Biology subjects

Kubala, L.

Publications and source records attributed to Kubala, L..

5 recordsLinked to original sources

bioRxiv2026

Characterization of a chronic UV-induced photoaging mouse model: insights into skin barrier dysfunction, extracellular matrix remodeling, and altered adipogenesis

Chronic exposure to ultraviolet (UV) radiation, known as photoaging, accelerates skin aging by inducing molecular, histological, and functional changes. This study established a mouse model using SKH-1 hairless mice to investigate chronic UV-induced photoaging over eight weeks. SKH-1 hairless mice were exposed to a combination of UVA and UVB, and the progression of skin damage was monitored through physical, histological, and molecular parameters, with a focus on erythema, transepidermal water loss, and collagen and hyaluronan (HA) metabolism. Significant reductions in HA content and alterations in DNA repair markers, such as {gamma}H2AX, were observed, highlighting the impact of chronic UV exposure on skin structure and function. Reactive adipogenesis and increased epidermal thickness were noted, reflecting adaptive responses to UV-induced damage. By investigating these parameters over the evaluation period, we provide a comprehensive time-course analysis of the progressive impact of UV-induced photoaging, offering insights into the underlying mechanisms and potential therapeutic targets to prevent or delay photoaging.

pathology↗

bioRxiv2026

Fibrin Selective Alteplase with Improved Thrombolysis and Inhibition Resistance Engineered by Rational Design

Thrombolytic enzymes represent an important class of proteolytic biocatalysts for medical applications, yet currently used FDA-approved variants, including alteplase and tenecteplase, remain limited by suboptimal catalytic efficiency, off-target activity, and susceptibility to inhibition. These limitations reflect the complexity of enzyme function in physiological environments, where therapeutic performance depends on the simultaneous optimization of multiple catalytic and biophysical properties. Here, we introduce a multi-objective enzyme engineering strategy for the design of next-generation thrombolytic proteases, explicitly targeting multiple properties required for therapeutic performance. Our approach combines computer-aided design, evolutionary reconstruction, and literature-guided mutation selection to improve catalytic activity, fibrin selectivity, inhibition resistance, and functional lifetime within a single workflow. This framework is coupled with systematic biochemical characterization, in vitro evaluation of clot penetration and fibrinolytic activity, and in vivo validation of efficacy and safety. By addressing multiple performance parameters simultaneously, this strategy enables efficient navigation of trade-offs that typically limit enzyme optimization. Using this approach, we identify Brnoteplase as a lead variant with enhanced fibrin selectivity, improved resistance to inhibition, and superior clot penetration, resulting in increased effective catalytic lifetime and enabling bolus administration. In vivo studies demonstrate enhanced thrombolysis and recanalization with reduced hemorrhagic complications. These findings provide a broadly applicable framework for designing proteolytic biocatalysts suitable for complex biological environments.

biochemistry↗

bioRxiv2026

Biochemical and Immunological Properties of Engineered Low-Immunogenic Staphylokinases for Next-Generation Thrombolytic Therapy

Staphylokinase (SAK) is a highly fibrin-specific plasminogen activator with significant potential as a safe and affordable thrombolytic. Yet, its clinical translation can be limited by potential immunogenicity. To accelerate the development of improved thrombolytics, a critical step is identifying the most suitable molecular template. Therefore, we performed a comparative analysis of biochemical and immunological properties of three engineered low-immunogenic variants (SAK SY155, SAK THR174, and SAK STAR FRIDA) and two wild-types (SAK STAR and SAK 42D), using a newly established panel of assays. All variants retained potent thrombolytic activity, with SAK SY155 displaying the highest catalytic efficiency and fibrin-clot permeability. However, this advantage did not fully translate into improved clot reduction under flow conditions. Comprehensive immunological profiling, including T lymphocyte proliferation, dendritic cell maturation, mouse immunization models, and human serum reactivity tests, confirmed decreased immunogenicity for two low-immunogenic variants. Overall, low-immunogenic SAK SY155 emerged as the most promising template for rational engineering of next-generation thrombolytics.

biochemistry↗

bioRxiv2025

Amplifying Inflammation: Lipid Remodeling and Cargo Transfer by Macrophage-Derived Extracellular Vesicles

Background: Inflammation drives the progression of acute and chronic diseases, including sepsis and peritonitis, yet the mechanisms by which bacterial stimuli alter intercellular inflammatory communication remain incompletely understood. Extracellular vesicles (EVs) are increasingly recognized as important mediators of immune signaling and may transmit stimulus-specific lipid and protein cargo between cells. We investigated whether bacterial stimulation remodels macrophage-derived EV composition and whether these changes are associated with altered EV uptake and inflammatory activity. Methods: EVs were isolated from RAW264.7 macrophages under basal conditions or following stimulation with bacterial lysate (BL) from Lacticaseibacillus rhamnosus CCM7091 or lipopolysaccharide (LPS). EV identity and quality were assessed according to MISEV recommendations. Lipidomic profiling was performed to define stimulus-associated lipid remodeling, while EV-associated inducible nitric oxide synthase (iNOS) and its enzymatic activity were evaluated using biochemical approaches. EV uptake and functional activity were assessed in macrophages and endothelial cells. EV-associated iNOS was further investigated in murine models of peritoneal inflammation and fibrosis and in EVs isolated from peritoneal exudates of patients with acute peritonitis. Results: Bacterial stimulation altered the molecular composition and biological activity of macrophage-derived EVs. BL-EVs exhibited enrichment of saturated fatty acids and ceramides, generating a distinct lipid signature associated with enhanced cellular uptake. Functionally, bacteria-stimulated EVs promoted macrophage activation, increasing nitric oxide and TNF production, while LPS-EVs additionally induced endothelial IL-6 and CCL5/RANTES production and ICAM-1 expression. Importantly, enzymatically active iNOS was identified in bacteria-stimulated EVs, demonstrating functional iNOS cargo in small EVs derived from a defined macrophage cell line. EV-associated iNOS was further detected in murine models of peritoneal inflammation and fibrosis and in patient-derived EVs from acute peritonitis, supporting its relevance beyond the in vitro system. Conclusions: Bacterial stimulation remodels macrophage-derived EV lipid and protein cargo and generates EV populations with altered uptake and inflammatory activity. These findings identify stimulus-dependent lipid remodeling as a feature of bacteria-stimulated EVs and establish enzymatically active iNOS as a previously unrecognized inflammatory EV cargo. The detection of EV-associated iNOS in experimental models and patients with peritoneal inflammation further supports its potential as a biomarker of inflammatory disease and for therapeutic modulation of EV-mediated immune signaling.

immunology↗

bioRxiv2022

A forskolin-mediated increase in cAMP promotes T helper cell differentiation into the Th1 and Th2 subsets rather than into the Th17 subset

The cyclic adenosine monophosphate (cAMP) signaling pathway is involved in various physiological and pathophysiological processes. Forskolin (FSK), a labdane diterpene well known as an activator of cAMP production, is suggested to possess significant immunomodulatory potential. However, the specific effects of elevated cAMP levels caused by the FSK-mediated activation of adenylate cyclase (AC) on T helper (Th) cell differentiation and functions are still unclear. We speculated that the increased levels of cAMP in Th cells affect the differentiation program of distinct Th populations differently, in particular the development of Th1, Th2, and Th17 subsets. Only minor changes in the expressions of isoforms of ACs and phosphodiesterases (PDE), enzymes responsible for the degradation of cAMP, were observed in differentiating human Th cells with prevailing ADCY1, ADCY3, ADCY7, and ADCY9 and PDE3B, PDE4A/B/D, PDE7A/B, and PDE8A isoforms. FSK mediated elevation in Th1-specific markers reinforcing the Th1 cell phenotype. The differentiation of Th2 was not altered by FSK, though cell metabolism was affected. In contrast, the Th17 immunophenotype was severely suppressed leading to the highly specific upregulation of the level of CXCL13. The causality between FSK-elicited cAMP production and the observed modulation of Th2 differentiation was proven by using cAMP inhibitor 2,5-dideoxyadenosine that reverted the FSK effects. Overall, an FSK-mediated cAMP increase has an effect on Th1, Th2 and Th17 differentiation and can contribute to the identification of novel therapeutic targets for the treatment of Th cell-related pathological processes.

cell biology↗