Journal of Biological Engineering

Fermentation of C1 gases is an emerging technology where waste gases are bio converted into value-added products. This study navigates the gas fermentation potential of Gordonia rubripertincta to produce carotenoids. The crucial carbon monoxide dehydrogenase (CODH) enzyme, necessary for gas uptake by the microbe, was found to be present in G. rubripertincta through blastp on NCBI website. The organism was then used for gas fermentation experiments in a continuous stirred tank reactor (CSTR) in different modes of reactor operation resulting in the production of about 500 mg pigment/g WCW (wet cell weight). Two important reactor parameters, molybdenum content and pH, were optimized for enhanced carotenoid production. Overall, G. rubripertincta was observed to be an efficient candidate organism for C1 gas fermentation. KEY HIGHLIGHTSO_LIGordonia rubripertincta synthesises aerobic carbon monoxide dehydrogenase enzyme. C_LIO_LIIt is a potential gas fermenting microbe that gives carotenoids as product. C_LIO_LIThe gas uptake efficiency of the microbe is more in fed-batch discontinued mode. C_LIO_LIIn FB-D, the resultant carotenoids are 500+9 mg/g wet cell weight (WCW). C_LIO_LIMo/pH of 20 mg/7.0 resulted in highest carotenoids, i.e., 134+41 mg/g WCW. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/722808v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@8b1185org.highwire.dtl.DTLVardef@2b6f90org.highwire.dtl.DTLVardef@1a9697dorg.highwire.dtl.DTLVardef@14c9dc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rapid and specific diagnosis of viral and bacterial infections is a significant challenge in medicine and veterinary science, especially in the case of epidemically dangerous pathogens. The African swine fever virus (ASFV), for example, causes annual outbreaks among livestock, resulting in significant economic losses for farmers. DNA aptamers have been identified as a promising tool for point-of-care diagnostics, being highly specific to the target and stable ambient temperatures during storage. In this study, we describe the selection of DNA aptamers targeting the p54 viral protein using a single-round selection process. These aptamers were able to bind both to recombinant protein and inactivated ASFV viral particles. Analysis of the newly generated aptamers revealed a dependence of affinity and thermal stability on Ni2+ content, which was a dopant in the selection process. In some cases, the affinity increased 100 times, and melting temperature increased by 30{degrees}C. We have identify two novel DNA motifs that bound 2-3 Ni2+ or Zn2+ ions.

IntroductionCancer progression is driven not only by tumor cells but also by interactions between the extracellular matrix (ECM), stromal cells, and immune cells within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are major drivers of ECM remodeling, assembling ECM with aberrant organization. Extra domain A fibronectin (EDA-FN), a cellular FN containing an extra type III domain, is upregulated in the TME. EDA-FN regulates cellular behavior and has been associated with poor patient prognosis. Macrophages are among the most abundant immune cells within the TME, where they contribute to TME remodeling and inflammation to promote cancer cell invasion and metastasis. However, how tumor-associated matrix-specific cues regulate macrophage behavior remains largely understudied. PurposeHere, we developed a fibroblast-derived matrix platform that captures the structural imprint of tumor-associated EDA-enriched matrices and investigated how matrix-specific cues regulate macrophage behavior in the absence of ongoing soluble factor cues. MethodHuman mammary fibroblasts (HMFs) preconditioned in incubated low-serum media (lNC, or control) and MDA-MB231 metastatic breast cancer cell-conditioned media (mTCM) were cultured on polyacrylamide gels of 2 kPa and 20 kPa, respectively, followed by decellularization. Matrix organization, including fiber alignment, width, and intrafibrillar spacing, was quantified from confocal images. Decellularized EDA-FN-enriched matrices were subsequently reseeded with macrophages to assess macrophage morphology, phenotype, and matrix interactions. ResultsThe combined effects of tumor-derived soluble factors and pathological stiffness induced a CAF-like phenotype in HMFs, accompanied by cytoskeletal reorganization and microarchitectural alterations of EDA-FN-enriched matrices. Tumor-associated matrices exhibited increased alignment, narrower fiber width, and enlarged intrafibrillar spacing compared to control matrices. These aberrant, tumor-associated matrix-derived features were associated with altered macrophage behavior, including heterogeneous morphology, enhanced localized EDA-FN matrix loss beneath the cell body, and a hybrid phenotype with a shift toward a CD206-dominant profile. ConclusionsThese findings demonstrate the feasibility of obtaining EDA-FN-enriched matrices to isolate matrix-specific cues for investigating macrophage-ECM interactions. Furthermore, this platform can be leveraged to identify matrix-targeting therapeutic approaches for modulating macrophage function within the TME.

Matrix-bound nanovesicles (MBVs) are a type of small extracellular vesicle (EV) embedded in the extracellular matrix (ECM) throughout the body. MBVs have been previously isolated from various tissues and in vitro-cultured cell sheets, demonstrating remarkable attributes in regenerative medicine. However, differences between MBVs and conditioned culture medium-derived EVs (liquid-EVs) have yet to be characterized, and the field currently lacks specific protein markers that can identify MBVs from other EV subtypes. Here, we isolate MBVs and liquid-EVs from bone marrow mesenchymal stem cell (MSC) sheets and define differences in size, protein, and zeta potential between these EVs. We show that there is a correlation between cell-driven ECM deposition and MBV and liquid-EV production. We also find that MBVs are smaller, contain less protein per particle, and possess lower zeta potential than liquid-EVs. Interestingly, MBVs also comprise a distinct tetraspanin profile compared to liquid-EVs, with MBVs containing more CD63 and little to no CD81. Finally, we define that CD63, LAMP1, Alix, ITG{beta}1, and GRP94 and their abundance, may be markers specifically used to identify MBVs from liquid-EVs. Our study paves the way for the characteristic differentiation between MBVs from liquid-EVs, elucidates their differences in biogenesis, and reveals a potential connection between EV and ECM production.

The slightly-alkaline (pH [~]8.5), boiling ([~]90{degrees}C) vent-water of a Trans-Himalayan geothermal spring, moderately-rich in dissolved solids ([~]1500 ppm), was explored six times over a year. 11 archaeal and 46 bacterial species were detected consistently, while nine bacteria occurred intermittently, in the vent-epicenter featuring a largely-stable physicochemical milieu. All 11 archaea were detected as metagenome-assembled genomes ascribable to Thermoproteota. Of the total 55 bacteria detected, 32 were retrieved as MAGs, 20 as isolates, and three in both forms. Four bacteria could not be classified below the domain-level; three and four belonged to hyperthermophilic (Aquificia) and thermophilic (Thermaceae and Thermoflexaceae) taxa respectively; 27 belonged to taxa having some moderately-thermophilic members; 17 belonged to mesophilic taxa. According to metagenomics, an Aquificia, followed by two Thermoprotei and one Thermoproteales, dominated the microbiome overwhelmingly. Metatranscriptomically, however, the Thermoproteales was most active. Metatranscriptomic signatures envisaged the in situ metabolic status of the 66 species discovered as follows. Among the 18 putative hyperthermophiles and thermophiles identified, 17 rendered wide-ranging activities including growth; one Thermoproteota species had considerable activities sans growth. One new-phylum-level bacterium rendered wide-ranging activities including growth, while three such entities had considerable/minimal activities sans growth. Among the 27 potential moderate-thermophiles discovered, two Armatimonadota and one Thermosynechococcus species rendered wide-ranging activities including growth, 20 had considerable/minimal activities sans growth, whereas four had zero activities. Among the 17 mesophiles identified, 16 rendered considerable/minimal activities sans growth, whereas one had zero activity. Molecular drivers were envisaged from the metatranscriptomic data to explain the trends of inequitable population ecology.

Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of few cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of{Delta} sodB mutant further confirmed its targeted deletion. Overall, CRISRPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. Key PointsO_LIRapid and efficient CRISPR/Cas12a editing established in Cyanothece PCC 7425. C_LIO_LIFully segregated knockout mutants obtained after single selection round. C_LIO_LIPlatform for nuclear waste bioremediation and other biotechnological applications. C_LI

BackgroundBone graft biomaterials play a critical role in bone regeneration by influencing osteoblast differentiation and mineralization. However, comparative data regarding the osteogenic potential of commonly used graft materials under standardized conditions remain limited. Method and materialIn this in vitro experimental study, osteoblast-like cells (MG-63) were cultured with four bone graft materials, including Bio-Oss, Cerasorb, Bio-Tiss Cerabone, and Pro Osteon. The relative mRNA expression of osteogenic markers (COL1 and OPN) was evaluated at 1, 7, 14, and 21 days using real-time PCR. Alkaline phosphatase (ALP) activity and mineralization capacity were also assessed using colorimetric assay and Alizarin Red staining. Data were analyzed using one-way ANOVA and Tukey post hoc test (P < 0.05). ResultsSignificant differences were observed among the tested materials across all evaluated parameters. Bio-Oss and Cerasorb demonstrated higher gene expression levels and ALP activity compared to Bio-Tiss Cerabone and Pro Osteon (P < 0.05). Mineralization analysis showed significantly greater calcium deposition in the Bio-Oss and Cerasorb groups, whereas Pro Osteon consistently exhibited the lowest osteogenic performance. ConclusionBone graft biomaterials significantly influence osteogenic activity in osteoblast-like cells. Bio-Oss and Cerasorb showed superior osteogenic potential, while Pro Osteon demonstrated weaker performance. These findings highlight the importance of material properties in optimizing bone regeneration.

Biofilms formed by soil microbes hold immense potential for bioremediation, carbon dioxide sequestration, and the development of sustainable cementitious materials. However, quantifying the complex temporal coupling among bacterial growth, extracellular matrix (ECM) production, and mineralization dynamics remains a significant challenge due to the inherent nonlinearity of these processes and signal noise in high-throughput assays. To address this, we utilized an automated real-time kinetic analysis framework integrating connectivity-based segmentation, automated baseline alignment, and robust sliding-window algorithms to quantify the biomineralization competence of Bacillus subtilis under varying calcium regimes. Crucially, our results demonstrate that calcium carbonate promotes microbial growth as effectively as the highly soluble calcium acetate, providing strong evidence that B. subtilis actively solubilizes this crystalline powder to facilitate its metabolic requirements. Despite this growth efficacy, we found that calcium carbonate is an inadequate source for macro-calcite production compared to organic salts. By quantifying the expression efficiency of the sinI reporter gene, we determined that calcium-acetate-driven ECM expression significantly enhances the structural compatibility required for robust biomineralization. Furthermore, kinetic modeling suggests that ECM overproduction can partially compensate for defects in crystal growth-when provided crystalline calcium carbonate powder. These findings, enabled by high-resolution automated signal processing, underscore the critical role of self-mediated carbonate supply and present new engineering pathways for upcycling mineral-rich construction waste.

Some long non-coding RNAs (lncRNAs) are known to regulate gene expression. However, the underlying temporal dynamics of lncRNAs influencing gene and epigenetic regulation and mechanisms of lncRNA regulation in trans are less understood. To investigate this, we genetically engineered 17 doxycycline-inducible lncRNA transgenes for ectopic expression at the H11 safe harbor locus in human pluripotent stem cells (hiPSCs), and we generated high-density temporal RNA-seq and ATAC-seq profiles. Most lncRNA transgenes were induced at 2 hours and maintained expression through the 96-hour time course. Surprisingly, when we sought to identify gene expression changes due to the lncRNAs, we found that the global transcriptional landscape was dominated by a strong systemic response triggered by doxycycline exposure. We rigorously defined this cohort of genes as a Doxycycline-Responsive Gene Signature (DRGS). The DRGS was also present in at least 28 public datasets from dox-inducible transgene studies involving diverse cell types. Next, we determined which lncRNAs exhibited trans-regulatory events. We identified DANCR, FENDRR, LINC00667, LINC00847, LNCPRESS1, and PNKY as lncRNAs that regulate specific transcript expression in trans. The downstream target genes encoded 53 mRNAs and 10 lncRNAs. None of the target lncRNAs altered gene expression proximal to their own loci (i.e., triggering secondary cis-effects). Surprisingly, the target genes of LINC00847 (transcribed from chromosome 22) were substantially enriched on chromosome 19, with a preponderance of target genes encoding RNA metabolism and RNA splicing factors. Collectively, our study provides a resource to discern artifacts in the doxycycline-inducible system and identifies temporally regulated targets of 6 lncRNAs for future mechanistic studies.

BackgroundThe use of autologous or allogeneic cell therapies has now entered to the clinical practice in several fields of medicine, especially in oncology and hematology. From this regard, 2D-cell manufacturing is complex and costly and bioreactors have attracted major interest for efficient and cost-effective mass production of cells. Bioreactors have several advantages such as homogeneous repartition of nutrients and gas, control of all culture parameters and increased yield. However, the important shear stress generated by those bioreactors is an important disadvantage as it can affect cell survival or cell quality. This important shear stress is the result of the mixing method using either blades (used in stirred-tanked bioreactors) or gas bubbles (used in airlift bioreactors). Another downside of the use of bioreactors is the difficulty to scale-up. As the volume increases, the shear stress generated by blades radically increases leading to cell death and a decrease of cell quality. DescriptionIn this study, we describe a bioreactor developed using a different mixing method effectively reducing the shear stress and facilitating scale-up. This bladeless method uses an inclination of the bioreactor as well as rotation to mix fluids in a container. Here we described different steps that led to the adaptation of this bioreactor, initially developed for fragile microalgae culture, for mammalian cell culture amplification. The bioreactor was tested to amplify a natural killer (NK) cell line NK92 which is an IL-2 dependent cell line used in clinical trials for cancer therapy. We have tested the influence of 1-The number of cells seeded; 2-The influence of the rotation speed on cell growth and viability; 3-The influence of the bioreactor angle on the above parameters; 4-The duration of the culture. ResultsCells were initially seeded at 2.5.105 / ml in a volume of 380 ml. According to the rotation speed of 15, 30, 45 and 60 rpm, we have observed an increase of cell numbers at day 3 (3-fold), day 5 (7-fold) and day 7 (10-fold) compared to seeding, the best expansion being obtained at day 7 with a rotation speed of 45 rpm. The optimal angle of rotation was found to be 3 degree, with an optimal amplification at day 7 versus day 3 (p < 0.01). The viability was also found to be optimal in the latter condition. ConclusionsThese preliminary results demonstrate that NK92 cells could be amplified using this bioreactor. In the best tested condition, neither cell viability nor cell growth was impacted. These results strongly suggest the potential use of this device in future clinically applicable conditions.

Adult hippocampal neurogenesis plays a central role in learning, memory formation, and adaptive neural plasticity, making it an attractive target for noninvasive neuromodulation strategies. Low-intensity focused ultrasound (LIFU) has emerged as a promising modality for modulating brain function, yet its effects on adult neurogenesis and the role of stimulation frequency remain incompletely understood. In this study, we evaluated whether transcranial LIFU applied to the dentate gyrus influences neurogenic and cognitive outcomes in a frequency-dependent manner. Adult rats received twice-weekly ultrasound stimulation for four weeks at 0.5, 1, or 5 MHz. Neurogenesis was assessed through BrdU incorporation and neuronal differentiation by BrdU/NeuN co-labeling, while expression of neurogenesis-associated markers (BDNF, FGF-2, and Sox-2) was quantified using qRT-PCR. Behavioral effects were examined using the novel object recognition task. Among the tested conditions, 0.5 MHz stimulation produced the most pronounced neurogenic response, with increased cellular proliferation in the dentate gyrus, elevated expression of neurogenic markers, and improved recognition memory relative to sham-treated animals. Higher stimulation frequencies yielded comparatively weaker effects. These findings identify stimulation frequency as a critical determinant of LIFU-driven neuroplastic responses and support the potential of focused ultrasound as a noninvasive approach for promoting hippocampal regeneration and functional recovery.

Pathologic arterial stiffening is a hallmark of vascular disease that contributes to maladaptive vascular remodeling and neointimal hyperplasia through vascular smooth muscle cell (VSMC) phenotypic switching. Yet, because vascular disease progression is governed by both biomechanical and extracellular matrix (ECM) alterations, existing in vitro models often fail to recapitulate the full complexity of the diseased vascular microenvironment. Here, we developed a bioactive decellularized extracellular matrix (dECM) and methacrylated hyaluronic acid (MeHA) composite scaffold platform with tunable stiffness that preserves native vascular ECM components while enabling controlled investigation of stiffness-dependent cell behavior. Proteomic analyses confirmed retention of key vascular matrisome components, including collagens and glycoproteins, following decellularization. Electrospun vascular dECM scaffolds maintained an aligned fibrous architecture and spanned stiffness ranges representative of healthy and pathologically stiffened arterial microenvironments. Within this matrix-preserving platform, human VSMCs cultured on stiff dECM scaffolds exhibited increased spreading, altered morphology, enhanced nuclear localization of YAP and survivin, and broad transcriptional changes consistent with a shift toward a proliferative, matrix-remodeling VSMC phenotype. Together, this bioactive, matrix-preserving platform enables mechanobiologically relevant modeling of stiffness-driven vascular remodeling and indicates YAP and survivin as candidate regulators of maladaptive VSMC mechanotransduction.

Understanding skeletal muscle metabolism involves real-time monitoring of key cellular parameters, such as calcium ions (Ca2+), adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP), and intracellular temperature. Fluorescent protein (FP)-based biosensors are used for live-cell imaging of these signals with high spatiotemporal resolution. Differentiated myotubes are in vitro models used for physiological muscle metabolism research. However, efficient transfection of FP-based biosensors into these cells is challenging. Here, we developed an electroporation-based strategy for delivering recombinant protein biosensors into fully differentiated myotubes. Biosensors for Ca2+, ATP, cAMP, and temperature were recombinantly produced using Escherichia coli and introduced into myotubes using electroporation. Electroporation conditions were optimised to maximise delivery efficiency, preserve cell viability, and minimise cellular damage. We established a robust intracellular delivery system that effectively demonstrated Ca2+, ATP, and temperature dynamics. Furthermore, we achieved the successful co-delivery of two biosensors that enabled dual imaging of Ca2+ and cAMP in response to stimulation.

Odd-chain carboxylates such as valerate and heptanoate are ecologically relevant metabolites and promising platform chemicals, yet the factors leading to their formation during secondary lactate fermentations remain poorly understood. Here, a continuous anaerobic bioreactor was operated for 297 days under mildly acidic conditions to evaluate how lactate:propionate molar ratios shape product spectrum in lactate fermentations. Valerate was the predominant odd-chain product under all conditions, reaching concentrations up to 110 mM, while heptanoate accumulated only at low levels (<10 mM). At low lactate concentrations (10-20 g/L), product selectivity strongly depended on the lactate:propionate ratio. When lactate:propionate ratios were around 1.2 mol/mol, odd-chain products were favored, whereas higher ratios (up to 4.8 mol/mol) shifted metabolism toward caproate and butyrate formation. However, this trend was not maintained at higher lactate concentrations (30-40 g/L; lactate not fully consumed), where odd-chain selectivities remained high even at lactate:propionate ratios of 4.8 mol/mol. Pathway analysis indicated that under high-lactate conditions up to 30% of lactate was redirected toward propionate and acetate formation, likely via the acrylate pathway. Microbial community analysis revealed a stable dominance of Caproiciproducens spp., that could be correlated to valerate production. Overall, this work provides mechanistic insights into the ecology of lactate fermentations and offers a framework for steering product selectivity in engineered anaerobic systems. HighlightsValerate was the dominant product, reaching up to 110 mM. Lactate:propionate ratios drive product selectivities. High lactate concentrations activated in situ propionate formation pathways. Caproiciproducens dominance was associated with sustained valerate production.

Inositol 1,4,5-trisphosphate (IP3) is a key second messenger that regulates diverse physiological processes. Visualization of IP3 dynamics in living cells is therefore important for understanding its signaling processes. In this study, we developed genetically encoded green fluorescent IP3 biosensors named Green iPenguins with distinct half-maximal effective concentrations (EC50) for IP3, enabling detection of IP3 signals over a range of concentrations. The biosensors displayed more than a 4-fold increase in fluorescence intensity upon IP3 and showed high specificity for IP3 over structurally related molecules. When expressed in HEK293T cells, the biosensors enabled visualization of IP3 dynamics involved in different signaling pathways. They were also compatible with dual-color imaging, allowing simultaneous monitoring of IP3 together with cAMP or Ca2+ signals. In addition, the hierarchical relationship between IP3 and Ca2+ signaling was visualized, providing insight into the temporal relationship between these two second messengers. The biosensors are expected to facilitate future studies of physiological processes involving IP3 signaling networks.

PurposeBacterial vaginosis (BV) is associated with disruption of the vaginal microbiome and extracellular matrix (ECM) remodeling, yet the contribution of host proteases to this process remains unclear. This study investigated whether expression and activity of cathepsins K, L, S, and V differ by BV diagnosis and community state type (CST). We hypothesized that BV and BV associated CSTs would exhibit increased expression and activity of collagen and elastin-degrading cathepsins. MethodsVaginal fluid samples were collected and classified by BV diagnosis and CST. Cathepsin expression was evaluated by Western blotting to distinguish inactive and active enzyme forms. Proteolytic activity was assessed using multiplex cathepsin zymography. Statistical analyses compared cathepsin expression and activity across diagnoses and CSTs. Principal component analysis and linear regression were performed to assess associations between cathepsin activity, microbial diversity, and CST. ResultsProcathepsin K expression was significantly increased in BV-positive and CST IV samples, while total cathepsin L expression was significantly elevated in samples with Nugent-intermediate scores. Cathepsins S and V showed variation in inactive and active forms in Nugent-intermediate and CST III samples. In contrast, total cathepsin activity, including cathepsins K and V, did not significantly differ across BV diagnoses or CSTs. Overall, cathepsin activity varied between individuals rather than by clinical classification. ConclusionsCathepsin expression and maturation state differ by microbiome composition, suggesting that the vaginal microbiome may regulate post-translational processing of cathepsins. As a result, cathepsin activity appears to be regulated at the individual level rather than strictly by BV diagnosis or CST. These findings link vaginal microbiome composition to ECM remodeling and potential adverse reproductive outcomes.

Bio-based materials are known for their excellent biodegradability and, in some cases, their potential to fix carbon dioxide. Owing to these properties, they are increasingly being utilized as environmentally friendly alternatives across various applications. In this study, we focused on using living cells themselves as material components, aiming to evaluate their potential as substitutes for conventional plastic-based thermal insulators. We selected two types of cells, photosynthetic purple non-sulfur bacterium Rhodovulum sulfidophilum and tobacco BY-2 plant suspension cells. After optimizing solidification conditions through the addition of pectin and cellulose nanofibers, we measured the thermal conductivity of the solidified cells under atmospheric pressure. The results showed that R. sulfidophilum exhibited 0.0553 W/m{middle dot}K, while BY-2 exhibited a thermal conductivity of 0.043 W/m{middle dot}K. Both values indicate relatively low thermal conductivity compared to existing bio-based materials, suggesting high insulation performance. Among the solidified cells, the solidified BY-2 cells showed minimal variation in thermal insulation performance under pressure changes, and had a low thermal emissivity as revealed by FT-IR analysis. Based on these findings, we propose that cell-derived materials can serve as potentially biodegradable bio-based thermal insulation materials.

BackgroundCurrent microphysiological models do not support long-term investigations into the chronic effects of vascular risk factors and the development of vascular diseases. Prolonged culture frequently leads to cellular senescence and loss of functional integrity, resulting in variability and inconsistency in modeling chronic vascular responses. Here we aimed to develop and sustain a long-term multicellular human vascular avatar, addressing the critical need for long-term disease modeling and drug testing. MethodsTo identify the optimal media for longevity, cell identity and function were assessed by morphology, qPCR, beta-gal staining, ELISA, bulk RNA-seq and single cell RNA-seq analysis. After optimizing the culture media, iPSCs-derived ECs and VSMCs from unaffected and Hutchinson-Gilford Progeria Syndrome (HGPS) donors were grown in Gravitational Lumen Patterning (GLP) Vessel- Chips for 1-6 months to generate a long-lived vascular avatar for the study of vascular aging. ResultsGuided by quantitative morphological analyses and bulk RNAseq profiling, we generated a novel optimized culture media VSL (VEGF, SB431542 as a TGF-{beta} inhibitor, low fetal bovine serum) that enhances the long-term health of vascular endothelial cells (ECs). Furthermore, we modified the VSL formulation (mVSL) by modulating 8Br-cAMP, FGF, PDGF, and a cell viability enhancer HMH1015 levels to enhance EC-VSMC (vascular smooth muscle cell) crosstalk and support long-term cellular viability. Subsequently, we maintained and characterized a human vascular avatar with a three-dimensional extracellular matrix environment and 3D vascular architecture for over 180 days. Finally, we demonstrated that this long-lived human vascular avatar enabled modeling vascular aging using iPSC-derived vascular cells from patients with Hutchinson-Gilford Progeria Syndrome (HGPS). ConclusionsWe have successfully engineered and maintained a human vascular avatar for over 180 days. The vascular avatar provides a robust platform for modeling disease-associated vascular aging and for evaluating therapeutic strategies targeting chronic vascular disorders.

Prodigiosin is a red pigment produced by various bacteria, including Serratia marcescens. Despite its wide and promising range of biological activities, the large-scale production of prodigiosin is currently limited by its high cost and low yields. Here we propose and optimize an innovative, low-cost, peanut-based solid culture medium that enhances the yield of prodigiosin produced by Serratia marcescens. Colorimetric assays revealed that peanut significantly stimulates prodigiosin synthesis. Further HPLC-MS analysis allowed us to unambiguously identify prodigiosin and shows that our medium specifically improves the yield of prodigiosin. Overall, our innovative culture medium could help lower prodigiosin production costs and, ultimately, open new industrial applications.

PurposeLeft ventricular hypertrophy (LVH) is a major complication of chronic hypertension and an independent risk factor for cardiovascular morbidity and mortality. There are currently no clinically validated markers available to identify hypertensive individuals at risk for developing LVH. In hearts of hypertensive rats, we previously described metabolic changes that precede LVH development, including in branched-chain amino acid (BCAA) metabolism. This study investigated whether cardiac leucine uptake, measured with dynamic 5-[18F]fluoroleucine positron emission tomography-computed tomography ([18F]FLE-PET/CT), was impaired and could serve as an in vivo marker for hypertension-induced LVH development. ProceduresWe synthesized [18F]FLE following established radiochemistry protocols and performed dynamic [18F]FLE-PET/CT imaging in 3-month-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) control rats (n = 4 per group). Cardiac magnetic resonance (CMR) imaging was conducted on the same animals for structural co-registration. A dual-output reversible two-tissue compartment model with spill-over (SP) and partial volume (PV) corrections was developed to quantify the first-pass rate constant (K1) and total distribution volume (Vt = K1/k2) for [18F]FLE. Protein expression of L-type amino acid transporter 1 (LAT1) and branched-chain keto acid dehydrogenase (BCKDH) phosphorylation status were assessed by immunoblotting of isolated heart tissue. ResultsSHR demonstrated markedly lower first-pass leucine uptake rates (K1) and total distribution volumes (Vt) compared with WKY rats, consistent with reduced cardiac BCAA uptake. Concurrently, LAT1 (SLC7A5) expression was significantly reduced in SHR hearts compatible with decreased leucine uptake. Elevated BCKDH phosphorylation at Ser293 in SHR hearts indicated diminished BCKDH enzymatic activity and impaired BCAA catabolism. ConclusionsDynamic cardiac [18F]FLE-PET imaging successfully detects decreased leucine uptake in hypertensive rat hearts at 3 months of age, before LVH is established at 5 months. Reduced cardiac leucine uptake may thus serve as a surrogate marker for impaired cardiac BCAA metabolism and early in vivo indicator of cardiometabolic dysfunction that precedes LVH. The imaging approach holds translational potential for identifying hypertensive patients at risk for LVH progression.