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The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca 2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21–bound transcriptome reveals strong interactions with the Rag1 3′-UTR. Arpp21–deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3′-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities. Regulation of thymocyte development by RNA-binding proteins is not fully characterized. Here the authors show the RBP ARPP21 interacting with the Rag1 3’-UTR to promote Rag1 expression, TCR rearrangement and an increased diversity of the TCR repertoire and that ARPP21 is down regulated by TCR stimulation.

Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies. Mutations in the RNA-binding proteins Roquin-1 or Regnase-1 cause systemic autoimmunity. Heissmeyer and colleagues show that Roquin-1 and Regnase-1 physically interact and thereby regulate CD4 + and CD8 + T cell metabolism and functionality.

Erstes Hauptziel dieser Arbeit war die Darstellung und Charakterisierung der zwei optischen Antipoden des P5-Deltacyclens sowie die Untersuchung deren Reaktivität und chiroptischer Eigenschaften. Die chiralen P5-Deltacyclene bestehen jeweils aus zwei Epimeren, die miteinander in einem dynamischen Gleichgewicht stehen und nicht voneinander zu trennen sind. Dies spielt eine große Rolle in den Folgereaktionen der P5-Deltacyclene, weil es den Käfigen erlaubt, selektiv mit verschiedenen Reagenzien zu reagieren. Umsetzungen eines optischen Antipoden mit [M(CO)5THF] (M= Cr, W) lieferten jeweils vier untrennbare Einkernkomplexe als Isomerengemische. Die erhaltenen Koordinationsisomere zeigten jeweils die gleiche Stereochemie wie ihr eingesetztes Epimerenpaar, was durch die Bestimmung der chiroptischen Eigenschaften bestätigt werden konnte. Reine Epimere und damit Enantiomere der optischen Antipoden des P5-Deltacyclens konnten durch selektive Koordination eines [(Benzol)RuCl2]-Fragments an ein P-Atom erzielt werden. Hierbei entstand jeweils nur ein einziges Produkt, bei dem die Konfiguration der P-H-Einheit der des freien Käfigepimers entspricht, das vorher deutlich benachteiligt war. Auch eine selektive Oxidationsreaktion der optischen Antipoden des P5-Deltacyclens lieferte ausschließlich jeweils enantiomerenreine Oxokäfige, wobei aufgrund kinetischer Resolution das Gleichgewicht wie beim Ruthenium vollständig auf die Seite der freien benachteiligten Käfigepimere verschoben wurde. Die Rutheniumkomplexe und Oxokäfige zeigten untereinander für die entsprechenden Käfigkonfigurationen analoge chiroptische Effekte und diese ähnelten auch denen der Ausgangsmaterialien. Eine basisch induzierte Umlagerung der optischen Antipoden des P5-Deltacyclens führte unter Inversion der Stereochemie zu den jeweils ebenfalls optisch aktiven iso-P5-Deltacyclenen, deren Umsatz mit Metallpentacarbonylen die entsprechenden M(CO)5-Komplexe (M= Cr, W) lieferten. Die iso-P5-Deltacyclene bildeten wieder Epimerenpaare, bei denen bei der Umlagerung ein P-Atom und eine CtBu-Einheit formal ihre Plätze getauscht haben und diese formale Rotation findet gleichzeitig mit einer Inversion gegenüber dem P4(CtBu)3-Restkäfig statt, es wird dabei die spiegelbildliche Variante des iso-Käfigs gebildet. Eine zweite basisch induzierte Umlagerung der P5-Deltacyclene führte zu den enantiomerenreinen Pentaphosphahomocuneanen, von denen ebenfalls die entsprechenden Wolframpentacarbonylkomplexe zugänglich waren. Dabei werden jeweils die iso-P5-Käfige als Intermediate postuliert. Die Reaktivität der P5-Deltacyclene erwies sich als äußerst vielfältig und hoch interessant. Als optisch aktive Käfigliganden lieferten sie weitere optisch aktive Käfigliganden. Bei allen chemischen Reaktionen blieb die optische Aktivität erhalten. Die hier vorgestellten chiralen P5-Deltacyclene haben ein hohes Potential als Steuerliganden in der asymmetrischen Katalyse Einsatz zu finden. Zweites Hauptziel dieser Arbeit war die Darstellung verschieden alkylierter Hexaphosphapentaprismane. Dabei sollten vor allem optisch reine Seitenketten addiert werden, um die resultierenden Diastereomere trennen zu können. Bislang wurde in der Literatur noch kein Versuch beschrieben, zwei optische Antipoden der Hexaphosphapentaprismanstruktur darzustellen. Dies konnte nun erstmalig ausgehend vom Diiodo-hexaphosphapentaprisman erreicht werden, wobei durch eine alternative Synthesemethode auch ein neuer [P6(CtBu)4]-Käfig, das Hexaphosphasnouten dargestellt und vollständig charakterisiert werden konnte. Das luft- und hydrolyseunempfindliche Diiodo-hexaphosphapentaprisman konnte erfolgreich mit verschiedenen Alkylierungsreagenzien umgesetzt werden. Neben achiralen Seitenketten, die zur Bildung von racemischen Käfigen führen, konnten auch chirale Seitenketten eingeführt werden, die Diastereomerenpaare als Produkte liefern, die bislang noch nicht voneinander getrennt werden konnten. Bei der Alkylierung entstehen Gemische der dialkylierten und der monoalkylierten Verbindungen. Dies stellt jedoch kein Problem dar, denn bei der Reaktion der Mischungen mit CODPdCl2 bzw. (CH3CN)2PtCl2 werden ausschließlich die gewünschten C2-symmetrischen Dialkly-MCl2-Komplexe (M= Pd, Pt) erhalten. Mit der optisch reinen Seitenkette (S)-2-Methylbutyl gelang eine Diastereomerentrennung für den Palladiumkomplex sowohl durch fraktionierende Kristallisation, als auch durch Säulenchromatographie mit CH2Cl2.

Purpose Mesonephric-like adenocarcinomas (MLA) of the female genital tract represent a rare and relatively recently described neoplasm exhibiting characteristic morphologic and immunohistochemical findings commonly associated with a KRAS -mutation. Most cases display an aggressive clinical behavior, but knowledge about treatment approaches is limited, especially for targeting KRAS . Methods We report a series of eight cases with a detailed molecular analysis for KRAS . These cases as well as the data of previously published cases with detailed information regarding KRAS -mutational events were reviewed for a potential targeted approach and its prognostic impact. Results Both the uterine and ovarian MLA harbor a somatic KRAS-mutation in about 85% of the reported cases, affecting the hotspot codons 12 and 13. 15.7% of the endometrial and 15.6% of ovarian MLA are wild type for KRAS . A p.G12A-alteration was seen in 5.6% (5/89) of the endometrial and in 6.2% (2/32) of the ovarian tumors, for p.G12C in 7.9% and 6.2%, for p.G12D in 32.6% and 34.5% and for p.G12V in 36% and 37.5%, respectively. Very limited data are available regarding the prognostic impact of different mutational sites within the KRAS-gene without significant prognostic impact. Conclusion Because of a specific p.G12C- KRAS somatic mutation, only the minority of MLA (7.9% with uterine and 6.2% with ovarian primary) are potentially targetable by sotarasib in that rare but aggressive subtype of adenocarcinoma of the female genital tract. Until now, the different location of a somatic KRAS -mutation is of no prognostic impact.

PurposeEndometrial mesonephric-like adenocarcinoma (ML-AC) represents a recently recognized subtype of endometrial adenocarcinoma (AC) associated with a subtle immunophenotype with a characteristic KRAS-mutation. Detailed clinico-pathologic analyses and prognostic data on ML-AC are limited.MethodsWe report a series of four cases with histopathological, immunohistochemical, and molecular analyses. These cases as well as the data of previously published cases were reviewed for clinico-pathologic variables and clinical follow-up information.ResultsForty cases of ML-AC were identified. ML-AC represents about 1% of all endometrial carcinomas. Similar to other types of endometrial AC, vaginal bleeding was the leading presenting symptom, and the mean age was 60.0 years (range 31–91). More than a half of the patients presented with locally advanced disease (≥ FIGO stage II) at time of diagnosis, developed a recurrence or died of the disease within a mean follow-up period of 24.7 months (range 3–144.5 months). The most common site of distant disease was pulmonary involvement. Microscopically, ML-ACs present with mixed morphology and show a co-expression of so-called mesonephric and Müllerian markers, suggesting a Müllerian origin of the tumors. Immunostaining for PD-L1 was negative in all tested cases, using different antibodies against PD-L1. Retained staining for mismatch repair proteins on immunohistochemistry and a POLE-mutation suggest a copy number low phenotype within the molecular classification of endometrial carcinomas. Almost all cases showed a KRAS-mutation at codon 12 (mostly G12V).ConclusionUterine ML-AC represents a distinct subtype of invasive endometrial AC, associated with KRAS-mutations and characteristic immunohistochemical findings. Clinically, ML-AC may show an aggressive behavior with a high rate of recurrent disease and a substantial risk for distant metastatic disease, especially to the lungs.

Background The design and internal layout of modern operating rooms (OR) are influencing the surgical team’s collaboration and communication, ergonomics, as well as intraoperative hygiene substantially. Yet, there is no objective method for the assessment and design of operating room setups for different surgical disciplines and intervention types available. The aim of this work is to establish an improved OR setup for common procedures in arthroplasty. Methods With the help of computer simulation, a method for the design and assessment of enhanced OR setups was developed. New OR setups were designed, analyzed in a computer simulation environment and evaluated in the actual intraoperative setting. Thereby, a 3D graphical simulation representation enabled the strong involvement of clinical stakeholders in all phases of the design and decision-making process of the new setup alternatives. Results The implementation of improved OR setups reduces the instrument handover time between the surgeon and the scrub nurse, the travel paths of the OR team as well as shortens the procedure duration. Additionally, the ergonomics of the OR staff were improved. Conclusion The developed simulation method was evaluated in the actual intraoperative setting and proved its benefit for the design and optimization of OR setups for different surgical intervention types. As a clinical result, enhanced setups for total knee arthroplasty and total hip arthroplasty surgeries were established in daily clinical routine and the OR efficiency was improved.

PurposeEpithelial-mesenchymal transition (EMT) is associated with increased metastatic spread and poor prognosis. Data on vulvar carcinoma are limited.MethodsThirty-two cases of squamous cell carcinoma of the vulva (16 with and 16 without inguinal lymph node metastases) and their lymph node deposits were evaluated for immunohistochemical expression of EMT markers (vimentin, cyclin D1, e-cadherin), p16, p53 and Ki-67. Results of EMT-immunostainings were compared to lymph node involvement and expression of p53 and p16. The micro-anatomical staining pattern for EMT markers comparing the tumor center with the front of invasion was analysed in each tumor.ResultsThere was no difference in the expression of EMT markers between node negative and node positive tumors. Staining for vimentin and cyclin D1 was seen within tumor cells at the front of invasion in 100 and 84.4% of the tumors, respectively. The majority of cases (68.7%) showed negative or reduced staining for e-cadherin in this micro-anatomical localization. Tumor cells within the lymph node metastases showed positive staining for e-cadherin in 75% and for cyclin D1 in 49% of the cells but were negative for vimentin in 13 out of 16 cases (81.3%). Tumors with aberrant p53 staining represented a non-significant higher vimentin but significantly higher cyclin D1 expression at the front of invasion than those with p53 wild-type pattern.ConclusionThe present study shows no differences in the expression of EMT markers between node positive and node negative vulvar cancers. The evaluation of immunostaining within the micro-anatomical context indicates that an EMT-phenotype is restricted to the tumor cells at the front of invasion. Paired analyses of vulvar carcinomas and their lymph node deposits suggest mesenchymal-epithelial transition (MET) in the metastatic deposits. Immunohistochemical staining results may suggest that EMT is more prevalent in vulvar cancer with aberrant p53 staining.

Vulvar carcinoma is a rare disease, meeting the criteria for a “rare cancer”, but its incidence is increasing, especially in women <60 years of age. Squamous cell carcinoma (VSCC) accounts for the overwhelming majority of vulvar carcinomas and is the focus of this review. As with many cancers, the increased understanding of molecular events during tumorigenesis has led to the emergence of the molecular subclassification of VSCC, which is subclassified into tumors that arise secondary to high-risk human papillomavirus infection (HPV-associated, or HPVa) and those that arise independently of HPV (HPVi), most commonly in the setting of a chronic inflammatory condition of the vulvar skin. This latter group of HPVi VSCC arises in most cases secondary to mutations in TP53, but recently, attention has focused on the uncommon TP53 wild-type HPVi VSCC. These three molecular subtypes of VSCC (HPVa, HPVi p53 abnormal, and HPVi p53 wild type), as well as their precursor lesions, cannot be diagnosed based on a routine histopathological examination or immunostaining for p53 and p16 as surrogate markers for TP53 mutation and high-risk HPV infection, respectively, are required. The molecular subtyping of VSCC shows high reproducibility and provides important prognostic information. HPVa VSCC has the most favorable prognosis, while HPVi VSCC with TP53 mutations (p53abn) has the worst prognosis, and HPVi VSCC with wild-type TP53 (p53wt) has an intermediate prognosis. In this review, we discuss the evidence supporting this molecular subclassification and its implications for the diagnosis and treatment of VSCC and its precursors.

PurposeTumor grade is one of the more controversial factors with limited prognostic information in squamous cell carcinomas (SCC) of the uterine cervix.MethodsHistologic slides of 233 surgically treated cervical SCC (FIGO IB1) were re-examined regarding the prognostic impact of the WHO-based grading system, using the different degree of keratinization, categorizing the tumors in G1, G2 and G3 (conventional tumor grade).Results45.1% presented with well-differentiated tumors (G1), 29.2% with moderate (G2) and 25.8% with poor differentiation (G3). Tumor grade significantly correlated with decreased recurrence-free and overall survival. However, detailed analyses between G1- and G2-tumors failed to show any correlation with either recurrence-free or overall survival. G1- and G2-tumors were therefore merged into low-grade tumors and were compared to the high-grade group (G3-tumors). This binary conventional grading system showed an improved 5-years recurrence-free (low-grade: 90.2% vs. high-grade: 71.6%; p = 0.001) and overall survival rates (low-grade: 89.9% vs. high-grade: 71.1%; p = 0.001) for low-grade tumors. On multivariate analysis adjusted for lymph node metastasis, high-grade tumors represented a hazard ratio of 2.4 (95% CI 1.3–4.7) for reduced recurrence-free and 2.4 (95% CI 1.2–4.6) for overall survival. High-grade tumors showed a significantly higher risk for pelvic lymph node involvement [OR 2.7 (95% CI 1.4–5.5); p = 0.003]. The traditional three-tiered grading system failed to predict pelvic lymph node metastases.ConclusionA binary grading model for the conventional tumor grade (based on the degree of keratinization) in SCC of the uterine cervix may allow a better prognostic discrimination than the traditionally used three-tiered system.