Biology subjects

Tuhkala, A.

Publications and source records attributed to Tuhkala, A..

5 recordsLinked to original sources

bioRxiv2026

A novel method for obtaining epileptic brain tissue for omic analyses using electrodes from a clinical stereoelectroencephalography study

Abstract Purpose: Developing a valid and practical method for obtaining brain tissue samples for transcriptomic and proteomic analyses using tissue adhered to stereoelectroencephalography (SEEG) electrodes, to enable investigation of the molecular mechanisms underlying chronic epilepsy. Method: Brain tissue samples adhered to SEEG electrodes were collected from six patients with drug-resistant epilepsy and preprocessed in a hospital environment after electrode removal. RNA extraction was initiated immediately, and proteomics samples were snap-frozen until subsequent analysis. The samples were categorized into three groups according to their electrophysiological profile: epileptogenic zone, propagation zone, and least-involved zone. The omic findings between these zones and anatomical brain areas were compared. Results: High-quality RNA and protein samples were obtained from tissue adhered to SEEG electrodes. Neuron- and brain-specific gene expression patterns and proteins were identified. Signs of activation of inflammatory mechanisms were most pronounced in the epileptogenic zone. Transcriptomic and proteomic findings demonstrated concordance. Conclusion: SEEG electrodes are a useful source for obtaining brain tissue for molecular characterization of chronic epilepsy. This method will enable the identification of shared and distinct molecular mechanisms in patients with varying etiologies of epilepsy.

neuroscience↗

bioRxiv2026

Efficient NK cell transduction with VSV-G-pseudotyped lentiviral vectors

The need for safe, allogeneic cell therapies for cancer is driving a growing interest in CAR-NK-based therapies, which, unlike CAR-T cell therapies, offer the potential for off-the-shelf administration. Lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) are commonly used for genetic modification of cell therapy products. Their use in NK cells, however, is limited by low transduction efficiency. This study explores the complexities of NK cell transduction using lentiviral vectors pseudotyped with VSV-G. We demonstrate that efficient transduction depends on multiple factors such as NK cell activation, construct design, lentivirus pseudotype selection, and the use of transduction enhancers. By optimizing these elements, we achieved effective transduction, facilitating the use of VSV-G-pseudotyped LVs for therapeutic NK cell production. Our optimized workflow comprises NK cell activation with interleukins, followed by transduction with a NK cell-specific CAR construct using VSV-G-pseudotyped LVs in the presence of BX795 and Retronectin, resulting in excellent transduction efficiency without compromising NK cell phenotype or growth. This allows for the use of a widely used gene transfer vector with an excellent safety record for producing therapeutic NK cell products.

immunology↗

bioRxiv2025

ISG15 Differentially Modulates Clade Ib and II MPXV Infection in MEF cells

The unprecedented human-to-human transmission of Clade IIb monkeypox virus (MPXV) during the 2022 outbreak has renewed focus on host determinants of viral fitness. Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein with broad immunomodulatory functions, yet its role in MPXV infection remains unclear. Using representative strains from recent and historical outbreaks spanning Clades I and II, we show that ISG15 deficiency enhances viral replication and protein production in murine cells. Given that rodents are considered potential natural reservoirs of MPXV, these findings highlight the importance of studying murine models to understand virus-host interactions. Notably, the 2024 Democratic Republic of Congo strain displays reduced sensitivity to ISG15, suggesting clade-specific adaptation. ISG15 also influences viral immune evasion, as knockout cells infected with Clade II viruses expressed fewer immunomodulatory proteins and exhibited marked reductions in host protein phosphorylation. These results identify ISG15 as a determinant of MPXV infection and underscore evolutionary differences between clades.

microbiology↗

bioRxiv2025

The non-catalytic ϵ DNA polymerase subunit is an NPF motif recognition protein

Short linear motifs (SLiMs) in disordered protein regions direct numerous protein-protein interactions, yet most remain uncharacterized. The Asn-Pro-Phe (NPF) motif is a well-known EH-domain ligand implicated in endocytosis, but here we reveal that the non-catalytic subunit of human DNA polymerase {varepsilon} (POLE2) also serves as a general NPF-motif receptor. Using a quantitative "native holdup" assay, we find that POLE2 selectively binds diverse NPF-containing peptides, including canonical EH-domain ligands (e.g., SYNJ1) and previously uncharacterized motifs. Biochemical measurements and mutational analysis show that NPF motifs interact with a shallow pocket near the POLE2 C-terminus, and AlphaFold predictions confirm key roles for Y513, E520, and S522 in motif coordination. Proteome-scale affinity screens identify NPF-containing nuclear proteins (e.g., WDHD1, DONSON, TTF2) that bind POLE2 with micromolar affinities, and their motif mutations abolish binding in cell extracts. Although POLE2 primarily tethers the catalytic POLE subunit to replication forks, these results indicate that it can also recruit various NPF-bearing partners involved in replication, DNA repair, and transcription regulation. Notably, NPF motifs optimized for EH-domain binding can still associate with POLE2, highlighting the inherent degeneracy of SLiM-mediated networks. Overall, these findings establish POLE2 as a central hub linking replication with other processes via broad NPF-motif recognition.

biochemistry↗

bioRxiv2024

T cell correction pipeline for Inborn Errors of Immunity

CRISPR/Cas9 gene editing technology is a promising tool for correcting pathogenic variants for autologous cell therapies for Inborn Errors of Immunity (IEI). The present IEI correction strategies mainly focus on the knock-in of therapeutic cDNAs, or knockout of the disease-causing gene when feasible. These strategies address many single-gene defects but may disrupt gene expression and require significant optimization for each newly discovered IEI-causing gene, highlighting the need for complementary platforms that can precisely correct diverse pathogenic variants. Here, we present a safe and efficient T cell single nucleotide variant (SNV) correction pipeline based on homology-directed repair (HDR), suitable for diverse monogenic mutations. By using founder mutations of Deficiency of ADA2 (DADA2), Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and Cartilage Hair Hypoplasia (CHH) as IEI models, we show that our pipeline can achieve up to 80% bi-allelic editing, with resultant functional correction of the disease phenotype in patient T cells. We do not find detectable pre-malignant off-target effects or karyotypic, transcriptomic or proteomic aberrations upon profiling patient T cells with GUIDE-seq, single cell RNA sequencing, PacBio based long-read whole genome sequencing, and high-throughput proteomics. This study demonstrates that HDR-based SNV editing is a safe and effective option for IEI T cell correction and that it could be developed to an autologous T cell therapy, as the presented protocol is scalable for a GMP-compatible workflow. This study is a step towards the development of gene correction platform that targets a broad number of monogenic mutations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/610811v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f1eb47org.highwire.dtl.DTLVardef@18dbcc2org.highwire.dtl.DTLVardef@63862dorg.highwire.dtl.DTLVardef@1fe1561_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

bioengineering↗