Maintenance Of Cell Viability Research Articles

Biodegradation of atrazine with biochar-mediated functional bacterial biofilm: Construction, characterization and mechanisms

The abuse and residue of herbicides in the black soil area had seriously affected the soil structure, function and crop growth, posing severe threats to agricultural soil environment and public health. Given the limitation of routine microbial remediation, innovative and eco-friendly functional bacterial biofilm which could adapt under adverse conditions was developed on the biochar to investigate its enhanced bioremediation and metabolic characteristics of typical herbicide atrazine. Results revealed that the atrazine degrading strain Acinetobacter lwoffii had competitive advantage in soil indigenous microorganisms and formed dense biofilms on the biochar which was beneficial to cell viability maintenance and aggregations. Metatranscriptomics and RT-qPCR analysis demonstrated that the biochar-mediated biofilm improved the frequency of intercellular communications through quorum sensing and two-component signal regulation systems, and enhanced the atrazine biodegradation efficiency through horizontal gene transfer in co-metabolism mode, providing important scientific basis for the biological remediation of farmland soil non-point source pollution.

Oxidative Stress Induced by Lipotoxicity and Renal Hypoxia in Diabetic Kidney Disease and Possible Therapeutic Interventions: Targeting the Lipid Metabolism and Hypoxia.

Oxidative stress, a hallmark pathophysiological feature in diabetic kidney disease (DKD), arises from the intricate interplay between pro-oxidants and anti-oxidants. While hyperglycemia has been well established as a key contributor, lipotoxicity emerges as a significant instigator of oxidative stress. Lipotoxicity encompasses the accumulation of lipid intermediates, culminating in cellular dysfunction and cell death. However, the mechanisms underlying lipotoxic kidney injury in DKD still require further investigation. The key role of cell metabolism in the maintenance of cell viability and integrity in the kidney is of paramount importance to maintain proper renal function. Recently, dysfunction in energy metabolism, resulting from an imbalance in oxygen levels in the diabetic condition, may be the primary pathophysiologic pathway driving DKD. Therefore, we aim to shed light on the pivotal role of oxidative stress related to lipotoxicity and renal hypoxia in the initiation and progression of DKD. Multifaceted mechanisms underlying lipotoxicity, including oxidative stress with mitochondrial dysfunction, endoplasmic reticulum stress activated by the unfolded protein response pathway, pro-inflammation, and impaired autophagy, are delineated here. Also, we explore potential therapeutic interventions for DKD, targeting lipotoxicity- and hypoxia-induced oxidative stress. These interventions focus on ameliorating the molecular pathways of lipid accumulation within the kidney and enhancing renal metabolism in the face of lipid overload or ameliorating subsequent oxidative stress. This review highlights the significance of lipotoxicity, renal hypoxia-induced oxidative stress, and its potential for therapeutic intervention in DKD.

Abstract A053: The dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 induces functional differentiation and splicing defects in preclinical models of acute myeloid leukemia (AML)

Abstract The BRG/BRM associated factor (BAF) complex is a chromatin remodeler critical for maintenance of cell viability in many cancers. This includes acute myeloid leukemia (AML), where BAF maintains the stem-like transcriptional state of blast cells. FHD-286 is a dual inhibitor of the BAF ATPase subunits BRG1 and BRM and is currently being investigated in relapsed or refractory AML and myelodysplastic syndrome. We have shown previously that FHD-286 induces the expression of the myeloid differentiation marker CD11b in preclinical models after treatment with sub-cytoreductive doses at long timepoints. Here we further characterized the differentiation phenotype induced by low-dose FHD-286 in AML cell lines through RNA-seq and a genome-wide CRISPR-Cas9 knockout screen. These datasets and mechanistic follow-up studies suggested AML cell lines treated with FHD-286 can functionally differentiate to gain the ability to produce superoxide anion and perform phagocytosis. We also found that FHD-286 treatment disrupts mRNA splicing and that this is a likely contributor to AML cell growth defects induced by BRG1/BRM inhibition. This study suggests multiple mechanisms by which FHD-286 is able to disrupt the stem-like transcriptional state of AML blasts to cause differentiation and ultimately cell death. Citation Format: Ashley K. Gartin, David N. Mayhew, David L. Lahr, GiNell Elliott, Heena Gandevia, Molly M. Wilson, Richard Centore, Mike Collins, Astrid Thomsen, Jessica Piel, Gabriel J. Sandoval, Martin Hentemann. The dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 induces functional differentiation and splicing defects in preclinical models of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A053.

A scalable human islet 3D-culture platform maintains cell mass and function long-term for transplantation

Present-day islet culture methods provide short-term maintenance of cell viability and function, limiting access to islet transplantation. Attempts to lengthen culture intervals remain unsuccessful. A new method was developed to permit the long-term culture of islets. Human islets were embedded in polysaccharide 3D-hydrogel in cell culture inserts or gas-permeable chambers with serum-free CMRL 1066 supplemented media for up to 8 weeks. The long-term cultured islets maintained better morphology, cell mass, and viability at 4 weeks than islets in conventional suspension culture. In fact, islets cultured in the 3D-hydrogel retained β cell mass and function on par with freshly isolated islets in vitro and, when transplanted into diabetic mice, restored glucose balance similar to fresh islets. Using gas-permeable chambers, the 3D-hydrogel culture method was scaled up over 10-fold and maintained islet viability and function, although the cell mass recovery rate was 50%. Additional optimization of scale-up methods continues. If successful, this technology could afford flexibility and expand access to islet transplantation, especially single-donor islet-after-kidney transplantation.

An innovative multi-enzymatic system for gluconic acid production from starch using Aspergillus niger whole-cells

The use of multi-enzymatic systems for the industrial production of chemical compounds is currently considered an important green tool in synthetic organic chemistry. Gluconic acid is a multi-functional organic acid widely used in the chemical, pharmaceutical, food, textile, and construction industries. Its industrial production from glucose by fermentation using Aspergillus niger has drawbacks including high costs related to cell growth and maintenance of cell viability. This study presents an innovative one-step multi-enzymatic system for gluconic acid production from starch using Aspergillus niger whole-cells in association with amylolytic enzymes. Using soluble starch as substrate, the following results were achieved for 96 h of reaction: 134.5 ± 4.3 g/L gluconic acid concentration, 98.2 ± 1.3 % gluconic acid yield, and 44.8 ± 1.4 gGA/gwhole-cells biocatalyst yield. Although the process has been developed using starch as raw material, the approach is feasible for any substrate or residue that can be hydrolyzed to glucose.

Comparative Evaluation of Maintenance of Cell Viability of an Experimental Transport Media "Ringer's Lactate" with Dextrose Normal Saline ORS Egg White and Infant Milk Formula for Transportation of an Avulsed Tooth.

The viability of the periodontal ligament (PDL) cells on the root surface of the avulsed tooth determines the prognosis of the replanted tooth, which in turn is determined by a suitable transport medium in which the tooth was stored. The aim of the present study is to evaluate and compare the effectiveness of Ringer's lactate (RL) as a storage medium for an avulsed tooth in maintaining the PDL cell viability with dextrose normal saline (DNS), oral rehydration salt (ORS), egg white (EW), and infant milk formula (IMF). A total of 85 freshly extracted human teeth were divided into five experimental groups and two control groups. The positive and negative controls corresponded to 0-minute and 8-hour dry time, respectively. The experimental teeth were stored dry for 30 minutes and then immersed in one of five experimental media (RL, DNS, ORS, EW, and IMF) for 45 minutes. The teeth were then treated with collagenase type III and trypsin for 10 minutes. The number of viable PDL cells was counted with a hemocytometer and analyzed. Statistical analysis showed that IMF, RL, and EW had no statistically significant differences among them in maintaining the viability of the PDL cells but were significantly better than DNS. No statistically significant difference between RL, EW, and ORS in the number of viable PDL cells. Infant milk formula (IMF), RL, and EW showed similar results within the parameters of the study; they can be used as alternative storage media for avulsed teeth. DNS showed poor results, and ORS could serve as short-term storage media if the other solutions are not readily available. The search for an appropriate storage media with favorable pH and osmolality along with easy availability is the basic thought behind this study. Kumar P, Kotumachagi SS, Fabi AJ, et al. Comparative Evaluation of Maintenance of Cell Viability of an Experimental Transport Media "Ringer's Lactate" with Dextrose Normal Saline ORS Egg White and Infant Milk Formula for Transportation of an Avulsed Tooth. Int J Clin Pediatr Dent 2023;16(3):453-458.

Alginate-Based 3D A549 Cell Culture Model to Study Paracoccidioides Infection

A three-dimensional (3D) lung aggregate model based on sodium alginate scaffolds was developed to study the interactions between Paracoccidioides brasiliensis (Pb) and lung epithelial cells. The suitability of the 3D aggregate as an infection model was examined using cell viability (cytotoxicity), metabolic activity, and proliferation assays. Several studies exemplify the similarity between 3D cell cultures and living organisms, which can generate complementary data due to the greater complexity observed in these designed models, compared to 2D cell cultures. A 3D cell culture system of human A549 lung cell line plus sodium alginate was used to create the scaffolds that were infected with Pb18. Our results showed low cytotoxicity, evidence of increased cell density (indicative of cell proliferation), and the maintenance of cell viability for seven days. The confocal analysis revealed viable yeast within the 3D scaffold, as demonstrated in the solid BHI Agar medium cultivation. Moreover, when ECM proteins were added to the alginate scaffolds, the number of retrieved fungi was significantly higher. Our results highlight that this 3D model may be promising for in vitro studies of host–pathogen interactions.

The ecl family gene ecl3+ is induced by phosphate starvation and contributes to sexual differentiation in fission yeast.

In Schizosaccharomyces pombe, ecl family genes are induced by several signals, such as starvation of various nutrients, including sulfur, amino acids and Mg2+, and environmental stress, including heat or oxidative stress. These genes mediate appropriate cellular responses and contribute to the maintenance of cell viability and induction of sexual differentiation. Although this yeast has three ecl family genes with overlapping functions, any environmental conditions that induce ecl3+ remain unidentified. We demonstrate that ecl3+ is induced by phosphate starvation, similar to its chromosomally neighboring genes, pho1+ and pho84+, which respectively encode an extracellular acid phosphatase and an inorganic phosphate transporter. ecl3+ expression was induced by the transcription factor Pho7 and affected by the cyclin-dependent kinase (CDK)-activating kinase Csk1. Phosphate starvation induced G1 arrest and sexual differentiation via ecl family genes. Biochemical analyses suggested that this G1 arrest was mediated by the stabilization of the CDK inhibitor Rum1, which was dependent on ecl family genes. This study shows that ecl family genes are required for appropriate responses to phosphate starvation and provides novel insights into the diversity and similarity of starvation responses.

Protective Effect of Dolichos biflorus Seed Extract on 3T3-L1 Preadipocyte Differentiation and High-Fat Diet-Induced Obesity in Rats

Obesity is known to be one of the severe health issues worldwide, as its prevalence continues to rise as well as its association with other chronic diseases worsens. Even though various approaches have been underway to prevent or treat obesity, alternative approaches are in need to combat this chronic condition because of the unsatisfactory effectiveness and adverse side effects of the existing approaches. Dolichos biflorus L. seeds have been employed as a weight-loss treatment in folk medicine. Considering the necessity to develop a safe alternative remedy to rising obesity, the current investigation has been set up to assess the antiobesity potential and the mode of action of the aqueous seed extract of D. biflorus (ASEDB) in a cell line (3T3-L1) and high-fat diet (HFD)-induced rats. For in-vitro studies, 3T3-L1 cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) augmented with adipogenic-inducing medium and the influence of the extract (10 µg/mL–500 µg/mL) on 3T3-L1 adipocyte viability, adipogenesis, and lipolysis was assessed. An in-vitro study revealed maintenance of cell viability, reduced triglycerides (TG) accumulation, and promoted lipolysis in 3T3-L1 cells by ASEDB. Following in-vitro analysis, the HFD-induced obese rats were treated with ASEDB at different concentrations (100 mg/kg, 200 mg/kg, and 300 mg/kg) for 60 days and the effect was evaluated through various anthropometric and biochemical parameters. The findings revealed a significant decrement in total body weight, organ weights, fat pad weights, and restoration of abnormal levels of glucose, leptin, insulin, lipid markers, and antioxidant system to normal by ASEDB treatment. Also, pancreatic lipase inhibition analysis of ASEDB revealed a modest level of inhibition with an IC50 value of 213.3 µg/mL. All these findings exposed that ASEDB possesses pronounced antiobesity potential and exhibits its protective effect by suppressing food intake, reducing fat digestion and absorption, limiting adipogenesis, enhancing lipolysis, and alleviating oxidative stress.

Downregulation of LRP/LR with siRNA inhibits several cancer hallmarks in lung cancer cells.

The incidence and mortality rates of cancer are growing rapidly worldwide, with lung cancer being the most commonly occurring cancer in males. Human carcinomas circumvent the inhibitory pathways induced by DNA damage and senescence through the upregulation of telomerase activity. The 37 kDa/67 kDa laminin receptor (LRP/LR) is a cell surface receptor which plays a role in several cancer hallmarks, including metastasis, angiogenesis, cell viability maintenance, apoptotic evasion, and mediating telomerase activity. We have previously shown that the knockdown of LRP/LR with an LRP-specific siRNA significantly impedes adhesion and invasion, induces apoptosis, and inhibits telomerase activity in various cancer cell lines invitro. Here, we investigated the effect of downregulating LRP/LR with LRP-specific siRNA in A549 lung cancer cells. Downregulation of LRP/LR resulted in a significant decrease in cell viability, migration potential, and telomerase activity, as well as a significant increase in apoptosis. Proteomic analysis further suggested the re-establishment of immune control over the lung cancer cells, a previously unidentified facet of LRP downregulation in cancer. Altogether, we suggest that targeting LRP/LR for downregulation may have therapeutic potential for inhibiting several cancer hallmarks.

Developing Stable Freeze-Dried Functional Ingredients Containing Wild-Type Presumptive Probiotic Strains for Food Systems

Designing stable dried functional food ingredients and foods containing live probiotic cultures maintaining high viable cell loads at the time of consumption is a challenge for the industry. The aim of the present study was the development of stable freeze-dried functional food ingredients with enhanced shelf-life during long storage. Zea flakes, pistachios, and raisins were used as immobilization supports for the wild-type presumptive probiotic strains Pediococcus acidilactici SK and Lactiplantibacillus plantarum F4, while L. plantarum B282 was used as a reference strain. Cell survival was monitored during storage at room and refrigerated temperatures for up to 6 months. Levels of freeze-dried cultures were maintained up to 7.2 logcfu/g after 6 months storage at room temperature and up to 8.5 logcfu/g at refrigerator temperature, in contrast to free cell levels that ranged <7 logcfu/mL, suggesting the positive effects of immobilization and freeze-drying on cell viability. Of note, levels of freeze-dried immobilized P. acidilactici SK cells on zea flakes and pistachios remained stable after 6 months of storage at 4 °C, ranging 8.1–8.5 logcfu/g (survival rates 98.2 and 99.7%, respectively). The technology developed presents important advantages for the maintenance of cell viability during storage, assuring stability of ready-to-use functional food ingredients that could be directly incorporated in food systems.

Mikrobiološki kvalitet probiotičkih proizvoda

Microorganisms used as probiotics should meet elementary safety aspects (non-toxicity, absence of antibiotic resistance genes and translocation) and functional/technological aspects (resistance and survival in the acid gastric environment, adhesiveness, stability, and cell viability). Probiotics with the health claim of being a dietary product or a pharmabiotic (drug category) should be clinically tested, validated, documented, and continuously controlled for quality. Important quality parameters include the identification of declared probiotic strains, the number of viable microorganisms (probiotic bacteria and/or fungi), and microbiological purity (absence of specified pathogenic/opportunistic pathogenic bacteria and fungi, and limitation of total unspecified contaminants such as aerobic bacteria, yeasts, and molds). Due to numerous reports of low-quality commercial probiotics marketed for human use, this review discusses the methods used to test the probiotic microorganism content, safety for the intended use, and proven health benefits of those probiotics whose microbiological quality deviates from the manufacturer's stated content, as well as the maintenance of cell viability, i.e., stability of the probiotic during the shelf life. In addition, the adverse effects of probiotics and the potential hazards to the health of the user are addressed.

OsPDIL1-1 controls ROS generation by modulating NADPH oxidase in developing anthers to alter the susceptibility of floret fertility to heat for rice

High temperature (HT) at meiosis induces heat injury to pollen viability and floret fertility, which is closely associated with HT-induced endoplasmic reticulum (ER) stress and ROS damage in developing anthers. Disulfide isomerase like proteins (PDILs) play an essential role in the formation, reduction, and isomerization of disulfide bonds in nascent secretory proteins for the maintenance of cell viability and ER homeostasis. However, the underlying mechanism by which HT induces ROS burst in rice anthers and its relation to ER stress for the varying existence of PDILs is largely unknown. In this paper, we investigated the action of PDILs in the regulation of heat injury to floret fertility and its association with HT-induced ROS generation in developing anthers under well-controlled climatic conditions. Results showed that knock-down of OsPDIL1-1 by RNAi enhanced the activity of NADPH oxidase and caused the excessive ROS accumulation in developing anthers, consequently the up-grading sensitivity of pollen viability and floret fertility to heat stress. RBOHb is the primary site where HT exposure affected NADPH oxidase activity and triggered ROS generation in rice anthers because OsPDIL1-1 was found to interact with RBOHb in the ER-PM junction. Furthermore, HT exposure triggered the RBOHb-mediated ROS generation in a Ca 2+ -dependent manner, while the induction of HT exposure to ER stress was not necessarily associated with ROS generation derived from NADPH oxidase. • PDIL1-1 was essential for maintaining floret fertility at elevated temperatures. • PDIL1-1 directly interacts with RBOHb to modulate ROS generation in rice anthers. • HT triggered the RBOHb-mediated ROS generation in a Ca 2+ -dependent manner. • HT-induced ER stress was not associated with NADPH oxidase-derived ROS. • PDIL1-1 was found to interact with RBOHb in the ER-PM junction.

Journey of the Probiotic Bacteria: Survival of the Fittest.

This review aims to bring a more general view of the technological and biological challenges regarding production and use of probiotic bacteria in promoting human health. After a brief description of the current concepts, the challenges for the production at an industrial level are presented from the physiology of the central metabolism to the ability to face the main forms of stress in the industrial process. Once produced, these cells are processed to be commercialized in suspension or dried forms or added to food matrices. At this stage, the maintenance of cell viability and vitality is of paramount for the quality of the product. Powder products requires the development of strategies that ensure the integrity of components and cellular functions that allow complete recovery of cells at the time of consumption. Finally, once consumed, probiotic cells must face a very powerful set of physicochemical mechanisms within the body, which include enzymes, antibacterial molecules and sudden changes in pH. Understanding the action of these agents and the induction of cellular tolerance mechanisms is fundamental for the selection of increasingly efficient strains in order to survive from production to colonization of the intestinal tract and to promote the desired health benefits.

Engineering Vascular Bioreactor Systems to Closely Mimic Physiological Forces In Vitro.

In vitro models of the vasculature play an important role in biomedical discovery research, with diverse applications in vascular biology, drug discovery, and tissue engineering. These models aim to replicate the conditions of the human vasculature including physical geometry, employing appropriate vascular cells exposed to physiological forces. However, vessel biology is complex, with multiple relevant cell types, precise three-dimensional (3D) architectural arrangement, an array of biological cues and pressure, flow rate, and shear stress stimulation that are difficult to replicate outside of the body. Vessel bioreactors typically comprise core modules, common to most systems: a 3D tubular scaffold to support cells, media and nutrient exchange for cell viability, a pumping module, and sensor arrays for monitoring. In our comprehensive review of the literature, foundational elements such as maintenance of cell viability, nutrient exchange with flow, use of 3D scaffolds, and basic sensing capabilities are well established. However, most bioreactor systems fail to adequately replicate combinations of physiologically relevant stimuli-including pressure, shear stress, and flow rate-independently, as system input parameters. At the root of this deficiency is the field's reliance on simple pumping systems designed for other applications, making it necessary to add resistors and compliance chambers to even approach human vascular conditions. As vascular biology research rapidly progressed it became increasingly clear that combinations of physical forces strongly influence cell phenotype, gene expression, and in turn can be drivers of pathology. We highlight the need for renewed innovation in vascular bioreactor development with a focus on the importance of providing appropriate physiological forces in the same system. Impact statement In vitro systems modeling aspects of the human vasculature are increasingly important in tissue engineering and biomedical research. Current systems maintain basic cell viability and facilitate nutrient exchange but poorly replicate physiological forces, reliant on simplistic pumping systems. Our review highlights the need to more accurately mimic arterial pressure, flow rate, and shear stress in the same system. Innovation in this area would improve in vitro modeling of the vasculature, significantly impacting tissue engineering and vascular biology in this area.

92 (PB082) - RP12146, a novel, selective, and potent small molecule inhibitor of PARP 1/2, synergizes with SOC therapy in preclinical models of solid tumors

Background: Poly (ADP-ribose) polymerase (PARP) activity involves synthesis of Poly-ADP ribose (PAR) polymers that recruit host DNA repair proteins leading to correction of DNA damage and maintenance of cell viability. Several PARP inhibitors have been approved for the treatment of BRCA-mutated ovarian, breast and pancreatic cancer. In addition to BRCA-mutated cancers, therapeutic role of PARP inhibitors as monotherapy agent or in combination with established therapy can be expanded to other types of solid tumors through ‘synthetic lethality’ mechanism. Herein, we describe the efficacy of RP12146 as a single agent and in combination with approved therapies in preclinical models of solid tumors. Material & Methods: Enzymatic potency was evaluated using a PARP Chemiluminescent Activity Assay Kit (BPS biosciences). Cell growth was determined following incubation with RP12146 as a single agent or in combination with approved agents in various solid tumor cell lines. Apoptosis & Cell cycle was evaluated following incubation of cell lines with compound for 48 or 72 h, subsequent staining with Annexin-V-PE and 7-AAD or Propidium Iodide, and analysis by flow cytometry. Anti-tumor potential of RP12146 as a single agent or in combination with chemotherapeutic agents was tested in OVCAR-3 and NCI-H69 Xenografts. Expression of downstream markers were determined in cell lines and xenograft tumor samples by Western blotting. Results: RP12146 inhibited PARP1 (0.6 nM) & PARP2 (0.5 nM) with equal potency with several fold selectivity over the other members of the PARP family. In addition, RP12146 demonstrated selective PARP1-Trapping with ∼20-fold selectivity over PARP2. RP12146 caused a dose-dependent growth inhibition of both BRCA mutant and non-mutant cancer cell lines with GI50 in the range of 0.04 μM to 9.6 μM. Combination of RP12146 with Temozolomide, SN-38, Topotecan, and Lurbinectedin demonstrated either additive or synergistic effects manifested by an inhibition in growth, induction of apoptosis and cell cycle arrest in Breast, Gastric, Glioma, Lung Cancer, and Ovarian cancer cell lines. RP12146 exhibited anti-tumor potential with TGI of 28% and 21.8% as a single agent in OVCAR-3 and NCI-H69 Xenograft model respectively. In NCI-H69 xenograft model, RP12146 in combination with cisplatin exhibited TGI of 69.3%. Conclusion: Data demonstrate the therapeutic potential of RP12146 as single agent and in combination in solid tumors. RP12146 is currently being evaluated in Phase 1 clinical trials in patients with locally advanced or metastatic solid tumors (NCT05002868). No conflict of interest.

N-acetylcysteine attenuates oxidative stress-mediated cell viability loss induced by dimethyl sulfoxide in cryopreservation of human nucleus pulposus cells: A potential solution for mass production.

Cell therapy is considered a promising strategy for intervertebral disc (IVD) regeneration. However, cell products often require long-term cryopreservation, which compromises cell viability and potency, thus potentially hindering commercialization and off-the-shelf availability. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant, however, DMSO is associated with cytotoxicity and cell viability loss. This study aimed to investigate the effects of DMSO on human nucleus pulposus cells (NPC) and the role of oxidative stress in DMSO-induced cytotoxicity. Furthermore, we examined the potential of antioxidant N-acetylcysteine (NAC) supplementation to mitigate the negative effects of DMSO. NPC were exposed to various concentrations of DMSO with or without a freezing cycle. Cell viability, cell apoptosis and necrosis rates, intracellular reactive oxygen species (ROS) levels, and gene expression of major antioxidant enzymes were evaluated. In addition, NAC was added to cryopreservation medium containing 10% DMSO and its effects on ROS levels and cell viability were assessed. DMSO concentrations ≤1% for 24 h did not significantly affect the NPC viability, whereas exposure to 5 and 10% DMSO (most commonly used concentration) caused cell viability loss (loss of 57% and 68% respectively after 24 h) and cell death in a dose- and time-dependent manner. DMSO increased intracellular and mitochondrial ROS (1.9-fold and 3.6-fold respectively after 12 h exposure to 10% DMSO) and downregulated gene expression levels of antioxidant enzymes in a dose-dependent manner. Tempering ROS through NAC treatment significantly attenuated DMSO-induced oxidative stress and supported maintenance of cell viability. This study demonstrated dose- and time-dependent cytotoxic effects of DMSO on human NPC. The addition of NAC to the cryopreservation medium ameliorated cell viability loss by reducing DMSO-induced oxidative stress in the freeze-thawing cycle. These findings may be useful for future clinical applications of whole cells and cellular products.

The aggregate distribution of Pseudomonas aeruginosa on biochar facilitates quorum sensing and biofilm formation

Biochar when applied into soil, together with soil clay minerals, may provide habitats for soil microbes and shift soil microbial community structure. Although several mechanisms have been proposed to explain the effects of biochar on microbial community, the impact of biochar on quorum sensing (QS) and QS-regulated behavior is poorly understood. In this study, we compared the effects of biochar and three common soil minerals (i.e., montmorillonite, kaolinite, and goethite) on QS and biofilm formation. Pseudomonas aeruginosa PAO1 with complete QS systems was selected as a model organism. Our results showed that biochar and goethite effectively promoted microbial QS and biofilm formation, while montmorillonite and kaolinite posed no significant effect. Live/Dead staining, SEM and density-dependent QS activity indicated that biochar was beneficial to cell viability maintenance and cell aggregations, which improved the efficiency of intercellular communications through QS. QS mutant strain experiments confirmed that biochar enhanced PAO1 biofilm formation by promoting QS. Goethite promoted biofilm formation with a different mechanism that cell debris induced by iron ions and positive charge on goethite surface provided raw materials for bacterial biofilm formation. Our findings provide evidence that the presence of biochar can enhance QS and biofilm formation through a feedforward loop of the QS system. This contributes to better understand biochar-mediated microbial cell to cell communications through QS.

Practical Considerations for Translating Mesenchymal Stromal Cell-Derived Extracellular Vesicles from Bench to Bed.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown potential for the treatment of tendon and ligament injuries. This approach can eliminate the need to transplant live cells to the human body, thereby reducing issues related to the maintenance of cell viability and stability and potential erroneous differentiation of transplanted cells to bone or tumor. Despite these advantages, there are practical issues that need to be considered for successful clinical application of MSC-EV-based products in the treatment of tendon and ligament injuries. This review aims to discuss the general and tissue-specific considerations for manufacturing MSC-EVs for clinical translation. Specifically, we will discuss Good Manufacturing Practice (GMP)-compliant manufacturing and quality control (parent cell source, culture conditions, concentration method, quantity, identity, purity and impurities, sterility, potency, reproducibility, storage and formulation), as well as safety and efficacy issues. Special considerations for applying MSC-EVs, such as their compatibility with arthroscopy for the treatment of tendon and ligament injuries, are also highlighted.

High expression of eIF4A1 predicts unfavorable prognosis in clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) is a worldwide malignancy with high morbidity and mortality. Translation initiation factor 4A1 (eIF4A1), which is an ATP-dependent RNA helicase as a part of eIF4F complex, has been linked to malignant transformation and progression, and a variety of cancers display dysregulation of this enzyme. However, its role in ccRCC remains unclear. In our study, we examined its potential effects in ccRCC. MethodsBased on Proteomic data, TCGA and ONCOMINE database, RCC cell lines and tissues, the expression of eIF4A1 between ccRCC and normal tissues were investigated. A correlation was evaluated between the prognostic model for OS and ccRCC progression. Analysis of functional enrichment and PPI network were performed. After examining differentially expressed genes between the eIF4A1 high and low-expression groups, we performed GSEA analysis. Furthermore, we investigated immune cell infiltration of eIF4A1. Then we determined eIF4A1 functions in the establishment and maintenance of cell viability, migration and invasion of cell lines. Flow cytometry was utilized to detect cell cycle. ResultsThe eIF4A1 was up-regulated in ccRCC tissues and cell lines. An increased level of eIF4A1 was linked to lower survival rates and impaired immunity. Depletion of eIF4A1 could arrest tumor cells in G1 phase, so as to seriously limit cell proliferation and weaken the capacity of cell migration. ConclusionccRCC patients with high eIF4A1 expression are at increased risk of poor prognosis, furthermore eIF4A1 plays a prominent role in facilitating tumor cell proliferation and migration which may further be a potential prognostic biomarker and therapeutic target.