Vacuoles Research Articles

Impact of polyethylene terephthalate microfibers on histopathology and molecular responses caused by cadmium in the polychaete, Perinereis aibuhitensis.

Heavy metals and microplastics have been found to co-exist in marine sediment environments. Nevertheless, the impact of microplastics on altering the toxicity of heavy metals to marine benthic organisms remains poorly understood. Herein, we investigated the impact of polyethylene terephthalate microfibers (PET MFs, 100 μm in diameter, 500 items·kg-1·dw) on the toxicities and individual/subcellular bioaccumulation of cadmium (Cd, 1, 10 and 100 μg·g-1·dw) to the benthic polychaete, Perinereis aibuhitensis, after 28 days of sediment exposure. Either Cd (10 and 100 μg·g-1) or PET MFs alone caused the intestinal inflammation response including cell vacuolation, villi exfoliation and cytolysis, and the presence of PET MFs significantly aggravated the inflammatory response at a Cd concentration of 1 μg·g-1. PET MFs also showed significant impact on oxidative stress biomarkers including lipid peroxidation (LPO), protein carbonylation (PC) and glutathione S-transferases (GST) activity at 500 items·kg-1. In addition, compared to the single Cd exposure, co-exposure significantly reduced LPO and PC levels while enhancing GST enzyme activity at a Cd concentration of 100 μg·g-1, suggesting decreased oxidative damage. Besides, co-exposure also significantly up- or down-regulated mRNA expressions of selected genes involved in stress response (CAT, SOD, HSP70, HSP90), metabolism (CYP4) and detoxification (MPⅡ) as shown by real-time q-PCR. However, Cd bioaccumulation at individual or subcellular level was not affected by PET MFs, suggesting the key role of toxicity contribution of PET MFs themselves in the combined toxicity. The potential adverse effects of the co-existence of MFs and heavy metals in sediment environments under long-term exposure scenarios still require further validation.

Kinase Inhibitor-Induced Cell-Type Specific Vacuole Formation in the Absence of Canonical ATG5-Dependent Autophagy Initiation Pathway.

Pyridinyl-imidazole class p38 MAPKα/β (MAPK14/MAPK11) inhibitors including SB202190 have been shown to induce cell-type specific defective autophagy resulting in micron-scale vacuole formation, cell death, and tumor suppression. We had earlier shown that this is an off-target effect of SB202190. Here we provide evidence that this vacuole formation is independent of ATG5-mediated canonical autophagosome initiation. While SB202190 interferes with autophagic flux in many cell lines parallel to vacuolation, autophagy-deficient DU-145 cells and CRISPR/Cas9 gene-edited ATG5-knockout A549 cells also undergo vacuolation upon SB202190 treatment. Late-endosomal GTPase RAB7 colocalizes with these compartments and RAB7 GTP-binding is essential for SB202190-induced vacuolation. A screen for modulators of SB202190-induced vacuolation revealed molecules including multi-kinase inhibitor sorafenib as inhibitors of vacuolation and sorafenib co-treatment enhanced cytotoxicity of SB202190. Moreover, VE-821, an ATR inhibitor was found to phenocopy the cell-type specific vacuolation response of SB202190. To identify the factors determining the cell-type specificity of vacuolation induced by SB-compounds and VE-821, we compared the transcriptomics data from vacuole-forming and non-vacuole-forming cancer cell lines and identified a gene expression signature that may define sensitivity of cells to these small-molecules. Further analyses using small molecule tools and the gene signature discovered here, could reveal novel mechanisms regulating this interesting anti-cancer phenotype.

The Secretome of Brain Endothelial Cells Exposed to the Pyrrolizidine Alkaloid Monocrotaline Induces Astrocyte Reactivity and Is Neurotoxic

Monocrotaline (MCT) has well-characterized hepatotoxic and pneumotoxic effects attributed to its active pyrrole metabolites. Studies have previously shown that astrocytes and neurons are targets of MCT, and that toxicity is attributed to astrocyte P450 metabolism to reactive metabolites. However, little is known about MCT toxicity and metabolism by brain endothelial cells (BECs), cells that, together with astrocytes, are specialized in xenobiotic metabolism and neuroprotection. Therefore, in the present study, we evaluated the toxicity of MCT in BECs, and the effects on astrocyte reactivity and neuronal viability in vitro. MCT was purified from Crotalaria retusa seeds. BECs, obtained from the brain of adult Wistar rats, were treated with MCT (1–500 µM), and cell viability and morphology were analyzed after 24–72 h of treatment. Astrocyte/neuron co-cultures were prepared from the cortex of neonatal and embryonic Wistar rats, and the cultures were exposed to conditioned medium (secretome) derived from BECs previously treated with MCT (100–500 µM, SBECM100/500). MCT was not toxic to BECs at the concentrations used and induced a concentration-dependent increase in cell dehydrogenase after 72 h of treatment, suggesting resistance to damage and drug metabolism. However, exposure of astrocyte/neuron co-cultures to the SBECM for 24 h induced changes in the cell morphology, vacuolization, and overexpression of GFAP in astrocytes, characterizing astrogliosis, and neurotoxicity with a reduction in the length of neurites labeled for β-III-tubulin, effects that were MCT concentration-dependent. These results support the hypothesis that MCT neurotoxicity may be due to products of its metabolism by components of the BBB such as BECs and astrocytes, which may be responsible for the brain lesions and symptoms observed after intoxication.

Duck Tembusu virus induced mitophagy in vacuolate spermatogenic cells is mediated by PINK1-Parkin pathway.

As a significant emerging and re-emerging pathogen in China, the widely spread of Duck Tembusu virus (DTMUV) caused enormous economic losses to poultry industry. On account of DTMUV diseases' main symptoms on haemorrhagic oophoritis, intensive attentions were focused on female reproductive organ. Nevertheless, the DTMUV infection of sperm and testis manifested that testis was an important vector for vertical transmission of DTMUV. In the present study, histopathology, immunofluorescence and transmission electron microscopy (TEM) analysis of DTMUV-infected duck testis revealed that DTMUV infection induced seminiferous epithelium injury via spermatogenic cells vacuolization. After DTMUV infection, the expression of autophagy-related genes and proteins in testis were significantly up-regulated. Further TEM analysis discovered that different stages of autophagic and mitophagy structures were visible in cytoplasm of spermatogenic cells after DTMUV infection. And more notably, the testicular protein expression of PINK1 and Parkin were significantly increased after DTMUV infection. In summary, our study discovered that, after DTMUV infection, PINK1-Parkin pathway mediated mitophagy were activated and then induced spermatogenic cells vacuolization.

Inhibition of mitochondrial energy production leads to reorganization of the plant endomembrane system.

Mitochondria have generated the bulk of ATP to fuel cellular activities, including membrane trafficking, since the beginning of eukaryogenesis. How inhibition of mitochondrial energy production will affect the form and function of the endomembrane system and whether such changes are specific in today's cells remain unclear. Here, we treated Arabidopsis thaliana with antimycin A (AA), a potent inhibitor of the mitochondrial electron transport chain (mETC), as well as other mETC inhibitors and an uncoupler. We investigated the effects of AA on different endomembrane organelles connected by vesicle trafficking via anterograde and retrograde routes that heavily rely on ATP and GTP provision for SNARE and RAB/GEF function, respectively, in root cells. Similar to previous reports, AA inhibited root growth mainly by shortening the elongation zone (EZ) in an energy- and auxin-dependent way. We found that PIN-FORMED 2 (PIN2) and REQUIRES HIGH BORON 1 (BOR1), key proteins for EZ establishment and cell expansion, undergo accelerated endocytosis and accumulate at enlarged multivesicular bodies (MVBs) after AA treatment. Such accumulation is consistent with the observation that the central vacuole becomes fragmented and spherical and that the Arabidopsis Rab7 homolog RABG3f, a master regulator of MVB and vacuolar function, localizes to the tonoplast, likely in a GTP-bound form. We further examined organelles and vesicle populations along the secretory pathway and found that the Golgi apparatus-in particular, the endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-cannot be maintained when mETC is inhibited. Our findings reveal the importance and specific impact of mitochondrial energy production on endomembrane homeostasis.

Intoxication of sheep by Astragalus arequipensis in northwestern Argentina.

Spontaneous intoxication by Astragalus arequipensis was diagnosed in a flock of 300 sheep in Jujuy province, northwestern Argentina, that grazed an area heavily invaded by this plant. The main clinical signs were intention tremors, ataxia, and progressive loss of condition. Autopsy of 2 affected animals revealed loss of body condition. The main microscopic changes were fine cytoplasmic vacuolation of cells in the cerebrum, cerebellum, thyroid and adrenal glands, kidney, liver, pancreas, urinary bladder, and lymph nodes, and swollen axons in the cerebellum. Ultrastructurally, the cytoplasmic vacuoles consisted of dilated secondary lysosomes. Composite leaf and stem samples of A. arequipensis analyzed by high-performance liquid chromatography contained 0.05% swainsonine. The diagnosis of intoxication by A. arequipensis was made based on the clinical history and signs; gross, microscopic, and ultrastructural changes; and detection of swainsonine in the plant.

A mouse model of immunosuppression facilitates oral Candida albicans biofilms, bacterial dysbiosis and dissemination of infection.

Opportunistic pathogens are a major threat to people, especially those with impaired immune systems. Two of the most important microbes in this category are the fungus Candida albicans and Gram-positive bacteria of the genus Enterococcus, which share overlapping niches in the oral cavity, gastrointestinal and urogenital tracts. The clinical importance of oral C. albicans biofilm and its interaction with the host under immunosuppressive conditions remains largely understudied. Here, we used a mouse model of oropharyngeal candidiasis (OPC) with cortisone acetate injection on alternate days and a continuous supply of C. albicans in drinking water for three days, resulting in immunosuppression. Results showed abundant growth of resident oral bacteria and a strong C. albicans biofilm on the tongue consisting of hyphae which damaged papillae, the epidermal layer, and invaded tongue tissue with the accumulation of inflammatory cells as demonstrated by Grocott's methenamine silver and hematoxylin and eosin staining, respectively. The dispersed microbes from the oral biofilm colonized the gastrointestinal (GI) tract and damaged its integrity, disseminating microbes to other organs. Although no visible damage was observed in the kidney and liver, except increased lipid vacuoles in the liver cells, C. albicans was found in the liver homogenate. Intriguingly, we found co-occurrence of Enterococcus faecalis in the tongue, liver, and stool of immunosuppressed control and C. albicans infected organs. Targeted 16S rRNA and ITS2 amplicon sequencing of microbes from the fecal samples of mice confirmed the above results in the stool samples and revealed an inverse correlation of beneficial microbes in the dysbiosis condition. Our study shows that mucosal-oral infection of C. albicans under immunosuppressed conditions causes tissue damage and invasion in local and distant organs; the invasion may be aided by the overgrowth of the resident endogenous Enterobacteriaceae and other members, including the opportunistic pathogen Enterococcus faecalis.

The Potassium Utilization Gene Network in Brassica napus and Functional Validation of BnaZSHAK5.2 Gene in Response to Potassium Deficiency.

Potassium, an essential inorganic cation, is crucial for the growth of oil crops like Brassica napus L. Given the scarcity of potassium in soil, enhancing rapeseed's potassium utilization efficiency is of significant importance. This study identified 376 potassium utilization genes in the genome of B. napus ZS11 through homologous retrieval, encompassing 7 functional and 12 regulatory gene families. These genes are unevenly distributed across 19 chromosomes, and the proteins encoded by these genes are mainly localized in the cell membrane, vacuoles, and nucleus. Microsynteny analysis highlighted the role of small-scale replication events and allopolyploidization in the expansion of potassium utilization genes, identifying 77 distinct types of cis-acting elements within their promoter regions. The regulatory mechanisms of potassium utilization genes were provided by analyses of transcription factors, miRNA, and protein interaction networks. Under low potassium stress, the potassium utilization genes, particularly those belonging to the KUP and CBL families, demonstrate pronounced co-expression. RNA-seq and RT-qPCR analysis identified the BnaZSHAK5.2 gene, which is a high-affinity potassium ion transporter, playing a crucial role in the stress response to potassium deficiency in B. napus, as its expression is strongly induced by low potassium stress. A functional complementation study demonstrates that the BnaZSHAK5.2 gene could rescue the primary root growth of the Athak5 mutant under low potassium conditions, confirming its role in response to low potassium stress by sustaining root development.

The Formation and Features of Massive Vacuole Induced by Nutrient Deficiency in Human Embryonic Kidney Cells.

Cellular vacuolization is a commonly observed phenomenon under physiological and pathological conditions. However, the mechanisms underlying vacuole formation remain largely unresolved. LysoTracker Deep Red probes and Enhanced Green Fluorescent Protein-tagged light chain 3B (LC3B) plasmids were employed to differentiate the types of massive vacuoles. By confocal microscopy, lysosome-like massive vacuoles (LysoTracker Deep Red+), autophagosome-like massive vacuoles (LC3B-enhanced green fluorescent protein (EGFP+)), and autolysosome-like massive vacuoles (LC3B-EGFP+ LysoTracker Deep Red+) in starved HEK293T cells were observed. In this study, we demonstrated that nutrient deficiency can induce the formation of massive vacuoles that appear highly electron-lucent in HEK293T cells. Additionally, these massive vacuoles, resulting from nutrient depletion, can originate from various organelles, including small vacuoles, autophagosomes, lysosomes, and autolysosomes. We found that massive vacuoles could form through two primary mechanisms: the accumulation of small vacuoles into larger vacuoles or the fusion of homogeneous or heterogeneous vacuoles. Further analysis revealed that the membranes of massive vacuoles, regardless of origin, were composed of a bilayer membrane structure. As the volume of the massive vacuoles increased, the cytoplasm and nucleus were displaced toward the periphery of the cells, leading to the formation of signet ring-like cells. Interestingly, we provided evidence that complete massive vacuoles or autophagosome-like massive vacuoles can be released and exist independently outside HEK293T cells. Nutrient deprivation induces the formation of heterogeneous, massive vacuoles in human embryonic kidney cells, some of which contribute to the development of signet ring cells, while others lead to extracellular vacuole formation.

Modulation of morphogenesis and metabolism by plant cell biomechanics: From model plants to traditional herbs

Abstract The quality of traditional herbs depends on organ morphogenesis and the accumulation of active pharmaceutical ingredients. While recent research highlights the significance of cell mechanobiology in model plant morphogenesis, our understanding of mechanical signal initiation and transduction in traditional herbs remains incomplete. Recent studies reveal a close correlation between cell wall (CW) biosynthesis and active ingredient production, yet the role of cell mechanics in balancing morphogenesis and secondary metabolism is often overlooked. This review explores how the cell wall, plasma membrane, cytoskeleton, and vacuole collaborate to regulate cell mechanics and respond to mechanical changes. We propose CW biosynthesis as a hub in connecting cell mechanics with secondary metabolism and emphasize that understanding the relationship between mechanical remodeling and secondary metabolism could provide new insights into plant cell mechanobiology and the breeding of high-quality herbs.

Cryo-SEM and large volume FIB-SEM of Arabidopsis cotyledons: Degradation of lipid bodies, biogenesis of glyoxysomes and reorganisation of organelles during germination.

Until recently, the lack of three-dimensional visualisation of whole cells at the electron microscopic (EM) level has led to a significant gap in our understanding of the interaction of cellular organelles and their interconnection. This is particularly true with regard to the role of the endoplasmic reticulum (ER). In this study, we perform three-dimensional reconstructions of serial FIB/SEM stacks and anaglyphs derived from volume rendering, cryo-scanning electron microscopy (cryo-SEM) and state-of-the-art electron microscopy immobilisation and imaging techniques. The results show that glyoxysomes are formed de novo in large numbers and in characteristic clusters on the ER upon germination in mesophyll cells of Arabidopsis cotyledons. The degradation of lipid bodies during germination occurs not only via the ER, which enlarges by taking up polar lipids resulting from enzymatic degradation by lipases, but also via glyoxysomes, which engulf lipid bodies. Dictyosomal (Golgi-derived) vesicles, which fuse with glyoxysomes or their precursors, also appear to be involved in the differentiation of glyoxysomes from segments of the ER. The formation of the central vacuole is the result of the fusion of protein storage vacuoles (protein bodies), which become complex three-dimensional structures during germination. Our observations also suggest that the vacuole plays a role in the degradation of glyoxysomes. The evidence provided in three dimensions shows that the endoplasmic reticulum plays a central role in the biogenesis and degradation of lipid bodies, the ontogeny of glyoxysomes and the development of plastids in the mesophyll cells of Arabidopsis cotyledons.

Newborn daughters get a fresh start through PI(3,5)P2-mediated vacuolar acidification.

Huda et al. (https://doi.org/10.1083/jcb.202406170) reveal a transient, cell cycle-dependent increase in PI(3,5)P2 levels at the lysosome-like vacuole of yeast daughter cells. The resulting lipid asymmetry alters vacuolar pH in both daughter and mother cells and will impact multiple downstream functions.

FRAP Assay to Trace Lipid Mixing of the Inner and Outer Leaflet of Yeast Vacuoles: Assessing the Fusion State in Live Cells.

Fluorescence recovery after photobleaching (FRAP) can be employed to investigate membrane lipid mixing of vacuoles in live budding yeast cells and distinguish the fused, hemi-fused or non-fused states of these organelles under physiological conditions. Here, we describe a protocol for labeling the outer and inner leaflets of vacuoles in live cells that allow to detect hemifusion intermediates and, thus, identify components necessary for fusion pore opening.

Influence of sulfur and selenium application on wheat growth in arsenic-contaminated soil.

Wheat could become poisoned when grown in soil with high arsenic (As) content. It is worthwhile to investigate the potential use of sulfur (S) and selenium (Se) for crop protection while detoxifying heavy metal(loid)s. In this study, a pot experiment was conducted under both single and combined application of the two elements. Their effects on wheat growth were analyzed based on As distribution in subcellular tissues and the variation in physiological and biochemical indicators. Despite wheat absorbing and enriching As under S and Se application, its growth status improved. Cell wall and vacuole sequestered majority of elevated As. Phytochelatins (PCs) content increased significantly in the roots, particularly when Se was applied alone. They could chelate with As using thiol groups. Superoxide dismutase (SOD) activity was found to be considerably lower in leaves and without any discernible increase in roots. Peroxidase (POD) activity in roots/stems and/or catalase (CAT) activity in stems increased, and exerted antioxidant effects. The leaf was well protected, and its chlorophyll content significantly increased. The application of S alone had a relatively weaker effect on reducing As content in grains, but the mixed application of Se could induce an inhibitory effect.

Cell-free hemoglobin released from hemolysis induces programmed cell death through iron overload and oxidative stress in grass carp (Ctenopharyngodon idella).

Intravascular hemolysis releases hemoglobin (Hb) from red blood cells under specific conditions, yet the effect of hemolysis in aquaculture systems remain poorly understood. In this study, a continuous hemolysis model for grass carp was established by injection of phenylhydrazine (PHZ) to investigate the mechanistic impacts of sustained hemolysis. PHZ-induced hemolysis altered liver color, and subsequent hematoxylin and eosin staining revealed substantial Hb accumulation in the head kidney, accompanied by inflammatory cell infiltration and vacuolization in liver tissue. Quantitative real-time PCR and western blotting confirmed that PHZ treatment significantly upregulated Real-time fluorescence quantitative PCR and Western blot confirmed that PHZ treatment significantly up-regulated the expression of iron metabolism-related genes and proteins, including transferrin (Tf), ferritin, ferroportin 1 (FPN1), transferrin receptor 1 (TfR1), nuclear receptor coactivator 4 (NCOA4), divalent metal transporter 1 (DMT1), and six-transmembrane epithelial antigen of prostate 3 (STEAP3). Further investigation of PHZ-induced hemolysis effects on tissues showed that inflammation- and antioxidant enzyme-related genes in the liver and head kidney were significantly upregulated, indicating that hemolysis activated the antioxidant system and intensified inflammatory responses. Perls' staining revealed iron deposition in the head kidney and liver at ten and fourteen days post-PHZ injection. Moreover, β-galactosidase staining and transmission electron microscopy showed increased cellular senescence and mitochondrial damage, respectively, as a result of PHZ-induced hemolysis. In vitro assays with hemin treatment demonstrated increased Fe2+ content in CIK and L8824cells, which induced oxidative stress, upregulated iron metabolism and inflammatory genes, and ultimately led to cell death. These findings suggest that excessive Hb release during sustained hemolysis leads to iron overload, elevates reactive oxygen species production, disrupts antioxidant balance, and ultimately causes cellular damage.

Efficacy of natural NF-κB inhibitors in the treatment of fibrosarcoma: an in vitro model study

IntroductionNF-κB plays a pivotal role in the progression of cancers, including myosarcomas such as fibrosarcoma. Plants possess considerable potential for the provision of chemotherapeutic effects against cancer. The present study assessed, among others, the cytotoxicity, migration capacity and DNA damage induced by several natural compounds (berberine, curcumin, biochanin A, cucurbitacin E (CurE) and phenethyl caffeic acid (CAPE)) in cancer cells (WEHI-164) and normal muscle cells (L6).MethodsIC50 parameter was determined for all substances after 24-hour incubation. Molecular docking studies were performed to assess compound binding to cytoskeletal proteins. Neutral comet assay and immunocytochemical analysis were used to assess the intensity of apoptosis, and transmission electron microscopy was employed to validate these results at the ultrastructural level.Results and DiscussionThe results showed that the tested compounds had a significantly increased cytotoxic effect on cancer cells compared to normal cells. Furthermore, molecular docking studies indicated that CAPE, biochanin A, and CurE could inhibit actin polymerization, suggesting their potential role in disrupting the cytoskeleton of cancer cells. Increased expression of caspase-3 and PARP-1 in WEHI-164 cells after treatment indicated the induction of apoptosis. Transmission electron microscopy confirmed the presence of cellular stress and vacuolation in cells treated with these compounds, with more pronounced effects observed in cancer cells compared to normal cells. The results indicate that natural NF-κB inhibitors may be capable of selectively targeting cancer cells, reducing their viability and inducing apoptosis while sparing normal cells. This selectivity is of great importance for the development of safer anticancer therapies. The results of this research support the hypothesis that these natural compounds may be effective anticancer agents, particularly in the treatment of fibrosarcoma. Further, in vivo studies and clinical trials are required to gain a full understanding of their mechanisms of action and potential synergies with existing chemotherapeutic agents.

Protocol to fluorescently stain vacuoles in Arabidopsis root cells.

Plant vacuoles are essential organelles that respond to developmental and environmental signals. Here, we present a protocol for staining the vacuole lumen in Arabidopsis root cells, enabling precise visualization of vacuolar dynamics. We describe steps for preparing plant material and staining with commonly used fluorescent dyes. We then detail procedures for visualizing vacuoles in the blue, green, and red emission spectra, allowing for their combined use with a variety of compatible fluorescent-tagged protein markers. For complete details on the use and execution of this protocol, please refer to Dubrovsky etal.,1 Fricker,2 Bassil etal.,3 Grzam etal.,4 and Stefano etal.5.

Vitamin D supplementation alleviates high fat diet-induced metabolic associated fatty liver disease by inhibiting ferroptosis pathway.

Recently, a significant negative correlation has been found between vitamin D (VD) and metabolic associated fatty liver disease (MAFLD), suggesting a potential beneficial role of VD in preventing of MAFLD, while underscoring the importance of exploring its mechanisms. The experiment comprised two parts: male C57BL/6J mice (6 weeks) were fed a high-fat diet (HFD) and intraperitoneally injected with vitamin D3 (VD3) (1.68 IU/g/week) for 16 weeks. Meanwhile, palmitic acid (PA)-induced HepG2 cells were treated with 1,25(OH)2D3 (10 nM). The general conditions of the mice were evaluated by measuring body weight, liver/body weight, serum biochemical parameters, and inflammation indices. Additionally, injury-associated indices and histopathology were used to assess the severity of liver injury. Furthermore, indicators of ferroptosis, including lipid peroxidation, iron aggregation, and the aberrant expression of related proteins, were determined using Prussian blue staining, ELISA assay, and Western blot. Long-term VD3 administration significantly reduced body weight gain and the liver/body weight ratio of HFD-induced MAFLD mice, while also improving serum lipid metabolism dysregulation and enhancing insulin sensitivity. The changes in the expressions of liver injury indices and histological manifestations due to VD3 treatment indicated that VD3 may exerts beneficial effects on liver injury through inhibiting inflammatory cell infiltration and vacuolation. Importantly, VD3 supplementation also inhibited ferroptosis by enhancing the body's antioxidant capacity, reducing local iron aggregation, and modulating the expression levels of ferroptosis-related proteins. These findings were further confirmed in a PA-induced HepG2 steatosis cell model, highlighting the pharmacological effects of VD. VD shows promise in mitigating HFD -induced liver injury by improving metabolic dysregulation and inhibiting ferroptosis, suggesting therapeutic potential in MAFLD.

The Flash Vacuum Expansion Process Increases the Bioaccessibility and Stability of Antioxidant Compounds in Papaya Puree During Digestion

Among all fruits, the papaya ranks among the most significant, occupying fourth place in terms of marketing volumes. The papaya encounters various deterioration issues throughout the marketing chain, which results in the loss of bioactive phytochemicals in the fruit’s pulp. Making puree is the best way to make papaya pulp last longer, but processing can break down antioxidants like phenolic compounds (which are mostly stored in cell vacuoles) and carotenoids (which are stored in chromoplasts). The flash vacuum expansion process (FVE) promotes an expansion of the water present in the vacuoles, which generates cell rupture and consequently, the release of intracellular components. Because cell rupture is promoted from within the cell, the expulsion of bioactive compounds is more efficient and can therefore increase their bioaccessibility. Our results show that the FVE process increased the antioxidant capacity of the purees (measured by TEAC, FRAP, and DPPH assays) before and during digestion. Our results show that the FVE process significantly enhances the bioaccessibility and stability of antioxidant compounds, providing a clear advantage over the conventional method. Fruit purees generated by FVE can be an ingredient that increases the functionality of foods (juices, nectars, purees, ice creams, and yogurt) aimed at people with digestive problems, baby food, or the use of fruits with low commercial value.

Localization of Secondary Metabolites in Relict Gymnosperms of the Genus Sequoia In Vivo and in Cell Cultures In Vitro, and the Biological Activity of Their Extracts.

In order to scientifically search for new sources of secondary metabolites with valuable qualities for phytopharmacognosy, tasks requiring a step-by-step solution were set. The primary task is the development of technologies for obtaining in vitro highly productive biomass of cells of relict gymnosperms of the genus Sequoia, capable of accumulating various classes of secondary metabolites. The study of the accumulation and localization of secondary metabolites allowed us to evaluate the biological activity and cytotoxicity of in vitro Sequoia cultures. In our study, histochemical methods were used to determine the localization of secondary compounds (phenolic and terpenoid in nature) in plant tissues. Secondary metabolites-polyphenols, catechins, and terpenoids-are mainly localized in the epidermal, parenchymal, and conductive tissues of Sequoia leaves and stems. In callus and suspension cultures of Sequoia, secondary metabolites were localized in cell walls and vacuoles. The mineral composition of the nutrient medium (MS and WPM), the light source (photoperiod), and the endogenous content of polyphenols in the primary explant influenced the initiation and growth characteristics of the in vitro culture of Sequoia plants. Inhibition of growth in suspension cultures on the WPM nutrient medium was noted. The cultivation of Sequoia cell lines at a 16 h photoperiod stimulated the formation of polyphenols but had a negative effect on the growth of callus cultures. Extractive substances obtained from intact and callus tissues of evergreen Sequoia demonstrate high biological (fungicidal) activity and cytotoxicity. The inhibitory effect on Fusarium oxisporum was noted when 200 mg/L of Sequoia extract was added to the nutrient medium. Extracts of redwood callus cultures were low in toxicity to normal FetMSC cells but inhibited the growth of lines of "immortal" cervical HeLa cancer cells and human glioblastoma A172. Intact tissues of Sequoia plants and cell cultures initiated from them in vitro are producers of secondary metabolites with high biological activity.