Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation.

In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

Barriers to research among residents in Otolaryngology - Head & Neck Surgery around the world.

OBJECTIVES: To document the challenges faced by residents in Otolaryngology - Head & Neck Surgery (OTL-HNS) around the world to successfully complete research projects. The second objective is to assess if the challenges are uniform worldwide. METHODS: A survey was sent to all OTL-HNS under 45 years old from the 2017 IFOS meeting. This survey was conducted by the YO-IFOS group (Young Otolaryngologists of the International Federation of Otolaryngological Societies). Data was collected for a period of 1 month. Demographic characteristics, information regarding research projects conducted and data concerning perceived barriers to completion of research projects were collected. RESULTS: Among the 2787 attendees, 928 responded to the survey (response rate=33.3%). Of these 928 answers, 267 responses were from residents/interns in OTL-HNS, while 635 responses were from certified otolaryngologists. The three most frequent obstacles to conducting research projects for trainees were limited dedicated time (64%), insufficient financial resources (55%) and lack of education in research (45%). There was no statistical difference in these barriers among the different countries (P>0.05). CONCLUSION: This is the first international study that provides insight on trainee's challenges to conduct research projects during residency. Despite the notion that research is essential for generating new knowledge to guide patient care, many residents fail to successfully incorporate research in their surgical curriculum. These obstacles must be addressed by Otolaryngology - Head & Neck Surgery programs in order to facilitate and support resident's research.

Challenges faced by young otolaryngologists-head & neck surgeons around the world.

OBJECTIVES: To document work-related stressors and to identify coping strategies employed by young board-certified otolaryngologists-head & neck surgeons (OTL-HNS) around the world. The second objective is to evaluate demographic and professional characteristics associated with a higher level of work-related stress. METHODS: A survey was sent to all OTL-HNS under 45 years old from the 2017 IFOS meeting. This survey was conducted by the YO-IFOS group (Young Otolaryngologists of the International Federation of Otolaryngological Societies). Data were collected for a period of 1 month. Demographic characteristics and information concerning challenges encountered by OTL-HNS during the early years of their career were collected. RESULTS: Among the 2787 attendees, 928 responded to the survey (response rate=33.3%). The three most frequent challenges faced by OTL-HNS in the early years of their career were related to administrative workload (45%), high patient quota (42%) and desire to achieve adequate work-life balance (42%). Practices used by OTL-HNS to cope with stress were physical activity (37%), recreational activities (35%) and self-organization (32%). Higher levels of stress were frequently found in participants who possessed five to ten years of experience (P=0.007) and who were employed by an academic institution (P=0.020). On the other hand, lower levels of stress were often encountered in participants who had 5 years or less of experience (P=0.002). CONCLUSION: This study provides insight on characteristics that are associated with various levels of stress. Moreover, it demonstrates the work-related stressors and the resilience techniques employed by OTL-HNS in early years of their career. Stress will always be present during the surgeon's career. Therefore, knowing how to recognize it and how to deal with it is key. More resources should be made available for OTL-HNS needing aid. Because surgeons must be in control of their stress if they want to provide high quality health care.

Global Professional Behaviours and Networking Preferences of Young Otolaryngologists - An International Survey.

AIMS: An international survey was conducted to explore the professional and regional spread of "young" otolaryngologists, their society membership and networking preferences, with relevance to global health and future initiatives. MATERIAL AND METHODS: Otolaryngologists under the age of 45 years who had attended the 2017 International Federation of Otorhinolaryngological Societies (IFOS) meeting were invited by e-mail to participate in an online survey. Basic demographic data and information regarding career geography and networking preferences was requested. RESULTS: A total of 928 responses (including 635 certified otolaryngologists and 268 trainees) were received from 2787 individuals invited to complete the survey. The overall response rate was 33.3%. Most otolaryngologists were based in an academic hospital. The spread of respondents likely reflects the European location of the meeting from which participants were identified; 61.2% of all respondents were based in Europe. International movement between career stages was evident. The principal preferred networking methods involved face-to-face contact whilst social media use was the method of choice for 13%. CONCLUSION: This survey offers a present-day snapshot and is hoped to serve as a platform for further work. Little is known on a global scale regarding the professional behaviours and networking preferences of otolaryngologists. A greater understanding will facilitate not only education and research but also enable networking and global health work.

Real time and in vivo pharmaceutical and environmental studies with SpiderMass instrument.

Remote Infrared Matrix Assisted Laser Desorption Ionization (Remote IR MALDI) system (SpiderMass) with endogenous water as matrix allows to perform real-time DMPK in vivo. In this work, SpiderMass was used to analyze the impact on metabolite production or release of invalidated pro-protein PC1/3 macrophages by Short RNA (shRNA) versus scramble shRNA with Paclitaxel. Time course in vivo experiments were then performed on the inner and outer faces of patients' forearms or comedo treated with Melascreen (Ducray) containing ascorbyl glucoside. Finally, the impact of car pollution (emitted soot) on skin was also investigated. Taken together, we demonstrate that the SpiderMass instrument opens the door to clinical, pharmaceutical and environmental domains for real-time, in vivo pharmacokinetic (Drug Metabolism and PharmacoKinetics, DMPK) analysis.

The proprotein convertase PC1/3 regulates TLR9 trafficking and the associated signaling pathways.

Endosomal TLR9 is considered as a potent anti-tumoral therapeutic target. Therefore, it is crucial to decipher the mechanisms controlling its trafficking since it determines TLR9 activation and signalling. At present, the scarcity of molecular information regarding the control of this trafficking and signalling is noticeable. We have recently demonstrated that in macrophages, proprotein convertase 1/3 (PC1/3) is a key regulator of TLR4 Myd88-dependent signalling. In the present study, we established that PC1/3 also regulates the endosomal TLR9. Under CpG-ODN challenge, we found that PC1/3 traffics rapidly to co-localize with TLR9 in CpG-ODN-containing endosomes with acidic pH. In PC1/3 knockdown macrophages, compartmentalization of TLR9 was altered and TLR9 clustered in multivesicular bodies (MVB) as demonstrated by co-localization with Rab7. This demonstrates that PC1/3 controls TLR9 trafficking. This clustering of TLR9 in MVB dampened the anti-inflammatory STAT3 signalling pathway while it promoted the pro-inflammatory NF-kB pathway. As a result, macrophages from PC1/3 KO mice and rat PC1/3-KD NR8383 macrophages secreted more pro-inflammatory cytokines such as TNF-α, IL6, IL1α and CXCL2. This is indicative of a M1 pro-inflammatory phenotype. Therefore, PC1/3 KD macrophages represent a relevant mean for cell therapy as "Trojan" macrophages.

Delivery of Alginate Scaffold Releasing Two Trophic Factors for Spinal Cord Injury Repair.

Spinal cord injury (SCI) has been implicated in neural cell loss and consequently functional motor and sensory impairment. In this study, we propose an alginate-based neurobridge enriched with/without trophic growth factors (GFs) that can be utilized as a therapeutic approach for spinal cord repair. The bioavailability of key GFs, such as Epidermal Growth factor (EGF) and basic Fibroblast Growth Factor (bFGF) released from injected alginate biomaterial to the central lesion site significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons (choline acetyltransferase positive motoneurons) and sensory fibres. In addition, we document enhanced outgrowth of corticospinal tract axons and presence of blood vessels at the central lesion. Tissue proteomics was performed at 3, 7 and 10 days after SCI in rats indicated the presence of anti-inflammatory factors in segments above the central lesion site, whereas in segments below, neurite outgrowth factors, inflammatory cytokines and chondroitin sulfate proteoglycan of the lectican protein family were overexpressed. Collectively, based on our data, we confirm that functional recovery was significantly improved in SCI groups receiving alginate scaffold with affinity-bound growth factors (ALG+GFs), compared to SCI animals without biomaterial treatment.

MALDI-MS and NanoSIMS imaging techniques to study cnidarian-dinoflagellate symbioses.

Cnidarian-dinoflagellate photosynthetic symbioses are fundamental to biologically diverse and productive coral reef ecosystems. The hallmark of this symbiotic relationship is the ability of dinoflagellate symbionts to supply their cnidarian host with a wide range of nutrients. Many aspects of this association nevertheless remain poorly characterized, including the exact identity of the transferred metabolic compounds, the mechanisms that control their exchange across the host-symbiont interface, and the precise subcellular fate of the translocated materials in cnidarian tissues. This lack of knowledge is mainly attributed to difficulties in investigating such metabolic interactions both in situ, i.e. on intact symbiotic associations, and at high spatial resolution. To address these issues, we illustrate the application of two in situ and high spatial resolution molecular and ion imaging techniques-matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and the nano-scale secondary-ion mass spectrometry (NanoSIMS) ion microprobe. These imaging techniques provide important new opportunities for the detailed investigation of many aspects of cnidarian-dinoflagellate associations, including the dynamics of cellular interactions.

Parafilm-assisted microdissection: a sampling method for mass spectrometry-based identification of differentially expressed prostate cancer protein biomarkers.

Mass spectrometry-based methods for prostate cancer biomarker discovery are hampered by their low-throughput capabilities because of extensive sample preparation. We present the parafilm-assisted microdissection technique coupled with label-free quantification and bioinformatics analysis as a means to evaluate directly protein expression changes on benign and tumor regions.

[A report of the SFGM-TC on the interactions between national transplant coordination and local coordinators in allogeneic stem cell transplantation activity]. / Interactions entre coordination nationale de greffe et coordinateurs locaux.

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the third annual series of workshops which brought together practitioners from all member centers and took place in October 2012 in Lille. The main aim of this session was to describe the relations between the national transplant coordination office of the French registry and local stem cell transplantation coordinators throughout France.

Prospective pilot study: efficacy of intravitreal dexamethasone and bevacizumab injections in the treatment of macular oedema associated with branch retinal vein occlusion.

BACKGROUND/AIMS: To compare the efficacy of intravitreal injections of dexamethasone implants (IVD) with those of bevacizumab (IVB) for the treatment of macular oedema associated with branch retinal vein occlusion. METHODS: A total of 19 patients (19 eyes) were included in this prospective pilot study. Initially, 8 eyes received three IVBs (group 1) and 11 received one IVD (group 2). All the patients underwent a 1-, 3-, 4- and 6-month follow-up visit. A repeated IVB (group 1) or IVD (group 2) was proposed at 4 months when necessary. RESULTS: The mean visual acuity was significantly better 1 month after treatment in group 2, while the mean central macular thickness was also significantly lower in group 2. However, there was no longer any difference between the two groups at 3, 4 and 6 months, neither in terms of visual acuity nor in terms of retinal thickness. More than three IVBs were needed in 3 of 10 patients in group 1 while two IVDs were required in 10 of 11 patients in group 2. CONCLUSION: There was no significant difference between the two treatment regimens at the 6-month follow-up visit. A more rapid functional and anatomical efficacy was noted with IVD during the first month; however, reinjection at 4 months seemed more frequent with IVD than with IVB treatment.

[Cornea imagery and keratitis caused by processionary caterpillar hairs]. / Apport de l'imagerie cornéenne dans la kératite à poils de chenilles processionnaires.

INTRODUCTION: With their ability to migrate into the cornea and release toxins, caterpillar hairs can induce different clinical presentations such as conjunctivitis, keratoconjunctivitis, uveitis, and less frequently vitreoretinal inflammation (hyalitis, papillitis, macular edema). OBSERVATION: We report a case that occurred in Alsace (France) in a 13-years-old boy presenting with keratitis caused by caterpillar hairs. We localized them in the cornea, for the first time, using confocal microscopy and anterior segment spectral optical coherence tomography. CONCLUSION: Confocal microscopy and spectral optical coherence tomography can be useful for diagnosis and follow-up of this disease.

Polymerase chain reaction and immunoassay--matrix assisted laser desorption mass spectrometry using tag-mass technology: new tools to break down quantification limits and multiplexes.

We present a new development of the Tag-Mass concept based on a photocleavable linker with tagged molecules for polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) quantification coupled to mass spectrometry. PCR-MS and immunosorbent assay-MS with tagged oligonucleotides, bases, and antibodies will allow the acquisition of multiplexed information from genomic, transcriptomic, and proteomic experiments. This is a novel application of Tag-Mass from tissue imaging to fluid quantification and will open doors to several clinical applications ranging from biomarker-driven gene modulation to use at the patient's bedside following treatment.

On-tissue N-terminal peptide derivatizations for enhancing protein identification in MALDI mass spectrometric imaging strategies.

Matrix-assisted laser desorption/ionization (MALDI) is a new tool that can acquire the localization of various compounds, including peptides and proteins, directly from tissue sections. Despite the important developments recently performed in the field of MALDI imaging in tissue, the precise identification of compounds still needs improvement. We have developed N-terminal chemical derivatization strategies to improve tissue identification of proteins, including de novo sequencing performance. We have first focused on sulfonation agents, such as 4-SPITC and 3-SBASE. These two derivatizations were optimized to be performed directly on tissue sections. By adding a negative charge at the N-terminus of a tryptic digest peptide, we were able to generate a complete y fragment series directly from the tissue. Of these derivatizations, 3-SBASE has shown to be more efficient, as loss of the derivative group is one of the major fragmentation pathways for 4-SPITC. 3-SBASE was optimized so that the derivatization reaction could be automatically performed using an automatic microspotting device. It was then included in an automatic process that included automated trypsin digestion and matrix deposition. Derivatizations allowed the acquisition to be easily interpretable by MS(2) spectra, leading to very precise identification as well as easy manual reading of sequences for de novo sequencing. It was observed that only arginine-terminated peptides were observed after derivatization, likely due to the high gas-phase basicity of such peptides compared to those that are lysine-terminated. We also observed a stop in the y fragmentation series for peptides presenting a miscleavage. We have now begun to study a different derivatization using N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP). This derivatization allows the orientating of a fragmentation toward a series of fragment ions, and thus it is independent of the presence of basic residues in the sequence. This derivatization can be performed at room temperature, which greatly facilitates the automation of the process. The TMPP derivatization therefore yields an advantageous new generation of derivatives suited for use in tissue.

MALDI imaging of formalin-fixed paraffin-embedded tissues: application to model animals of Parkinson disease for biomarker hunting.

A common technique for the long-term storage of tissues in hospitals and clinical laboratories is preservation in formalin-fixed paraffin-embedded (FFPE) blocks. Such tissues stored for more than five years have not been useful for proteomic studies focused on biomarker discovery. Recently, MS-based proteomic analyses of FFPE showed positive results on blocks stored for less than 2 days. However, most samples are stored for more than one year, and thus our objective was to establish a novel strategy using as a model system 6-hydroxydopamine (6-OHDA) treated rat brain tissues stored in FFPE blocks for more than 9 years. We examined MALDI tissue profiling combining the use of automatic spotting of the MALDI matrix with in situ tissue enzymatic digestion. On adjacent sections, the identification of compounds is carried out by tissue digestion followed by nanoLC/MS-MS analysis. The combination of these approaches provides MALDI direct analysis, MALDI/MS imaging, as well as the localization of a large number of proteins. This method is validated since the analyses confirmed that ubiquitin, trans-elongation factor 1, hexokinase, and the Neurofilament M are down-regulated as previously shown in human or Parkinson animal models. In contrast, peroxidoredoxin 6, F1 ATPase, and alpha-enolase are up-regulated. In addition, we uncovered three novel putative biomarkers, the trans-elongation factor 1 (eEF1) and the collapsin response mediator 1 and 2 from protein libraries. Finally, we validate the CRMP-2 protein using immunocytochemistry and MALDI imaging based on the different ions from trypsic digestion of the protein. The access to archived FFPE tissue using MALDI profiling and imaging opens a whole new area in clinical studies and biomarker discovery from hospital biopsy libraries.

Tag-mass: specific molecular imaging of transcriptome and proteome by mass spectrometry based on photocleavable tag.

MALDI tissue imaging of tissues has become a promising technique for tracking biomarkers while determining their location and structural characterization. We have now developed specific targeting probes (oligonucleotides, antibodies), named Tag-Mass. This approach is based on probes modified with a photocleavable linker coupled with a tag cleaved and detected using mass spectrometry. Tag-Mass development is the key for a rapid, sensitive, and accurate approach to correlate levels of expression of different mRNA or proteins in diseases.

Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections.

Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffin-embedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.

MALDI-MS direct tissue analysis of proteins: Improving signal sensitivity using organic treatments.

Direct tissue analysis using MALDI-MS allows the generation of profiles while maintaining the integrity of the tissue, displaying cellular localizations and avoiding tedious extraction and purification steps. However, lower spectral quality can result from direct tissue analysis due to variations in section thickness, the nature of the tissue, and the limited access to peptides/proteins due to high lipid content. To improve signal sensitivity, we have developed a tissue-washing procedure using organic solvents traditionally used for lipid extraction, i.e., CHCl3, hexane, toluene, acetone, and xylene. The increased detection for peptides/proteins (m/z 5000-30,000) is close to 40% with chloroform or xylene, and 25% with hexane, while also improving sample reproducibility for each solvent used in the present study. This strategy improved matrix cocrystallization with tissue peptides/proteins and more importantly with cytoplasmic proteins without delocalization. The extracted lipids were characterized by nanoESI-QqTOF/MS/MS using the precursor ion mode, lithium adducts, or both and were identified as phospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and lysophosphatidylinositol, confirming membrane lipid extraction from the tissues.