Abstract Genome sequencing is the first-line diagnostic method for primary mitochondrial diseases (PMDs), yet its effectiveness is limited by variants of uncertain significance or unresolved genetic findings. We systematically evaluated the clinical performance of fibroblast-based functional testing, comprised of respiratory chain enzyme assays, blue native polyacrylamide gel electrophoresis with in-gel activity staining (BN-PAGE), complex I assembly assay, and targeted protein abundance assessments, in a cohort of 204 genetically confirmed PMD patients, 51 healthy controls, and 53 patients with differential diagnoses. Individually, enzyme assays, BN-PAGE, and complex I assembly assay showed sensitivities of 46%, 40%, and 49%, with specificities of 93%, 98%, and 99%, respectively. Combined, the assays achieved an overall sensitivity of 76%, a specificity 93%, a positive predictive value 96%, and a negative predictive value of 67%. Sensitivity was highest for isolated respiratory chain deficiencies, nuclear DNA-encoded mitochondrial translation defects, cofactor deficiencies, and mitochondrial aminoacyl-tRNA synthetase disorders, whereas mitochondrial DNA variants and maintenance defects remained challenging. Secondary mitochondrial dysfunction was rare. The strong clinical utility of comprehensive fibroblast functional testing improves PMD diagnosis when used complementary to genomic sequencing.
Abstract Short-chain fatty acids (SCFA) protect against central nervous system (CNS) autoimmunity, but the receptor-mediated mechanisms underlying these effects remain poorly understood, limiting their therapeutic exploitation. Here, we show that selective oral activation of free fatty acid receptor 2 (FFAR2) with the synthetic agonist Cpd1 effectively ameliorates established experimental autoimmune encephalomyelitis (EAE), demonstrating therapeutic efficacy after the onset of the autoimmune response. Cpd1 treatment promoted regulatory T cells and type 3 innate lymphoid cells in the intestine while reducing inflammatory infiltration in the CNS, indicating that modulation of intestinal immunity is sufficient to restrain neuroinflammation. Transcriptomic analysis of intestinal CD4⁺ T cells revealed broad suppression of pathways controlling T-cell activation and proliferation. Mechanistically, the therapeutic effects of Cpd1 required interleukin-22 (IL-22), as antibody-mediated IL-22 neutralization abolished disease protection, identifying IL-22 as a key downstream effector of FFAR2 signaling. Together, these findings establish selective FFAR2 agonism as a tractable strategy to therapeutically modulate the gut-CNS immune axis and identify FFAR2 as a promising target for the treatment of multiple sclerosis and other autoimmune diseases.
Abstract Despite decades of intensive research and substantial clinical gains, malaria remains a major global health challenge exacerbated by the continued emergence of drug-resistant strains of the causative agent, Plasmodium parasites. Encouragingly, recent advances in functional genomics, chemical biology and computational science are reshaping antimalarial drug discovery. In this review, we examine the discovery and development of next-generation antimalarials, including advances in phenotypic and target-based screening, omics-enabled target discovery and emerging therapeutic modalities such as long-acting agents, targeted covalent inhibitors and host-directed therapies. We further evaluate the opportunities and limitations of drug repurposing and discuss how artificial intelligence and data-driven approaches are reshaping target identification, compound optimisation and clinical development. Finally, we argue that future success will depend not only on scientific innovation but also on interdisciplinary collaboration, equitable partnerships, open science and the development of accessible therapies tailored to malaria-endemic populations.
Abstract Respiratory syncytial virus (RSV) remains a major cause of severe respiratory disease, and stabilization of the prefusion (preF) conformation of the F glycoprotein is central for vaccine development. Here, we report a structure-guided engineering strategy that enhances preF stability by reducing the hydrophobic cavity within the trimeric F protein. Targeted modifications at metastability-associated sites generated RVF-88, a disulfide-free stabilized preF immunogen that preserves key neutralizing epitopes, including antigenic site Ø, while exhibiting improved structural integrity and long-term storage stability. Formulated as an unadjuvanted bivalent vaccine, RVF-88 elicited potent neutralizing antibody responses and durable immune protection lasting up to 5 months in mice. Vaccination also protected both mice and cotton rats against RSV challenge. Structural analyses confirmed the intended cavity-reduction design, revealing a reduced apical hydrophobic cavity volume and surface area while maintaining the prefusion architecture. Together, these findings establish hydrophobic cavity reduction as a rational strategy for stabilizing prefusion RSV F and provide a promising next-generation vaccine candidate with improved stability and immunogenicity for further clinical development.
Abstract Triple-negative breast cancer (TNBC) has a high incidence of metastasis and limited therapeutic options. Here, we identify a multimodal and targetable miR-342-E2F network that regulates metastatic outgrowth in TNBC. Through integrating clinical and experimental datasets, we uncover miR-342 as a suppressor of TNBC metastasis. Temporal re-expression of miR-342 significantly inhibited metastatic progression in both immunocompetent and xenograft TNBC models by specifically limiting the outgrowth of disseminated tumour cells. Using multi-omics profiling, we identified the global complement of miR-342 targets, demonstrating that it directly suppresses transcriptional and post-transcriptional networks that converge to dampen E2F signalling. Single-cell analysis of matched primary and metastatic patient-derived TNBC samples reveals activation of E2F signalling in metastasis that corresponds with reduced miR-342 host gene EVL expression. Furthermore, pharmacologic inhibition of this pathway with the CDK4/6 inhibitor palbociclib specifically reduces metastatic outgrowth of pre-established TNBC lesions. Our findings reveal that TNBCs with low miR-342/high E2F signalling have increased metastatic competency and may be amenable to CDK4/6 inhibitor therapy, offering a potential strategy for targeted intervention to limit TNBC metastasis.
