Rapid simulation of glycoprotein structures by grafting and steric exclusion of glycan conformer libraries.

Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins.

An N-glycopeptide MS/MS data analysis workflow leveraging two complementary glycoproteomic software tools for more confident identification and assignments.

Complete coverage of all N-glycosylation sites on the SARS-CoV2 spike protein would require the use of multiple proteases in addition to trypsin. Subsequent identification of the resulting glycopeptides by searching against database often introduces assignment errors due to similar mass differences between different permutations of amino acids and glycosyl residues. By manually interpreting the individual MS2 spectra, we report here the common sources of errors in assignment, especially those introduced by the use of chymotrypsin. We show that by applying a stringent threshold of acceptance, erroneous assignment by the commonly used Byonic software can be controlled within 15%, which can be reduced further if only those also confidently identified by a different search engine, pGlyco3, were considered. A representative site-specific N-glycosylation pattern could be constructed based on quantifying only the overlapping subset of N-glycopeptides identified at higher confidence. Applying the two complimentary glycoproteomic software in a concerted data analysis workflow, we found and confirmed that glycosylation at several sites of an unstable Omicron spike protein differed significantly from those of the stable trimeric product of the parental D614G variant.

Consistent MYC and FLT4 gene amplification in radiation-induced angiosarcoma but not in other radiation-associated atypical vascular lesions.

Angiosarcoma (AS) is a distinct group of sarcomas characterized by upregulation of vascular-specific receptor tyrosine kinases, including TIE1, KDR, TEK, and FLT1. In keeping with the clinical heterogeneity, gene-expression profiling distinguishes two AS genomic clusters, which correlate with anatomical location and prior exposure to radiation. Furthermore, a high percentage of secondary AS, but not primary AS, shows distinct 8q24 chromosomal gains, due to MYC amplification. In this study, we mined the transcriptional output of 10 secondary and 11 primary AS to better define the dichotomy in the pathogenesis of these two clinical subsets. The oncogenic role of MYC was investigated further in secondary AS as well as in radiation-induced atypical vascular lesions (AVL) and other radiation-associated sarcomas. High-level MYC amplification was found in 100% of secondary AS, but in none of the AVL or other radiation-associated sarcomas. Coamplification of FLT4 (encoding VEGFR3) was identified in 25% of secondary AS, but not in other types. Our findings reinforce the distinct pathogenesis of AS subtypes, with MYC amplification being an early, but necessary event in secondary AS. Secondary genetic hits, such as FLT4 gene coamplification or KDR mutations, may play a role in tumor progression as well as potential therapeutic targeting.

EWSR1-POU5F1 fusion in soft tissue myoepithelial tumors. A molecular analysis of sixty-six cases, including soft tissue, bone, and visceral lesions, showing common involvement of the EWSR1 gene.

The diagnosis of myoepithelial (ME) tumors outside salivary glands remains challenging, especially in unusual clinical presentations, such as bone or visceral locations. A few reports have indicated EWSR1 gene rearrangement in soft tissue ME tumors, and, in one case each, the fusion partner was identified as either PBX1 or ZNF444. However, larger studies to investigate whether these genetic abnormalities are recurrent or restricted to tumors in soft tissue locations are lacking. Sixty-six ME tumors mainly from soft tissue (71%), but also from skin, bone, and visceral locations, characterized by classic morphological features and supporting immunoprofile were studied. Gene rearrangements in EWSR1, FUS, PBX1, and ZNF444 were investigated by fluorescence in situ hybridization. EWSR1 gene rearrangement was detected in 45% of the cases. A EWSR1-POU5F1 fusion was identified in a pediatric soft tissue tumor by 3'Rapid Amplification of cDNA Euds (RACE) and subsequently confirmed in four additional soft tissue tumors in children and young adults. An EWSR1-PBX1 fusion was seen in five cases, whereas EWSR1-ZNF444 and FUS gene rearrangement was noted in one pulmonary tumor each. In conclusion, EWSR1 gene rearrangement is a common event in ME tumors arising outside salivary glands, irrespective of anatomical location. EWSR1-negative tumors were more often benign, superficially located, and showed ductal differentiation, suggesting the possibility of genetically distinct groups. A subset of soft tissue ME tumors with clear cell morphology harbor an EWSR1-POU5F1 fusion, which can be used as a molecular diagnostic test in difficult cases. These findings do not support a pathogenetic relationship between soft tissue ME tumors and their salivary gland counterparts.

KDR activating mutations in human angiosarcomas are sensitive to specific kinase inhibitors.

Angiosarcomas (AS) represent a heterogeneous group of malignant vascular tumors occurring not only in different anatomic locations but also in distinct clinical settings, such as radiation or associated chronic lymphedema. Although representing only 1% to 2% of soft tissue sarcomas, vascular sarcomas provide unique insight into the general process of tumor angiogenesis. However, no molecular candidates have been identified to guide a specific therapeutic intervention. By expression profiling, AS show distinct up-regulation of vascular-specific receptor tyrosine kinases, including TIE1, KDR, SNRK, TEK, and FLT1. Full sequencing of these five candidate genes identified 10% of patients harboring KDR mutations. A KDR-positive genotype was associated with strong KDR protein expression and was restricted to the breast anatomic site with or without prior exposure to radiation. Transient transfection of KDR mutants into COS-7 cells showed ligand-independent activation of the kinase, which was inhibited by specific KDR inhibitors. These data provide a basis for the activity of vascular endothelial growth factor receptor-directed therapy in the treatment of primary and radiation-induced AS.

Comparison of suprapubic versus transobturator surgical treatments of female stress urinary incontinence.

OBJECTIVE: The aim of this study was to retrospectively compare surgical outcomes of the suprapubic arc sling (SPARC) and transobturator suburethral tape (MONARC) for treatment of stress urinary incontinence (SUI) in women. MATERIALS AND METHODS: A total of 74 women treated for SUI using MONARC were compared with 32 treated using SPARC. Postoperative evaluations were conducted at 3-6 months, including 1-hour pad test, cough stress test, uroflowmetry and residual urine volume. Subjective outcomes were evaluated using telephone interviews. Perioperative complications were investigated from chart records. RESULTS: No significant difference was found between the SPARC and MONARC treatment groups in terms of the objective cure rate at 3 months postoperatively (90.6% vs. 80.6%; p = 0.258). However, a significant difference was found between the subjective cure rates of the SPARC and MONARC groups (77.4% vs. 97.3%; p < 0.05). CONCLUSION: Both MONARC and SPARC are safe and effective for surgical treatment of SUI.

Are there any predictors for failed Burch colposuspension?

OBJECTIVE: To investigate early postoperative complications of Burch colposuspension and the risk factors that may be associated with failure. METHODS: We retrospectively reviewed the chart records of all patients who underwent Burch colposuspension from October 1997 to September 2002. Indications for colposuspension included urodynamic stress incontinence with bladder neck hypermobility and adequate vaginal capacity. The occurrence of early postoperative complications related to the operation was documented. Subjective failure of the operation was defined as patient dissatisfaction and/or persistent urinary leakage. Chi-square test or Fisher's exact test was used to determine the association among risk factors, early postoperative complications, and the failure rate of Burch colposuspension. RESULTS: A total of 258 patients (92.5%) reported satisfaction with the surgical outcome. The age, parity, menopausal status, use of hormone replacement therapy, previous hysterectomy, and occurrence of early postoperative complications did not significantly influence the failure rate. CONCLUSION: Our results demonstrate that Burch colposuspension is an effective and, according to our patients, highly satisfactory procedure for the treatment of urodynamic stress incontinence.