ZBTB46 coordinates angiogenesis and immunity to control tumor outcome.

Tumor angiogenesis and immunity show an inverse correlation in cancer progression and outcome1. Here, we report that ZBTB46, a repressive transcription factor and a widely accepted marker for classical dendritic cells (DCs)2,3, controls both tumor angiogenesis and immunity. Zbtb46 was downregulated in both DCs and endothelial cells by tumor-derived factors to facilitate robust tumor growth. Zbtb46 downregulation led to a hallmark pro-tumor microenvironment (TME), including dysfunctional vasculature and immunosuppressive conditions. Analysis of human cancer data revealed a similar association of low ZBTB46 expression with an immunosuppressive TME and a worse prognosis. In contrast, enforced Zbtb46 expression led to TME changes to restrict tumor growth. Mechanistically, Zbtb46-deficient endothelial cells were highly angiogenic, and Zbtb46-deficient bone marrow progenitors upregulated Cebpb and diverted the DC program to immunosuppressive myeloid lineage output, potentially explaining the myeloid lineage skewing phenomenon in cancer4. Conversely, enforced Zbtb46 expression normalized tumor vessels and, by suppressing Cebpb, skewed bone marrow precursors toward immunostimulatory myeloid lineage output, leading to an immune-hot TME. Remarkably, Zbtb46 mRNA treatment synergized with anti-PD1 immunotherapy to improve tumor management in preclinical models. These findings identify ZBTB46 as a critical factor for angiogenesis and for myeloid lineage skewing in cancer and suggest that maintaining its expression could have therapeutic benefits.

The genetic and phenotypic spectra of adult genetic leukoencephalopathies in a cohort of 309 patients.

Genetic leukoencephalopathies (gLEs) are a highly heterogeneous group of rare genetic disorders. The spectrum of gLEs varies among patients of different ages. Distinct from the relatively more abundant studies of gLEs in children, only a few studies that explore the spectrum of adult gLEs have been published, and it should be noted that the majority of these excluded certain gLEs. Thus, to date, no large study has been designed and conducted to characterize the genetic and phenotypic spectra of gLEs in adult patients. We recruited a consecutive series of 309 adult patients clinically suspected of gLEs from Beijing Tiantan Hospital between January 2014 and December 2021. Whole-exome sequencing, mitochondrial DNA sequencing and repeat analysis of NOTCH2NLC, FMR1, DMPK and ZNF9 were performed for patients. We describe the genetic and phenotypic spectra of the set of patients with a genetically confirmed diagnosis and summarize their clinical and radiological characteristics. A total of 201 patients (65%) were genetically diagnosed, while 108 patients (35%) remained undiagnosed. The most frequent diseases were leukoencephalopathies related to NOTCH3 (25%), NOTCH2NLC (19%), ABCD1 (9%), CSF1R (7%) and HTRA1 (5%). Based on a previously proposed pathological classification, the gLEs in our cohort were divided into leukovasculopathies (35%), leuko-axonopathies (31%), myelin disorders (21%), microgliopathies (7%) and astrocytopathies (6%). Patients with NOTCH3 mutations accounted for 70% of the leukovasculopathies, followed by HTRA1 (13%) and COL4A1/2 (9%). The leuko-axonopathies contained the richest variety of associated genes, of which NOTCH2NLC comprised 62%. Among myelin disorders, demyelinating leukoencephalopathies (61%)-mainly adrenoleukodystrophy and Krabbe disease-accounted for the majority, while hypomyelinating leukoencephalopathies (2%) were rare. CSF1R was the only mutated gene detected in microgliopathy patients. Leukoencephalopathy with vanishing white matter disease due to mutations in EIF2B2-5 accounted for half of the astrocytopathies. We characterized the genetic and phenotypic spectra of adult gLEs in a large Chinese cohort. The most frequently mutated genes were NOTCH3, NOTCH2NLC, ABCD1, CSF1R and HTRA1.

Variation of CI-2 Conformers upon Addition of Methanol to Water: An IMS-MS-Based Thermodynamic Analysis.

Chymotrypsin inhibitor 2 (CI-2) is a well-studied, textbook example of a cooperative, two-state, native ↔ denatured folding transition. A recent hybrid ion mobility spectrometry (IMS)/mass spectrometry (MS) thermal denaturation study of CI-2 (the well-studied truncated 64-residue model) in water reported evidence that this two-state transition involves numerous (∼41) unique native and non-native (denatured) solution conformations. The characterization of so many, often low-abundance, states is possible because of the very high dynamic range of IMS-MS measurements of ionic species that are produced upon electrospraying CI-2 solutions from a variable temperature electrospray ionization source. A thermodynamic analysis of these states revealed large changes in enthalpy (ΔH) and entropy (ΔS) at different temperatures, and it was suggested that such variation might arise because of temperature-dependent conformational changes of the protein in response to changes in the conformational entropy and the dielectric permeability of water, which drops from a value of ε ∼ 79 at 24 °C to ∼ 60 at 82 °C. Herein, we examine how adding methanol to water influences the distributions of CI-2 conformers and their ensuing stabilities. The dielectric constant of a 60:40 water:methanol (MeOH) drops from ε ∼ 60 at 24 °C to ∼ 51 at 64 °C. Although the same set of conformers observed in water appears to be present in 60:40 water:MeOH, the abundance of each is substantially altered by the presence of methanol. Relative free energy values (ΔG) and thermodynamic values [ΔH and ΔS and heat capacities (ΔCp)] are derived from a Gibbs-Helmholtz analysis. A comparison of these data from water and water:MeOH systems allows rare insight into how variations in solvation and temperature affect many-state protein equilibria. While these studies confirm that variations in solvent dielectric constant with temperature affect the distributions of conformers that are observed, our findings suggest that other solvent differences may also affect abundances.

Autonomic dysfunction-dominant phenotype in a Chinese family with biallelic GGC repeat expansions in NOTCH2NLC.

The GGC repeat expansions in the NOTCH2NLC gene are associated with multiple neurodegenerative disorders. Herein, we report the clinical phenotype in a family with biallelic GGC expansions in NOTCH2NLC. Autonomic dysfunction was a prominent clinical manifestation in three genetically confirmed patients without dementia, parkinsonism, and cerebellar ataxia for > 12 years. A 7-T brain magnetic resonance imaging in two patients revealed a change in the small cerebral veins. The biallelic GGC repeat expansions may not modify the disease progression in neuronal intranuclear inclusion disease. Autonomic dysfunction-dominant may expand the clinical phenotype of NOTCH2NLC.

Targeted inhibition of thrombin attenuates murine neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants and an orphan disease with no specific treatment. Most patients with confirmed NEC develop moderate-severe thrombocytopenia requiring one or more platelet transfusions. Here we used our neonatal murine model of NEC-related thrombocytopenia to investigate mechanisms of platelet depletion associated with this disease [K. Namachivayam, K. MohanKumar, L. Garg, B. A. Torres, A. Maheshwari, Pediatr. Res. 81, 817-824 (2017)]. In this model, enteral administration of immunogen trinitrobenzene sulfonate (TNBS) in 10-d-old mouse pups produces an acute necrotizing ileocolitis resembling human NEC within 24 h, and these mice developed thrombocytopenia at 12 to 15 h. We hypothesized that platelet activation and depletion occur during intestinal injury following exposure to bacterial products translocated across the damaged mucosa. Surprisingly, platelet activation began in our model 3 h after TNBS administration, antedating mucosal injury or endotoxinemia. Platelet activation was triggered by thrombin, which, in turn, was activated by tissue factor released from intestinal macrophages. Compared to adults, neonatal platelets showed enhanced sensitivity to thrombin due to higher expression of several downstream signaling mediators and the deficiency of endogenous thrombin antagonists. The expression of tissue factor in intestinal macrophages was also unique to the neonate. Targeted inhibition of thrombin by a nanomedicine-based approach was protective without increasing interstitial hemorrhages in the inflamed bowel or other organs. In support of these data, we detected increased circulating tissue factor and thrombin-antithrombin complexes in patients with NEC. Our findings show that platelet activation is an important pathophysiological event and a potential therapeutic target in NEC.

Peptide-Based Nanoparticles for Systemic Extrahepatic Delivery of Therapeutic Nucleotides.

Peptide-based nanoparticles (PBN) for nucleotide complexation and targeting of extrahepatic diseases are gaining recognition as potent pharmaceutical vehicles for fine-tuned control of protein production (up- and/or down-regulation) and for gene delivery. Herein, we review the principles and mechanisms underpinning self-assembled formation of PBN, cellular uptake, endosomal release, and delivery to extrahepatic disease sites after systemic administration. Selected examples of PBN that have demonstrated recent proof of concept in disease models in vivo are summarized to offer the reader a comparative view of the field and the possibilities for clinical application.

Simultaneous Inhibition of Thrombosis and Inflammation Is Beneficial in Treating Acute Myocardial Infarction.

Myocardial ischemia reperfusion injury (IRI) in acute coronary syndromes is a condition in which ischemic/hypoxic injury to cells subtended by the occluded vessel continues despite successful resolution of the thrombotic obstruction. For decades, most efforts to attenuate IRI have focused on interdicting singular molecular targets or pathways, but none have successfully transitioned to clinical use. In this work, we investigate a nanoparticle-based therapeutic strategy for profound but local thrombin inhibition that may simultaneously mitigate both thrombosis and inflammatory signaling pathways to limit myocardial IRI. Perfluorocarbon nanoparticles (PFC NP) were covalently coupled with an irreversible thrombin inhibitor, PPACK (Phe[D]-Pro-Arg-Chloromethylketone), and delivered intravenously to animals in a single dose prior to ischemia reperfusion injury. Fluorescent microscopy of tissue sections and 19F magnetic resonance images of whole hearts ex vivo demonstrated abundant delivery of PFC NP to the area at risk. Echocardiography at 24 h after reperfusion demonstrated preserved ventricular structure and improved function. Treatment reduced thrombin deposition, suppressed endothelial activation, inhibited inflammasome signaling pathways, and limited microvascular injury and vascular pruning in infarct border zones. Accordingly, thrombin inhibition with an extraordinarily potent but locally acting agent suggested a critical role for thrombin and a promising therapeutic strategy in cardiac IRI.

Rapamycin Perfluorocarbon Nanoparticle Mitigates Cisplatin-Induced Acute Kidney Injury.

For nearly five decades, cisplatin has played an important role as a standard chemotherapeutic agent and been prescribed to 10-20% of all cancer patients. Although nephrotoxicity associated with platinum-based agents is well recognized, treatment of cisplatin-induced acute kidney injury is mainly supportive and no specific mechanism-based prophylactic approach is available to date. Here, we postulated that systemically delivered rapamycin perfluorocarbon nanoparticles (PFC NP) could reach the injured kidneys at sufficient and sustained concentrations to mitigate cisplatin-induced acute kidney injury and preserve renal function. Using fluorescence microscopic imaging and fluorine magnetic resonance imaging/spectroscopy, we illustrated that rapamycin-loaded PFC NP permeated and were retained in injured kidneys. Histologic evaluation and blood urea nitrogen (BUN) confirmed that renal structure and function were preserved 48 h after cisplatin injury. Similarly, weight loss was slowed down. Using western blotting and immunofluorescence staining, mechanistic studies revealed that rapamycin PFC NP significantly enhanced autophagy in the kidney, reduced the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as decreased the expression of the apoptotic protein Bax, all of which contributed to the suppression of apoptosis that was confirmed with TUNEL staining. In summary, the delivery of an approved agent such as rapamycin in a PFC NP format enhances local delivery and offers a novel mechanism-based prophylactic therapy for cisplatin-induced acute kidney injury.

COMMD10 inhibits HIF1α/CP loop to enhance ferroptosis and radiosensitivity by disrupting Cu-Fe balance in hepatocellular carcinoma.

BACKGROUND & AIMS: Copper (Cu) is an essential trace element whose serum levels have been reported to act as an effective indicator of the efficacy of radiotherapy. However, little is known about the role of Cu in radiotherapy. In this study we aimed to determine this role and investigate the precise mechanism by which Cu or Cu-related proteins regulate the radiosensitivity of hepatocellular carcinoma (HCC). METHODS: The expression and function of Cu and copper metabolism MURR1 domain 10 (COMMD10) were assessed via a Cu detection assay, immunostaining, real-time PCR, western blot, a radiation clonogenic assay and a 5-ethynyl-2'-deoxyuridine assay. Ferroptosis was determined by detecting glutathione, lipid peroxidation, malondialdehyde and ferrous ion (Fe) levels. The in vivo effects of Cu and COMMD10 were examined with Cu/Cu chelator treatment or lentivirus modification of COMMD10 expression in radiated mouse models. RESULTS: We identified a novel role of Cu in promoting the radioresistance of HCC cells. Ionizing radiation (IR) induced a reduction of COMMD10, which increased intracellular Cu and led to radioresistance of HCC. COMMD10 enhanced ferroptosis and radiosensitivity in vitro and in vivo. Mechanistically, low expression of COMMD10 induced by IR inhibited the ubiquitin degradation of HIF1α (by inducing Cu accumulation) and simultaneously impaired its combination with HIF1α, promoting HIF1α nuclear translocation and the transcription of ceruloplasmin (CP) and SLC7A11, which jointly inhibited ferroptosis in HCC cells. In addition, elevated CP promoted HIF1α expression by reducing Fe, forming a positive feedback loop. CONCLUSIONS: COMMD10 inhibits the HIF1α/CP loop to enhance ferroptosis and radiosensitivity by disrupting Cu-Fe homeostasis in HCC. This work provides new targets and treatment strategies for overcoming radioresistance in HCC. LAY SUMMARY: Radiotherapy benefits patients with unresectable or advanced hepatocellular carcinoma (HCC), but its effectiveness is hampered by radioresistance. Herein, we uncovered a novel role for copper in promoting the radioresistance of HCCs. This work has revealed new targets and potential treatment strategies that could be used to sensitize HCC to radiotherapy.

SFTPA1 is a potential prognostic biomarker correlated with immune cell infiltration and response to immunotherapy in lung adenocarcinoma.

Pulmonary surfactant protein A1 (SFTPA1) is a member of the C-type lectin subfamily that plays a critical role in maintaining lung tissue homeostasis and the innate immune response. SFTPA1 disruption can cause several acute or chronic lung diseases, including lung cancer. However, little research has been performed to associate SFTPA1 with immune cell infiltration and the response to immunotherapy in lung cancer. The findings of our study describe the SFTPA1 expression profile in multiple databases and was validated in BALB/c mice, human tumor tissues, and paired normal tissues using an immunohistochemistry assay. High SFTPA1 mRNA expression was associated with a favorable prognosis through a survival analysis in lung adenocarcinoma (LUAD) samples from TCGA. Further GeneOntology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that SFTPA1 was involved in the toll-like receptor signaling pathway. An immune infiltration analysis clarified that high SFTPA1 expression was associated with an increased number of M1 macrophages, CD8+ T cells, memory activated CD4+ T cells, regulatory T cells, as well as a reduced number of M2 macrophages. Our clinical data suggest that SFTPA1 may serve as a biomarker for predicting a favorable response to immunotherapy for patients with LUAD. Collectively, our study extends the expression profile and potential regulatory pathways of SFTPA1 and may provide a potential biomarker for establishing novel preventive and therapeutic strategies for lung adenocarcinoma.