Phylogenomics including new sequence data of phytoplankton-infecting chytrids reveals multiple independent lifestyle transitions across the phylum.

Parasitism is the most common lifestyle on Earth and has emerged many times independently across the eukaryotic tree of life. It is frequently found among chytrids (Chytridiomycota), which are early-branching unicellular fungi that feed osmotrophically via rhizoids as saprotrophs or parasites. Chytrids are abundant in most aquatic and terrestrial environments and fulfil important ecosystem functions. As parasites, they can have significant impacts on host populations. They cause global amphibian declines and influence the Earth's carbon cycle by terminating algal blooms. To date, the evolution of parasitism within the chytrid phylum remains unclear due to the low phylogenetic resolution of rRNA genes for the early diversification of fungi, and because few parasitic lineages have been cultured and genomic data for parasites is scarce. Here, we combine transcriptomics, culture-independent single-cell genomics and a phylogenomic approach to overcome these limitations. We newly sequenced 29 parasitic taxa and combined these with existing data to provide a robust backbone topology for the diversification of Chytridiomycota. Our analyses reveal multiple independent lifestyle transitions between parasitism and saprotrophy among chytrids and multiple host shifts by parasites. Based on these results and the parasitic lifestyle of other early-branching holomycotan lineages, we hypothesise that the chytrid last common ancestor was a parasite of phytoplankton.

Impact of the Reduction Time-Dependent Electrical Conductivity of Graphene Nanoplatelet-Coated Aligned Bombyx mori Silk Scaffolds on Electrically Stimulated Axonal Growth.

Graphene-based nanomaterials, renowned for their outstanding electrical conductivity, have been extensively studied as electroconductive biomaterials (ECBs) for electrically stimulated tissue regeneration. However, using eco-friendly reducing agents like l-ascorbic acid (l-Aa) can result in lower conductive properties in these ECBs, limiting their full potential for smooth charge transfer in living tissues. Moreover, creating a flexible biomaterial scaffold using these materials that accurately mimics a specific tissue microarchitecture, such as nerves, poses additional challenges. To address these issues, this study developed a microfibrous scaffold of Bombyx mori (Bm) silk fibroin uniformly coated with graphene nanoplatelets (GNPs) through a vacuum coating method. The scaffold's electrical conductivity was optimized by varying the reduction period using l-Aa. The research systematically investigated how different reduction periods impact scaffold properties, focusing on electrical conductivity and its significance on electrically stimulated axonal growth in PC12 cells. Results showed that a 48 h reduction significantly increased surface electrical conductivity by 100-1000 times compared to a shorter or no reduction process. l-Aa contributed to stabilizing the reduced GNPs, demonstrated by a slow degradation profile and sustained conductivity even after 60 days in a proteolytic environment. ß (III) tubulin immunostaining of PC12 cells on varied silk:GNP scaffolds under pulsed electrical stimulation (ES, 50 Hz frequency, 1 ms pulse width, and amplitudes of 100 and 300 mV/cm) demonstrates accelerated axonal growth on scaffolds exhibiting higher conductivity. This is supported by upregulated intracellular Ca2+ dynamics immediately after ES on the scaffolds with higher conductivity, subjected to a prolonged reduction period. The study showcases a sustainable reduction approach using l-Aa in combination with natural Bm silk fibroin to create a highly conductive, mechanically robust, and stable silk:GNP-based aligned fibrous scaffold. These scaffolds hold promise for functional regeneration in electrically excitable tissues such as nerves, cardiac tissue, and muscles.

Environmental DNA, hydrochemistry and stable water isotopes as integrative tracers of urban ecohydrology.

Urbanization and the persistent environmental changes present a major challenge for urban freshwaters and availability of water for humans and wildlife. In order to increase understanding of urban ecohydrology, we investigated the variability of planktonic bacteria and benthic diatoms - as two key biological indicators - coupled with insights from hydrochemistry and stable water isotopes across four urban streams characterized by different dominant water sources in Berlin, the German capital, over a period of one year (2021-2022). DNA metabarcoding results show that substantial spatio-temporal variability exists across urban streams in terms of microbial diversity and richness, with clear links to abiotic factors and nutrient concentrations. Bacterial communities showed clear distinction between effluent-impacted and non-effluent impacted streams as well as clear seasonal turnover. In-stream benthic diatom assemblages also showed robust seasonal variation as well as high species diversity. Our multiple-tracer approach is relevant for emerging questions regarding the increased use of treated effluent to supplement declining baseflows, the assessment of stream restoration projects and the impact of storm drainage and surface pollution on aquatic ecosystem health. eDNA analysis allows analysis of spatial and temporal patterns not feasibly studied with traditional analyses of macroinvertebrates. This can ultimately be leveraged for future water resource management and restoration planning and monitoring of urban freshwater systems across metropolitan areas.

2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified.

BACKGROUND: Human mesenchymal stem cells (hMSCs) utilize discrete biosynthetic pathways to self-renew and differentiate into specific cell lineages, with undifferentiated hMSCs harbouring reliance on glycolysis and hMSCs differentiating towards an osteogenic phenotype relying on oxidative phosphorylation as an energy source. METHODS: In this study, the osteogenic differentiation of hMSCs was assessed and classified over 14 days using a non-invasive live-cell imaging modality-two-photon fluorescence lifetime imaging microscopy (2P-FLIM). This technique images and measures NADH fluorescence from which cellular metabolism is inferred. RESULTS: During osteogenesis, we observe a higher dependence on oxidative phosphorylation (OxPhos) for cellular energy, concomitant with an increased reliance on anabolic pathways. Guided by these non-invasive observations, we validated this metabolic profile using qPCR and extracellular metabolite analysis and observed a higher reliance on glutaminolysis in the earlier time points of osteogenic differentiation. Based on the results obtained, we sought to promote glutaminolysis further by using lactate, to improve the osteogenic potential of hMSCs. Higher levels of mineral deposition and osteogenic gene expression were achieved when treating hMSCs with lactate, in addition to an upregulation of lactate metabolism and transmembrane cellular lactate transporters. To further clarify the interplay between glutaminolysis and lactate metabolism in osteogenic differentiation, we blocked these pathways using BPTES and α-CHC respectively. A reduction in mineralization was found after treatment with BPTES and α-CHC, demonstrating the reliance of hMSC osteogenesis on glutaminolysis and lactate metabolism. CONCLUSION: In summary, we demonstrate that the osteogenic differentiation of hMSCs has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture using 2P-FLIM. Furthermore, extracellular lactate is shown as an essential metabolite and metabolic fuel to ensure efficient osteogenic differentiation and as a signalling molecule to promote glutaminolysis. These findings have significant impact in the use of 2P-FLIM to discover potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite-driven osteogenesis.

Single-cell genomics reveals new rozellid lineages and supports their sister relationship to Microsporidia.

The phylum Rozellomycota has been proposed for a group of early-branching holomycotan lineages representing obligate parasites and hyperparasites of zoosporic fungi, oomycotes or phytoplankton. Given their predominantly intracellular lifestyle, rozellids are typically known from environmental ribosomal DNA data, except for the well-studied Rozella species. To date, the phylogenetic relationship between rozellids and microsporidians (Microsporidia) is not fully understood and most reliable hypotheses are based on phylogenomic analyses that incorporate the only publicly available rozellid genome of Rozella allomycis. Here, we provide genomic data of three new rozellid lineages obtained by single-cell sequencing from environmental samples and show with a phylogenomic approach that rozellids form a monophyletic group that is sister to microsporidians, corroborating the previously proposed phylum Rozellomycota. Whereas no mitochondrial genes coding for the respiratory Complex I could be found, we discovered a gene coding for a nucleotide phosphate transporter in one of the three draft genomes. The scattered absence of Complex I genes and scattered presence of nucleotide transporter genes across diverse microsporidian and rozellid lineages suggest that these adaptations to a parasitic lifestyle, which reduce the parasite's capability to synthesize ATP but enables it to steal ATP from its host, evolved independently in microsporidians and rozellids.

Thou shall not heal: Overcoming the non-healing behaviour of diabetic foot ulcers by engineering the inflammatory microenvironment.

Diabetic foot ulcers (DFUs) are a devastating complication for diabetic patients that have debilitating effects and can ultimately lead to limb amputation. Healthy wounds progress through the phases of healing leading to tissue regeneration and restoration of the barrier function of the skin. In contrast, in diabetic patients dysregulation of these phases leads to chronic, non-healing wounds. In particular, unresolved inflammation in the DFU microenvironment has been identified as a key facet of chronic wounds in hyperglyceamic patients, as DFUs fail to progress beyond the inflammatory phase and towards resolution. Thus, control over and modulation of the inflammatory response is a promising therapeutic avenue for DFU treatment. This review discusses the current state-of-the art regarding control of the inflammatory response in the DFU microenvironment, with a specific focus on the development of biomaterials-based delivery strategies and their cargos to direct tissue regeneration in the DFU microenvironment.

Persisting roadblocks in arthropod monitoring using non-destructive metabarcoding from collection media of passive traps.

Background: Broad-scale monitoring of arthropods is often carried out with passive traps (e.g., Malaise traps) that can collect thousands of specimens per sample. The identification of individual specimens requires time and taxonomic expertise, limiting the geographical and temporal scale of research and monitoring studies. DNA metabarcoding of bulk-sample homogenates has been found to be faster, efficient and reliable, but the destruction of samples prevents a posteriori validation of species occurrences and relative abundances. Non-destructive metabarcoding of DNA extracted from collection medium has been applied in a limited number of studies, but further tests of efficiency are required with different trap types and collection media to assess the consistency of the method. Methods: We quantified the detection rate of arthropod species when applying non-destructive DNA metabarcoding with a short (127-bp) fragment of mitochondrial COI on two combinations of passive traps and collection media: (1) water with monopropylene glycol (H2O-MPG) used in window-flight traps (WFT, 53 in total); (2) ethanol with monopropylene glycol (EtOH-MPG) used in Malaise traps (MT, 27 in total). We then compared our results with those obtained for the same samples using morphological identification (for WFTs) or destructive metabarcoding of bulk homogenate (for MTs). This comparison was applied as part of a larger study of arthropod species richness in silver fir (Abies alba Mill., 1759) stands across a range of climate-induced tree dieback levels and forest management strategies. Results: Of the 53 H2O-MPG samples from WFTs, 16 produced no metabarcoding results, while the remaining 37 samples yielded 77 arthropod MOTUs in total, of which none matched any of the 343 beetle species morphologically identified from the same traps. Metabarcoding of 26 EtOH-MPG samples from MTs detected more arthropod MOTUs (233) than destructive metabarcoding of homogenate (146 MOTUs, 8 orders), of which 71 were shared MOTUs, though MOTU richness per trap was similar between treatments. While we acknowledge the failure of metabarcoding from WFT-derived collection medium (H2O-MPG), the treatment of EtOH-based Malaise trapping medium remains promising. We conclude however that DNA metabarcoding from collection medium still requires further methodological developments and cannot replace homogenate metabarcoding as an approach for arthropod monitoring. It can be used nonetheless as a complementary treatment when enhancing the detection of soft-bodied arthropods like spiders and Diptera.

A Workflow to Produce a Low-Cost In Vitro Platform for the Electric-Field Pacing of Cellularised 3D Porous Scaffolds.

Endogenous electrically mediated signaling is a key feature of most native tissues, the most notable examples being the nervous and the cardiac systems. Biomedical engineering often aims to harness and drive such activity in vitro, in bioreactors to study cell disease and differentiation, and often in three-dimensional (3D) formats with the help of biomaterials, with most of these approaches adopting scaffold-free self-assembling strategies to create 3D tissues. In essence, this is the casting of gels which self-assemble in response to factors such as temperature or pH and have capacity to harbor cells during this process without imparting toxicity. However, the use of materials that do not self-assemble but can support 3D encapsulation of cells (such as porous scaffolds) warrants consideration given the larger repertoire this would provide in terms of material physicochemical properties and microstructure. In this method and protocol paper, we detail and provide design codes and assembly instructions to cheaply create an electrical pacing bioreactor and a Rig for Stimulation of Sponge-like Scaffolds (R3S). This setup has also been engineered to simultaneously perform live optical imaging of the in vitro models. To showcase a pilot exploration of material physiochemistry (in this aspect material conductivity) and microstructure (isotropy versus anisotropy), we adopt isotropic and anisotropic porous scaffolds composed of collagen or poly(3,4-ethylene dioxythiophene):polystyrenesulfonate (PEDOT:PSS) for their contrasting conductivity properties yet similar in porosity and mechanical integrity. Electric field pacing of mouse C3H10 cells on anisotropic porous scaffolds placed in R3S led to increased metabolic activity and enhanced cell alignment. Furthermore, after 7 days electrical pacing drove C3H10 alignment regardless of material conductivity or anisotropy. This platform and its design, which we have shared, have wide suitability for the study of electrical pacing of cellularized scaffolds in 3D in vitro cultures.

Probing organoid metabolism using fluorescence lifetime imaging microscopy (FLIM): The next frontier of drug discovery and disease understanding.

Organoid models have been used to address important questions in developmental and cancer biology, tissue repair, advanced modelling of disease and therapies, among other bioengineering applications. Such 3D microenvironmental models can investigate the regulation of cell metabolism, and provide key insights into the mechanisms at the basis of cell growth, differentiation, communication, interactions with the environment and cell death. Their accessibility and complexity, based on 3D spatial and temporal heterogeneity, make organoids suitable for the application of novel, dynamic imaging microscopy methods, such as fluorescence lifetime imaging microscopy (FLIM) and related decay time-assessing readouts. Several biomarkers and assays have been proposed to study cell metabolism by FLIM in various organoid models. Herein, we present an expert-opinion discussion on the principles of FLIM and PLIM, instrumentation and data collection and analysis protocols, and general and emerging biosensor-based approaches, to highlight the pioneering work being performed in this field.

An assessment of the response of human MSCs to hydrostatic pressure in environments supportive of differential chondrogenesis.

Mechanical stimulation can modulate the chondrogenic differentiation of stem/progenitor cells and potentially benefit tissue engineering (TE) of functional articular cartilage (AC). Mechanical cues like hydrostatic pressure (HP) are often applied to cell-laden scaffolds, with little optimization of other key parameters (e.g. cell density, biomaterial properties) known to effect lineage commitment. In this study, we first sought to establish cell seeding densities and fibrin concentrations supportive of robust chondrogenesis of human mesenchymal stem cells (hMSCs). High cell densities (15*106 cells/ml) were more supportive of sGAG deposition on a per cell basis, while collagen deposition was higher at lower seeding densities (5*106 cells/ml). Employment of lower fibrin (2.5 %) concentration hydrogels supported more robust chondrogenesis of hMSCs, with higher collagen type II and lower collagen type X deposition compared to 5 % hydrogels. The application of HP to hMSCs maintained in identified chondro-inductive culture conditions had little effect on overall levels of cartilage-specific matrix production. However, if hMSCs were first temporally primed with TGF-ß3 before its withdrawal, they responded to HP by increased sGAG production. The response to HP in higher cell density cultures was also associated with a metabolic shift towards glycolysis, which has been linked with a mature chondrocyte-like phenotype. These results suggest that mechanical stimulation may not be necessary to engineer functional AC grafts using hMSCs if other culture conditions have been optimised. However, such bioreactor systems can potentially be employed to better understand how engineered tissues respond to mechanical loading in vivo once removed from in vitro culture environments.

Melt electrowriting of a biocompatible photo-crosslinkable poly(D,L-lactic acid)/poly(ε-caprolactone)-based material with tunable mechanical and functionalization properties.

The use of polymeric biomaterials to create tissue scaffolds using additive manufacturing techniques is a well-established practice, owing to the incredible rapidity and complexity in design that modern 3D printing methods can provide. One frontier approach is melt electrowriting (MEW), a technique that takes advantage of electrohydrodynamic phenomena to produce fibers on the scale of 10's of microns with designs capable of high resolution and accuracy. Poly(ε-caprolactone) (PCL) is a material that is commonly used in MEW due to its favorable thermal properties, high stability, and biocompatibility. However, one of the drawbacks of this material is that it lacks the necessary chemical groups which allow covalent crosslinking of additional elements onto its structure. Attempts to functionalise PCL structures therefore often rely on the functional units to be applied externally via coatings or integrally mixed elements. Both can be extremely useful depending on their applications, but can add extra difficulties into the use of the resulting structures. Coatings require careful design and application to prevent rapid degradation, while elements mixed into the polymer melt must deal with the possibilities of phase separation and changes to MEW properties of the unadulterated polymer. With this in mind, this study sought to imbibe functionality to MEW-printed scaffolds using the approach of adding functional units directly, via covalent bonding of functional groups to the polymer itself. To this end, this study employs a recently developed class of polymers called acrylate-endcapped urethane-based polymers (AUPs). The polymer backbone of the specific AUP used consists of a poly(D,L-lactic acid) (PDLLA)/PCL copolymer chain, which is functionalized with 6 acrylate groups, 3 at either end. Through blending of the AUP with PCL, various concentrations of this mixture were used with MEW to produce scaffolds that possessed acrylate groups on their surface. Using UV crosslinking, these groups were tagged with Fluorescein-o-Acrylate to verify that PDLLA/PCL AUP/PCL blends facilitate the direct covalent bonding of external agents directly onto the MEW material. Blending of the AUP with PCL increases the scaffold's stiffness and ultimate strength. Finally, blends were proven to be highly biocompatible, with cells attaching and proliferating readily at day 3 and 7 post seeding. Through this work, PDLLA/PCL AUP/PCL blends clearly demonstrated as a biocompatible material that can be processed using MEW to create functionalised tissue scaffolds.

Macrophage metabolic profile is altered by hydroxyapatite particle size.

Since the recent observation that immune cells undergo metabolic reprogramming upon activation, there has been immense research in this area to not only understand the basis of such changes, but also to exploit metabolic rewiring for therapeutic benefit. In a resting state, macrophages preferentially utilise oxidative phosphorylation to generate energy; however, in the presence of immune cell activators, glycolytic genes are upregulated, and energy is generated through glycolysis. This facilitates the rapid production of biosynthetic intermediates and a pro-inflammatory macrophage phenotype. While this is essential to mount responses to infectious agents, more evidence is accumulating linking dysregulated metabolism to inappropriate immune responses. Given that certain biomaterials are known to promote an inflammatory macrophage phenotype, this prompted us to investigate if biomaterial particulates can impact on macrophage metabolism. Using micron and nano sized hydroxyapatite (HA), we demonstrate for the first time that these biomaterials can indeed drive changes in metabolism, and that this occurs in a size-dependent manner. We show that micronHA, but not nanoHA, particles upregulate surrogate markets of glycolysis including the glucose transporter (GLUT1), hexokinase 2 (HK2), GAPDH, and PKM2. Furthermore, we demonstrate that micronHA alters mitochondrial morphology and promotes a bioenergetic shift to favour glycolysis. Finally, we demonstrate that glycolytic gene expression is dependent on particle uptake and that targeting glycolysis attenuates the pro-inflammatory profile of micronHA-treated macrophages. These results not only further our understanding of biomaterial-based macrophage activation, but also implicate immunometabolism as a new area for consideration in intelligent biomaterial design and therapeutic targeting. STATEMENT OF SIGNIFICANCE: Several recent studies have reported that immune cell activation occurs concurrently with metabolic reprogramming. Furthermore, metabolic reprogramming of innate immune cells plays a prominent role in determining cellular phenotype and function. In this study we demonstrate that hydroxyapatite particle size alters macrophage metabolism, in turn driving their functional phenotype. Specifically, the pro-inflammatory phenotype promoted by micron-sized HA-particles is accompanied by changes in mitochondrial dynamics and a bioenergetic shift favouring glycolysis. This effect is not seen with nano-HA particles and can be attenuated upon inhibition of glycolysis. This study therefore not only identifies immunometabolism as a useful tool for characterising the immune response to biomaterials, but also highlights immunometabolism as a targetable aspect of the host response for therapeutic benefit.

Spatial and phylogenetic structure of Alpine stonefly assemblages across seven habitats using DNA-species.

Stream ecosystems are spatially heterogeneous, with many different habitat patches distributed within a small area. The influence of this heterogeneity on the biodiversity of benthic insect communities is well documented; however, studies of the role of habitat heterogeneity in species coexistence and assembly remain limited. Here, we investigated how habitat heterogeneity influences spatial structure (beta biodiversity) and phylogenetic structure (evolutionary processes) of benthic stonefly (Plecoptera, Insecta) communities. We sampled 20 sites along two Alpine rivers, including seven habitats in four different reaches (headwaters, meandering, bar-braided floodplain, and lowland spring-fed). We identified 21 morphological species and delineated 52 DNA-species based on sequences from mitochondrial cox1 and nuclear ITS markers. Using DNA-species, we first analysed the patterns of variation in richness, diversity, and assemblage composition by quantifing the contribution of each reach and habitat to the overall DNA-species diversity using an additive partition analysis and distance-based redundancy analysis. Using gene-tree phylogenies, we assessed whether environmental filtering could lead to the co-occurrence of DNA-species using a two-step analysis to detect a phylogenetic signal. All four reaches significantly contributed to DNA-species richness, with the meandering reach having the highest contribution. Habitats had an effect on DNA-species diversity, where glide, riffle and, pool influenced the spatial structure of stonefly assemblage possibly due to the high habitat heterogeneity. Among the habitats, the pool showed significant phylogenetic clustering, suggesting high levels of evolutionary adaptation and strong habitat filtering. This assemblage structure may be caused by long-term stability of the habitat and the similar requirements for co-occurring species. Our study shows the importance of different habitats for the spatial and phylogenetic structure of stonefly assemblage and sheds light on the habitat-specific diversity that may help improve conservation practices.

Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis.

Background: Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with MSTN genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C > T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse gluteus medius, were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for ex vivo skeletal muscle isolated from Thoroughbred horses with each genotype. Results: Primary myoblasts conditionally immortalised with the temperature sensitive SV40TtsA58 lentivirus vector successfully proliferated and could revert to their primary cell phenotype and differentiate into multinucleated myotubes. Skeletal muscle fibre type, MSTN gene expression, mitochondrial abundance, and mitochondrial function of the three MSTN genotype cell lines, were consistent with equivalent characterisation of ex vivo skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q10 (CoQ10) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ10 in the diet. Conclusions: The observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to ex vivo skeletal muscle indicates that this in vitro system will enable efficient and cost-effective analyses of equine skeletal muscle for a range of different applications including understanding metabolic function, testing of nutritional supplements, drug test development and gene doping test development. In the multi-billion-euro international Thoroughbred horse industry research advances in the biological function of skeletal muscle are likely to have considerable impact. Furthermore, this novel genotype-specific system may be adapted and applied to human biomedicine to improve understanding of the effects of myostatin in human physiology and medicine.

MXene functionalized collagen biomaterials for cardiac tissue engineering driving iPSC-derived cardiomyocyte maturation.

Electroconductive biomaterials are gaining significant consideration for regeneration in tissues where electrical functionality is of crucial importance, such as myocardium, neural, musculoskeletal, and bone tissue. In this work, conductive biohybrid platforms were engineered by blending collagen type I and 2D MXene (Ti3C2Tx) and afterwards covalently crosslinking; to harness the biofunctionality of the protein component and the increased stiffness and enhanced electrical conductivity (matching and even surpassing native tissues) that two-dimensional titanium carbide provides. These MXene platforms were highly biocompatible and resulted in increased proliferation and cell spreading when seeded with fibroblasts. Conversely, they limited bacterial attachment (Staphylococcus aureus) and proliferation. When neonatal rat cardiomyocytes (nrCMs) were cultured on the substrates increased spreading and viability up to day 7 were studied when compared to control collagen substrates. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were seeded and stimulated using electric-field generation in a custom-made bioreactor. The combination of an electroconductive substrate with an external electrical field enhanced cell growth, and significantly increased cx43 expression. This in vitro study convincingly demonstrates the potential of this engineered conductive biohybrid platform for cardiac tissue regeneration.

Non-invasive classification of macrophage polarisation by 2P-FLIM and machine learning.

In this study, we utilise fluorescence lifetime imaging of NAD(P)H-based cellular autofluorescence as a non-invasive modality to classify two contrasting states of human macrophages by proxy of their governing metabolic state. Macrophages derived from human blood-circulating monocytes were polarised using established protocols and metabolically challenged using small molecules to validate their responding metabolic actions in extracellular acidification and oxygen consumption. Large field-of-view images of individual polarised macrophages were obtained using fluorescence lifetime imaging microscopy (FLIM). These were challenged in real time with small-molecule perturbations of metabolism during imaging. We uncovered FLIM parameters that are pronounced under the action of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which strongly stratifies the phenotype of polarised human macrophages; however, this performance is impacted by donor variability when analysing the data at a single-cell level. The stratification and parameters emanating from a full field-of-view and single-cell FLIM approach serve as the basis for machine learning models. Applying a random forests model, we identify three strongly governing FLIM parameters, achieving an area under the receiver operating characteristics curve (ROC-AUC) value of 0.944 and out-of-bag (OBB) error rate of 16.67% when classifying human macrophages in a full field-of-view image. To conclude, 2P-FLIM with the integration of machine learning models is showed to be a powerful technique for analysis of both human macrophage metabolism and polarisation at full FoV and single-cell level.

A preclinical model of cutaneous melanoma based on reconstructed human epidermis.

Malignant melanoma is among the tumor entities with the highest increase of incidence worldwide. To elucidate melanoma progression and develop new effective therapies, rodent models are commonly used. While these do not adequately reflect human physiology, two-dimensional cell cultures lack crucial elements of the tumor microenvironment. To address this shortcoming, we have developed a melanoma skin equivalent based on an open-source epidermal model. Melanoma cell lines with different driver mutations were incorporated into these models forming distinguishable tumor aggregates within a stratified epidermis. Although barrier properties of the skin equivalents were not affected by incorporation of melanoma cells, their presence resulted in a higher metabolic activity indicated by an increased glucose consumption. Furthermore, we re-isolated single cells from the models to characterize the proliferation state within the respective model. The applicability of our model for tumor therapeutics was demonstrated by treatment with a commonly used v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib. This selective BRAF inhibitor successfully reduced tumor growth in the models harboring BRAF-mutated melanoma cells. Hence, our model is a promising tool to investigate melanoma development and as a preclinical model for drug discovery.

Phylogenomic insights into the early diversification of fungi.

Phylogenomic analyses have boosted our understanding of the evolutionary trajectories of all living forms by providing continuous improvements to the tree of life.1-5 Within this tree, fungi represent an ancient eukaryote group,6 having diverged from the animals ∼1.35 billion years ago.7 Estimates of the number of extant species range between 1.5 and 3.8 million.8,9 Recent reclassifications and the discovery of the deep-branching Sanchytriomycota lineage10 have brought the number of proposed phyla to 20,11 21 if the Microsporidia are included.12-14 Uncovering how the diverse and globally distributed fungi are related to each other is fundamental for understanding how their lifestyles, morphologies, and metabolic capacities evolved. To date, many of the proposed relationships among the phyla remain controversial and no phylogenomic study has examined the entire fungal tree using a taxonomically comprehensive dataset and suitable models of evolution. We assembled and curated a 299-protein dataset with a taxon sampling broad enough to encompass all recognized fungal diversity with available data, but selective enough to run computationally intensive analyses using best-fitting models. Using a range of reconstruction methods, we were able to resolve many contested nodes, such as a sister relationship of Chytridiomyceta to all other non-Opisthosporidia fungi (with Chytridiomycota being sister to Monoblepharomycota + Neocallimastigomycota), a branching of Blastocladiomycota + Sanchytriomycota after the Chytridiomyceta but before other non-Opisthosporidia fungi, and a branching of Glomeromycota as sister to the Dikarya. Our up-to-date fungal tree of life will serve as a springboard for future investigations on the early evolution of fungi.

Cholesterol crystals drive metabolic reprogramming and M1 macrophage polarisation in primary human macrophages.

BACKGROUND AND AIMS: Metabolic reprogramming of innate immune cells is emerging as a key player in the progression of a number of chronic diseases, including atherosclerosis, where high rates of glycolysis correlate with plaque instability. This study aimed to investigate if cholesterol crystals, which are key atherosclerosis-associated DAMPs (damage/danger-associated molecular patterns), alter immune cell metabolism and whether this, in turn, impacts on macrophage phenotype and function. METHODS AND RESULTS: Primary human macrophages were treated with cholesterol crystals and expression of M1 (CXCL9, CXCL10) and M2-associated (MRC1, CCL13) macrophage markers, alarmins, and inflammatory cytokines were assessed either by real-time PCR or ELISA. Cholesterol crystal-induced changes in glycolytic markers were determined using real-time PCR and western blotting, while changes in cellular respiration and mitochondrial dynamics were examined via Seahorse analysis, Fluorescence Lifetime Imaging Microscopy (FLIM) and confocal microscopy. Treatment of macrophages with cholesterol crystals upregulated mRNA levels of CXCL9 and CXCL10, while concomitantly downregulating expression of MRC1 and CCL13. Cholesterol crystal--treated macrophages also exhibited a significant shift in metabolism to favour glycolysis, accompanied by the expression of key glycolytic markers GLUT1, Hexokinase 2, HIF1α, GAPDH and PFKFB3. Furthermore, we show that these effects are mediated upstream by the glycolytic enzyme, PKM2, and that direct inhibition of glycolysis or PKM2 nuclear localisation leads to a significant reduction in cholesterol crystal-induced inflammatory readouts. CONCLUSIONS: This study not only provides further insight into how atherosclerosis-associated DAMPs impact on immune cell function, but also highlights metabolic reprogramming as a potential therapeutic target for cholesterol crystal-related inflammation.

Large-scale sampling of the freshwater microbiome suggests pollution-driven ecosystem changes.

Freshwater microbes play a crucial role in the global carbon cycle. Anthropogenic stressors that lead to changes in these microbial communities are likely to have profound consequences for freshwater ecosystems. Using field data from the coordinated sampling of 617 lakes, ponds, rivers, and streams by citizen scientists, we observed linkages between microbial community composition, light and chemical pollution, and greenhouse gas concentration. All sampled water bodies were net emitters of CO2, with higher concentrations in running waters, and increasing concentrations at higher latitudes. Light pollution occurred at 75% of sites, was higher in urban areas and along rivers, and had a measurable effect on the microbial alpha diversity. Genetic elements suggestive of chemical stress and antimicrobial resistances (IntI1, blaOX58) were found in 85% of sites, and were also more prevalent in urban streams and rivers. Light pollution and CO2 were significantly related to microbial community composition, with CO2 inversely related to microbial phototrophy. Results of synchronous nationwide sampling indicate that pollution-driven alterations to the freshwater microbiome lead to changes in CO2 production in natural waters and highlight the vulnerability of running waters to anthropogenic stressors.