Low Cell Density Research Articles

Development and validation of a nomogram for predicting the risk of obstructive coronary artery disease in rheumatoid arthritis patients based on LDL-C, Th17 cells, and IL-17

ObjectiveThis study aims to develop and validate a nomogram model for predicting the risk of obstructive coronary artery disease (CAD) in patients with rheumatoid arthritis (RA), incorporating low-density lipoprotein cholesterol (LDL-C), Th17 cells, and interleukin (IL)-17 levels. The proposed model seeks to enable personalized cardiovascular risk assessment for RA patients, thereby optimizing clinical management strategies.MethodsA total of 120 patients with rheumatoid arthritis (RA) who were treated at the Second Hospital of Shanxi Medical University between January 2019 and September 2023 were enrolled in this study. Based on coronary angiography results, patients were categorized into the RA-obstructive CAD group and the RA-non-obstructive CAD group. Additionally, 53 healthy controls (HC group) were included. Clinical characteristics, laboratory parameters, peripheral blood lymphocyte subsets, and cytokine levels were collected for analysis. Univariate logistic regression was used to identify risk factors associated with RA-obstructive CAD. These variables were further refined using a random forest model for optimal selection. Finally, multivariate logistic regression analysis was performed with the selected variables to develop a nomogram model, which was subsequently validated to assess its performance.ResultsCompared with the RA-non-obstructive CAD group, the RA-obstructive CAD group demonstrated significantly elevated levels of immune cell subsets, such as Th17 cells, and cytokines, including IL-17, IL-2, and IL-4, along with a reduction in Treg cells. (2) In the training cohort, univariate and multivariate logistic regression analyses identified LDL-C (OR = 0.04, P < 0.001), Th17 cells (OR = 0.76, P = 0.005), and IL-17 (OR = 0.75, P = 0.001) as independent risk factors for obstructive CAD in RA patients. Subsequently, a predictive nomogram model for RA-obstructive CAD risk was developed based on these indicators, incorporating LDL-C, Th17 cells, and IL-17.ConclusionThis study developed a predictive nomogram for RA-obstructive CAD by combining traditional risk factors, such as LDL-C, with immune biomarkers Th17 and IL-17. The model demonstrated robust predictive accuracy, enabling more precise risk assessment of CAD in RA patients. It offers clinicians a valuable tool for advancing cardiovascular risk management in RA, underscoring its significant potential for clinical application.

High cell density cultivation by anaerobic respiration

BackgroundOxygen provision is a bottleneck in conventional aerobic high cell density culturing (HCDC) of bacteria due to the low O2 solubility in water. An alternative could be denitrification: anaerobic respiration using nitrogen oxides as terminal electron acceptors. Denitrification is attractive because NO3− is soluble in water, the end-product (N2) is harmless, and denitrification is widespread among bacteria, hence suitable organisms for most purposes can be found. The pH must be controlled by injection of an inorganic acid to compensate for the pH increase by NO3−-consumption, resulting in salt accumulation if feeding the bioreactor with NO3− salt. We avoid this with our novel pH–stat approach, where the reactor is supplied with 5 M HNO3 to compensate for the alkalization, thus sustaining NO3−-concentration at a level determined by the pH setpoint. Here we present the first feasibility study of this method, growing the model strain Paracoccus denitrificans anaerobically to high densities with glucose as the sole C-source and NO3− as the N-source and electron acceptor.ResultsOur fed-batch culture reached 20 g cell dry weight L−1, albeit with slower growth rates than observed in low cell density batch cultures. We explored reasons for slow growth, and the measured trace element uptake indicates it is not a limiting factor. Bioassays with spent medium excluded accumulation of inhibitory compounds at high cell density as the reason for the slow growth. The most plausible reason is that high metabolic activity led to CO2/H2CO3 accumulation, thus suppressing pH, leading to a paucity in HNO3-feeding until N2-sparging had removed sufficient CO2. The three free intermediates in the denitrification pathway (NO3− → NO2− → NO → N2O → N2) can all reach toxic concentrations if the electron flow is unbalanced, and this did occur if cells were glucose-limited. On the other hand, accumulation of polyhydroxyalkanoates occurred if the cells were NO3−-limited. Carefully balancing glucose provision according to the HNO3 injected is thus crucial.ConclusionsThis work provides a proof of concept, while also identifying CO2/H2CO3 accumulation as a hurdle that must be overcome for further development and optimization of the method.

Hydrogen peroxide concentration as an indicator of cyanobacterial response to diurnal variation in light intensity

We measured diurnal variations in oxidative stress conditions of cyanobacteria utilizing field observations and laboratory experiments in order to evaluate photoinhibition effects. On clear summer days, transparent bottles filled with surface water were set up at several depths and were collected every three hours together with the measurement of the photosynthetically active radiation (PAR). In the laboratory experiment, two cyanobacterial species were exposed to gradually increasing and then decreasing light intensities. The samples were analyzed with the PAR-induced (H2O2), along with the total hydrogen peroxide concentrations (total H2O2), the catalase activities (CAT), OD730, protein (Protein), and chlorophyll a (Chl a) contents, and so on. Protein was significantly proportionate with OD730 and Chl a, and was used as an indicator of cell biomass. Increasing PAR, H2O2 concentration increased proportionately with the PAR intensity. Then, an oxidative stress indicator in a cell, H2O2/Protein is given by the PAR divided by cell volume, evaluated by Protein. CAT activity in a cell, far largest among antioxidant activities, solely followed total H2O2/Protein. The prediction model for H2O2/Protein was developed with the sufficient agreement with the experimental and field observation results. The model elucidated that the maximum H2O2/Protein in a day was larger with lower cell density even at the water surface, indicating that the higher photoinhibition was imposed at low density, in addition to the lower attenuation of PAR. These results indicate that H2O2/Protein is an effective biomarker to indicate the stress level of cyanobacteria; the observed levels of H2O2 to freshwater may prove useful in designing the criteria for cyanobacteria management.

The effects of Cefixime and Aspirin on phytoplankton community structure and dynamics: A Mesocosm approach

Pharmaceuticals are emerging contaminants of interest due to their ability to induce changes in the environment. These chemical compounds are discharged into aquatic ecosystems with a high likelihood of affecting non-target organisms such as phytoplankton. However, there is a gap in knowledge regarding the effects of pharmaceuticals on phytoplankton. This study used the mesocosm approach to investigate the effects of cefixime, a cephalosporin antibiotic and aspirin, a nonsteroidal anti-inflammatory drug on the community structure (species diversity, richness, and abundance) of phytoplankton. The mesocosm approach studied the effects of three treatments of pharmaceuticals: cefixime, aspirin and a combination of cefixime and aspirin, alongside a control experiment for 21 days. A total of 31 phytoplankton species belonging to five groups were identified; with diatoms and green algae having higher diversity compared to the other algae groups. Scenedesmus sp., Pediastrum sp. and Zygnema sp. were the top three recurring species in all the samples taken throughout the experimental period. Navicula sp. were the most recurring diatoms. The exposure of phytoplankton to cefixime, aspirin, and a combination of cefixime and aspirin significantly reduced the diversity, richness, and abundance of all species by the 21st day of the experiment. The control had the highest cell density (8,910 cells mL) of phytoplankton species on the 21st day, while the cefixime treatment had the lowest cell density (48 cells mL) f phytoplankton species on the seventh day of the study. This study demonstrated that cefixime and aspirin had major impacts on the phytoplankton community.

Controlling spheroid attachment improves pancreatic beta cell differentiation from human iPS cells.

Regenerative medicine using human induced pluripotent stem cells (hiPSCs) is available for treating type 1 diabetes; however, the efficiency and maturation of hiPSC differentiation into pancreatic beta cells requires improvement. Various protocols, including three-dimensional (3D) culture, have been developed to improve differentiation efficiency and maturation. Several methods for 3D culture have been reported; however, they require costly and complicated equipment, special materials, and complicated operations. To solve these problems, we developed a simple 3D culture method under static conditions using a cyclo-olefin polymer (COP) characterized by high moisture barrier properties, low surface energy, and hydrophobicity. Using this 3D method and our simple and low-cost protocol, we found that differentiation into the definitive endoderm (DE) was better when the spheroids were attached. Therefore, upon the addition of Y-27632, attached spheroids with unique shapes and cavities were formed, and the differentiation efficiency into DE increased. During DE differentiation, the attachment of spheroids to the substrate and their subsequent floating improved differentiation efficiency. We found that the amount of C-peptide in spheroids differentiated using COP dishes was greater than that in rotary culture. Furthermore, INSULIN was highly expressed in areas with low cell density, suggesting that the unique shape of the spheroids made from COP dishes improved differentiation efficiency. Our study suggests that a device-free, simple 3D culture method that controls spheroid attachment improves the efficiency of hiPSC differentiation into pancreatic beta cells.

Edible electrospun materials for scalable cultivated beef production

Cultivated meats are a direct response to an ever-increasing global demand for meat, that will alleviate the negative impacts of animal farming on the environment and food security. Despite recent advances, however, challenges regarding scalability and costs remain, impeding the availability and affordability of these novel foods. Consequently, this study aims to design novel edible and biocompatible scaffolds for the expansion of bovine mesenchymal stem cells, using silk fibroin from degummed Bombyx mori cocoons. The scaffolds were created from 12 % (w/w) silk fibroin in formic acid via two different methods of electrospinning, a needle-based laboratory set-up and a needleless configuration with the ability to produce non-woven fabrics at industrial scale. The supports were further treated with methanol or ethanol, which induced β-sheet crystallisation and preserved their fibrous nature in an aqueous environment for at least 2 weeks, with <10 % total weight loss. Although the highly porous nanofibrous morphology was maintained in all cases (98–166 nm fibre diameters), the alcohol treatments increased the stiffness, strength and brittleness of the materials by 6-fold, 5-fold and 3-fold, respectively. When different seeding densities (1500, 3000 and 5000 cells/cm2) of bovine mesenchymal stem cells were investigated, there were no signs of cytotoxicity, and the best growth was achieved at the lowest cell density, yielding a 9-fold expansion, with a 0.018 h−1 specific growth rate and 44 h doubling time over 7 days. These findings provide novel insights into electrospun materials and may support future developments in cultivated meats.

Molecular dynamics simulations of a multicellular model with cell-cell interactions and Hippo signaling pathway.

The transcriptional coactivator Yes-associated protein (YAP)/transcriptional co-activator with PDZ binding motif (TAZ) induces cell proliferation through nuclear localization at low cell density. Conversely, at extremely high cell density, the Hippo pathway, which regulates YAP/TAZ, is activated. This activation leads to the translocation of YAP/TAZ into the cytoplasm, resulting in cell cycle arrest. Various cancer cells have several times more YAP/TAZ than normal cells. However, it is not entirely clear whether this several-fold increase in YAP/TAZ alone is sufficient to overcome proliferation inhibition (contact inhibition) under high-density conditions, thereby allowing continuous proliferation. In this study, we construct a three-dimensional (3D) mathematical model of cell proliferation incorporating the Hippo-YAP/TAZ pathway. Herein, a significant innovation in our approach is the introduction of a novel modeling component that inputs cell density, which reflects cell dynamics, into the Hippo pathway and enables the simulation of cell proliferation as the output response. We assume such 3D model with cell-cell interactions by solving reaction and molecular dynamics (MD) equations by applying adhesion and repulsive forces that act between cells and frictional forces acting on each cell. We assume Lennard-Jones (12-6) potential with a softcore character so that each cell secures its exclusive domain. We set cell cycles composed of mitotic and cell growth phases in which cells divide and grow under the influence of cell kinetics. We perform mathematical simulations at various YAP/TAZ levels to investigate the extent of YAP/TAZ increase required for sustained proliferation at high density. The results show that a twofold increase in YAP/TAZ levels of cancer cells was sufficient to evade cell cycle arrest compared to normal cells, enabling cells to continue proliferating even under high-density conditions. Finally, this mathematical model, which incorporates cell-cell interactions and the Hippo-YAP/TAZ pathway, may be applicable for evaluating cancer malignancy based on YAP/TAZ levels, developing drugs to suppress the abnormal proliferation of cancer cells, and determining appropriate drug dosages. The source codes are freely available.

NIMG-59. COMPARATIVE ANALYSIS OF LONG-TERM GLIOBLASTOMA SURVIVORS USING MACHINE LEARNING GENERATED RADIO-PATHOMIC METRICS

Abstract BACKGROUND Glioblastoma (GBM) remains the most devastating central nervous system malignancy with poor overall survival (OS), even with gross total resection (GTR) and chemoradiation. Identifying factors associated with long-term survival (LTS) in GBM patients is challenging but crucial for improving prognostic models. This study compares an internal dataset of the top 10% long-term survivors of GBM (n=14) against a combined external cohort of 528 GBM patients who underwent GTR. METHODS We used a previously published machine learning-generated radio-pathomic model based on autopsy tissue as ground-truth to generate whole-brain maps of cell density and tumor probability maps (TPM) using conventional preoperative MRI sequences in both our internal dataset and a combined external cohort of GBM patients who underwent GTR from the UCSF-PDGM-v2 and UPenn-GBM databases. We then analyzed clinical and radio-pathomic metrics, including cell density and TPM values within contrast-enhancing (CE) and FLAIR hyperintense regions. Statistical comparisons were performed using t-tests. RESULTS Cell density metrics showed significant differences, with lower cell density in contrast-enhancing regions (t-value: 4.709, p&amp;lt;0.001; median: 1559 vs. 1959) and in FLAIR hyperintense regions (t-value: 3.585, p&amp;lt;0.001; median: 1356 vs. 1568) within the LTS group. TPM metrics also showed significant differences, with lower TPM values in contrast-enhancing regions (t-value: 3.312, p&amp;lt;0.001; median: 0.71 vs. 0.72) and in non-enhancing regions (t-value: 2.606, p&amp;lt;0.01; median: 0.68 vs. 0.69) in the LTS group. CONCLUSION Our comparative analysis reveals distinct radio-pathomic characteristics associated with long-term survival in GBM patients. Reduced cell density and lower TPM values in both contrast-enhancing and FLAIR hyperintense regions are significantly correlated with long-term survival. These findings suggest that integrating radio-pathomic data may enhance prognostic models and warrants further studies evaluating the relationship of these markers and OS.

Multivariate Environmental Factors and Seasonal Spatial Dynamics Affecting the Phytoplankton Community in Yazhou Bay, South China Sea

This study investigated phytoplankton and water environmental factors in Yazhou Bay, South China Sea, during the winter, spring, and summer of 2023. It examined phytoplankton community structure, subgroup heterogeneity, and key environmental drivers. Phytoplankton abundance ranged from 0.08 to 14.30 × 10⁴ cells·L−1, with high concentrations in estuary and nearshore zones. In summer, currents carry phytoplankton offshore, with stratification leading to high sedimentation in southern offshore waters. RDA results indicated that in winter and spring, inorganic nitrogen mainly influences phytoplankton distribution, while silicate is the primary factor in summer. Although seasonal differences in total phytoplankton abundance are minimal, significant horizontal and vertical distribution variations exist. Diverse preferences of different phytoplankton species for temperature, salinity, nitrogen, and phosphorus result in high species diversity. The Shannon–Wiener diversity index (H′) averages 3.96 ± 0.09, and the Pielou evenness index (J) averages 0.82 ± 0.01. Dominant species include Pseudo-nitzschia pungens, Skeletonema costatum, and Rhizosolenia sinica. Influenced by external oceanic water masses, estuary input, and islands, phytoplankton subgroups show regional and seasonal variations. Despite recorded harmful algal blooms (HABs) in adjacent waters, Yazhou Bay’s high biodiversity and low cell density suggest a low HAB risk, though future risks due to climate change and human activities remain.

Systematic insights into cell density-dependent transcriptional responses upon medium replacements

Understanding the molecular mechanisms governing transfection efficiency and particle secretion in high cell density cultures is critical to overcome the cell density effect upon transient gene expression. The effect of different conditioned media in HEK293 transcriptome at low and high cell density is explored. A systematic pair-wise comparative study was performed to shed light on the effect on previous phenotypical characteristics of different media conditions: fresh, exhausted and media depleted from extracellular vesicles (EVs) as well as associated proteins and RNAs. The obtained results suggest that restorative effects observed in transfection efficiency when employing EV-depleted media may arise predominantly from physicochemical alterations rather than cellular processes. A significant downregulation of genes associated with nucleocytoplasmic transport for the conditions involving the use of exhausted or EV-depleted media was observed. Moreover, upregulation of histone-related genes in EV-depleted media suggest a role for histone signaling in response to cellular stress or growth limitations, thereby highlighting the potential of manipulating histone levels as a promising strategy to enhance transient transfection. It was also corroborated that the accumulation of extracellular matrix proteins upon cell growth may inhibit transfection by an already-known competitive effect between cell membrane-bound and free proteoglycans. Proteomic characterization of EV-depleted media further unveiled enrichment of pathways associated with infection response and double-strand DNA breaks, suggesting that HEK293 cells undergo an induced infection-like state that disrupts cellular processes. Importantly, the study reveals that EV-depleted media stimulates virion release pathways underscoring the complex interplay between extracellular vesicles and viral budding.

Dual-directional regulation of extracellular respiration in Shewanella oneidensis for intelligently treating multi-nuclide contamination

Radionuclide contamination has become a global environmental concern due to the high mobility and toxicity of certain isotopes. Bioreduction mediated by electrochemically active bacteria (EAB) with unique extracellular electron transfer (EET) capability is recognized as a promising approach for nuclear waste treatment. However, it is difficult to achieve bidirectional regulation of EET pathway through traditional genetic manipulation, limiting the bioremediation application of EAB. Here, we designed and optimized a novel Esa quorum sensing (EQS) system for highly efficient gene expression and interleaved cellular functional output. By promoting dimethyl sulfoxide reductase at low cell density and increasing the synthesis of electron conductive complex and flavins at high cell density, the EQS system dynamically switched the extracellular respiratory pathway of Shewanella oneidensis MR-1 according to cell density. The engineered strain exhibited precise switching and substantial improvement in the extracellular remediation of multiple nuclides, sequentially increasing the reduction of iodine IO3- and uranium U(VI) by 2.51- and 2.05-fold compared with the control, respectively. Furthermore, a mobile bacterial biofilm material was fabricated for collecting uranium precipitates coupled with U(VI) reduction. This work clearly demonstrates that EQS system contributes to the bidirectional regulation of EET pathway in EAB, providing an effective and refined strategy for bioremediation of multi-nuclide contamination.

Advanced imaging reveals enhanced malignancy in glioblastomas involving the subventricular zone: evidence of increased infiltrative growth and perfusion.

Glioblastoma's infiltrative growth and heterogeneity are influenced by neural, molecular, genetic, and immunological factors, with the precise origin of these tumors remaining elusive. Neurogenic zones might serve as the tumor stem cells' nest, with tumors in contact with these zones exhibiting worse outcomes and more aggressive growth patterns. This study aimed to determine if these characteristics are reflected in advanced imaging, specifically diffusion and perfusion data. In this monocentric retrospective study, 137 glioblastoma therapy-naive patients (IDH-wildtype, grade 4) with advanced preoperative MRI, including perfusion and diffusion imaging, were analyzed. Tumors and neurogenic zones were automatically segmented. Advanced imaging metrics, including cerebral blood volume (CBV) from perfusion imaging, tissue volume mask (TVM), and free water corrected fractional anisotropy (FA-FWE) from diffusion imaging, were extracted. SVZ infiltration positively correlated with CBV, indicating higher perfusion in tumors. Significant CBV differences were noted between high and low SVZ infiltration cases at specific percentiles. Negative correlation was observed with TVM and positive correlation with FA-FWE, suggesting more infiltrative tumor growth. Significant differences in TVM and FA-FWE values were found between high and low SVZ infiltration cases. Glioblastomas with SVZ infiltration exhibit distinct imaging characteristics, including higher perfusion and lower cell density per voxel, indicating a more infiltrative growth and higher vascularization. Stem cell-like characteristics in SVZ-infiltrating cells could explain the increased infiltration and aggressive behavior. Understanding these imaging and biological correlations could enhance the understanding of glioblastoma evolution.

Escherichia coli Survival on Dry Bulb Onions Treated with Crop Protection Sprays Prepared using Contaminated Water in the Treasure Valley Growing Region

Contaminated agricultural water has been implicated in produce-associated outbreaks, including dry bulb onions (Allium cepa). This study was designed to quantify risks associated with contaminated water used to prepare crop protection sprays applied immediately before the onset of field curing of dry bulb onions. Laboratory experiments determining the behavior of Salmonella and Escherichia coli in crop protection chemical solutions were performed to guide selection for field use. Field trials were conducted (2022, 2023) in eastern Oregon (Treasure Valley) using two onion cultivars (‘Red Wing’ and ‘Cometa’) inoculated with a rifampicin-resistant E. coli cocktail (3–4 log CFU/100 mL) suspended in fungicide solution or clay suspension, and applied with a backpack sprayer at the end of the growing season. Onions were sampled through the next 4 weeks of field curing and after 1 and 4–5 mos of postharvest storage. In 2022, onions were initially contaminated at a maximum cell density of 48 MPN/onion (Geometric mean (GM): 3.7 MPN/onion). At the end of curing, a single onion (out of 320) tested positive at 2 MPN/onion. In 2022, E. coli was not detected during postharvest storage (n = 160). In 2023, the application of contaminated sprays resulted in a maximum contamination of 275 MPN/onion (GM: 8.6 MPN/onion). At the end of the 2023 curing period, three out of 320 onions (0.9%) had detectable levels of E. coli (1–2 MPN/onion). Three ‘Cometa’ onions from the same plot that were treated with fungicide were positive for E. coli after 5 months of postharvest storage (2, 11, and 83 MPN/onion). These field trials indicate field curing conditions in the Treasure Valley help mitigate risks associated with contaminated water used for applying crop protection sprays. E. coli was detected on a small percentage of onions at low cell density after curing. The single onion with elevated E. coli populations after postharvest storage had internal damage characteristic of bacterial rot.

Developing microalgae harvesting: Enhanced efficiency with magnetite-urea nanocomposites for sustainable astaxanthin biorefinery

Harvesting costs, accounting for 15–30 % of total expenses, are a major bottleneck in microalgae biorefinery due to low cell density and energy-intensive processes. This study investigates Fe3O4-Urea (Fe@urea) nanocomposites (NCs) to replace conventional methods and improve the harvesting efficiency (HE) of Chlorella zofingiensis, known for its high lipid and astaxanthin content. Urea-coated magnetite nanoparticles (NPs) with additional functional groups significantly improved harvesting performance across all pH levels compared to conventional Fe3O4 NPs. HE was evaluated using both optical density and cell counting methods, with the latter proving superior results by eliminating interference from unreacted salts and debris. Optimization of nanocomposite concentrations and culture pH with Fe@urea achieves 95 % HE at 800 mg L−1 and pH 3–4. The Langmuir and Freundlich models confirmed the nanocomposite's multifaceted capabilities. Characterization techniques included SEM, FTIR, EDX, zeta potential analysis, and TEM. This innovative approach can revolutionize large-scale microalgae biorefineries, enhancing sustainable biofuel and bioproduct production and supporting the Sustainable Development Goal of Affordable and Clean Energy (SDGs 7).

Quantitative evaluation of DNA damage repair dynamics to elucidate predictors of autism vs. cancer in individuals with germline PTEN variants.

Persons with germline variants in the tumor suppressor gene phosphatase and tensin homolog, PTEN, are molecularly diagnosed with PTEN hamartoma tumor syndrome (PHTS). PHTS confers high risks of specific malignancies, and up to 23% of the patients are diagnosed with autism spectrum disorder (ASD) and/or developmental delay (DD). The accurate prediction of these two seemingly disparate phenotypes (cancer vs. ASD/DD) for PHTS at the individual level remains elusive despite the available statistical prevalence of specific phenotypes of the syndrome at the population level. The pleiotropy of the syndrome may, in part, be due to the alterations of the key multi-functions of PTEN. Maintenance of genome integrity is one of the key biological functions of PTEN, but no integrative studies have been conducted to quantify the DNA damage response (DDR) in individuals with PHTS and to relate to phenotypes and genotypes. In this study, we used 43 PHTS patient-derived lymphoblastoid cell lines (LCLs) to investigate the associations between DDR and PTEN genotypes and/or clinical phenotypes ASD/DD vs. cancer. The dynamics of DDR of γ-irradiated LCLs were analyzed using the exponential decay mathematical model to fit temporal changes in γH2AX levels which report the degree of DNA damage. We found that PTEN nonsense variants are associated with less efficient DNA damage repair ability resulting in higher DNA damage levels at 24 hours after irradiation compared to PTEN missense variants. Regarding PHTS phenotypes, LCLs from PHTS individuals with ASD/DD showed faster DNA damage repairing rate than those from patients without ASD/DD or cancer. We also applied the reaction-diffusion partial differential equation (PDE) mathematical model, a cell growth model with a DNA damage term, to accurately describe the DDR process in the LCLs. For each LCL, we can derive parameters of the PDE. Then we averaged the numerical results by PHTS phenotypes. By performing simple subtraction of two subgroup average results, we found that PHTS-ASD/DD is associated with higher live cell density at lower DNA damage level but lower cell density level at higher DNA damage level compared to LCLs from individuals with PHTS-cancer and PHTS-neither.

Geometric constraint of mechanosensing by modification of hydrogel thickness prevents stiffness-induced differentiation in bone marrow stromal cells.

Extracellular matrix (ECM) stiffness is fundamental in cell division, movement and differentiation. The stiffness that cells sense is determined not only by the elastic modulus of the ECM material but also by ECM geometry and cell density. We hypothesized that these factors would influence cell traction-induced matrix deformations and cellular differentiation in bone marrow stromal cells (BMSCs). To achieve this, we cultivated BMSCs on polyacrylamide hydrogels that varied in elastic modulus and geometry and measured cell spreading, cell-imparted matrix deformations and differentiation. At low cell density BMSCs spread to a greater extent on stiff compared with soft hydrogels, or on thin compared with thick hydrogels. Cell-imparted matrix deformations were greater on soft compared with stiff hydrogels or thick compared with thin hydrogels. There were no significant differences in osteogenic differentiation relative to hydrogel elastic modulus and thickness. However, increased cell density and/or prolonged culture significantly reduced matrix deformations on soft hydrogels to levels similar to those on stiff substrates. This suggests that at high cell densities cell traction-induced matrix displacements are reduced by both neighbouring cells and the constraint imposed by an underlying stiff support. This may explain observations of the lack of difference in osteogenic differentiation as a function of stiffness.

GefB, a GGDEF domain-containing protein, affects motility and biofilm formation of Vibrio parahaemolyticus and is regulated by quorum sensing regulators

Vibrio parahaemolyticus (V. parahaemolyticus) stands as the predominant etiological agent responsible for gastroenteritis associated with the consumption of seafood. Cyclic di-guanosine monophosphate (c-di-GMP), a secondary messenger in bacteria, controls multiple bacterial behaviors including pathogenesis, the development of biofilms, and motility. The protein GefB (VPA1478), characterized by the presence of a GGDEF domain, inhibits the swarming motility of V. parahaemolyticus. In this study, we showed that deletion of gefB remarkably reduced cellular c-di-GMP level and biofilm formation by V. parahaemolyticus, but significantly enhanced the swimming and swarming motility. In addition, GefB inhibited the polar and lateral flagellar genes but activated genes associated with exopolysaccharide production of V. parahaemolyticus. The data also demonstrated that vpa1477 and gefB are co-transcribed as a single transcriptional unit, designated as vpa1477-gefB. Transcription of vpa1477-gefB was under the collective regulation of the master quorum sensing (QS) regulators AphA and OpaR, which function at low (LCD) and high cell density (HCD), respectively. AphA positively regulated vpa1477-gefB transcription at LCD, whereas OpaR negatively regulated its transcription at HCD. The findings significantly enhance our comprehension of the metabolism and regulatory mechanisms of c-di-GMP in V. parahaemolyticus.

Abstract B081: In situ multi-modal characterization of pancreatic ductal adenocarcinoma reveals tumor cell identity as a defining factor of the surrounding microenvironment

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is heterogeneous with low tumor purity, prominent microenvironment and complex architecture, which preclude identification of shared tumor intrinsic biology within and across patients. We overcame these challenges by applying complementary spatial omics approaches – providing necessary resolution and context – to primary untreated PDAC tumors from 39 patients capturing 341,949 low-bulk and 531,718 single-cell spatial transcriptomes. Of these, 59,403 low-bulk profiles and 205,665 single-cells represented tumor cells. We leveraged this data to build on prior reports of tumor cell subtypes in PDAC. We identify 5 distinct malignant subtypes, excluding ductal-like and intraepithelial neoplasia cells, that span the classical-to-basal spectrum. One tumor subtype is highly proliferative and enriched for the DNA synthesis-condensation-mitosis transcriptional network suggesting this subset disproportionately contribute to tumor growth. Strikingly, the spectrum of tumor cell subtypes was present within all patients, suggesting that subtype-based treatment stratification may need refinement. We find that each subtype has its own distinct regulators and histology. The classical subtype has extensive cell intrinsic regulation, higher cell density and lower extracellular matrix content. In contrast, the basal subtype phenotype results from a combination of cell intrinsic and tumor microenvironment (TME) interactions, wherein the basal subtype has a lower cell density, is surrounded by activated stroma and dense collagen matrix, and is poised for hypoxic adaptation. We derive the composition of cellular neighborhoods providing an explanation for the complex architecture seen in PDAC. Tumor cell neighborhoods stratified based on which side of the classical-to-basal spectrum they fell. For example, cells towards the classical-side of the spectrum had more homogenous tumor neighborhoods that generally lacked intermediate and basal tumor cells. In contrast, cells towards the basal-side of the spectrum were likely to contain classical neighbors. More broadly, neighborhoods along this continuum also contained different proportions of immune and stromal cells, cell densities, collagen deposition, and TME adaptation. These observations support a model where tumor subtypes exist within disparate niches. Lastly, our atlas unifies the catalog of previously reported stromal cells (fibroblasts and stellate cells versus myofibroblastic and inflammatory cancer associated fibroblasts (CAF)) and describes a novel interferon stimulated gene (ISG) resistance signature (ISG.RS) fibroblast. We map the location of these various stromal cells relative to tumor subtypes. For example, myofibroblast CAFs and ISG.RS fibroblasts are enriched near the basal tumor subtype whereas inflammatory CAF abundance increases with distance away from tumor cells. In sum, our atlas provides a lens for stratifying tumor complexity in a cancer cell centric manner and provides a path for testing therapeutic hypothesis in the right cell subtype and context. Citation Format: Brian Rabe, Anna Lyubetskaya, Andrew Kavran, Yulong Bai, Andrew Fisher, Hannah Pliner, Alba Font-Tello, Anne Lewin, Ruifeng Hu, Alexandre P Alloy, Yelena Cheng, Chao Dai, Yunfan Fan, Constance Brett, Todd Brett, Lauren Giampapa, Soeren Stahlschmidt, Fayaz Seifuddin, Steven Vasquez Grinnell, Daniel Carrera, Carlos Rios, Tom Lila, Pradeep Kar, Abhishek Shukla, Rachael Bashford Rogers, Lara Heij, Mike Mason, Ryan Golhar, Isaac Neuhaus, Enas Abu Shah, Shivan Sivakumar, Jimena Trillo Tinoco, Benjamin J Chen, Konstantinos J Mavrakis, Eugene Drokhlyansky. In situ multi-modal characterization of pancreatic ductal adenocarcinoma reveals tumor cell identity as a defining factor of the surrounding microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B081.

Patterning in stratified epithelia depends on cell-cell adhesion.

Epithelia consist of proliferating and differentiating cells that often display patterned arrangements. However, the mechanism regulating these spatial arrangements remains unclear. Here, we show that cell-cell adhesion dictates multicellular patterning in stratified epithelia. When cultured keratinocytes, a type of epithelial cell in the skin, are subjected to starvation, they spontaneously develop a pattern characterized by areas of high and low cell density. Pharmacological and knockout experiments show that adherens junctions are essential for patterning, whereas the mathematical model that only considers local cell-cell adhesion as a source of attractive interactions can form regions with high/low cell density. This phenomenon, called cell-cell adhesion-induced patterning (CAIP), influences cell differentiation and proliferation through Yes-associated protein modulation. Starvation, which induces CAIP, enhances the stratification of the epithelia. These findings highlight the intrinsic self-organizing property of epithelial cells.

Huxin Formula Inhibits Oxidized low-Density Lipoprotein-Induced Foam Cell Formation in THP-1 Macrophages via the LOX-1/NF-κB Pathway

Background Macrophage-derived foam cells are essential in the progression of atherosclerosis (AS). Based on our previous study, the Huxin Formula (HXF), a traditional Chinese medicine formula, demonstrates potential in anti-atherosclerosis. Nevertheless, it is still unknown how HXF affects the formation of foam cells derived from THP-1 macrophages. Purpose This research aims to examine the preventive role of HXF in the development of foam cells and its underlying molecular mechanism. Methods THP-1 derived macrophages and THP-1 cells overexpressing LOX-1 (LV-OLR1) were exposed to ox-LDL to establish foam cell models, and then treated with HXF. Meantime, Oil red O staining was used to detect lipid droplet production. ELISA kit was performed to measure intracellular levels of IL-6 and TGF-β. RT-qPCR and Western Blot were then utilized to determine the LOX-1 and NF-κB mRNA/protein levels. Results The findings indicated that HXF treatment potently reduced the lipid accumulation, downregulated IL-6 levels and upregulated TGF-β levels. However, this impact was almost reversed when LOX-1 was overexpressed in THP-1 cells stimulated with ox-LDL. Moreover, THP-1 were treated with HXF markedly reduced the levels of LOX-1 and NF-κB mRNA/protein, whereas overexpressing LOX-1 significantly reversed this effect. Conclusion HXF reduced the formation of foam cell in ox-LDL-stimulated THP-1 macrophages via inhibiting lipid accumulation and inflammation through regulating the LOX-1/ NF-κB pathway. These present findings further indicate a potential beneficial role of HXF in ameliorating atherosclerosis and foam cell formation, while provide a novel potential therapeutic strategy for preventing atherosclerosis.