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Small-angle-scattering tensor tomography is a technique for studying anisotropic nanostructures of millimetre-sized samples in a volume-resolved manner. It requires the acquisition of data through repeated tomographic rotations about an axis which is subjected to a series of tilts. The tilt that can be achieved with a typical setup is geometrically constrained, which leads to limits in the set of directions from which the different parts of the reciprocal space map can be probed. Here, we characterize the impact of this limitation on reconstructions in terms of the missing wedge problem of tomography, by treating the problem of tensor tomography as the reconstruction of a three-dimensional field of functions on the unit sphere, represented by a grid of Gaussian radial basis functions. We then devise an acquisition scheme to obtain complete data by remounting the sample, which we apply to a sample of human trabecular bone. Performing tensor tomographic reconstructions of limited data sets as well as the complete data set, we further investigate and validate the missing wedge problem by investigating reconstruction errors due to data incompleteness across both real and reciprocal space. Finally, we carry out an analysis of orientations and derived scalar quantities, to quantify the impact of this missing wedge problem on a typical tensor tomographic analysis. We conclude that the effects of data incompleteness are consistent with the predicted impact of the missing wedge problem, and that the impact on tensor tomographic analysis is appreciable but limited, especially if precautions are taken. In particular, there is only limited impact on the means and relative anisotropies of the reconstructed reciprocal space maps., (open access.)

For vulvar cancers, radiotherapy is targeting cancer cells, but also affects the host immune system. As this may affect treatment outcome, in this prospective study, we characterized the individual T cell immune milieu induced by surgery and adjuvant radio +/- chemotherapy (aRT) systemically in the blood of vulvar cancer patients and found increased frequencies of Interleukin (IL)-17-producing CD4 + and CD8 + T cells after aRT while frequencies of Th1 and perforin-producing CD8 + killer cells were strongly diminished. Phenotypic characterization revealed enhanced expression of the ectonucleotidase CD39 on Th17 and Tc17 cells as well as CD8 + perforin + cells after aRT. Furthermore, the aRT cohort exhibited increased proportions of Programmed Cell Death Protein (PD-1) expressing cells among Th1 and CD8 + perforin + cells, but not among Th17 and Tc17 cells. High post-therapeutic levels of Th17 and Tc17 cells and low proportions of Th1 and CD8 + perforin + cells expressing PD-1 was associated with reduced recurrence free survival on follow-up. In conclusion, our study defines individual therapy-induced changes in the cellular immune milieu of patients and their association with cancer relapse. Our results may help to explain differences in the individual courses of disease of vulvar cancer patients and suggest PD-1 and IL-17 as targets for immunotherapy in vulvar cancer., (© 2023 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Parkinson's disease (PD) emerges as a complex, multifactorial disease. While there is increasing evidence that dysregulated T cells play a central role in PD pathogenesis, elucidation of the pathomechanical changes in related signaling is still in its beginnings. We employed time-resolved RNA expression upon the activation of peripheral CD4+ T cells to track and functionally relate changes on cellular signaling in representative cases of patients at different stages of PD. While only few miRNAs showed time-course related expression changes in PD, we identified groups of genes with significantly altered expression for each different time window. Towards a further understanding of the functional consequences, we highlighted pathways with decreased or increased activity in PD, including the most prominent altered IL-17 pathway. Flow cytometric analyses showed not only an increased prevalence of Th17 cells but also a specific subtype of IL-17 producing γδ-T cells, indicating a previously unknown role in PD pathogenesis., (© 2023. The Author(s).)

Cervical cancer therapy is still a major clinical challenge, as patients substantially differ in their response to standard treatments, including chemoradiotherapy (CRT). During cervical carcinogenesis, T-helper (Th)-17 cells accumulate in the peripheral blood and tumor tissues of cancer patients and are associated with poor prognosis. In this prospective study, we find increased Th17 frequencies in the blood of patients after chemoradiotherapy and a post-therapeutic ratio of Th17/CD4 + T cells > 8% was associated with early recurrence. Furthermore, Th17 cells promote resistance of cervical cancer cells toward CRT, which was dependent on the AKT signaling pathway. Consistently, patients with high Th17 frequencies in pretherapeutic biopsies exhibit lower response to primary CRT. This work reveals a key role of Th17 cells in CRT resistance and elevated Th17 frequencies in the blood after CRT correspond with early recurrence. Our results may help to explain individual treatment responses of cervical cancer patients and suggest evaluation of Th17 cells as a novel predictive biomarker for chemoradiotherapy responses and as a potential target for immunotherapy in cervical cancer., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

T cells are central to the immune response against various pathogens and cancer cells. Complex networks of transcriptional and post-transcriptional regulators, including microRNAs (miRNAs), coordinate the T cell activation process. Available miRNA datasets, however, do not sufficiently dissolve the dynamic changes of miRNA controlled networks upon T cell activation. Here, we established a quantitative and time-resolved expression pattern for the entire miRNome over a period of 24 h upon human T-cell activation. Based on our time-resolved datasets, we identified central miRNAs and specified common miRNA expression profiles. We found the most prominent quantitative expression changes for miR-155-5p with a range from initially 40 molecules/cell to 1600 molecules/cell upon T-cell activation. We established a comprehensive dynamic regulatory network of both the up- and downstream regulation of miR-155. Upstream, we highlight IRF4 and its complexes with SPI1 and BATF as central for the transcriptional regulation of miR-155. Downstream of miR-155-5p, we verified 17 of its target genes by the time-resolved data recorded after T cell activation. Our data provide comprehensive insights into the range of stimulus induced miRNA abundance changes and lay the ground to identify efficient points of intervention for modifying the T cell response., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Micro(mi)RNAs are increasingly recognized as central regulators of immune cell function. While it has been predicted that miRNAs have multiple targets, the majority of these predictions still await experimental confirmation. Here, miR-34a, a well-known tumor suppressor, is analyzed for targeting genes involved in immune system processes of leucocytes., Methods: Using an in-silico approach, we combined miRNA target prediction with GeneTrail2, a web tool for Multi-omics enrichment analysis, to identify miR-34a target genes, which are involved in the immune system process subcategory of Gene Ontology., Results: Out of the 193 predicted target genes in this subcategory we experimentally tested 22 target genes and confirmed binding of miR-34a to 14 target genes including VAMP2, IKBKE, MYH9, MARCH8, KLRK1, CD11A, TRAFD1, CCR1, PYDC1, PRF1, PIK3R2, PIK3CD, AP1B1, and ADAM10 by dual luciferase assays. By transfecting Jurkat, primary CD4 + and CD8 + T cells with miR-34a, we demonstrated that ectopic expression of miR-34a leads to reduced levels of endogenous VAMP2 and CD11A, which are central to the analyzed subcategories. Functional downstream analysis of miR-34a over-expression in activated CD8 + T cells exhibits a distinct decrease of PRF1 secretion., Conclusions: By simultaneous targeting of 14 mRNAs miR-34a acts as major hub of T cell regulatory networks suggesting to utilize miR-34a as target of intervention towards a modulation of the immune responsiveness of T-cells in a broad tumor context.

NF-κB functions as modulator of T cell receptor-mediated signaling and transcriptional regulator of miR-34a. Our in silico analysis revealed that miR-34a impacts the NF-κB signalosome with miR-34a binding sites in 14 key members of the NF-κB signaling pathway. Functional analysis identified five target genes of miR-34a including PLCG1, CD3E, PIK3CB, TAB2, and NFΚBIA. Overexpression of miR-34a in CD4 + and CD8 + T cells led to a significant decrease of NFΚBIA as the most downstream cytoplasmic NF-κB member, a reduced cell surface abundance of TCRA and CD3E, and to a reduction of T cell killing capacity. Inhibition of miR-34a caused an increase of NFΚBIA, TCRA, and CD3E. Notably, activation of CD4 + and CD8 + T cells entrails a gradual increase of miR-34a. Our results lend further support to a model with miR-34a as a central NF-κB regulator in T cells.

Treatment decisions in oncology are based on a balance between the efficacy of therapy and its side effects. Patients with metastases and patients with a limited prognosis are a particular challenge, since communication about the disease situation and the expected therapeutic benefit is difficult not only for patients, but also for physicians. The aim of this study was therefore to compare the benefits expected of therapy by patients and physicians. Questionnaires were sent to 9,000 breast cancer patients and to 6,938 physicians. The questionnaires described 10 cases of breast cancer in the metastatic setting. The patients and physicians were asked to state the treatment benefit they would require to decide for the therapy options chemotherapy, endocrine therapy, antibody therapy, radiotherapy, and bisphosphonates. Additionally, the participants provided data on patient and physician characteristics. Expected treatment benefits were compared between patients and physicians, and influencing factors that modified the expected benefit were identified. Patients expected much greater benefits from the therapies offered than the physicians. For all treatment modalities, about 50 % or more of patients expected more than a 12-month increase in overall survival from all therapies. Among the doctors, this proportion ranged from 7 to 30 %. Among patients, previous experience of side effects and having young children in the family were the strongest influencing factors. Among the doctors, age and level of education had a strong influence on the expected prognostic improvement to indicate a therapy option. As expectations of treatment differ greatly between patients and doctors, a structured approach to solving this conflict is required. There appear to be some indicators that might help address the problem, such as the physicians' level of training and experience and the patients' specific social circumstances.

Several factors can influence individual perceptions of the expected benefit of recommended adjuvant treatment for breast cancer. This study investigated differences between patients and physicians with regard to the required efficacy of treatment and the factors influencing patients' and physicians' willingness to accept different therapeutic options. A total of 9,000 questionnaires were distributed to patients with breast cancer, and 6,938 questionnaires were distributed to physicians treating breast cancer patients. The patients were asked for personal information and about their medical history and experiences during treatment. The physicians were asked about personal information and their specialty and work environment. The treatment efficacy required by the two groups was assessed using six virtual cases of breast cancer and the treatment regimens proposed, with specific benefits and side effects. A total of 2,155 patients and 527 physicians responded to the questionnaire (return rates of 23.9 and 7.6 %). Significantly different ratings between patients and physicians with regard to the expected benefit of certain treatment options were observed. The differences were noted not only for chemotherapy but also for antihormonal and antibody treatments. Whereas physicians had a quite realistic view of the expected treatment benefits, the patients' expectations were varied. Approximately one-fifth of the patients were willing to accept treatment regimens even with marginal anticipated benefits, whereas one-third required unrealistic treatment benefits. Several influencing factors that were significantly associated with the quality rating of treatment regimens in the groups of breast cancer patients and physicians were also identified. In contrast to physicians, many breast cancer patients required treatment benefits beyond what was realistically possible, although a large group of patients were also satisfied with minimal benefits. Individual factors were also identified in both groups that significantly influence thresholds for accepting adjuvant treatment, independently of risk estimates and therapy guidelines.