Godanti bhasma (anhydrous CaSO4) induces massive cytoplasmic vacuolation in mammalian cells: A model for phagocytosis assay.

Phagocytosis is an essential physiological mechanism; its impairment is associated with many diseases. A highly smart particle is required for understanding detailed sequential cellular events in phagocytosis. Recently, we identified an Indian traditional medicine named Godanti Bhasma (GB), a bioactive calcium sulfate particle prepared by thermo-transformation ofgypsum. Thermal processing of the gypsum transforms its native physicochemical properties by removing water molecules into the anhydrous GB, which was confirmed by Raman and FT-IR spectroscopy. GB particle showed a 0.5-5 µm size range and a neutral surface charge. Exposure of mammalian cells to GB particles showed a rapid cellular uptake through phagocytosis and induced massive cytoplasmic vacuolation in cells. Interestingly, no cellular uptake and cytoplasmic vacuolation were observed with the parent gypsum particle. The presence of the GB particles in intra-vacuolar space was confirmed using FESEM coupled with EDX. Flow cytometry analysis and live tracking of GB-treated cells showed particle internalization, vacuole formation, particle dissolution, and later vacuolar turnover. Quantification of GB-induced vacuolation was done using neutral red uptake assay in cells. Treatment of lysosomal inhibitors (BFA1 or CQ) with GB could not induce vacuolation, suggesting the requirement of an acidic environment for the vacuolation. In the mimicking experiment, GB particle dissolution in acidic cell-free solution suggested that degradation of GB occurs by acidic pH inside the cell vacuole. Vacuole formation generally accompanies with cell death, whereas GB-induced massive vacuolation does not cause cell death. Moreover, the cell divides and proliferates with the vacuolar process, intra-vacuolar cargo degradation, and eventually vacuolar turnover. Taken together, the sequential cellular events in this study suggest that GB can be used as a smart particle for phagocytosis assay development in animal cells.

Loss of Prom1 impairs autophagy and promotes epithelial-mesenchymal transition in mouse retinal pigment epithelial cells.

Mutations in the Prominin-1 (Prom1) gene disrupt photoreceptor disk morphogenesis, leading to macular dystrophies. We have shown that human retinal pigment epithelial (RPE) homeostasis is under the control of Prom1-dependent autophagy, demonstrating that Prom1 plays different roles in the photoreceptors and RPE. It is unclear if retinal and macular degeneration caused by the loss of Prom1 function is a cell-autonomous feature of the RPE or a generalized disease of photoreceptor degeneration. In this study, we investigated whether Prom1 is required for mouse RPE (mRPE) autophagy and phagocytosis, which are cellular processes essential for photoreceptor survival. We found that Prom1-KO decreases autophagy flux, activates mTORC1, and concomitantly decreases transcription factor EB (TFEB) and Cathepsin-D activities in mRPE cells. In addition, Prom1-KO reduces the clearance of bovine photoreceptor outer segments in mRPE cells due to increased mTORC1 and reduced TFEB activities. Dysfunction of Prom1-dependent autophagy correlates with both a decrease in ZO-1 and E-cadherin and a concomitant increase in Vimentin, SNAI1, and ZEB1 levels, consistent with induction of epithelial-mesenchymal transition (EMT) in Prom1-KO mRPE cells. Our results demonstrate that Prom1-mTORC1-TFEB signaling is a central driver of cell-autonomous mRPE homeostasis. We show that Prom1-KO in mRPE leads to RPE defects similar to that seen in atrophic age-related macular degeneration and opens new avenues of investigation targeting Prom1 in retinal degenerative diseases.

Impact of Pb Toxicity on the Freshwater Pearl Mussel, Lamellidens marginalis: Growth Metrics, Hemocyto-Immunology, and Histological Alterations in Gill, Kidney, and Muscle Tissue.

Pb is one of the most extensively used harmful heavy metals in Bangladesh, and its occurrence in waters affects aquatic organisms significantly. The tropical pearl mussel, Lamellidens marginalis, was exposed to different concentrations (T1 21.93 mgL-1, T2 43.86 mgL-1, and T3 87.72 mgL-1) of Pb(NO3)2 and was evaluated against a control C 0 mgL-1 of Pb(NO3)2, followed by a 96 h acute toxicity test. The LC50 value was recorded as 219.32 mgL-1. The physicochemical parameters were documented regularly for each treatment unit. The values of % SGR, shell weight, soft tissue wet weight, and weight gain remained statistically higher for the control group in comparison with the treatment. No mortality was noted for control units, while a gradually decreased survival rate was recorded for the different treatment groups. Fulton's condition factor was recorded as highest in the control and lowest in the T3 unit, while the condition indices did not vary between the control and treatment groups. The hemocyte was accounted as maximum in the control and T1, while minimum in T2 and T3. The serum lysosomal parameters also followed a similar pattern, and a significantly low level of lysosomal membrane stability, and serum lysosome activity was noted for T3 and T2 units in comparison to the control group. The histology of the gill, kidney, and muscle was well structured in the control group, while distinct pathologies were observed in the gill, kidney, and muscle tissue of different treatment groups. The quantitative comparison revealed that the intensity of pathological alteration increased as the dosage of Pb increased. The current study, therefore, indicated that intrusion of Pb(NO3)2 in the living medium significantly alters growth performance and hemocyte counts, and chronic toxicity induces histomorphological abnormalities in vital organs.

San-wei-tan-xiang capsule attenuates atherosclerosis by increasing lysosomal activity in adipose tissue macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Dyslipidemia is the leading risk factor of atherosclerosis (AS). Adipose tissue macrophages (ATMs) can regulate postprandial cholesterol levels via uptake and hydrolyzation of lipids and regulation of macrophage cholesterol efflux (MCE). San-wei-tan-xiang (SWTX) capsule, a Traditional Chinese medicine, exerts clinical benefits in patients with atherosclerotic cardiovascular diseases. AIM OF THE STUDY: This work is aimed to evaluate the chemical ingredients and mechanisms of SWTX in anti-AS. MATERIALS AND METHODS: The chemical ingredients of SWTX identified by liquid chromatography coupled with tandem mass spectrometry were used for network pharmacological analysis. The atheroprotective function of SWTX was evaluated in ApoE-/- mice fed a cholesterol-enriched diet. RESULTS: The chemical ingredients identified in SWTX were predicated to be important for lipid metabolism and AS. Animals studies suggested that SWTX effectively attenuated the atherosclerotic plaque growth, elevated postprandial HDL cholesterol levels, elevated the proportion of Tim4 and CD36-expressed ATMs, and upregulated the uptake of lipid and lysosomal activity in ATMs. SWTX-induced elevation of postprandial HDL cholesterol levels was dependent on increased lysosomal activity, since chloroquine, an inhibitor of lysosomal function, blocked the effect of SWTX. Lastly, some predicated bioactive compounds in SWTX can elevate lysosomal activity in vitro. CONCLUSION: SWTX could attenuate atherosclerotic plaque formation by elevating lysosomal activity and enhancing MCE in ATMs.

Caveolin-1 influences mitochondrial plasticity and function in hepatic stellate cell activation.

Caveolin-1 (Cav-1) is an integral membrane protein present in all organelles, responsible for regulating and integrating multiple signals as a platform. Mitochondria are extremely adaptable to external cues in chronic liver diseases, and expression of Cav-1 may affect mitochondrial flexibility in hepatic stellate cells (HSCs) activation. We previously demonstrated that exogenous expression of Cav-1 was sufficient to increase some classical markers of activation in HSCs. Here, we aimed to evaluate the influence of exogenous expression and knockdown of Cav-1 on regulating the mitochondrial plasticity, metabolism, endoplasmic reticulum (ER)-mitochondria distance, and lysosomal activity in HSCs. To characterize the mitochondrial, lysosomal morphology, and ER-mitochondria distance, we perform transmission electron microscope analysis. We accessed mitochondria and lysosomal networks and functions through a confocal microscope and flow cytometry. The expression of mitochondrial machinery fusion/fission genes was examined by real-time polymerase chain reaction. Total and mitochondrial cholesterol content was measured using Amplex Red. To define energy metabolism, we used the Oroboros system in the cells. We report that GRX cells with exogenous expression or knockdown of Cav-1 changed mitochondrial morphometric parameters, OXPHOS metabolism, ER-mitochondria distance, lysosomal activity, and may change the activation state of HSC. This study highlights that Cav-1 may modulate mitochondrial function and structural reorganization in HSC activation, being a potential candidate marker for chronic liver diseases and a molecular target for therapeutic intervention.

Structurally diverse diterpenoids from Isodon oresbius and their bioactivity.

Twelve new diterpenoids, isoresbins A-L (1-12), together with twenty-eight known ones, were isolated from the aerial parts of Isodon oresbius. Their diverse structures included 6,7-seco-ent-kaurane, 7,20-epoxy-ent-kaurane, 6,7:8,15-diseco-ent-kaurane, and abietanes skeletons, which were elucidated by spectroscopic data interpretation, single-crystal X-ray diffraction, and quantum chemical calculation. Isoresbins A (1) and B (2) possessed a new rearranged 15(8 â†’ 11)-abeo-6,7-seco-ent-kaurane skeleton. 1 and 5 promoted lysosomal function, which was evaluated by LysoTracker Red staining and DQ-ovalbumin dequenching assay. 1 showed cytotoxicity against six human tumor cell lines with IC50 values in 2.07-4.04 µM range. Moreover, 1 induced damage of mitochondrial membrane potential, G2/M cell cycle arrest and apoptosis in SW480 cells.

Thrombospondin-1 aggravates colonic mucosal inflammatory injuries via promoting the differentiation of CD11c+ macrophages with lysosomal activity limited in colitis.

BACKGROUND: Increased CD11c+ Mφ aggravates colonic mucosal injuries in ulcerative colitis (UC) with TSP1 protein increased. The thrombospondin-1 (TSP1) protein which could activate Mφ is closely related to the colonic mucosal damage in UC. Here, we investigated the role of TSP1 in the differentiation of CD11c+ Mφ and the mechanism. METHODS: We analyzed the population characteristics of TSP1 genes using the Genotype-Tissue Expression (GTEx) database, and human serum TSP1 protein was detected with ELISA. DSS-induced colitis rats were used to explore the effects of TSP1 on colonic mucosal inflammation. We analyzed the serum cytokines and tissue histopathology to evaluate the severity of UC. Furthermore, we analysed the main source of TSP1 in colon tissue. In vitro, lamina propria mononuclear cells (LPMC) and CD11c+ lamina propria macrophages (LPMP) was isolated from model rats in vivo. The target of TSP1 protein was assessed by LSKL, CD36 and CD47 interfering plasmids. The proteins, the lysosome, lysosomal activity and Cathepsin E activity, and the migration were detected by western blotting, test kits and Transwell. RESULTS: The expression of TSP1 was significantly higher in younger, male, and in the rectum and sigmoid than that in older, females, and colon tissues, and was closely related to the severity of UC. Compared with normal rats, the worse disease activity index (DAI) score, more histological damage, CD11c+ Mφ infiltration, and increased expression of several proinflammatory cytokines was displayed in colitis rats with the elevation of serum TSP1 protein. In vitro, TSP1 protein derived from cmMφ and endothelial cells promoted the migration and the differentiation of CD11c+ Mφ via binding on CD36, rather than the cell proliferation. Furthermore, PRKCQ/NF-κB signaling pathway was activated by CD36. However, the effect of TSP1 protein could be reversed by LSKL in vivo, and LSKL and anti-TSP1 antibody in vitro. CONCLUSIONS: TSP1 promotes the migration and the differentiation of CD11c+ LPMP with lysosomal activity limited via activating the CD36-PRKCQ/NF-κB signaling pathway, which aggravates the colonic mucosal inflammatory injuries in UC.

The Effect of the Chorion on Size-Dependent Acute Toxicity and Underlying Mechanisms of Amine-Modified Silver Nanoparticles in Zebrafish Embryos.

As the worldwide application of nanomaterials in commercial products increases every year, various nanoparticles from industry might present possible risks to aquatic systems and human health. Presently, there are many unknowns about the toxic effects of nanomaterials, especially because the unique physicochemical properties of nanomaterials affect functional and toxic reactions. In our research, we sought to identify the targets and mechanisms for the deleterious effects of two different sizes (~10 and ~50 nm) of amine-modified silver nanoparticles (AgNPs) in a zebrafish embryo model. Fluorescently labeled AgNPs were taken up into embryos via the chorion. The larger-sized AgNPs (LAS) were distributed throughout developing zebrafish tissues to a greater extent than small-sized AgNPs (SAS), which led to an enlarged chorion pore size. Time-course survivorship revealed dose- and particle size-responsive effects, and consequently triggered abnormal phenotypes. LAS exposure led to lysosomal activity changes and higher number of apoptotic cells distributed among the developmental organs of the zebrafish embryo. Overall, AgNPs of ~50 nm in diameter exhibited different behavior from the ~10-nm-diameter AgNPs. The specific toxic effects caused by these differences in nanoscale particle size may result from the different mechanisms, which remain to be further investigated in a follow-up study.

Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation.

The skin is a multilayered and primary defensive organ. Intimate intercellular communication in the skin is necessary to ensure effective surveillance. Extracellular vesicles (EVs) are being explored for their involvement in intercellular skin communication. The aim of this study was to evaluate how human dermal fibroblasts (HDFs) accelerate EV production during senescence and the effects of senescence-associated EVs on epidermal homeostasis. Replicative senescent HDFs were assessed with senescence-associated ß-galactosidase staining and the expression of senescence-related markers. Isolated EVs were characterized by dynamic light scattering and EV marker expression. EVs secreted from untreated young or senescent HDFs, or from those treated with a nSMase inhibitor, antioxidant, and lysosomal activity regulators, were determined by sandwich ELISA for CD81. Human epidermal keratinocytes were treated with young- and senescent HDF-derived EVs. Compared to young HDFs, senescent HDFs produced relatively high levels of EVs due to the increased nSMase activity, oxidative stress, and altered lysosomal activity. The nSMase inhibitor, antioxidant, and agents that recovered lysosomal activity reduced EV secretion in senescent HDFs. Relative to young HDF-derived EVs, senescent HDF-derived EVs were less supportive in keratinocyte differentiation and barrier function but increased proinflammatory cytokine IL-6 levels. Our study suggests that dermis-derived EVs may regulate epidermal homeostasis by reflecting cellular status, which provides insight as to how the dermis communicates with the epidermis and influences skin senescence.

Iron-Induced Apoptotic Cell Death and Autophagy Dysfunction in Human Neuroblastoma Cell Line SH-SY5Y.

Iron accumulation plays a major role in neuronal cell death which has severe effects on mental health like neurodegenerative disorders. The present work aims to explore the involvement of molecular pathways involved in iron-mediated neuronal cell death using Ferric Ammonium Citrate (FAC) as a source of iron to treat neuroblastoma SH-SY5Y cells. In this study, it was found that cytotoxicity induced by iron treatment is highly correlated with enhanced intracellular reactive oxygen species (ROS) generation and loss of mitochondrial integrity. Appearance of early and late apoptotic cells with altered nuclear morphology and increased expression of effector proteins, i.e., cleaved Caspase 3 and cleaved PARP (Poly-ADP-ribose Polymerase), clearly confirmed iron-induced apoptotic cell deaths. Furthermore, excess accumulation of acidic vesicles and microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3II/I expressions were observed. Simultaneously, ultrastructural studies of SH-SY5Y cells demonstrated the accumulation of a large number of autophagosomes, autophagic vacuolization, and swollen mitochondria which further confirmed the induction of autophagy concomitant with mitochondrial damage. Furthermore, increased incorporation of lysosome-specific dye, LysoTracker Deep Red, and the red fluorescence retention of LC3-GFP-RFP constructs indicates the incomplete autophagy or autophagy dysfunction due to altered lysosomal activity. Hence, the present work unveiled the interruption in autophagy progression caused by the plausible suppression of lysosomal activity due to iron treatment resulting in autophagic cell death in SH-SY5Y cell lines. In general, both apoptotic and autophagic pathways were prominent and each of the pathways played their prospective roles, in iron-mediated neuronal cell death.

Diet Modulation Restores Autophagic Flux in Damaged Skeletal Muscle Cells.

OBJECTIVES: Autophagy is a physiological and highly regulated mechanism, crucial for cell homeostasis maintenance. Its impairment seems to be involved in the onset of several diseases, including muscular dystrophies, myopathies and sarcopenia. According to few papers, chemotherapeutic drug treatment is able to trigger side effects on skeletal muscle tissue and, among these, a defective autophagic activation, which leads to the persistence of abnormal organelles within cells and, finally, to myofiber degeneration. The aim of this work is to find a strategy, based on diet modulation, to prevent etoposide-induced damage, in a model of in vitro skeletal muscle cells. METHODS: Glutamine supplementation and nutrient deprivation have been chosen as pre-treatments to counteract etoposide effect, a chemotherapeutic drug known to induce oxidative stress and cell death. Cell response has been evaluated by means of morpho-functional, cytofluorimetric and molecular analyses. RESULTS: Etoposide treated cells, if compared to control, showed dysfunctional mitochondria presence, ER stress and lysosomal compartment damage, confirmed by molecular investigations. CONCLUSIONS: Interestingly, both dietary approaches were able to rescue myofiber from etoposide-induced damage. Glutamine supplementation, in particular, seemed to be a good strategy to preserve cell ultrastructure and functionality, by preventing the autophagic impairment and partially restoring the normal lysosomal activity, thus maintaining skeletal muscle homeostasis.

Ultrastructural analysis of human umbilical cord derived MSCs at undifferentiated stage and during osteogenic and adipogenic differentiation.

Mesenchymal stem cells (MSCs) are considered as an important tool for regenerative medicine and experimental treatments. Unveiling the ultrastructural changes during the differentiation of MSCs might help us to understand the nature of the process and to develop novel therapeutic approaches. For this purpose, human umbilical cord (hUC) was chosen as MSC source. In the first place, MSCs were isolated from sub-amniotic, intervascular and perivascular areas of hUC by enzymatic and tissue explant method to determine the most favorable region of hUC and technique for further processing. Therefore, microscopic and growth kinetics analyses showed that there was no clear difference in the morphologies and proliferation rates among the hUC-MSC groups. Flow cytometric analysis showed that CD44 and CD90 MSC markers were highly expressed, while CD34 and CD45 hematopoietic stem cells markers were expressed at low degree. Because our preliminary results showed that there was no conspicuous superiority among the hUC-MSCs groups, whole UC was utilized as a source, and tissue explant method was applied to isolate MSCs for further differentiation analysis. At the 1st and 3rd week of osteogenic and adipogenic differentiation, ultrastructural analysis showed an increase in the number of secondary lysosomes in comparison with the undifferentiated status. Increase in the mitochondrial content was also detected at the 1st week of adipogenic differentiation. Consequently, ultrastructural changes including increase in the number of mitochondria and secondary lysosomes during the adipogenic and osteogenic differentiation could be attributed to the switch in energy metabolism of the MSCs and increment in the lysosomal activity respectively.

Four New Steroidal Glycosides, Protolinckiosides A - D, from the Starfish Protoreaster lincki.

Four new steroidal glycosides, protolinckiosides A - D (1 - 4, resp.), were isolated along with four previously known glycosides, 5 - 8, from the MeOH/EtOH extract of the starfish Protoreaster lincki. The structures of 1 - 4 were elucidated by extensive NMR and ESI-MS techniques as (3ß,4ß,5α,6ß,7α,15α,16ß,25S)-4,6,7,8,15,16,26-heptahydroxycholestan-3-yl 2-O-methyl-ß-d-xylopyranoside (1), (3ß,5α,6ß,15α,24S)-3,5,6,8,15-pentahydroxycholestan-24-yl α-l-arabinofuranoside (2), sodium (3ß,6ß,15α,16ß,24R)-29-(ß-d-galactofuranosyloxy)-6,8,16-trihydroxy-3-[(2-O-methyl-ß-d-xylopyranosyl)oxy]stigmast-4-en-15-yl sulfate (3), and sodium (3ß,6ß,15α,16ß,22E,24R)-28-(ß-d-galactofuranosyloxy)-6,8,16-trihydroxy-3-[(2-O-methyl-ß-d-xylopyranosyl)oxy]ergosta-4,22-dien-15-yl sulfate (4). The unsubstituted ß-d-galactofuranose residue at C(28) or C(29) of the side chains was found in starfish steroidal glycosides for the first time. Compounds 1 - 4 significantly decreased the intracellular reactive oxygen species (ROS) content in RAW 264.7 murine macrophages at induction by proinflammatory endotoxic lipopolysaccharide (LPS) from E. coli.

Sodium phenylbutyrate reverses lysosomal dysfunction and decreases amyloid-ß42 in an in vitro-model of inclusion-body myositis.

Sporadic inclusion-body myositis (s-IBM) is a severe, progressive muscle disease for which there is no enduring treatment. Pathologically characteristic are vacuolated muscle fibers having: accumulations of multi-protein aggregates, including amyloid-ß(Aß) 42 and its toxic oligomers; increased γ-secretase activity; and impaired autophagy. Cultured human muscle fibers with experimentally-impaired autophagy recapitulate some of the s-IBM muscle abnormalities, including vacuolization and decreased activity of lysosomal enzymes, accompanied by increased Aß42, Aß42 oligomers, and increased γ-secretase activity. Sodium phenylbutyrate (NaPB) is an orally bioavailable small molecule approved by the FDA for treatment of urea-cycle disorders. Here we describe that NaPB treatment reverses lysosomal dysfunction in an in vitro model of inclusion-body myositis, involving cultured human muscle fibers. NaPB treatment improved lysosomal activity, decreased Aß42 and its oligomers, decreased γ-secretase activity, and virtually prevented muscle-fiber vacuolization. Accordingly, NaPB might be considered a potential treatment of s-IBM patients.

Neospora caninum: infection induces high lysosomal activity.

Neospora caninum is a protozoan that causes abortion in cattle and neuromuscular lesions in dogs, making it an important target of veterinary medicine. Lysosomes are cellular organelles responsible for important biological functions as cellular defense mechanisms. The aim of this work was to evaluate the lysosomal stability of rat gliocytes infected in vitro with N. caninum. Rat glial cultures were infected at a ratio of 1:1 (cell/parasite). The enzymatic activity of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was assayed in the medium of control and infected cell cultures. The activity observed at 24h of incubation was 0.4±0.08mU/mg/min for control cells and 1.3±0.5mU/mg/min for infected cells. After 72h, control and infected cells exhibited activities of 1.3±0.5 and 4.1±0.9mU/mg/min, respectively. These results suggested that lysosomal compartment plays an important role in the mechanisms of cellular infection by N. caninum.