Deubiquitylases (DUBs) are crucial in cell signaling and are often regulated by interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signaling by cleaving K63-linked polyubiquitin chains on type I interferon receptors (IFNAR1). As a Zn2+-dependent JAMM/MPN (JAB1, MOV34, MPR1, Pad1 N-terminal) DUB, BRCC36 is challenging to target with selective inhibitors. Here, we discover first-in-class inhibitors, termed BRISC molecular glues (BLUEs), which stabilize a 16-subunit human BRISC dimer in an autoinhibited conformation, blocking active sites and interactions with the targeting subunit, serine hydroxymethyltransferase 2. This unique mode of action results in selective inhibition of BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. BLUE treatment reduced interferon-stimulated gene expression in cells containing wild-type BRISC and this effect was abolished when using structure-guided, inhibitor-resistant BRISC mutants. Additionally, BLUEs increase IFNAR1 ubiquitylation and decrease IFNAR1 surface levels, offering a potential strategy to mitigate type I interferon-mediated diseases. Our approach also provides a template for designing selective inhibitors of large protein complexes by promoting rather than blocking protein-protein interactions.

Hydrogen peroxide-induced oxidative stress disrupts cytosolic Ca²⁺ homeostasis, a critical indicator of pollen fertility. Thus, measuring Ca²⁺ levels provides valuable insights into the role of antioxidants in protecting plant reproductive processes. Given the established link between high temperatures and oxidative stress, this study evaluates the response of selenium (Se)-biofortified pollen from Olea europaea L. (Leccino cultivar) to combined thermal and oxidative challenges. Pollen from untreated and Se-sprayed trees was incubated for 48 h at 20 °C, 30 °C, and 40 °C, with or without 10 mM hydrogen peroxide. The results show that disruptions to Ca²⁺ homeostasis across these temperatures reflect an additive effect beyond that of hydrogen peroxide alone. Se-biofortified pollen significantly mitigated these disruptions, suggesting protective mechanisms that were particularly evident in germination assessments. At 40 °C without hydrogen peroxide, Se-biofortified pollen achieved 22

Disruption of alternative splicing frequently causes or contributes to human diseases and disorders. Consequently, there is a need for efficient and sensitive reporter assays capable of screening chemical libraries for compounds with efficacy in modulating important splicing events. Here, we describe a screening workflow employing dual Nano and Firefly luciferase alternative splicing reporters that affords efficient, sensitive, and linear detection of small molecule responses. Applying this system to a screen of ~95,000 small molecules identified compounds that stimulate or repress the splicing of neuronal microexons, a class of alternative exons often disrupted in autism and activated in neuroendocrine cancers. One of these compounds rescues the splicing of several analyzed microexons in the cerebral cortex of an autism mouse model haploinsufficient for Srrm4, a major activator of brain microexons. We thus describe a broadly applicable high-throughput screening system for identifying candidate splicing therapeutics, and a resource of small molecule modulators of microexons with potential for further development in correcting aberrant splicing patterns linked to human disorders and disease.

BackgroundTargeting the enzyme L-Arginine:glycine amidinotransferase (AGAT) to reduce the formation of guanidinoacetate (GAA) in patients with guanidinoacetate methyltransferase (GAMT) deficiency, we attempted to identify drugs for repurposing that reduce the expression of AGAT via transcriptional inhibition.Research Design and MethodsThe authors applied a HeLa cell line stably expressing AGAT promoter and firefly luciferase reporter for high-content screening and secondary screening. For further assessment, the authors integrated Nanoluc luciferase as a reporter into the endogenous AGAT gene in HAP1 cell lines and used the human immortalized cell line RH30 as model of GAMT deficiency.ResultsScreening 6,000 drugs and drug-like compounds, the authors identified 43 and 34 high-score candidates as inhibitors and inducers of AGAT promoter-reporter expression, respectively. After further deselection considering dose response, drug toxicity, topical formulations, price, and accessibility, the authors assessed seven candidates and found none of them demonstrating efficacy in HAP1 and RH30 cells and warranting further assessment.ConclusionThe selection of the test models is crucial for screening of gene repressor drugs. Almost all drugs with an impact on gene expression had off-target effects. It is unlikely to find drugs that are selective inhibitors of AGAT expression, rendering pharmacological AGAT gene repression a risky approach for the treatment of GAMT deficiency.

Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.

Background: S100B and Tau are implicated with both brain growth and injury. Their urinary levels in 30-to-40-day-old full-term, preterm, IUGR, and preterm-IUGR subjects were measured to investigate their possible relationship with future delayed neurodevelopment. Methods: Values were related to the neuro-behavioral outcome at two years of age, as well as to brain volumes and urinary NGF assessed at the same postnatal time point. Results: Using the Griffiths III test, cognitive and motor performances were determined to establish subgroups characterized by either normal or impaired neuro-behavior. The latter included preterm, IUGR, and preterm-IUGR individuals who exhibited significantly higher and lower S100B and Tau levels, respectively, along with markedly reduced cerebral volumes and urinary NGF, as previously demonstrated. Contrary to NGF, however, Tau and S100B displayed a weak correlation with brain volumes. Conclusions: Delayed cognitive and motor performances observed in two-year-old preterm and IUGR-born individuals were also found to be associated with anomalous urinary levels of S100B and Tau, assessed at 30–40 days of the postnatal period, and their changes did not correlate with brain growth. Thus, our data suggests that, in addition to cerebral volumes and NGF, urinary S100B and Tau can also be considered as valuable parameters for the early detection of future neurodevelopmental abnormalities.

Supplementary Figure 1 from Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

Supplementary Data from High-Throughput Screening Identifies Cardiac Glycosides as Potent Inhibitors of Human Tissue Kallikrein Expression: Implications for Cancer Therapies

Supplementary Figures 1-4 from Complex <i>N</i>-Glycan and Metabolic Control in Tumor Cells

Supplementary Figure 3 from Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

High-throughput screening (HTS) is a vaunted technology in drug discovery, and drug repositioning a celebrated strategy with famous examples of successful stories; however, repositioned drugs have primarily resulted from serendipitous observations, retrospective studies, and pharmacological analyses as opposed to experimental routes. This observation points to a methodological paradox, considering that academic laboratories of the post-genomic era have benefited from unprecedented technological progress, and a facilitated access to powerful resources that, historically, were a prerogative of the pharma industry. This disconnect is exacerbated by financial, practical, and regulatory complexities affecting drug repositioning; however, the pivotal significance of stringent and rigorous data is what unconditionally sits at the crossroad of go/no-go decisions concerning the therapeutic significance, or predictive validity, of selected drugs. Here, I propose a visionary approach, to which I assigned the term labsourcing, to dramatically enhance efficiency and clinical relevance of academic drug screens and, ultimately, generate contextual and reproducible data for correct interpretations and reliable selection of drug candidates. The overall concept implies intra- and intermural aggregation of expertise (e.g., assay development, cell biology, statistics, bioinformatics) to perform multiple bioassays, under multiple conditions and readouts, using a common screening collection. Advantages of high input screens can be manifold: (i) to tackle discrepancies that may arise from the screens of libraries of variable size and content and assay types and conditions too narrow in scope; (ii) the opportunity to generate massive amounts of data applicable for multiple publications and funding requests; (iii) the educational benefits for students and post-docs collegially exposed to long-term programs; and (iv) the opportunity to democratize research and recruit small labs that could not otherwise join screening programs due to costs, timelines, and risks.

Legend for Supplementary Figures S1-S9.

Selenium (Se) is not essential for plant nutrition, however, growing evidence suggests a role of this element against oxidative stress. Here, we investigated the antioxidant effect of Se in an experimental system consisting of suspensions of olive callus cells exposed to hydrogen peroxide. Due to the disruptive capacity of oxidative stress on cytosolic Ca 2+ homeostasis, Se treatments were simply and effectively assessed with cytosolic Ca 2+ measurements. Se did not alter cell morphology, nor interfered with fluorometric determinations, suggesting a suitable model for monitoring alterations of cytosolic Ca 2+ . Furthermore, we used thapsigargin (TG), a known inhibitor of the Ca 2+ -ATPases of the endoplasmic reticulum, to establish whether Se had a specific impact on oxidative stress. We found that Se antagonized H 2 O 2 -mediated perturbations of cytosolic Ca 2+ , but was unable to offset TG-mediated disruptions. We conclude that Se merits consideration in strategies designed to tackle abiotic stress leading to oxidative insults.

Supplementary Figures S1-S9. Avocatin B is the most active avocado lipid analogue (S1); Kinetics of avocatin B-induced death (S2); Cell cycle analysis of avocatin B treated TEX cells (S3); Avocatin B increases DCFH-DA and DHE (S4); Avocatin B's activity is neutralized by co-incubation with PEG-SOD (S5); Jurkat T cells cultured in ethidium bromide medium have reduced mitochondria with decreased function (S6); Avocatin B is absent in mitochondria and cytosolic fractions of vehicle control treated TEX cells (S7); Avocatin B calibration curves and estimated concentraiton in cytosolic and mitochondrial fractions (S8); Avocatin B decreases levels of NADH and NADPH and increases ROS in OCI-AML2 cells (S9).

Supplementary Tables S1-S3. Summary of EC50 values for avocatin B in a panel of AML cell lines (S1); AML patient sample details used for annexin/PI (S2); AML patient sample details used for colony formation assays (S3).

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.