Prion diseases are a group of fatal neurodegenerative diseases that proceed through the templated conversion of the normal PrPC protein to a self-propagating and infectious form termed PrPSc. This conversion process is central to the disease progression. However, because of difficulties in producing functional PrPSc molecules that can be selectively modified with chemical probes, many aspects of PrPSc biology cannot be directly studied. To overcome this limitation, we substituted p-azido-l-phenylalanine (AzF), a small click chemistry-reactive amino acid, for tryptophan residue 99 of PrPC. The W99AzF PrPC substrate can efficiently and faithfully propagate either infectious or noninfectious PrPSc conformers in vitro. Critically, W99AzF PrPSc remains amenable to click chemistry by various ligands after the prion conversion process. Through the combination of site-specific substitution, the modularity of click chemistry, and the functional diversity of click labels, a multitude of modified prions can now be produced to ask targeted questions about the biochemical and biological bases of prion infectivity.
Acute coronary syndrome (ACS) is a time-critical medical emergency in which early guideline-based prehospital diagnosis and treatment are crucial for the subsequent care pathway. The aim of this study was to compare documented adherence to selected prehospital ACS process indicators between two provider structures operating within the same municipal EMS system. As part of the retrospective, bicentric observational study MONAH-1, all prehospital physician missions with typical ACS diagnoses in Magdeburg between 2014 and 2018 were analysed. This prespecified intra-urban subgroup analysis compared one EMS physician base staffed by MD1 with two EMS physician bases staffed by MD2. Because case retrieval was diagnosis-targeted from archived protocols rather than based on a prospectively maintained screening registry, a full flow diagram of all EMS missions could not be reconstructed reliably; endpoint-specific denominators are therefore reported in the text and tables. Multivariable analyses were adjusted for age and gender only and should be interpreted as partially adjusted exploratory models. A total of 1,438 emergency physician interventions were evaluated (MD1: n = 661; MD2: n = 777). MD1 showed documented higher rates of 12-lead ECGs (76.9% vs. 43.5%; aOR 4.24 [95% CI 3.36-5.35]), ASA administration (91.4% vs. 70.9%; aOR 4.38 [3.19-6.00]) and heparin administration (92.6% vs. 68.0%; aOR 5.86 [4.21-8.16]). In the descriptive indication-positive subgroup with documented VAS ≥ 4, morphine was documented more often at MD1 (70.6% vs. 54.5%); the exploratory adjusted morphine model was based on missions with documented pain assessment (aOR 2.67 [2.04-3.50]). No significant differences were found for indication-based nitro-glycerine and oxygen administration. Prehospital dwell time was longer at MD1 (median 34 vs. 29 min; p < 0.001). Documented adherence to selected prehospital ACS process indicators differed between the two providers. MD1 showed higher documented rates for several process measures, but the retrospective design, heterogeneous documentation formats, limited case-mix adjustment, and the possibility of reverse causation for dwell time preclude causal inference or conclusions about patient benefit. The findings are hypothesis-generating and primarily relevant for local quality assurance and prospective validation. The study was registered retrospectively in the German Clinical Trials Register (DRKS00036944) on 27 August 2025.
Recent neutral randomized controlled trials have created clinical equipoise around the treatment of obstructive sleep apnea (OSA) for the secondary prevention of cardiovascular disease. These findings are in part limited by poor compliance, lack of treatment optimization and personalization strategies with OSA therapy. This pilot study aims to assess the feasibility and impact of a personalized OSA treatment program on therapy compliance and cardiovascular health in patients with acute coronary syndrome (ACS) diagnosed with OSA. Patients with ACS and OSA (oxygen desaturation index 3% > 10 events/hour) will be recruited and randomized to receive either personalized OSA therapy (PT) or usual care (UC) with six-month follow-up. The PT group will receive standard medical care for ACS plus the choice of continuous positive airway pressure and/or mandibular advancement device therapy for OSA. Treatment optimization strategies including treatment acclimatization, augmented support and patient engagement tools will be implemented. The UC group will receive standard medical care for ACS but no treatment for OSA. The primary outcome is the feasibility of personalized treatment for OSA assessed by study and treatment acceptance, and treatment compliance. Secondary outcomes include treatment effectiveness, cardiovascular health markers and patient reported outcomes across the six-months intervention. This study will provide novel findings on the impact of personalized and optimized OSA therapy on patient compliance, preliminary treatment effectiveness and health outcomes in patients with established cardiovascular disease. Such novel data will inform the design and delivery of future large scale randomized clinical trials to assess cardiovascular disease benefit in patients with OSA. ACTRN 12620000050954. Registered 22 January 2020, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=378536&isReview=true.
Lipid mediators can control the inflammatory response in infectious diseases, including leishmaniasis. However, these mediators may promote antagonistic roles in the Leishmania-host interaction depending on the species involved in the infection. Herein, we analyzed the role of mediators in the Leishmania-host interaction in the experimental and clinical context, aiming to identify the main cell types studied, as well as the main eicosanoids and their influence during the infection. The main lipid mediators studied were the eicosanoids LTB4 and PGE2, which are related to the inflammatory response in cutaneous and visceral leishmaniasis. In vitro models using macrophages and neutrophils infection reveal that LTB4 plays a fundamental role in reducing the parasite load, while PGE2 and PGF2α suppress the immune response, favoring the survival of the parasite in the host. In the in vivo infection, PGE2 is related to the visceralization process of the disease and the persistence of tegumentary lesions. An emerging role in pathophysiology has been pointed out for the mediators of the HETE class and for Resolvin D1, which act favoring Leishmania infection and are associated with more severe cases of the disease. Thus, it can be concluded that lipid mediators play crucial roles in the Leishmania-host interaction, modulating the inflammatory response and disease progression. Studies exploring the contribution of intervention in the production of lipid mediators during the course of the disease are still needed.
Dental caries is among the most prevalent infectious chronic diseases worldwide, yet localized acidogenic activity associated with cariogenic processes often remains difficult to visualize at the whole-dentition level. This highlights the urgent need for analytical tools that enable imaging of localized acidity-related changes. However, current methods, such as salivary test strips and hand-held pH meters, remain limited to single-point or averaged measurements, lacking the spatial resolution required to visualize the distribution of acidic microenvironments. Herein, we present a conceptual approach that moves from single-point sensing to spatially resolved imaging through an AI-enhanced optical imaging platform for visualizing tooth-surface acidity patterns. The method integrates an imprint-based colorimetric assay using a food-derived pH indicator, with an AI-assisted image analysis workflow guided by dentist-labeled annotations, and explores the feasibility of supervised, annotation-guided image analysis under pilot conditions. By mapping localized acidogenic activity as an indicator associated with cariogenic bacteria, the method provides interpretable spatial readouts of acidic microenvironments. The method allows visual detection of lactic acid at concentrations as low as 5 mM under the tested conditions, and provides multisite results within 5 min, showing qualitative spatial correspondence with clinical findings within this pilot cohort. This approach demonstrates the initial feasibility of treating the dentition as an analytically accessible surface, and explores an AI-assisted analytical concept for relating spatial pH patterns to clinician-identified caries-associated regions on biological surfaces. By combining accessible materials with intelligent data interpretation, this study presents a proof-of-concept pathway toward low-cost visualization of microenvironments for at-home and low-resource settings, offering particular value for children, the elderly, and individuals in remote areas.
To describe the epidemiology of local signs at intravascular catheter insertion sites across catheter types and care settings, to compare the identification of redness between local investigators and experts, and to evaluate the association between redness and blood culture positivity in patients with suspected infection. DeepCath was a prospective multicentre cohort study conducted between September 2022 and December 2023 in France and Switzerland. Adult patients with short-term central venous catheters (CVCs), arterial catheters (ACs), peripheral venous catheters (PVCs), peripherally inserted central catheters (PICCs), or midlines were included. Local investigators captured one photograph per insertion site and recorded local signs. Four experts independently reassessed all images, serving as the reference standard. Agreement between local investigators and experts on redness was evaluated using Cohen's kappa. Among patients with suspected infection, the association between redness and blood culture results was evaluated. Among 5,164 collected images, 2,670 eligible catheters (one image per catheter) were analysed: 1,447 PVCs (54.2%), 608 CVCs (22.8%), 296 ACs (11.1%), and 319 PICCs/midlines (11.9%). Local investigators reported signs of local infection in 257/2,670 catheters (9.6%), mainly redness (218/2,670, 8.2%). Experts identified local infection signs in 376/2,670 catheters (14.1%), including redness in 364/2,670 (13.6%). Redness prevalence varied by catheter type and was highest for PICCs/midlines (82/319, 25.7% by expert assessment). Overall agreement for redness was fair (Cohen's kappa of 0.40, 95% CI 0.35-0.46) and was observed in 2,358/2,670 images (88.3%). Among 732 patients with suspected infection who underwent blood cultures diagnostic, redness was more frequent in those with positive cultures (23/71, 32.4%) than in those with negative (15/225, 6.7%) or pending cultures (40/436, 9.2%; p<0.0001). Local signs at catheter insertion sites are frequent and differ across catheter type. Redness is under-recognised in routine clinical assessment compared with expert review, supporting the need for standardized, potentially artificial intelligence-assisted, approaches in daily practice.
Bacterial infection is now a significant obstacle to the effective repair and regeneration of both soft tissues and bone in clinical settings. Managing infected sites is challenging due to multiple antimicrobial resistance, abnormal inflammatory responses, and an excessive inflammatory microenvironment. To meet the complex healing needs following soft tissue or bone, an immunomodulatory thermosensitive hydrogel was designed and synthesized. The composite hydrogel (CQDs@UPDA@gel) combines carbon quantum dots (CQDs) as the second near-infrared window (NIR-II) photothermal agents (PTAs) and ultrasmall polydopamine nanoparticles (UPDA NPs) as reactive oxygen species (ROS) scavengers and immunomodulators, embedded within a chitosan matrix coordinated with Cu2+ that enhances the good swelling performance and stable rheological properties of the hydrogel. This synergistic approach simultaneously inhibits bacterial infection and alters the immunosuppressive microenvironment, thereby promoting coordinated soft tissue or bone regeneration. In vivo studies have shown that the hydrogel can encourage the regeneration of soft tissue or bone under NIR-II laser irradiation for the treatment of infectious wounds, subcutaneous abscesses, and infectious bone defects in the maxillofacial region. Overall, this project offers a general strategy for creating multifunctional hydrogels that both eradicate pathogens and modulate the immune microenvironment to support the repair of complex soft tissue or bone infections.
Early and accurate diagnosis of Chikungunya virus (CHIKV) infection is critical for controlling its outbreaks. CRISPR/Cas-based detection offers promise for pathogen identification, yet one-step CRISPR/Cas systems are limited by suboptimal sensitivity, field-deployability, and adaptability to complex clinical samples, hindering their use in rapid outbreak response. Here, we developed a CRISPR/Cas13a-based One-Step System for Rapid Detection of Emerging Viruses (CRISPR-CORE) and applied it during the CHIKV outbreak in Guangdong Province, China. Multidimensional optimizations enabled the CRISPR-CORE system to achieve a limit of detection of 5 copies/μL within 40 min. An extraction-free RNA release protocol for CHIKV in blood samples and a premixed reagent approach were implemented. Furthermore, a portable fluorescence detector was used to enhance user-friendliness in point-of-care (POC) settings. The clinical CHIKV genomic information was identified through hybrid capture sequencing, informing the design of highly specific CRISPR RNA (crRNA). Clinical validation across three regions yielded 92.6% sensitivity and 100% specificity, underscoring the applicability and reliability of the CRISPR-CORE system. Our system demonstrates its suitability for CHIKV outbreak detection. It facilitates rapid and POC testing for emerging viruses in resource-limited settings. Furthermore, it provides a universal strategy for the prevention and control of infectious diseases.
With zoonotic outbreaks on the rise, rapid and accurate infectious disease diagnostics are critical for both human and animal health. Traditional lateral flow assays lack sensitivity and specificity, while isothermal amplification methods like LAMP, though rapid, can be hindered by optical detection issues. We present an electrochemical nucleic acid amplification test (NAAT) that combines isothermal amplification with real-time voltammetric detection. This novel method relies on monitoring of amplification-associated proton release through redox probing of a pH-sensing compound. The method shows high concordance with RT-qPCR under the tested conditions and demonstrates a limit of detection (LOD) of 50 copies/reaction for IAV. In RNA purified from clinical samples, 231/234 (98.7%) are concordant with RT-qPCR for SARS-CoV-2, IAV, IBV, or RSV A. In a proof-of-concept extraction-free workflow, 14/14 equine nasal swab samples are correctly classified for EHV-5 relative to qPCR. Together, these data support the feasibility of real-time voltammetric LAMP across several sample types, while broader point-of-care validation across additional matrices and prospective cohorts remains to be established.
Extending our research on the design, synthesis, and pharmacological evaluation of antitrypanosomatid nitroheterocyclic aromatic adamantanes, we have identified hydrazone derivatives conjugated with 5-nitrothiophene that have submicromolar activity against trypanosomes. Key structural features, namely, the incorporation of the adamantane ring into the skeleton of the new compounds, the nature and the length of the hydrazone linker, and the nitroheterocycle moiety were evaluated to optimize both efficacy and safety profiles. It is also shown that these compounds function as prodrugs that are bioactivated by parasite-specific nitroreductase enzymes, primarily NTR-1. These findings suggest that nitroheterocyclic aromatic adamantanohydrazones represent promising candidates for further development as antitrypanosomal agents.
Current standard of care, artemisinin-based therapies for malaria, are threatened by emerging drug resistance. Developing antimalarials with novel mechanisms of action and low propensity for resistance is of the highest priority. Here, we explore the target landscape of MMV022224, a promising antimalarial that is active against multiple stages of Plasmodium falciparum and refractory to resistance generation. Using two orthogonal chemical proteomics approaches, chemical pulldown and thermal proteome profiling, we demonstrate that MMV022224 binds selectively and with high affinity to the genetically essential P. falciparum protein kinase 6 (PfPK6), as well as to several additional Plasmodium kinases. Enzymatic studies verify that MMV022224 inhibits PfPK6; however, PfPK6 knockdown does not affect parasite compound susceptibility, confirming that PfPK6 inhibition is not the sole driver of antimalarial activity and that MMV022224 may act through broader, kinase-focused polypharmacology. Employing the same chemical proteomics strategies, we demonstrate that the structurally related azaindole, TCMDC-135051, is a selective inhibitor of the cyclin-dependent kinase PfCLK3. Collectively, these studies demonstrate the value of chemical proteomics for antimalarial drug target deconvolution.
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Atherosclerotic cardiovascular disease (ASCVD) is a leading cause of death, with substantial residual risk persisting despite current lipid-lowering, antithrombotic, antihypertensive, weight-management, and anti-inflammatory therapies. This unmet clinical need reflects the multifactorial and heterogeneous nature of ASCVD, which is not fully captured by traditional discovery approaches. Recent advances in large-scale datasets, multi-omics technologies, polygenic risk scores, and artificial intelligence offer unprecedented opportunities to disentangle disease complexity and identify novel therapeutic targets and biomarkers. However, translation into clinically actionable strategies requires robust validation in models that faithfully recapitulate human disease. Conventional two-dimensional cell cultures and standard murine models have provided important mechanistic insights but often fail to reflect human-specific features such as lipid metabolism, hemodynamics, and plaque destabilization. To address these limitations, advanced in vitro and ex vivo platforms are emerging, including induced pluripotent stem cell-derived vascular systems, microphysiological vessel-on-chip devices, vascularized organoids, and ex vivo human tissue models. These systems offer controlled, human-relevant microenvironments for scalable perturbation testing and support personalized therapeutic development. Nevertheless, in vivo models remain essential for capturing systemic physiology, inter-organ crosstalk, and pharmacokinetic and pharmacodynamic responses, underscoring the need for complementary, rather than replacement, use of model systems. In this review, we propose an integrated framework linking data-driven target and biomarker discovery to validation in human-relevant experimental models, supported by selective use of in vivo systems. By aligning multi-omics and AI-based discovery with advanced preclinical platforms, this approach aims to improve translational success and accelerate the development of precision therapies for ASCVD.
Indole 4-carboxamides are promising prodrugs that liberate 4-aminoindole, which poisons tryptophan biosynthesis in Mycobacterium tuberculosis (Mtb). Limiting enthusiasm for these compounds is the high rate of emergence of resistance since the amidase, which liberates the 4-aminoindole core, is nonessential. To overcome this limitation, we designed bifunctional β-lactams that, upon hydrolytic opening of the β-lactam ring, liberate 4-aminoindole. These bifunctional molecules had good potency against Mtb, which could be rescued by exogenous l-tryptophan addition. In contrast to the parental indole 4-carboxamides, resistant mutants to these bifunctional compounds developed at a much lower frequency than the parental compounds in vitro, supporting this strategy as a means to protect these agents from rapid development of resistance.
Short-chain fatty acids (SCFAs) are bacterial metabolites with crucial roles in host homeostasis and immune system modulation. Given their benefits, they have been proposed as markers of healthy microbiota. However, accurate SCFA quantification typically requires gas chromatography coupled with mass spectrometry (GC-MS), which is time-consuming, expensive, and requires specialized personnel and equipment, limiting its routine use for stool quality assessment in clinical contexts. In this initial feasibility study, we explored the use of Fourier transform infrared (FT-IR) spectroscopy as a rapid metabolic screening approach for stool samples. Analysis of SCFA-associated spectral windows enhanced discrimination between healthy and dysbiotic stool samples with Clostridioides difficile infection using principal component analysis. FT-IR is not intended to replace GC-MS for precise SCFA quantification but rather to provide a rapid screening of metabolically relevant differences. Although additional validation is still needed, the present study provides a robust proof-of-concept demonstrating the feasibility of applying FT-IR spectroscopy to clinical stool samples. Combined with the widespread availability of this technology in most hospitals, these advantages highlight its potential for future development as a tool for routine screening in clinical laboratories.
Antimicrobial resistance represents a critical global health challenge, necessitating deeper insights into bacterial regulatory networks that govern adaptive survival and can be exploited as novel targets for drug discovery. Among these, nucleotide-based second messengers have emerged as central modulators of bacterial physiology and stress responses. Cyclic dinucleotides, including c-di-GMP and c-di-AMP, alongside the alarmone (p)ppGpp, orchestrate diverse cellular processes required for bacterial survival, pathogenesis, and metabolic regulation. Accumulating evidence highlights their pivotal role in shaping antimicrobial susceptibility through biofilm formation, transcriptional regulation, influencing target accessibility, efflux pump expression, persistence, and tolerance phenotypes. Dysregulation of these signaling pathways could promote the evolution of resistance, either directly or indirectly, by modulating fitness landscapes and stress-induced mutagenesis. This review summarizes the current knowledge of global regulators (c-di-GMP, c-di-AMP, and (p)ppGpp) with respect to their contributions in governing AMR in priority pathogens, emphasizing their potential as promising targets for novel antimicrobial and antibiofilm strategies.
The assembly of enveloped viruses is a highly orchestrated process that depends on the coupling of multiple protein-protein interactions within a membrane environment. To gain mechanistic insight into this process, we use Chikungunya virus as a model system to study Alphavirus assembly, focusing on the interplay between core-spike and spike-spike interactions. We begin with coarse-grained molecular dynamics simulations to systematically explore how the symmetry of the nucleocapsid core, together with the relative strengths of spike-core and spike-spike interactions, influences budding efficiency and the emergence of icosahedral particle symmetry. Building on these computational results, we performed site-directed mutagenesis on Chikungunya virus 181/25 and examined the consequences for particle assembly and budding in cultured cells, as well as the impact of these mutations during in-cellulo assembly. Our results revealed that canonical core-spike interactions, while necessary, were not sufficient for successful assembly. Instead, lateral interactions among glycoproteins emerged as critical determinants of efficient budding, particle stability, and the maintenance of icosahedral symmetry. Together, these findings provided an integrated computational and experimental framework for understanding the molecular principles governing Alphavirus assembly.
Reactive arthritis induced by Mycobacterium tuberculosis (M.tb) causes severe cartilage degradation, yet the underlying mechanisms remain elusive. This study investigated the molecular mechanisms and potential therapeutic targets for M.tb-induced cartilage damage. A mouse model of BCG-induced tuberculous arthritis was established. Cartilage matrix degradation, chondrocyte apoptosis, and metabolic balance were evaluated histologically and biochemically. Activation of the NF-κB p65 and STAT3 signaling pathways was assessed, followed by pharmacological inhibition. BCG infection significantly reduced cartilage matrix content and promoted chondrocyte apoptosis, disrupting the metabolic balance between matrix synthesis and degradation. Mechanistically, BCG markedly activated the NF-κB p65 and STAT3 pathways in articular cartilage. Pharmacological inhibition of these pathways effectively prevented cartilage matrix loss and reduced chondrocyte apoptosis at the protein level. BCG induces cartilage damage through activation of NF-κB p65 and STAT3 pathways, leading to chondrocyte apoptosis and matrix catabolism. Targeting these signaling cascades represents a promising therapeutic strategy for preventing cartilage degeneration in tuberculous arthritis.
Bacterial cells are surrounded by a dynamic cell wall which is made up of a mesh-like peptidoglycan (PG) layer that provides the cell with structural integrity and resilience. In Gram-positive bacteria, this layer is thick and robust, whereas in Gram-negative bacteria, it is thinner and flexible as the cell is supported by an additional outer membrane. PG undergoes continuous turnover, with degradation products being recycled to maintain cell wall homeostasis. Some Gram-negative species can bypass de novo PG biosynthesis, relying instead on PG recycling to sustain growth and division. Legionella pneumophila (hereafter Legionella), the causative agent of Legionnaires' disease, encodes such recycling machinery within its genome. This study investigates the biochemical, genetic, and pathogenic roles of PG recycling in Legionella. Here, two PG recycling gene homologues in the Legionella genome lpg0296 (amgK) and lpg0295 (murU) were identified; chemical biology strategies were used to illuminate incorporation of "click"-PG-probes into whole PG. Copper-free click chemistry with ultrafast tetrazine-NAM probes enabled live-cell PG labeling further supported the use of recycling programs in Legionella. Deletion of amgK abolished PG labeling, while genetic complementation restored labeling. The data suggest that under conditions where de novo peptidoglycan synthesis is blocked, amgK plays a critical role in maintaining cell wall integrity, as its deletion led to increased antibiotic susceptibility and impaired survival in host alveolar macrophages. An intracellular survival assay demonstrated that while PG recycling is not essential for internalization, survival of Legionella within MH-S murine alveolar macrophages requires functional amgK. These findings underscore the essential role of AmgK in Legionella's intracellular survival, emphasizing the importance of PG recycling in pathogenicity, and establishing a foundation for developing novel Legionella-specific antibiotic strategies.
Staphylococcus aureus exploits host extracellular matrix components to promote tissue invasion and dissemination. Here, we identify the serine-aspartate repeat protein C (SdrC) as a previously unrecognized plasminogen-binding protein on the S. aureus surface. Using recombinant domains and isogenic mutants, we show that SdrC is a major determinant of plasminogen recruitment at the cellular level. Biochemical and biophysical analyses demonstrate that plasminogen recognition is enhanced by the cooperative action of the SdrC N2 and N3 domains, which together bind plasminogen with submicromolar to low-micromolar affinity. This interaction is lysine-dependent and is selectively inhibited by lysine and 6-aminocaproic acid, with measurable IC50 values, and requires plasminogen kringle domain 4. Importantly, SdrC-bound plasminogen remains readily activatable by host plasminogen activators, generating active plasmin capable of degrading fibrinogen. Consistently, heterologous expression of SdrC enhances plasminogen binding and promotes plasmin activity at the bacterial surface. These findings link a defined staphylococcal adhesin to localized engagement of the host fibrinolytic system and suggest that SdrC-mediated plasminogen recruitment may contribute to persistence and tissue dissemination during invasive infection. Overall, our results establish the SdrC-plasminogen axis as a mechanistically characterized and pharmacologically tractable antivirulence target.