The ß-Carboline Harmine Has a Protective Immunomodulatory Role in Nonhealing Cutaneous Leishmaniasis.

Cutaneous leishmaniasis affects 1 million people worldwide annually. Although conventional treatments primarily target the parasite, there is growing interest in host immune modulation. In this study, we investigated the impact of synthetic ß-carboline harmine (ACB1801), previously shown to be immunoregulatory in cancer, on the pathology caused by a drug-resistant Leishmania major strain causing persistent cutaneous lesions. Exposure to ACB1801 in vitro had a modest impact on parasite burden within host macrophages. Moreover, it significantly increased major histocompatibility complex II and costimulatory molecule expression on infected dendritic cells, suggesting an enhanced immune response. In vivo, ACB1801 monotherapy led to a substantial reduction in lesion development and parasite burden in infected C57BL/6 mice, comparable with efficacy of amphotericin B. Transcriptomics analysis further supported ACB1801 immunomodulatory effects, revealing an enrichment of TNF-α, IFN-γ, and major histocompatibility complex II antigen presentation signatures in the draining lymph nodes of treated mice. Flow cytometry analysis confirmed an increased frequency (1.5×) of protective CD4+IFN-γ+TNF-α+ T cells and a decreased frequency (2×) in suppressive IL-10+FoxP3- T cells at the site of infection and in draining lymph nodes. In addition, ACB1801 downregulated the aryl hydrocarbon receptor signaling, known to enhance immunosuppressive cytokines. Thus, these results suggest a potential use for ACB1801 alone or in combination therapy for cutaneous leishmaniasis.

Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons.

Dopa-responsive dystonia (DRD) and Parkinson's disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites.

New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of Spike remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single-molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.

Salmonella-based platform for efficient delivery of functional binding proteins to the cytosol.

Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules.

Dicer1 imparts essential survival cues in Notch-driven T-ALL via miR-21-mediated tumor suppressor Pdcd4 repression.

The modulatory function of individual microRNAs (miRNAs) in Notch-driven T-cell acute lymphoblastic leukemias (T-ALLs) has recently been established. Although protumorigenic and tumor-suppressive miRNAs are implicated in disease onset in murine models of Notch-driven T-cell leukemia, whether Dicer1-processed miRNAs are essential for Notch-driven T-ALL is currently unknown. Here we used conditional and inducible genetic loss-of-function approaches to test whether the development and maintenance of Notch-driven T-ALL was dependent on Dicer1 function. Mice with specific inactivation of both Dicer1 alleles in the T-cell lineage did not develop Notch-driven T-ALL. In contrast, loss of 1 functional Dicer1 allele did not significantly perturb T-ALL onset and tumor progression. Inducible inactivation of Dicer1 in early stage polyclonal T-ALL cells was sufficient to abrogate T-ALL progression in leukemic mice, whereas late-stage monoclonal T-ALL cells were counterselected against loss of Dicer1. Lineage-tracing experiments revealed that Dicer1 deficiency led to the induction of apoptosis in T-ALL cells, whereas cell cycle progression remained unaltered. Through microarray-based miRNA profiling, we identified miR-21 as a previously unrecognized miRNA deregulated in both mouse and human T-ALL. Herein, we demonstrate that miR-21 regulates T-ALL cell survival via repression of the tumor suppressor Pdcd4.