Advanced Biology

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

The estimation of the biological sex of archeological remains is crucial information in bioarchaeology and forensic anthropology. In recent years, proteomics based on molecular sexual dimorphism have emerged as a preferred method, particularly because of its minimally-invasive approach to extracting amelogenin X and Y proteins from tooth enamel. However, there is an increasing demand to accelerate this process while facilitating the analysis of large archaeological assemblages. This study presents a novel high-throughput targeted paleoproteomics method for biological sex estimation using MALDI-CASI-FTICR mass spectrometry. This approach combines the strengths of existing methods, including ultra-high resolution, significantly reduced processing times, targeted analysis, and scalability to large archaeological sample sets. The method was initially validated on modern individuals with known sex and subsequently applied to 130 adult and juvenile individuals from medieval Great Moravia (present-day Czech Republic). Biological sex was successfully estimated for all but one of the individuals. The results not only provide a more efficient biological sex estimation but also help to resolve a few errors in sex assessment previously encountered with osteomorphological and tooth morphometric techniques. The implementation of this method significantly improves the accuracy and efficiency of biological sex estimation, offering a powerful tool for anthropological research. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/706309v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1ede7e6org.highwire.dtl.DTLVardef@13d2f5org.highwire.dtl.DTLVardef@17ee44dorg.highwire.dtl.DTLVardef@1be9dd9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mitochondrial membrane potential ({Delta}{Psi}m) is central to ATP production, ion homeostasis, and cell survival, reflecting the functional state of the inner mitochondrial membrane and oxidative phosphorylation. Accurate assessment of {Delta}{Psi}m is therefore essential for understanding mitochondrial physiology and dysfunction in health, ageing, and disease. Lipophilic cationic fluorescent dyes, such as TMRM and TMRE, are widely used to monitor {Delta}{Psi}m in live cells, enabling high-temporal-resolution imaging of both steady-state membrane potential and dynamic fluctuations. Beyond stable bioenergetic measurements, live-cell imaging reveals transient, reversible depolarisation events, known as mitochondrial "flickers." These events, observed across multiple cell types and imaging platforms, are often associated with brief openings of the mitochondrial permeability transition pore (mPTP) and may represent regulated mitochondrial excitability, rather than irreversible damage. While excessive or synchronised depolarisations may signal mitochondrial injury, transient flickers are increasingly viewed as potential signalling mechanisms within the mitochondrial network. This work discusses methodological considerations for {Delta}{Psi}m imaging, the biological significance of mitochondrial flickers, and the importance of distinguishing physiological events from probe- and light-induced artefacts, highlighting the emerging concept of mitochondria as dynamic and communicative bioenergetic networks.

Green and blue are rare colours in bivalve shells. Where such colours occur, it is unclear whether they result from shared pigments across taxa, as similar appearances may arise from different compounds and are not necessarily homologous. Identifying their biochemical basis is therefore crucial to understanding the evolution and possible metabolic costs of these unusual traits. We used Raman spectroscopy to analyse resin-embedded cross-sections of valves from 15 bivalve species with green or blue shells. Species represented diversity in shell colour in terms of both phylogeny and morphological traits, including colour location (organic periostracum, organic layers in the calcareous shell or inorganic calcareous matrix). Green colour consistently occurred in organic layers rather than calcareous shell, while blue colour occurs in both calcareous shell and organic layers. Blue colour appears to be due to carotenoid-based pigments, similar to other pigments observed in bivalve shells. Green colour, however, is due either to novel pigments, not previously identified in mollusc shells, which are only weakly Raman active or nanostructures that produce structural colour in the absence of green pigments. Within bivalves, to date structural colour has been reported only in one genus, underscoring its evolutionary significance.

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes, play a critical role for maintaining bone homeostasis. Although, aging impairs MSC function and contributes to several complications, the effects of aging on subcellular structure and related gene expression need further investigation. Here we established an in vitro system to study MSCs isolated from bones of 5, 12, and 24 months old mice which showed significantly decreased bone volume and exercise performance with age. RNA sequencing revealed downregulation of genes related to cell-matrix interactions, cell metabolism, and division. Functionally MSCs extracted from 24mo showed significantly increased adipogenesis and reduced osteogenesis compared to 5mo, which was accompanied by reduced levels of cell proliferative marker Ki67 and increased expression of senescence protein p16. Data-driven segmentation of F-actin architecture revealed no cell-wide or nuclei-associated alteration, while 24mo MSCs showed decreased nuclear volume and increased spreading and higher nuclear stiffness compared to 5mo. Mitochondria from 12mo and 24mo showed increased length and volume of fibers when compared to 5mo, which was accompanied by gene expression changes associated with mitochondrial inflammation and oxidative phosphorylation. To test the functional consequences, MSCs were subjected to mechanical stress for 72 hours using 90Hz, 07g low-intensity vibration (LIV). While 5mo MSCs showed a robust fusing of individual mitochondrial fibers, LIV response of 12mo and 24mo MSCs were progressively less, indicating an already stressed mitochondria compromised to mechanical challenge. In summary, this study has established an in vitro assay system, revealing age-associated impairments in differentiation, mitochondrial function, and mechanotransduction capacity.

Astroglia can counteract the harmful effects of -synuclein (-SYN) protofibrils and reverse premature cellular senescence by promoting tunneling nanotubes (TNTs). However, the mechanism behind this recovery is unknown. This study is the first to examine TNT-mediated mechanical stability in senescent astroglial recovery. We demonstrate that disruption of Lamin A/C in -SYN-protofibrils-treated senescent cells reduces actin-cytoskeleton stress, as measured by nucleus flatness index and isometric scale factor from quantitative microscopy. ROCK (Rho-associated kinase) inhibition, which is crucial for reducing actin-cytoskeleton tension, promotes TNTs. Small molecules like Cytochalasin-D, Nocodazole, and Jasplakinolide, which inhibit TNTs by altering actin tension other than ROCK pathway, cannot reverse senescence. RNA-sequence heatmaps reveal changes in senescence-, integrin-, and ROCK-pathway genes; STRING links these to the Hippo pathway. Experimental results show that cytosolic YAP translocation, a key regulator of Hippo pathway, is vital for TNT formation and actin-based stability in U87-MG astrocytoma and primary astrocytes. Interestingly, TNTs form between two cells with different actin tensions: one exhibits low actin tension with Hippo signaling on, while the other has higher actin tension with Hippo signaling off. The most notable observation is the high abundance of YAP inside the TNTs, along with actin. The study shows that TNTs maintain mechanical stability through Lamin A/C integrity and actin tension in -SYN-induced senescent astroglia, thereby protecting the cells, reversing senescence. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/711517v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@192307dorg.highwire.dtl.DTLVardef@ad8b75org.highwire.dtl.DTLVardef@19ece78org.highwire.dtl.DTLVardef@1056395_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cellular organelles are not just static structures; they are highly dynamic and directly linked to cellular functions. Changes in their morphology can be early indicators of diseases. Recent advancements in light microscopy techniques have transformed organelle research from qualitative descriptions to precise, quantitative measurements, enabling nanoscale resolution, high-throughput image analysis, and live-cell compatibility. This enables accurate measurement of organelle morphology, dynamics, and spatial organization using modern imaging and analysis techniques. By quantifying organelles, we go beyond simply visualizing to measuring and statistically comparing cellular features across different samples. This protocol addresses a wide range of cellular organelles across all major experimental systems, specifically mentioning mitochondria, myofibers, actin filaments, endoplasmic reticulum, and Golgi apparatus, by integrating experimental design, optimized sample preparation, high-resolution imaging, and validated Fiji/ImageJ-based analysis workflows. For each organelle, step-by-step methods specify reagents, equipment, acquisition parameters, and expected results. While recent advances, such as expansion microscopy, correlative light-electron microscopy, and AI-powered segmentation, offer gains in throughput and resolution, this workflow demonstrates that Fiji-based analysis remains fully capable of delivering high-precision organelle quantification. The entire workflow can be completed within 2-4 weeks, from initial design through validation and the production of measurements suitable for cross-study comparisons. Overall, this protocol establishes a flexible approach to standardize organelle quantification to understand multiple organelles simultaneously in their cellular contexts. Basic Protocol 1: Mitochondrial Quantification Basic Protocol 2: Myofibril Quantification Basic Protocol 3: Golgi Apparatus Morphometry Basic Protocol 4: Endoplasmic Reticulum Network Analysis Alternate Protocol 1: Super-Resolution Imaging Protocol

SUN5 is a testis-specific SUN domain protein essential for connecting the sperm tail to the nucleus. However, until now, its precise localization, intracellular dynamics, and membrane topology during spermiogenesis have remained controversial. To address these discrepancies, we applied ultrastructure expansion microscopy (U-ExM) to systematically track SUN5 redistribution throughout spermiogenesis. This approach enabled a detailed reconstruction of SUN5 localization across developmental stages and revealed previously undescribed enrichment at the perinuclear ring (PNR) and the microtubule manchette, suggesting secondary functions at the PNR or a potential role in intra-manchette transport (IMT). Complementary immunogold labelling using the Tokuyasu method, together with biochemical assays, demonstrated that SUN5 adopts a membrane localization and topology consistent with that of classical SUN domain proteins. Quantitative measurements of the nuclear envelope architecture at the head-to-tail coupling apparatus (HTCA) further enabled us to present a refined structural model of SUN5 positioning at the head-tail junction. Overall, our findings resolve previous discrepancies in the field and provide a coherent framework for understanding SUN5 organization and its role in mammalian spermiogenesis. Summary StatementIn the presented study, we analyzed the dynamic redistribution of SUN5 during mammalian spermiogenesis and resolved its topology in developing spermatids to gain insights concerning the proteins molecular function in head-tail coupling.

The retinohypothalamic tract (RHT) is the primary pathway for circadian photoentrainment. Rodent models exhibit a significant translational gap for human physiology due to their nocturnal nature. To overcome this, we developed a functional human RHT assembloid by fusing human pluripotent stem cell (hPSC) derived retinal and hypothalamus organoids. Characterization revealed mature retinal brush borders and the preservation of melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) integrated via excitatory glutamatergic synapses. Multielectrode array (MEA) analysis confirmed synchronized network activity across the interface. The development of the human RHT assembloid represents a significant leap forward in chronobiology. The "gold standard" for circadian models--self-sustained gene expression oscillations--was demonstrated using a PER2::Luciferase reporter, showing robust 20- 30 hour rhythms. This validates the hypothalamic component as a functional "clock in a dish". This platform provides a readout to screen drugs or test light-pulse effects on circadian phase, directly modeling jet lag or phase-shifting. Overall, this model offers a high-fidelity system for investigating human-specific chronobiological mechanisms in vitro. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/700761v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1327027org.highwire.dtl.DTLVardef@61183corg.highwire.dtl.DTLVardef@7e280eorg.highwire.dtl.DTLVardef@77e7ce_HPS_FORMAT_FIGEXP M_FIG C_FIG

Extracellular vesicles (EVs) hold great promise as therapeutic delivery vehicles, leveraging their natural role as mediators of intercellular communication in all organisms studied. However, many barriers must be overcome to realize their full potential. Saccharomyces cerevisiae is an attractive chassis organism to explore solutions: It is used for drug biomanufacturing, it is amenable to complex genetic engineering, and their EVs can drive responses in human cells. To further develop this prospect, we sought to genetically modify S. cerevisiae EVs by devising a research framework amenable to iterative design, build, test, learn cycles - a core principle of synthetic biology. Using this approach, we focused on identifying new scaffolds - proteins that load cargoes into EVs - from a small pool of candidates. We first optimized a modular cloning strategy, called "EVclo", for plasmid and genome-integrated candidate gene expression. Candidate genes were fused to EGFP, and after confirming expression in cells, we showed that scaffold-EFGP proteins colocalized with mRuby2-tagged Nhx1, a biomarker of multivesicular bodies, presumed sites of EV biogenesis. We triggered release of EVs by heat stress, isolated these EVs by ultrafiltration and size exclusion chromatography, and confirmed the presence of exosome-sized EVs in all samples. We find that candidate scaffold proteins did not affect EV size, morphology or titers. Further analysis of these samples indicated that some EGFP-tagged scaffolds are present in EVs: Bro1, a yeast ortholog of ALIX, was most abundant and ExoSignal showed highest enrichment of the human candidates. In all, we conclude that Bro1 is a good scaffold for future engineering strategies, and that human proteins can be sorted into yeast EVs suggesting conservation of the sorting machinery and demonstrating that yeast EVs can be humanized. This synthetic biology-based, proof-of-concept study establishes S. cerevisiae as a platform to engineer and bioproduce designer EVs for many applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/710173v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1cf61dcorg.highwire.dtl.DTLVardef@21f412org.highwire.dtl.DTLVardef@11ecde9org.highwire.dtl.DTLVardef@160b3f7_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS AND TOC BLURBO_LIsynthetic biology-based system was optimized to engineer EVs in S. cerevisiae C_LIO_LIEV scaffolds can be sorted to yeast EVs C_LIO_LIis an efficient scaffold to sort proteins into yeast EVs C_LIO_LIS. cerevisiae can be used to engineer designer EVs for drug delivery C_LI Extracellular vesicles (EVs) are a promising new modality for drug delivery. However, designer EVs must be engineered to broaden applications and improve efficacy. Here, Bouffard et al. optimize methods rooted in synthetic biology to genetically engineer EVs in S. cerevisiae, a yeast commonly used to manufacture biological drugs. They find that ectopically expressed human EV scaffolds (CD63, ExoSignal, PDGFR) can be sorted to yeast EVs, but Bro1 - the yeast ortholog of ALIX - was most efficient at sorting GFP into EVs. This proof-of-concept study demonstrates a single DBTL (design-build-test-learn) cycle that can be used to develop designer EVs for therapeutic applications.

Live-cell imaging (LCI) provides researchers the opportunity to understand biological phenomena at a temporal resolution and is achieved using dedicated imaging systems. These studies enable insight into dynamic phenotypic changes occurring in cells, which may otherwise be missed when studying fixed samples. Access to advanced microscopy is disproportionately available to researchers in high-income countries, whereas researchers in low-to middle-income countries (LMICs) are severely underrepresented in the adoption of such technologies. A major barrier to the dissemination of advanced microscopy centres around economic inequalities, with the cost of high-end imaging systems often being prohibitively expensive. Recognition of such disparities has motivated the wider microscopy community to manufacture frugal microscopes that are accessible to researchers in resource-constrained settings. The OpenFlexure Microscope (OFM) is an open source, customisable, 3D-printed microscope suitable for medical research and field-diagnostics. We have made adaptations to the OFM to enable its use for live-cell imaging in humid tissue culture incubators. By moving major electronic components outside of the microscope, we remove the risk of corrosion of the Raspberry Pi and Sangaboard used to operate the instrument. We tested four common 3D-printing polymer materials for increased thermal robustness and found ASA is the best plastic to print the main body of the microscope, offering both durability and image stability in 24- to 48-hour time course experiments. We have also created an optional 3D-printable weighted-hammock system to reduce external vibration artefacts during image acquisition. Critically, electronic modifications included custom extension cables from the motors and camera to the Raspberry Pi and Sangaboard, and the inclusion of 22 ohm ({Omega}) resistors to reduce the current to the stepper motors, preventing detrimental temperature increases inside sealed incubators during prolonged powering of the instrument. To remove dependence on WiFi connections for setting up timelapse experiments, we generated a simple application with a graphical user interface (GUI) that can be installed locally on a Raspberry Pi and is specifically designed for setting up timelapse experiments without extensive computational knowledge or experience. We validated our LCI-OFM adaptations with a 48-hour treatment of MDA-MB-231 breast cancer cells with the chemotherapeutic drug docetaxel, showcasing how the modified microscope can seamlessly feed into established bioimaging pipelines and generate biologically meaningful results. For researchers in LMICs, this adapted LCI-OFM provides new opportunities to study locally-relevant health challenges with timelapse microscopy, enabling deeper insight into biological dynamics and supporting the generation of preliminary data critical for securing grant funding and access to more advanced imaging systems in purpose-built regional imaging hubs.

Automated transmission electron microscopy (TEM) generates large datasets that challenge traditional qualitative analysis of cellular ultrastructure. Quantitative assessment of structural differences between different samples remains difficult due to structural variability in thin sections of organelles. Here we applied supervised machine learning (sML) to segment, quantify and compare cellular structures -including nuclei, chromatin, mitochondria, rough endoplasmic reticulum, and endocytic vesicles- in large TEM images of wild-type macrophages versus those with altered cellular physiology due to deficiency in O-GlcNAc Transferase (OGT). sML revealed that OGT knockout macrophages are larger and more oval, with increased euchromatin, nucleoli size, and relative mitochondrial and rER surface areas. Comparison with six TEM experts showed sML provides more objective and sensitive quantification of subtle differences, while expert consensus is only achieved for larger structural variations. These findings demonstrate that sML enhances quantitative TEM analysis and complements human expertise in ultrastructural studies.

The clitoris is one of the least studied organs of the human body. The detailed anatomy of the clitoris is challenging to address through a gross dissection, as most of its parts are embedded internally, surrounded by pubic bone and several pelvic organs. While clinical imaging methods such as magnetic resonance imaging can capture the gross 3D morphology, they lack the spatial resolution required to resolve the detailed structures. In this study, we generated micron-scale computed tomography images of the female pelvises, leveraging a synchrotron radiation X-ray source. This unique data revealed the complex trajectory of the dorsal nerve of the clitoris, the main sensory nerve of the clitoris. Notably, the nerve trunks within the clitoral glans were revealed, with the maximum diameter ranging from 0.2 to 0.7 mm. They showed a tree-like branching pattern projecting towards the surface of the glans. We also revealed that some branches of the dorsal nerve of the clitoris ramify to innervate the clitoral hood and mons pubis. Finally, the posterior labial nerve, a branch of the perineal nerves, was shown to innervate the surroundings of the clitoris and the labial structures. These findings have an immediate impact on operations performed around the vulva area, such as gender-affirmation surgery and reconstruction surgery after genital mutilation.

AimsThe development of a method for isolating mitochondria from a specific cell type within a given tissue, while preserving their structural and functional integrity to the greatest possible extent, remains an ongoing challenge. The aim of this study was to establish a protocol for the isolation of mitochondria from rodent cardiomyocytes, characterized by minimal contamination with other cell types and a high yield of mitochondrial fractions originating from distinct subcellular regions of cardiomyocytes. Methods and resultsIn the present study, cardiomyocytes from guinea pig and rat hearts were isolated using a standard enzymatic digestion protocol in a Langendorff heart perfusion system. Traditionally, the isolation of organelles, including mitochondria, from whole cardiac tissue as well as from cardiomyocytes has relied primarily on mechanical tissue homogenization These conventional approaches involve the localized application of high pressure to cells, which may potentially damage delicate organelles, particularly mitochondria. Moreover, such homogenization preferentially releases mitochondria located in the subsarcolemmal region of cardiomyocytes rather than representing the entire mitochondrial population. In our study, we employed an alternative approach based on the gentle mechanical disruption of cardiomyocytes by passing the cell suspension through selected cell strainers using a cell scraper. This strategy facilitated mild disruption of cellular structures, significantly increasing the yield of mitochondria released from interfibrillar regions while preserving mitochondrial functionality. Moreover, this method decrease probability of sample contamination with mitochondria from other cells, based on cell size differences. The effectiveness of this method was confirmed by transmission electron microscopy, and high-resolution respirometry, which revealed no evidence of outer mitochondrial membrane damage, as indicated by the lack of response to the addition of exogenous cytochrome c to the incubation chamber. Moreover, mitochondrial oxygen consumption increased by 7.39 {+/-} 1.25-fold following the addition of 100 {micro}M ADP, reflecting efficient ADP-stimulated respiration. Furthermore, fluorescence measurements were performed. to assess changes in the mitochondrial inner membrane potential ({Delta}{Psi}). The isolated mitochondria were also suitable for electrophysiological studies using the single-channel patch-clamp technique. Additionally, mitochondria isolated using the protocol developed in our laboratory exhibited a high capacity for transplantation into H9c2 cells. ConclusionIn summary, our mitochondrial isolation method is rapid, efficient, and yields functionally competent mitochondria. These preparations are suitable for a wide range of downstream applications, including patch-clamp electrophysiology, analyses of oxygen consumption under various pharmacological conditions, as well as mitochondrial transplantation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/716092v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@613495org.highwire.dtl.DTLVardef@1c34338org.highwire.dtl.DTLVardef@722900org.highwire.dtl.DTLVardef@e1f7a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biocompatible fluorosurfactants are essential for many droplet microfluidic workflows but are often obtained from commercial sources because published syntheses of perfluoropolyether (PFPE)-based surfactants typically require acid chloride intermediates and chemistry-oriented purification methods. These requirements can limit access for biology and clinical laboratories seeking low-cost or customizable surfactant systems. Here we describe a practical method for preparing functional PFPE-based fluorosurfactant materials by direct carbodiimide coupling of functionalized PFPE carboxylic acids(Krytox 157 FSH) to amine-containing head groups under laboratory-accessible conditions. Using this approach, we prepared a PFPE-polyethylene-glycol (PFPE-PEG) material from Jeffamine ED900 and a PFPE-Tris material from Tris base. Because these products were not fully structurally characterized, we present them as functional reaction products and evaluate them by use in biomicrofluidic workflows rather than by definitive compositional assignment. PFPE-Tris was useful for generating relatively uniform small droplets, whereas the PFPE-PEG preparation supported a broader range of biological applications. These materials were used in genomic library screening for {beta}-glucosidase activity, thermocycling-associated droplet workflows, and protein crystallization experiments. In addition, the PFPE-PEG preparation improved emulsion behavior in many protein crystallization screens that were unstable with a commercial droplet oil used in our laboratory. This method reduces the practical barrier to in-house fluorosurfactant preparation and allows biology-focused laboratories to explore head-group chemistry, oil composition, and operating conditions without complete reliance on commercial reagents. The results support this workflow as a useful entry point for biomicrofluidics laboratories, while also highlighting the need for careful interpretation of thermocycled droplet assays and for future analytical characterization of the resulting materials. Significance statementDroplet microfluidics relies on fluorosurfactants that are often costly and difficult to synthesize outside of chemistry-focused settings. We describe a simple, biology-laboratory-compatible approach for generating functional perfluoropolyether-based fluorosurfactant materials using direct carbodiimide coupling and straightforward cleanup. The resulting materials supported multiple biomicrofluidic workflows in our laboratory, including enzymatic screening and protein crystallization, and provide a practical route for groups seeking lower-cost and more customizable surfactant systems.

3T3-L1 adipocyte spheroids are widely used three-dimensional adipose models that undergo pronounced physical property changes during adipogenic maturation, including progressive buoyancy driven by lipid accumulation. We quantitatively profiled the time-resolved evolution of bulk spheroid density over 60 days, revealing a 6.7% reduction from 1.022 to 0.954 g/cm3 associated with intracellular lipid accumulation. This buoyancy-driven displacement hinders standardized interrogation of spheroid physical properties during long-term culture. To enable longitudinal and reproducible analysis of density remodeling, we introduced an anti-buoyancy spheroid trap (AS-Trap) that mechanically stabilizes spheroids during extended culture. Morphometric analysis revealed that lipid droplet areas in mature spheroids (790.68{+/-}513.84 m{superscript 2}) closely approximated in vivo adipocytes (846.34{+/-}257.28 m{superscript 2}) and were substantially larger than those in 2D cultures, providing structural context for the observed density reduction. Notably, the terminal spheroid density converged toward reported values for native white adipose tissue, underscoring the physiological relevance of the material state achieved during maturation. Together, this work establishes density as a quantitative physical phenotype of adipogenic maturation and enables standardized long-term interrogation of adipose spheroids as living materials.

The present study introduces a novel design and analysis of a sensor based on a terahertz metamaterial absorber (MMA) to identify Glioblastoma cell by employing microwave imaging techniques. Terahertz (THz) frequencies offer unique advantages for biomedical applications. Computer Simulation Technology (CST) employs a finite integration (FIT) approach to simulate the suggested structure in the resonant frequency (RF) range of 4.5 THz to 6 THz. The crystal structure displays three distinct absorption peaks at resonance frequencies. The MTA can absorb energy in three specific spectral bands: 4.782 THz, 5.30 THz, and 5.7319 THz. At these frequencies, the MTM achieves exceptionally high absorption, reaching 99.99%, 99.98%, and 99.68% peak absorption, respectively. The electric field (E), magnetic field (H), and surface current of the MTM are also examined. Finally, detecting Glioblastoma cells is also being investigated by analyzing the E-field H-field using microwave imaging. The suggested biosensor features a high-quality factor of 143.63, a frequency shifts per refractive index of 1.45 THz/RIU. In this study, the PCR value is measured at 0.95 at a frequency of 4.782 THz, indicating a high efficiency in polarization conversion. Polarization Conversion Ratio (PCR) quantifies the efficiency of a metamaterial in converting the polarization of an incident electromagnetic wave. Extensive simulation studies confirm the sensors capability to distinguish between healthy and cancerous cervical tissue. The suggested MMA-based sensor has numerous advantages and can be utilized for Glioblastoma cell detection.

Building a high-fidelity computational model of the whole human brain will require preservation of the ultrastructure at the level of the entire organ, post-mortem. For such a model to reflect as closely as possible the brain in the living state, artifacts that arise during both the agonal phase and the postmortem interval will need to be minimized. This is potentially feasible if a terminally-ill patient donates their brain for research following physician-assisted death. In this paper, we modify a protocol for aldehyde-stabilized cryopreservation to make it compatible with physician-assisted death. We use pigs as a model, which resemble humans in cardiovascular and brain anatomy. Aldehyde-stabilized cryopreservation was designed to provide superior structural preservation of brains of any size, across all anatomical scales, compatible with diverse analytical assays and long-term storage without ultrastructural degradation. We demonstrate, with light microscopy and volume electron microscopy, that our brain preservation protocol results in connectomically traceable whole brains and propose an economically feasible storage modality that is expected to maintain stability of ultrastructure and macromolecules in the brain even for thousands of years. Most importantly, we establish that 14 min is the approximate length of the perfusability window--the time after the cardiac arrest during which blood washout needs to be initiated so that the brain ultrastructure is preserved.

Droplet sorting technology has the potential to revolutionize the biotechnology sector as it provides massive high-throughput screening capacity, but the technology remains not accessible for a wider audience yet. There is a need for more affordable droplet sorting platforms to design cell factories and screen cell libraries. In here we demonstrate our droplet cytometry/sorter platform for single-cell screening of yeast cells based on their fluorescence signal.

Glycated hemoglobin (HbA1c) is a central biomarker for long-term glycemic control and diabetes management, traditionally quantified using laboratory-intensive chromatographic or immunochemical assays. As the global burden of diabetes continues to rise, there is growing interest in alternative, scalable approaches capable of rapid biochemical assessment. Fourier-transform infrared (FTIR) spectroscopy offers a reagent-free method that captures molecular signatures of protein glycation, but translating complex spectra into clinically interpretable HbA1c values requires robust analytical frameworks. Here, we present a complementary multi-model strategy for predicting HbA1c from FTIR spectra of whole blood. Using 685 blood samples with matched reference HbA1c measurements, we evaluated three analytically distinct yet synergistic approaches: partial least squares regression (PLSR), peak-resolved curve fitting based on pseudo-Voigt functions combined with H2O AutoML, and a convolutional neural network (CNN). PLSR and CNN models were trained on biologically informative spectral regions (800-1800 cm-{superscript 1} and 2800-3400 cm-{superscript 1}), while curve fitting focused on the fingerprint region (1000-1720 cm-{superscript 1}) to extract interpretable biochemical parameters. PLSR achieved the highest predictive accuracy (R{superscript 2} = 0.76), closely followed by the CNN (R{superscript 2} = 0.73), reflecting their ability to capture global linear and nonlinear spectral relationships. Although curve fitting yielded lower predictive performance (R{superscript 2} = 0.59), its peak-level decomposition enabled mechanistic interpretation of glycation-related changes. Explainable AI analysis using SHAP identified lipid- and protein-associated vibrations, carbohydrate-linked glycation bands, and amide-region structural features as key contributors to HbA1c prediction. Rather than treating these approaches as competing alternatives, our results demonstrate that their integration provides a more informative framework than any single model alone. By combining predictive performance with biochemical interpretability, this multi-model FTIR strategy highlights a scalable and mechanistically grounded pathway toward non-invasive HbA1c assessment and broader metabolic screening in diabetes monitoring. The code for this study is freely available at https://github.com/MelnychenkoM/ftir-hba1c-prediction.