Biology subjects

Kallio, P.

Publications and source records attributed to Kallio, P..

9 recordsLinked to original sources

bioRxiv2026

Overexpression of flavodiiron protein Flv3 in engineered Synechocystis stimulates sucrose production and growth by altering cellular redox balance through enhanced sulfur metabolism

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

biochemistry↗

bioRxiv2026

Site-Dependent Decoupling of Drug-Biomarker Associations in Clear Cell Renal Cell Carcinoma Revealed by Functional Profiling of Patient-Derived Cell Models

Clear cell renal cell carcinoma (ccRCC) frequently exhibits primary and acquired resistance to standard-of-care therapies, underscoring the need for improved strategies to predict therapeutic response and prioritize patient-specific treatments. Although recent multi-omic and single-cell studies have provided insight into the molecular landscape of ccRCC, molecular alterations alone incompletely predict drug sensitivity. We prospectively profiled tumors from 28 patients with localized and metastatic ccRCC by integrating molecular characterization, functional drug sensitivity and resistance testing in patient-derived cell models, and longitudinal clinical data. Although genomic biomarkers suggested potentially actionable therapies in 27/28 patients, functional testing revealed discordance between genomic actionability and ex vivo drug sensitivity, whereas linear mixed-effects modelling uncovered 16 novel copy-number-based features associated with sensitivity to 11 drugs. Genotype-drug response associations were largely preserved between primary tumors and vena cava thrombi but frequently disrupted in distant metastases. Integrating functional drug testing with multi-omic profiling reveals vulnerabilities not apparent from genomic data alone, refines therapeutic actionability, captures interpatient and intersite heterogeneity in ccRCC, and provides a scalable framework for individualized treatment prioritization.

cancer biology↗

bioRxiv2026

Hypoxia and Associated Acidosis Generate Cell-Type Specific Myeloid Responses in Glioblastoma

Hypoxia is a defining feature of glioblastoma (GBM), yet how it cooperates with hypoxia-associated acidosis to shape microglia and infiltrating monocyte-derived macrophages (MDM) remains poorly understood. We integrated cyclic immunohistochemistry, single-cell RNA sequencing, spatial transcriptomics, in vitro cell cultures, and DNA methylation profiling to outline hypoxia-driven responses in up to 136 GBMs. These hypoxic niches were selectively enriched for MDMs that activated carbonic anhydrase (CA) mediated pH buffering and other metabolic adaptation programs, enabling survival in acidic hypoxia, increasingly interacted with cancer cells, and show polarization toward immunosuppressive myeloid-derived suppressor cell (MDSC)-like states. In contrast, microglia were depleted in hypoxic areas, lacked compensatory CA isoenzymes, and developed TNF-linked stress responses and loss of homeostatic identity in acidic hypoxia. These findings identify metabolic adaptation to hypoxia-associated microenvironmental stress as a key determinant of GBM immune architecture, driving myeloid cell fates, spatial TME reorganization and the emergence of immunosuppressive tumor ecosystems.

cancer biology↗

bioRxiv2026

Optimizing photoautotrophic production of soluble 3-hydroxybutyrate in Synechocystis sp. PCC 6803 through combinatorial translational tuning

While photosynthetic cyanobacteria are potential biotechnological hosts for light-driven production of sustainable chemicals from CO2, engineering more efficient strains is critical for the development of competitive industrial processes. This study demonstrates significantly enhanced production of the soluble bioplastic precursor (R)-3-hydroxybutyrate (3HB) that has been engineered based on the polyhydroxybutyrate (PHB) pathway in photoautotrophic cyanobacterium Synechocystis sp PCC 6803. As the key novelty, we generated a library of engineered 3HB pathway variants that express the three key heterologous pathway enzymes PhaA, PhaB and TesB at varying efficiencies, followed by the screening of most efficient 3HB producers. This was achieved by placing each of the pathway enzymes under the translational regulation of three alternative RBSs in different combinations, resulting in strains with wide dynamic range of 3HB productivities. The best strains accumulated over 5 gl-1 under 200 mol photons m-2s-1 and 3% CO2 in a 14-day flask batch culture, with the highest titer reaching 12 gl-1, corresponding to nearly 3 gl-1 d-1 during the peak production phase. These are the highest 3HB production levels reported so far in cyanobacteria, and comparable to those previously established in heterotrophic production systems. Proteomic comparison of selected strains revealed that the different RBS combinations result in varying expression patterns of the pathway proteins, and that the strain-specific enzyme levels remained relatively constant over the monitored six-day period. The results show that altering the levels of the target pathway enzymes can dramatically improve product yield in Synechocystis, while even very small quantitative differences in the strain-specific expression profiles can have marked effects on the production efficiency. This could be used as a general tool for optimizing engineered pathways in cyanobacteria, provided that the flux to the end-product is not critically restricted by substrate availability but rather determined by the balance between the consecutive pathway steps.

synthetic biology↗

bioRxiv2025

Over four months of ethylene production: Unlocking the potential of solid-state photosynthetic cell factories

This study demonstrates the feasibility of employing solid-state photosynthetic cell factories (solid-state PCFs) as a proof-of-concept platform for long-term ethylene production using sodium bicarbonate as the carbon source. Solid-state PCFs were constructed by entrapping Synechocystis sp. PCC 6803 (efe mutant, strain S5), specifically engineered for ethylene biosynthesis, within TEMPO-oxidized cellulose nanofiber (TCNF) matrices. Two distinct formulations were tested: (i) Ca2+-PVA-TCNF, in which TCNF was crosslinked with Ca2+ and polyvinyl alcohol to produce hydrogel films approximately 200 m thick; and (ii) an all-polysaccharide-based Ca2+-MLG-TCNF formulation, in which TCNF was crosslinked with Ca2+ and mixed-linkage glucan. The latter films were fabricated using an osmotic dehydration approach, yielding mechanically robust, fully biodegradable structures with a thickness of approximately 2 mm. The integration of engineered cells with TCNF matrices created a biocatalytic system that improved the distribution of light, nutrients, and substrates to the cells, while facilitating ethylene separation, thereby supporting the fitness of immobilized cells and enhancing their metabolic performance. Using a custom-designed photobiofilm reactor optimized for semi-wet cultivation, the solid-state PCFs sustained ethylene production for over four months, representing the longest reported continuous ethylene production by cyanobacteria to date. Notably, the solid-state PCFs achieved up to a twofold increase in ethylene yield compared to the continuous-flow suspension culture. Importantly, the suspension-based system also represented the first demonstration of four-month ethylene production under continuous-flow operation. In addition, biodegradability assessments confirmed the environmental compatibility of the TCNF-based matrices, with the all-polysaccharide formulation being particularly advantageous due to its exclusively nature-based composition. Together, these results demonstrate the potential of solid-state PCFs as a scalable and sustainable platform for photosynthetic ethylene production.

bioengineering↗

bioRxiv2025

Biomimetic hydrogel platform reveals active force transduction from retinal pigment epithelium to photoreceptors

In the eye, the retinal pigment epithelium (RPE) maintains the functionality and welfare of retinal photoreceptors and forms a tight, interlocked structure with photoreceptor outer segments (POSs). The RPE-retina interaction is difficult to recapitulate in vitro, limiting the studies addressing the retinal maintenance functions of the RPE. To overcome this challenge, we constructed a retina-mimicking structure using a soft polyacrylamide hydrogel coated with Matrigel. This structure was introduced to RPE cells apical side to model the RPE-retina interface in vitro. As a result, RPE cells attached to the hydrogels during culture, enabling further studies of cell adhesion and force transduction between the RPE-hydrogel with rheology and traction force microscopy. These methods were applied to a critical interactive process between the retina and the RPE: phagocytosis of the aged tips of POSs enabling their renewal. During phagocytosis, RPE cells imposed considerable traction forces to the POS particles. The force generation was actin-dependent, and the forces were significantly reduced by the disruption of RPEs actin cytoskeleton. These results add another layer to the diverse interaction mechanisms between the RPE and the neural retina and pave the way for further studies of the RPE-retina interplay.

biophysics↗

bioRxiv2025

Human iPSC-based coculture model reveals neuroinflammatory crosstalk between microglia and astrocytes

BackgroundMicroglia and astrocytes have been implicated as central mediators of neuroinflammatory processes in several neurodegenerative diseases. However, their intricate crosstalk and contributions to pathogenesis remain elusive, highlighting the need for innovative in vitro approaches for investigating glial interactions in neuroinflammation. The aim of this study was to develop advanced human-based glial coculture models to explore the inflammatory roles and interactions of microglia and astrocytes in vitro. MethodsWe utilized human induced pluripotent stem cell (iPSC)-derived microglia and astrocytes cultured both in conventional culture dishes and in a compartmentalized microfluidic chip coculture platform. This novel platform features separate compartments for both cell types, enabling the creation of fluidically isolated microenvironments with spontaneous migration of microglia toward astrocytes through interconnecting microtunnels. To induce inflammatory activation, glial cultures were stimulated with lipopolysaccharide (LPS), a combination of tumor necrosis factor- (TNF-) and interleukin-1{beta} (IL-1{beta}), or interferon-{gamma} (IFN-{gamma}) for 24 hours. The glial activation and crosstalk were analyzed with immunocytochemistry, the secretion of inflammatory factors from the culture media was measured, and microglial migration was quantified. ResultsMicroglia-astrocyte cocultures were successfully generated in both conventional cultures and the microfluidic chip platform. Inflammatory stimulation with LPS and TNF-/IL-1{beta} elicited cell type-specific responses in microglia and astrocytes, respectively. Notably, the levels of secreted inflammatory mediators were altered under coculture conditions, revealing significant glial crosstalk. Utilization of our microfluidic coculture platform facilitated the study of microglial migration and glial activation within distinct inflammatory microenvironments. Microglia migrated efficiently toward the astrocyte compartment, and the chemoattractant adenosine diphosphate (ADP) notably increased microglial migration within this platform. Furthermore, inflammatory stimulation of the microfluidic chip cocultures successfully recapitulated glial crosstalk, revealing unique responses. This crosstalk was associated with elevated levels of complement component C3 in the cocultures, emphasizing the intricate interplay between microglia and astrocytes under inflammatory conditions. ConclusionsOur results depict an elaborate molecular crosstalk between inflammatory microglia and astrocytes, providing evidence of how glial cells orchestrate responses during neuroinflammation. Importantly, we demonstrate that the microfluidic coculture platform developed in this study for microglia and astrocytes provides a more functional and enhanced setup for investigating inflammatory glial interactions in vitro.

neuroscience↗

bioRxiv2021

M1-linked ubiquitination facilitates NF-κB activation during sterile inflammation

Methionine 1 (M1)-linked ubiquitination plays a key role in the regulation of inflammatory nuclear factor-{kappa}B (NF-{kappa}B) signalling and is important for clearance of pathogen infection in Drosophila melanogaster. M1-linked ubiquitin (M1-Ub) chains are assembled by the linear ubiquitin E3 ligase (LUBEL) in flies. Here, we have studied the role of LUBEL in sterile inflammation induced by different types of cellular stresses. We have found that LUBEL catalyses formation of M1-Ub chains in response to hypoxic, oxidative and mechanical stress conditions. LUBEL is shown to be important for flies to survive low oxygen conditions and paraquat-induced oxidative stress. This protective action seems to be driven by stress-induced activation of the NF-{kappa}B transcription factor Relish via the Immune deficiency (Imd) pathway. In addition to LUBEL, the intracellular mediators of Relish activation, including the Drosophila inhibitor of apoptosis (IAP) Diap2, the I{kappa}B kinase {gamma} (IKK{gamma}) Kenny and the initiator caspase Death-related ced-3/Nedd2-like protein (Dredd), but not the membrane receptor peptidoglycan recognition protein (PGRP)-LC, are shown to be required for sterile inflammatory response and survival. Finally, we showed that the stress-induced upregulation of M1-Ub chains in response to hypoxia, oxidative and mechanical stress is also induced in mammalian cells. Taken together, our results suggest that M1-Ub chains are important for NF-{kappa}B signalling in inflammation induced by stress conditions often observed in chronic inflammatory diseases and cancer.

cell biology↗

bioRxiv2020

PROX1 transcription factor is a master regulator of myogenic and oncogenic features of rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue cancer in need for novel therapies. Here we show that the PROX1 transcription factor, which is essential for normal myoblast differentiation, is highly expressed in RMS tumors. We demonstrate that PROX1 is needed for RMS cell stemness and growth in vitro, and for RMS tumor formation in mouse xenograft models. In addition, we unveil that PROX1 is an essential for myogenic properties in RMS. PROX1 depletion reprogrammed the RMS transcriptome to resemble benign mesenchymal stem cells and repressed many of the previously identified RMS effector transcripts and myogenic genes. By using proximity labeling and mass spectrometry, we found that PROX1 interacts with the NuRD and CoREST complexes containing class I HDACs. Our studies reveal a major role of PROX1-HDAC interaction in RMS and give insights that inhibiting this interaction could be a promising therapeutic approach.

cancer biology↗