Search Results (11)

Droplet-based microfluidics (DBM) has emerged as a powerful tool for a wide range of biochemical applications, from single-cell analysis and drug screening to diagnostics and tissue engineering. This review provides a comprehensive overview of the latest advancements in droplet generation and trapping techniques, highlighting both passive and active approaches. Passive methods—such as co-flow, cross-flow, and flow-focusing geometries—rely on hydrodynamic instabilities and capillary effects, offering simplicity and integration with compact devices, though often at the cost of tunability. In contrast, active methods exploit external fields—electric, magnetic, thermal, or mechanical—to enable on-demand droplet control, allowing for higher precision and throughput. Furthermore, we explore innovative trapping mechanisms such as hydrodynamic resistance networks, microfabricated U-shaped wells, and anchor-based systems that enable precise spatial immobilization of droplets. In the final section, we also examine active droplet sorting strategies, including electric, magnetic, acoustic, and thermal methods, as essential tools for downstream analysis and high-throughput workflows. These manipulation strategies facilitate in situ chemical and biological analyses, enhance experimental reproducibility, and are increasingly adaptable to industrial-scale applications. Emphasis is placed on the design flexibility, scalability, and biological compatibility of each method, offering critical insights for selecting appropriate techniques based on experimental needs and operational constraints. Full article

GSK-3β plays a critical role in regulating the Wnt/β-catenin signaling pathway, and manipulating GSK-3β in dendritic cells (DCs) has been shown to improve the antitumor efficacy of DC vaccines. Since the inhibition of GSK-3β leads to the activation of β-catenin, we hypothesize that blocking GSK-3β in DCs negatively regulates DC-mediated CD8 T cell immunity and antitumor immunity. Using CD11c-GSK-3β^−/− conditional knockout mice in which GSK-3β is genetically deleted in CD11c-expressing DCs, we surprisingly found that the deletion of GSK-3β in DCs resulted in increased antitumor immunity, which contradicted our initial expectation of reduced antitumor immunity due to the presumed upregulation of β-catenin in DCs. Indeed, we found by both Western blot and flow cytometry that the deletion of GSK-3β in DCs did not lead to augmented expression of β-catenin protein, suggesting that GSK-3β exerts its function independent of β-catenin. Supporting this notion, our single-cell RNA sequencing (scRNA-seq) analysis revealed that GSK-3β-deficient DCs exhibited distinct gene expression patterns with minimally overlapping differentially expressed genes (DEGs) compared to DCs with activated β-catenin. This suggests that the deletion of GSK-3β in DCs is unlikely to lead to upregulation of β-catenin at the transcriptional level. Consistent with enhanced antitumor immunity, we also found that CD11c-GSK-3β^−/− mice exhibited significantly augmented cross-priming of antigen-specific CD8 T cells following DC-targeted vaccines. We further found that the deletion of GSK-3β in DCs completely abrogated memory CD8 T cell responses, suggesting that GSK-3β in DCs also plays a negative role in regulating the differentiation and/or maintenance of memory CD8 T cells. scRNA-seq analysis further revealed that although the deletion of GSK-3β in DCs positively regulated transcriptional programs for effector differentiation and function of primed antigen-specific CD8 T cells in CD11c-GSK-3β^−/− mice during the priming phase, it resulted in significantly reduced antigen-specific memory CD8 T cells, consistent with diminished memory responses. Taken together, our data demonstrate that GSK-3β in DCs has opposite functions in regulating cross-priming and memory CD8 T cell responses, and GSK-3β exerts its functions independent of its regulation of β-catenin. These novel insights suggest that targeting GSK-3β in cancer immunotherapies must consider its dual role in CD8 T cell responses. Full article

Many protein expression systems are primarily utilised to produce a single, specific recombinant protein. In contrast, most biological processes such as virus assembly rely upon a complex of several interacting proteins rather than the activity of a sole protein. The high complexity of the baculovirus genome, coupled with a multiphase replication cycle incorporating distinct transcriptional steps, made it the ideal system to manipulate for high-level expression of a single, or co-expression of multiple, foreign proteins within a single cell. We have developed and utilised a series of recombinant baculovirus systems to unravel the sequential assembly process of a complex non-enveloped model virus, bluetongue virus (BTV). The high protein yields expressed by the baculovirus system not only facilitated structure–function analysis of each viral protein but were also advantageous to crystallography studies and supported the first atomic-level resolution of a recombinant viral protein, the major BTV capsid protein. Further, the formation of recombinant double-shelled virus-like particles (VLPs) provided insights into the structure–function relationships among the four major structural proteins of the BTV whilst also representing a potential candidate for a viral vaccine. The baculovirus multi-gene expression system facilitated the study of structurally complex viruses (both non-enveloped and enveloped viruses) and heralded a new generation of viral vaccines. Full article

Among the available therapeutics for the conservative treatment of osteoarthritis (OA), mesenchymal stromal cells (MSCs)-based products appear to be the most promising. Alongside minimally manipulated cell-based orthobiologics, where MSCs are the engine of the bioactive properties, cell expansion under good manufacturing practice (GMP) settings is actively studied to obtain clinical-grade pure populations able to concentrate the biological activity. One of the main characteristics of GMP protocols is the use of clinical-grade reagents, including the recently released serum-free/xeno-free (SFM/XFM) synthetic media, which differ significantly from the traditional reagents like those based on fetal bovine serum (FBS). As SFM/XFM are still poorly characterized, a main lack is the notion of reliable housekeeping genes (HKGs) for molecular studies, either standalone or in combination with standard conditions. Indeed, the aim of this work was to test the stability of five commonly used HKGs (ACTB, EF1A, GAPDH, RPLP0, and TBP) in adipose-derived MSCs (ASCs) cultivated in two commercially available SFM/XFM and to compare outcomes with those obtained in FBS. Four different applets widely recognized by the scientific community (NormFinder, geNorm, comparative ΔCt method, and BestKeeper) were used and data were merged to obtain a final stability order. The analysis showed that cells cultured in both synthetic media had a similar ranking for HKGs stability (GAPDH being best), albeit divergent from FBS expanded products (EF1A at top). Moreover, it was possible to identify specific HKGs for side by side studies, with EF1A/TBP being the most reliable normalizers for single SFM/XFM vs. FBS cultured cells and TBP the best one for a comprehensive analysis of all samples. In addition, stability of HKGs was donor-dependent. The normalization effect on selected genes coding for factors known to be involved in OA pathology, and whose amount should be carefully considered for the selection of the most appropriate MSC-based treatment, showed how HKGs choice might affect the perceived amount for the different media or donor. Overall, this work confirms the impact of SFM/XFM conditions on HKGs stability performance, which resulted similarly for both synthetic media analyzed in the study. Full article

The brain network consists of ten billion neurons and is the most complex structure in the universe. Understanding the structure of complex brain networks and neuronal functions is one of the main goals of modern neuroscience. Since the seminal invention of Golgi staining, single-cell labeling methods have been among the most potent approaches for dissecting neuronal structures and neural circuits. Furthermore, the development of sparse single-cell transgenic methods has enabled single-cell gene knockout studies to examine the local functions of various genes in neural circuits and synapses. Here, we review non-transgenic single-cell labeling methods and recent advances in transgenic strategies for sparse single neuronal labeling. These methods and strategies will fundamentally contribute to the understanding of brain structure and function. Full article

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with dire consequences and in urgent need of improved therapies. Compelling evidence indicates that damage or dysfunction of AT2s is of central importance in the development of IPF. We recently identified a novel AT2 subpopulation characterized by low SFTPC expression but that is enriched for PD-L1 in mice. These cells represent quiescent, immature AT2 cells during normal homeostasis and expand upon pneumonectomy (PNX) and were consequently named injury-activated alveolar progenitors (IAAPs). FGF10 is shown to play critical roles in lung development, homeostasis, and injury repair demonstrated in genetically engineered mice. In an effort to bridge the gap between the promising properties of endogenous Fgf10 manipulation and therapeutic reality, we here investigated whether the administration of exogenous recombinant FGF10 protein (rFGF10) can provide preventive and/or therapeutic benefit in a mouse model of bleomycin-induced pulmonary fibrosis with a focus on its impact on IAAP dynamics. C57BL/6 mice and Sftpc^CreERT2/+; tdTomato^flox/+ mice aged 8–10 weeks old were used in this study. To induce the bleomycin (BLM) model, mice were intratracheally (i.t.) instilled with BLM (2 μg/g body weight). BLM injury was induced after a 7-day washout period following tamoxifen induction. A single i.t. injection of rFGF10 (0.05 μg/g body weight) was given on days 0, 7, 14, and 21 after BLM injury. Then, the effects of rFGF10 on BLM-induced fibrosis in lung tissues were assessed by H&E, IHC, Masson’s trichrome staining, hydroxyproline and Western blot assays. Immunofluorescence staining and flow cytometry was used to assess the dynamic behavior of AT2 lineage-labeled Sftpc^Pos (IAAPs and mature AT2) during the course of pulmonary fibrosis. We observed that, depending on the timing of administration, rFGF10 exhibited robust preventive or therapeutic efficacy toward BLM-induced fibrosis based on the evaluation of various pathological parameters. Flow cytometric analysis revealed a dynamic expansion of IAAPs for up to 4 weeks following BLM injury while the number of mature AT2s was drastically reduced. Significantly, rFGF10 administration increased both the peak ratio and the duration of IAAPs expansion relative to EpCAM^Pos cells. Altogether, our results suggest that the administration of rFGF10 exhibits therapeutic potential for IPF most likely by promoting IAAP proliferation and alveolar repair. Full article

Fetal bovine serum (FBS) is the only known stimulus for the migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis as-says to be incorporated in a test battery for reproductive and developmental toxicity. We con-firmed here in an optimized transwell assay that FBS triggers directed migration along a con-centration gradient. The responsible factor was found to be a protein in the 30–100 kDa size range. In a targeted approach, we tested a large panel of serum constituents known to be chem-otactic for NCCs in animal models (e.g., VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs. We then examined, whether human cells would produce any fac-tor able to trigger NCC migration in a broad screening approach. We found that HepG2 hepa-toma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as a direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease sensitivity suggested also a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants. Full article

An increasing number of surface-exposed ligands and receptors acting on immune cells are being considered as a starting point in drug development applications. As they are dedicated to manipulating a wide range of immune responses, accurately predicting their molecular interactions will be necessary for the development of safe and effective therapeutics to enhance immune responses and vaccination. Here, we focused on the characterization of human CD160 and HVEM immune receptors, whose mutual engagement leads to bidirectional signaling (e.g., T cell inhibition, natural killer cell activation or mucosal immunity). In particular, our study reports on the molecule preparation, characterization and initial crystallographic analysis of the CD160–HVEM complex and both HVEM and CD160 in the absence of their binding partner. Despite the importance of the CD160–HVEM immune signaling and its therapeutic relevance, the structural and mechanistic basis underlying CD160–HVEM engagement has some controversial evidence. On one hand, there are studies reporting on the CD160 molecule in monomeric form that was produced by refolding from bacterial cells, or as a covalently linked single-chain complex with its ligand HVEM in insect cells. On the other hand, there are older reports providing evidence on the multimeric form of CD160 that acts directly on immune cells. In our study, the native non-linked CD160–HVEM complex was co-expressed in the baculovirus insect host, purified to homogeneity by anion-exchange chromatography to provide missing evidence of the trimeric form in solution. Its trimeric existence was also confirmed by the initial crystallographic analysis. The native CD160–HVEM complex crystallized in the orthorhombic space group with unit cell parameters that could accommodate one trimeric complex (3:3) in an asymmetric unit, thus providing ample space for the multimeric form. Crystals of the CD160–HVEM complex, CD160 trimer and HVEM monomer (reported in two space groups) diffracted to a minimum Bragg spacing of 2.8, 3.1 and 1.9/2.1 Å resolution, respectively. The obtained data will lead to elucidating the native structure of the complex. Full article

Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein’s function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties. Full article

Objective: The aim of this study was to compare treatment methods of the knee joint degenerative osteoarthritis, using autologous bone marrow-derived mononuclear cells and hyaluronic acid injections and observe prevalence of adverse effects in both groups. Materials and methods: A prospective randomized controlled clinical trial was carried out. The analysis of pain and changes in osteoarthritis symptoms after a single intra-articular bone marrow-derived mononuclear cell injection into the knee joint in the Kellgren– Lawrence stage II–III osteoarthritis during the 12-month period were performed. The results were compared with the control group treated routinely by hyaluronic acid injections therapy. A therapy group of patients (n = 28) received single bone marrow-derived mononuclear cell intra-articular injections. A control group of patients (n = 28) was treated with a total of three sodium hyaluronate intra-articular injections each one performed a week apart. The clinical results were obtained using the Knee Osteoarthritis Outcome Score (KOOS) and the Knee Society Score (KSS) before and 3, 6, and 12 months after injection.Results: A statistically significant improvement was observed in the mononuclear cell group over the starting point in all scores. At the endpoint at month 12, the KOOS score improved significantly (P < 0.05) on the pain subscale (+25.44), activity and daily living subscale (+21.36), quality of life subscale (+28.83), and total KOOS (+18.25). The KSS score also demonstrated a significant improvement on the symptoms subscale (+25.42) and the function subscale (+38.32) (P < 0.001). The KOOS symptoms and sports subscales improved without statistical significance. The difference between the control group treated with hyaluronic acid versus the bone marrow-derived mononuclear cells group at time points 6 and 12 months demonstrated a statistically significant (P < 0.05) superiority in the KOOS pain subscale over the hyaluronic acid group. In both groups serious adverse effects were not observed.Conclusions: The intra-articular injection of bone marrow-derived mononuclear cells is a safe manipulation with no side effects during the 12-month period. This treatment provides statistically significant clinical improvement between the starting point and 1, 3, 6, and 12 months after. When compared to hyaluronic acid treatment, better pain relief in the longterm period of mononuclear cell group was observed. Full article

In recent years, there has been rapidly growing interest in developing hand held, sensitive and cost-effective on-chip biosensing systems that directly translate the presence of certain bioanalytes (e.g., biomolecules, cells and viruses) into an electronic signal. The impressive and rapid progress in micro- and nanotechnology as well as in biotechnology enables the integration of a variety of analytical functions in a single chip. All necessary sample handling and analysis steps are then performed within the chip. Microfluidic systems for biomedical analysis usually consist of a set of units, which guarantees the manipulation, detection and recognition of bioanalytes in a reliable and flexible manner. Additionally, the use of magnetic fields for performing the aforementioned tasks has been steadily gaining interest. This is because magnetic fields can be well tuned and applied either externally or from a directly integrated solution in the biosensing system. In combination with these applied magnetic fields, magnetic nanoparticles are utilized. Some of the merits of magnetic nanoparticles are the possibility of manipulating them inside microfluidic channels by utilizing high gradient magnetic fields, their detection by integrated magnetic microsensors, and their flexibility due to functionalization by means of surface modification and specific binding. Their multi-functionality is what makes them ideal candidates as the active component in miniaturized on-chip biosensing systems. In this review, focus will be given to the type of biosening systems that use microfluidics in combination with magnetoresistive sensors and detect the presence of bioanalyte tagged with magnetic nanoparticles. Full article