Cell Suspension Research Articles

Whole-pathway stimulation of anthocyanin biosynthesis by UDP-glycose: flavonoid glycosyltransferase gene (UFGT) providing new insights into natural product development based on plant cell factory

Anthocyanins are a type of plant natural products (PNPs) that play a crucial role in various fields, including food, medicine, cosmetics, and dyes. However, their primary source is plant extraction, leading to significant ecological damage and resource wastage issues. To address the challenge of achieving ecologically sustainable high-throughput anthocyanin production, this study proposes a plant cell factory (PCF) strategy based on suspension cell culture (SCC). In this study, the key gene VdUFGT involved in anthocyanin biosynthesis was cloned from Vitis davidii, and gene overexpression was conducted using Agrobacterium-mediated methods. Notably, the vgt3 transgenic cell line exhibited 4.4-fold increase in anthocyanin content, reaching 286.9 μg/g (FW) in solid medium, along with enhanced levels of flavonoids and proanthocyanidins. Integrative analysis of transcriptomics and metabolomics demonstrated that VdUFGT significantly promoted the expression of genes involved in anthocyanin biosynthesis, leading to the enhanced accumulation of various flavonoids, including anthocyanins. A small-scale plant cell factory was established, achieving an impressive production of 4.11 mg/g (DW) of anthocyanin within just 12 days, which is 8.5 times higher than that of the WT cell line, with the yield of purified freeze-dried anthocyanin powder reaching 3.17%. The plant cell factory, employing genetic engineering and suspension cell culture, offers a safe, ecological, economical, and efficient approach for the high-throughput production of plant natural products such as anthocyanins.

A CH4-Driven Ion Cloud-Stretched Approach Enables ICP-qMS for Multiplex Single-Cell Analysis.

In the last 40 years, inductively coupled plasma quadrupole (q) mass spectrometry (ICP-qMS) has been recognized as one of the best tools for the quantification of multiple elements/isotopes and even the biomolecules they labeled in a homogeneous solution sample. However, it meets a tough challenge when acquiring multi-m/z signals from an intact single-cell dispersed in a cell suspension, since the single-cell ion cloud generated in ICP presents an intermittently transient event with a duration time of hundreds of microseconds while the dwell time plus settling time of the q is at the similar time scale when peak-hopping between different m/z. Herein, we report CH4 is able to stretch the single-cell ion cloud duration time to more than 7,000 μs in collision-reaction-cell (CRC), allowing multi-m/z signals acquisition by ICP-qMS. Quantification of single-cell's multiple phenotype protein markers can thus be achieved on ICP-(CH4-CRC)-qMS, not only revealing the heterogeneity between the single cells but also enabling an unambiguous cell-classification of their subtypes. CH4-driven ion cloud-stretched approach breaks through the long-standing bottleneck limited single-cell multiplex analysis on ICP-qMS, paving a path for more important applications of ICP-qMS in the fields related to single-cell analysis.

Establishment of Betalain-Producing Cell Line and Optimization of Pigment Production in Cell Suspension Cultures of Celosia argentea var. plumosa

The prevalence of synthetic colorants in commercial products has raised concerns regarding potential risks, including allergic reactions and carcinogenesis, associated with their use or consumption. Natural plant extracts have gained attention as potential alternatives. This research focuses on callus induction and the establishment of cell suspension cultures from Celosia argentea var. plumosa. Friable callus was successfully induced using hypocotyl explants cultured on semi-solid Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg/L 6-benzylaminopurine (BAP). The friable callus cell line was used to establish a suspension culture. The effects of sucrose, BAP, and tyrosine concentrations on betalain production were investigated using response surface methodology (RSM) based on central composite design (CCD). Optimal conditions (43.88 g/L sucrose, 0.15 mg/L tyrosine, and 0.77 mg/L BAP) yielded 43.87 mg/L total betalain content after 21 days, representing a threefold increase compared to the control. BAP had a significant positive impact on betalain production, and increasing BAP and sucrose concentrations generally led to higher betalain production. However, tyrosine was not a significant factor for betalain production in cell suspension cultures. Additionally, antioxidant assays showed that suspension-cultured cells (SCCs) under optimized conditions exhibited free radical scavenging activity comparable to that observed in C. argentea var. plumosa flower extract. This study indicates the potential for further research on betalain production using C. argentea var. plumosa cell cultures, which may have commercial applications.

Physicochemical Rationale of Matrix Effects Involved in the Response of Hydrogel-Embedded Luminescent Metal Biosensors

There is currently a critical need for understanding how the response and activity of whole-cell bacterial reporters positioned in a complex biological or environmental matrix are impacted by the physicochemical properties of their micro-environment. Accordingly, a comprehensive analysis of the bioluminescence response of Cd(II)-inducible PzntA-luxCDABE Escherichia coli biosensors embedded in silica-based hydrogels is reported to decipher how metal bioavailability, cell photoactivity and ensuing light bioproduction are impacted by the hydrogel environment and the associated matrix effects. The analysis includes the account of (i) Cd speciation and accumulation in the host hydrogels, in connection with their reactivity and electrostatic properties, and (ii) the reduced bioavailability of resources for the biosensors confined (deep) inside the hydrogels. The measurements of the bioluminescence response of the Cd(II) inducible-lux biosensors in both hydrogels and free-floating cell suspensions are completed by those of the constitutive rrnB P1-luxCDABE E. coli so as to probe cell metabolic activity in these two situations. The approach contributes to unraveling the connections between the electrostatic hydrogel charge, the nutrient/metal bioavailabilities and the resulting Cd-triggered bioluminescence output. Biosensors are hosted in hydrogels with thickness varying between 0 mm (the free-floating cell situation) and 1.6 mm, and are exposed to total Cd concentrations from 0 to 400 nM. The partitioning of bioavailable metals at the hydrogel/solution interface following intertwined metal speciation, diffusion and Boltzmann electrostatic accumulation is addressed by stripping chronopotentiometry. In turn, we detail how the bioluminescence maxima generated by the Cd-responsive cells under all tested Cd concentration and hydrogel thickness conditions collapse remarkably well on a single plot featuring the dependence of bioluminescence on free Cd concentration at the individual cell level. Overall, the construction of this master curve integrates the contributions of key and often overlooked processes that govern the bioavailability properties of metals in 3D matrices. Accordingly, the work opens perspectives for quantitative and mechanistic monitoring of metals by biosensors in environmental systems like biofilms or sediments.

HPB SO33 - Transcriptomic characterisation of the diabetogenic pancreatic ductal adenocarcinoma phenotype

Abstract Background New-onset diabetes mellitus (NOD) can manifest as a consequence of pancreatic ductal adenocarcinoma (PDAC), although not all PDAC patients develop NOD. For many patients, hyperglycaemia begins 3 years prior to PDAC diagnosis, with 30% of PDAC patients experiencing NOD by the time their cancer is diagnosed. The mechanisms underlying the onset of diabetes mellitus (DM) in PDAC and its consequences for disease development, detection and treatment are not well understood. The aim of this study was to characterise the transcriptomic differences between PDAC tumours that present with NOD compared to those that do not. Method Eight tumour samples from patients undergoing surgery for PDAC (4 without DM (HbA1c <35), 4 with NOD (HbA1c >65, within 3 years of cancer diagnosis) underwent single cell-RNA sequencing (scRNAseq) after taking intra-operative biopsies using Chromium 10X Genomics technology. Unique cell clusters were identified using CellRanger and cell types annotated using scATOMIC and singleR datasets. Manual annotation was undertaken when results were not agreeable after consulting the literature. Differential expression between groups was performed using DESeq2. Significant genes were identified as having a log2 fold-change >1.5 and p<0.05. Gene set enrichment was performed using Reactome. Analysis was conducted in RStudio. Results Single cell suspensions were created with a mean cell count of 1.2 million cells and a mean viability of 90%. PDAC cells and the full complement of tumour microenvironmental cells were recovered. Significant differential expression was seen in the PDAC cells themselves, CD4+ cells, macrophages, mast cells and fibroblasts. Cancer associated fibroblasts (CAFs) seemed not to differ. PDAC cells with NOD seemed to be enriched in MYC oncogenes whereas those without DM seemed to be enriched in KRAS mutations. There was a mixture of “basal” and “classical” transcriptional subtypes between the two groups. Conclusion Differences between diabetogenic PDAC and non-diabetogenic PDAC have been observed at the transcriptional level. Diabetogenic tumours seem to be promoted by MYC oncogenes and be more inflammatory than non-diabetogenic tumours. These findings require validation which will be done on independent samples using spatial transcriptomics to allow endocrine-exocrine crosstalk to be determined. Understanding the molecular pathways in diabetogenic pancreatic tumours could enhance future strategies to detect PDAC earlier and to target diabetes-associated vulnerabilities for therapeutic purposes.

DDDR-22. USE OF BIOPSY-DERIVED TUMOR ORGANOIDS FOR PERSONALIZED DRUG TESTING IN RECURRENT GLIOBLASTOMA

Abstract To date, there are no effective systemic treatment options for recurrent glioblastoma (GBM). Furthermore, often surgical resection is not possible and tumors have acquired an increased chemo- or radioresistance. Patient-derived tumor organoids (PDTOs) provide a new exciting tool to test individual treatment responses. Aim of this study was to assess the feasibility of a personalized PDTO-based drug testing even from rare material such as stereotactic biopsies. PDTOs were prepared based on single cell suspensions from tumor material obtained either from open GBM resections (reference data set, n = 37) or from stereotactic biopsies (n = 8). Drug response curves (6-9 doses) were performed for 9 drugs including commonly used drugs such as temozolomide, lomustine, etoposide and temsirolimus. Viability was measured using CellTiterGlo (Promega) to assess half-maximal inhibitory concentrations (IC50). Medical records were reviewed for survival and molecular information such as the MGMT methylation status. Drug testing on PDTOs from open resections allowed to adjust the test range for each drug. Additionally, a significant association between increased temozolomide sensitivity of PDTOs and both, MGMT methylation and increased survival was observed. However, in 59% of the cases, no sensitivity to any of the drugs was seen, and only 17% were highly sensitive to more than one drug. Subsequent drug testing on PDTOs from biopsies of recurrent GBM revealed sensitivity in 3/8 cases against one (n=2) or two (n=1) of the drugs. However, sensitivity against drugs varied substantially in a patient-individual manner. We successfully developed a workflow to test drug sensitivity on PDTO even from low starting material obtained from stereotactic biopsies. Association of clinical outcome and MGMT methylation with temozolomide sensitivity corroborated the strength of this approach. Furthermore, patient-individual treatment responses strongly suggest for a future personalized drug testing.

EXTH-53. THE PHARMACOLOGICAL ATLAS OF MENINGIOMAS

Abstract No systemic treatment options exist for recurrent or refractory meningioma patients. This study aimed to create a pharmacological atlas of all FDA-approved oncology drugs in a large cohort of patient-derived meningioma organoids (TOs). TOs were generated from single cell suspensions from freshly resected meningiomas and were treated with semi-logarithmic concentrations spanning from 10 nM to 30 µM by the automated liquid handler Hamilton MicroLAB STAR®. The drug library AODX from the National Cancer Institute consisted of 179 FDA-approved anticancer drugs. The viability was measured with the RealTimeGlo (Promega). Half-maximal inhibitory concentrations (IC50) were calculated by GraphPad Prism software. We created a pharmacological atlas of meningiomas by generating 8,413 dose-response curves of 179 current FDA-approved oncology drugs in patient-derived tumor organoids from 47 meningioma patients. The cohort consisted of 35 grade 1, 11 grade 2, and 1 grade 3 meningiomas, including 10 recurrent tumors. In total, 21% (n=39/179) of the drugs exhibited sensitivity (median IC50 < 30 µM), with significant enrichment for topoisomerase, RNA/protein synthesis, proteasome, and HDAC inhibitors. The top five drugs with the lowest IC50 values were romidepsin, dactinomycin, carfilzomib, plicamycin, and omacetaxine with median values of 10, 31, 145, 303, and 400 nM. Hierarchical clustering of IC50 data revealed at least two distinct drug response clusters defined by sensitivity to tyrosine kinase inhibitors. When considering the Cmax/IC50 ratio as a potential predictive marker of treatment response (>1), the HDAC inhibitors belinostat (74.5), romidepsin (69.7), the proteasome inhibitor carfilzomib (34.6), and the anthracyclines epirubicin (2.6), and doxorubicin (1.8), and the PI3K inhibitor idelalisib (1.6) are the most promising drug candidates for further in vivo evaluation. We provide the first comprehensive insight into the pharmacological landscape of meningiomas. This data might serve as the foundation for future clinical studies on the systemic treatment of aggressive meningiomas.

BIOM-04. SINGLE-CELL RNA SEQUENCING PROTOCOL OF THE HUMAN METASTATIC PROSTATE SPINE TUMOR MICROENVIRONMENT

Abstract INTRODUCTION 90% of spine tumor patients can develop metastases with debilitating complications, such as sensorimotor dysfunction, paralysis, and spinal instability. As spine tumor patients have limited eligibility to participate in clinical trials and have heterogenous tumor pathology, there is a distinct lack of therapeutic targets and prognostication markers of disease. Thus, there is a distinct need for molecular profiling of spine tumors. OBJECTIVE We describe an OR-to-bench-top processing of spine tumors for single-cell omic studies, with samples from a metastatic prostate cancer patient to demonstrate the ability to compare molecular markers and microenvironments of difficult-to-process spine tumors and adjacent normal tissue. METHODS Under IRB protocol #Pro00101198, patients undergoing spine surgery with the Department of Neurosurgery at Duke University were consented for tissue collection. Vertebral osseous metastases and adjacent normal tissue were confirmed histologically (H&E, immunohistochemistry) and radiologically (MRI, PET, CT, and x-ray). Tissues were manually and enzymatically homogenized in serum-free media with collagenase A and DNAse I to obtain a single cell suspension. Cells were then filter sterilized, erythrolyzed, counted to determine live-death ratio, resuspended to 10-20 million cells per mL, then cryopreserved for downstream standard library preparation for single-cell sequencing using the 10X genomics platform. Downstream analysis was performed using Seurat pipeline, InferCNV analysis, and SingleR. RESULTS P404S and PSA expression confirmed tumor was prostate in origin. Radiological imaging and H&E staining confirmed the presence of T7 osseous metastasis. Uniform manifold approximation and project (UMAP) plots identified 12 unique clusters of immune cell subpopulations between normal and tumorous vertebrae, with their own subsets of differentially expressed genes. CONCLUSION We have developed a protocol to generate granular single cell molecular profiling for spine tumors and metastases. Applications of this protocol can aid in identifying therapeutic targets, molecular predictors of disease, and potentiate increased eligibility of patients for clinical trials.

EXTH-54. COMBINATORIAL DRUG SCREENING IDENTIFIES CARFILZOMIB AND ENZALUTAMIDE FOR TREATING AGGRESSIVE MENINGIOMAS

Abstract The goal of combinatorial therapy is to improve the effectiveness of treatment by targeting multiple aspects of cancer cells or overcoming resistance to single drugs. Therefore, we performed an automated combinatorial drug screening in meningioma cell lines and patient-derived tumor organoids (TOs). Three grade 3 meningioma cell lines (NCH93, IOMM-Lee, and KT21-MG1) were stably transduced with blue, green, and red fluorescence proteins. The cell lines were multiplexed into 384-well plates and treated in 5x5 dose-response (0-1000 nmol/l) matrices for 48h by the automated liquid handler Hamilton MicroLAB STAR®. The drug library consisted of 166 FDA-approved anticancer drugs. The viability of each cell line was demultiplexed by using fluorescence signals and CellTiterGlo. TOs were established from single cell suspensions of freshly resected meningioma tissue (n=20). The ZIP model was used to determine synergism. This drug screening effort generated 13,695 unique drug-drug combinations per cell line. Most synergistic combinations were observed in IOMM-Lee (1.14%), followed by KT21-MG1 (0.88%), and NCH93 (0.54%). The 110 most effective drug combinations were validated in wild-type meningioma cells. 75 drug combinations demonstrated a positive most synergistic area (MSA) score, indicating synergistic effects. Based on synergy scores and literature evidence, the 16 most promising combinations were selected for screening in TOs. The proteasome inhibitor carfilzomib in combination with the androgen receptor inhibitor enzalutamide exhibited the highest synergy in TOs. In 40% (n=8/20) of the cases, the combination showed strong synergism (MSA>10) resulting in an overall average MSA of 7.36, followed by carfilzomib and neratinib, and romidepsin and gemcitabine with average MSAs of 5.49, and 4.32, respectively. Furthermore, carfilzomib and enzalutamide induced apoptosis in TOs assessed by Caspase-3/7. This comprehensive combinatorial drug screening identified the synergistic combination of the proteasome inhibitor carfilzomib and androgen receptor inhibitor enzalutamide that can be an effective treatment option for selected meningioma patients.

IMMU-30. IGV-001: A BIOLOGIC-DEVICE COMBINATION PRODUCT ELICITS POTENT ANTI-GBM RESPONSES AS A MONOTHERAPY AND IN COMBINATION WITH CHECK POINT INHIBITION

Abstract INTRODUCTION Imvax is developing GoldspireTM, a personalized immunotherapy that combines whole tumor-derived cells with an antisense oligonucleotide against insulin-like growth factor 1 receptor (IGF-1R; IMV-001) in biodiffusion chambers (BDCs; 0.1 µm pore). BDCs are irradiated and implanted at abdominal sites for ~48h to deliver an antigenic payload and immunostimulatory factors that together train the immune system to attack tumor cells. The lead product, IGV-001, was evaluated in newly diagnosed glioblastoma (ndGBM) patients in a phase 1b study. Median overall survival (OS) of highest exposure IGV-001-treated Stupp-eligible patients (n=10) was 38.2 mos compared with 16.2 mos in standard-of-care-treated patients (p=0.044; ClinicalTrials.gov NCT02507583). A Phase 2b study in ndGBM is currently underway (ClinicalTrials.gov NCT04485949), where a primary endpoint of progression-free survival and key secondary endpoints of overall survival and safety will be evaluated. METHODS The orthotopically implanted GBM (luciferase-expressing GL261-luc2) model was utilized to evaluate the efficacy of mIGV-001 (the murine version of IGV-001) alone or in combination with anti-PD-1 immunotherapy. The following four groups were compared: mIGV-001 or BDC placebo + isotype or anti-PD-1 antibody (4 doses total every 3-4 days). GL261-luc2 cells were treated with IGF-1R antisense IMV-001. The treated cell suspension was loaded into clinical-grade BDCs (0.1-µm pore size, polyvinylidene difluoride membrane) at a density of 1 × 106 cells per BDC and then irradiated with 5–6 Gy. mIGV-001 or BDCs filled with saline were administered 28 days prior to tumor challenges and tumor progression and survival were monitored for an additional 100 days. RESULTS Preventative treatment with mIGV-001 limited tumor progression and extended OS in mice orthotopically (i.e., intracranially) challenged with GL261-luc2 cells. The therapeutic benefit of mIGV-001 was enhanced with anti-PD-1 treatment and the combination therapy was superior to either monotherapy.

Anti –Inflammatory Activity of Propolis Trigona sp. Water Extract from North Lombok with Red Blood Cell Membrane Stability Method

Propolis is a bee product that can be used as an anti-inflammatory. Cultivation of propolis-producing bees is also carried out in North Lombok. However, propolis has not been utilized optimally by the people of North Lombok. Apart from that, testing of North Lombok propolis is still limited to the chemical content and antioxidant activity of propolis extracted with ethanol solvent. Therefore, this study aims to determine the activity and effective concentration of North Lombok propolis water extract as an anti-inflammatory using the red blood cell membrane stability method. Groups include control groups such as positive control (diclofenac sodium), negative control (distilled water), and test groups (propolis water extract concentrations of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, and 30%). In The first step human blood was centrifuged of human blood, and then part of the red blood cells (sediment) was taken and saline was added to obtain a red blood cell suspension. Next, mixing the test solution was carried out in the order of 0.5 ml of red blood cell suspension, then 0.5 ml of extract was added (0.1% w/v Na-diclofenac solution in the positive control, distilled water in the negative control), 1 mL of buffer solution and 2 mL of hyposaline solution, then homogenized. Each group was incubated at 37°C for 30 minutes, then centrifuged for 10 minutes at 3000 rpm. The supernatant was taken, and the absorbance was read with a UV-Vis spectrophotometer at 560 nm. Next, the percentage value of red blood cell hemolysis protection was calculated. The data obtained were tested statistically using One-way ANOVA and post-hoc (LSD) tests SPSS version 29. The results showed that propolis water extract concentrations were 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, and 30% have anti-inflammatory activity because they can increase the stability of the red blood cell membrane with a percentage of hemolysis protection of respectively 57.92%, 59.99%, 60.99%, 61.99%, 64.31%, 69.59%, 75.07%, 79.77% and 84.45%. Propolis water extract concentrations of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, and 25% had anti-inflammatory effects that were not significantly different from the positive control (p>0.05). The 27.5 % and 30% concentrations had a higher percent hemolysis protection value than the positive control (p<0.05).

Phytochemical Profiling and Biological Activities of Extracts from Bioreactor-Grown Suspension Cell Cultures of Schisandra henryi

Phytochemical Profiling and Biological Activities of Extracts from Bioreactor-Grown Suspension Cell Cultures of Schisandra henryi

Highly Parallel and High‐Throughput Nanoliter‐Scale Liquid, Cell, and Spheroid Manipulation on Droplet Microarray

AbstractThe droplet microarray (DMA) platform is a powerful tool for high‐throughput biological and chemical applications, enabling miniaturization and parallelization of experimental processes. Capable of holding hundreds of nanoliter droplets, it facilitates the screening and analysis of samples, such as cells, bacteria, embryos, and spheroids. Handling thousands of small volumes in parallel presents significant challenges. In this study, we utilize the open format of the DMA for controlled, parallel high‐throughput liquid manipulations using the sandwich technique. We demonstrate high‐throughput medium replacement at nanoliter‐scale, maintaining high cell viability on DMA for up to 7 days; for HeLa‐CLL2 cells (adherent) and SU‐DHL4 cells (suspension), and up to 14 days for HEK293 spheroids. Additionally, we achieve highly parallel aliquot uptake from nanoliter droplets, enabling non‐destructive cell viability assessments. Furthermore, the presented method enables the parallel transfer of cell spheroids between different DMAs, allowing transfer and pooling of spheroids in seconds without damage. These advances significantly enhance the capabilities of the DMA platform, enabling long‐term cell culture in nanoliter droplets and parallel sampling for high‐throughput cell or spheroid manipulation. This broadens the scope of DMA's potential applications in fields such as cell‐based high‐throughput screening, formation of complex 3D cell models for drug screening, and microtissue engineering.

Cardioimmunology in Health and Diseases: Impairment of the Cardio-Spleno-Bone Marrow Axis Following Myocardial Infarction in Diabetes Mellitus.

A comprehensive understanding of the cardio-spleen-bone marrow immune cell axis is essential for elucidating the alterations occurring during the pathogenesis of diabetes mellitus (DM). This study investigates the dynamics of immune cell kinetics in DM after myocardial infarction (MI) over time. MI was induced in diabetic and healthy control groups using C57BL/N6 mice, with sacrifices occurring at days 1, 3, 7, and 28 post-MI to collect heart, peripheral blood (PB), spleen, and bone marrow (BM) samples. Cell suspensions from each organ were isolated and analyzed via flow cytometry. Additionally, the endothelial progenitor cell-colony-forming assay (EPC-CFA) was performed using mononuclear cells derived from BM, PB, and the spleen. The results indicated that, despite normal production in BM and the spleen, CD45+ cells were lower in the PB of DM mice at days 1 to 3. Further analysis revealed a reduction in total and pro-inflammatory neutrophils (N1s) in PB at days 1 to 3 and in the spleen at days 3 to 7 in DM mice, suggesting that DM-induced alterations in splenic neutrophils fail to meet the demand in PB and ischemic tissues. Infiltrating macrophages (total, M1, M2) were reduced at day 3 in the DM-ischemic heart, with total and M1 (days 1-3) and M2 (days 3-7) macrophages being significantly decreased in DM-PB compared to controls, indicating impaired macrophage recruitment and polarization in DM. Myeloid dendritic cells (mDCs) in the heart were higher from days 1 to 7, which corresponded with the enhanced recruitment of CD8+ cells from days 1 to 28 in the DM-infarcted myocardium. Total CD4+ cells decreased in DM-PB at days 1 to 3, suggesting a delayed adaptive immune response to MI. B cells were reduced in PB at days 1 to 3, in myocardium at day 3, and in the spleen at day 7, indicating compromised mobilization from BM. EPC-CFA results showed a marked decrease in definitive EPC colonies in the spleen and BM from days 1 to 28 in DM mice compared to controls in vitro, highlighting that DM severely impairs EPC colony-forming activity by limiting the differentiation of EPCs from primitive to definitive forms. Taking together, this study underscores significant disruptions in the cardio-spleen-bone marrow immune cell axis following MI in DM, revealing delayed innate and adaptive immune responses along with impaired EPC differentiation.

A High-Fidelity Computational Model for Predicting Blood Cell Trafficking and 3D Capillary Hemodynamics in Retinal Microvascular Networks.

To present a first principle-based, high-fidelity computational model for predicting full three-dimensional (3D) and time-resolved retinal microvascular hemodynamics taking into consideration the flow and deformation of individual blood cells. The computational model is a 3D fluid-structure interaction model based on combined finite volume/finite element/immersed-boundary methods. Three in silico microvascular networks are built from high-resolution in vivo motion contrast images of the superficial capillary plexus in the parafoveal region of the human retina. The maximum tissue area represented in the model is approximately 500 × 500 µm2, and vessel lumen diameters ranged from 5.5 to 25 µm covering capillaries, arterioles, and venules. Blood is modeled as a suspension of individual blood cells, namely, erythrocytes (RBC), leukocytes (WBC), and platelets in plasma. An accurate and detailed biophysical modeling of each blood cell and their flow-induced deformation is considered. A physiological, pulsatile boundary condition corresponding to an average cardiac cycle of 0.9 second is used. Detailed quantitative data and analysis of 3D retinal microvascular hemodynamics are presented, and their relationship to RBC flow dynamics is illustrated. Blood velocity is shown to have temporal oscillations superimposed on the background pulsatile variation, which arise because of the way RBCs partition at vascular junctions, causing repeated clogging and unclogging of vessels. Temporal variations in RBC velocity and hematocrit are anti-correlated in a given vessel, but their time-averaged distributions are positively correlated across the network. Whole blood velocity is 65% to 85% of RBC velocity, with the discrepancy related to the formation of an RBC-free region, adjacent to the vascular endothelium and typically 0.8 to 1.8 µm thick. The 3D velocity and RBC concentration profiles are shown to be oppositely skewed with respect to each other, because of the way that RBCs "hug" the apex of each bifurcation. RBC deformation is predicted to have biphasic behavior with respect to vessel diameter, with minimal cell length for vessels approximately 7 µm in diameter. The wall shear stress (WSS) exhibits a strongly 3D distribution with local regions of high value and gradient spanning a range of 10 to 80 dyn/cm2. WSS is highest where there is faster flow, greater curvature of the vessel wall, capillary bifurcations, and at locations of RBC crowding and associated thinning of the cell-free layer. This study highlights the usefulness of high-fidelity cell-resolved modeling to obtain accurate and detailed 3D, time-resolved retinal hemodynamic parameters that are not readily available through noninvasive imaging approaches. The results presented are expected to complement and enhance the interpretation of in vivo data, as well as open new avenues to study retinal hemodynamics in health and disease.

Elicitors Mediated Enhancement of Picrosides and Their Pathway Precursors in Dedifferentiated Cell Suspension Culture of Picrorhiza kurroa Royle ex Benth

Elicitors Mediated Enhancement of Picrosides and Their Pathway Precursors in Dedifferentiated Cell Suspension Culture of Picrorhiza kurroa Royle ex Benth

Cell Culture of a Swine Genotype 4 Hepatitis E Virus Strain.

HEV infection has become a global health concern. The study of HEV pathogenicity has been hindered by the lack of a suitable in vitro culture system. In the present research, we systematic demonstration of efficient replication of swine GT4 HEV in A549 cells, Huh-7 cells, and HepG2/C3A cell lines. The results of the immunofluorescence assay and immunofluorescence confocal assay showed that swine GT4 HEV is efficiently replicated in three cell lines at 72 h postinoculation. Meanwhile, we also detected the virus titer quantified were increased at 2-, 6,- and 11-days postinoculation. Moreover, we successfully observed HEV virus particles in the cell suspension at 6 days postinoculation. This finding holds significance for advancing in vitro HEV studies.

The codon optimised gene produces an active human basic fibroblastic growth factor in rice cell suspension culture

The coding sequence of human basic fibroblast growth factor (hbFGF) was optimised for expression in rice. An expression cassette was constructed by fusing the PCR-amplified RAmy3D promoter, along with its 5’UTR, 3’UTR, and terminator sequences, to the codon-optimised hbFGF sequence. This cassette was inserted into the pCAMBIA1304 shuttle vector, which also contained the RAmy3D signal peptide. Agrobacterium tumefaciens strain LBA 4404 was used to transform rice callus. Among the transformed lines, the callus expressing the highest level of bFGF (38.1 mg/kg fresh weight) was identified via ELISA and selected for establishing a cell suspension culture. Expression and secretion of the recombinant bFGF into the culture medium were observed three days after incubating the transgenic rice cells in sucrose-free medium. The presence of recombinant bFGF was confirmed through Western blot and SDS-PAGE analyses. Furthermore, the rice-derived bFGF effectively stimulated the proliferation of NIH/3T3 cells, demonstrating a comparable biological activity to that of commercial bFGF.

Production of phenolic compounds, flavonoids, and lupeol by Lycium schweinfurthii suspension cultures in shake flasks and a rocking-motion bioreactor

AbstractLycium schweinfurthii, a wild shrub of the Solanaceae family, has received increasing attention in the last decade for its therapeutic potential in traditional medicine due to its diverse array of secondary metabolites, including phenolic substances and terpenoids. The aim of this study was to investigate the accumulation of phenolics, flavonoids, and the terpenoid lupeol in L. schweinfurthii cell suspension shake flask cultures and a single-use 2-dimensional rocking motion bioreactor. Three different media formulations were compared for in vitro cell cultures. Various parameters, such as biomass accumulation, settled cell volume, cell viability (assessed via a 2,3,5-triphenyl tetrazolium chloride assay), and sucrose consumption were determined as indicators of cell activity and growth. Total phenolic and flavonoid contents were estimated spectrophotometrically, lupeol was quantified via High-Performance Thin Layer Chromatography (HPTLC). Although a higher fresh biomass concentration of 464 g L− 1 was obtained in MS medium supplemented with a combination of each, 1 mg L− 1 of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-Naphthaleneacetic acid (NAA), the rocking-motion bioreactor cultivation was performed with 2 mg L− 1 NAA due to its superior reproducibility in viability, productivity, and content of bioactive compounds (e.g., phenolics, flavonoids, lupeol). A final fresh biomass concentration of 185 g L− 1 was achieved in a 16 L cultivation scale with a notable increase in the concentration of phenolics (1.4-fold) and flavonoids (1.7-fold). Most importantly, the concentration of lupeol, a pentacyclic triterpenoid known for its anti-inflammatory, antibacterial, and anti-atherogenic properties, exhibited a remarkable 5.5-fold increase in the bioreactor cultivation (585 µg g− 1) compared to shake flask cultivations (106 µg g− 1). The current study demonstrated the profound impact of media composition and non-limited fed-batch conditions in a rocking-motion bioreactor on the accumulation of bioactive compounds. The findings are also relevant to other plant cell cultures.

“Comparative evaluation of cytotoxicity of three herbal endodontic irrigants at three intervals of time” – An in vitro study

Aim: The aim of the study was to evaluate and compare the cytotoxicity of 25% of neem leaf extract (Azadirachta indica), 20% of guava leaf extracts (Psidium guajava), and 20% of cinnamon extract (Cinnamomum zeylanicum) irrigants at three intervals of time. Methodology: Four groups were formed (n = 15), Group 1 (control group) – normal saline solution, Group 2 – 25% of neem extract, Group 3 – 20% of guava extract, and Group 4 – 20% of cinnamon extract. Each group was further divided into three subgroups based on intervals (n = 5). Subgroup A – at 10 min, Subgroup B – at 20 min, and Subgroup C – at 30 min. One hundred microliters of each irrigant was added to 2 mL of the diluted red blood cells suspension obtained from a human volunteer. Hemoglobin (Hb) estimation was done with an automated hematology analyzer after incubating the test samples at 10, 20, and 30 min intervals. Results: The reduction in the mean Hb values was not statistically significant in the normal saline, guava, and cinnamon groups. However, in the neem extract group, the mean Hb values reduced significantly at P < 0.001. Among the subgroups, Subgroup A (10 min) showed the least cytotoxicity. Conclusion: In the present study, 20% guava extract had the lowest cytotoxicity and cytotoxicity increased with time. Hence, 20% guava extract can be used as an alternative to conventional irrigants as it has been shown to have the least cytotoxicity.