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SUMMARYDeficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway, and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial and most efforts aimed to identify BRCA1/BARD1 ubiquitination substrates rely on indirect evidence. Here, we observed that the BRCA1/BARD1 ubiquitin E3 activity was not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identified substrates for BRCA1/BARD1. We found that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes replication fork stability epistatically to BRCA1 S114 phosphorylation, addressing the controversy about the function of BRCA1/BARD1 E3 activity in Homologous Recombination.

Objective Seasonal allergic rhinitis (SAR) is an exaggerated immunological reaction to allergens (pollen) in the air. In a small subgroup of patients, SAR can be difficult to control with first‐line therapy. Intramuscular corticosteroid injections (IMCIs) are an additional treatment in this subgroup of SAR patients. The aim of this systematic review is to investigate the efficacy and safety of IMCIs in SAR. Methods Titles and abstracts were independently screened, followed by full‐text screening based on predefined criteria. Included articles were critically appraised using the Cochrane Risk of Bias 2 (RoB 2) tool. The primary outcome is reported as the final conclusion about efficacy that was stated in the included studies. The secondary outcome is the safety of IMCIs with regard to long lasting side‐effects. Results The search yielded 2139 records, of which 10 were relevant and valid for our clinical question. Critical appraisal showed high risk of bias, which was due to unclear description of methods. Four out of four placebo‐controlled, randomized controlled trials reported a significant and relevant difference in efficacy in favor of IMCIs compared with placebo. The occurrence of side‐effects was not different between IMCIs and placebo or oral corticosteroids (OCs). Conclusion The outcome of this systematic review on trials concerning intramuscular steroid injections, despite being based on individual studies claiming favorable outcome with their use, is “inconclusive.” This is because of the epidemiological high risk of bias in these studies that were mostly executed more than 30 years ago. The “inconclusive” rating allows for a description as an “optional therapy” for severe cases in guideline formation.

Human leukocyte antigen (HLA) molecules present small peptide antigens to T cells, thereby allowing them to recognize pathogen-infected and cancer cells. A central dogma over the last 50+ y is that peptide binding to HLA molecules is mediated by the docking of side chains of particular amino acids in the peptide into pockets in the HLA molecules in a conserved N- to C-terminal orientation. Whether peptides can be presented in a reversed C- to N-terminal orientation remains unclear. Here, we performed large-scale identification of peptides bound to HLA-DP molecules and observed that in addition to peptide binding in an N- to C-terminal orientation, in 9 out of 14 HLA-DP allotypes, reverse motifs are found, compatible with C- to N-terminal peptide binding. Moreover, we isolated high-avidity human cytomegalovirus (CMV)-specific HLA-DP–restricted CD4 + T cells from the memory repertoire of healthy donors and demonstrate that such T cells recognized CMV-derived peptides bound to HLA-DPB1*01:01 or *05:01 in a reverse C- to N-terminal manner. Finally, we obtained a high-resolution HLA-DPB1*01:01-CMVpp65 (142–158) peptide crystal structure, which is the molecular basis for C- to N-terminal peptide binding to HLA-DP. Our results point to unique features of HLA-DP molecules that substantially broaden the HLA class II bound peptide repertoire to combat pathogens and eliminate cancer cells.

Proteases comprise the class of enzymes that catalyze the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility towards protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors. We developed a new method which combines the high diversity of a combinatorial synthetic peptide library with the sensitivity and detection power of mass spectrometry to determine protease cleavage specificity. We applied this method to study a group of bacterial metalloproteases that have the unique specificity to cleave between two prolines, i.e. Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-side specificity of PPEP-1 and PPEP-2, but also revealed some new unexpected peptide substrates. Moreover, we have characterized a new PPEP (PPEP-3) which has a prime-side specificity that is very different from that of the other two PPEPs. Importantly, the approach that we present in this study is generic and can be extended to investigate the specificity of other proteases.

Importance Tympanic membrane retraction (TMR) is a relatively common otological finding. However, no consensus on its management exists. We are looking especially for a treatment strategy in the military population who are unable to attend frequent follow-up visits, and who experience relatively more barotrauma at great heights and depths and easily suffer from otitis externa from less hygienic circumstances. Objective To assess and summarize the available evidence for the effectiveness of surgical interventions and watchful waiting policy in patients with a tympanic membrane retraction. Evidence review The protocol for this systematic review was published at Prospero (207859). PubMed, Embase, and the Cochrane Database of Systematic Reviews were systematically searched from inception up to September 2020 for published and unpublished studies. We included randomized trials and observational studies that investigated surgical interventions (tympanoplasty, ventilation tube insertion) and wait-and-see policy. The primary outcomes of this study were clinical remission of the tympanic membrane retraction, tympanic membrane perforations and cholesteatoma development. Findings In total, 27 studies were included, consisting of 1566 patients with TMRs. We included data from 2 randomized controlled trials (76 patients) and 25 observational studies (1490 patients). Seven studies (329 patients) investigated excision of the TMR with and without ventilation tube placement, 3 studies (207 patients) investigated the wait-and-see policy and 17 studies (1030 patients) investigated tympanoplasty for the treatment of TMRs. Conclusions and relevance This study provides all the studies that have been published on the surgical management and wait-and-policy for tympanic membrane retractions. No high level of evidence comparative studies has been performed. The evidence for the management of tympanic membrane retractions is heterogenous and depends on many factors such as the patient population, location and severity of the TMR and presence of other ear pathologies (e.g., perforation, risk of cholesteatoma and serous otitis media).

Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2 and E3 enzymes with E3s providing substrate specificity. Mass spectrometry-based approaches have enabled the identification of more than 60,000 acceptor sites for ubiquitin and 40,000 acceptor sites for SUMO2/3. However, E3-to-target wiring is poorly understood. The limited number of SUMO E3s provides the unique opportunity to systematically study E3-substrate wiring. We developed SUMO Activated Target Traps (SATTs) and systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL(ZNF451-3) and ZMIZ2. SATTs enabled us to identify 590 SUMO1 and 1195 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference, even at the substrate isoform level. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool (https://amsterdamstudygroup.shinyapps.io/PolaRVolcaNoseR/) to browse the dataset in an interactive manner, increasing the accessibility of this resource for the community. Overall, we uncover E3-to-target wiring of 1681 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.

BackgroundTranscription factor Wilms’ tumor gene 1 (WT1) is an ideal tumor target based on its expression in a wide range of tumors, low-level expression in normal tissues and promoting role in cancer progression. In clinical trials, WT1 is targeted using peptide-based or dendritic cell-based vaccines and T-cell receptor (TCR)-based therapies. Antitumor reactivities were reported, but T-cell reactivity is hampered by self-tolerance to WT1 and limited number of WT1 peptides, which were thus far selected based on HLA peptide binding algorithms.MethodsIn this study, we have overcome both limitations by searching in the allogeneic T-cell repertoire of healthy donors for high-avidity WT1-specific T cells, specific for WT1 peptides derived from the HLA class I associated ligandome of primary leukemia and ovarian carcinoma samples.ResultsUsing broad panels of malignant cells and healthy cell subsets, T-cell clones were selected that demonstrated potent and specific anti-WT1 T-cell reactivity against five of the eight newly identified WT1 peptides. Notably, T-cell clones for WT1 peptides previously used in clinical trials lacked reactivity against tumor cells, suggesting limited processing and presentation of these peptides. The TCR sequences of four T-cell clones were analyzed and TCR gene transfer into CD8+ T cells installed antitumor reactivity against WT1-expressing solid tumor cell lines, primary acute myeloid leukemia (AML) blasts, and ovarian carcinoma patient samples.ConclusionsOur approach resulted in a set of naturally expressed WT1 peptides and four TCRs that are promising candidates for TCR gene transfer strategies in patients with WT1-expressing tumors, including AML and ovarian carcinoma.

Painful neuromas are a devastating condition that is notoriously difficult to treat. The large number of techniques that have been attempted suggest that no one technique is superior. Neuromas often occur in the extremities, but iatrogenically caused pain in the head and neck area has also been described. This article describes 3 consecutive patients diagnosed with traumatic neuroma who underwent transection of the causative nerve, followed by capping of the nerve stump with a Neurocap. With a follow-up of 7 to 24 months, our results show a marked reduction in the pain scores of all 3 patients. The preliminary results indicate that this technique might be a viable treatment option for patients with a suspected neuroma in the head and neck area.

TIPIC-syndrome (Transient Perivascular Inflammation of the Carotid Artery) as a cause of a painful lump in the neck is not often described in the literature. This self-limiting perivascular inflammation of the carotid bifurcation is relatively unknown.We describe a 50-year old patient, in whom a CT-scan that was performed because of unilateral pain in the neck and paranasal sinuses revealed the unexpected diagnosis of TIPIC-syndrome.Knowledge of the symptoms at presentation and the following clinical course of TIPIC-syndrome can prevent unnecessary use of additional tests.

The type A glycan modification found in human pathogen Clostridioides difficile consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This structure has previously been described on the flagellar protein flagellin C of several C. difficile strains and is important for bacterial motility. The study of post-translational modifications often relies on some type of enrichment strategy; however, a procedure for enrichment of this modification has not yet been demonstrated. In this study, we show that an approach that is commonly used in phosphoproteomics, Fe3+-immobilized metal affinity chromatography, also enriches for peptides with this unique post-translational modification. Using LC-MS/MS analyses of immobilized metal affinity chromatography-captured tryptic peptides, we observed not only type A-modified C. difficile flagellin peptides but also a variety of truncated/modified type A structures on these peptides. Using an elaborate set of mass spectrometry analyses, we demonstrate that one of these modifications consists of a type A structure containing a phosphonate (2-aminoethylphosphonate), a modification that is rarely observed and has hitherto not been described in C. difficile. In conclusion, we show that a common enrichment strategy results in reliable identification of peptides carrying a type A glycan modification, and that the results obtained can be used to advance models about its biosynthesis.