Soluble Mesothelin-Related Peptide as a Prognosticator in Pleural Mesothelioma Patients Receiving Checkpoint Immunotherapy.

OBJECTIVE: Immune checkpoint therapy (ICT) has significantly impacted malignant pleural mesothelioma (MPM) treatment. Despite some promising results from combination therapies, nearly half of MPM patients do not benefit, underscoring the urgent need for reliable predictive biomarkers. This study assesses the prognostic value of serum soluble mesothelin-related peptide (SMRP) and PD-L1 levels in MPM patients receiving ICT. METHODS: We conducted a retrospective analysis of 125 MPM patients treated with ICT by measuring pre-ICT serum levels of SMRP and PD-L1. We also examined the correlation of these serum levels with tumor mRNA expressions of MSLN and PD-L1. Both univariable and multivariable Cox regression analyses were used to determine independent prognosticators for overall survival (OS). A prospective ICT clinical trial and our historical cohort were included for validation. RESULTS: Seventy-seven patients (62%) were treated with either anti-PD-(L)1 monotherapy, and the remaining 38% were given combination ICT. Higher pre-ICT SMRP levels were observed in epithelioid versus non-epithelioid MPM. Serum PD-L1 levels did not show significant differences between the groups. Univariable analysis identified durable clinical benefit, development of immune-related adverse events, and SMRP levels as significantly associated with OS. Multivariable analysis confirmed SMRP as an independent prognostic factor, with lower levels (≤1.35 nmol/L) correlating with improved OS. The association of high SMRP with worse prognosis was validated in the prospective ICT clinical trial cohort and not in our historical cohort treated without ICT. CONCLUSIONS: SMRP is a promising serum biomarker for predicting survival in MPM patients treated with ICT and warrants prospective investigation.

Role of Poly(A)-Binding Protein Cytoplasmic 1, a tRNA-Derived RNA Fragment-Bound Protein, in Respiratory Syncytial Virus Infection.

Respiratory Syncytial Virus (RSV) is a significant cause of lower respiratory tract infections (LRTI) across all demographics, with increasing mortality and morbidity among high-risk groups such as infants under two years old, the elderly, and immunocompromised individuals. Although newly approved vaccines and treatments have substantially reduced RSV hospitalizations, accessibility remains limited, and response to treatment varies. This underscores the importance of comprehensive studies on host-RSV interactions. tRNA-derived RNA fragments (tRFs) are recently discovered non-coding RNAs, notable for their regulatory roles in diseases, including viral infections. Our prior work demonstrated that RSV infection induces tRFs, primarily derived from the 5'-end of a limited subset of tRNAs (tRF5), to promote RSV replication by partially targeting the mRNA of antiviral genes. This study found that tRFs could also use their bound proteins to regulate replication. Our proteomics data identified that PABPC1 (poly(A)-binding protein cytoplasmic 1) is associated with tRF5-GluCTC, an RSV-induced tRF. Western blot experimentally confirmed the presence of PABPC1 in the tRF5-GluCTC complex. In addition, tRF5-GluCTC is in the anti-PABPC1-precipitated immune complex. This study also discovered that suppressing PABPC1 with its specific siRNA increased RSV (-) genome copies without impacting viral gene transcription, but led to less infectious progeny viruses, suggesting the importance of PABPC1 in virus assembly, which was supported by its interaction with the RSV matrix protein. Additionally, PABPC1 knockdown decreased the production of the cytokines MIP-1α, MIP-1ß, MCP-1, and TNF-α. This is the first observation suggesting that tRFs may regulate viral infection via their bound proteins.

One-year Safety and Effectiveness of Ustekinumab in Patients With Crohn's Disease: The K-STAR Study.

BACKGROUND: This study investigated the safety and effectiveness of ustekinumab (UST) in Korean patients with Crohn's disease (CD). METHODS: Adult patients with CD treated with UST were prospectively enrolled in the K-STAR (Post-MarKeting Surveillance for Crohn's Disease patients treated with STelARa) study between April 2018 and April 2022. Both the clinical effectiveness and adverse effects of UST therapy were analyzed. Missing data were handled using nonresponder imputation (ClinicalTrials.gov Identifier: NCT03942120). RESULTS: Of the 464 patients enrolled from 44 hospitals across Korea, 457 and 428 patients (Crohn's disease activity index ≥150) were included in the safety analysis and effectiveness analysis sets, respectively. At weeks 16 to 20 after initiating UST, clinical response, clinical remission, and corticosteroid-free remission rates were 75.0% (321 of 428), 64.0% (274 of 428), and 61.9% (265 of 428), respectively. At week 52 to 66, clinical response, clinical remission, and corticosteroid-free remission rates were 62.4% (267 of 428), 52.6% (225 of 428), and 50.0% (214 of 428), respectively. Combined effectiveness (clinical response + biochemical response) was achieved in 40.0% (171 of 428) and 41.6% (178 of 428) at week 16 to 20 and week 52 to 66, respectively. Biologic-naïve patients exhibited significantly higher rates of combined effectiveness than biologic-experienced patients (50.3% vs 30.7% at week 16-20, P < .001; 47.7% vs 36.0% at week 52-66, P = .014). No additional benefits were observed with the concomitant use of immunomodulators. Ileal location was independently associated with a higher probability of clinical remission compared with colonic or ileocolonic location at week 52 to 66. Adverse and serious adverse events were observed in 28.2% (129 of 457) and 12.7% (58 of 457), respectively, with no new safety signal associated with UST treatment. CONCLUSIONS: Ustekinumab was well-tolerated, effective, and safe as induction and maintenance therapy for CD in Korea.

Ustekinumab was well-tolerated and safe for Koran patients with Crohn's disease with no new safety signal as induction and maintenance therapy. Biologic-naïve patients exhibited better effectiveness outcomes, whereas combination therapy with immunomodulators was not superior to ustekinumab monotherapy.

Age Group-Specific Improvement of Vertebral Scoliosis after the Surgical Release of Congenital Muscular Torticollis.

Background Congenital muscular torticollis (CMT) is a common musculoskeletal disorder in children. Secondary scoliosis can occur in patients with CMT; however, the extent of inclination and improvement of scoliosis after surgical correction of CMT have not been adequately studied. In this study, we aimed to evaluate and measure the improvement in vertebral tilting after surgical correction according to age at the time of surgery. Methods Between June 2007 and January 2020, 831 patients with CMT underwent sternocleidomastoid release. Among them, 426 patients were enrolled, and their medical records were retrospectively reviewed. Ultimately, 210 patients available for radiological evaluation and analysis were enrolled in this study. The patients were divided into four groups according to age at the time of surgery to determine the relationship between age and changes in scoliosis. Results Our findings showed an improvement in scoliosis in all age groups after surgery. The results for follow-up after 1 year confirmed long-term improvement in vertebral tilting. The degree of improvement in scoliosis was significantly higher in the younger age group than in patients aged 18 years or older. Conclusion The effect of surgical release on scoliosis was significant in all age groups. The findings of this study suggest that CMT should be corrected before the age of 3 years to ensure an optimal surgical mitigation of scoliosis. Furthermore, in cases of neglected CMT, surgical release should be actively attempted because there is significant improvement.

Serum Extracellular Vesicle Protein Profiling for Prediction of Corneal Transplant Rejection.

BACKGROUND: Corneal transplantation is the most common transplant procedure worldwide. Despite immune and angiogenic privilege of the cornea, 50% to 70% of corneal transplants fail in high-risk recipients, primarily because of immune rejection. Therefore, it is crucial to identify predictive biomarkers of rejection to improve transplant survival. METHODS: In search for predictive biomarkers, we performed proteomics analysis of serum extracellular vesicles (EVs) in a fully major histocompatibility complex-mismatched (C57BL/6-to-BALB/c) murine corneal transplantation model, wherein 50% of transplants undergo rejection by day 28 following transplantation. RESULTS: Our time course study revealed a decrease in the number of serum EVs on day 1, followed by a gradual increase by day 7. A comparative analysis of proteomics profiles of EVs from transplant recipients with rejection (rejectors) and without rejection (nonrejectors) found a distinct enrichment of histocompatibility 2, Q region locus 2, which is a part of major histocompatibility complex-class I of donor C57BL/6 mice, in day 7 EVs of rejectors, compared with nonrejectors, syngeneic controls, or naïve mice. In contrast, serum amyloid A2, a protein induced in response to injury, was increased in day 7 EVs of nonrejectors. CONCLUSIONS: Our findings offer noninvasive EV-based potential biomarkers for predicting corneal allograft rejection or tolerance.

The Hepatokine Orosomucoid 2 Mediates Beneficial Metabolic Effects of Bile Acids.

Bile acids (BAs) are pleiotropic regulators of metabolism. Elevated levels of hepatic and circulating BAs improve energy metabolism in peripheral organs, but the precise mechanisms underlying the metabolic benefits and harm still need to be fully understood. In the current study, we identified orosomucoid 2 (ORM2) as a liver-secreted hormone (i.e., hepatokine) induced by BAs and investigated its role in BA-induced metabolic improvements in mouse models of diet-induced obesity. Contrary to our expectation, under a high-fat diet (HFD), our Orm2 knockout (Orm2-KO) exhibited a lean phenotype compared with C57BL/6J control, partly due to the increased energy expenditure. However, when challenged with a HFD supplemented with cholic acid, Orm2-KO eliminated the antiobesity effect of BAs, indicating that ORM2 governs BA-induced metabolic improvements. Moreover, hepatic ORM2 overexpression partially replicated BA effects by enhancing insulin sensitivity. Mechanistically, ORM2 suppressed interferon-γ/STAT1 activities in inguinal white adipose tissue depots, forming the basis for anti-inflammatory effects of BAs and improving glucose homeostasis. In conclusion, our study provides new insights into the molecular mechanisms of BA-induced liver-adipose cross talk through ORM2 induction.

Incongruity between T cell receptor recognition of breast cancer hotspot mutations ESR1 Y537S and D538G following exogenous peptide loading versus endogenous antigen processing.

T cell receptor engineered T cell (TCR T) therapies have shown recent efficacy against certain types of solid metastatic cancers. However, to extend TCR T therapies to treat more patients across additional cancer types, new TCRs recognizing cancer-specific antigen targets are needed. Driver mutations in AKT1, ESR1, PIK3CA, and TP53 are common in patients with metastatic breast cancer (MBC) and if immunogenic could serve as ideal tumor-specific targets for TCR T therapy to treat this disease. Through IFN-γ ELISpot screening of in vitro expanded neopeptide-stimulated T cell lines from healthy donors and MBC patients, we identified reactivity towards 11 of 13 of the mutations. To identify neopeptide-specific TCRs, we then performed single-cell RNA sequencing of one of the T cell lines following neopeptide stimulation. Here, we identified an ESR1 Y537S specific T cell clone, clonotype 16, and an ESR1 Y537S/D538G dual-specific T cell clone, clonotype 21, which were HLA-B*40:02 and HLA-C*01:02 restricted, respectively. TCR Ts expressing these TCRs recognized and killed target cells pulsed with ESR1 neopeptides with minimal activity against ESR1 WT peptide. However, these TCRs failed to recognize target cells expressing endogenous mutant ESR1. To investigate the basis of this lack of recognition we performed immunopeptidomics analysis of a mutant-overexpressing lymphoblastoid cell line and found that the ESR1 Y537S neopeptide was not endogenously processed, despite binding to HLA-B*40:02 when exogenously pulsed onto the target cell. These results indicate that stimulation of T cells that likely derive from the naïve repertoire with pulsed minimal peptides may lead to the expansion of clones that recognize non-processed peptides, and highlights the importance of using methods that selectively expand T cells with specificity for antigens that are efficiently processed and presented.

Reflection-Boosted Wearable Ring-Type Pulse Oximeters for SpO2 Measurement with High Sensitivity and Low Power Consumption.

In this study, we demonstrated a Monte Carlo simulation to model a finger structure and to calculate the intensity of photons passing through tissues, in order to determine optimal angular separation between a photodetector (PD) and a light-emitting diode (LED), to detect SpO2. Furthermore, our model was used to suggest a mirror-coated ring-type pulse oximeter to improve the sensitivity by up to 80% and improve power consumption by up to 65% compared to the mirror-uncoated structure. A ring-type pulse oximeter (RPO) is widely used to detect photoplethysmography (PPG) signals for SpO2 measurement during sleep and health-status monitoring. Device sensitivity and the power consumption of an RPO, which are key performance indicators, vary greatly with the geometrical arrangement of PD and LED within the inner surface of an RPO. We propose a reflection-boosted design of an RPO to achieve both high sensitivity and low power consumption, and determine an optimal configuration of a PD and LED by performing a 3D Monte Carlo simulation and confirming its agreement with experimental measurement. In order to confirm the reflection-boosted performance in terms of signal-to-noise ratio, R ratio, and perfusion index (PI), RPOs were fabricated with and without a highly reflective coating, and then used for SpO2 measurement from eight participants. Our simulation allows the numerical calculation of the intensity of photon passing and scattering through finger tissues. The reflection-boosted RPO enables reliable measurement with high sensitivity, resulting in less power consumption for the LED and longer device usage than conventional RPOs without any reflective coating, in order to maintain the same level of SNR and PI. Compared to the non-reflective reference RPO, the reflection-boosted RPO design greatly enhanced both detected light intensity (67% in dc and 322% in ac signals at a wavelength λ1 = 660 nm, and also 81% and 375% at λ2 = 940 nm, respectively) and PI (23.3% at λ1 and 25.5% at λ2). Thus, the reflection-boosted design not only enhanced measurement reliability but also significantly improved power consumption, i.e., by requiring only 36% and 30% power to drive the LED sources with λ1 and λ2, respectively, to produce the device performance of a non-reflective RPO reference. It is expected that our proposed RPO provides long-term monitoring capability with low power consumption and an enhanced PI for SpO2 measurement.

Purification and Characterization of Mitochondrial Mg2+-Independent Sphingomyelinase from Rat Brain.

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg2+-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg2+, Mn2+, N2+, Cu2+, Zn2+, Fe2+, and Fe3+ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg2+-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

A Phase II Window of Opportunity Study of Neoadjuvant PD-L1 versus PD-L1 plus CTLA-4 Blockade for Patients with Malignant Pleural Mesothelioma.

PURPOSE: We report the results of a phase II, randomized, window-of-opportunity trial of neoadjuvant durvalumab versus durvalumab plus tremelimumab followed by surgery in patients with resectable malignant pleural mesothelioma (MPM; NCT02592551). PATIENTS AND METHODS: The primary objective was alteration of the intratumoral CD8/regulatory T cell (Treg) ratio after combination immune checkpoint blockade (ICB) therapy. Secondary and exploratory objectives included other changes in the tumor microenvironment, survival, safety, tumor pathologic response (PR), and systemic immune responses. RESULTS: Nine patients received monotherapy and 11 received combination therapy. Seventeen of the 20 patients (85%) receiving ICB underwent planned thoracotomy. Both ICB regimens induced CD8 T-cell infiltration into MPM tumors but did not alter CD8/Treg ratios. At 34.1 months follow-up, patients receiving combination ICB had longer median overall survival (not reached) compared with those receiving monotherapy (14.0 months). Grade ≥3 immunotoxicity occurred in 8% of patients in the monotherapy group and 27% of patients in the combination group. Tumor PR occurred in 6 of 17 patients receiving ICB and thoracotomy (35.3%), among which major PR (>90% tumor regression) occurred in 2 (11.8%). Single-cell profiling of tumor, blood, and bone marrow revealed that combination ICB remodeled the immune contexture of MPM tumors; mobilized CD57+ effector memory T cells from the bone marrow to the circulation; and increased the formation of tertiary lymphoid structures in MPM tumors that were rich in CD57+ T cells. CONCLUSIONS: These data indicate that neoadjuvant durvalumab plus tremelimumab orchestrates de novo systemic immune responses that extend to the tumor microenvironment and correlate with favorable clinical outcomes.

Uridine Diphosphate Glucuronosyl Transferase 2B28 (UGT2B28) Promotes Tumor Progression and Is Elevated in African American Prostate Cancer Patients.

Prostate cancer (PCa) is the second most diagnosed cancer in the United States and is associated with metabolic reprogramming and significant disparities in clinical outcomes among African American (AA) men. While the cause is likely multi-factorial, the precise reasons for this are unknown. Here, we identified a higher expression of the metabolic enzyme UGT2B28 in localized PCa and metastatic disease compared to benign adjacent tissue, in AA PCa compared to benign adjacent tissue, and in AA PCa compared to European American (EA) PCa. UGT2B28 was found to be regulated by both full-length androgen receptor (AR) and its splice variant, AR-v7. Genetic knockdown of UGT2B28 across multiple PCa cell lines (LNCaP, LAPC-4, and VCaP), both in androgen-replete and androgen-depleted states resulted in impaired 3D organoid formation and a significant delay in tumor take and growth rate of xenograft tumors, all of which were rescued by re-expression of UGT2B28. Taken together, our findings demonstrate a key role for the UGT2B28 gene in promoting prostate tumor growth.

α-Iso-cubebene attenuates neointima formation by inhibiting HMGB1-induced monocyte to macrophage differentiation via suppressing ROS production.

α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis, a medicinal herb used to improve cardiovascular symptoms. To investigate the mechanisms involved, the effects of ICB on cellular production of reactive oxygen species (ROS) was determined using cultured human THP-1 cells. When THP-1 cells were stimulated with HMGB1, cellular concentration of ROS was increased in dose- and time-dependent manners. These increases were significantly attenuated in cells pretreated with NADPH oxidase inhibitors, diphenyleneiodonium chloride and apocynin, but not by other inhibitors related to ROS generation in monocytes. The expression of constitutively expressed NADPH oxidase (NOX) subunits including NOX1, NOX2, NOX4 and NOX5 was not affected by HMGB1, but HMGB1-induced ROS production was exclusively attenuated in NOX2-deficient cells using siRNA, suggesting an enhanced NOX2 complex assembly. When cells were stimulated with HMGB1, p47phox phosphorylation at ser345, ser359 and ser370 was increased in dose- and time-dependent manners, which were significantly attenuated in ICB (3-10 µg/mL)-pretreated cells. In addition, HMGB1-induced monocyte-macrophage differentiation (MMD) in bone marrow-derived cells isolated from mice were significantly attenuated in cells treated with apocynin and ICB. Also, macrophage infiltration and intimal hyperplasia in the wire-injured femoral artery were significantly attenuated in ICB-treated mice compared to wild-type control mice. The results of this study show that ICB inhibits HMGB1-induced MMD by suppressing ROS production in monocytes, thus suggest that ICB has therapeutic potential for vascular inflammation with subsequent intimal hyperplasia related to vascular injury.

Therapeutic Targeting of Macrophage Plasticity Remodels the Tumor-Immune Microenvironment.

SIGNIFICANCE: Comprehensive single-cell proteomics analyses of lung adenocarcinoma progression reveal the role of tumor-associated macrophages in resistance to PD-1 blockade therapy. See related commentary by Lee et al., p. 2515.

PDGFR-ß signaling mediates HMGB1 release in mechanically stressed vascular smooth muscle cells.

Mechanically stressed vascular smooth muscle cells (VSMCs) have potential roles in the development of vascular complications. However, the underlying mechanisms are unclear. Using VSMCs cultured from rat thoracic aorta explants, we investigated the effects of mechanical stretch (MS) on the cellular secretion of high mobility group box 1 (HMGB1), a major damage-associated molecular pattern that mediates vascular complications in stressed vasculature. Enzyme-linked immunosorbent assay (ELISA) demonstrated an increase in the secretion of HMGB1 in VSMCs stimulated with MS (0-3% strain, 60 cycles/min), and this secretion was markedly and time-dependently increased at 3% MS. The increased secretion of HMGB1 at 3% MS was accompanied by an increased cytosolic translocation of nuclear HMGB1; the acetylated and phosphorylated forms of this protein were significantly increased. Among various inhibitors of membrane receptors mediating mechanical signals, AG1295 (a platelet-derived growth factor receptor (PDGFR) inhibitor) attenuated MS-induced HMGB1 secretion. Inhibitors of other receptors, including epidermal growth factor, insulin-like growth factor, and fibroblast growth factor receptors, did not inhibit this secretion. Additionally, MS-induced HMGB1 secretion was markedly attenuated in PDGFR-ß-deficient cells but not in cells transfected with PDGFR-α siRNA. Likewise, PDGF-DD, but not PDGF-AA, directly increased HMGB1 secretion in VSMCs, indicating a pivotal role of PDGFR-ß signaling in the secretion of this protein in VSMCs. Thus, targeting PDGFR-ß-mediated secretion of HMGB1 in VSMCs might be a promising therapeutic strategy for vascular complications associated with hypertension.

The clock modulator Nobiletin mitigates astrogliosis-associated neuroinflammation and disease hallmarks in an Alzheimer's disease model.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, and there is a pressing need to identify disease-modifying factors and devise interventional strategies. The circadian clock, our intrinsic biological timer, orchestrates various cellular and physiological processes including gene expression, sleep, and neuroinflammation; conversely, circadian dysfunctions are closely associated with and/or contribute to AD hallmarks. We previously reported that the natural compound Nobiletin (NOB) is a clock-enhancing modulator that promotes physiological health and healthy aging. In the current study, we treated the double transgenic AD model mice, APP/PS1, with NOB-containing diets. NOB significantly alleviated ß-amyloid burden in both the hippocampus and the cortex, and exhibited a trend to improve cognitive function in these mice. While several systemic parameters for circadian wheel-running activity, sleep, and metabolism were unchanged, NOB treatment showed a marked effect on the expression of clock and clock-controlled AD gene expression in the cortex. In accordance, cortical proteomic profiling demonstrated circadian time-dependent restoration of the protein landscape in APP/PS1 mice treated with NOB. More importantly, we found a potent efficacy of NOB to inhibit proinflammatory cytokine gene expression and inflammasome formation in the cortex, and immunostaining further revealed a specific effect to diminish astrogliosis, but not microgliosis, by NOB in APP/PS1 mice. Together, these results underscore beneficial effects of a clock modulator to mitigate pathological and cognitive hallmarks of AD, and suggest a possible mechanism via suppressing astrogliosis-associated neuroinflammation.

Pillar-Based Mechanical Induction of an Aggressive Tumorigenic Lung Cancer Cell Model.

Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 µm diameter and 16 µm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 µm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and in vivo tumor colonization assays. Moreover, the pillar-induced morphological deformation in nuclei was accompanied by cell-cycle arrest in the S phase, leading to suppressed proliferation. While these marked traits of morphology-migration-proliferation support more aggressive characteristics of metastatic cancer cells, typical indices of epithelial-mesenchymal transition were not found, but instead, remarkable traces of amoeboidal transition were confirmed. Our study also emphasizes the importance of mechanical stimuli from the microenvironment during pathogenesis and how gained traits can be passed onto subsequent generations, ultimately affecting their pathophysiological behavior. Furthermore, this study highlights the potential use of pillar-based mechanical stimuli as an in vitro cell culture strategy to induce more aggressive tumorigenic cancer cell models.

Cancer stem cells, not bulk tumor cells, determine mechanisms of resistance to SMO inhibitors.

The emergence of treatment resistance significantly reduces the clinical utility of many effective targeted therapies. Although both genetic and epigenetic mechanisms of drug resistance have been reported, whether these mechanisms are stochastically selected in individual tumors or governed by a predictable underlying principle is unknown. Here, we report that the dependence of cancer stem cells (CSCs), not bulk tumor cells, on the targeted pathway determines the molecular mechanism of resistance in individual tumors. Using both spontaneous and transplantable mouse models of sonic hedgehog (SHH) medulloblastoma (MB) treated with an SHH/Smoothened inhibitor, sonidegib/LDE225, we show that genetic-based resistance occurs only in tumors that contain SHH-dependent CSCs (SD-CSCs). In contrast, SHH MBs containing SHH-dependent bulk tumor cells but SHH-independent CSCs (SI-CSCs) acquire resistance through epigenetic reprogramming. Mechanistically, elevated proteasome activity in SMOi-resistant SI-CSC MBs alters the tumor cell maturation trajectory through enhanced degradation of specific epigenetic regulators, including histone acetylation machinery components, resulting in global reductions in H3K9Ac, H3K14Ac, H3K56Ac, H4K5Ac, and H4K8Ac marks and gene expression changes. These results provide new insights into how selective pressure on distinct tumor cell populations contributes to different mechanisms of resistance to targeted therapies. This insight provides a new conceptual framework to understand responses and resistance to SMOis and other targeted therapies.

Nuclear S-nitrosylation impacts tissue regeneration in zebrafish.

Despite the importance of nitric oxide signaling in multiple biological processes, its role in tissue regeneration remains largely unexplored. Here, we provide evidence that inducible nitric oxide synthase (iNos) translocates to the nucleus during zebrafish tailfin regeneration and is associated with alterations in the nuclear S-nitrosylated proteome. iNos inhibitors or nitric oxide scavengers reduce protein S-nitrosylation and impair tailfin regeneration. Liquid chromatography/tandem mass spectrometry reveals an increase of up to 11-fold in the number of S-nitrosylated proteins during regeneration. Among these, Kdm1a, a well-known epigenetic modifier, is S-nitrosylated on Cys334. This alters Kdm1a binding to the CoRest complex, thus impairing its H3K4 demethylase activity, which is a response specific to the endothelial compartment. Rescue experiments show S-nitrosylation is essential for tailfin regeneration, and we identify downstream endothelial targets of Kdm1a S-nitrosylation. In this work, we define S-nitrosylation as an essential post-translational modification in tissue regeneration.

A Pivotal Role for AP-1-Mediated Osteopontin Expression in the Increased Migration of Vascular Smooth Muscle Cells Stimulated With HMGB1.

Migration of vascular smooth muscle cells (VSMCs) plays an essential role in the development of vascular remodeling in the injured vasculatures. Previous studies have identified high-mobility group box 1 (HMGB1) as a principal effector mediating vascular remodeling; however, the mechanisms involved have not been fully elucidated. Thus, this study investigated the role of HMGB1 on VSMC migration and the underlying molecular mechanisms involved. VSMCs were ex plant cultured using rat thoracic aorta, and the cellular migration was measured using wound-healing assay. Osteopontin (OPN) mRNA and protein were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. The OPN promoter was cloned into pGL3 basic to generate a pLuc-OPN-2284 construct. Migration of VSMCs stimulated with HMGB1 (100ng/ml) was markedly increased, which was significantly attenuated in cells pretreated with MPIIIB10 (100-300ng/ml), a neutralizing monoclonal antibody for OPN as well as in cells deficient of OPN. In VSMCs stimulated with HMGB1, OPN mRNA and protein levels were significantly increased in association with an increased promotor activity of OPN gene. Putative-binding sites for activator protein 1 (AP-1) and CCAAT/enhancer-binding protein beta (C/EBPß) in the indicated promoter region were suggested by TF Search, and the HMGB1-induced expression of OPN was markedly attenuated in cells transfected with siRNA for AP-1. VSMC stimulated with HMGB1 also showed an increased expression of AP-1. Results of this study suggest a pivotal role for AP-1-induced OPN expression in VSMC migration induced by HMGB1. Thus, the AP-1-OPN signaling axis in VSMC might serve as a potential therapeutic target for vascular remodeling in the injured vasculatures.

Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function.

Astrocytes display extraordinary morphological complexity that is essential to support brain circuit development and function. Formin proteins are key regulators of the cytoskeleton; however, their role in astrocyte morphogenesis across diverse brain regions and neural circuits is unknown. Here, we show that loss of the formin protein Daam2 in astrocytes increases morphological complexity in the cortex and olfactory bulb, but elicits opposing effects on astrocytic calcium dynamics. These differential physiological effects result in increased excitatory synaptic activity in the cortex and increased inhibitory synaptic activity in the olfactory bulb, leading to altered olfactory behaviors. Proteomic profiling and immunoprecipitation experiments identify Slc4a4 as a binding partner of Daam2 in the cortex, and combined deletion of Daam2 and Slc4a4 restores the morphological alterations seen in Daam2 mutants. Our results reveal new mechanisms regulating astrocyte morphology and show that congruent changes in astrocyte morphology can differentially influence circuit function.