Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

SIRT6 Suppresses Pancreatic Cancer through Control of Lin28b.

Chromatin remodeling proteins are frequently dysregulated in human cancer, yet little is known about how they control tumorigenesis. Here, we uncover an epigenetic program mediated by the NAD(+)-dependent histone deacetylase Sirtuin 6 (SIRT6) that is critical for suppression of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies. SIRT6 inactivation accelerates PDAC progression and metastasis via upregulation of Lin28b, a negative regulator of the let-7 microRNA. SIRT6 loss results in histone hyperacetylation at the Lin28b promoter, Myc recruitment, and pronounced induction of Lin28b and downstream let-7 target genes, HMGA2, IGF2BP1, and IGF2BP3. This epigenetic program defines a distinct subset with a poor prognosis, representing 30%-40% of human PDAC, characterized by reduced SIRT6 expression and an exquisite dependence on Lin28b for tumor growth. Thus, we identify SIRT6 as an important PDAC tumor suppressor and uncover the Lin28b pathway as a potential therapeutic target in a molecularly defined PDAC subset. PAPERCLIP.

Condensates induced by transcription inhibition localize active chromatin to nucleoli.

The proper function of the genome relies on spatial organization of DNA, RNA, and proteins, but how transcription contributes to the organization is unclear. Here, we show that condensates induced by transcription inhibition (CITIs) drastically alter genome spatial organization. CITIs are formed by SFPQ, NONO, FUS, and TAF15 in nucleoli upon inhibition of RNA polymerase II (RNAPII). Mechanistically, RNAPII inhibition perturbs ribosomal RNA (rRNA) processing, releases rRNA-processing factors from nucleoli, and enables SFPQ to bind rRNA. While accumulating in CITIs, SFPQ/TAF15 remain associated with active genes and tether active chromatin to nucleoli. In the presence of DNA double-strand breaks (DSBs), the altered chromatin compartmentalization induced by RNAPII inhibition increases gene fusions in CITIs and stimulates the formation of fusion oncogenes. Thus, proper RNAPII transcription and rRNA processing prevent the altered compartmentalization of active chromatin in CITIs, suppressing the generation of gene fusions from DSBs.

Chromatin-bound RB targets promoters, enhancers, and CTCF-bound loci and is redistributed by cell-cycle progression.

The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.

Reconstructing and reprogramming the tumor-propagating potential of glioblastoma stem-like cells.

Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor-propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, and OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements and are sufficient to fully reprogram differentiated GBM cells to "induced" TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies candidate therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies. PAPERCLIP:

Binding of TMPRSS2-ERG to BAF Chromatin Remodeling Complexes Mediates Prostate Oncogenesis.

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.

Epigenome editing of microsatellite repeats defines tumor-specific enhancer functions and dependencies.

Various types of repetitive sequences are dysregulated in cancer. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 induces chromatin features typical of active enhancers at GGAA microsatellite repeats, but the function of these sites has not been directly demonstrated. Here, by combining nascent transcription profiling with epigenome editing, we found that a subset of GGAA microsatellite repeats is transcriptionally active in Ewing sarcoma and that silencing individual repeats abolishes local nascent transcription and leads to markedly reduced expression of putative target genes. Epigenome silencing of these repeat sites does not affect gene expression in unrelated cells, can prevent the induction of gene expression by EWS-FLI1, and, in the case of a GGAA repeat that controls SOX2 expression from a distance of 470 kb, is sufficient to impair the growth of Ewing sarcoma xenografts. Using an experimental approach that is broadly applicable to testing different types of repetitive genomic elements, our study directly demonstrates that specific repeat microsatellites can have critical gene regulation functions in cancer and thus represent tumor-specific vulnerabilities that may be exploited to develop new therapies.

Resolving medulloblastoma cellular architecture by single-cell genomics.

Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

Ureteral stent after PCNL: is leaving the threads through the percutaneous tract safe and better tolerated?

OBJECTIVE: To assess safety, urinary symptoms, and feasibility of JJ stent removal with exteriorized threads through the percutaneous tract after percutaneous nephrolithotomy (PCNL). MATERIALS AND METHODS: Prospective, transversal, comparative, experimental, randomized 1-to-1 cohort study in 52 patients who underwent "tubeless" PCNL from October 2020 to November 2022. Group A with threads through the urethra and Group B through the percutaneous tract. The validated USSQ (Ureteral Stent Symptom Questionnaire) was applied in the Urology office a week after the procedure, and the JJ stent was withdrawn by pulling the threads. Hemoglobin and urine culture, and pre- and post-surgery were evaluated. RESULTS: There is a statistically significant difference in favor of group B when comparing urinary symptoms (p = 0.008), body pain (p = 0.009), and general condition (p = 0.042), mainly for non-urgency incontinence, frequency of analgesic use, and dysuria. There were significant differences between groups (p = 0.028, p = 0.026, p = 0.027, respectively). There is no association with urinary infections (p = 0.603) nor an increased risk of bleeding (p = 0.321). CONCLUSION: The removal of the JJ stent with exteriorized threads through the percutaneous tract after PCNL in the office is a feasible and safe procedure if it is removed before 8 days and has better tolerance regarding the urinary symptoms.

Risk factors of death or chronic renal replacement therapy requirements in patients with thrombotic microangiopathies without ADAMTS-13 deficiency.

Thrombotic microangiopathy (TMA), characterized by microangiopathic hemolytic anemia, thrombocytopenia, and multisystem organ dysfunction, is a life-threatening disease. Patients with TMA who do not exhibit a severe ADAMTS-13 deficiency (defined as a disintegrin-like and metalloprotease with thrombospondin type 1 motif no. 13 activity ≥10%: TMA-13n) continue to experience elevated mortality rates. This study explores the prognostic indicators for augmented mortality risk or necessitating chronic renal replacement therapy (composite outcome: CO) in TMA-13n patients. We included 42 TMA-13n patients from January 2008 to May 2018. Median age of 41 years and 60% were female. At presentation, 62% required dialysis, and 57% warranted intensive care unit admission. CO was observed in 45% of patients, including a 9-patient mortality subset. Multivariate logistic regression revealed three independent prognostic factors for CO: early administration of eculizumab (median time from hospitalization to eculizumab initiation: 5 days, range 0-19 days; odds ratio [OR], 0.14; 95% confidence interval [CI], 0.02-0.94), presence of neuroradiological lesions (OR, 6.67; 95% CI, 1.12-39.80), and a PLASMIC score ≤4 (OR, 7.39; 95% CI, 1.18-46.11). In conclusion, TMA-13n patients exhibit a heightened risk of CO in the presence of low PLASMIC scores and neuroradiological lesions, while early eculizumab therapy was the only protective factor.

A Novel Experimental Apparatus for Characterizing Flow Regime in Mechanically Stirred Tanks through Force Sensors.

Pressure fluctuations in a mixing tank can provide valuable information about the existing flow regime within the tank, which in turn influences the degree of mixing that can be achieved. In the present work, we propose a prototype for identifying the flow regime in mechanically stirred tanks equipped with four vertical baffles through the characterization of pressure fluctuations. Our innovative proposal is based on force sensors strategically placed in the baffles of the mixing tank. The signals coming from the sensors are transmitted to an electronic module based on an Arduino UNO development board. In the electronic module, the pressure signals are conditioned, amplified and sent via Bluetooth to a computer. In the computer, the signals can be plotted or stored in an Excel file. In addition, the proposed system includes a moving average filtering and a hierarchical bottom-up clustering analysis that can determine the real-time flow regime (i.e., the Reynolds number, Re) in which the tank was operated during the mixing process. Finally, to demonstrate the versatility of the proposed prototype, experiments were conducted to identify the Reynolds number for different flow regimes (static, laminar, transition and turbulent), i.e., 0≤Re≤ 42,955. Obtained results were in agreement with the prevailing consensus on the onset and developed from different flow regimes in mechanically stirred tanks.

Clinical Implications and Treatment Strategies for ESR1 Fusions in Hormone Receptor-Positive Metastatic Breast Cancer: A Case Series.

In hormone receptor-positive metastatic breast cancer (HR+ MBC), endocrine resistance is commonly due to genetic alterations of ESR1, the gene encoding estrogen receptor alpha (ERα). While ESR1 point mutations (ESR1-MUT) cause acquired resistance to aromatase inhibition (AI) through constitutive activation, far less is known about the molecular functions and clinical consequences of ESR1 fusions (ESR1-FUS). This case series discusses 4 patients with HR+ MBC with ESR1-FUS in the context of the existing ESR1-FUS literature. We consider therapeutic strategies and raise the hypothesis that CDK4/6 inhibition (CDK4/6i) may be effective against ESR1-FUS with functional ligand-binding domain swaps. These cases highlight the importance of screening for ESR1-FUS in patients with HR+ MBC while continuing investigation of precision treatments for these genomic rearrangements.

Performance of point charge embedding schemes for excited states in molecular organic crystals.

Modeling excited state processes in molecular crystals is relevant for several applications. A popular approach for studying excited state molecular crystals is to use cluster models embedded in point charges. In this paper, we compare the performance of several embedding models in predicting excited states and S1-S0 optical gaps for a set of crystals from the X23 molecular crystal database. The performance of atomic charges based on ground or excited states was examined for cluster models, Ewald embedding, and self-consistent approaches. We investigated the impact of various factors, such as the level of theory, basis sets, embedding models, and the level of localization of the excitation. We consider different levels of theory, including time-dependent density functional theory and Tamm-Dancoff approximation (TDA) (DFT functionals: ωB97X-D and PBE0), CC2, complete active space self-consistent field, and CASPT2. We also explore the impact of selection of the QM region, charge leakage, and level of theory for the description of different kinds of excited states. We implemented three schemes based on distance thresholds to overcome overpolarization and charge leakage in molecular crystals. Our findings are compared against experimental data, G0W0-BSE, periodic TDA, and optimally tuned screened range-separated functionals.

Sexual orientation knowledge and attitudes and its association with therapy satisfaction among lesbian, gay, and bisexual + Hispanic Puerto Ricans.

This study aimed to examine the difference in therapy satisfaction between lesbian, gay, and bisexual + (LGB +) individuals and heterosexual individuals, and to identify the association between therapy satisfaction and the perception of knowledge and attitudes of their last therapist among the LGB + participants. Through an exploratory design with a comparative group, 125 LGB + and 75 heterosexual participants were recruited online by availability. Results indicate that the participants' sexual orientation has no significant relation on therapy satisfaction. However, there was a significant positive association between satisfaction with therapy and the LGB + participants' perception that their therapist demonstrated knowledge and positive attitudes. This research highlights the importance for continuous education and curriculum efforts on LGB + issues.

Measurement of Cutting Temperature in Interrupted Machining Using Optical Spectrometry.

This research presents an experimental study focused on measuring temperature at the tool flank during the up-milling process at high cutting speed. The proposed system deals with emissivity compensation through a two-photodetector system and during calibration. A ratio pyrometer composed of two photodetectors and a multimode fiber-optic coupler is employed to capture the radiation emitted by the cutting insert. The pyrometer is calibrated using an innovative calibration system that addresses theoretical discrepancies arising from various factors affecting the measurement of cutting temperature. This calibration system replicates the milling process to generate a calibration curve. Experimentally, AISI 4140 steel is machined with coated tungsten carbide inserts, using cutting speeds of 300 and 400 m/min, and feed rates of 0.08 and 0.16 mm/tooth. The results reveal a maximum recorded cutting temperature of 518 °C and a minimum of 304 °C. The cutting temperature tends to increase with higher cutting speeds and feed rates, with cutting speed being the more influential factor in this increase. Both the pyrometer calibration and experimental outcomes yield satisfactory results. Finally, the results showed that the process and the device prove to be a convenient, effective, and precise method of measuring cutting temperature in machine processes.

Implementation and Clinical Adoption of Precision Oncology Workflows Across a Healthcare Network.

BACKGROUND: Precision oncology relies on molecular diagnostics, and the value-proposition of modern healthcare networks promises a higher standard of care across partner sites. We present the results of a clinical pilot to standardize precision oncology workflows. METHODS: Workflows are defined as the development, roll-out, and updating of disease-specific molecular order sets. We tracked the timeline, composition, and effort of consensus meetings to define the combination of molecular tests. To assess clinical impact, we examined order set adoption over a two-year period (before and after roll-out) across all gastrointestinal and hepatopancreatobiliary (GI) malignancies, and by provider location within the network. RESULTS: Development of 12 disease center-specific order sets took ~9 months, and the average number of tests per indication changed from 2.9 to 2.8 (P = .74). After roll-out, we identified significant increases in requests for GI patients (17%; P < .001), compliance with testing recommendations (9%; P < .001), and the fraction of "abnormal" results (6%; P < .001). Of 1088 GI patients, only 3 received targeted agents based on findings derived from non-recommended orders (1 before and 2 after roll-out); indicating that our practice did not negatively affect patient treatments. Preliminary analysis showed 99% compliance by providers in network sites, confirming the adoption of the order sets across the network. CONCLUSION: Our study details the effort of establishing precision oncology workflows, the adoption pattern, and the absence of harm from the reduction of non-recommended orders. Establishing a modifiable communication tool for molecular testing is an essential component to optimize patient care via precision oncology.

Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma.

Although human tumours are shaped by the genetic evolution of cancer cells, evidence also suggests that they display hierarchies related to developmental pathways and epigenetic programs in which cancer stem cells (CSCs) can drive tumour growth and give rise to differentiated progeny. Yet, unbiased evidence for CSCs in solid human malignancies remains elusive. Here we profile 4,347 single cells from six IDH1 or IDH2 mutant human oligodendrogliomas by RNA sequencing (RNA-seq) and reconstruct their developmental programs from genome-wide expression signatures. We infer that most cancer cells are differentiated along two specialized glial programs, whereas a rare subpopulation of cells is undifferentiated and associated with a neural stem cell expression program. Cells with expression signatures for proliferation are highly enriched in this rare subpopulation, consistent with a model in which CSCs are primarily responsible for fuelling the growth of oligodendroglioma in humans. Analysis of copy number variation (CNV) shows that distinct CNV sub-clones within tumours display similar cellular hierarchies, suggesting that the architecture of oligodendroglioma is primarily dictated by developmental programs. Subclonal point mutation analysis supports a similar model, although a full phylogenetic tree would be required to definitively determine the effect of genetic evolution on the inferred hierarchies. Our single-cell analyses provide insight into the cellular architecture of oligodendrogliomas at single-cell resolution and support the cancer stem cell model, with substantial implications for disease management.

Machine learned calibrations to high-throughput molecular excited state calculations.

Understanding the excited state properties of molecules provides insight into how they interact with light. These interactions can be exploited to design compounds for photochemical applications, including enhanced spectral conversion of light to increase the efficiency of photovoltaic cells. While chemical discovery is time- and resource-intensive experimentally, computational chemistry can be used to screen large-scale databases for molecules of interest in a procedure known as high-throughput virtual screening. The first step usually involves a high-speed but low-accuracy method to screen large numbers of molecules (potentially millions), so only the best candidates are evaluated with expensive methods. However, use of a coarse first-pass screening method can potentially result in high false positive or false negative rates. Therefore, this study uses machine learning to calibrate a high-throughput technique [eXtended Tight Binding based simplified Tamm-Dancoff approximation (xTB-sTDA)] against a higher accuracy one (time-dependent density functional theory). Testing the calibration model shows an approximately sixfold decrease in the error in-domain and an approximately threefold decrease in the out-of-domain. The resulting mean absolute error of ∼0.14 eV is in line with previous work in machine learning calibrations and out-performs previous work in linear calibration of xTB-sTDA. We then apply the calibration model to screen a 250k molecule database and map inaccuracies of xTB-sTDA in chemical space. We also show generalizability of the workflow by calibrating against a higher-level technique (CC2), yielding a similarly low error. Overall, this work demonstrates that machine learning can be used to develop a cost-effective and accurate method for large-scale excited state screening, enabling accelerated molecular discovery across a variety of disciplines.

COX-2 mediates tumor-stromal prolactin signaling to initiate tumorigenesis.

Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression.