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The Gleason score is one of the most important parameters for therapeutic decision-making in prostate cancer patients. Gleason growth patterns are defined by their histological features on 4- to 5-µm cross sections, and little is known about their three-dimensional architecture. Our objective was to characterize the three-dimensional architecture of prostate cancer growth patterns. Intact tissue punches ( n  = 46) of representative Gleason growth patterns from radical prostatectomy specimens were fluorescently stained with antibodies targeting Keratin 8/18 and Keratin 5 for the detection of luminal and basal epithelial cells, respectively. Punches were optically cleared in benzyl alcohol–benzyl benzoate and imaged using a confocal laser scanning microscope up to a depth of 500 µm. Gleason pattern 3, poorly formed pattern 4, and cords pattern 5 all formed a continuum of interconnecting tubules in which the diameter of the structures and the lumen size decreased with higher grades. In fused pattern 4, the interconnections between the tubules were markedly closer together. In these patterns, all tumor cells were in direct contact with the surrounding stroma. In contrast, cribriform Gleason pattern 4 and solid pattern 5 demonstrated a three-dimensional continuum of contiguous tumor cells, in which the vast majority of cells had no contact with the surrounding stroma. Transitions between cribriform pattern 4 and solid pattern 5 were seen. There was a decrease in the number and size of intercellular lumens from cribriform to solid growth pattern. Glomeruloid pattern 4 formed an intermediate structure consisting of a tubular network with intraluminal epithelial protrusions close to the tubule splitting points. In conclusion, three-dimensional microscopy revealed two major architectural subgroups of prostate cancer growth patterns: (1) a tubular interconnecting network including Gleason pattern 3, poorly formed and fused Gleason pattern 4, and cords Gleason pattern 5, and (2) serpentine contiguous epithelial proliferations including cribriform Gleason pattern 4 and solid Gleason pattern 5.

In July 2022, the ongoing monkeypox (MPX) outbreak was declared a public health emergency of international concern. Modified vaccinia Ankara—Bavarian Nordic (MVA-BN, also known as Imvamune, JYNNEOS or Imvanex) is a third-generation smallpox vaccine that is authorized and in use as a vaccine against MPX. To date, there are no data showing MPX virus (MPXV)-neutralizing antibodies in vaccinated individuals nor vaccine efficacy against MPX. Here we show that MPXV-neutralizing antibodies can be detected after MPXV infection and after historic smallpox vaccination. However, a two-shot MVA-BN immunization series in non-primed individuals yields relatively low levels of MPXV-neutralizing antibodies. Dose-sparing of an MVA-based influenza vaccine leads to lower MPXV-neutralizing antibody levels, whereas a third vaccination with the same MVA-based vaccine significantly boosts the antibody response. As the role of MPXV-neutralizing antibodies as a correlate of protection against disease and transmissibility is currently unclear, we conclude that cohort studies following vaccinated individuals are necessary to assess vaccine efficacy in at-risk populations. Historic smallpox vaccination and monkeypox virus (MPXV) infection elicit MPXV-neutralizing antibodies, but MPXV-neutralizing antibodies are less frequent and of lower magnitude after vaccination with MVA-BN—the vaccine approved and in use for protection against MPXV and smallpox.

Extracellular vesicles (EVs) are a family of small membrane vesicles that carry information about cells by which they are secreted. Growing interest in the role of EVs in intercellular communication, but also in using their diagnostic, prognostic and therapeutic potential in (bio) medical applications, demands for accurate assessment of their biochemical and physical properties. In this review, we provide an overview of available technologies for EV analysis by describing their working principles, assessing their utility in EV research and summarising their potential and limitations. To emphasise the innovations in EV analysis, we also highlight the unique possibilities of emerging technologies with high potential for further development.

Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (∼ 0.7 s) and the other half for longer time periods (∼ 2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (≤ 1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å 2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo , and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor. The androgen receptor is crucial for the development and physiology of reproductive organs. Here the authors present the structure of the androgen receptor ligand-binding domain bound to dihydrotestosterone, identifying a homodimerization interface that is crucial for receptor activity in vivo .

Urinary extracellular vesicles (EVs) have gained increased interest as a biomarker source. Clinical implementation on a daily basis requires protocols that inevitably includes short-term storage of the clinical samples, especially when collected at home. However, little is known about the effect of delayed processing on the urinary EVs concentration and proteome. We evaluated two storage protocols. First, urine stored at 4 °C. Secondly a protocol compatible with at-home collection, in which urine was stored with the preservative EDTA at room temperature (RT). EVs were isolated using the ME-kit (VN96-peptide). For both conditions we explored the effect of storage duration (0, 2, 4 and 8 days) on EV concentration and proteome using EVQuant and data-independent acquisition mass spectrometry, respectively. The urinary EV concentration and proteome was highly stable using both protocols, in terms of protein number and quantitative changes. Furthermore, EDTA does not affect the urinary EV concentration or global proteome. In conclusion, urine can be stored either at 4 °C or with EDTA at RT for up to 8 days without any significant decay in EV concentration or a notable effect on the EV-proteome. These findings open up biomarker studies in urine collected via self-sampling at home.

The precise regulation of RNA Polymerase II (Pol II) transcription after genotoxic stress is crucial for proper execution of the DNA damage-induced stress response. While stalling of Pol II on transcription-blocking lesions (TBLs) blocks transcript elongation and initiates DNA repair in cis, TBLs additionally elicit a response in trans that regulates transcription genome-wide. Here we uncover that, after an initial elongation block in cis, TBLs trigger the genome-wide VCP-mediated proteasomal degradation of promoter-bound, P-Ser5-modified Pol II in trans. This degradation is mechanistically distinct from processing of TBL-stalled Pol II, is signaled via GSK3, and contributes to the TBL-induced transcription block, even in transcription-coupled repair-deficient cells. Thus, our data reveal the targeted degradation of promoter-bound Pol II as a critical pathway that allows cells to cope with DNA damage-induced transcription stress and enables the genome-wide adaptation of transcription to genotoxic stress. DNA damage inhibits elongating RNA polymerase II, but also initiates genome-wide transcriptional responses. Here the authors reveal that particularly promoter-bound Pol II is degraded upon DNA damage in a GSK3 signaling-mediated response.

Background Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA). Methods For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR. Results Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA , KRAS and BRAF . Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (> 20%) of circulating tumor DNA (ctDNA). Expression profiling showed that the EVs from patients resemble healthy donors more than tumor cell lines supporting that most EVs are derived from healthy tissue. Conclusions We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients’ blood plasma. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.

Urinary extracellular vesicles (uEVs) are emerging as non‐invasive biomarkers for various kidney diseases, but it is unknown how differences in nephron mass impact uEV excretion. To address this, uEV excretion was measured before and after human kidney donor nephrectomy and rat nephrectomy. In male and female donors, uEVs were quantified in cell‐free spot and 24‐h urine samples using nanoparticle tracking analysis (NTA), EVQuant, and CD9‐time‐resolved fluorescence immunoassay. Female donors had significantly lower total kidney volume (TKV) and excreted 49% fewer uEVs than male donors. uEV excretion correlated positively with estimated glomerular filtration rate (eGFR), creatinine clearance, and TKV (R's between 0.6 and 0.7). uEV excretion rate could also be predicted from spot urines after multiplying spot uEV/creatinine by 24‐h urine creatinine. Donor nephrectomy reduced eGFR by 36% ± 10%, but the excretion of uEVs by only 16% (CD9+ uEVs ‐37%, CD9‐ uEVs no decrease). Donor nephrectomy increased the podocyte marker WT‐1 and the proximal tubule markers NHE3, NaPi‐IIa, and cubilin in uEVs two‐ to four‐fold when correcting for the nephrectomy. In rats, the changes in GFR and kidney weight correlated with the changes in uEV excretion rate (R = 0.46 and 0.60, P < 0.01). Furthermore, the estimated degree of hypertrophy matched the change in uEV excretion rate (1.4‐ to 1.5‐fold after uninephrectomy and four‐fold after 5/6th nephrectomy). Taken together, our data show that uEV excretion depends on nephron mass, and that nephrectomy reduces uEV excretion less than expected based on nephron loss due to compensatory hypertrophy. The major implication of our findings is that a measure for nephron mass or uEV excretion rate should be included when comparing uEV biomarkers between individuals.

Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA–DDB1–CUL4A–RBX1 cullin–RING ubiquitin ligase complex (CRL CSA ). Despite its vital role in TC-NER, little is known about the regulation of the CRL CSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin. TRiC’s binding to CSA ensures its stability and DDB1-dependent assembly into the CRL CSA complex. Consequently, loss of TRiC leads to mislocalization and depletion of CSA, as well as impaired transcription recovery following UV damage, suggesting defects in TC-NER. Furthermore, Cockayne syndrome (CS)-causing mutations in CSA lead to increased TRiC binding and a failure to compose the CRL CSA complex. Thus, we uncover CSA as a TRiC substrate and reveal that TRiC regulates CSA-dependent TC-NER and the development of CS. An integrated network of chaperones and protein degradation machineries called the proteostasis network (PN) is required to maintain protein homeostasis. Here the authors show that one of the components of the PN, the chaperonin TRiC, interacts with the core transcription-coupled nucleotide excision repair protein CSA to ensure its assembly into the CRLCSA complex.