BioVendor (Czechia)

Abstract AIMS Thrombolytic enzymes represent an accessible and streamlined treatment for acute ischemic stroke, yet current FDA-approved agents, alteplase and tenecteplase, are limited by incomplete recanalization, hemorrhagic risk, and neurotoxicity. We aimed to develop a multidisciplinary workflow for rational design and multi-level testing of thrombolytic proteins to generate a next-generation thrombolytic with improved potency and safety. METHODS AND RESULTS Our pipeline integrated in silico design, biochemical characterization, in vitro thrombolysis, and in vivo validation. Computational screening included rational mutagenesis to reduce side effects, reconstructed ancestral sequences, and database-mined homologs. Selected candidates were biochemically assessed for enzymatic activity, fibrin stimulation, selectivity and affinity, inhibition resistance, and clot penetrability. The most promising variants underwent in vitro clot thrombolysis and in vivo efficacy and safety studies in rats. Brnoteplase emerged as the lead candidate, exhibiting 80-fold enhanced fibrin selectivity, 4-fold higher inhibition resistance, and superior clot penetrability compared to alteplase. In vitro , Brnoteplase demonstrated deeper intrathrombus penetration and a progressive dose–response profile, unlike alteplase, which declined at higher concentrations. In vivo , Brnoteplase achieved higher thrombolysis rates and recanalization frequency across all doses, with extended biological half-life enabling bolus administration. Safety assessments revealed the lowest incidence of severe hemorrhagic transformation and minimal hemispheric asymmetry. CONCLUSIONS We established a robust pipeline for efficient generation and selection of novel thrombolytics and identified Brnoteplase as a promising candidate combining improved fibrin selectivity, inhibition resistance, biological half-life, and clot penetration. These features translate into higher thrombolytic efficacy and a promising safety profile in vivo , warranting further preclinical evaluation to confirm its potential as a next-generation thrombolytic for acute ischemic stroke.

Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients’ general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated (Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and three caries-associated (oral Lactobacilli, Streptococcus mutans, Streptococcus sobrinus). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk’s potential and suitability for inclusion in larger prospective implementation studies in dental care settings.

Recent studies show that the haptoglobin phenotype in individuals with diabetes mellitus is an important factor for predicting the risk of myocardial infarction, cardiovascular death, and stroke. Current methods for haptoglobin phenotyping include PCR and gel electrophoresis. A need exists for a reliable method for high-throughput clinical applications. Mass spectrometry (MS) can in principle provide fast phenotyping because haptoglobin α 1 and α 2, which define the phenotype, have different molecular masses. Because of the complexity of the serum matrix, an efficient and fast enrichment technique is necessary for an MS-based assay. MALDI plates were functionalized by ambient ion landing of electrosprayed antihaptoglobin antibody. The array was deposited on standard indium tin oxide slides. Fast immunoaffinity enrichment was performed in situ on the plate, which was further analyzed by MALDI-TOF MS. The haptoglobin phenotype was determined from the spectra by embedded software script. The MALDI mass spectra showed ion signals of haptoglobin α subunits at m/z 9192 and at m/z 15 945. A cohort of 116 sera was analyzed and the reliability of the method was confirmed by analyzing the identical samples by Western blot. One hundred percent overlap of results between the direct immunoaffinity desorption/ionization MS and Western Blot analysis was found. MALDI plates modified by antihaptoglobin antibody using ambient ion landing achieve low nonspecific interactions and efficient MALDI ionization and are usable for quick haptoglobin phenotyping.

Fibroblast growth factor-21 (FGF21) is a novel endocrine and paracrine regulator of metabolic homeostasis. The aim of our study was to measure its serum concentrations in patients with obesity, obesity and type 2 diabetes mellitus (T2DM) and healthy subjects (C), and to assess the changes of its circulating levels and mRNA expression after dietary and pharmacological interventions. We measured biochemical parameters, serum FGF21, adiponectin, leptin and insulin levels by commercial ELISA and RIA kits, and mRNA expression in the liver, subcutaneous and visceral fat by RT PCR in 26 obese patients, 11 T2DM patients and 32 control subjects. The interventions were acute hyperinsulinaemia during isoglycaemic-hyperinsulinaemic clamp, very low calorie diet (VLCD) and 3 months treatment with PPAR-alpha agonist fenofibrate. Baseline serum FGF21 levels were significantly higher in both obese and T2DM patients relative to healthy controls. FGF21 levels in obesity did not significantly differ from T2DM group. Both 3 weeks of VLCD and 3 months of fenofibrate treatment significantly increased FGF21 levels. FGF21 mRNA expression in visceral fat was twofold higher in obesity relative to C group, while it did not differ in subcutaneous fat. VLCD significantly increased FGF21 mRNA expression in subcutaneous fat of obesity. 3-h hyperinsulinaemia during the clamp increased FGF21 levels in T2DM but not in C group. An increase in FGF21 levels after VLCD and fenofibrate treatment may contribute to positive metabolic effect of these interventions and suggests the possibility of direct positive metabolic effects of FGF21 in humans.