were noted. Files were separated into four categories. RESULTS: In the postmenopausal bleeding category, all files complied with the SOGC. We found hyperplasia or neoplasia in 13% of patients. In the asymptomatic endometrial thickening category, 9% of the files did not show sufficient indication for biopsy. None of the patients presented hyperplasia or neoplasia. In the abnormal uterine bleeding (AUB) - under 41 years old category, there was no indication for biopsy in 23% of the files. We found hyperplasia or neoplasia in 13% of patients, but only in patients with an indication for biopsy. In patients with AUB - over 40, non-compliance with SOGC was 3%. But according to international guidelines, 42% of patients with AUB between 41 and 45 years old did not have an indication for biopsy and none showed hyperplasia or neoplasia. CONCLUSION: We demonstrated clinically significant overinvestigation in patients with AUB. Indications should be reviewed carefully before performing an endometrial biopsy in women under 41. In addition, the value of endometrial biopsies in patients between 41 and 45 years old with menorrhagia and no additional risk factor should be reevaluated.

Dynamics of Endothelial Cell Responses to Laminar Shear Stress on Surfaces Functionalized with Fibronectin-Derived Peptides.

Surface endothelialization could improve the long-term performance of vascular grafts and stents. We previously demonstrated that aerosol-generated fibronectin-derived peptide micropatterns consisting of GRGDS spots over a WQPPRARI background increase endothelial cell yields in static cultures. We developed a novel fluorophore-tagged RGD peptide (RGD-TAMRA) to visualize cell-surface interactions in real-time. Here, we studied the dynamics of endothelial cell response to laminar flow on these peptide-functionalized surfaces. Endothelial cells were exposed to 22 dyn/cm2 wall shear stress while acquiring time-lapse images. Cell surface coverage and cell alignment were quantified by undecimated wavelet transform multivariate image analysis. Similar to gelatin-coated surfaces, surfaces with uniform RGD-TAMRA distribution led to cell retention and rapid cell alignment (∼63% of the final cell alignment was reached within 1.5 h), contrary to the micropatterned surfaces. The RGD-TAMRA peptide is a promising candidate for endothelial cell retention under flow, and the spray-based micropatterned surfaces are more promising for static cultures.

Necrolytic Migratory Erythema: A Forgotten Paraneoplastic Condition.

BACKGROUND: Necrolytic migratory erythema (NME) is most commonly a paraneoplastic condition. It is the dermatologic manifestation classically associated with glucagonoma pancreatic neuroendocrine tumour. Glucagonoma syndrome has been defined by the constellation of secreting tumour associated with overproduction by the α-cells in the pancreatic islets of Langerhans, abnormally elevated blood level of glucagon, and skin findings of NME. OBJECTIVE: Although rare, all dermatologists must know and recognise NME promptly to request useful investigations for the diagnosis of this characteristic neuroendocrine tumour. METHODS AND RESULTS: We report a case of a middle-aged woman seen in our dermatology clinic with longstanding skin findings suggestive of NME revealing a glucagonoma. Surgical removal was associated with complete resolution of the cutaneous and systemic features. CONCLUSION: NME is often the first clinical finding of an occult neuroendocrine pancreatic neoplasia. Dermatologists must be aware of this condition since they can be the first physician to suspect it and allow multidisciplinary management, which influences the prognostic value. Surgical removal is the first-line therapy if early diagnosis is done before liver metastases occur.

Kaposi Sarcoma Associated With Iatrogenic Immunosuppression: A Rare Complication of Bullous Pemphigoid Treatment.

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune bullous disease requiring immunosuppressive therapy. Kaposi sarcoma (KS) is an angioproliferative tumor associated with the opportunistic viral infection human herpes virus 8 (HHV-8). It is a well-known condition associated with longstanding human immunodeficiency virus infection, but it may also occur in the context of iatrogenic immunosuppression. OBJECTIVE: Although a rare complication, all dermatologists dealing with immunosuppressors must be aware and have a high index of suspicion when a patient presents with rapidly progressive violaceous papules. METHODS AND RESULTS: We describe an Italian male patient treated for bullous pemphigoid with topical and systemic corticosteroids and mycophenolate mofetil (MMF) who developed multifocal cutaneous KS after a few months of therapy. CONCLUSION: To our knowledge, KS onset associated with MMF and corticosteroids intake for BP treatment has not been reported previously. KS associated with iatrogenic immunosuppression can have an aggressive course, and it must be promptly recognised since cessation of immunosuppression therapy can lead to complete resolution. Immune restoration is the key to control this viral infection.

Plesiomonas shigelloides: An Unusual Cause of Septic Abortion.

Plesiomonas shigelloides, the only oxidase-positive Enterobacteriaceae, is an inhabitant of freshwater and estuary ecosystems. We report the first possible case of Plesiomonas shigelloides-induced septic abortion. This 24-year-old female was successfully treated by dilatation and curettage as well as antimicrobial therapy.

Microstructured human fibroblast-derived extracellular matrix scaffold for vascular media fabrication.

In the clinical and pharmacological fields, there is a need for the production of tissue-engineered small-diameter blood vessels. We have demonstrated previously that the extracellular matrix (ECM) produced by fibroblasts can be used as a scaffold to support three-dimensional (3D) growth of another cell type. Thus, a resistant tissue-engineered vascular media can be produced when such scaffolds are used to culture smooth muscle cells (SMCs). The present study was designed to develop an anisotropic fibroblastic ECM sheet that could replicate the physiological architecture of blood vessels after being assembled into a small diameter vascular conduit. Anisotropic ECM scaffolds were produced using human dermal fibroblasts, grown on a microfabricated substrate with a specific topography, which led to cell alignment and unidirectional ECM assembly. Following their devitalization, the scaffolds were seeded with SMCs. These cells elongated and migrated in a single direction, following a specific angle relative to the direction of the aligned fibroblastic ECM. Their resultant ECM stained for collagen I and III and elastin, and the cells expressed SMC differentiation markers. Seven days after SMCs seeding, the sheets were rolled around a mandrel to form a tissue-engineered vascular media. The resulting anisotropic ECM and cell alignment induced an increase in the mechanical strength and vascular reactivity in the circumferential direction as compared to unaligned constructs. Copyright © 2016 John Wiley & Sons, Ltd.

Tissue-Engineered Tubular Heart Valves Combining a Novel Precontraction Phase with the Self-Assembly Method.

Recently, the tubular shape has been suggested as an effective geometry for tissue-engineered heart valves, allowing easy fabrication, fast implantation, and a minimal crimped footprint from a transcatheter delivery perspective. This simple design is well suited for the self-assembly method, with which the only support for the cells is the extracellular matrix they produce, allowing the tissue to be completely free from exogenous materials during its entire fabrication process. Tubular constructs were produced by rolling self-assembled human fibroblast sheets on plastic mandrels. After maturation, the tubes were transferred onto smaller diameter mandrels and allowed to contract freely. This precontraction phase thickened the tissue and prevented further contraction, while improving fusion between the self-assembled layers and aligning the cells circumferentially. When mounted in a pulsed-flow bioreactor, the valves showed good functionality with large leaflets coaptation and opening area. Although physiological aortic flow conditions were not reached, the leaflets could withstand a 1 Hz pulsed flow with a 300 mL/s peak flow rate and a 70 mmHg peak transvalvular pressure. This study shows that the self-assembly method, which has already proven its potential for the production of small diameter vascular grafts, could also be used to achieve functional tubular heart valves.

In Vivo Remodeling of Fibroblast-Derived Vascular Scaffolds Implanted for 6 Months in Rats.

There is a clinical need for tissue-engineered small-diameter (<6 mm) vascular grafts since clinical applications are halted by the limited suitability of autologous or synthetic grafts. This study uses the self-assembly approach to produce a fibroblast-derived decellularized vascular scaffold (FDVS) that can be available off-the-shelf. Briefly, extracellular matrix scaffolds were produced using human dermal fibroblasts sheets rolled around a mandrel, maintained in culture to allow for the formation of cohesive and three-dimensional tubular constructs, and decellularized by immersion in deionized water. The FDVSs were implanted as an aortic interpositional graft in six Sprague-Dawley rats for 6 months. Five out of the six implants were still patent 6 months after the surgery. Histological analysis showed the infiltration of cells on both abluminal and luminal sides, and immunofluorescence analysis suggested the formation of neomedia comprised of smooth muscle cells and lined underneath with an endothelium. Furthermore, to verify the feasibility of producing tissue-engineered blood vessels of clinically relevant length and diameter, scaffolds with a 4.6 mm inner diameter and 17 cm in length were fabricated with success and stored for an extended period of time, while maintaining suitable properties following the storage period. This novel demonstration of the potential of the FDVS could accelerate the clinical availability of tissue-engineered blood vessels and warrants further preclinical studies.

Mechanical properties of endothelialized fibroblast-derived vascular scaffolds stimulated in a bioreactor.

There is an ongoing clinical need for tissue-engineered small-diameter (<6mm) vascular grafts since clinical applications are restricted by the limited availability of autologous living grafts or the lack of suitability of synthetic grafts. The present study uses our self-assembly approach to produce a fibroblast-derived decellularized vascular scaffold that can then be available off-the-shelf. Briefly, scaffolds were produced using human dermal fibroblasts sheets rolled around a mandrel, maintained in culture to allow for the formation of cohesive and three-dimensional tubular constructs, and then decellularized by immersion in deionized water. Constructs were then endothelialized and perfused for 1week in an appropriate bioreactor. Mechanical testing results showed that the decellularization process did not influence the resistance of the tissue and an increase in ultimate tensile strength was observed following the perfusion of the construct in the bioreactor. These fibroblast-derived vascular scaffolds could be stored and later used to deliver readily implantable grafts within 4weeks including an autologous endothelial cell isolation and seeding process. This technology could greatly accelerate the clinical availability of tissue-engineered blood vessels.

Comparison of the direct burst pressure and the ring tensile test methods for mechanical characterization of tissue-engineered vascular substitutes.

Tissue engineering provides a promising alternative for small diameter vascular grafts, especially with the self-assembly method. It is crucial that these grafts possess mechanical properties that allow them to withstand physiological flow and pressure without being damaged. Therefore, an accurate assessment of their mechanical properties, especially the burst pressure, is essential prior to clinical release. In this study, the burst pressure of self-assembled tissue-engineered vascular substitutes was first measured by the direct method, which consists in pressurizing the construct with fluid until tissue failure. It was then compared to the burst pressure estimated by Laplace׳s law using data from a ring tensile test. The major advantage of this last method is that it requires a significantly smaller tissue sample. However, it has been reported as overestimating the burst pressure compared to a direct measurement. In the present report, it was found that an accurate estimation of the burst pressure may be obtained from a ring tensile test when failure internal diameter is used as the diameter parameter in Laplace׳s law. Overestimation occurs with the method previously reported, i.e. when the unloaded internal diameter is used for calculations. The estimation of other mechanical properties was also investigated. It was demonstrated that data from a ring tensile test provide an accurate estimate of the failure strain and the stiffness of the constructs when compared to measurements with the direct method.