Cellular Metabolic Activity Research Articles

Thioredoxin Is a New Target for the Phytotoxicity of Small Lactone Mycotoxins, Patulin and Penicillic Acid on Maize Seedlings

The phytotoxic mechanisms of patulin (PAT) and penicillic acid (PA) have not been identified unambiguously. This study aims to clarify their effects on thioredoxins (Trxs). Aflatoxin B1 (AFB1), PAT and PA were isolated by solvent extraction and chromatographic techniques from the cultures of Aspergillus flavus Z2 (LC171449), Penicillium vulpinum CM1 and Aspergillus ochraceus EMCC516, respectively. The three mycotoxins showed phytotoxicity to the germination of maize seeds, which was manifested by inhibiting radicle and coleoptile emergence, in addition to their toxic effects on fresh weights and root and shoot lengths. The phytotoxicity of AFB1 and PAT was greater than that of PA. Due to the central roles exhibited by plant Trxs in cellular metabolic activities, they were tested as target proteins for PAT and PA using AFB1 as positive control. In vivo studies showed that the mycotoxins significantly reduced Trx activity measured in the roots and shoots of maize seedlings. PAT showed greater Trx-inhibiting activity than PA and AFB1. In vitro studies of the mycotoxins on Trx y1 (from Arabidopsis thaliana) and thioredoxin reductase (Tr, from rat liver) activities confirm the results of in vivo studies. The inactivation of Trx with PAT and PA was reduced in the presence of glutathione (GSH). Data obtained suggest that lactone mycotoxins are more highly reactive with simple low-molecular-weight thiols (like GSH) than with complex ones (like Trx).

Microporous/Macroporous Polycaprolactone Scaffolds for Dental Applications.

This study leverages the advantages of two fabrication techniques, namely, melt-extrusion-based 3D printing and porogen leaching, to develop multiphasic scaffolds with controllable properties essential for scaffold-guided dental tissue regeneration. Polycaprolactone-salt composites are 3D-printed and salt microparticles within the scaffold struts are leached out, revealing a network of microporosity. Extensive characterization confirms that multiscale scaffolds are highly tuneable in terms of their mechanical properties, degradation kinetics, and surface morphology. It can be seen that the surface roughness of the polycaprolactone scaffolds (9.41 ± 3.01 µm) increases with porogen leaching and the use of larger porogens lead to higher roughness values, reaching 28.75 ± 7.48 µm. Multiscale scaffolds exhibit improved attachment and proliferation of 3T3 fibroblast cells as well as extracellular matrix production, compared with their single-scale counterparts (an approximate 1.5- to 2-fold increase in cellular viability and metabolic activity), suggesting that these structures could potentially lead to improved tissue regeneration due to their favourable and reproducible surface morphology. Finally, various scaffolds designed as a drug delivery device were explored by loading them with the antibiotic drug cefazolin. These studies show that by using a multiphasic scaffold design, a sustained drug release profile can be achieved. The combined results strongly support the further development of these scaffolds for dental tissue regeneration applications.

Reduction of S0 deposited on electroactive biofilm under an oxidative potential

The inevitable deposition of S0 on the electroactive biofilm (EAB) via anodic sulfide oxidation affects the stability of bioelectrochemical systems (BESs) when an accidental discharge of sulfide occurred, leading to the inhibition of electroacitivity, because the potential of anode (e.g., 0 V versus Ag/AgCl) is ~500 mV more positive than the redox potential of S2−/S0. Here we found that S0 deposited on the EAB can be spontaneously reduced under this oxidative potential independent of microbial community variation, leading to a self-recovery of electroactivity (> 100 % in current density) with biofilm thickening (~210 μm). Transcriptomics of pure culture indicated that Geobacter highly expressed genes involving in S0 metabolism, which had an additional benefit to improve the viability (25 % – 36 %) of bacterial cells in biofilm distant from the anode and the cellular metabolic activity via electron shuttle pair of S0/S2−(Sx2−). Our findings highlighted the importance of spatially heterogeneous metabolism to its stability when EABs encountered with the problem of S0 deposition, and that in turn improved the electroactivity of EABs.

Synthesis, characterization, DNA interaction, antimicrobial activity, DFT analysis and molecular docking studies of copper(II) complex with triazine and neocuproine

In this paper, a new copper (II) complex with neocuproine and triazine has been synthesized and characterised. In the complex [Cu (Neocup)(Triazine)H2O](NO3)2 (where neocup=2,9-dimethyl-1;10-phenanthroline,Triazine=3-(2-pyridyl)5,6-diphenyl-1.2,4-triazine) is characterized by using UV Spectra, FT-IR, EPR, and thermal analyses. UV spectra, fluorescence spectra, and cyclic voltammetry techniques were used to determine the binding mechanism of the Cu (II) complex with calf thymus DNA (CT-DNA). The stability of the prepared Cu (II) complex has been evaluated through the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy gab. In addition, the molecular docking studies were indicating the Cu (II) complex into the base pairs of CT-DNA. Furthermore, the Cu (II) complex was exposed to human liver cancer cell in vitro cytotoxicity assays, using the MTT assay, one can assess cellular metabolic activity as a sign of cell viability, proliferation, and cytotoxicity. The resultant complex shows potent cytotoxic behaviour against human cell lines (HepG2). The IC50 and R2 values of an optimized complex of Cu (II) complexes are noted as 60.81 µg/ml, 19.19 µg/ml and 0.991%, 0.789% respectively. The results revealed that copper complex [Cu(Neocup)(Triazine) H2O](NO3)2 showed high potent cytotoxicity effects against human cell line (HepG2) than [Cu(phen)(tryp)I]. The complex has been implied for antibacterial properties against the staphylococcusaureus (Gram-negative (+Ve)) and the klebsiella pneumonia, Escherichia coli (Gram-negative (-Ve)) bacterial strains and the antifungal activity against C.albicanspecies, respectively. The optimized results revealed that the Cu (II) complex exhibits excellent antibacterial and antifungal activity.

Luteolin: A Phytochemical to Mitigate S. Typhimurium Flagellin-Induced Inflammation in a Chicken In Vitro Hepatic Model.

The use of natural feed supplements is an alternative tool to diminish the damage caused by certain bacteria, improving animal health and productivity. The present research aimed to investigate the proinflammatory effect of flagellin released from the bacterial flagellum of Salmonella enterica serovar Typhimurium and to attenuate the induced inflammation with luteolin as a plant-derived flavonoid on a chicken primary hepatocyte-non-parenchymal cell co-culture. Cells were cultured in a medium supplemented with 250 ng/mL flagellin and 4 or 16 µg/mL luteolin for 24 h. Cellular metabolic activity, lactate dehydrogenase (LDH) activity, interleukin-6, 8, 10 (IL-6, IL-8, IL-10), interferon-α, γ (IFN-α, IFN-γ), hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations were determined. Flagellin significantly increased the concentration of the proinflammatory cytokine IL-8 and the ratio of IFN-γ/IL-10, while it decreased the level of IL-10, indicating that the model served adequate to study inflammation in vitro. Luteolin treatment at 4 µg/mL did not prove to be cytotoxic, as reflected by metabolic activity and extracellular LDH activity, and significantly reduced the flagellin-triggered IL-8 release of the cultured cells. Further, it had a diminishing effect on the concentration of IFN-α, H2O2 and MDA and restored the level of IL-10 and the ratio of IFN-γ/IL-10 when applied in combination with flagellin. These results suggest that luteolin at lower concentrations may protect hepatic cells from an excessive inflammatory response and act as an antioxidant to attenuate oxidative damage.

Engineering Modular, Oxygen-Generating Microbeads for the In Situ Mitigation of Cellular Hypoxia.

Insufficient oxygenation is a key obstacle in the design of clinically scalable tissue-engineered grafts. In this work, an oxygen-generating composite material, termed OxySite, is created through the encapsulation of calcium peroxide (CaO2 ) within polydimethylsiloxane and formulated into microbeads for ease in tissue integration. Key material parameters of reactant loading, porogen addition, microbead size, and an outer rate-limiting layer are modulated to characterize oxygen generation kinetics and their suitability for cellular applications. In silico models are developed to predict the local impact of different OxySite microbead formulations on oxygen availability within an idealized cellular implant. Promising OxySite microbead variants are subsequently coencapsulated with murine β-cells within macroencapsulation devices, resulting in improved cellular metabolic activity and function under hypoxic conditions when compared to controls. Additionally, the coinjection of optimized OxySite microbeads with murine pancreatic islets within a confined transplant site demonstrates ease of integration and improved primary cell function. These works highlight the broad translatability delivered by this new oxygen-generating biomaterial format, whereby the modularity of the material provides customization of the oxygen source to the specific needs of the cellular implant.

Methamphetamine and Designer Stimulants Modulate Tonic Human Cerebrovascular Smooth Muscle Contractility: Relevance to Drug-Induced Neurovascular Stress.

To avoid criminal prosecution, clandestine chemists produce designer stimulants that mimic the pharmacological and psychoactive effects of conventional stimulants, such as methamphetamine. Following persistent or high-dose exposure, both acute vasoconstriction and loss of vascular homeostasis are reported dangers of conventional stimulants, and designer stimulants may pose even greater dangers. To compare the effects of a conventional stimulant and two designer stimulants on vascular contraction, this study examined the direct effects of 1,3-benzodioxolylbutanamine (BDB) and N-butylpentylone in comparison to methamphetamine on the function of human brain vascular smooth muscle cells (HBVSMCs). HBVSMCs suspended in collagen gels were exposed to varying concentrations of each drug, and the degree of constriction was assessed over one week. The MTT assay was used to measure the impact of the three drugs on the cellular metabolic activity as a marker of cellular toxicity. The highest concentration tested of either methamphetamine or N-butylpentylone produced a loss of HBVSMC contractility and impaired cellular metabolism. BDB showed a similar pattern of effects, but, uniquely, it also induced vasoconstrictive effects at substantially lower concentrations. Each drug produced direct effects on HBVSMC contraction that may be a mechanism by which the cardiovascular system is damaged following high-dose or persistent exposure, and this could be exacerbated by any sympathomimetic effects of these compounds in whole organisms. BDB appears to impact HBVSMC function in ways distinct from methamphetamine and N-butylpentylone, which may present unique dangers.

THE POTENTIAL OF MAGNETIC CELL SHEET PATCHES IN TENDON REGENERATION

Tissue engineering and regenerative medicine (TERM) hold the promise to provide therapies for injured tendons despite the challenging cues of tendon niche and the lack of specific factors to guide regeneration. The emerging potential of magnetic responsiveness and magnetic nanoparticles (MNPs) functionalities offers new perspectives to tackle TERM challenges. Moreover, pulsed electromagnetic field (PEMF) is FDA approved for orthopaedics with potential to control inflammation upon injury. We previously demonstrated that magnetic cell-sheets assisted by PEMF trigger the inflammation resolution by modulating cytokine-enriched environments [1]. To further understand the potential of magnetically assisted living patches, we have recently conducted in vivo studies using a rat patellar defect model.After labeling of human adipose stem cells with iron oxide MNPs for 16h, magCSs were cultured up to 3 days in α-MEM medium under non-magnetic or PEMF conditions. MagCSs were evaluated by immunocytochemistry, and real time RT-PCR for tendon markers. Cell metabolic activity was also assessed by MTS and ECM proteins quantified by Sirius Red/Fast Green.The MagCSs effect in ameliorating healing was assessed after implantation in window defects created in the patellar tendon of rats. PEMF was externally applied (3mT, 70Hz) 3d/week for 1h (magnetotherapy). After 4 and 8w, tendons were histologically characterized for immune-detection of tendon and inflammatory markers, and for Perls van Gieson and HE stains. Blood and detoxification organs were screened for inflammatory mediators and biodistribution of MNPs, respectively.In vitro results suggest that PEMF stimulates cellular metabolic activity, influences protein synthesis and the deposition of collagen and non-collagenous proteins is significantly increased compared to non-magnetic conditions. No adverse reactions, as infection or swelling, were observed after surgery or during follow-up. After 8w, magCSs remained at the implantation site and no MNPs were detected on detoxification organs. Plasma levels of IL1α, β, IL6 and TNFα assessed by multiplex assay were below detectable values (<12.5pg/ml).Thus, the combination of cell sheets and magnetic technologies hold promise for the development of living tendon substitutes.Acknowledgement to ERC-COG MagTendon772817, H2020 Achilles 810850, FCT - 2020.01157.CEECIND.

Biological Role of Zinc in Liver Cirrhosis: An Updated Review

Liver cirrhosis is a complication usually due to the consequence of persistent chronic liver disease. It is associated with different mechanisms, including hypoalbuminemia, impaired amino acid turnover, and micronutrient deficiencies. Consequently, cirrhotic patients can develop progressive complications like ascites, hepatic encephalopathy, and hepatocellular carcinoma. The liver is a vital organ that regulates the different metabolic pathways and transportation of trace elements. Zn is an indispensable micronutrient trace element involved in its crucial functions in cellular metabolic activity. Zn mediates its action by binding to a wide range of proteins; therefore, it imparts numerous biological effects, including cellular division, differentiation, and growth. It is also involved in critical processes for the biosynthesis of structural proteins and regulation of transcription factors and acts as a co-factor for the various enzymatic processes. As the liver is a significant regulator of Zn metabolism, its abnormalities lead to Zn deficiency, which has consequences on cellular, endocrine, immune, sensory, and skin dysfunctions. Conversely, Zn deficiency may modify the functions of hepatocytes and immune responses (acute phase protein production) in inflammatory liver diseases. This review has concisely stated the evolving indication of the critical role of Zn in biological processes and complications associated with liver cirrhosis pathogenesis due to Zn deficiency.

Abstract 3677: DNA damage signaling activates de novo GTP synthesis to promote chemoradiation resistance in glioblastoma

Abstract Glioblastoma (GBM) is uniformly fatal due to inherent radiation (RT) and chemotherapy resistance. We have found this therapeutic resistance is mediated by alterations in tumor cellular metabolic activity. Our group and others have found that metabolites can regulate DNA repair and RT resistance in brain tumors, but little is known about how DNA damage regulates metabolic pathway activity in cancer. Here, we show that DNA damage acutely increases guanine-containing purine metabolites in multiple in vitro and intracranial GBM models. By interrogating metabolic fluxes in vitro using a variety of stable isotope tracers, we confirmed RT-induced elevation in guanylates was due to increased de novo purine synthesis (DNPS) rather than activation of purine salvage. By developing and using novel stable isotope tracing methods to directly measure DNPS in awake, unrestrained mice, we confirmed that orthotopic GBMs have higher DNPS rates than adjacent cortical tissue that further increase after treatment with RT. Neither salvage synthesis of purines nor pyrimidine synthesis were impacted by RT in any intracranial tissues. With these findings, we opened a clinical study to directly measure purine synthesis in patients, and we found that human GBMs have similarly high purine synthesis rates compared to normal brain tissue. Because DNA damage activated DNPS without affecting purine salvage or pyrimidine synthesis, we reasoned that active signaling may be involved. Indeed, therapy-induced DNPS increases are lost in vitro and in vivo upon pharmacological or genomic inhibition of the DNA-damage sensing kinase DNA-PK. Moreover, RT and DNA-PK have direct influence over the spatial organization of DNPS enzymes, including IMPDH, the rate-limiting step in guanylate synthesis. Because purines can promote DNA repair, these findings suggest that DNA-PK signaling helps promote DNA repair in part by causing the spatial reorganization of DNPS enzymes, thereby activating purine synthesis. To determine if disrupting this regulation can augment GBM treatment efficacy, we combined an FDA-approved inhibitor of purine synthesis with chemoradiation in a variety of mouse models of GBM. Critically, targeting GTP synthesis improved the efficacy of both RT alone and chemoradiation in multiple patient-derived and syngeneic intracranial models, suggesting a potential therapeutic targeting opportunity in patients. In this study, we have developed novel methodology to directly measure purine synthesis in brain tumors in mice and humans. With these tools, we discovered that after DNA damage, DNA-PK mediates a novel pathway controlling the spatial reorganization of purine synthesis enzymes and subsequent DNPS increases. The resulting elevation of GTP levels promotes therapy resistance in tumors, and we are now directly measuring and inhibiting this molecular activity in patients with GBM in an effort to improve standard therapy. Citation Format: Andrew J. Scott, Alexandra M. O'Brien, Weihua Zhou, Vidhi Pareek, Zhou Sha, Sravya Palavalasa, Ayesha U. Kothari, Kari Wilder-Romans, Li Zhang, Anthony C. Andren, Sriram Chandrasekaran, Jason Heth, Yoshie Umemura, Nathan Qi, John Woulfe, Sriram Venneti, Meredith A. Morgan, Theodore S. Lawrence, Wajd N. Al-Holou, Costas A. Lyssiotis, Daniel R. Wahl. DNA damage signaling activates de novo GTP synthesis to promote chemoradiation resistance in glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3677.

Gene Expression in Glenoid Articular Cartilage Varies Across Acute Instability, Chronic Instability, and Osteoarthritis.

Shoulder instability is a common pathology associated with an elevated risk of osteoarthritis (OA). Little is known about gene expression in the cartilage of the glenohumeral joint after dislocation events, particularly as it relates to the risk of posttraumatic OA. This study tested the hypothesis that gene expression in glenoid cartilage varies among acute instability (<3 dislocations), chronic instability (≥3 dislocations), and OA. Articular cartilage was collected from the anteroinferior glenoid of consenting patients undergoing shoulder stabilization surgery (n = 17) or total shoulder arthroplasty (n = 16). Digital quantitative polymerase chain reaction was used to assess the relative expression of 57 genes (36 genes from OA risk allele studies, 21 genes from differential expression studies), comparing (1) OA versus instability (acute and chronic combined), (2) acute versus chronic instability, (3) OA versus acute instability, and (4) OA versus chronic instability. The expression of 11 genes from OA risk allele studies and 9 genes from differential expression studies was significantly different between cartilage from patients with instability and those with OA. Pro-inflammatory genes from differential expression studies and genes from OA risk allele studies were more highly expressed in cartilage in the OA group compared with the instability group, which expressed higher levels of extracellular matrix and pro-anabolic genes. The expression of 14 genes from OA risk allele studies and 4 genes from differential expression studies, including pro-inflammatory genes, anti-anabolic genes, and multiple genes from OA risk allele studies, was higher in the acute instability group compared with the chronic instability group. Cartilage in the OA group displayed higher expression of CCL3, CHST11, GPR22, PRKAR2B, and PTGS2 than cartilage in the group with acute or chronic instability. Whereas cartilage in both the acute and chronic instability groups had higher expression of collagen genes, cartilage in the OA group had expression of a subset of genes from OA risk allele studies or from differential expression studies that was lower than in the acute group and higher than in the chronic group. Glenoid cartilage has an inflammatory and catabolic phenotype in shoulders with OA but an anabolic phenotype in shoulders with instability. Cartilage from shoulders with acute instability displayed greater (cellular) metabolic activity compared with shoulders with chronic instability. This exploratory study identified genes of interest, such as CCL3, CHST11, GPR22, PRKAR2B, and PTGS2, that have elevated expression in osteoarthritic glenoid cartilage. These findings provide new biological insight into the relationship between shoulder instability and OA, which could lead to strategies to predict and potentially modify patients' risk of degenerative arthritis due to shoulder instability.

Differential cytotoxic effects of fenbendazole on mouse lymphoma EL-4 cells and spleen cells

Fenbendazole (FBZ) is a benzimidazole anthelmintic widely used to treat parasitic infections. The anticancer effect of FBZ has been recently highlighted leading to its consideration as a potential anticancer agent. Although previous studies have demonstrated the effect of FBZ on cancer cells, there is a paucity of studies on the effect of FBZ on lymphoma cells and normal immune cells. Herein, we investigated the effects of FBZ on a mouse lymphoma cell line, EL-4 cells, and spleen cells, using vincristine as a positive control. The cellular metabolic activity of EL-4 cells was decreased by FBZ, but that of the spleen cells was not decreased. Moreover, FBZ reduced the mitochondrial membrane potential and induced reactive oxygen species production in EL-4 cells, but not in spleen cells. FBZ induced G2/M phase arrest and increased the sub G0/G1 phase ratio, indicating apoptosis. Furthermore, compared to the control cells, the reactivity of spleen cells pretreated with FBZ to lipopolysaccharide was maintained. In summary, FBZ is cytotoxic to EL-4 cells, but not to spleen cells. This study provides experimental evidence that FBZ exerts an anticancer effect, and less cytotoxic effects and functional damage to normal spleen cells.

Reactive Oxygen Species and Antioxidant System in Selected Skin Disorders.

The skin has a solid protective system that includes the stratum corneum as the primary barrier and a complete antioxidant defence system to maintain the skin's normal redox homeostasis. The epidermal and dermal cells are continuously exposed to physiological levels of reactive oxygen species (ROS) originating from cellular metabolic activities. Environmental insults, such as ultraviolet (UV) rays and air pollutants, also generate ROS that can contribute to structural damage of the skin. The antioxidant defence system ensures that the ROS level remains within the safe limit. In certain skin disorders, oxidative stress plays an important role, and there is an established interplay between oxidative stress and inflammation in the development of the condition. Lower levels of skin antioxidants indicate that oxidative stress may mediate the pathogenesis of the disorder. Accordingly, the total antioxidant level was also found to be lower in individuals with skin disorders in individuals with normal skin conditions. This review attempts to summarise the skin oxidant sources and antioxidant system. In addition, both skin and total antioxidant status of individuals with psoriasis, acne vulgaris, vitiligo and atopic dermatitis (AD), as well as their associations with the progression of these disorders will be reviewed.

Lactate transport and metabolism in rumen epithelial cells in SARA condition

Lactate, a main product of rumen microbial fermentation from dietary starch, has been recognised as an important mediator of rumen microbiota regulation in rumen acidosis. However, the mechanism of lactate transport and metabolism in the rumen remains unclear. This study aimed to evaluate the significance of the absorption, transport, and metabolism of lactic acid in the pH and LPS-induced subacute ruminal acidosis (SARA) model of rumen epithelial cells (RECs). In the current study, RECs were obtained from two adult Saanen goats (44.5 ± 4.6 kg BW) and using Lipopolysaccharide (LPS) and low pH to co-stimulate RECs to construct a microenvironment under SARA conditions, to further analyse the lactate transport and metabolism by RECs. The results showed that the LPS and low pH could significantly reduce cell viability by 29.36% (p < 0.05), induce inflammation in RECs, destroy tight junctions between RECs, and increase cell permeability. In addition, stimulation leads to changes in cellular metabolic activity and inhibits the expression of lactate transporters (p < 0.05), promoting an increased risk of lactate accumulation in the intracellular and intestinal lumen. This study hopefully provides a better understanding of lactate metabolic regulation and new perspectives for the nutritional prevention and treatment of SARA. HIGHLIGHTS Lactic acid is an important fermentation product in the rumen of ruminants, and its transport and metabolic regulation mechanisms are still unclear. To understand transport uptake in the rumen epithelium, a SARA model was therefore used. Low pH and high LPS trigger epithelial inflammation, increasing the risk of lactic acid entering the bloodstream.

Water exchange rates measure active transport and homeostasis in neural tissue.

For its size, the brain is the most metabolically active organ in the body. Most of its energy demand is used to maintain stable homeostatic physiological conditions. Altered homeostasis and active states are hallmarks of many diseases and disorders. Yet there is currently no direct and reliable method to assess homeostasis and absolute basal activity of cells in the tissue noninvasively without exogenous tracers or contrast agents. We propose a novel low-field, high-gradient diffusion exchange nuclear magnetic resonance (NMR) method capable of directly measuring cellular metabolic activity via the rate constant for water exchange across cell membranes. Exchange rates are s under normal conditions in viable ex vivo neonatal mouse spinal cords. High repeatability across samples suggest that values are absolute and intrinsic to the tissue. Using temperature and drug (ouabain) perturbations, we find that the majority of water exchange is metabolically active and coupled to active transport by the sodium-potassium pump. We show that this water exchange rate is sensitive primarily to tissue homeostasis and provides distinct functional information. In contrast, the apparent diffusion coefficient (ADC) measured with submillisecond diffusion times is sensitive primarily to tissue microstructure but not activity. Water exchange appears independently regulated from microstructural and oxygenation changes reported by ADC and relaxation measurements in an oxygen-glucose deprivation model of stroke; exchange rates remain stable for 30-40 min before dropping to levels similar to the effect of ouabain and never completely recovering when oxygen and glucose are restored.

Coffee-Derived Exosome-Like Nanoparticles: Are They the Secret Heroes?

Regular coffee consumption has beneficial and preventative effects on liver and chronic neurodegenerative diseases. However, the studies performed with the ingredients found in coffee beverages have not clarified the responsible mechanisms. Exosomes are small, membrane-coated cargo packages secreted by prokaryote and eukaryote cells. Exosomes regulate intercellular communication and affect cellular metabolic activities even among different species. In this study, we aimed to isolate and characterize the edible plant-derived exosome-like nanoparticles from roasted hot coffee beverages, hypothesizing that the edible plant-derived exosome-like nanoparticles were responsible for the beneficial effects of coffee. Size exclusion chromatography and commercial kits were used for the isolation process. Efficient coffee edible plant-derived exosome-like nanoparticle fractions were determined by an ultraviolet-visible spectrophotometer. Harvested coffee edible plant-derived exosome-like nanoparticles were characterized by transmission electron microscopy. The quantification procedure was performed using a commercial kit. Coffee edible plant-derived exosome-like nanoparticles' proliferative effects on human hepatic stellate cells and human hepatocellular carcinoma cells were studied using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Whole-exosome RNA sequencing was performed. Transmission electron microscopy scanning analysis indicated round-shaped nanoparticles with sizes ranging from 40 to 100 nm. Both size exclusion chromatography and kit-isolated edible plant-derived exosome-like nanoparticle samples showed maximum absorbance at 227.5 nm in ultraviolet-visible spectrophotometer analysis. Regarding the quantitation results, kit isolation was more efficient than the size exclusion chromatography method when the harvested particle numbers were compared. An important MTT assay finding confirmed the observed beneficial effects of coffee beverages: coffee edible plant-derived exosome-like nanoparticles significantly suppressed hepatocellular carcinoma cell proliferation. As a result of sequencing, we identified 15 mature miRNAs. A MapReduce-based MicroRNA Target Prediction Method (The DIANA tools' MR-microT algorithm) highlighted 2 genes specifically associated with the miRNAs that we obtained: KMT2C and ZNF773. For the first time in the literature, coffee edible plant-derived exosome-like nanoparticles were identified. These nanoparticles may have therapeutic effects on chronic liver diseases. Experimental studies, therefore, should be performed on disease models to demonstrate their efficacy.

Insights in opiates toxicity: impairment of human vascular mesenchymal stromal cells

The most common pulmonary findings in opiate-related fatalities are congestion and oedema, as well as acute and/or chronic alveolar haemorrhage, the cause of which is thought to be a damage to the capillary endothelium related to ischemia. Human vascular mesenchymal stromal cells (vMSCs) play a fundamental role in tissue regeneration and repair after endothelial cell injury, and they express opioid receptors. The aim of this study was to assess the effect of in vitro morphine exposure on the physiological activity and maintenance of human vMSCs. vMSCs were obtained from abdominal aorta fragments collected during surgery repair and were exposed to incremental doses (0.1 mM, 0.4 mM, 0.8 mM and 1 mM) of morphine sulphate for 7 days. The effect was investigated through cell viability assessment, proliferation assay, reactive oxygen species (ROS) detection assay, senescence-associated β-galactosidase assay, senescent-related markers (p21WAF1/CIP1 and p16INK4) and the apoptosis-related marker caspase 3. Moreover, an ultrastructural analysis by transmission electron microscopy and in vitro vascular differentiation were evaluated. Results showed a decrease of the cellular metabolic activity, a pro-oxidant and pro-senescence effect, an increase in intracellular ROS and the activation of the apoptosis signalling, as well as ultrastructural modifications and impairment of vascular differentiation after morphine treatment of vMSC. Although confirmation studies are required on real fatal opiate intoxications, the approach based on morphological and immunofluorescence methodologies may have a high potential also as a useful tool or as a complementary method in forensic pathology. The application of these techniques in the future may lead to the identification of new markers and morphological parameters useful as complementary investigations for drug-related deaths.

Study on Performance Simulation of Vascular-like Flow Channel Model Based on TPMS Structure

In medical validation experiments, such as drug testing and clinical trials, 3D bioprinted biomimetic tissues, especially those containing blood vessels, can be used to replace animal models. The difficulty in the viability of printed biomimetic tissues, in general, lies in the provision of adequate oxygen and nutrients to the internal regions. This is to ensure normal cellular metabolic activity. The construction of a flow channel network in the tissue is an effective way to address this challenge by both allowing nutrients to diffuse and providing sufficient nutrients for internal cell growth and by removing metabolic waste in a timely manner. In this paper, a three-dimensional TPMS vascular flow channel network model was developed and simulated to analyse the effect of perfusion pressure on blood flow rate and vascular-like flow channel wall pressure when the perfusion pressure varies. Based on the simulation results, the in vitro perfusion culture parameters were optimised to improve the structure of the porous structure model of the vascular-like flow channel, avoiding perfusion failure due to unreasonable perfusion pressure settings or necrosis of cells without sufficient nutrients due to the lack of fluid passing through some of the channels, and the research work promotes the development of tissue engineering in vitro culture.

Untangling the inseparable life of an ABC transporter and a potassium channel: Towards understanding the structure-function relationship in the KATP channel complex.

KATP channels are unique protein complexes of a potassium channel, Kir6.1 or Kir6.2, and an ABC transporter, SUR1, SUR2A or SUR2B (splice variants of SUR2), in a 4:4 stoichiometry. Broadly expressed in many cell types and tissues, KATP channels couple changes in cellular metabolic activity with membrane excitability to control a wide range of physiological processes, including hormone secretion, control of vascular tone, learning and memory, and cardiac and neuronal protection against ischemic insults. In pancreatic β-cells KATP channels formed by Kir6.2 and SUR1 control insulin secretion, and gain- or loss-of-function mutations in these channels cause neonatal diabetes or congenital hyperinsulinism, respectively. Physiological activity of pancreatic KATP channels is regulated by intracellular ATP and ADP. Non-hydrolytic binding of ATP to Kir6.2 inhibits channel activity, while MgATP and MgADP binding to the nucleotide binding domains (NBDs) of SUR1 stimulates channel activity. Besides ATP and ADP, membrane phospholipids in particular PIP2 interacts with Kir6.2 and stimulates channel activity. In addition, pharmacological inhibitors and activators act on SUR1 to modulate channel activity. Although Kir6.2 and SUR1 harbor the primary binding sites for specific physiological and pharmacological ligands, both subunits participate in ligand regulation. How do the potassium channel and the silent ABC transporter work together to decipher signals from different ligands for gating has long been a major question in the KATP channel field. Here, I present structural and functional evidence that SUR1 contributes directly to ATP and PIP2 binding via its TMD0 and L0 linker to enhance Kir6.2 sensitivity to both ligands. Moreover, SUR1 regulates Kir6.2 activity allosterically via its dynamic interaction with the N-terminus of Kir6.2.

Investigating the relationship between cytotoxic T lymphocyte mechanical output and metabolism.

In order to execute their cytolytic functions, cytotoxic T lymphocytes (CTLs) must first migrate into the peripheral tissues and then form immune synapses (IS) on target antigen-presenting cells. Both migration and IS formation involve mechanical force generation that is sustained by actin cytoskeletal rearrangement, a dynamic process of polymerizing G-actin monomers into F-actin filaments. Actin dynamics are important for generating structures such as the lamellipodia of migrating cells, the microvilli and mobile signalsomes at the IS, as well as synaptic pushing-and-pulling forces that have been implicated in maximizing cytolytic efficiency. However, little is known of the energy sources that fuel such mechanical activities. Actin dynamics are expected to be energetically demanding, given that actin is an ATPase and each G-actin hydrolyzes one ATP during assembly into filaments. In this study, we observed CTLs migrating and forming IS under various metabolic stresses. We discovered that during long-term migration, CTLs utilize nutrients in the environment, as real-time exchange of media into salt solutions within a perfusion chamber led to cells slowing down significantly. To measure force exertion at the IS, CTLs were treated with various inhibitors of metabolic pathways while forming synapses against deformable hydrogel balls. Using a custom computational pipeline, we found that synaptic forces depend on multiple metabolic pathways such as glycolysis and oxidative phosphorylation. Our study highlights a hitherto unexplored connection between cellular metabolism and mechanical activities integral to T-cell function and immune protection. This is of particular interest in the context of T-cell exhaustion, whereby CTLs chronically stimulated in metabolically inhospitable environments, such as tumors, develop into hypofunctional exhausted T cells. We suspect that immune hypo-functionality in these contexts may also involve defects in migration and IS formation due to the metabolic demand of these mechanical activities.