Advanced Biology

Slab photonic crystals, nanomaterials characterized by periodic pores for manipulating light, have applications in advanced optical technologies. Remarkably, similar materials have been identified in the silica shell of diatoms, in particular the girdle bands. Despite the potential applications and significance for diatom biology, their prevalence remains uncertain due to limited observations across a few species. In this study of 393 SEM girdle band micrographs across major taxonomic groups, we identified slab photonic crystals using Fast Fourier Transform (FFT) analysis. A correlation analysis of these properties on a phylogenetic tree revealed their distribution across the diversity of species and taxonomic groups. Square and hexagonal lattice varieties are prevalent in earlier-diverging groups, and linked to phytoplanktonic lifestyles. More recently-diverged clades lack these structures entirely in their girdle bands. Numerical analysis indicates that square lattice types exhibit anticipated photonic properties (stopbands) in the visible spectrum, while hexagonal lattice types are primarily linked to the near to mid-infrared range. This suggests that girdle band slab photonic crystal morphologies 1) originate from quasi-periodic photonic structures, 2) are primarily found in evolutionarily older clades (Coscinodiscophyceae and Mediophyceae), 3) lost square lattice types through diversification in the Mediophyceae, and 4) are absent in more recent clades (Fragilariophyceae and Bacillariophyceae). The limited inter-species distribution of slab photonic crystals may offer experimental cues to study their biological functionality. While these data suggest that stopband functionalities are a derived frustule trait, the ultimate purpose of slab photonic crystals in nature remains a mystery.

Antifungal drug resistance is a major problem in healthcare and agriculture. Synthesizing new drugs is one of the major mitigating strategies for overcoming this problem. In this context, carbon-dots (CDs) are a newer category of nanoparticles that have wide applications, potentially including use as antibiotics. However, there is a lack of understanding of the effect of long-term use of CDs as antimicrobials, particularly the ability of microbes to evolve resistance to antibiotic CDs. In this study, we synthesized novel florescent the bottom-up method using two antifungal drugs fluconazole and nourseothricin sulphate (ClonNAT). We first extensively characterized the physical properties of the newly synthesized carbon dots, Flu-Clo CDs. We measured the cytotoxicity of Flu-Clo CDs on budding yeast Saccharomyces cerevisiae and determined that it had comparable antifungal inhibition with extensively used drug fluconazole. Furthermore, we demonstrate that Flu-CLO CDs are not cytotoxic to human fibroblasts cell lines. Then, we quantified the ability of yeast to evolve resistance to Flu-Clo CDs. We evolved replicate laboratory yeast populations for 250 generations in the presence of Flu-Clo CDs or aqueous fluconazole. We found that yeast evolved resistance to Flu-Clo CDs and aqueous fluconazole at similar rates. Further, we found that resistance to Flu-Clo CDs conferred cross-resistance to aqueous fluconazole. Overall, the results demonstrate the efficacy of CDs as potential antifungal drugs. We can conclude that yeast populations can adapt quickly to novel antibiotics including CD based antibiotics, including CD-based antibiotics indicating the importance of proper use of antimicrobials in combating infections.

Brain-derived neurotrophic factor (BDNF), via activation of tropomyosin receptor kinase B (TrkB), plays a critical role in neuronal proliferation, differentiation, survival, and death. Dysregulation of TrkB signaling is implicated in neurodegenerative disorders and cancers. Precise activation of TrkB receptors with spatial and temporal resolution is greatly desired to study the dynamic nature of TrkB signaling and its role in related diseases. Here we develop different optogenetic approaches that use light to activate TrkB receptors. Utilizing the photosensitive protein Arabidopsis thaliana cryptochrome 2 (CRY2), the light-inducible homo-interaction of the intracellular domain of TrkB (iTrkB) in the cytosol or on the plasma membrane is able to induce the activation of downstream MAPK/ERK and PI3K/Akt signaling as well as the neurite outgrowth of PC12 cells. Moreover, we prove that such strategies are generalizable to other optical homo-dimerizers by demonstrating the optical TrkB activation based on the light-oxygen-voltage domain of aureochrome 1 from Vaucheria frigida. The results open up new possibilities of many other optical platforms to activate TrkB receptors to fulfill customized needs. By comparing all the different strategies, we find that the CRY2-integrated approach to achieve light-induced cell membrane recruitment and homo-interaction of iTrkB is most efficient in activating TrkB receptors. The optogenetic strategies presented are promising tools to investigate BDNF/TrkB signaling with tight spatial and temporal control.

The bottom-up constructed artificial cells help to understand the cell working mechanism and provide the evolution clues for organisms. Cyanobacteria are believed to be the ancestors of chloroplasts according to endosymbiosis theory. Herein we demonstrate an artificial cell containing cyanobacteria to mimic endosymbiosis phenomenon. The cyanobacteria sustainably produce glucose molecules by converting light energy into chemical energy. Two downstream "metabolic" pathways starting from glucose molecules are investigated. One involves enzyme cascade reaction to produce H2O2 (assisted by glucose oxidase) first, followed by converting Amplex red to resorufin (assisted by horseradish peroxidase). The more biological one involves nicotinamide adenine dinucleotide (NADH) production in the presence of NAD+ and glucose dehydrogenase. Further, NADH molecules are oxidized into NAD+ by pyruvate catalyzed by lactate dehydrogenase, meanwhile, lactate is obtained. Therefore, the sustainable cascade cycling of NADH/NAD+ is built. The artificial cells built here simulate the endosymbiosis phenomenon, meanwhile pave the way for investigating more complicated sustainable energy supplied metabolism inside artificial cells.

Mitochondrial function is critical for cellular health, with dysfunction contributing to human diseases. Structural changes in mitochondria, such as size and shape, reflect alterations in bioenergetics, fission-fusion dynamics, and metabolic homeostasis. Existing morphological quantification is outdated, can be biased, technologically limited, or overly complex. This study presents a high content system for quantifying morphology using open-access resources and widely available equipment. Fibroblasts were stained with PKmitoTM Dye Deep Red, imaged via automated confocal microscopy, and analyzed with CellProfiler and KNIME(R). We tested different imaging conditions and found live-cell confocal imaging at 60x magnification provided the most precise measurements. Using this system, we found that human Huntington Disease fibroblast mitochondria were significantly smaller and more circular, suggesting increased fission. To confirm our results, we employed other mitochondrial assays and found elevated expression of the fission protein Drp1, reduced respiration, impaired iron uptake, and increased membrane potential. This system offers a robust, unbiased high content approach to studying mitochondrial morphology in disease.

Eph receptors are ubiquitous class of transmembrane receptors that mediate cell-cell communication, proliferation, differentiation, and migration. EphA1 receptors specifically play an important role in angiogenesis, fetal development, and cancer progression; however, studies of this receptor can be challenging as its ligand, ephrinA1, binds and activates several EphA receptors simultaneously. Optogenetic strategies could be applied to circumvent this requirement for ligand activation and enable selective activation of the EphA1 subtype. In this work, we designed and tested several iterations of an optogenetic EphA1 - Cryptochrome 2 (Cry2) fusion, investigating their capacity to mimic EphA1-dependent signaling in response to light activation. We then characterized the key cell signaling target of MAPK phosphorylation activated in response to light stimulation. The optogenetic regulation of Eph receptor RTK signaling without the need for external stimulus promises to be an effective means of controlling individual Eph receptor-mediated activities and creates a path forward for the identification of new Eph-dependent functions.

A method to measure telomere length in S. cerevisiae was developed based on bioluminescence resonance energy transfer (BRET). The system uses energy transfer between a luciferase-Rif2 fusion protein and fluorescently tagged Rap1. The study demonstrates that the BRET ratio correlates with the Rap1/Rif2 complex at the telomeres and thus the availability of telomeric Rap1 binding sites. This enables the measurement of telomere length in living cells. The system was able to reproduce reported deviations in telomere length in mutants lacking telomere length regulators, cells treated with telomere length modifying compounds and strains expressing inducible telomerase. The BRET ratio linearly correlated with the average number of telomeric nucleotides derived from long-read sequencing data using a novel algorithm for telomere length calculation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/711003v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1850c4dorg.highwire.dtl.DTLVardef@1ead295org.highwire.dtl.DTLVardef@1a76358org.highwire.dtl.DTLVardef@6b3183_HPS_FORMAT_FIGEXP M_FIG C_FIG

In this paper, we report for the first time, the synthesis of a semiconducting biofilm. Photosynthetic bacterial biofilm has been used to weave together MoS2 nanosheets into an adherent film grown on interdigitated electrodes. Liquid-phase exfoliation of bulk MoS2 powder was used to obtain MoS2 nanosheets. A synchronous-fluorescence scan revealed the presence of two emission maxima at 682nm and 715nm for the MoS2 suspension. Such maxima with bandgap energy 1.82 and 1.73 eV corresponded to the single and double layer of MoS2. The presence of such single and multi-layered structures was confirmed by Raman spectroscopy, FTIR studies, and electron microscopy. The current-voltage (I-V) studies of such a bio-nano hybrid revealed the emergence of the gated nature of the current flow. This Schottky diode like behavior, reported earlier for Graphene-biofilm junctions, is also observed in this case. Gating voltage depended on the composition of the biofilm. The semiconductor biofilms, when studied using electrochemical impedance spectroscopy, revealed characteristic Nyquist and Bode plots, suggesting special circuit-equivalence for each film. While Mos2 was marked with stability with respect to variations in RMS voltage and bias voltage, the graphene biofilm was unique by the absence of any Warburg element.

Spheroids have become principal three-dimensional biological models to study cancer, developmental processes, and drug efficacy. For spheroid generation, ultra-low attachment plates are noteworthy due to their simplicity, compatibility with automation, and experimental and commercial accessibility. Nonetheless, it is unknown whether and to what degree the plate type impacts spheroid formation and biology. This study employed automated brightfield microscopy to systematically compare the size and eccentricity of spheroids formed in six different plate types using four distinct human cell lines, i.e., CCD-1137Sk fibroblasts, HaCaT keratinocytes, and MDA-MB-231 and HT-29 cancer cells. Results showed that all plate types exhibited similar sphe-roid-forming capabilities, and the gross patterns of growth or shrinkage during four days after seeding were comparable. Yet, size and eccentricity varied systematically among specific cell lines and plate types. A confocal wholemount analysis by a novel pipeline of AI-based 3D-image analysis procedures revealed changes in cell proliferation, cell number, nuclear volume, and keratino-cyte differentiation, which were accompanied by altered YAP1-signals. The findings show that the plate type may influence the outcome of experimental campaigns. It is advisable to scan different plate types for the optimal configuration for a specific investigation instead of using one standard plate for all kinds of applications.

Cytochrome c (Cyt c) is a key protein that is needed to maintain life (respiration) and cell death (apoptosis). The dual-function of Cyt c comes from its capability to act as mitochondrial redox carrier that transfers electrons between the membrane-embedded complexes III and IV and to serve as a cytoplasmic apoptosis-triggering agent, activating the caspase cascade.1-6 However, the precise roles of Cyt c in mitochondria, cytoplasm and extracellular matrix under normal and pathological conditions are not completely understood.7-9 To date, no pathway of Cyt c release that results in caspase activation has been compellingly demon-strated in any invertebrate.10 The significance of mitochondrial dysfunctionality has not been studied in ductal carcinoma to the best of our knowledge.1 Here we show that proper concentration of monounsaturated fatty acids, saturated fatty acids, cardi-olipin and Cyt c is critical in the correct breast ductal functioning and constitutes an important parameter to assess breast epithelial cells integrity and homeostasis. We look inside human breast ducts answering fundamental questions about location and distribution of various biochemical components inside the lumen, epithelial cells of the duct and the extracellular matrix around the cancer duct during cancer development in situ. We found in histopathologically controlled breast cancer duct that Cyt c, cardi-olipin, and palmitic acid are the main components inside the lumen of cancerous duct in situ. The pre-sented results show direct evidence that Cyt c is released to the lumen from the epithelial cells in can-cerous duct. In contrast the lumen in normal duct is empty and free of Cyt c. Our results demonstrate how Cyt c is likely to function in cancer development. We anticipate our results to be a starting point for more sophisticated in vitro and in vivo animal models. For example, the correlation between concentration of Cyt c and cancer grade could be tested in various types of cancer. Furthermore, Cyt c is a target of anti-cancer drug development 11,12 and a well-defined and quantitative Raman based assay for oxidative phosphorylation and apoptosis will be relevant for such developments.

Magnetogenetics is a promising field of biology devoted to the manipulation of biological objects by magnetic fields, where magnetic nanoparticles usually act as actuators. However, studying the response of biological objects is hampered by the fact that microscopes are usually made of ferromagnetic materials, making it difficult to work with them in powerful magnetic fields. This article focuses on building a DIY 3d printed microscope based on the ESPressoscope platform, integrated into a gradient magnetic system of permanent magnets. The article reveals the design, choice of materials and, using the example of cellular spheroids saturated with magnetic nanoparticles, demonstrates the application of MagnEspScope for the observation of magnetophoresis of biological objects.

Genetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca2+ and voltage sensors are available to study the signaling behavior of entire neural circuits. There is a pressing need for improved sensors to properly interrogate these systems. Improving them is challenging because testing them involves low throughput, labor-intensive processes. Our goal was to create a live cell system in HEK293 cells that use a simple, reproducible, optogenetic process for testing prototypes of genetically encoded biosensors. In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP [1]. In turn, the cAMP opens a cAMP gated ion channel (olfactory cyclic nucleotide-gated channel, CNG, or the hyperpolarization-activated cyclic nucleotide-gated channel, HCN2). This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we added the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. This is a modular system in which the types of channels, and their relative amounts, can be tuned to produce the cellular behavior that is crucial for screening biosensors. The result is a highly reproducible, high-throughput live cell system that can be used to screen voltage and Ca2+ sensors in multiple fluorescent wavelengths simultaneously.

Vertebrate vision is accomplished by two phenotypically distinct types of photoreceptors in the retina: the saturation-resistant cones for the detection of bright light and the highly sensitive rods for dim light conditions [1]. The current dogma is that, during development, all vertebrates initially feature a cone-dominated retina, and rods are added later [2, 3]. By studying the ontogeny of vision in three species of deep-sea fishes, we show that their larvae express cone-specific genes in photoreceptors with rod-like morphologies. Through development, these fishes either retain this rod-like cone retina (Maurolicus mucronatus) or switch to a retina with true rod photoreceptors with expression of rod-specific genes and transcription factors (Vinciguerria mabahiss and Benthosema pterotum). In contrast to the larvae of most marine fishes, which inhabit the bright upper layer of the open ocean, the larvae of deep-sea fishes occur deeper, exposing them to a dimmer light environment [4-7]. Spectral maxima predictions from molecular dynamics simulations and environmental light estimations suggest that using transmuted photoreceptors that combine the characteristics of both cones and rods maximises visual performance in these dimmer light conditions. Our findings provide molecular, morphological, and functional evidence for the evolution of an alternative developmental pathway for vertebrate vision.

Transmission electron microscopy (TEM) is a powerful imaging technique, yielding ultrastructural investigation of organic and non-organic samples. Despite its ability to reach nanoscale resolutions, conventional TEM presents a major disadvantage by only acquiring two-dimensional snapshots, thus hindering our volumetric understanding of samples. Electron tomography (ET) overcomes this limitation by offering detailed views of a thin specimen in 3 dimensions (3D). This technique is widely used in biology and has expanded our understanding of mitochondrial structure or synaptic organization. Proper brain functioning is highly reliable on a constant nutritional support through its microvasculature lined by endothelial cells. These unique cells form a selective and protective barrier, known as the blood-brain barrier (BBB), which limits the entrance of blood-borne molecules into the brain. In pathological conditions, the BBB is disrupted, resulting in neuronal damage. Understanding the fine changes underlying BBB disruption requires advanced imaging tools such as ET, to detect the finest changes in endothelial ultrastructure. This manuscript briefly explains how TEM and ET function, and then provides a detailed, didactic method for sample preparation, tomogram generation and 3D segmentation of brain endothelial cells using ET.

1.Cell motility, shape supporting, and intracellular signaling are followed by changes in cell morphology and cytoskeleton. The cell reaction and the reorganization of the cytoskeleton occurs in a single volume of the cytoplasm and affects all components of the cytoskeleton: intermediate filaments, microtubules and microfilaments. A promising way to manipulate cells is magnetic nanoparticles that control cellular physiology. This approach is called magnetogenetics and has found application in various fields of cell and molecular biology. Using a magnetic field, it is possible to non-invasively regulate biochemical processes, migration and changes in the morphology of cells with magnetic nanoparticles. Our work opens up new possibilities for spatial manipulation of individual cytoskeletal components in vitro and operates biochemical pathways associated with individual cytoskeletal components.

When studying the split-gill fungus Schizophyllum commune, we observed that the growing colonies displayed endogenous spikes of electrical potential similar to the action potentials of neurons. In order to investigate the impact of light on the electrical activities of these colonies, we exposed them to intermittent stimulation with cold light (5800k) and later with blue (c. 470nm), red (c. 642nm) and green (c. 538nm) light. Our findings revealed spiking activity can be influenced using this input including observable responses with patterns of spiking at relatively high average amplitudes (>1mV) appearing consistently upon illumination of the sample. The response is likely related to the activity of fungal photoreceptors, including potential sensitisation to blue light in the cellular signalling pathways facilitated by white collar proteins (WC-1, WC-2) in S. commune. Based on these findings, we suggest that fungal photosensors and photonic computing substrates have the potential to enable applications beyond the scope of conventional electronics via relatively fast spiking responses to light tuned by external input stimulation. Further work should focus on identifying the signal transduction pathway for responses to different wavelengths of light and its role in translation into engineered ELMs to extend existing studies in fungal photobiology.

1.Nanoelectrospray can be used to generate a layered structure consisting of bipolar lipids, detergent-solubilized membrane proteins, and glycerol that self-assembles upon detergent extraction into one extended layer of a protein containing membrane. This manuscript presents the first evidence that this method might allow membrane protein complexes to assemble in this process.

Phospholipid synthesis is a fundamental process that promotes cell propagation and, presently, is the most challenging issue in artificial cell research aimed at reconstituting living cells from biomolecules. Here, we constructed a cell-free phospholipid synthesis system that combines in vitro fatty acid synthesis and a cell-free gene expression system that synthesizes acyltransferases for phospholipid synthesis. Fatty acids were synthesized from acetyl-CoA and malonyl-CoA, then continuously converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of synthetic intermediates that suppress the reaction, the yield of phospholipid has significantly improved from previous schemes (up to 400 {micro}M). Additionally, by adding enzymes for recycling CoA, we synthesized phosphatidic acids from acetic acid and bicarbonate as carbon sources. The constructed system is available to express the genes from pathogenic bacteria and to analyze the synthesized phospholipids. By encapsulating our system inside giant vesicles, it would be possible to construct the artificial cells in which the membrane grows and divides sustainably.

The production of giant unilamellar vesicles (GUVs) plays a pivotal role in various scientific disciplines, particularly in the development of synthetic cells. While numerous methods exist for GUV preparation, the modified continuous droplet interface crossing encapsulation (cDICE) method offers the advantages of simplicity and high encapsulation efficiency. However, a significant limitation of this technique is the generation of vesicles with a broad size distribution and the inability to control the desired size range. This raises a key question: Can the modified cDICE method be optimized to produce GUVs with controlled size distribution? In this study, we examined the effects of two experimental parameters--rotation time (tROT) and the angular frequency ({omega}) of the cDICE chamber--on the size distribution of GUVs. Our results show that reducing either the angular frequency or rotation time shifts the size distribution toward larger vesicles, enabling effective size selection. These findings are further supported by a physical model, which provides insights into the mechanisms underlying size selection. This work demonstrates that control over GUV size distribution can be achieved through straightforward adjustments of system parameters. The ability to fine-tune vesicle size offers researchers a powerful tool for developing customizable experimental systems for synthetic biology and related fields.

3.1Birds are one of the most colourful animal groups in the world and there are multiple ways by which they achieve this feature. Mechanisms of colour production range from pigmentary (pigment deposition) to structural (nanostructural arrangements), or the combination of both. Despite the huge breadth of colour gamut, the basic components of feathers are shared across all of them (keratin, air and presence of pigments in accordance with the colour produced). It has been shown that in some instances, colour evolution between pigmentary and structural colours can proceed by rearrangement of the nano-structural elements of feathers. Here, we investigated evolutionary transitions between pigmentary grey and non-iridescent structural blue. We focus on the Thraupidae (tanagers and allies) that display a variety of blues and greys including a potential transition state that we refer to as slate. We used digitally calibrated images of birds to quantify colour and determine the distinctiveness of slate colour in colourspace. Following, we identify the most likely pathway for the evolution of the colour blue: from grey via slate colour. Our research reveals a new pathway in the evolution of blue colour.