Biology subjects

Jirak, D.

Publications and source records attributed to Jirak, D..

5 recordsLinked to original sources

bioRxiv2026

Universal functionalization of extracellular vesicles with nanobody adapters

Extracellular vesicles (EVs) have emerged as a powerful platform for targeted therapies due to their intrinsic capacity for intercellular communication and low immunogenicity. In addition to their desirable natural properties, EVs can be engineered to programmably display targeting moieties on their surface, leading to enhanced specificity. Current methods for EV engineering rely on genetic engineering of parental cells, which is robust but labor-intensive due to the requirement to generate stable cell lines for each targeting protein. To address this hurdle, we introduce the Nanobody-Tag-Ligand system (NaTaLi), in which anti-ALFA tag nanobodies are anchored to the EV surface, enabling flexible and nearly covalent attachment of ALFA-tagged proteins. Crucially, NaTaLi allows stable and uniform functionalization of isolated EVs with any tagged protein, removing the need for further mammalian cell engineering. We demonstrate that NaTaLi enables simultaneous display of multiple functional moieties, allowing for precise tunability. In a murine model of breast cancer, NaTaLi-engineered EVs exhibited specific, high-efficiency delivery to tumor cells in vivo. Thus, NaTaLi is a versatile, plug-and-play system that may accelerate the development of targeted EV therapeutics and open the door to readily engineering complex, multispecific EVs. Graphical AbstractA schematic representation of the NaTaLi delivery system. EVs are engineered to display ALFA nanobodies on their surface (ALFA-EVs). ALFA-tagged proteins of choice are isolated and purified from bacteria. Mixing ALFA-EVs with ALFA-tagged proteins creates EVs functionalized with proteins of choice. For examples, ALFA-EVs can be functionalized with tumor-targeting proteins for in vivo targeting of tumors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/708726v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@189c052org.highwire.dtl.DTLVardef@b14f76org.highwire.dtl.DTLVardef@d7dab1org.highwire.dtl.DTLVardef@156b9ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Long-Term Expression and Safety of AAV1-Mediated PI3Kδ Overexpression in the Adult Rat Cortex

Mature CNS neurons are incapable of sufficiently regenerating their axons following spinal cord injury (SCI). This is largely due to developmental changes in epigenetic control leading to suppression of axon growth transcriptomic profile, leading to a shift towards synapse function support. Recently, manipulating the PI3K/Akt/mTOR pathway through PI3K{delta} overexpression in cortical neurons enhanced axonal regeneration of corticospinal tract axons, which was accompanied by functional recovery monitored for up to 16 weeks. However, PI3K is more widely known for its role as an oncogene, and since overexpression is achieved by the use of AAVs, valid safety concerns are raised as it is unknown what the long-term consequences of sustained PI3K{delta} expression in the brain are, which may be necessary to achieve complete re-establishment of the motor pathway. In this study, AAV1-hSYN-PIK3CD was injected into the motor cortex of rats, which survived for 1 year. Comparison with uninjected control animals reveal stable PI3K{delta} expression and sustained pathway activation through increased pS6. PI3K{delta}-treated animals show absence of tumour formation, neural soma hypertrophy, glial cell activation, or haematological or biochemical abnormalities. Thus, long-term neuronal PI3K{delta} expression appears to be well tolerated and may provide a safe and durable strategy to promote functional repair following SCI.

neuroscience↗

bioRxiv2024

Plectin-mediated cytoskeletal crosstalk as a target for inhibition of hepatocellular carcinoma growth and metastasis.

The most common primary malignancy of the liver, hepatocellular carcinoma (HCC), is a heterogeneous tumor entity with high metastatic potential and complex pathophysiology. Increasing evidence suggests that tissue mechanics plays a critical role in tumor onset and progression. Here we show that plectin, a major cytoskeletal crosslinker protein, plays a crucial role in mechanical homeostasis and mechanosensitive oncogenic signaling that drives hepatocarcinogenesis. Our expression analyses revealed elevated plectin levels in liver tumors, which correlated with poor prognosis for HCC patients. Using autochthonous and orthotopic mouse models we demonstrated that genetic and pharmacological inactivation of plectin potently suppressed the initiation and growth of HCC. Moreover, plectin targeting potently inhibited the invasion potential of human HCC cells and reduced their metastatic outgrowth in the lung. Proteomic and phosphoproteomic profiling linked plectin-dependent disruption of cytoskeletal networks to attenuation of oncogenic FAK, MAPK/Erk, and PI3K/AKT signatures. Importantly, by combining cell line-based and murine HCC models, we show that plectin inhibitor plecstatin-1 (PST) is well-tolerated and potently inhibits HCC progression. In conclusion, our study demonstrates that plectin-controlled cytoarchitecture is a key determinant of HCC development and suggests that pharmacologically induced disruption of mechanical homeostasis may represent a new therapeutic strategy for HCC treatment.

cancer biology↗

bioRxiv2023

Spatiotemporal transcriptomic map of ischemic brain injury

The role of non-neuronal cells in the resolution of cerebral ischemia remains to be fully understood. To decode key cellular processes that occur after ischemia, we performed spatial and single-cell transcriptomic profiling of mouse brain tissue during the first week of injury. Cortical gene expression was severely disrupted, being defined by inflammation and cell death in the lesion core, and glial scar formation on the periphery. For each of the three major glial populations, an inflammatory-responsive state, resembling the reactive states observed in neurodegenerative contexts, was documented. The recovered spectrum of ischemia-induced oligodendrocyte states supports the emerging hypothesis that oligodendrocytes actively respond to and modulate the neuroinflammatory stimulus. Thus, we present a landmark transcriptomic dataset that provides a comprehensive view of spatiotemporal organization of processes in the post-ischemic brain and documents the conservation of glial response in CNS pathology.

neuroscience↗

bioRxiv2020

Plectin Ensures Intestinal Epithelial Integrity and Protects Colon Against Colitis

Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in intestinal epithelial cells (IEC; Ple{Delta}IEC) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal 6{beta}4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the Ple{Delta}IEC colon.

molecular biology↗