Search Results (39)

The 2x2 Demonstrator, a prototype for the Deep Underground Neutrino Experiment (DUNE) liquid argon (LAr) Near Detector, was exposed to the Neutrinos from the Main Injector (NuMI) neutrino beam at Fermi National Accelerator Laboratory (Fermilab). This detector is a prototype of a new modular design for a liquid argon time-projection chamber (LArTPC), comprising a two-by-two array of four modules, each further segmented into two optically isolated LArTPCs. The 2x2 Demonstrator features a number of pioneering technologies, including a low-profile resistive field shell to establish drift fields, native 3D ionization pixelated imaging, and a high-coverage dielectric light readout system. The 2.4-tonne active mass detector is flanked upstream and downstream by supplemental solid-scintillator tracking planes, repurposed from the MINERvA experiment, which track ionizing particles exiting the argon volume. The antineutrino beam data collected by the detector over a 4.5 day period in 2024 include over 30,000 neutrino interactions in the LAr active volume—the first neutrino interactions reported by a DUNE detector prototype. During its physics-quality run, the 2x2 Demonstrator operated at a nominal drift field of 500 V/cm and maintained good LAr purity, with a stable electron lifetime of approximately 1.25 ms. This paper describes the detector and supporting systems, summarizes the installation and commissioning, and presents the initial validation of collected NuMI beam and off-beam self-triggers. In addition, it highlights observed interactions in the detector volume, including candidate muon antineutrino events. Full article

The concept of united airway disease (UAD) highlights the bidirectional relationship between inflammatory disorders of the upper airways—such as allergic rhinitis and chronic rhinosinusitis with or without nasal polyps (CRSwNP/CRSsNP)—and lower airway diseases, most notably asthma. This paradigm is supported by epidemiological, embryological, and immunological evidence demonstrating that airway inflammation represents a single, interconnected process rather than isolated compartmental pathology. Central to many UAD phenotypes is type 2 (T2) inflammation, driven by cytokines including IL-4, IL-5, and IL-13, and mediated by effector cells such as eosinophils and group 2 innate lymphoid cells (ILC2s). Epithelial barrier dysfunction often serves as the initiating trigger for this shared inflammatory cascade by production of TSLP, IL-25 and IL-33. Optimal diagnosis and management of UAD require an integrated, multidisciplinary framework. Clinical evaluation remains essential for patient characterization but must be complemented by pheno-endotypic assessment using imaging (CT), allergy testing, biomarker profiling (FeNO, blood eosinophils, IgE), and pulmonary function testing (spirometry, impulse oscillometry). Therapeutic strategies are layered, targeting both symptom control and inflammation across airway compartments. Standard approaches include intranasal and inhaled corticosteroids as well as saline irrigations, while severe T2-high disease increasingly benefits from biologic therapies (anti-IL-5/IL-5R, anti-IL-4R, anti-TSLP), which reduce dependence on systemic corticosteroids and surgical interventions such as endoscopic sinus surgery (ESS). Emerging precision-medicine models, particularly the “treatable traits” approach, further underscore the need to view the airway as a unified system. Collectively, these insights reinforce the clinical imperative of addressing upper and lower airway disease as a continuum, ensuring that inflammation in one district is neither overlooked nor treated in isolation. Full article

Regulating tumor invasion and metastasis is pivotal for improving cancer patient prognosis. While cell migration is a key factor in these processes, the non-targeted effects of chemoradiotherapy on cell motility remain poorly understood. In this study, we employed HeLa-FUCCI cells—a cervical cancer-derived HeLa cell line integrated with the Fluorescent Ubiquitination-Based Cell Cycle Indicator (FUCCI) probe, enabling the visualization of cell cycle phases—to investigate the radiation-induced impacts, including non-targeted effects, on cell migration. To create irradiated (In-field) and non-irradiated (out-of-field) regions, half of the culture dish was shielded with a lead block during irradiation. Cells were then exposed to 2 Gy X-rays, with or without cisplatin. Following irradiation, the cells were subjected to time-lapse imaging at 15 min intervals for 24 h, and the acquired data were analyzed using cell segmentation and tracking algorithms, Cellpose 2.0 and TrackMate 7. Without cisplatin, the migration velocity and total distance traveled of Out-of-field cells were significantly reduced compared to controls, suggesting a suppressive bystander signal. In contrast, with cisplatin treatment, these parameters significantly increased in both In-field and Out-of-field cells. This suggests that chemoradiotherapy may inadvertently enhance tumor cell motility outside the target volume, a critical finding with significant implications for therapeutic outcomes. Full article

Cancer-associated fibroblasts (CAFs) restructure collagen hydrogels via actomyosin-driven fibril bundling and crosslinking, increasing polymer density to generate mechanical stress that accelerates tumor proliferation. Conventional hydrogel models lack spatial heterogeneity, thus obscuring how localized stiffness gradients regulate cell cycle progression. To address this, we developed a collagen hydrogel-based microtissue platform integrated with programmable microstrings (single/double tethering), enabling real-time quantification of gel densification mechanics and force transmission efficiency. Using this system combined with FUCCI cell cycle biosensors and molecular perturbations, we demonstrate that CAF-polarized contraction increases hydrogel stiffness (350 → 775 Pa) and reduces pore diameter (5.0 → 1.9 μm), activating YAP/TAZ nuclear translocation via collagen–integrin–actomyosin cascades. This drives a 2.4-fold proliferation increase and accelerates G1/S transition in breast cancer cells. Pharmacological inhibition of YAP (verteporfin), actomyosin (blebbistatin), or collagen disruption (collagenase) reversed mechanotransduction and proliferation. Partial rescue upon CYR61 knockdown revealed compensatory effector networks. Our work establishes CAF-remodeled hydrogels as biomechanical regulators of tumor growth and positions gel-based mechanotherapeutics as promising anti-cancer strategies. Full article

Background: The COVID-19 pandemic caused by SARS-CoV-2 continues to pose a significant threat worldwide, with severe cases leading to hospitalization and death. This study aims to evaluate the potential use of serum nucleocapsid antigen (NAg) and Krebs von den Lungen-6 glycoprotein (KL-6) as biomarkers of severe COVID-19 and to investigate their correlation with clinical, radiological, and biochemical parameters. Methods: This retrospective study included 128 patients with confirmed SARS-CoV-2 infection admitted to a Neapolitan hospital in Italy between October 2020 and July 2021. Demographic, clinical, and laboratory data were collected, including serum levels of NAg and KL-6. The Chung et al. Computed Tomography Severity Score (TSS) was used to assess the severity of pneumonia, and outcomes were classified as home discharge, rehabilitation, and death. Statistical analyses were performed to compare Group I (home discharge and rehabilitation) and Group II (death, sub-intensive care, and ICU stay) based on demographic data, laboratory parameters, and TSS. Results: Group II patients showed worse outcomes with higher levels of NAg, KL-6, and inflammatory markers, including interleukin-6 (IL-6), interleukin-2 receptor (IL-2R), and adrenomedullin. TSS was also significantly higher in Group II, with a positive correlation between TSS and NAg and KL-6 levels. Group I patients had higher values of hemoglobin (Hb) and platelets (PLT), while Group II patients had higher values of C-reactive protein (CRP), procalcitonin (PCT), D-Dimer, and glycemia. No significant difference was observed in gender distribution. Conclusions: Serum NAg and KL-6 levels are potential biomarkers of severe COVID-19 pneumonia, with higher levels indicating greater inflammation and organ damage. NAg may help identify infected patients at an increased risk of severe COVID-19 and ensure their admission to the most appropriate level of care. KL-6 may help predict interstitial lung damage and the severity of clinical features. Further studies are needed to establish a decision-making cut-off for these biomarkers in COVID-19. Full article

(1) Background: Colorectal cancer (CRC) is the third most common malignant tumor worldwide and the second most common cause of cancer death. However, effective anti-CRC drugs are still lacking in clinical settings. This article investigated the anti-proliferative effect of involucrasin B on CRC Caco-2 cells. (2) Methods: This study employed a sulforhodamine B (SRB) method, colony formation experiments, flow cytometry, FastFUCCI assay, dual luciferase assay, and Western blot analysis for the investigation. (3) Results: The SRB method and colony formation experiments showed that involucrasin B exhibited an inhibitory effect on the Caco-2 cells cultured in vitro. Subsequently, the flow cytometry, FastFUCCI assay, and Western blotting results showed that involucrasin B induced cell cycle arrest in the G1 phase dose-dependently. Involucrasin B significantly enhanced the TGFβ RII protein level and SMAD3 phosphorylation, thus inhibiting the expression of CDK4 and cyclin D1 and causing G1 cell cycle arrest. (4) Conclusion: This study shows that involucrasin B exerts its anti-proliferative effect by regulating the TGFβ/SMAD2-3-4 pathway to cause G1 cycle arrest in Caco-2 cells. Full article

In the model system for genetics, Drosophila melanogaster, sexual differentiation and male courtship behavior are controlled by sex-specific splicing of doublesex (dsx) and fruitless (fru). In vitro and in vivo studies showed that female-specific Transformer (TRA) and the non-sex-specific Transformer 2 (TRA2) splicing factors interact, forming a complex promoting dsx and fru female-specific splicing. TRA/TRA2 complex binds to 13 nt long sequence repeats in their pre-mRNAs. In the Mediterranean fruitfly Ceratitis capitata (Medfly), a major agricultural pest, which shares with Drosophila a ~120 million years old ancestor, Cctra and Cctra2 genes seem to promote female-specific splicing of Ccdsx and Ccfru, which contain conserved TRA/TRA2 binding repeats. Unlike Drosophila tra, Cctra autoregulates its female-specific splicing through these putative regulatory repeats. Here, a yeast two-hybrid assay shows that CcTRA interacts with CcTRA2, despite its high amino acid divergence compared to Drosophila TRA. Interestingly, CcTRA2 interacts with itself, as also observed for Drosophila TRA2. We also generated a three-dimensional model of the complex formed by CcTRA and CcTRA2 using predictive approaches based on Artificial Intelligence. This structure also identified an evolutionary and highly conserved putative TRA2 recognition motif in the TRA sequence. The Y2H approach, combined with powerful predictive tools of three-dimensional protein structures, could use helpful also in this and other insect species to understand the potential links between different upstream proteins acting as primary sex-determining signals and the conserved TRA and TRA2 transducers. Full article

A large proportion of infertile men do not receive a clear diagnosis, being considered as idiopathic or unexplained cases due to infertility diagnosis based on standard semen parameters. Particularly in unexplained cases, the search for new indicators seems mandatory to provide specific information. In the etiopathogenesis of male infertility oxidative stress displays important roles by negatively affecting sperm quality and function. In this study, performed in a population of 34 idiopathic infertile men and in 52 age-matched controls, redox parameters were assessed in blood, leukocytes, spermatozoa, and seminal fluid and related to semen parameters. The main findings indicate that blood oxidative stress markers reflect seminal oxidative stress. Interestingly, blood leukocyte ROS production was significantly correlated to sperm ROS production and to semen parameters. Overall, these results suggest the potential employ of blood redox markers as a relevant and adjunctive tool for sperm quality evaluation aimed to preconception care. Full article

True genetically encoded monomeric fluorescent timers (tFTs) change their fluorescent color as a result of the complete transition of the blue form into the red form over time. Tandem FTs (tdFTs) change their color as a consequence of the fast and slow independent maturation of two forms with different colors. However, tFTs are limited to derivatives of the mCherry and mRuby red fluorescent proteins and have low brightness and photostability. The number of tdFTs is also limited, and there are no blue-to-red or green-to-far-red tdFTs. tFTs and tdFTs have not previously been directly compared. Here, we engineered novel blue-to-red tFTs, called TagFT and mTagFT, which were derived from the TagRFP protein. The main spectral and timing characteristics of the TagFT and mTagFT timers were determined in vitro. The brightnesses and photoconversions of the TagFT and mTagFT tFTs were characterized in live mammalian cells. The engineered split version of the TagFT timer matured in mammalian cells at 37 °C and allowed the detection of interactions between two proteins. The TagFT timer under the control of the minimal arc promoter, successfully visualized immediate-early gene induction in neuronal cultures. We also developed and optimized green-to-far-red and blue-to-red tdFTs, named mNeptusFT and mTsFT, which were based on mNeptune-sfGFP and mTagBFP2-mScarlet fusion proteins, respectively. We developed the FucciFT2 system based on the TagFT-hCdt1-100/mNeptusFT2-hGeminin combination, which could visualize the transitions between the G1 and S/G2/M phases of the cell cycle with better resolution than the conventional Fucci system because of the fluorescent color changes of the timers over time in different phases of the cell cycle. Finally, we determined the X-ray crystal structure of the mTagFT timer and analyzed it using directed mutagenesis. Full article

Replication stress has been suggested to be an ultimate trigger of carcinogenesis. Oncogenic signal, such as overexpression of CyclinE, has been shown to induce replication stress. Here, we show that various biological stresses, including heat, oxidative stress, osmotic stress, LPS, hypoxia, and arsenate induce activation of Chk1, a key effector kinase for replication checkpoint. Some of these stresses indeed reduce the fork rate, inhibiting DNA replication. Analyses of Chk1 activation in the cell population with Western analyses showed that Chk1 activation by these stresses is largely dependent on Claspin. On the other hand, single cell analyses with Fucci cells indicated that while Chk1 activation during S phase is dependent on Claspin, that in G1 is mostly independent of Claspin. We propose that various biological stresses activate Chk1 either directly by stalling DNA replication fork or by some other mechanism that does not involve replication inhibition. The former pathway predominantly occurs in S phase and depends on Claspin, while the latter pathway, which may occur throughout the cell cycle, is largely independent of Claspin. Our findings provide evidence for novel links between replication stress checkpoint and other biological stresses and point to the presence of replication-independent mechanisms of Chk1 activation in mammalian cells. Full article

The sirtuin 1 (SIRT1) activator resveratrol has emerged as a promising candidate for the prevention of vascular oxidative stress, which is a trigger for endothelial dysfunction. However, its clinical use is limited by low oral bioavailability. In this work, we have applied a previously developed computational protocol to identify the most promising derivatives from our in-house chemical library of resveratrol derivatives. The most promising compounds in terms of SIRT1 activation and oral bioavailability, predicted in silico, were evaluated for their ability to activate the isolated SIRT1 enzyme. Then, we assessed the antioxidant effects of the most effective derivative, compound 3d, in human umbilical vein endothelial cells (HUVECs) injured with H₂O₂ 100 µM. The SIRT1 activator 3d significantly preserved cell viability and prevented an intracellular reactive oxygen species increase in HUVECs exposed to the oxidative stimulus. Such effects were partially reduced in the presence of a sirtuin inhibitor, sirtinol, confirming the potential role of sirtuins in the activity of resveratrol and its derivatives. Although 3d appeared less effective than resveratrol in activating the isolated enzyme, the effects exhibited by both compounds in HUVECs were almost superimposable, suggesting a higher ability of 3d to cross cell membranes and activate the intracellular target SIRT1. Full article

The PCTAIRE subfamily belongs to the CDK (cyclin-dependent kinase) family and represents an understudied class of kinases of the dark kinome. They exhibit a highly conserved binding pocket and are activated by cyclin Y binding. CDK16 is targeted to the plasma membrane after binding to N-myristoylated cyclin Y and is highly expressed in post-mitotic tissues, such as the brain and testis. Dysregulation is associated with several diseases, including breast, prostate, and cervical cancer. Here, we used the N-(1H-pyrazol-3-yl)pyrimidin-4-amine moiety from the promiscuous inhibitor 1 to target CDK16, by varying different residues. Further optimization steps led to 43d, which exhibited high cellular potency for CDK16 (EC₅₀ = 33 nM) and the other members of the PCTAIRE and PFTAIRE family with 20–120 nM and 50–180 nM, respectively. A DSF screen against a representative panel of approximately 100 kinases exhibited a selective inhibition over the other kinases. In a viability assessment, 43d decreased the cell count in a dose-dependent manner. A FUCCI cell cycle assay revealed a G2/M phase cell cycle arrest at all tested concentrations for 43d, caused by inhibition of CDK16. Full article

DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and may contribute to the increased biological effectiveness of heavy ions in radiotherapy. To investigate the resection regulation of complex DSBs, we exposed G1 cells depleted for different candidate factors to heavy ions or α-particle radiation. Immunofluorescence microscopy was used to monitor the resection marker RPA, the DSB marker γH2AX and the cell-cycle markers CENP-F and geminin. The Fucci system allowed to select G1 cells, cell survival was measured by clonogenic assay. We show that in G1 phase the ubiquitin ligase RNF138 functions in resection regulation. RNF138 ubiquitinates the resection factor CtIP in a radiation-dependent manner to allow its DSB recruitment in G1 cells. At complex DSBs, RNF138′s participation becomes more relevant, consistent with the observation that also resection is more frequent at these DSBs. Furthermore, deficiency of RNF138 affects both DSB repair and cell survival upon induction of complex DSBs. We conclude that RNF138 is a regulator of resection that is influenced by DSB complexity and can affect the quality of DSB repair in G1 cells. Full article

Advanced prostate cancer (PCa) patients with bone metastases are treated with androgen pathway directed therapy (APDT). However, this treatment invariably fails and the cancer becomes castration resistant. To elucidate resistance mechanisms and to provide a more predictive pre-clinical research platform reflecting tumor heterogeneity, we established organoids from a patient-derived xenograft (PDX) model of bone metastatic prostate cancer, PCSD1. APDT-resistant PDX-derived organoids (PDOs) emerged when cultured without androgen or with the anti-androgen, enzalutamide. Transcriptomics revealed up-regulation of neurogenic and steroidogenic genes and down-regulation of DNA repair, cell cycle, circadian pathways and the severe acute respiratory syndrome (SARS)-CoV-2 host viral entry factors, ACE2 and TMPRSS2. Time course analysis of the cell cycle in live cells revealed that enzalutamide induced a gradual transition into a reversible dormant state as shown here for the first time at the single cell level in the context of multi-cellular, 3D living organoids using the Fucci2BL fluorescent live cell cycle tracker system. We show here a new mechanism of castration resistance in which enzalutamide induced dormancy and novel basal-luminal-like cells in bone metastatic prostate cancer organoids. These PDX organoids can be used to develop therapies targeting dormant APDT-resistant cells and host factors required for SARS-CoV-2 viral entry. Full article

Glioblastoma is a lethal brain cancer that commonly recurs after tumor resection and chemotherapy treatment. Depolarized resting membrane potentials and an acidic intertumoral extracellular pH have been associated with a proliferative state and drug resistance, suggesting that forced hyperpolarization and disruption of proton pumps in the plasma membrane could be a successful strategy for targeting glioblastoma overgrowth. We screened 47 compounds and compound combinations, most of which were ion-modulating, at different concentrations in the NG108-15 rodent neuroblastoma/glioma cell line. A subset of these were tested in the U87 human glioblastoma cell line. A FUCCI cell cycle reporter was stably integrated into both cell lines to monitor proliferation and cell cycle response. Immunocytochemistry, electrophysiology, and a panel of physiological dyes reporting voltage, calcium, and pH were used to characterize responses. The most effective treatments on proliferation in U87 cells were combinations of NS1643 and pantoprazole; retigabine and pantoprazole; and pantoprazole or NS1643 with temozolomide. Marker analysis and physiological dye signatures suggest that exposure to bioelectric drugs significantly reduces proliferation, makes the cells senescent, and promotes differentiation. These results, along with the observed low toxicity in human neurons, show the high efficacy of electroceuticals utilizing combinations of repurposed FDA approved drugs. Full article