We present a protocol to evaluate natural killer (NK)-tumor interactions using an ex ovo chick embryo system. We describe procedures to culture GFP-labeled SH-SY5Y neuroblastoma cells, expand and Hoechst-label human NK cells, and perform sequential intracardiac injections. We use fluorescence imaging to visualize NK and tumor cell distribution in vivo and live imaging to assess interactions in vitro. This approach provides a rapid and accessible platform to quantify NK cell activity in a living vertebrate model.

BACKGROUND:Diabetic patients are particularly vulnerable to heat exposure due to impaired thermoregulation and reduced sweating ability. The impact of heat on skin cell function, particularly keratinocytes, is poorly understood. Recent studies highlight the critical role of nitric oxide (NO) in thermoregulation and heat stress responses, but its specific involvement in keratinocyte responses and metabolic profiles remains unexplored. OBJECTIVE:This proof-of-concept study investigates the metabolic profiles of HaCat keratinocytes under normal and high-glucose conditions during varied heat exposures. METHODS:We conducted experiments using a metabolomics approach, NO levels assessments, western blot analysis, and evaluations of mitochondrial morphology. RESULTS:Our findings indicate that acute heat exposure over 90 minutes significantly alters metabolic pathways, particularly amino acid metabolism (including arginine, valine, leucine, and serine), the pyrimidine metabolite uracil, and glycolysis, notably lactate production. Arginine metabolism was uniquely affected by high glucose combined with heat, aligning with previous clinical observations. Furthermore, we discovered that changes in NO production correlated with heat exposure duration, and that NO levels in extracellular vesicles (EVs) from HaCat cells were inversely related to intracellular NO levels. Additionally, we observed alterations in HSP-70 protein expression and mitochondrial morphology, supporting cellular adaptation to thermal stress. CONCLUSION:This study is the first to demonstrate heat-induced metabolic changes in keratinocytes involving arginine and NO, highlighting their potential as clinical biomarkers for thermal stress adaptation, with implications for both healthy individuals and diabetic patients.

Human gut microbiota is associated with obesity. Gut microbiota-derived extracellular vesicles (EVs), lipid coated nanoparticles secreted by bacteria, have been suggested as a communication mechanism between gut microbiota and the host. This study characterized the effect of Roux-en-Y gastric bypass (RYGB) on gut microbiota and gut microbiota-derived EVs in patients with obesity. Fecal samples were collected from 30 recruited patients at baseline and 6 months after surgery. EVs were isolated from fecal samples, and their origin and protein content were analyzed. The number of unique proteins was increased in gut microbiota-derived EVs after the surgery as compared to baseline. A significant difference in both microbiota composition (p = 0.001; PERMANOVA) and microbiota-derived EVs (p = 0.001; PERMANOVA) was observed in response to surgery. Based on 16S rRNA gene sequencing data, a random forest classifier accurately classified both gut microbiota (AUC = 0.93) and EVs (AUC = 0.80) to baseline and after surgery groups. This study found that gastric bypass surgery altered both the composition and characteristics of gut microbiota and gut microbiota-derived EVs in patients with obesity. Thus, gut microbiota-derived EVs may play a role in obesity and influence the health effects of bariatric surgery beyond the gut. Trial Registration: ClinicalTrials.gov identifier: NCT00950003.

Morphological knowledge refers to the ability to recognize and use morphemes correctly in syntactic contexts and word formation. This is crucial for learning a morphologically rich language like Finnish, which features both agglutinative and fusional morphology. In Finnish, agglutination occurs in forms like aamu: aamu+lla (‘morning: in the morning’), where a suffix is transparently added. Fusional features, as seen in ilta: illa+lla (‘evening: in the evening’), involve allomorphic stem changes that reduce transparency. We investigated the challenges posed by stem allomorphy for word recognition in isolation and in context for L2 learners and L1 speakers of Finnish. In a lexical decision task, L2 speakers had longer response times and higher error rates for semitransparent inflections, while L1 speakers showed longer response times for both transparent and semitransparent inflection types. In sentence reading, L2 speakers exhibited longer fixation times for semitransparent forms, whereas L1 speakers showed no significant effects. The results suggest that the challenges in L2 inflectional processing are more related to fusional than agglutinative features of the Finnish language.

The establishment of cell fate and boundaries between cell types is an essential step in development and organogenesis. In the mammalian cochlea, a distinct boundary exists between a medial region of non-sensory cells and a lateral region of sensory cells. We report that Wnt4 and sFRP2 act in combination to modulate the sensory cell differentiation of the organ of Corti. The hair cell inhibitory effects of Wnt4 in the inner ear are mediated through the activation of the non-canonical Wnt/Calcium/PKC pathway. We show that Wnt4 stimulates the activation of PKC in the cochlea, and that the inhibition of PKC rescues the ectopic Wnt4 activity phenotype. Finally, we demonstrate that modification at a PKC target site on Atoh1 diminishes its ability to induce hair cell formation. Ultimately, we identify a new Wnt/Calcium/PKC non-canonical signaling pathway that is involved in proper hair cell and organ of Corti formation in the developing mammalian cochlea.

Solar ultraviolet B (UVB) radiation-induced DNA damage is a well-known initiator of skin carcinomas. The UVB-induced DNA damage response (DDR) involves series of signaling cascades that are activated to maintain cell integrity. Among the different biological processes, little is known about the role of energy metabolism in the DDR.We sought to determine whether UVB-induced nuclear and/or mitochondrial cyclobutane pyrimidine dimers (CPDs) alter cellular energy metabolism. To gain insight into this question, we took advantage of keratinocytes expressing nuclear or mitochondrial CPD photolyase. Applying a quantitative proteomic approach and targeted metabolomics, we observed biphasic alterations in multiple metabolic pathways and in the abundance of various metabolites, largely influenced by the presence of genomic CPDs. The heightened oxygen consumption rate post-irradiation, along with mitochondrial structural rearrangements, was found to be dependent on both mitochondrial and nuclear CPDs.Understanding the influence of nuclear and mitochondrial DNA damage on keratinocyte responses to UVB irradiation deepens current knowledge regarding skin cancer prevention, initiation, and therapy.

Extracellular vesicles (EVs), nanoparticles secreted by both gram-negative and gram-positive bacteria, carry various biomolecules and cross biological barriers. Gut microbiota-derived EVs are currently being investigated as a communication mechanism between the microbiota and the host. Few clinical studies, however, have investigated gut microbiota-derived EVs. Here, we show that machine learning models were able to accurately distinguish gut microbiota and respective microbiota-derived EV samples according to their taxonomic composition both within each data set (area under the curve [AUC] 0.764-1.00) and in a cross-study setting (AUC 0.701-0.997). These results show that gut microbiota-derived EVs form a distinct taxonomic entity from gut microbiota. Thus, conventional gut microbiota composition may not correctly reflect communication between the gut microbiota and the host unless microbiota-derived EVs are reported separately.IMPORTANCEGut microbiota-derived extracellular vesicles (EVs) have been suggested to be a communication mechanism between the gut microbiota and the human body. However, the data on EV secretion from the gut microbiota remain limited. To investigate and compare the composition of gut microbiota-derived EVs to gut microbiota composition, we used a machine learning approach to classify 16S rRNA gene sequencing data in seven clinical data sets incorporating both gut microbiota and gut microbiota-derived EV samples. The results of the study show that microbiota-derived EVs form a separate taxonomic entity from the gut microbiota. Gut microbiota-derived EVs should be included in clinical studies that investigate gut microbiota to gain more comprehensive insight into gut microbiota-host communication.

The field of extracellular vesicles (EVs) is rapidly advancing, offering promising applications in diagnostics, therapeutics, and drug delivery. However, the translation of EV-based technologies to the clinic faces significant challenges related to heterogeneity, scalable biomanufacturing, and regulatory compliance. To address these issues, the European Innovation Council (EIC), in collaboration with the Horizon2020-funded BOW project, organized the "Extracellular Vesicle EIC Cluster Meeting," bringing together researchers, startups, and regulatory stakeholders across Europe. Discussions focused on overcoming bottlenecks in EV production, standardization, and clinical readiness. Key outcomes included the need for application-specific benchmarks, robust manufacturing pipelines, and regulatory frameworks tailored to EVs. The event emphasized the importance of interdisciplinary collaboration, coopetition, and continued EU funding to drive innovation and strengthen Europe's leadership in the EV field.

Branching morphogenesis is a characteristic feature of many essential organs, such as the lung and kidney, and most glands, and is the net result of two tissue behaviors: branch point initiation and elongation. Each branched organ has a distinct architecture customized to its physiological function, but how patterning occurs in these ramified tubular structures is a fundamental problem of development. Here, we use quantitative 3D morphometrics, time-lapse imaging, manipulation of ex vivo cultured mouse embryonic organs and mice deficient in the planar cell polarity component Vangl2 to address this question in the developing mammary gland. Our results show that the embryonic epithelial trees are highly complex in topology owing to the flexible use of two distinct modes of branch point initiation: lateral branching and tip bifurcation. This non-stereotypy was contrasted by the remarkably constant average branch frequency, indicating a ductal growth invariant, yet stochastic, propensity to branch. The probability of branching was malleable and could be tuned by manipulating the Fgf10 and Tgfβ1 pathways. Finally, our in vivo data and ex vivo time-lapse imaging suggest the involvement of tissue rearrangements in mammary branch elongation.

Plant-derived nanovesicles such as bilberries nanovesicles (BNVs) show immense promise as next-generation biotherapeutics and functional food ingredients; however, their isolation, purification, and storage on a large scale remain a challenge. In this study, biocompatible and nanostructured composite all-cellulose membranes are introduced as a scalable and straightforward approach to the isolation of BNV. The membranes consisting of a cellulose acetate matrix infused with anionic or cationic nanocelluloses promoted selective capture of BNVs through electrostatic and size-exclusion-mediated depth filtration. Furthermore, the surface of the composite membrane acted as a storage matrix for BNVs, ensuring their prolonged stability at 4 °C. The BNVs stored in the membrane could be promptly released through elution assisted by low-pressure vacuum filtration or diffusion in liquid media. The morphology, bioactivity, and stability of the extracted BNVs were preserved, and the release rate of BNVs in different cell cultures could be regulated, facilitating their use for local therapy. Consequently, this approach paves the way for the scalable production, purification, and storage of nanovesicles and advances their use in biotherapeutics and functional foods.

Kidney diseases such as glomerulopathy and nephron dysfunction are estimated to grow to more than 900 million cases by 2030, in 45% of which kidney transplantation will be required, representing a major challenge for biomedicine. A wealth of progress has been made to model human diseases using induced pluripotent stem cells (iPSCs) in vitro differentiated to a variety of organoids, including kidney organoids, and in developing various microfluidics-based organ-on-a-chip (OoC) systems based on them. With the combination of targeted gene editing capacities, relevant polymorphic genetic variants can be established in such organoid models to advance evidence-based medicine. However, the major drawback of the current organoid disease models is the lack of functional endothelial vasculature, which especially concerns the kidney, the function of which is strongly associated with blood flow. The design of novel medical devices using tissue engineering approaches such as kidney organoids is also strongly dependent on the understanding of the fundamental principles of nephrogenesis and the vascularization of organs and tissues. Developmental vascularization of the kidney has been an area of intense research for decades. However, there is still no consensus among researchers on how exactly the vascularization of the kidney occurs in normal and pathological conditions. This lack of consensus is partly due to the lack of an appropriate model system to study renal vascularization during nephrogenesis. In this review, we will describe recent progress in the areas of kidney vasculature development, kidney organoids in general and assembled on microfluidic devices in particular. We will focus on the in vitro vasculature of kidney organoids in microfluidic OoC model systems to study kidney diseases and on the perspectives of tissue engineering for the modeling of kidney diseases and the design of bioartificial medical devices. We also aim to summarize the information related to the key mechanisms of intercellular communication during nephrogenesis and the formation of the renal vasculature in an OoC setup.

Isolation of extracellular vesicles (EV) has been developing rapidly in parallel with the interest in EVs. However, commonly utilized protocols may not suit more challenging sample matrixes and could potentially yield suboptimal results. Knowing and assessing the pitfalls of isolation procedure to be used, should be involved to some extent for EV analytics. EVs in cow milk are of great interest due to their abundancy and large-scale availability as well as their cross-species bioavailability and possible use as drug carriers. However, the characteristics of milk EVs overlap with those of other milk components. This makes it difficult to isolate and study EVs individually. There exists also a lack of consensus for isolation methods. In this study, we demonstrated the differences between various differential centrifugation-based approaches for isolation of large quantities of EVs from cow milk. Samples were further purified with gradient centrifugation and size exclusion chromatography (SEC) and differences were analyzed. Quality measurements were conducted on multiple independent platforms. Particle analysis, electron microscopy and RNA analysis were used, to comprehensively characterize the isolated samples and to identify the limitations and possible sources of contamination in the EV isolation protocols. Vesicle concentration to protein ratio and RNA to protein ratios were observed to increase as samples were purified, suggesting co-isolation with major milk proteins in direct differential centrifugation protocols. We demonstrated a novel size assessment of vesicles using a particle mobility analyzer that matched the sizing using electron microscopy in contrast to commonly utilized nanoparticle tracking analysis. Based on the standards of the International Society for Extracellular Vesicles and the quick checklist of EV-Track.org for EV isolation, we emphasize the need for complete characterization and validation of the isolation protocol with all EV-related work to ensure the accuracy of results and allow further analytics and experiments.

Sweat contains biomarkers for real-time non-invasive health monitoring, but only a few relevant analytes are currently used in clinical practice. In the present study, we investigated whether sweat-derived extracellular vesicles (EVs) can be used as a source of potential protein biomarkers of human and bacterial origin. Methods: By using ExoView platform, electron microscopy, nanoparticle tracking analysis and Western blotting we characterized EVs in the sweat of eight volunteers performing rigorous exercise. We compared the presence of EV markers as well as general protein composition of total sweat, EV-enriched sweat and sweat samples collected in alginate skin patches. Results: We identified 1209 unique human proteins in EV-enriched sweat, of which approximately 20% were present in every individual sample investigated. Sweat derived EVs shared 846 human proteins (70%) with total sweat, while 368 proteins (30%) were captured by medical grade alginate skin patch and such EVs contained the typical exosome marker CD63. The majority of identified proteins are known to be carried by EVs found in other biofluids, mostly urine. Besides human proteins, EV-enriched sweat samples contained 1594 proteins of bacterial origin. Bacterial protein profiles in EV-enriched sweat were characterized by high interindividual variability, that reflected differences in total sweat composition. Alginate-based sweat patch accumulated only 5% proteins of bacterial origin. Conclusion: We showed that sweat-derived EVs provide a rich source of potential biomarkers of human and bacterial origin. Use of commercially available alginate skin patches selectively enrich for human derived material with very little microbial material collected.

Particle cross-stream migration in electrohydrodynamic microfluidic systems exhibits intriguing behaviors, which makes it interesting when viewed from a fundamental perspective and promising for nanoparticle focusing and separation applications. So far, particle behavior in such systems has been explained with the slip-induced lift force model (Saffman model), which predicts particle central or side focusing based on the direction of electric field and fluid flow. However, in our previous work, we observed particle migration patterns that did not adhere to the prediction of the Saffman model. In this work, we further studied this novel particle lateral migration behavior, which we termed the “anti-Saffman” behavior. We experimentally investigated how changing the conductivity of the suspending medium influences particle behavior and quantitatively measured the net lateral force experienced by the particles. Then, we compared this net force with the prediction of the relevant lift force models in the literature. We concluded that the anti-Saffman behavior is positively correlated with medium conductivity and shear rate (∝γ̇2). Furthermore, the comparison with the existing force models revealed that none of them can predict the experimentally observed particle lift. The net lift predicted by hydrodynamic lift models indicated that the underlying mechanism behind our experiments also potentially has a hydrodynamic origin. We believe this phenomenon offers the possibility of manipulating and separating nanoparticles suspended in standard aqueous electrolyte solutions, which makes it applicable to various biological samples.

LASTU is a tool for searching for Finnish language stimulus words for psycholinguistic studies. The tool allows the user to query a number of properties, including forms, lemmas, frequencies, and morphological features. It also includes two new measures for quantifying lemma and form ambiguity. The tool is written in Python and is available for Windows and macOS platforms. It is available at https://osf.io/j8v6b/ . Included with the tool is a database based on a massive corpus of dependency-parsed Finnish language data crawled from the Internet (over 5 billion tokens). While LASTU has been developed for researchers working on the Finnish language, the openly available implementation can also be applied to other languages.

The renal tubular epithelial cells (TEC) have a strong capacity for repair after acute injury, but when this mechanism becomes uncontrollable, it leads to chronic kidney diseases (CKD). Indeed, in progress toward CKDs, the TECs may dedifferentiate, undergo epithelial-to-mesenchyme transition (EMT), and promote inflammation and fibrosis. Given the critical role of Wnt4 signaling in kidney ontogenesis, we addressed whether changes in this signaling are connected to renal inflammation and fibrosis by taking advantage of a knock-in Wnt4mCh/mCh mouse. While the Wnt4mCh/mCh embryos appeared normal, the corresponding mice, within one month, developed CKD-related phenotypes, such as pro-inflammatory responses including T-cell/macrophage influx, expression of fibrotic markers, and epithelial cell damage with a partial EMT. The Wnt signal transduction component β-catenin remained unchanged, while calcium signaling is induced in the injured TECs involving Nfat and Tfeb transcription factors. We propose that the Wnt4 signaling pathway is involved in repairing the renal injury, and when the signal is overdriven, CKD is established.

Lignin is a phenolic polymer in plants that rigidifies the cell walls of water-conducting tracheary elements and support-providing fibers and stone cells. Different mechanisms have been suggested for the transport of lignin precursors to the site of lignification in the cell wall. Extracellular vesicle (EV)-enriched samples isolated from a lignin-forming cell suspension culture of Norway spruce (Picea abies L. Karst.) contained both phenolic metabolites and enzymes related to lignin biosynthesis. Metabolomic analysis revealed mono-, di-, and oligolignols in the EV isolates, as well as carbohydrates and amino acids. In addition, salicylic acid (SA) and some proteins involved in SA signaling were detected in the EV-enriched samples. A proteomic analysis detected several laccases, peroxidases, beta-glucosidases, putative dirigent proteins, and cell wall-modifying enzymes, such as glycosyl hydrolases, transglucosylase/hydrolases, and expansins in EVs. Our findings suggest that EVs are involved in transporting enzymes required for lignin polymerization in Norway spruce, and radical coupling of monolignols can occur in these vesicles. Extracellular vesicles transport enzymes putatively involved in lignin polymerization in Norway spruce, and radical coupling of monolignols can occur in these vesicles.

Aims Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply translating ribosome affinity purification sequencing to profile SMC-specific gene expression directly from tissue.Methods and results To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing enhanced green fluorescent protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific alpha SMA promoter. These mice were further crossed with the atherosclerosis model Ldlr-/-, ApoB100/100 to generate SMCTRAP-AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip, Lum, Mfge8, Spp1, and Serpina3, which are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4, Cd276, inter-alpha-trypsin inhibitor-4 (Itih4), Myof, Pcdh11x, Rab31, Serpinb6b, Slc35e4, Slc8a3, and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease through the colocalization of genome-wide association studies, splice quantitative trait loci (QTL), and protein QTL signals.Conclusion We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality.

Genome studies heavily rely on valuable sources of nucleic acids found in biological fluids. Sweat, an easily collectible and cost-effective biofluid, has emerged as a promising material for genomic research. However, the suitability of sweat DNA for genome sequencing compared to venous blood DNA requires further investigation. Sweat samples were collected from four individuals, and a statistical comparison was conducted between sweat DNA and healthy blood DNA reads. The study focused on four key aspects: genome coverage, fragment lengths, mapping quality, telomere sequences, and chaos game representation (CGR) analysis. The length, quality, and genome coverage of all DNA fragments were examined, and tandem repeats in the telomere sequences of the study samples were identified in this article. The statistical analysis revealed significant similarities in genomic study outcomes between sweat and blood samples. The results suggest that sweat has the potential to be an alternative substitute for genomic analysis, offering a cost-effective and easily accessible avenue for future investigations. Our study highlights the potential utility of sweat as a valuable source of nucleic acids for genomic research. While significant similarities were observed between sweat and blood samples regarding genomic outcomes, further studies are warranted to explore the full capabilities of sweat DNA in genome sequencing. Using sweat in genome research could lead to cost-effective and accessible approaches for future genomic investigations.

Renal development is a complex process in which two major processes, tubular branching and nephron development, regulate each other reciprocally. Our previous findings have indicated that collagen XVIII (ColXVIII), an extracellular matrix protein, affects the renal branching morphogenesis. We investigate here the role of ColXVIII in nephron formation and the behavior of nephron progenitor cells (NPCs) using isoform-specific ColXVIII knockout mice. The results show that the short ColXVIII isoform predominates in the early epithelialized nephron structures whereas the two longer isoforms are expressed only in the later phases of glomerular formation. Meanwhile, electron microscopy showed that the ColXVIII mutant embryonic kidneys have ultrastructural defects at least from embryonic day 16.5 onwards. Similar structural defects had previously been observed in adult ColXVIII-deficient mice, indicating a congenital origin. The lack of ColXVIII led to a reduced NPC population in which changes in NPC proliferation and maintenance and in macrophage influx were perceived to play a role. The changes in NPC behavior in turn led to notably reduced overall nephron formation. In conclusion, the results show that ColXVIII has multiple roles in renal development, both in ureteric branching and in NPC behavior.