Advanced Biology

A cnidarian photosymbiont alga, Breviolum minutum, is an emerging model to study symbiosis due its ability to colonise host in absence of light, and amenability to genetic and physiological manipulations. This alga undergoes subcellular reorganisation in response to stress conditions such as elevated temperature and nutrient deprivation. However, subcellular visualisation of this alga is challenging because of its broad spectrum autofluorescence (400-700 nm) and relatively small size (6-8 m). We developed a super resolution imaging, Expansion Microscopy (ExM) workflow - a hydrogel-based technique for mechanical enlargement of cells, that reveals previously inaccessible subcellular features in B. minutum. This ExM workflow presents a set of thermic and enzymatic conditions which enables 4-fold expansion of B. minutum, optical clearing of autofluorescence as well as the removal of its thick cellulose rich cell wall. We implemented a recently described platinum (II)-based tri-functional linker 1, to retain in situ hybridised oligonucleotides targeted to 18S rRNA within ExM hydrogel and exploited its azide reactive group for post-ExM fluorophore labelling (DBCO modification). We observed actin patches (a cytoskeletal feature) and calmodulin (a calcium binding signalling protein) that are not previously visualised in B. minutum. This approach overcomes some of the long-standing problems in visualisation of B. minutum using commonly available reagents and commercially available low-cost ExM compatible chemistries. The broader uptake of this tool for the visualisation of diverse species of photosymbionts will pave the way for fundamental discoveries underpinning cellular reorganisation in formation and breakdown of symbiosis.

Monitoring pH and extracellular acidification rate (ECA) in biological samples containing live mammalian cells can provide valuable information on the glycolytic activity and bioenergetic status of cells. Compared to pH electrodes, optochemical pH sensors look more advantageous, since they allow rapid, non-invasive parallel analysis of multiple samples with stable readout of pH. We have developed new fluorescent pH sensors based on hydrophobic protonable metal-free porphyrins,OEP and OEPK, embedded in a plasticized PVC matrix containing a proton transfer agent. These pH sensors provide internally-referenced calibration-free operation, both in ratiometric intensity and lifetime-based detection modes. Sensor development included optimization of the indicator dye and its photophysical characteristics, screening of different proton transfer agents to minimize sensor toxicity, tuning of the protonation range and pKa, long-term storage stability and response time studies. Optimised pH sensor coatings were then deposited on plastic substrates (96-well microplates) and used for real-time monitoring of Extracellular Acidification Rate (ECAR) for cultured cancer cells and 3D spheroid structures on standard laboratory equipment (multi-label plate reader and confocal FLIM microscope). The advanced pH sensors tailored for use with biological samples have high potential for cell analysis and related applications.

Understanding how aging reshapes retinal metabolism requires methods that can resolve molecular and structural changes across the retinas highly organized cellular layers. Here, we applied a nonlinear multimodal imaging platform that integrates fluorescence lifetime imaging microscopy (FLIM), second-harmonic generation (SHG), hyperspectral stimulated Raman scattering (HS-SRS), and deuterium oxide-based stimulated Raman scattering (DO-SRS) to map age-associated metabolic and compositional alterations in young and aged mouse retinas. FLIM analysis of the outer nuclear layer (ONL) revealed increased free NADH and NADPH fractions in aged retinas, consistent with reduced oxidative phosphorylation and enhanced lipid anabolic activity. SHG imaging of the sclera showed pronounced age-related remodeling of collagen organization, including increased fiber density, elevated anisotropy, and the emergence of densely crosslinked bundles in the central sclera. DO-SRS further demonstrated elevated lipid turnover in rod photoreceptor outer segments and the retinal pigment epithelium (RPE) with aging which was confirmed by lipidomic analysis. Complementary HS-SRS analysis revealed reduced triacylglycerol and cholesterol content together with localized sphingosine accumulation in the RPE. Together, these findings provide a spatially resolved view of metabolic remodeling in the aging retina and establish multimodal optical imaging as a powerful framework for studying alterations associated with age-related retinal disease.

Live imaging of plant subcellular structures is key to deciphering the spatiotemporal bases of cellular processes, and their functional impact on growth and morphogenesis at various biological scales. Live imaging of plant cells essentially relies on expression of fluorescent markers labeling cells or subcellular structures of interest. Simultaneous multi-channel imaging of several markers is still not routine practice in plant cell biology, owing to issues linked to genetic or spectral compatibility of markers, differences in expression levels, silencing, toxicity, etc. Here we designed a three-color marker in Arabidopsis thaliana and Capsella rubella, enabling high-resolution live imaging of plant morphogenesis, including labeling of the cell membrane, the nucleus and the microtubule cytoskeleton. Detection of MT arrays involved the development of a MAP4-MBD-based microtubule marker optimized for plant cells. The three-color marker allows visualization of the three-dimensional organization and dynamics of plant microtubules within the intracellular space with unprecedented precision, in various organs including the root and shoot meristems, the leaf, anther, and gynoecium. Our results demonstrate the potential of such single-construct strategy for cell biology studies in plants.

The effect of microcurrents on facultative microbial pathogens remains controversial. Solid cultures in Sabouraud Glucose Agar of the ubiquitous mold Aspergillus fumigatus were repeatedly treated with a commercially available device performing wireless conductive microelectrostimulation by 3.5 A microcurrent routed by spraying negatively charged air particles onto solid cultures in modified petri dishes. The treated cultures displayed increased growth compared to standard ones, but only as a function of mycelial density and total surface; the radial growth rate of the mycelium remained unaltered. The increased growth was positively related to the duration of the treatment. At the same time, secondary development (new mycelial loci within the dish) was greatly upheld due to treatment, as the spraying created microairstreams dislocating the fungal spores. These results imply perplexed kinetics of mycelial growth both with and without treatment, since the folding of the mycelial mat is observed regularly. Both the fungus response to the ES and the possible revision of growth kinetics create prospects for biotechnological and bioremediation applications but also imply biomedical considerations, regarding infection dynamics of mycelial fungi and their in situ resistance to immune responses and treatment.

Summary/AbstractTelomere length has a significant impact on human health. Short telomeres cause age-related diseases, including pulmonary fibrosis, immunodeficiency, and bone marrow failure, while long telomeres predispose to cancer. Given the impact on human health, accurately measuring telomere length is important. A variety of methods have been developed over the past 40 years to measure telomere length. Many of these methods report only on the mean length of all of the telomere in the cell. Here, we describe the Telomere Profiling protocol using Oxford Nanopore Technologies (ONT) based on long read sequencing that can accurately measure chromosome specific telomere length.

The ultrastructure of endothelial cells (ECs) "in situ" is of great interest due to their involvement in many physiological processes. In some organs, these cells form transcellular pores or fenestrae, allowing for the rapid exchange of molecules between blood and interstitium. Despite their importance, no optical images of these dynamic morphological structures have yet been acquired in situ. Major obstacles to their in-situ imaging are the lack of specifical labels for fenestrae and their size well below the optical diffraction limit. Here, we report how we have overcome these challenges and managed to visualize the EC ultrastructure in situ in 25 {micro}m thick liver sections. To enable this, a lipophilic, fluorescent membrane dye was infused into the portal vein of murine livers to stain the sinusoidal ECs before the organ was harvested. Tissue sections were subsequently imaged using a novel, super-resolution optical-sectioning structured illumination microscope (OS-SIM), providing approx. 170 nm spatial resolution with significantly faster image acquisition compared to confocal microscopy.

BackgroundContinuous glucose monitoring (CGM) systems are used not only for retrospective assessment of the glycemic profile but also for real-time decision-making, including automated insulin delivery. Accordingly, CGM performance characterization must capture not only the agreement of individual paired values but also the systems ability to reproduce the direction, rate, amplitude, and shape of glucose concentration change. Summary metrics, most notably MARD, cannot establish whether an observed deviation reflects an error in the formation of the test profile itself, a constant sensor offset, amplitude compression, a change in response rate, temporal misalignment, or hysteresis. ObjectiveTo adapt a programmable flow-based in vitro platform for the separate assessment of the experimentally delivered glucose profile and the dynamic response of CGM systems, and to propose a set of metrics that decomposes dynamic error into its components. MethodsGLU profiles were generated by programmable mixing of solutions at a constant total flow rate of 2 mL/min. Actual GLU concentration was independently measured with a SUPER GL2 glucose analyzer. Four static levels, three repeats of a 5.5[->]12.0[->]5.5 mmol/L profile, three repeats of a 6.0[->]3.0[->]6.0 mmol/L hypoglycemic profile, three 5.0[->]15.0[->]5.0 mmol/L profiles at different rates, one complex 4[->]18[->]3[->]12[->]5.5 mmol/L profile, and two proof-of-concept sensor experiments at 100- and 200-min transitions were investigated. Dynamic response was characterized by bias, MAE, RMSE, MARD, amplitude transfer coefficient K_A, rate transfer coefficients K_up and K_down, normalized shape RMSE, residual shift, and hysteresis loop area. ResultsAt the static levels, measured GLU exceeded the programmed value by 0.234-0.780 mmol/L. In the repeated 5.5[->]12.0[->]5.5 profiles, the ratio of actual to programmed rate was 0.978-1.083 on the rising phase and 0.987-1.157 on the falling phase, while the amplitude transfer coefficient was 0.967-1.066. In the hypoglycemic profile, minimum GLU was 2.55- 2.96 mmol/L, and time below 3.0 mmol/L was 15.2-72.6 min. The measured rates of 0.0519, 0.1045, and 0.2027 mmol/L/min preserved the intended ratio of approximately 1:2:4. In the complex profile, the programmed plateau of 18 mmol/L was not reached: mean measured GLU was 16.20 mmol/L. For CGM-A, K_A was 0.682 and 0.650, and K_up/K_down were 0.666/0.730 and 0.634/0.626; the corresponding values for CGM-B were 1.228 and 1.128, and 1.564/1.328 and 1.276/1.145. Hysteresis loop area differed 5- to 10-fold between the two sensor responses, exceeding an order of magnitude at the 100-min transition. ConclusionThe programmed concentration should be treated as a control setpoint, rather than as a reference measurement. The "programmed trajectory -- measured glucose -- CGM output" cascade first allows quantitative assessment of the agreement between the programmed and actually realized profile and only then separate characterization of sensor response. Decomposition of dynamic error into amplitude, rate, shape, and hysteresis components reveals differences that a single MARD value or correlation coefficient cannot capture.

Introduction: Advanced biomaterials for regenerative medicine are increasingly expected to combine multifunctionality and compatibility with tissue-specific cellular processes. In this context, hydroxyapatite-based platforms modified through ionic substitution represent promising candidates, as they may integrate structural similarity to bone mineral with additional biological functionality and luminescent properties, enabling diagnostic applications and real-time monitoring. In this study, we evaluated whether silicate-phosphate substituted calcium hydroxyapatite Ca10(PO4)6-x(SiO4)x(OH)2 (where x = 1.5) co-doped with lithium(I), europium(III), and gadolinium(III) ions (Si-HAp-LEG) affects the osteogenic, chondrogenic, and adipogenic differentiation potential of human bone marrow stromal/stem cells (BMSCs). Methods: Human BMSCs were cultured under lineage-specific differentiation conditions in the presence of undoped silicate-substituted phosphate hydroxyapatite (abbr. as Si-HAp), which served as a control, and two distinct Si-HAp-LEG formulations differing in gadolinium(III) (Gd3+) as well as lithium (Li+) and europium(III) (Eu3+) ion concentrations: Si-HAp-LEG-221 (1 mol% Gd3+ ion) and Si-HAp-LEG-222 (2 mol% Gd3+ ion). Differentiation-associated phenotypic outcomes, including extracellular matrix formation and lipid accumulation, were evaluated using Safranin O, Alizarin Red, and Oil Red O staining. In parallel, biomaterial-induced molecular responses were characterized at the transcriptomic and protein levels using RT-qPCR for selected coding and non-coding RNAs and Western blot analysis for representative lineage-associated proteins. Results: Histochemical evaluation confirmed that, across all tested biomaterial groups, BMSCs retained the ability to form mineralized calcium deposits, proteoglycan-rich extracellular matrix, and intracellular lipid accumulation under osteogenic, chondrogenic, and adipogenic conditions, respectively. Quantitative staining analysis revealed no significant Si-HAp-LEG-dependent enhancement of terminal differentiation outcomes compared with undoped Si-HAp. In turn, the molecular response differed between biomaterials modifications. Si-HAp-LEG-222 induced the most prominent changes in transcriptional and post-transcriptional regulators, particularly within BMP/SMAD-associated pathways under osteogenic and chondrogenic conditions, underlying a potential link between gadolinium concentration and osteogenic lineage commitment. However, these transcriptomic responses were not mirrored by consistent changes at the protein level. The results suggest that silicate-phosphate substituted hydroxyapatite co-doped with Li+, Eu3+, and Gd3+ ions primarily affects the early regulatory pathways associated with BMSCs differentiation rather than enhancing their terminal maturation. Discussion: In conclusion, the collective data indicate that Li+, Eu3+, and Gd3+ ions LEG co-doping broadens the multifunctional potential of Si-HAp by introducing imaging-related properties while preserving its underlying pro-regenerative character. Li+, Eu3+, and Gd3+ ions co-doped LEG-substituted Si-HAp may therefore be considered a compatible biomaterial platform that maintains BMSC cellular plasticity and supports balanced, differentiation-dependent modulation of lineage-associated molecular responses.

Gait is a scalable biomarker of functional, physical, and brain health, but most studies rely on gait speed alone. Here, we developed and validated gait age clocks that estimate age from multidimensional gait features and quantify deviations as gait age gaps, with gaps >0 (<0) for accelerated (delayed) aging. We included data from 5,681 participants, including healthy controls and clinical groups (Parkinson's disease, neurodegenerative diseases, stroke, diabetes, fallers, and frailty). Normative models trained in healthy controls showed robust age prediction (r=0.851, p<0.001), and full gait models outperformed gait speed alone ({Delta}R2=0.175). Gaps captured accelerated aging across neurological and physical conditions, tracked Parkinson's disease severity, and were associated with frailty, physical performance, white matter hyperintensities, and geriatric depression. Gait age gaps are also related to brain aging, risk/protective lifestyle factors, and mortality risk. These findings support gait age gaps as an interpretable biomarker for aging, risk stratification, and clinical monitoring.

Electrical stimulation (ES) and transepithelial/transendothelial electrical resistance (TEER) measurements are essential techniques in cell biology and tissue engineering, yet commercial devices for these applications cost between USD 2,500-9,000 and typically offer only one functionality. We present LATEER (Low-cost Arduino-based TEER and Electrical stimulation device), an open-source hardware platform that combines both ES and TEER measurement capabilities at a total cost below USD 100. The device features four independent channels, configurable pulsatile signals (amplitude up to 8.2 V, frequency 0.1-500 Hz, pulse width [&ge;]0.1 ms), and a resistance measurement range of 300 {Omega} to 1 M{Omega}, with <5% error for R {gtrsim} 4.7 k{Omega}. LATEER uses commercially available graphite pencil leads as electrodes ([~]USD 2 vs. USD 350 for commercial Ag/AgCl electrodes), which demonstrated excellent biocompatibility in cell culture. The system includes 3D-printed electrode holders compatible with standard 12-well and 24-well plates, allowing microscope visualization without electrode removal, and a Python-based graphical user interface for parameter configuration and real-time data acquisition. Because the electrodes remain fixed in the plate lid and only a single cable enters the incubator, both stimulation and resistance measurement can run continuously under standard culture conditions (37 {degrees}C, 5% CO2) without removing the plate or repositioning the electrodes, avoiding the temperature excursions and placement variability inherent to manual chopstick measurements. Validation with human pluripotent stem cell-derived cardiomyocytes demonstrated reliable frequency capture (electrical pacing) of the contracting monolayer, with a capture threshold between 250 and 400 mV/mm and controlled pacing across the 0.5-5 Hz range. TEER functionality was verified with mesenchymal stem cells, where the device resolved cell-density-dependent differences in electrical resistance in real time. All design files, firmware, and software are freely available under the CERN-OHL-S v2 license, enabling replication and customization by research laboratories worldwide. HighlightsO_LIAn open-source device combines electrical stimulation and TEER measurement under $100 C_LIO_LIGraphite electrodes offer biocompatibility at 0.6% cost of commercial alternatives C_LIO_LIFour independent channels with configurable parameters and real-time data logging. C_LIO_LIContinuous run setup in-incubator; no electrode repositioning needed C_LIO_LIValidated with stem cell-derived cardiomyocytes, achieving frequency capture (threshold 250-400 mV/mm) C_LIO_LI3D-printed holders enable microscope visualization without electrode removal C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/743263v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@98f9deorg.highwire.dtl.DTLVardef@13c73aborg.highwire.dtl.DTLVardef@1cdf099org.highwire.dtl.DTLVardef@16ed0cf_HPS_FORMAT_FIGEXP M_FIG C_FIG Specifications Table O_TBL View this table: org.highwire.dtl.DTLVardef@4ef802org.highwire.dtl.DTLVardef@7c651borg.highwire.dtl.DTLVardef@d20013org.highwire.dtl.DTLVardef@102fc89org.highwire.dtl.DTLVardef@111b927_HPS_FORMAT_FIGEXP M_TBL C_TBL

Two-photon polymerization (2PP) enables fabrication of hydrogel constructs with submicron, cell-scale resolution, but hydrogel-based bioinks are markedly more sensitive to process variability than conventional photoresists, and this sensitivity is further amplified when living cells are embedded in the resin. Post-processing evaluation, performed only after development, occurs too late to enable any corrective action. A full-factorial design of experiments across laser power and scan speed shows that fabrication outcome depends on both parameter choice and cell presence, with cells shifting and broadening the range of conditions yielding structurally sound constructs. However, substantial variability persists within each nominal condition and cannot be resolved by parameter refinement alone, indicating that outcome is governed by what occurs during each individual print rather than by the parameters set. To capture this, a layer-wise polymerization score is derived from pairwise comparisons of same-layer coaxial images, grounded in the psychophysics of relative judgment, and assembled into a Layer-wise Image Trajectory (LIT) for each print. Applied to both acellular and cell-laden formulations, LIT curves separate cleanly by post-processing outcome without any outcome label used in training, showing that fabrication quality can be predicted early in the build. Building on this signal, individual LIT curves are compared against statistical control limits derived from confirmed successful prints, enabling early detection of anomalous fabrication behavior at early-to-mid layers, well before development. To the best of the authors knowledge, this is the first application of in situ quality prediction and anomaly detection to cell-laden two-photon polymerization.

Cardiolipin (CL) is a phospholipid found in the inner mitochondrial membrane (IMM) where it increases the efficiency of ATP regeneration. We have investigated the hypothesis that this increase may result in part from CL concentrating H+ at the IMM surface through electrostatic interactions as the CL polar head is a dianion at physiological pH. To this aim, we compared the concentrations and movements of H+ at the surface of giant planar phosphatidylcholine (PC) membranes and 20% CL enriched PC membranes by recording their surface pH with the membrane-grafted pH probe fluorescein DHPE. CL enrichment of the membranes increased their surface H+ activity by a ~4 factor. Moreover, we observed non-gaussian spatial H+ concentration profiles with distance from a point H+ source with both PC and CL membranes suggesting that both lipids also induce interactions between probe molecules. A whole bath pH variation revealed that these interactions allow the traveling of reversible acidification fronts with constant speed over the membrane between high and low pH states. A reaction-diffusion model of these observations suggests that membranes support these fronts through a mechanism of autocatalytic (de)protonation of the membrane surface. In mitochondria, these fronts would result in transitions between high and low pH states, the low one having a larger H+ concentration in CL-enriched regions of the IMM. Such an increase at the inner leaflet of the IMM may increase efficiency of the respiratory chain whereas the increase at the outer leaflet may boost the ATP synthase rate.

Sensory and motor neurons differ significantly in their morphology, structural organization, functional properties, and mode of action. However, despite this heterogeneity, many in vitro studies focus only on a single neuronal subtype, mainly being sensory neurons, limiting the translational potential and relevance of these studies for the evaluation of therapeutic options for spinal cord injury. In this study, we investigate the differentiation, maturation, and neuronal outgrowth of human induced pluripotent stem cell (iPSC)-derived motor and sensory neurospheres using polyethylene glycol (PEG)-microgels with various bioactive coatings. Our results show subtype-specific responses to the PEG-microgel scaffolds, with respect to motor and sensory neurosphere morphology and size. Furthermore, we compare the formation of the PEG-microgel/scaffolds when starting from iPSCs-derived precursor neuron spheres versus undifferentiated iPSCs. We observe notable differences in structural organization, maturity, and neuronal outgrowth between the two approaches, as well as between motor and sensory neurospheres. Together, these results underline the importance of studying motor and sensory neurons separately and highlight the need for a controlled, tunable culture platform to assess the impact of the microenvironment and to improve the physiological relevance of in vitro platforms for neuron-based research.

Keratinocyte proliferation and differentiation are essential to produce the stratified structure of the epidermis and maintain its barrier function. These processes are regulated by complex mechanisms including epigenetic regulation. In this study, we evaluated the role of HDAC4/5, two class IIa histone deacetylases, in the epigenetic regulation of proliferative and differentiated human keratinocytes using dedicated 2D and 3D in vitro models. Our findings demonstrate that chemical inhibition or shRNA-mediated knock-down of HDAC4 impair keratinocyte proliferation notably through increased H3K27 acetylation and subsequent transcriptional activation of the cell cycle inhibitor gene BTG2. Interestingly, HDAC4/5 inhibition alters H3K27 acetylation landscape in proliferating keratinocytes, whereas the epigenetic identity of differentiated keratinocytes is much less affected. Inhibiting HDAC4/5 in 3D epidermis models resulted in reduced epidermal thickness and impaired barrier function linked to alteration in the lipid composition of the stratum corneum. Furthermore, analysis of several well-established skin aging markers revealed that reconstructed human epidermis treated with the HDAC4/5 inhibitor exhibit molecular and functional characteristics consistent with an aged-epidermis. Collectively, our results demonstrate that HDAC4/5 are essential for maintaining epidermal homeostasis and pave the way for the development of innovative models of skin aging based on the modulation of histone acetylation.

Controlling cell physiology is difficult, not only because of cells complexity, but also their capacity for real-time adaptation to interventions, leading to challenges such as drug resistance and transgene silencing. Accumulating evidence suggests that this adaptivity resembles classical forms of learning defined in behavioral science. However, a lack of appropriate platforms has led to gaps in our understanding of cells capacity for adaptive problem-solving in physiological and transcriptional space. Here, we present a device, the Cell Trainer, capable of performing a wide variety of automated training experiments on non-neural mammalian cells, using timed drug pulses as the stimulus, and a mobile fluorescence microscope to capture images of responses, across replicate cultures. The Cell Trainer can operate in either an open-loop (feedforward) or closed-loop (feedback-controlled) mode, and our image analysis pipeline can report the behaviors of individual cells throughout each experiment and quantify population heterogeneity. We showcase the ability of the Cell Trainer to execute experimental protocols and perform single-cell analyses in both modes. We first demonstrate with a feedforward experiment in which myoblasts are repeatedly pulsed with dimethyl sulfoxide (DMSO) and their discrete calcium responses are analyzed, revealing sensitization-like dynamics. Next, we demonstrate a feedback control scheme wherein the fluorescence of a pH/voltage reporter in kidney cells is maintained below a threshold level with controlled pulses of acid. To accelerate research in the field of cell training, learning, and memory, we are openly sharing the Cell Trainer schematics and software with the research community. This platform provides a flexible tool for studying how cellular physiological states can be shaped by patterned stimulation and feedback control through approaches that work with the native adaptive competencies of cells.

Three-dimensional (3D) spheroid culture provides a physiologically relevant alternative to conventional two-dimensional culture, but reliable spheroid formation in microwells depends on limiting cell-substrate adhesion. Pluronic F127 is an amphiphilic triblock copolymer that forms a hydrated surface layer, reducing protein adsorption. Here, we evaluated whether this intrinsic anti-fouling property could restore an anti-adhesive surface in used microwell plates to promote spheroid formation. Using chondrogenic ATDC5 and pancreatic {beta}-cell INS-1 cells, we characterized spheroid assembly kinetics, F127 cytotoxicity, surface hydrophilicity, protein adsorption, and spheroid morphology including size and shape factor. Both cell types formed compact spheroids within 24 hours on commercial anti-adhesive microwells. F127 coating markedly reduced water contact angle and protein adsorption, confirming increased surface hydrophilicity and reduced protein fouling. In microwells stripped of their original surface coating, F127 coating amounts of approximately 0.011-0.045 mg/cm2 consistently promoted spheroid formation across both cell types. Soluble F127 concentrations were confirmed to be non-cytotoxic up to 0.625% (w/v), while even complete dissolution of the highest tested coating amount would correspond to only 0.025% (w/v) F127. This simple, reproducible, and low-cost surface-modification strategy may provide an accessible approach for re-functionalizing microwell platforms for 3D cell culture.

Vanadium (V) is a potential insulinomimetic that can modulate carbohydrate metabolism, but its biological effects are sensitive to chemical form, concentration, and sex. Chelation of vanadium with organic ligands, in particular citrate, allows to increase its bioavailability and optimize pharmacokinetic properties. The aim of the study was to evaluate tissue-, dose-, and sex-dependent changes in physiological parameters and activity of the key glycolytic enzyme - lactate dehydrogenase (LDH) - under the influence of vanadium citrate. The study was conducted on 6-week-old Wistar rats of both sexes. The animals received vanadium citrate orally for 36-38 days at doses of 3, 12.5, and 50 g VCit/kg body weight. LDH activity in skeletal muscle, liver, kidney, and pancreas was investigated. No pronounced toxic effect on physiological parameters was detected: body weight dynamics corresponded to age norms, no behavioral changes were observed. LDH activity demonstrated pronounced sexual dimorphism and depended on the dose received. It was established that the optimal dose, which provides a modulating effect without signs of metabolic stress, for females is 12.5 g VCit/kg, while for males - 3 g VCit/kg. The most significant changes in LDH activity were recorded in the pancreas at a dose of 50 g V/kg, where the indicators decreased from 0.81 to 0.31 mol/(min x mg protein) in females and from 1.02 to 0.28 mol/(min x mg protein) in males. The effect of vanadium citrate on carbohydrate metabolism, as well as its dose-, tissue- and sex-specific nature, is likely determined by a dual action: the insulin-like effect of vanadium (redirecting pyruvate to oxidation) and the allosteric inhibition of glycolysis by the citrate ligand (substrate limitation for LDH). The obtained results emphasize the importance of considering sex and dose in the research and development of metabolically active compounds.

Purpose Prevention and early detection of osteoporosis remains a global challenge, more so in regions like the Philippines where screening barriers exist. Chest x-rays meanwhile are relatively inexpensive, and more frequently done, and therefore can be used for opportunistic screening. This study aimed to develop a deep learning model for osteoporosis detection from chest x-rays using DXA as the gold standard. Methods A convolutional neural network called Osteo-AI was developed using 406 pairs of chest x-rays and DXA scans of Filipino patients aged 50 and above. With data augmentation, the training set expanded to 6,300 pairs. Gradient-weighted class activation mapping technique was applied to localize and identify patterns and areas in the chest x-ray images correlating with osteoporosis. Results Training data consisted of 369 female patients and 37 males. Ages of the patients ranged from 50 to 89 with a mean age of 63 years old. Initial testing yielded promising results, with Osteo-AI achieving a diagnostic accuracy of 85.71%, easily outperforming a benchmark of 33.33% Conclusion Our findings suggest the potential of Osteo-AI to enhance osteoporosis screening accessibility, aiding in early intervention to prevent fragility fractures. Further research involving larger datasets is warranted to refine and optimize the model, potentially improving detection accuracy and expanding its utility in global healthcare settings.

Cellular senescence contributes to the progression of many age related musculoskeletal diseases. Cellular senescence is a biological state that arises from replicative exhaustion and various cellular stressors, including elevated oxidative stress, mitochondrial dysfunction, mechanical overload, and chronic exposure to pro-inflammatory cytokines and proteases. Although senolytic agents show promise for eliminating senescent cells, their translation has been hindered by the lack of physiologically relevant and scalable in vitro screening methods. In the present study, we developed a standardized, physiologically relevant senescence-induction model and validated a metabolic activity assay as a rapid, scalable method for screening senolytic compounds. We used primary human intervertebral disc cells (IVD) as an example, but the workflow applies to many other cell types. To mimic inflammatory and oxidative stress, we used a combination of TLR-2 activation (Pam2CSK4) and tert-butyl hydroperoxide (tBHP), a potent ROS generator. Senescence induction was validated by quantifying {beta}-galactosidase fluorescence intensity, {beta}-gal enzymatic activity, and the expression of the p16 senescence marker across 3 IVD cell types: nucleus pulposus (NP), inner annulus fibrosus (iAF), and outer annulus fibrosus (oAF) cells. The combined Pam2CSK4 + tBHP exposure generated a robust senescent phenotype across all 3 IVD cell types, with oAF cells exhibiting the strongest increases in {beta}-gal fluorescence, {beta}-gal enzymatic activity, and p16 expression. We then used oAF cells to evaluate if the metabolic activity assay (Alamar Blue) could be used to determine both cytotoxicity of senolytic drugs in non-senescent cells and senolytic activity in a mixed population of senescent and non-senescent cells. We validate the method by comparing metabolic activity results with {beta}-gal enzymatic activity and p16 expression in induced and noninduced cells following exposure to three known senolytics (o-Vanillin, RG-7112, and ABT-199). The metabolic activity assay reliably identified a therapeutic window in which the three senolytics were non-toxic to non-senescent cells while selectively reducing metabolic activity in a mixed population of senescent and non-senescent cells. The reductions in metabolic activity in the mixed population correlated with decreases in SA {beta}-gal enzymatic activity and p16 expression, validating metabolic activity as a sensitive and scalable senolytic readout.