Y chromosome loss in cancer drives growth by evasion of adaptive immunity.

Loss of the Y chromosome (LOY) is observed in multiple cancer types, including 10-40% of bladder cancers1-6, but its clinical and biological significance is unknown. Here, using genomic and transcriptomic studies, we report that LOY correlates with poor prognoses in patients with bladder cancer. We performed in-depth studies of naturally occurring LOY mutant bladder cancer cells as well as those with targeted deletion of Y chromosome by CRISPR-Cas9. Y-positive (Y+) and Y-negative (Y-) tumours grew similarly in vitro, whereas Y- tumours were more aggressive than Y+ tumours in immune-competent hosts in a T cell-dependent manner. High-dimensional flow cytometric analyses demonstrated that Y- tumours promote striking dysfunction or exhaustion of CD8+ T cells in the tumour microenvironment. These findings were validated using single-nuclei RNA sequencing and spatial proteomic evaluation of human bladder cancers. Of note, compared with Y+ tumours, Y- tumours exhibited an increased response to anti-PD-1 immune checkpoint blockade therapy in both mice and patients with cancer. Together, these results demonstrate that cancer cells with LOY mutations alter T cell function, promoting T cell exhaustion and sensitizing them to PD-1-targeted immunotherapy. This work provides insights into the basic biology of LOY mutation and potential biomarkers for improving cancer immunotherapy.

Cutting Edge: TNF Is Essential for Mycobacteria-Induced MINCLE Expression, Macrophage Activation, and Th17 Adjuvanticity.

TNF blockade is a successful treatment for human autoimmune disorders like rheumatoid arthritis and inflammatory bowel disease yet increases susceptibility to tuberculosis and other infections. The C-type lectin receptors (CLR) MINCLE, MCL, and DECTIN-2 are expressed on myeloid cells and sense mycobacterial cell wall glycolipids. In this study, we show that TNF is sufficient to upregulate MINCLE, MCL, and DECTIN-2 in macrophages. TNF signaling through TNFR1 p55 was required for upregulation of these CLR and for cytokine secretion in macrophages stimulated with the MINCLE ligand trehalose-6,6-dibehenate or infected with Mycobacterium bovis bacillus Calmette-Guérin. The Th17 response to immunization with the MINCLE-dependent adjuvant trehalose-6,6-dibehenate was specifically abrogated in TNF-deficient mice and strongly attenuated by TNF blockade with etanercept. Together, interference with production or signaling of TNF antagonized the expression of DECTIN-2 family CLR, thwarting vaccine responses and possibly increasing infection risk.

[se-atlas.de-Medical care atlas for people with rare diseases]. / se-atlas.de ­ Versorgungsatlas für Menschen mit Seltenen Erkrankungen.

In the European Union a disease is classified as rare if it affects no more than 5 out of 10,000 people. Currently, there are more than 6000 rare diseases, consisting of a large and heterogeneous number of different diseases that are complex in their symptomatology, multidimensional and therefore difficult to classify in everyday medical practice. This complicates the diagnosis and treatment as well as finding a suitable contact person, as there are only a few experts for each individual rare disease. The medical care atlas for rare diseases www.se-atlas.de enables the search for care facilities and patient organizations for specific rare diseases by disease name and presents the search results geographically. It also provides an overview of all German centers for rare diseases, which are a contact point for patients with an unclear diagnosis. The se-atlas serves as a compass through the heterogeneous amount of information on care facilities for rare diseases and provides low-threshold information for a broad user group, from affected persons to members of the medical care team.

Comparative studies on similarities and differences of cyclodipeptide oxidases for installation of C-C double bonds at the diketopiperazine ring.

Cyclodipeptide oxidases (CDOs) perform dehydrogenations on diketopiperazines and play an important role in the cyclodipeptide diversification. In this study, we investigated the two known CDOs AlbA/B and Ndas_1146/7 and one new member, CDO-Np. LC-MS monitoring of 32 cyclodipeptide biotransformations in E. coli revealed good consumption of cyclodipeptides containing aromatic amino acids. Cyclodipeptides consisting solely of aliphatic amino acids were poor substrates. In vitro assays of 34 substrates with crude enzyme extracts and product identification proved that the CDO-Np-containing extract catalyzes the formation of two C-C double bonds in many cases. The extracts containing the two other enzymes had lower activities and catalyzed mainly didehydrogenations. For didehydrogenation, the phenylalanyl or tyrosyl site was usually preferred. No or very low acceptance of benzodiazepinediones and a 2,6-diketopiperazine proved the importance of the 2,5-diketopiperazine ring. N-Methylation at the diketopiperazine ring or prenylation of the tryptophan-containing cyclodipeptides influences the enzyme activity and product spectrum. KEY POINTS: • Comparison of catalytic activities of three enzymes; Diverse cyclodipeptides and derivatives as substrates; Determination of double bond formation using2H-labeled substrates; Product identification also by interpretation of MS2fragmentation pattern.

Ancylobacter pratisalsi sp. nov. with plant growth promotion abilities from the rhizosphere of Plantago winteri Wirtg.

A Gram-negative bacterium, designated E130T, was isolated from rhizospheric soil of Plantago winteri Wirtg. from a natural salt meadow as part of an investigation on rhizospheric bacteria from salt-resistant plant species and evaluation of their plant growth-promoting abilities. Cells were rods, non-motile, aerobic, and oxidase and catalase positive, grew in a temperature range of between 4 and 37 °C, and in the presence of 0.5-5 % NaCl (w/v). Based on 16S rRNA gene sequence analysis, strain E130T is affiliated within the genus Ancylobacter, sharing the highest similarity with Ancylobacter rudongensis DSM 17131T (97.6 %), Ancylobacter defluvii CCUG 63806T (97.5 %) and Ancylobacter dichloromethanicus DSM 21507T (97.4 %). The DNA G+C content of strain E130T was 65.1 mol%. Its respiratory quinones were Q-9 and Q-10 and its major polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and unidentified phospholipid. Major fatty acids of the strains E130T were C12 : 0, C16 : 0, C18 : 1ω7c and C19 : 0cycloω8c. The DNA-DNA relatedness of E130T to A. rudongensis DSM 17131T, A. defluvii CCUG 63806T and A. dichloromethanicus DSM 21507T was 29.2, 21.2 and 32.2 % respectively. On the basis of our polyphasic taxonomic study the new isolate represents a novel species, for which the name Ancylobacter pratisalsi sp. nov. is proposed. The type strain is E130T (LMG 29367T=DSM 102029T).

Emotion Regulation Strategies in Depressive and Anxiety Symptoms in Youth: A Meta-Analytic Review.

The role of emotion regulation in subclinical symptoms of mental disorders in adolescence is not yet well understood. This meta-analytic review examines the relationship between the habitual use of prominent adaptive emotion regulation strategies (cognitive reappraisal, problem solving, and acceptance) and maladaptive emotion regulation strategies (avoidance, suppression, and rumination) with depressive and anxiety symptoms in adolescence. Analyzing 68 effect sizes from 35 studies, we calculated overall outcomes across depressive and anxiety symptoms as well as psychopathology-specific outcomes. Age was examined as a continuous moderator via meta-regression models. The results from random effects analyses revealed that the habitual use of all emotion regulation strategies was significantly related to depressive and anxiety symptoms overall, with the adaptive emotion regulation strategies showing negative associations (i.e., less symptoms) with depressive and anxiety symptoms whereas the maladaptive emotion regulation strategies showed positive associations (i.e., more symptoms). A less frequent use of adaptive and a more frequent use of maladaptive emotion regulation strategies were associated with depressive and anxiety symptoms comparably in the respective directions. Regarding the psychopathology-specific outcomes, depressive and anxiety symptoms displayed similar patterns across emotion regulation strategies showing the strongest negative associations with acceptance, and strongest positive associations with avoidance and rumination. The findings underscore the relevance of adaptive and also maladaptive emotion regulation strategies in depressive and anxiety symptoms in youth, and highlight the need to further investigate the patterns of emotion regulation as a potential transdiagnostic factor.

Emotional Awareness in Depressive and Anxiety Symptoms in Youth: A Meta-Analytic Review.

Emotion regulation is assumed to play an important role in depressive and anxiety symptoms in youth. However, the role of core components of emotion regulation, such as emotional awareness, is not well understood so far. Thus this meta-analysis aimed to examine the relationship between depressive and anxiety symptoms with emotional awareness in youth. A systematic literature search (PsycINFO, Medline, Google Scholar) identified 21 studies, from which 34 effect sizes were extracted. Results from random effects models showed that difficulties in emotional awareness were significantly correlated with a medium effect size for each, depressive and anxiety symptoms separately, and for their combined effects (overall outcome). Additionally, further analyses revealed that age was a significant moderator of the relationship between emotional awareness with depressive and anxiety symptoms, with younger samples (mean age ≤ 12 years) showing a stronger association between difficulties in emotional awareness and depressive and anxiety symptoms as compared to older samples (mean age > 12 years). The results suggest that emotional awareness may be of relevance for depressive and anxiety symptoms in youth. Future work is required to examine longitudinal developments, moderators, and mediators in multi-method approaches. Moreover, children and adolescents may benefit from interventions that aim to enhance emotional awareness.

PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors.

INTRODUCTION: Triple negative breast cancer (TNBC) is a heterogeneous collection of biologically diverse cancers, which contributes to variable clinical outcomes. Previously, we identified a TNBC subtype that has a luminal phenotype and expresses the androgen receptor (AR+). TNBC cells derived from these luminal AR + tumors have high frequency phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations. The purpose of this study was to determine if targeting phosphoinositide 3-kinase (PI3K) alone or in combination with an AR antagonist is effective in AR + TNBC. METHODS: We determined the frequency of activating PIK3CA mutations in AR + and AR- TNBC clinical cases. Using AR + TNBC cell line and xenograft models we evaluated the effectiveness of PI3K inhibitors, used alone or in combination with an AR antagonist, on tumor cell growth and viability. RESULTS: PIK3CA kinase mutations were highly clonal, more frequent in AR + vs. AR- TNBC (40% vs. 4%), and often associated with concurrent amplification of the PIK3CA locus. PI3K/mTOR inhibitors had an additive growth inhibitory effect when combined with genetic or pharmacological AR targeting in AR + TNBC cells. We also analyzed the combination of bicalutamide +/- the pan-PI3K inhibitor GDC-0941 or the dual PI3K/mTOR inhibitor GDC-0980 in xenograft tumor studies and observed additive effects. CONCLUSIONS: While approximately one third of TNBC patients respond to neoadjuvant/adjuvant chemotherapy, recent studies have shown that patients with AR + TNBC are far less likely to benefit from the current standard of care chemotherapy regimens and novel targeted approaches need to be investigated. In this study, we show that activating PIK3CA mutations are enriched in AR + TNBC; and, we show that the growth and viability of AR + TNBC cell line models is significantly reduced after treatment with PI3K inhibitors used in combination with an AR antagonist. These results provide rationale for pre-selection of TNBC patients with a biomarker (AR expression) to investigate the use of AR antagonists in combination with PI3K/mTOR inhibitors.

Redefining the catalytic HECT domain boundaries for the HECT E3 ubiquitin ligase family.

There are 28 unique human members of the homologous to E6AP C-terminus (HECT) E3 ubiquitin ligase family. Each member of the HECT E3 ubiquitin ligases contains a conserved bilobal HECT domain of approximately 350 residues found near their C-termini that is responsible for their respective ubiquitylation activities. Recent studies have begun to elucidate specific roles that each HECT E3 ubiquitin ligase has in various cancers, age-induced neurodegeneration, and neurological disorders. New structural models have been recently released for some of the HECT E3 ubiquitin ligases, but many HECT domain structures have yet to be examined due to chronic insolubility and/or protein folding issues. Building on these recently published structural studies coupled with our in-house experiments discussed in the present study, we suggest that the addition of ∼50 conserved residues preceding the N-terminal to the current UniProt defined boundaries of the HECT domain are required for isolating soluble, stable, and active HECT domains. We show using in silico bioinformatic analyses coupled with secondary structural prediction software that this predicted N-terminal α-helix found in all 28 human HECT E3 ubiquitin ligases forms an obligate amphipathic α-helix that binds to a hydrophobic pocket found within the HECT N-terminal lobe. The present study brings forth the proposal to redefine the residue boundaries of the HECT domain to include this N-terminal extension that will likely be critical for future biochemical, structural, and therapeutic studies on the HECT E3 ubiquitin ligase family.

Sex-biased adaptive immune regulation in cancer development and therapy.

The cancer research field is finally starting to unravel the mystery behind why males have a higher incidence and mortality rate than females for nearly all cancer types of the non-reproductive systems. Here, we explain how sex - specifically sex chromosomes and sex hormones - drives differential adaptive immunity across immune-related disease states including cancer, and why males are consequently more predisposed to tumor development. We highlight emerging data on the roles of cell-intrinsic androgen receptors in driving CD8+ T cell dysfunction or exhaustion in the tumor microenvironment and summarize ongoing clinical efforts to determine the impact of androgen blockade on cancer immunotherapy. Finally, we outline a framework for future research in cancer biology and immuno-oncology, underscoring the importance of a holistic research approach to understanding the mechanisms of sex dimorphisms in cancer, so sex will be considered as an imperative factor for guiding treatment decisions in the future.

Androgen conspires with the CD8+ T cell exhaustion program and contributes to sex bias in cancer.

Sex bias exists in the development and progression of nonreproductive organ cancers, but the underlying mechanisms are enigmatic. Studies so far have focused largely on sexual dimorphisms in cancer biology and socioeconomic factors. Here, we establish a role for CD8+ T cell-dependent antitumor immunity in mediating sex differences in tumor aggressiveness, which is driven by the gonadal androgen but not sex chromosomes. A male bias exists in the frequency of intratumoral antigen-experienced Tcf7/TCF1+ progenitor exhausted CD8+ T cells that are devoid of effector activity as a consequence of intrinsic androgen receptor (AR) function. Mechanistically, we identify a novel sex-specific regulon in progenitor exhausted CD8+ T cells and a pertinent contribution from AR as a direct transcriptional transactivator of Tcf7/TCF1. The T cell-intrinsic function of AR in promoting CD8+ T cell exhaustion in vivo was established using multiple approaches including loss-of-function studies with CD8-specific Ar knockout mice. Moreover, ablation of the androgen-AR axis rewires the tumor microenvironment to favor effector T cell differentiation and potentiates the efficacy of anti-PD-1 immune checkpoint blockade. Collectively, our findings highlight androgen-mediated promotion of CD8+ T cell dysfunction in cancer and imply broader opportunities for therapeutic development from understanding sex disparities in health and disease.

Selective crystallization of indigo B by a modified sublimation method and its redetermined structure.

Good-quality single crystals of the title compound, indigo B [systematic name: 2-(3-oxoindolin-2-yl-idene)indolin-3-one], C(16)H(10)N(2)O(2), have been prepared with high selectivity by a sublimation process. The previous structure of indigo B [Süsse & Wolf (1980 ▶). Naturwissenschaften, 67, 453], which showed that the complete mol-ecule is generated by crystallographic inversion symmetry has been confirmed, but the present study reports more realistic geometrical parameters and modern standards of precision (e.g. σ for C-C bonds = 0.002-0.003 Å). Each mol-ecule features two intra-molecular N-H⋯O hydrogen bonds. In the crystal, mol-ecules are linked by strong face-to-face π-π stacking inter-actions involving both the six- and five-membered rings [centroid-centroid separations = 3.6290 (14) and 3.6506 (14) Å] and inter-molecular N-H⋯O hydrogen bonds.

Acquisition of aneuploidy drives mutant p53-associated gain-of-function phenotypes.

p53 is mutated in over half of human cancers. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. To study mutant p53 GOF mechanisms and phenotypes, we genetically engineered non-transformed and tumor-derived WT p53 cell line models to express endogenous missense mutant p53 (R175H and R273H) or to be deficient for p53 protein (null). Characterization of the models, which initially differed only by TP53 genotype, revealed that aneuploidy frequently occurred in mutant p53-expressing cells. GOF phenotypes occurred clonally in vitro and in vivo, were independent of p53 alteration and correlated with increased aneuploidy. Further, analysis of outcome data revealed that individuals with aneuploid-high tumors displayed unfavorable prognoses, regardless of the TP53 genotype. Our results indicate that genetic variation resulting from aneuploidy accounts for the diversity of previously reported mutant p53 GOF phenotypes.

Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures.

MYC family members, MYC, MYCN, and MYCL, are oncogenic transcription factors that regulate the expression of genes involved in normal development, cell growth, proliferation, metabolism, and survival. While MYC is amplified and/or overexpressed across a variety of tissue types, MYCN is often overexpressed in tumors of the nervous system (neuroblastoma and medulloblastoma) or with neuroendocrine features (neuroendocrine prostate cancer). Given recent reports that MYCN expression is also deregulated in a variety of non-neuronal tissue types, we investigated whether MYCN was also deregulated in triple-negative breast cancer (TNBC). In contrast to previous individual immuno-fluorescence (IF) stains against higher expressing MYC family isoform protein, we developed an IF stain to simultaneously detect both MYCN- and MYC-expressing cells within the same tumor cell population. Our methodology allows for the detection of low level MYCN and MYC expression and can be multiplexed with additional protein probes. Herein, using tyramide signal amplification (TSA), we present two protocols for the IF detection of MYCN and MYC on formalin-fixed paraffin embedded (FFPE) tumor sections and in cell lines fixed in situ after growth as adherent cultures on chambered microscope slides.

Targeting MYCN-expressing triple-negative breast cancer with BET and MEK inhibitors.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that does not respond to endocrine therapy or human epidermal growth factor receptor 2 (HER2)-targeted therapies. Individuals with TNBC experience higher rates of relapse and shorter overall survival compared to patients with receptor-positive breast cancer subtypes. Preclinical discoveries are needed to identify, develop, and advance new drug targets to improve outcomes for patients with TNBC. Here, we report that MYCN, an oncogene typically overexpressed in tumors of the nervous system or with neuroendocrine features, is heterogeneously expressed within a substantial fraction of primary and recurrent TNBC and is expressed in an even higher fraction of TNBCs that do not display a pathological complete response after neoadjuvant chemotherapy. We performed high-throughput chemical screens on TNBC cell lines with varying amounts of MYCN expression and determined that cells with higher expression of MYCN were more sensitive to bromodomain and extraterminal motif (BET) inhibitors. Combined BET and MEK inhibition resulted in a synergistic decrease in viability, both in vitro and in vivo, using cell lines and patient-derived xenograft (PDX) models. Our preclinical data provide a rationale to advance a combination of BET and MEK inhibitors to clinical investigation for patients with advanced MYCN-expressing TNBC.

Membrane capacitance recordings resolve dynamics and complexity of receptor-mediated endocytosis in Wnt signalling.

Receptor-mediated endocytosis is an essential process in signalling pathways for activation of intracellular signalling cascades. One example is the Wnt signalling pathway that seems to depend on endocytosis of the ligand-receptor complex for initiation of Wnt signal transduction. To date, the roles of different endocytic pathways in Wnt signalling, molecular players and the kinetics of the process remain unclear. Here, we monitored endocytosis in Wnt3a and Wnt5a-mediated signalling with membrane capacitance recordings of HEK293 cells. Our measurements revealed a swift and substantial increase in the number of endocytic vesicles. Extracellular Wnt ligands specifically triggered endocytotic activity, which started immediately upon ligand binding and ceased within a period of ten minutes. By using specific inhibitors, we were able to separate Wnt-induced endocytosis into two independent pathways. We demonstrate that canonical Wnt3a is taken up mainly by clathrin-independent endocytosis whereas noncanonical Wnt5a is exclusively regulated via clathrin-mediated endocytosis. Our findings show that membrane capacitance recordings allow the resolution of complex cellular processes in plasma membrane signalling pathways in great detail.

Experimental Cutaneous Leishmaniasis: Mouse Models for Resolution of Inflammation Versus Chronicity of Disease.

Experimental cutaneous leishmaniasis of mice is a valuable model to study the immune response to the protozoan pathogen Leishmania and to define mechanisms of parasite control and resolution of inflammation as well as of parasite evasion and chronicity of disease. In addition, over many years Leishmania-infected mice have been successfully used to analyze the function of newly discovered immune cell types, transcription factors, cytokines, and effector mechanisms in vivo. In this chapter we present detailed protocols for the culture, propagation, and inoculation of Leishmania promastigotes, the monitoring of the course of cutaneous infection, the determination of the tissue parasite burden and for the phenotyping of the ensuing immune response. The focus lies on the L. major mouse model, but an overview on other established models of murine cutaneous leishmaniasis is also provided.