Vacuole Formation Research Articles

Differentiation between Bulinus truncatus and Bulinus hexaploidus by morphological characters, chromosomal study and compatibility with Schistosoma haematobium.

Schistosomiasis is a snail-born, neglected tropical disease (NTD) caused by blood flukes (trematode worms) of the genusSchistosoma. It is the second most socioeconomically devastating parasitic disease after malaria. Urogenital schistosomiasis is caused by Schistosoma haematobium which is transmitted by snail intermediate host of the genus Bulinus. This genus is a model system for the study of polyploidy in animals. This study aims to investigate ploidy levels existing among the Bulinus species and their compatibility with S. haematobium. The specimens were collected from two governorates in Egypt. Chromosomal preparation was made from gonad tissue (ovotestis). This study found two ploidy levels (tetraploid, n=36 and hexaploid, n=54) of B. truncatus/tropicus complex in Egypt. Tetraploid B. truncatus was found in El-Beheira governorate while-unexpectedly and for the first time in Egypt, the hexaploid population was found in Giza governorate. This identification focused on shell morphology, chromosomal count, and spermatozoa of each species. Afterward, all species were exposed to S. haematobium miracidia where B. hexaploidus snails were the only refractory species. The histopathological study showed early destruction and abnormal development of S. haematobium in B. hexaploidus tissues. In addition, the hematological investigation showed increasing in the total hemocyte count, the formation of vacuoles, several pseudopodia, and more dense granules in the hemocytes of infected B. hexaploidus snails. In conclusion, there were two types of snails one was refractory and the other was susceptible.

Biogenic silver nanoparticle exhibits schistosomicidal activity in vitro and reduces the parasitic burden in experimental schistosomiasis mansoni

Schistosomiasis is a neglected tropical parasitic disease that affects millions of people, being the second most prevalent parasitic disease worldwide. The current treatment has limited effectiveness, drug-resistant strains, and is not effective in different stages of the disease. This study investigated the antischistosomal activity of biogenic silver nanoparticles (Bio-AgNp) against Schistosoma mansoni. Bio-AgNp presented direct schistosomicidal activity on newly transformed schistosomula causing plasma membrane permeabilization. In S. mansoni adult worms, reduced the viability and affected the motility, increasing oxidative stress parameters, and inducing plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid bodies accumulation, and autophagic vacuoles formation. During the experimental schistosomiasis mansoni model, Bio AgNp restored body weight, reduced hepatosplenomegaly, and decrease the number of eggs and worms in feces and liver tissue. The treatment also ameliorates liver damage and reduces macrophage and neutrophil infiltrates. A reduction in count and size was evaluated in the granulomas, as well as a change to an exudative-proliferative phase, with a local increase of IFN-γ. Together our results showed that Bio-AgNp is a promising therapeutic candidate for studies of new therapeutic strategies against schistosomiasis.

The structure of prevacuolar compartments in Neurospora crassa as observed with super-resolution microscopy.

The hyphal tips of Neurospora crassa have prevacuolar compartments (PVCs) of unusual size and shape. They appear to function as late endosomes/multivesicular bodies. PVCs are highly variable in size (1-3 microns) and exhibit rapid changes in structure. When visualized with tagged integral membrane proteins of the vacuole the PVCs appear as ring or horseshoe-shaped structures. Some soluble molecules that fill the lumen of mature spherical vacuoles do not appear in the lumen of the PVC but are seen in the ring or horseshoe-shaped structures. By using super-resolution microscopy I have achieved a better understanding of the structure of the PVCs. The PVC appears to form a pouch with an open end. The walls of the pouch are composed of small vesicles or tubules, approximately 250 nm in diameter. The shape of the PVC can change in a few seconds, caused by the apparent movement of the vesicles/tubules. In approximately 85% of the PVCs dynein and dynactin were observed as poorly defined lumps inside the pouch-shaped PVCs. Within the PVCs they were not attached to microtubules nor did they appear to be in direct contact with the vesicles and tubules that formed the PVCs. In the future, the structure and relatively large size of the Neurospora PVC may allow us to visualize protein-sorting events that occur in the formation of vacuoles.

The presence of vacuoles in blastocysts is negatively associated with euploidy and live birth rates

To investigate whether the presence of vacuoles in biopsied blastocysts is associated with the likelihood of aneuploidy and clinical outcomes. Retrospective observational study. This study retrospectively analyzed data obtained through preimplantation genetic testing for aneuploidy (PGT-A) performed on 3351 blastocysts from 826 patients at a single reproductive center between August 2018 and July 2020. Ultimately, 167 single euploid blastocyst transfers were performed in these patients. Vacuoles existing in the trophectoderm or inner cell mass were observed using blastocyst biopsy. After biopsy, all blastocysts were vitrified, and embryo transfer was performed in a subsequent treatment cycle. Presence versus absence of vacuoles. The associations between vacuoles and euploidy or live birth rates were assessed using logistic regression models and estimated adjusted odds ratios [ORs] and 95% confidence intervals (CIs). Of the 3351 blastocysts from 826 patients, 903 (26.9%) were discovered to have vacuoles. The vacuole-positive group had a significantly lower percentage of euploid blastocysts after TE biopsy than did the vacuole-negative group (28.8% vs. 35.5%; p < 0.001). Embryos with vacuoles were significantly more likely to be poor quality (30.6% vs. 18.2%, p < 0.001). Logistic regression analyses revealed that euploid blastocysts were positively associated with the absence of vacuoles, maternal age, and good embryo quality (vacuole-negative group: adjusted OR= 1.291, 95% CI = 1.089-1.530; age < 38 years: adjusted OR= 1.989, 95% CI = 1.692-2.337; good embryo quality: adjusted OR = 1.703, 95% CI = 1.405-2.064). The implantation rate and live birth rate were significantly lower for the transferred single euploid blastocysts with vacuoles than for the blastocysts without vacuoles (35.5% vs. 56.6%, p = 0.033; 29.0% vs. 52.2%, p = 0.020, respectively). The live birth rate was positively associated with the absence of vacuoles (adjusted OR = 2.792, 95% CI = 1.180-6.608). The formation of vacuoles in blastocysts is associated with lower rates of euploidy and live birth. Blastocysts without vacuoles should thus be prioritized for embryo transfer in in vitro fertilization cycles.

Vacuole membrane contact sites regulate liquid-ordered domain formation during glucose starvation.

Liquid-ordered (Lo) membrane domains have been proposed to play important roles in a wide variety of biological processes, such as protein sorting and cell signaling. However, the mechanisms by which they are formed and maintained remain poorly understood. Lo domains are formed in the vacuolar membrane of yeast in response to glucose starvation. Here, we show that the deletion of proteins that localize to vacuole membrane contact sites (MCSs) caused a marked decrease in the number of cells with Lo domains. In addition to Lo domain formation, autophagy is induced upon glucose starvation. However, the deletion of core autophagy proteins did not inhibit Lo domain formation. Thus, we propose that vacuolar Lo domain formation during glucose restriction is regulated by MCSs but not by autophagy.

Abstract 483: Hederacolchiside A1 inhibits autophagy via CTSC inhibition and inhibits growth in colon cancer

Abstract Background: Autophagy is a normal regulatory process that degrades non-functional or unnecessary cellular components. The degradation of components provides materials for the synthesis of cellular components and, during tumor development, may provide energy. Therefore, autophagy regulation may have potential therapeutic uses in oncology. The triterpenoid saponin hederacolchiside A1 (HA1) has potent antitumor effects; however, its role in autophagy is still unknown. Methods: In the present study, we used human colon cancer cells (SW480 and HT29) and mouse colon cancer cells (CT26) for the main experiments, and several additional cancer cell lines for other experiments. To assess the autophagy dysregulation caused by HA1, we evaluated the protein expressions of two autophagy markers (LC3 and SQSTM1) using Western blotting and the changes in organelles using LC3 puncta assay and transmission electron microscopy. We compared the effects of HA1 and other autophagy inhibitors. Additionally, we compared the HA1-induced decrease in cathepsin C (CTSC) expression in tumors and non-tumors using publicly available data. The effect of HA1 on tumor growth was evaluated in vitro using a three-dimensional spheroid model and patient-derived colon organoids, and in vivo based on tumor growth in a subcutaneous injection mouse model. Results: HA1 treatment resulted in marked vacuolization of colon and other cancer cells. Additionally, autophagy was induced by the accumulation of LC3-II and SQSTM1. These findings were supported by increased LC3 puncta and abnormal organelle morphology. Compared to treatment with other autophagy inhibitors, HA1 treatment resulted in the formation of distinct vacuoles and increased LC3 and SQSTM1, similar to chloroquine treatment. The publicly available data showed that the lysosomal protease CTSC was increased in several cancers, including colon cancer, compared to the normal tissues. Furthermore, HA1 decreased the CTSC expression and enzymatic activity independently of the proteasome degradation, resulting in autophagy dysregulation and, subsequently, autophagy flux inhibition. HA1 inhibited cell growth and induced cell cycle arrest in the two-dimensional culture, three-dimensional spheroids, patient-derived colon cancer organoids, and the in vivo experiments. Conclusion: HA1 inhibits cell growth and induces autophagy dysregulation. Therefore, HA1 may be a potential therapeutic agent in colon cancer. Citation Format: Solbi Kim, Eunji Kim, Minju Han, Youn Seo Oh, Sora Kang, Heung Jin Jeon, Hyo Jin Lee. Hederacolchiside A1 inhibits autophagy via CTSC inhibition and inhibits growth in colon cancer [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 483.

Repurposing picropodophyllin as a potential thyroid eye disease treatment via delaying mitotic clonal expansion through a patient‐derived preclinical platform

Repurposing picropodophyllin as a potential thyroid eye disease treatment via delaying mitotic clonal expansion through a patient‐derived preclinical platform

Meroterpenoids from Gongolaria abies-marina against Kinetoplastids: In Vitro Activity and Programmed Cell Death Study.

Leishmaniasis and Chagas disease affect millions of people worldwide. The available treatments against these parasitic diseases are limited and display multiple undesired effects. The brown alga belonging to the genus Gongolaria has been previously reported as a source of compounds with different biological activities. In a recent study from our group, Gongolaria abies-marine was proven to present antiamebic activity. Hence, this brown alga could be a promising source of interesting molecules for the development of new antiprotozoal drugs. In this study, four meroterpenoids were isolated and purified from a dichloromethane/ethyl acetate crude extract through a bioguided fractionation process targeting kinetoplastids. Moreover, the in vitro activity and toxicity were evaluated, and the induction of programmed cell death was checked in the most active and less toxic compounds, namely gongolarone B (2), 6Z-1'-methoxyamentadione (3) and 1'-methoxyamentadione (4). These meroterpenoids triggered mitochondrial malfunction, oxidative stress, chromatin condensation and alterations of the tubulin network. Furthermore, a transmission electron microscopy (TEM) image analysis showed that meroterpenoids (2-4) induced the formation of autophagy vacuoles and ER and Golgi complex disorganization. The obtained results demonstrated that the mechanisms of action at the cellular level of these compounds were able to induce autophagy as well as an apoptosis-like process in the treated parasites.

BMP9 promotes autophagy and inhibits migration and invasion in breast cancer cells through the c-Myc/SNHG3/mTOR signaling axis

We previously reported that BMP9 inhibited breast cancer progression. However, the precise molecular mechanism is still unknown. Based on our RNA-sequencing (RNA-seq) results, BMP9 significantly down-regulated the expression of long non-coding RNA SNHG3. Exogenous BMP9 promoted autophagy and inhibited migration and invasion in MDA-MB-231 cells, which was effectively blunted by SNHG3 overexpression. Interestingly, SNHG3 was negatively connected with autophagy. Knockdown of SNHG3 induced autophagy by increasing the formation of autophagic vacuoles and thus inhibited the migration and invasion of MDA-MB-231 cells. Mechanically, BMP9-SNHG3 activated AMPK, AKT and mTOR signaling pathways to induce autophagy and inhibit migration and invasion. Meanwhile, BMP9 regulated SNHG3 transcription by suppressing c-Myc entry into the nucleus. In conclusion, BMP9 promotes autophagy and inhibits migration and invasion in breast cancer cells through the c-Myc/SNHG3/mTOR signaling axis, which might offer a fresh perspective on BMP9's breast cancer-inhibiting properties.

LncRNA PRKCQ-AS1 regulates paclitaxel resistance in triple-negative breast cancer cells through miR-361-5p/PIK3C3 mediated autophagy.

Paclitaxel (PTX) resistance is a key cause of chemotherapy failure in patients with triple negative breast cancer (TNBC). The aim of this study is to investigate the effect and mechanism of long non-coding RNA (lncRNA) on the PTX resistance of TNBC cells through autophagy. MDA-MB-231 cells are used to induce the PTX-resistant TNBC cell line MDA-MB-231.PR (MDR) by increasing dose intermittently. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the mRNA levels of phosphoinositide-3-kinase class 3 (PIK3C3), miR-361-5p and lncRNA PRKCQ-AS1 in the cells, and Western blot analysis was used to detect the protein expressions of PIK3C3, autophagy-related, drug-resistant and apoptosis-related genes. MDC staining detected the formation of autophagic vacuoles. The interactions between miR-361-5p and PIK3C3 and between lncRNA PRKCQ-AS1 and miR-361-5p were verified by dual-luciferase assay. Cell viability, apoptosis, migration and invasion were assessed by performing MTT, flow cytometry assay, and transwell assay. The mRNA level of miR-361-5p and the autophagy and drug resistance levels of TNBC PTX-resistant cells were significantly up-regulated. miR-361-5p could target autophagy-related gene PIK3C3, and overexpression of miR-361-5p could down-regulate PIK3C3 protein expression and autophagy level and PTX resistance of MDR cells. LncRNA PRKCQ-AS1 was selected through bioanalysis, and miR-361-5p could target lncRNA PRKCQ-AS1. In addition, lncRNA PRKCQ-AS1 level was up-regulated in TNBC PTX-resistant cells, and knockdown of lncRNA PRKCQ-AS1 could weaken autophagy and drug resistance level and could promote cell apoptosis. Overexpression of lncRNA PRKCQ-AS1 reversed the pro-apoptotic effect and down-regulation of autophagy and resistance levels was induced by miR-361-5p. In vivo experiments were performed to verify the role of lncRNA PRKCQ-AS1. We demonstrate that down-regulation of lncRNA PRKCQ-AS1 weakened PTX resistance and promoted cell apoptosis by miR-361-5p/PIK3C3 mediated autophagy.

MESENCHYMAL STEM-LIKE STROMAL CELLS FROM HUMAN SUBCUTANEOUS FAT AND POLYVINYLPYRROLIDONE-BASED POLYMERIC HYDROGELS: TOXICITY AND ADHESION

Relevance. Mesenchymal stem-like stromal cells (MSCs) represent a promising material for the therapy of restoration or regeneration of bone and cartilage tissues in the treatment of patients with injuries and diseases of the musculoskeletal system. The use of hydrogels for cultivating MSCs will make it possible to protect them after being introduced into the body from to the potentially “hostile” environment of damaged or diseased tissues. To date, the properties of MSCs in their cultivation on matrices based on natural and synthetic materials have not been sufficiently studied. The aim of this work is to evaluate the toxicity and adhesive properties of hydrogels based on polyvinylpyrrolidone (PVP), obtained by two technolo-gies, for MSCs from human adipose tissue (AT) in vitro. Material and methods. Four sterile PVP hydrogels were used in the experiment, differing in the technology of preparation, the presence or absence of antibiotics. Toxicity was assessed by the number of vital cells (stained with 0.1% trypan blue solution) after adding the MSC suspension to the gels after 24 h. The behavior of MSCs (AT) was studied in dynamics (on days 2 and 5 of cultivation) in terms of the rate and quality of the formed cell monolayer. Morphological analysis of cells, the state of chromatin in the nucleus, and the presence of cytoplasmic inclusions were performed in sam-ples stained with Giemsa dye. Results. As a result, it was found that human MSCs (AT) are able to attach to the surface of all 4 matrices, represented by a physicochemically modi-fied artificial material based on PVP. Non-toxicity of all 4 hydrogels and their suitability for short-term cell culture were revealed. MSCs (AT) on PVP hy-drogels with the addition of antibiotics showed slower growth, while morphological changes were observed in the form of vacuoles in the protoplasm and pycnosis in the chromatin of the cell nucleus. Conclusion. Thus, the results obtained can be used for further research and improvement of methods for analyzing the cytotoxicity of the drugs un-der development.

Mechanosensitive channel MscL induces non-apoptotic cell death and its suppression of tumor growth by ultrasound.

Mechanosensitive channel of large conductance (MscL) is the most thoroughly studied mechanosensitive channel in prokaryotes. Owing to its small molecular weight, clear mechanical gating mechanism, and nanopore forming ability upon opening, accumulating studies are implemented in regulating cell function by activating mechanosensitive channel of large conductance in mammalian cells. This study aimed to investigate the potentials of mechanosensitive channel of large conductance as a nanomedicine and a mechano-inducer in non-small cell lung cancer (NSCLC) A549 cells from the view of molecular pathways and acoustics. The stable cytoplasmic vacuolization model about NSCLC A549 cells was established via the targeted expression of modified mechanosensitive channel of large conductance channels in different subcellular organelles. Subsequent morphological changes in cellular component and expression levels of cell death markers are analyzed by confocal imaging and western blots. The permeability of mitochondrial inner membrane (MIM) exhibited a vital role in cytoplasmic vacuolization formation. Furthermore, mechanosensitive channel of large conductance channel can be activated by low intensity focused ultrasound (LIFU) in A549 cells, and the suppression of A549 tumors in vivo was achieved by LIFU with sound pressure as low as 0.053MPa. These findings provide insights into the mechanisms underlying non-apoptotic cell death, and validate the nanochannel-based non-invasive ultrasonic strategy for cancer therapy.

Hederacolchiside A1 Suppresses Autophagy by Inhibiting Cathepsin C and Reduces the Growth of Colon Cancer.

While autophagy degrades non-functional or unnecessary cellular components, producing materials for synthesizing cellular components, it can also provide energy for tumor development. Hederacolchiside A1 (HA1) derived from anemone raddeana has anticancer effects on several carcinomas by inducing apoptosis or exhibiting cytotoxicity, but the relationship with autophagy has not been studied. We investigated the association between HA1 and autophagy and evaluated its anticancer effect on colon cancer. HA1 induced accumulation of the autophagy-related markers LC3B and SQSTM1, with distinct vacuolar formation, unlike other autophagy inhibitors; the effects were similar to those of chloroquine. In addition, HA1 decreased the expression and proteolytic activity of lysosomal protein cathepsin C, reduced the growth of colon cancer cells in vitro, and inhibited tumor growth in vivo. It also reduced the expression of Ki-67 and cathepsin C in mouse tissues and reduced the growth of spheroids and organoids composed of cancer cells. Taken together, these results imply that HA1 regulates cell growth and autophagy and has potential as a promising therapeutic agent in colon cancer.

Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis.

In Arabidopsis thaliana, female gametophyte (FG) development is accompanied by the formation and expansion of the large vacuole in the FG; this is essential for FG expansion, nuclear polar localization, and cell fate determination. Arabidopsis VACUOLELESS GAMETOPHYTES (VLG) facilitates vesicular fusion to form large vacuole in the FG, but the regulation of VLG remains largely unknown. Here, we found that gain-of-function mutation of BRASSINOSTEROID INSENSITIVE2 (BIN2) (bin2-1) increases VLG abundance to induce the vacuole formation at stage FG1, and leads to abortion of FG. Loss-of-function mutation of BIN2 and its homologs (bin2-3 bil1 bil2) reduced VLG abundance and mimicked vlg/VLG phenotypes. Knocking down VLG in bin2-1 decreased the ratio of aberrant vacuole formation at stage FG1, whereas FG1-specific overexpression of VLG mimicked the bin2-1 phenotype. VLG partially rescued the bin2-3 bil1 bil2 phenotype, demonstrating that VLG acts downstream of BIN2. Mutation of VLG residues that are phosphorylated by BIN2 altered VLG stability and a phosphorylation mimic of VLG causes similar defects as did bin2-1. Therefore, BIN2 may function by interacting with and phosphorylating VLG in the FG to enhance its stability and abundance, thus facilitating vacuole formation. Our findings provide mechanistic insight into how the BIN2-VLG module regulates the spatiotemporal formation of the large vacuole in FG development.

LncR-GAS5 upregulates the splicing factor SRSF10 to impair endothelial autophagy, leading to atherogenesis.

Long noncoding RNAs (lncRNAs) play a critical role in the regulation of atherosclerosis. Here, we investigated the role of the lncRNA growth arrest-specific 5 (lncR-GAS5) in atherogenesis. We found that the enforced expression of lncR-GAS5 contributed to the development of atherosclerosis, which presented as increased plaque size and reduced collagen content. Moreover, impaired autophagy was observed, as shown by a decreased LC3II/LC3I protein ratio and an elevated P62 level in lncR-GAS5-overexpressing human aortic endothelial cells. By contrast, lncR-GAS5 knockdown promoted autophagy. Moreover, serine/arginine-rich splicing factor 10 (SRSF10) knockdown increased the LC3II/LC3I ratio and decreased the P62 level, thus enhancing the formation of autophagic vacuoles, autolysosomes, and autophagosomes. Mechanistically, lncR-GAS5 regulated the downstream splicing factor SRSF10 to impair autophagy in the endothelium, which was reversed by the knockdown of SRSF10. Further results revealed that overexpression of the lncR-GAS5-targeted gene miR-193-5p promoted autophagy and autophagic vacuole accumulation by repressing its direct target gene, SRSF10. Notably, miR-193-5p overexpression decreased plaque size and increased collagen content. Altogether, these findings demonstrate that lncR-GAS5 partially contributes to atherogenesis and plaque instability by impairing endothelial autophagy. In conclusion, lncR-GAS5 overexpression arrested endothelial autophagy through the miR-193-5p/SRSF10 signaling pathway. Thus, miR-193-5p/SRSF10 may serve as a novel treatment target for atherosclerosis.

Autophagy induced macrophages by α-alumina(α-AL2O3) conjugated cysteine peptidase, enhances the cytotoxic activity of CD8+ T lymphocytes against Leishmania major.

Induction of a protective immune response against Leishmania major requires the activation of both TH1 and CD8+ T lymphocytes. Because L. major is an intra-phagosomal parasite, its antigens do not have access to MHC-I. The present study aimed to evaluate the effect of cysteine peptidase A (CPA)/cysteine peptidase B (CPB) conjugated to α-AL2O3 on autophagy induction in L. major infected macrophages and subsequent activation of cytotoxic CD8+ T lymphocytes. Recombinant CPA and CPB of L. major were produced in expression vectors and purified. Aldehyde functionalized α-AL2O3 were conjugated to hydrazine-modified CPA/CPB by a chemical bond was confirmed by Fourier-transform infrared spectroscopy (FTIR). The High efficient internalization of α-AL2O3 conjugated CPA/CPB to macrophages was confirmed using a fluorescence microscope and flowcytometry. Induction of the acidic autophagosome and LC3 conversion in macrophages was determined by acridine orange (AO) staining and western blot. Autophagy-activated macrophages were used for CD8+ T cell priming. Cytotoxic activity of the primed CD8+ T cell against L. major infected macrophages was measured using apoptosis assay. α-AL2O3 conjugated CPA/CPB enhances macrophages antigen uptake and increases acidic vacuole formation and LC-3I to LC-3II conversion. Co-culture of autophagy-activated macrophages with CD8+ T cells augmented CD8+ T cells priming and proliferation more than in other study groups. These primed CD8+ T cells induce significant apoptotic death of L. major infected macrophages compared with non-primed CD8+ T cells. α-AL2O3 nanoparticles enhance the cross-presentation of L. major antigens to CD8+ T cells by inducing autophagy. This finding supports the positive role of autophagy and encourages the use of α-AL2O3 in vaccine design.

Early Subcellular Hepatocellular Alterations in Mice Post Hydrodynamic Transfection: An Explorative Study.

Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10-20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane.

HMGCS2-Induced Autophagic Degradation of Tau Involves Ketone Body and ANKRD24.

Accumulation of hyperphosphorylated Tau (pTau) contributes to the formation of neurofibrillary tangles in Alzheimer's disease (AD), and targeting Tau/pTau metabolism has emerged as a therapeutic approach. We have previously reported that mitochondrial 3-hydroxy-3-methylglutaryl-COA synthase 2 (HMGCS2) is involved in AD by promoting autophagic clearance of amyloid-β protein precursor via ketone body-associated mechanism, whether HMGCS2 may also regulate Tau metabolism remains elusive. The present study was to investigate the role of HMGCS2 in Tau/p degradation. The protein levels of Tau and pTau including pT217 and pT181, as well as autophagic markers LAMP1 and LC3-II were assessed by western blotting. The differentially regulated genes by HMGCS2 were analyzed by RNA sequencing. Autophagosomes were assessed by transmission electron microscopy. HMGCS2 significantly decreased Tau/pTau levels, which was paralleled by enhanced formation of autophagic vacuoles and prevented by autophagic regulators chloroquine, bafilomycin A1, 3-methyladenine, and rapamycin. Moreover, HMGCS2-induced alterations of LAMP1/LC3-II and Tau/pTau levels were mimicked by ketone body acetoacetate or β-hydroxybutyrate. Further RNA-sequencing identified ankyrin repeat domain 24 (ANKRD24) as a target gene of HMGCS2, and silencing of ANKRD24 reduced LAMP1/LC3-II levels, which was accompanied by the altered formation of autophagic vacuoles, and diminished the effect of HMGCS2 on Tau/pTau. HMGCS2 promoted autophagic clearance of Tau/pTau, in which ketone body and ANKRD24 played an important role.

Polysucrose hydrogel loaded with natural molecules/extracts for multiphase-directed sustainable wound healing.

Natural molecules/extracts have numerous beneficial effects on wound healing processes which are challenged by appropriate use and non-toxic dosage. Polysucrose-based (PSucMA) hydrogels have been synthesized with in situ loading of one or more natural molecules/extracts namely Manuka honey (MH), Eucalyptus honey (EH1, EH2), Ginkgo biloba (GK), thymol (THY) and metformin (MET). EH1 presented low amounts of hydroxymethylfurfural and methylglyoxal compared to MH indicating that EH1 was not temperature-abused. It also showed high diastase activity and conductivity. GK was added to PSucMA solution along with other additives including MH, EH1 and MET and crosslinked to form dual loaded hydrogels. The in vitro release profiles of EH1, MH, GK and THY from the hydrogels followed the exponential Korsmeyer-Peppas equation, with a release exponent value of less than 0.5 indicating a quasi-Fickian diffusion mechanism. The IC50 values of these natural products using L929 fibroblasts and RAW 264.7 macrophages indicated that EH1, MH and GK were cytocompatible at relatively high concentrations compared to MET, THY and curcumin used as a control. MH and EH1 induced high IL6 concentration compared to GK. In vitro studies were modelled to mimic the overlapping wound healing phases using human dermal fibroblasts (HDFs), macrophages and human umbilical endothelial cells (HUVECs) in dual culture. HDFs showed a highly interconnected cellular network on GK loaded scaffolds. EH1 loaded scaffolds were seen to induce formation of spheroids which increased in number and size in co-culture studies. The SEM images of HDF/HUVEC seeded GK, GKMH and GKEH1 loaded hydrogels indicated formation of vacuoles and lumen structures. These results indicated that a combination of GK and EH1 in the hydrogel scaffold would accelerate tissue regeneration by acting on the four overlapping phases of wound healing.

Digestive enzymes (amylase and invertase) analysis and histopathological changes in Oreochromis mossambicus treated with tricyclazole (75%) fungicide

Despite being efficient against rice blast fungal diseases when applied to agricultural fields, tricyclazole 75% fungicide has a variety of drawbacks that outweigh its benefits for the aquatic environment. Shortterm exposure to a sublethal dose of tricyclazole, fish O. mossambicus displayed altered digestive enzymes (amylase and invertase) as well as histological abnormalities in tissue organs like the skin, muscle, gills, liver, intestine, and brain tissues. The activity of amylase and invertase was found to decreasing and increasing in various concentrations. Histopathological changes on exposure to tricyclazole fungicides was found to increase in its pathological lesions of the tissue organs studied skin, muscle, gills, liver, intestine, and brain tissues of O. mossambicus noticed with necrosis, vacuolar degeneration, fusion and atrophy of the primary and secondary gill lamellae, degeneration of the cytoplasm in hepatocytes, atrophy, formation of vacuoles, rupture in blood vessels, damage in the dermis, concentrated melanophores, damaged blood vessels, and enlarged muscle fibers with increasing concentrations. Enzymatic alterations and pathological lesions in the tissue of O. mossambicus exposed to a toxicant at a sub-lethal concentration are a functional response of organisms that offers information on the nature of the toxicant. Hence, in the present study, in order to evaluate the environmental/aquatic pollution under experimental settings, an evaluation of the fish’s histological alterations and enzyme levels was carried out. In conclusion, fish are among the most susceptible aquatic species to harmful compounds; buildup of the fungicide in the fish may cause pathological problems that spread to people through the food chain.