Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics.

Phosphotyrosine (pY) enrichment is critical for expanding the fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY can handle up to 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic, and tyrosine-specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.

Coisolation of Peptide Pairs for Peptide Identification and MS/MS-Based Quantification.

Stable-isotope labeling with amino acids in cell culture (SILAC)-based metabolic labeling is a widely adopted proteomics approach that enables quantitative comparisons among a variety of experimental conditions. Despite its quantitative capacity, SILAC experiments analyzed with data-dependent acquisition (DDA) do not fully leverage peptide pair information for identification and suffer from undersampling compared to label-free proteomic experiments. Herein, we developed a DDA strategy that coisolates and fragments SILAC peptide pairs and uses y-ions for their relative quantification. To facilitate the analysis of this type of data, we adapted the Comet sequence database search engine to make use of SILAC peptide paired fragments and developed a tool to annotate and quantify MS/MS spectra of coisolated SILAC pairs. This peptide pair coisolation approach generally improved expectation scores compared to the traditional DDA approach. Fragment ion quantification performed similarly well to precursor quantification in the MS1 and achieved more quantifications. Lastly, our method enables reliable MS/MS quantification of SILAC proteome mixtures with overlapping isotopic distributions. This study shows the feasibility of the coisolation approach. Coupling this approach with intelligent acquisition strategies has the potential to improve SILAC peptide sampling and quantification.

IsobaricQuant enables cross-platform quantification, visualization, and filtering of isobarically-labeled peptides.

In mass spectrometry (MS)-based quantitative proteomics, labeling with isobaric mass tags such as iTRAQ and TMT can substantially improve sample throughput and reduce peptide missing values. Nonetheless, the quantification of labeled peptides tends to suffer from reduced accuracy due to the co-isolation of co-eluting precursors of similar mass-to-charge. Acquisition approaches such as multistage MS3 or ion mobility separation address this problem, yet are difficult to audit and limited to expensive instrumentation. Here we introduce IsobaricQuant, an open-source software tool for quantification, visualization, and filtering of peptides labeled with isobaric mass tags, with specific focus on precursor interference. IsobaricQuant is compatible with MS2 and MS3 acquisition strategies, has a viewer that allows assessing interference, and provides several scores to aid the filtering of scans with compression. We demonstrate that IsobaricQuant quantifications are accurate by comparing it with commonly used software. We further show that its QC scores can successfully filter out scans with reduced quantitative accuracy at MS2 and MS3 levels, removing inaccurate peptide quantifications and decreasing protein CVs. Finally, we apply IsobaricQuant to a PISA dataset and show that QC scores improve the sensitivity of the identification of protein targets of a kinase inhibitor. IsobaricQuant is available at https://github.com/Villen-Lab/isobaricquant.

Clinical features associated with the invasive component in lentigo maligna of the head and neck: A retrospective study of 175 cases.

BACKGROUND: Lentigo maligna (LM) can develop into lentigo maligna melanoma (LMM) with risk of metastatic dissemination. LMM may be underestimated on the basis of the initial biopsy. The invasion may affect both the therapeutic options and the prognosis. OBJECTIVES: To identify the clinical features associated with invasive forms of LM and factors associated with its recurrence. METHODS: A retrospective, single-centre study of consecutive LM and LMM histologically confirmed and treated by surgery between 2009 and 2014. RESULTS: In total, 175 patients with LM/LMM were surgically treated in our establishment. In men, lesions were more likely to be in the "peripheral zone" (41.8%), while in women they were seen more often in the "central zone" (P=0.001). In multivariate analysis, only the peripheral zone was found to be associated with a risk of invasion (P=0.008). The rate of recurrence was 9% and lesions were more likely to be primary LMM (P=0.0006) excised with clear margins. CONCLUSION: The treatment of choice in LM with non-clear margins must be re-excision, especially for lesions situated in the peripheral zone. Close follow-up is recommended due to risk of recurrence, even in the case of clear margins.

DIAmeter: matching peptides to data-independent acquisition mass spectrometry data.

MOTIVATION: Tandem mass spectrometry data acquired using data independent acquisition (DIA) is challenging to interpret because the data exhibits complex structure along both the mass-to-charge (m/z) and time axes. The most common approach to analyzing this type of data makes use of a library of previously observed DIA data patterns (a 'spectral library'), but this approach is expensive because the libraries do not typically generalize well across laboratories. RESULTS: Here, we propose DIAmeter, a search engine that detects peptides in DIA data using only a peptide sequence database. Although some existing library-free DIA analysis methods (i) support data generated using both wide and narrow isolation windows, (ii) detect peptides containing post-translational modifications, (iii) analyze data from a variety of instrument platforms and (iv) are capable of detecting peptides even in the absence of detectable signal in the survey (MS1) scan, DIAmeter is the only method that offers all four capabilities in a single tool. AVAILABILITY AND IMPLEMENTATION: The open source, Apache licensed source code is available as part of the Crux mass spectrometry analysis toolkit (http://crux.ms). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

[Dermatofibrosarcoma protuberans: Surgical margins using Slow-Mohs micrographic surgery. A clinical retrospective study about 20 cases]. / Marges d'exérèse des dermatofibrosarcomes cervico-faciaux par technique de Slow-Mohs : étude clinique rétrospective sur 20 cas.

OBJECTIVES: The main objective of this study is to determine the necessary surgical margins to obtain a complete R0 resection for head and neck dermatofibrosarcoma protuberans (DFSP) using Slow-Mohs micrographic surgery. The secondary objective is to study the recurrence rate of these tumors. PATIENTS AND METHODS: Slow-Mohs micrographic surgery was used for patients included between 2005 and 2015 at Bordeaux universitary hospital. For each patient the age, the sex and death occurrence, the initial surgical margins, the surgical margins for complete R0 resection, the occurrence of local or general recurrence during follow-up were reported. Surgery was realized under local anesthesia. The closure of the tumor site was realized secondarily using a skin graft or local flap. RESULTS: Twenty patients were included in the study. Initial surgical margins were 10mm (9 patients) or 15mm (11 patients). Complete resection was obtained from the first surgery for fifteen patients (75%). The average surgical margin for a complete R0 resection was 15,25±5,7mm (10-25). None of the patients presented recurrences during the entire follow-up (38 months) CONCLUSION: A complete R0 resection of head and neck DFSP is obtained from the first surgery in 75% of the cases, with minimum surgical margins (12,75±2,55mm) using the Slow-Mohs micrographic surgery. This allows a reduction of surgical margins and local recurrences. This technique provides a preservation of soft-tissues, which plays a key role for head and neck surgery.

Human papillomavirus does not play a role in the Barrett esophagus: a French cohort.

The role of human papillomavirus (HPV) in Barrett's esophagus (BE) has been examined but remains unclear. The purpose of the study is to dispute the connection between HPV and BE in a prospective case-control study. Biopsies were performed above and inside the Barrett's segment for BE patients and in the distal third of the esophagus for control patients for histological interpretation and for virological analysis. Biopsies for virological analysis were placed in a virus transport medium and immediately frozen in liquid nitrogen. Virological analysis involved real-time PCR using the SyBr® green protocol with modified SPF10 general primers. A total of 180 patients (119 control and 61 BE, respectively) were included. In BE patients, 31, 18, and 12 patients had, respectively, no dysplasia, low-grade dysplasia, and high grade dysplasia. Overall, nine were found to be HPV positive: five were control patients and four BE patients. HPV positive status was not associated with BE. No factors were associated with HPV, in particular the degree of BE dysplasia. HPV infection appears unlikely to be significant in the etiology of BE compared with control patients. (ClinicalTrials.gov, Number NCT02549053).

Diagnostic value of ultrasonography in elbow trauma in children: Prospective study of 34 cases.

INTRODUCTION: Among the various elbow injuries in children that initially have normal radiographs, a certain number of occult fractures are only diagnosed correctly after the fact, during a follow-up visit. PURPOSE: This study evaluated the diagnostic contribution of ultrasonography in the treatment of acute elbow injuries in children and the strategic and economic impact of using this tool alongside radiography. MATERIALS AND METHODS: During this prospective study performed between January 1 and April 1 2014, elbow ultrasonography was performed within 6 days in all children under 15 years of age with a suspected occult fracture. The ultrasonography exam looked for lipohemarthrosis, the posterior fat pad sign and cortical disruption. If no fracture was visible on ultrasonography, a removable splint was given to the patient to relieve pain, and no radiological or clinical follow-up was scheduled. The patients were contacted again at least 15 days later to determine whether an undetected fracture was present. Lastly, we evaluated the cost of treatment with and without ultrasonography in the cases where no fracture was diagnosed. RESULTS: In 13 cases, ultrasonography revealed lipohemarthrosis and a fat pat sign, with cortical disruption also present in 11 of these cases. In two cases, the diagnosis was made based solely on the presence of lipohemarthrosis and a fat pat sign. There were seven lateral condyle fractures, two medial epicondyle fractures and two supracondylar fractures. Among the 21 patients with normal ultrasonography, no fracture was diagnosed later on. In patients without a fracture, using ultrasonography resulted in a cost savings of €29.10 per patient versus not using it. CONCLUSION: In our study, ultrasonography is a sensitive examination for the diagnosis of occult elbow fractures in children. When the radiography and ultrasonography are both normal, the possibility of fracture can be rule out definitively, which reduces the need for immobilization, follow-up and treatment costs. The findings of this preliminary study should be validated with a larger prospective study.

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1.

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T37/T46, followed by T70 and S65. Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, (54)YXXXXLΦ(60) (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m(7)GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP150-84). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S65 and T70). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1-mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.

Sentinel lymph node biopsy for head and neck Merkel cell carcinoma: a preliminary study.

INTRODUCTION: A retrospective preliminary study assessed the feasibility of sentinel lymph node biopsy (SLNB) in the management of Merkel cell carcinoma (MCC) of the head and neck. PATIENTS AND METHODS: Twelve patients with stage I or II head and neck MCC underwent SLNB over a 4-year period. RESULTS: Only 1 of the 12 patients had a positive SLNB. The sentinel node was not identified in 3 patients. Two of the 8 patients with negative SLNB showed regional lymph node recurrence within 2years. One patient died during follow-up. CONCLUSION: Merkel cell carcinoma is an uncommon but highly aggressive pathology; management protocols have been based on small series. The role of SLNB in the management of MCC remains to be defined.

[When should an orthoptic evaluation be prescribed in the management of orbital floor fracture? A prospective study of 47 fractures]. / Quand demander un examen orthoptique dans la prise en charge des fractures du plancher de l'orbite ?

INTRODUCTION: We evaluated the indication of orthoptic evaluation for the management of orbital floor fractures in a prospective series. MATERIAL AND METHOD: Forty-seven patients presenting with an orbital floor fracture were included in our prospective study. Consultations in orthoptics and maxillo-facial surgery were regularly carried out. Diplopia and motility were systematically assessed as well as a coordimetric examination according to Hess-Lees's technique. RESULTS: Nineteen percent of coordimetric motility disorders were observed among asymptomatic patients, after trauma. No diplopia or clinical motility disorder were observed 1 and 2 months after trauma, and coordimetric examinations came back to normal 2 and 3 months after trauma respectively for non-operated (26) and operated (21) patients. CONCLUSIONS: An orthoptic evaluation is necessary for the management of orbital floor fractures to diagnose the type of diplopia, motility disorders, and to indicate a coordimetric examination if diplopia is present. We suggest this orthoptic evaluation for patients presenting with diplopia between 5 and 10 days following trauma, 1 month after trauma for non-surgical treatment and 2 months after trauma for surgical treatment.

Genetic parameters of juvenile osteochondral conditions (JOCC) in French Trotters.

Juvenile osteochondral conditions (JOCC) have been defined as lesions resulting from biomechanical influences (compressive, tensional or shear forces) on the developing and growing musculoskeletal system. They include different types of osteochondrosis, osteochondral fragmentation of the articular surface or of the periarticular margins, juvenile subchondral bone cysts, osteochondral collapse, avulsion fractures of epiphyseal (or metaphyseal) ossifying bone and 'physitis'. The aim of this study was to estimate heritability of JOCC in a sample of 2106 French Trotters from four different sources, comprising representative samples of the Trotter population, as well as material from auctions. Horses were aged 6-24months and were either not yet in training or just beginning training. Radiographs were taken of fore and hind feet, including proximal interphalangeal (pastern) joints, metacarpophalangeal and metatarsophalangeal (fetlock) joints, tarsocrural (hock) joints, carpi and femoropatellar (stifle) joints. The threshold model used included sex, age, region and month of birth, sampling group and sire (n=159) with all inter-sire relationships. The main results were a moderate heritability for findings in the hind fetlock (0.29) and the hock (0.19). There was a weak genetic correlation between findings in fetlocks and hocks (0.26). Higher heritability was found for findings in the hock (0.37 for findings in the distal row and 0.49 for the proximal row of tarsal bones) in that part of the data (699 horses) in which it was possible to integrate the grade, bilateral occurrence or not, and distal or proximal location of the lesions. It is possible to use these genetic parameters in breeding selection with more efficiency when detailed phenotypes are considered.

[Tongue reconstruction with a bilateral infrahyoid flap innervated by Ansa Cervicalis after total glossectomy]. / Le lambeau infrahyoïdien bilatéral innervé par l'Ansa Cervicalis pour la reconstruction des glossectomies totales.

INTRODUCTION: Reconstruction after total glossectomy remains a functional challenge. It must provide a large volume to ensure adequate phonation and swallowing. We present the larynx sparing bilateral infrahyoid flap reconstruction procedure after total glossectomy. PATIENTS AND METHOD: Three patients managed for an epidermoid carcinoma of the tongue, classified T4N0, underwent total glossectomy. The tongue was reconstructed with a bilateral infrahyoid flap pedicled on two superior thyroid arteries and innervated by Ansa Cervicalis. RESULTS: Oral food intake was resumed after 8 to 20 days. No false route was observed. The muscular flap mobility was clinically satisfactory. It was assessed by EMG in one case. Esophageal transit confirmed the absence of stasis and false route for one patient. DISCUSSION: This short series proves the feasibility of bilateral innervated and pedicled infrahyoid flap procedure. It is an alternative to volumetric and functional reconstruction after total glossectomy. The indications are rare and restricted to patients without IIa nodal region invasion. Our results are still limited and need to be confirmed by a larger series and by a more systematic assessment.

A multirecurrent ameloblastoma metastatic to the lung.

INTRODUCTION: The ameloblastoma is a rare tumor of odontogenic epithelial origin. It is a neoplasm in which ameloblastic features are revealed by the primary growth in jaws and by any metastatic growth. Recurrences are usually local and distant metastases are rare. We present a case of a multirecurrent ameloblastoma of the mandible metastatic to the lung. OBSERVATION: We present a case of a mandibular malignant ameloblastoma in a 42-year old man with widespread pulmonary metastases. Some of these lesions were treated surgically. DISCUSSION: Ameloblastoma metastasis often occurs in the lung. The curative treatment is surgical. The results of palliative chemotherapy and radiotherapy are not always efficient.

[276 cases of horizontal infrahyoid myocutaneous flap]. / Le lambeau musculocutané infrahyoïdien à palette cutanée horizontale : à propos de 276 cas.

INTRODUCTION: The infrahyoid myocutaneous flap was described by Wang et al. in 1986. The horizontal design of the skin paddle is a modification of this technique allowing for a smaller scar. We have been systematically using this modified technique for 10 years. We had for aim to describe the interest of the horizontal infrahyoid myocutaneous flap for cervicofacial carcinology. PATIENTS AND METHODS: A horizontal infrahyoid myocutaneous flap procedure was performed in 276 cervicofacial carcinology patients for lesions of the mouth floor, the mandibular gum, the oropharynx and the tongue between March 1997 and March 2007. RESULTS: No complications were observed in 252 patients. No patient presented with total flap necrosis. DISCUSSION: Modifying the infrahyoid myocutaneous flap technique with a horizontal design of the skin paddle does not modify the reliability of the flap and prevents more extensive scars. The main indications of this technique are defects of the mouth floor, the tongue and the oropharynx.

[Prognostic value of infiltration of squamous cell carcinoma of the floor of the mouth]. / Valeur pronostique de l'infiltration des carcinomes épidermoïdes du plancher buccal.

OBJECTIVE: The aim of this preliminary retrospective study was to evaluate the prognostic value of tumor thickness in squamous cell carcinoma of the floor of the mouth. MATERIALS AND METHODS: A total of 61 patients with previously untreated T1N0, T2N0 or T3N0 squamous cell carcinoma of the floor of the mouth were included in this preliminary study. An optical micrometer was used to determine the maximum of tumor thickness. We studied the relation between the overall survival at two and five years and tumor thickness. RESULTS: The cohort comprised 52 male and nine female patients. The mean and the median thickness were 7.2 and 6mm, respectively. Overall survival was 79.5 and 36.7% at two and five years, respectively. There was no significant association between thickness and overall survival (p=0.71) and between thickness and disease-free survival (p=0.63). CONCLUSION: The prognostic value of tumor thickness was not demonstrated in this preliminary study. We are currently conducting a study involving a larger patient population in our unit and we believe that tumor thickness should be considered in the management plan for patients with oral carcinoma.

Design and synthesis of FAJANU: a de novo C(2) symmetric cyclopeptide family.

A novel cyclic peptide has been designed from several potent marine cytotoxic peptides, including IB-01212, luzopeptin, triostin, and thiocoraline. The FAJANU scaffold maintains C 2 symmetry, cyclic structure, and the construction of aromatic and aliphatic character at the N- and C-terminal extremes. A first six-member family was previously synthesized and evaluated biologically. Several analogues presented greater activity than IB-01212. Furthermore, on the basis of the most active candidate, we have performed a more exhaustive synthetic and structural analysis: (i) structure-activity relationship provided clues about the key elements in the framework, (ii) NMR assignment confirmed C 2 symmetry, and (iii) confocal images revealed its penetration and cellular localization.

[The horizontal infrahyoid myocutaneous flap: surgical technique]. / Le lambeau musculocutané infrahyoïdien à palette cutanée horizontale. Technique chirurgicale.

The infrahyoid myocutaneous flap technique was described by Wang in 1986, the skin pad being orientated vertically. Its blood supply comes from the superior thyroid artery. This flap consists of the sternohyoid muscle, the sternothyroid muscle, and the superior belly of the omohyoid muscle. The harvesting of a horizontal skin flap does not modify its reliability and avoids additional scars. The donor site anatomy and flap vascularization are briefly described as well as the flap features and harvesting technique.

[Head and neck reconstruction with antebrachial flap concerning 100 patients]. / La reconstruction par lambeau antébrachial au niveau de la sphère cervicofaciale : revue d'une série de 100 cas.

We studied the files of head and neck reconstruction with antebrachial flap used in 100 patients between May 1996 and March 2007 in the department of Maxillofacial Surgery of the CHU of Bordeaux. Flap harvesting and vascular anastomosis were performed by the same operator. Antebrachial flap was used to cover the defect after resection of head and neck cancer in 89 patients and after shotgun injuries of the face in 11 patients. This flap, by its smoothness and its plasticity, makes it possible to rebuild all the oral cavity localizations, even the most complex, by covering the defects without blocking deglutition and enunciation. It brings to the patient a better quality of life by decreasing the functional after-effects that can be seen with the traditional myocutaneous flaps. This flap is highly reliable, not requiring specific care after the operation as other micro-surgeries, which simplifies the postoperative quality of life of the aged and debilitated patients.