Viable Cell Population Research Articles

Antifungal Properties of Polycephalomyces nipponicus (Ascomycetes) against Candida albicans: Potential for Novel Therapeutic Development

<i>Candida albicans</i> has the potential to turn pathogenic and cause mild to severe infections, particularly in people with weakened immune systems. Novel therapeutics are required due to its morphological alterations, biofilm development, and resistance to antifungal drugs. <i>Polycephalomyces nipponicus</i>, a traditional East Asian medicinal fungus, has shown potential as an antifungal agent. In this study, 15 <i>P. nipponicus</i> isolates were cultivated and their mycelial extracts were evaluated against <i>C. albicans</i> NCYC854 using agar well diffusion, broth microdilution, scanning electron microscope (SEM), and time-kill assays. Eight isolates exhibited significant antifungal activity, with inhibition zones from 11.25 ± 1.50 mm to 18 ± 0.82 mm, notably Cod-MK1209 surpassing amphotericin B. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) ranged from 125 to 500 μg/mL, with extracts from Cod-MK1206 and Cod-MK1209 showing the lowest MFC at 125 μg/mL. The results of time-kill experiments showed fungistatic effects by drastically lowering viable cell populations at 1× and 2× MIC concentrations within 24 h. The SEM analysis also indicated evidence of degradation to the cellular wall and membrane. These findings highlight the potential of <i>P. nipponicus</i> extracts as powerful antifungal medicines that target <i>C. albicans</i> selectively. Further research efforts have to focus on the identification and description of bioactive components, enhancement of extraction techniques, and advancement towards preclinical and clinical studies to validate their potential for therapeutic use.

DDDR-36. LOOKING AROUND THE CORNER: PREDICTING SYNERGY OR FUTILITY TO COMBINATION OF ARSENIC TRIOXIDE PLUS MNK1 INHIBITORS IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most prevalent type of malignant tumor within the central nervous system. The five-year survival rate of 6.8% highlights the challenges inherent to exceptionally heterogeneous tumors that grow behind the blood brain barrier. The protracted dearth in new agents approved for GBM begs innovative strategies by which to approach clinical trials. Arsenic trioxide (ATO) is the standard of care for myelodysplasia and relapsed or refractory acute promyelocytic leukemia. ATO has an idiopathic mechanism of action, but has shown beneficial effects in other solid tumors. Across six GBM PDX models a 10-fold difference in sensitivity manifests, indicative of underlying innate sensitivity or resistance to ATO. This resistance has been previously shown to correlate with MNK1 expression. In order to sensitize PDX models innately resistant to ATO we utilized combination therapy with the MNK1 inhibitor AUM001 (AUM Biosciences, Singapore; NCT05462236). In high MNK1 expressing PDX models the combination of ATO + AUM001 induces cytotoxic synergy. Additionally, when narrowly measuring effects on the glioma stem cell population (ELDA), strong synergy was observed in models, including those failing to show such using bulk cell population viability assay. GSEA analysis revealed differential high enrichment in gene sets related to oxidative phosphorylation in the models failing to show synergy. Such criteria could act as exclusionary criteria (futility) for clinical trials by excluding such putative non-responder patients from the treatment population. Synergistic models showed enrichment in gene sets related to epithelial/mesenchymal transition, skeletal and sensory organ development, and regionalization. These signatures could serve to prioritize patient inclusion in clinical trials of ATO in combination with AUM001, enriching the fraction of responsive glioma patients. Such molecular signatures may bring precision medicine in early-stage clinical trials to advance promising new agents and combinations of agents to full FDA approval.

Seasonal changes in the viability and abundance of bacterial cells in the snowpack ecosystem of a High Arctic ice cap

ABSTRACT Microbes play an essential role in nutrient turnover within Arctic environments, and their contribution to biogeochemical cycles can depend on several factors, including but not limited to cell viability. In this study, we employed the SYBR-PI dual cell stain to epifluorescence microscopy to enumerate proportions of potentially viable and non-viable bacterial cell populations within a melting snowpack on an ice cap, Foxfonna in Svalbard. Non-viable cells dominated on Foxfonna (2.5 ± 0.36 × 107 cells m−2) during the June to early July period, when biological production was usually at its peak. Furthermore, non-viable cells also dominated the total cell abundance within superimposed ice (223 ± 242 cells mL−1) and glacial ice (695 ± 717 cells mL−1) beneath the snow. We propose that the rapid, early loss of cell viability was caused by a number of abiotic and biotic factors. Hence, necromass (dead cell residue) contributed to the export of organic matter to downstream ecosystems.

Enumeration and gentle sorting of immune cells on chip, key to next generation advanced therapies in outpatient setting

BackgroundA thorough understanding of immune-oncology and molecular medicine has been vital in the development of cell therapeutics. At the basis of this translational research and its future implementation into a medicinal product, lies the availability of pure and viable cell populations. Currently, FACS and magnetic bead isolation are successfully used but suffer to fulfill all requirements. FACS is costly and difficult to upscale due to the limitation of shear stress, especially fragile, cells can handle. Therefore, magnetic bead isolation is often used as it is gentler, but it lacks the multiparametric aspect to isolate more complex cellular profiles. AimsWe aim to develop a versatile technology able of multi marker detection and isolation of complex cell types with high purity, viability and throughput. MethodsWe have developed a gentle sorting mechanism based on a jet flow created by micro vapor bubbles, enabling a closed microfluidic cell isolation platform capable of multiparametric sorting with high viability, purity and throughput. In this work we compared the purity, recovery and viability of sorted CD4+ CD14- cells to magnetic isolation, most often used for other cell manufacturing approaches. Futhermore, we cultured the sorted cells of both isolation strategies and compared their growth curve and expression of activation-induced IL2 and IFN-γ. ResultsWe demonstrate that this tool can achieve a pure population of CD4+ CD14- cells with high viability after sorting without compromising the recovery. On top of the viability also the growth and activation potential of sorted cells is unhampered by comparison to the benchmark gentle magnetic isolation. ConclusionsOur technology allows for the development of a compact system which sets it apart from other efforts intended to create automated cell therapeutic solutions.

Different Combinations of Nitrogen and Carbon Sources Influence the Growth and Postbiotic Metabolite Characteristics of Lactiplantibacillus plantarum Strains Isolated from Malaysian Foods.

Postbiotic metabolites produced by Lactiplantibacillus plantarum strains isolated from Malaysian food have been extensively reported for their positive effects on health. Understanding the effects of different combinations of carbon and nitrogen sources on the growth and corresponding characteristics of postbiotic metabolites produced by different strains of L. plantarum is important for various applications. Hence, the effects of different combinations of carbon (glucose, lactose, sucrose and dextrose) and nitrogen (X-SEED Kat, X-SEED Peptone, X-SEED Nucleo Advanced, Nucel875 MG, FM888 and FM902) sources on the growth of six strains of L. plantarum (RG11, RG14, RI11, RS5, TL1 and UL4) and the functional characteristics (bacteriocin inhibitory activity, antioxidant activity and lactic acid concentration) of their respective postbiotic metabolites were investigated in this study. UL4 produced the highest viable cell population with sucrose and Nucel875 nitrogen source. The UL4 strain also produced the strongest bacteriocin inhibitory activity with dextrose and FM888 nitrogen source. In comparison, the RI11 strain produced the highest lactic acid concentration with dextrose and Nucel875 nitrogen source and the highest reducing power of RS5 and TL1 postbiotic metabolites was achieved with MRS medium. In the combination of sucrose and X-Seed KAT nitrogen source, RG14 produce the highest hydroxyl radical scavenging activity. The effects of different combinations of carbon and nitrogen sources on the viable cell population of L. plantarum strains and the respective functional characteristics of postbiotic metabolites were strain dependent. The current study also revealed that fermentation media were an important factor that greatly impacted the functionalities of postbiotic metabolites due to the presence of various bioactive compounds that contributed to high antioxidant and antimicrobial properties. The results of this study will facilitate the subsequent medium design and optimisation for the development and production of specific postbiotic metabolites produced by the respective L. plantarum strain for their applications in various industries.

The molecular impact of sonoporation: A transcriptomic analysis of gene regulation profile

Sonoporation has long been known to disrupt intracellular signaling, yet the involved molecules and pathways have not been identified with clarity. In this study, we employed whole transcriptome shotgun sequencing (RNA-seq) to profile sonoporation-induced gene responses after membrane resealing has taken place. Sonoporation was achieved by microbubble-mediated ultrasound (MB-US) exposure in the form of 1 MHz ultrasound pulsing (0.50 MPa peak negative pressure, 10 % duty cycle, 30 s exposure period) in the presence of microbubbles (1:1 cell-to-bubble ratio). Using propidium iodide (PI) and calcein respectively as cell viability and cytoplasmic uptake labels, post-exposure flow cytometry was performed to identify three viable cell populations: 1) unsonoporated cells, 2) sonoporated cells with low uptake, and 3) sonoporated cells with high uptake. Fluorescence-activated cell sorting was then conducted to separate the different groups followed by RNA-seq analysis of the gene expressions in each group of cells. We found that sonoporated cells with low or high calcein uptake showed high similarity in the gene responses, including the activation of multiple heat shock protein (HSP) genes and immediate early response genes mediating apoptosis and transcriptional regulation. In contrast, unsonoporated cells exhibited a more extensive gene expression alteration that included the activation of more HSP genes and the upregulation of diverse apoptotic mediators. Four oxidative stress-related and three immune-related genes were also differentially expressed in unsonoporated cells. Our results provided new information for understanding the intracellular mobilization in response to sonoporation at the molecular level, including the identification of new molecules in the sonoporation-induced apoptosis regulatory network. Our data also shed light on the innovative therapeutic strategy which could potentially leverage the responses of viable unsonoporated cells as a synergistic effector in the microenvironment to favor tumor treatment.

Comparison of cation and anion-mediated resolution enhancement of bioprinted hydrogels for membranous tissue fabrication.

Fabrication of engineered thin membranous tissues (TMTs) presents a significant challenge to researchers, as these structures are small in scale, but present complex anatomies containing multiple stratified cell layers. While numerous methodologies exist to fabricate such tissues, many are limited by poor mechanical properties, need for post-fabrication, or lack of cytocompatibility. Extrusion bioprinting can address these issues, but lacks the resolution necessary to generate biomimetic, microscale TMT structures. Therefore, our goal was to develop a strategy that enhances bioprinting resolution below its traditional limit of 150 μm and delivers a viable cell population. We have generated a system to effectively shrink printed gels via electrostatic interactions between anionic and cationic polymers. Base hydrogels are composed of gelatin methacrylate type A (cationic), or B (anionic) treated with anionic alginate, and cationic poly-L-lysine, respectively. Through a complex coacervation-like mechanism, the charges attract, causing compaction of the base GelMA network, leading to reduced sample dimensions. In this work, we evaluate the role of both base hydrogel and shrinking polymer charge on effective print resolution and cell viability. The alginate anion-mediated system demonstrated the ability to reach bioprinting resolutions of 70 μm, while maintaining a viable cell population. To our knowledge, this is the first study that has produced such significant enhancement in extrusion bioprinting capabilities, while also remaining cytocompatible.

Isolation Procedure for Rat Pancreatic Ductal Cells.

Isolating pancreatic ductal cells from rats is a critical procedure in pancreatic research, offering valuable insights into pancreatic function, pathology, and potential treatments. The process involves several key steps, beginning with the proper removal of the rat's pancreas, followed by the initiation of the ductal cell isolation procedure. This aims to obtain pure and viable ductal cell populations for further experimentation and analysis.

Interactions Between Silver Nanoparticles and Culture Medium Biomolecules with Dose and Time Dependencies.

The interaction between silver nanoparticles (AgNPs) and molecules producing coronas plays a key role in cytotoxicity mechanisms. Once adsorbed coronas determine the destiny of nanomaterials in vivo, their effective deployment in the biomedical field requires a comprehensive understanding of the dynamic interactions of biomolecules with nanoparticles. In this work, we characterized 40nm AgNPs in three different nutritional cell media at different molar concentrations and incubation times to study the binding mechanism of molecules on surface nanoparticles. In addition, their cytotoxic effects have been studied in three cell lineages used as tissue regeneration models: FN1, HUV-EC-C, RAW 264.7. According to the data, when biomolecules from DMEM medium were in contact with AgNPs, agglomeration and precipitation occurred. However, FBS medium proteins indicated the formation of coronas over the nanoparticles. Nonetheless, little adsorption of molecules around the nanoparticles was observed when compared to DMEM supplemented with 10% FBS. These findings indicate that when nanoparticles and bioproteins from supplemented media interact, inorganic salts from DMEM contribute to produce large bio-coronas, the size of which varies with the concentration and time. The static quenching mechanism was shown to be responsible for the fluorescence quenching of the bioprotein aggregates on the AgNPs surface. The calculated bioprotein-nanoparticle surface binding constants were on the order of 105M-1 at 37°C, with hydrophobic interactions driven by enthalpy and entropy playing a role, as confirmed by thermodynamic analysis. Cytotoxicity data showed a systematic degrowth in the viable cell population as the number of nanoparticles increased and the diameter of coronas decreased. Cytotoxic intervals associated with half decrease of cell population were established for AgNPs molar concentration of 75µM for 24h and 50µM for 48h. In summary, through the cytotoxicity mechanism of bio-coronas we are able to manipulate cells' expansion rates to promote specific processes, such inflammatory mechanisms, at different time instants.

Investigating cell death responses associated with histotripsy ablation of canine osteosarcoma

Background Osteosarcoma (OS) is the most frequently occurring primary bone tumor in dogs and people and innovative treatment options are profoundly needed. Histotripsy is an emerging tumor ablation modality, and it is essential for the clinical translation of histotripsy to gain knowledge about the outcome of nonablated tumor cells that could remain postablation. The objective of this study was to characterize the cell death genetic signature and proliferation response of canine OS cells post a near complete histotripsy ablation (96% ± 1.5) and to evaluate genetic cell death signatures associated with histotripsy ablation and OS in vivo. Methods In the current study, we ablated three canine OS cell lines with a histotripsy dose that resulted in near complete ablation to allow for a viable tumor cell population for downstream analyses. To assess the in vivo cell death genetic signature, we characterized cell death genetic signature in histotripsy-ablated canine OS tumors collected 24-h postablation. Results Differential gene expression changes observed in the 4% viable D17 and D418 cells, and histotripsy-ablated OS tumor samples, but not in Abrams cells, were associated with immunogenic cell death (ICD). The 4% viable OS cells demonstrated significantly reduced proliferation, compared to control OS cells, in vitro. Conclusion Histotripsy ablation of OS cell lines leads to direct and potentially indirect cell death as evident by, reduced proliferation in remaining viable OS cells and cell death genetic signatures suggestive of ICD both in vitro and in vivo.

Comparison of Antibacterial Activity of Phytochemicals against Common Foodborne Pathogens and Potential for Selection of Resistance.

Antimicrobial resistance is now commonly observed in bacterial isolates from multiple settings, compromising the efficacy of current antimicrobial agents. Therefore, there is an urgent requirement for efficacious novel antimicrobials to be used as therapeutics, prophylactically or as preservatives. One promising source of novel antimicrobial chemicals is phytochemicals, which are secondary metabolites produced by plants for numerous purposes, including antimicrobial defence. In this report, we compare the bioactivity of a range of phytochemical compounds, testing their ability to directly inhibit growth or to potentiate other antimicrobials against Salmonella enterica Typhimurium, Pseudomonas aeruginosa, Listeria monocytogenes, and Staphylococcus aureus. We found that nine compounds displayed consistent bioactivity either as direct antimicrobials or as potentiators. Thymol at 0.5 mg/mL showed the greatest antimicrobial effect and significantly reduced the growth of all species, reducing viable cell populations by 66.8%, 43.2%, 29.5%, and 70.2% against S. enterica Typhimurium, S. aureus, P. aeruginosa, and L. monocytogenes, respectively. Selection of mutants with decreased susceptibility to thymol was possible for three of the pathogens, at a calculated rate of 3.77 × 10-8, and characterisation of S. enterica Typhimurium mutants showed a low-level MDR phenotype due to over-expression of the major efflux system AcrAB-TolC. These data show that phytochemicals can have strong antimicrobial activity, but emergence of resistance should be evaluated in any further development.

Involvement of oxidative stress and pro-inflammatory cytokines in copper sulfate-induced depression-like disorders and abnormal neuronal morphology in mice.

Epidemiological studies have implicated copper as one of the key environmental risk factors for the pathogenesis of depression. However, the precise mechanism by which copper contribute to the genesis of depression particularly the involvement of oxidative stress-driven neuroinflammation is yet to be fully investigated. Thus, this study was designed to evaluate the effects of copper sulfate (CuSO4) on depression-like behaviors and the role of oxidative stress and pro-inflammatory cytokines in mice. Forty male Swiss mice were distributed into control and three test groups (n = 10), and were treated orally with distilled water (10mL/kg) or CuSO4(25, 50 and 100mg/kg) daily for 28days. Afterwards, the tail suspension, forced swim, and sucrose splash tests were used for the detection of depression-like effects. The animals were then euthanized and the brains were processed for the estimation of biomarkers of oxidative stress and pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6). The histomorphological features and neuronal viability of the prefrontal cortex, hippocampus and striatum were also determined. Mice exposed to CuSO4 displayed depression-like features when compared with controls. The brain concentrations of malondialdehyde, nitrite and pro-inflammatory cytokines were elevated in CuSO4-treated mice. Mice exposed to CuSO4 also had reduced brain antioxidant status (glutathione, glutathione-s-transferase, total thiols, superoxide-dismutase and catalase), as well as altered histomorphological features, and decreased population of viable neuronal cells. These findings suggest that CuSO4 increases oxidative stress and pro-inflammatory cytokines to elicit depression-like effects in mice.

Serum- and xeno-free culture of human umbilical cord perivascular cells for pediatric heart valve tissue engineering

BackgroundConstructs currently used to repair or replace congenitally diseased pediatric heart valves lack a viable cell population capable of functional adaptation in situ, necessitating repeated surgical intervention. Heart valve tissue engineering (HVTE) can address these limitations by producing functional living tissue in vitro that holds the potential for somatic growth and remodelling upon implantation. However, clinical translation of HVTE strategies requires an appropriate source of autologous cells that can be non-invasively harvested from mesenchymal stem cell (MSC)-rich tissues and cultured under serum- and xeno-free conditions. To this end, we evaluated human umbilical cord perivascular cells (hUCPVCs) as a promising cell source for in vitro production of engineered heart valve tissue.MethodsThe proliferative, clonogenic, multilineage differentiation, and extracellular matrix (ECM) synthesis capacities of hUCPVCs were evaluated in a commercial serum- and xeno-free culture medium (StemMACS™) on tissue culture polystyrene and benchmarked to adult bone marrow-derived MSCs (BMMSCs). Additionally, the ECM synthesis potential of hUCPVCs was evaluated when cultured on polycarbonate polyurethane anisotropic electrospun scaffolds, a representative biomaterial for in vitro HVTE.ResultshUCPVCs had greater proliferative and clonogenic potential than BMMSCs in StemMACS™ (p < 0.05), without differentiation to osteogenic and adipogenic phenotypes associated with valve pathology. Furthermore, hUCPVCs cultured with StemMACS™ on tissue culture plastic for 14 days synthesized significantly more total collagen, elastin, and sulphated glycosaminoglycans (p < 0.05), the ECM constituents of the native valve, than BMMSCs. Finally, hUCPVCs retained their ECM synthesizing capacity after 14 and 21 days in culture on anisotropic electrospun scaffolds.ConclusionOverall, our findings establish an in vitro culture platform that uses hUCPVCs as a readily-available and non-invasively sourced autologous cell population and a commercial serum- and xeno-free culture medium to increase the translational potential of future pediatric HVTE strategies.Graphical This study evaluated the proliferative, differentiation and extracellular matrix (ECM) synthesis capacities of human umbilical cord perivascular cells (hUCPVCs) when cultured in serum- and xeno-free media (SFM) against conventionally used bone marrow-derived MSCs (BMMSCs) and serum-containing media (SCM). Our findings support the use of hUCPVCs and SFM for in vitro heart valve tissue engineering (HVTE) of autologous pediatric valve tissue. Figure created with BioRender.com.

Evaluation of a Viable-Cell Detection Assay for Xanthomonas fragariae with Latent Class Analysis

Most molecular diagnostic assays are designed to detect the simple presence of a protein or nucleic acid without regard to whether that protein or nucleic acid originated from a viable and presumably pathogenic organism at the time of isolation. We recently developed a viable-cell detection assay (propidium monoazide [PMA]-qPCR) for specific detection of viable (living) cells of Xanthomonas fragariae, the pathogen causing angular leaf spot of strawberry, and herein describe a unique set of statistical analyses for validation of this assay. For any detection assay or test, calculation of its sensitivity and specificity is essential for determining its diagnostic capabilities, particularly when evaluating competing assays or tests. This is often achieved by running competing tests concurrently on a set of samples with known pathogen density or disease status and cross-tabulating results. However, the PMA-qPCR assay is unique among PCR-based diagnostics because it is designed specifically to detect DNA from living cells only, whereas most traditional PCR assays are designed to detect DNA from the target organism regardless of its state of viability. Thus, one cannot directly compare the results from a cell-viability assay with those from a general assay to gauge the performance of either assay because the assays target two different but overlapping populations (i.e., the viable-cell population and the viable- plus nonviable-cell population). To address this challenge, two standard statistical approaches to diagnostic test evaluation were used jointly to estimate the test performance of the PMA-qPCR assay relative to two common assays for detection of X. fragariae. In both analyses, the PMA-qPCR outperformed the qPCR for detection of viable cells under a range of conditions. Viability testing is extremely useful in certification and disease management applications, and with the information on test performance generated here, the test can be put to practical use to design sampling strategies to account for the errors in testing. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Long term culturing of CHO cells: phenotypic drift and quality attributes of the expressed monoclonal antibody

Establishing cell lines with enhanced protein production requires a deep understanding of the cellular dynamics and cell line stability. The aim of the study is to investigate the impact of long term culturing (LTC) on cell morphology and altered cellular functions possibly leading to phenotypic drift, impacting product yield and quality. Study highlights the orthogonal cellular and analytical assay toolbox to define cell line stability for optimal culture performance and product quality. We investigated recombinant monoclonal antibody (mAb) expressing CHO cells for 60 passages or 180 generations and assessed the cell growth characteristics and morphology by confocal and scanning electron microscopy. Quality attributes of expressed mAb is accessed by performing charge variants, glycan, and host cell protein analysis. We observed a 1.65-fold increase in viable cell population and 1.3-fold increase in cell specific growth rate. A 2.5-fold decrease in antibody titer and abatement of actin filament indicate cellular phenotypic drift. Mitochondrial membrane potential (∆ΨM) signified cell health and metabolic activity during LTC. Host cell protein production is reduced by 1.8-fold. Charge heterogeneity was perturbed with 12.5% and 43% reduction in abundance of acidic and basic charge variants respectively. Glycan profile indicated a decline in fucosylation with 17% increase in galactosylated species as compared with early passaged cells. LTC impinges on cellular phenotype as well as the quality of the expressed antibody, suggesting a defined subculturing limit to retain stable protein expression and cell morphology to achieve consistent product quality. Study signifies the changes in cellular and metabolic markers, suggesting cellular and analytical toolbox which could play a significant role in defining cell characteristics and ensured product quality.

Photodynamic Therapy of Aluminum Phthalocyanine Tetra Sodium 2-Mercaptoacetate Linked to PEGylated Copper-Gold Bimetallic Nanoparticles on Colon Cancer Cells.

This work reports for the first time on the synthesis, characterization, and photodynamic therapy efficacy of the novel aluminium (III) chloride 2(3), 9(10), 16(17), 23(24)-tetrakis-(sodium 2-mercaptoacetate) phthalocyanine (AlClPcTS41) when alone and when conjugated to PEGylated copper-gold bimetallic nanoparticles (PEG-CuAuNPs) as photosensitizers on colon cancer cells (Caco-2). The novel AlClPcTS41 was covalently linked to the PEG-CuAuNPs via an amide bond to form AlClPcTS41-PEG-CuAuNPs. The amide bond was successfully confirmed using FTIR while the crystal structures were studied using XRD. The morphological and size variations of the PEG-CuAuNPs and AlClPcTS41-PEG-CuAuNPs were studied using TEM, while the hydrodynamic sizes and polydispersity of the particles were confirmed using DLS. The ground state electron absorption spectra were also studied and confirmed the typical absorption of metallated phthalocyanines and their nanoparticle conjugates. Subsequently, the subcellular uptake, cellular proliferation, and PDT anti-tumor effect of AlClPcTS41, PEG-CuAuNPs, and AlClPcTS41-PEG-CuAuNPs were investigated within in vitro Caco-2 cells. The designed AlClPcTS41 and AlClPcTS41-PEG-CuAuNPs demonstrated significant ROS generation abilities that led to the PDT effect with a significantly decreased viable cell population after PDT treatment. These results demonstrate that the novel AlClPcTS41 and AlClPcTS41-PEG-CuAuNPs had remarkable PDT effects against Caco-2 cells and may trigger apoptosis cell death pathway, indicating the potential of the AlClPcTS41 and AlClPcTS41-PEG-CuAuNPs in enhancing the cytotoxic effect of PDT treatment.

Investigation of the synergistic effect of glycolipid biosurfactant produced by Shewanella algae with some antibiotics against planktonic and biofilm forms of MRSA and antibiotic resistant Acinetobacter baumannii.

To tackling antibiotic resistance and the appearance of multidrug-resistant (MDR) strains, one current approach is the combined use of biosurfactants with antibiotics to increase their efficacy. The antimicrobial ability of biosurfactant produced by Shewanella algae strain B12 was examined using the agar well diffusion method versus some resistant Gram-negative and Gram-positive bacteria. The Minimum Inhibitory Concentration (MIC) of Glycolipid-Biosurfactant of B12 (GBB12) was performed by the broth dilution technique. The inhibition of biofilm formation, disruption of biofilm, and reducing the population of viable cells in biofilm were evaluated by the microtiter plate method. Finally, Scanning Electron Microscopy (SEM) analysis was used to confirm the disruption of the cell membrane by GBB12. In all experiments, when GBB12 was added to antibiotics (except Amikacin), the antimicrobial activity was increased. The synergistic effects of GBB12 and antibiotics (Ciprofloxacin and Gentamycin) against Methicillin-Resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii were confirmed by the Fractional Inhibitory Concentration Index (FICI). GBB12-Gentamycin mixture almost completely inhibits the formation of A. baumannii biofilm, reaching 99.8% inhibition. Also, the rate of MRSA biofilm inhibition treated with GBB12-Ciprofloxacin mixture was found to be 99.4%. biosurfactant-antibiotic mixture could be adequate replacements for traditional antibiotics in the near future. This study shows the potential of GBB12 as antimicrobial and antibiofilm agent.

Role of oxidative stress in modulating CHO cell culture performance: Impact on titer and quality attributes of a monoclonal antibody therapeutic

AbstractBACKGROUNDThe enormous success of antibody therapeutics has resulted in attempts to enhance protein expression which requisites high energy demand which leads to oxidative stress as characterized by significant levels of intracellular reactive oxygen species (ROS), disturbing many cellular functions. This study delineates the mechanistic role of oxidants and ROS scavengers in relation to cell viability, apoptosis and mitochondrial membrane potential (MMP), as well as critical quality attributes such as charge heterogeneity.RESULTSAddition of ROS scavengers such as glutathione, N‐acetylcysteine (NAC) and ascorbic acid to culture result in decreased levels of ROS (52%), increased MMP (47%) and viable cell populations (42%), as well as reduced cell apoptosis (69%) and a decrease in lysosome activity (38%). These effects rescue cells from damage caused by oxidative stress and DNA fragmentation during the production phase. Cells use the peroxiredoxin reductive pathway for regulation of ROS levels in response to oxidative stress environments. Enzymes involved in ROS reduction were found to be significantly high in antioxidant‐treated cells. Treatment with oxidants resulted in decreased titer (47%) and an increase in acidic species (18%), whereas addition of antioxidants resulted in enhancement of titer (38%) as well as abundance in basic variants (24%) of recombinant IgG1.CONCLUSIONAntioxidants improve cell culture performance by alleviating the viable cell pool through activating the intracellular reductive mechanism for maintaining high ∆ᴪm, thus reducing apoptosis and enhancing antibody production. Media supplementation with a combination of antioxidants during the production phase is likely to enhance antibody titer and quality. © 2022 Society of Chemical Industry (SCI).

Testis-on-chip platform to study ex vivo primate spermatogenesis and endocrine dynamics

Here, we report a testis-on-chip platform for the ex vivo culture of seminiferous tubules isolated from human and non-human primate testis. Tissues are cultured in a dedicated chamber with continuous perfusion via a vascular-like channel. The platform is fabricated from PDMS using a 3D printed mold, after design has been optimized, e.g., for the barrier between the culture chamber and the perfusion channel. COMSOL modeling revealed no direct negative impact of the flow on the tissues for the applied flowrate in the device, shear rate remaining in the physiological range. Culture experiments were performed using adult human seminiferous tubules from gender dysphoria patients and prepubertal seminiferous tubules from a 6-month-old marmoset for up to 11 and 9 days, respectively. First, microscopic, and live imaging revealed the presence of viable cell populations in both types of samples. Next, marmoset tissues were exposed to stimulatory conditions through perfusion of gonadotropins at different doses. The tissue response was characterized by histological analysis after their recovery from the device and testosterone and estradiol analysis in the effluent. Histological observations suggested improved maintenance of marmoset testicular tissues under stimulatory conditions, which similarly resulted in an increase in both testosterone and estradiol production, with yet different patterns for the low-dose and high-dose stimulation. The herein reported testis-on-chip platform shows great promise to evaluate endocrine and toxic effects on the testis.

Expression Profiles of Immune-Related Genes and Apoptosis Study of Avian Intraepithelial-Natural Killer Cells in Chickens Inoculated with Vaccine Strain of Newcastle Disease Virus (NDV) and Challenged with Virulent NDV.

In spite of the available information on the role of natural killer (NK) cells in several viral infections, the interactions between chicken intraepithelial-NK (IEL-NK) cells and Newcastle disease virus (NDV) are poorly understood. In this study, we investigated these interactions following the inoculation of chickens with NDV vaccine strain LaSota and subsequent challenge with velogenic NDV (vNDV) genotype VII (GVII) and VIII (GVIII), through quantification of IEL-NK cell's apoptosis and expression profiling of its surface receptors. Specific-pathogen-free chickens were randomly divided into six groups, as follows: one group of an uninfected control, one group infected with NDV LaSota, two groups each infected with either GVII or GVIII, and two groups inoculated with NDV LaSota and challenged with either GVII (LaSota-genotype VII [LSGVII]) or GVIII (LaSota-genotype VIII [LSGVIII]). Avian intraepithelial lymphocytes (IEL) were isolated from the duodenal loops, and CD3- cells were characterized. Immunophenotyping and apoptosis analysis of CD3-/CD25+/CD45+IEL NK cells were conducted using a flow cytometer. In addition, a gene expression study was conducted using real-time quantitative PCR. Data were analyzed using two-way analysis of variance. The results showed that vNDV GVII or GVIII caused apoptosis of IEL-NK cells; however, following inoculation of LSGVII or LSGVIII, the effect of vNDV GVII and GVIII to cause a reduction in the population of viable IEL-NK cells was significantly reduced. Furthermore, the expression profiles of activating receptors CD69, NK-lysin, and IFN-γ, were generally upregulated in chickens inoculated with LSGVII or LSGVIII. In contrast, B-NK, an inhibitory receptor, was downregulated in these treatment groups. In NDV GVII- and GVIII-challenged groups, however, B-NK was upregulated, whereas the other receptors were generally downregulated. The findings of this study showed that NDV vaccine strain LaSota may prevent apoptosis and cause upregulation of activating receptors of chicken IEL-NK cells in velogenic virus-challenged settings.