Oral Exposure to Titanium Dioxide E171 and Zinc Oxide Nanoparticles Induces Multi-Organ Damage in Rats: Role of Ceramide.

Food-grade titanium dioxide (E171) and zinc oxide nanoparticles (ZnO NPs) are common food additives for human consumption. We examined multi-organ toxicity of both compounds on Wistar rats orally exposed for 90 days. Rats were divided into three groups: (1) control (saline solution), (2) E171-exposed, and (3) ZnO NPs-exposed. Histological examination was performed with hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). Ceramide (Cer), 3-nitrotyrosine (NT), and lysosome-associated membrane protein 2 (LAMP-2) were detected by immunofluorescence. Relevant histological changes were observed: disorganization, inflammatory cell infiltration, and mitochondrial damage. Increased levels of Cer, NT, and LAMP-2 were observed in the liver, kidney, and brain of E171- and ZnO NPs-exposed rats, and in rat hearts exposed to ZnO NPs. E171 up-regulated Cer and NT levels in the aorta and heart, while ZnO NPs up-regulated them in the aorta. Both NPs increased LAMP-2 expression in the intestine. In conclusion, chronic oral exposure to metallic NPs causes multi-organ injury, reflecting how these food additives pose a threat to human health. Our results suggest how complex interplay between ROS, Cer, LAMP-2, and NT may modulate organ function during NP damage.

Targeting Lysosomes in Colorectal Cancer: Exploring the Anticancer Activity of a New Benzo[a]phenoxazine Derivative.

Colorectal cancer (CRC) has been ranked as one of the cancer types with a higher incidence and one of the most mortal. There are limited therapies available for CRC, which urges the finding of intracellular targets and the discovery of new drugs for innovative therapeutic approaches. In addition to the limited number of effective anticancer agents approved for use in humans, CRC resistance and secondary effects stemming from classical chemotherapy remain a major clinical problem, reinforcing the need for the development of novel drugs. In the recent years, the phenoxazines derivatives, Nile Blue analogues, have been shown to possess anticancer activity, which has created interest in exploring the potential of these compounds as anticancer drugs. In this context, we have synthetized and evaluated the anticancer activity of different benzo[a]phenoxazine derivatives for CRC therapy. Our results revealed that one particular compound, BaP1, displayed promising anticancer activity against CRC cells. We found that BaP1 is selective for CRC cells and reduces cell proliferation, cell survival, and cell migration. We observed that the compound is associated with reactive oxygen species (ROS) generation, accumulates in the lysosomes, and leads to lysosomal membrane permeabilization, cytosolic acidification, and apoptotic cell death. In vivo results using a chicken embryo choriollantoic membrane (CAM) assay showed that BaP1 inhibits tumor growth, angiogenesis, and tumor proliferation. These observations highlight that BaP1 as a very interesting agent to disturb and counteract the important roles of lysosomes in cancer and suggests BaP1 as a promising candidate to be exploited as new anticancer lysosomal-targeted agent, which uses lysosome membrane permeabilization (LMP) as a therapeutic approach in CRC.

Fe-N Co-Doped Titanium Dioxide Nanoparticles Induce Cell Death in Human Lung Fibroblasts in a p53-Independent Manner.

The advancement of nanotechnology in the last decade has developed an abundance of novel and intriguing TiO2-based nanomaterials that are widely used in many sectors, including industry (as a food additive and colorant in cosmetics, paints, plastics, and toothpaste) and biomedicine (photoelectrochemical biosensing, implant coatings, drug delivery, and new emerging antimicrobial agents). Therefore, the increased use of engineered nanomaterials in the industry has raised serious concern about human exposure and their unexpected cytotoxic effects. Since inhalation is considered the most relevant way of absorbing nanomaterials, different cell death mechanisms induced in MRC-5 lung fibroblasts, following the exposure to functionalized TiO2 NPs, were investigated. Long-term exposure to TiO2 nanoparticles co-doped with 1% of iron and nitrogen led to the alteration of p53 protein activity and the gene expression controlled by this suppressor (NF-kB and mdm2), DNA damage, cell cycle disruptions at the G2/M and S phases, and lysosomal membrane permeabilization and the subsequent release of cathepsin B, triggering the intrinsic pathway of apoptosis in a Bax- and p53-independent manner. Our results are of major significance, contributing to the understanding of the mechanisms underlying the interaction of these nanoparticles with in vitro biological systems, and also providing useful information for the development of new photocatalytic nanoparticles that are active in the visible spectrum, but with increased biocompatibility.

Lysosomal membrane permeabilization and cell death.

Lysosomes are membrane-enclosed organelles that mediate the intracellular degradation of macromolecules. They play an essential role in calcium regulation and have emerged as key signaling hubs in controlling the nutrient response. Maintaining lysosomal integrity and function is therefore crucial for cellular homeostasis. Different forms of stress can induce lysosomal membrane permeabilization (LMP), resulting in the translocation to the cytoplasm of intralysosomal components, such as cathepsins, inducing lysosomal-dependent cell death (LDCD). Here, we review recent advances that have furthered our understanding of the molecular mechanisms of LMP and the methods used to detect it. We discuss several endolysosomal damage-response mechanisms that mediate the repair or elimination of compromised lysosomes and summarize the role of LMP and cathepsins in LDCD and other cell death pathways. Finally, with the emergence of lysosomes as promising therapeutic targets for several human diseases, we review a variety of therapeutic strategies that seek to either destabilize lysosomes or to maintain, enhance or restore lysosomal function.

Lysosomal membrane permeabilization as a cell death mechanism in cancer cells.

Lysosomes are acidic organelles that contain hydrolytic enzymes that mediate the intracellular degradation of macromolecules. Damage of these organelles often results in lysosomal membrane permeabilization (LMP) and the release into the cytoplasm of the soluble lysosomal contents, which include proteolytic enzymes of the cathepsin family. This, in turn, activates several intracellular cascades that promote a type of regulated cell death, called lysosome-dependent cell death (LDCD). LDCD can be inhibited by pharmacological or genetic blockade of cathepsin activity, or by protecting the lysosomal membrane, thereby stabilizing the organelle. Lysosomal alterations are common in cancer cells and may increase the sensitivity of these cells to agents that promote LMP. In this review, we summarize recent findings supporting the use of LDCD as a means of killing cancer cells.

Investigation into the distinct subcellular effects of docosahexaenoic acid loaded low-density lipoprotein nanoparticles in normal and malignant murine liver cells.

BACKGROUND: Recent studies have shown that low density lipoproteins reconstituted with the natural omega 3 fatty acid docosahexaenoic acid (LDL-DHA) is selectively cytotoxic to liver cancer cells over normal hepatocytes. To date, little is known about the subcellular events which transpire following LDL-DHA treatment. METHODS: Herein, murine noncancer and cancer liver cells, TIB-73 and TIB-75 respectively, were investigated utilizing confocal microscopy, flow cytometry and viability assays to demonstrate differential actions of LDL-DHA nanoparticles in normal versus malignant cells. RESULTS: Our studies first showed that basal levels of oxidative stress are significantly higher in the malignant TIB-75 cells compared to the normal TIB-73 cells. As such, upon entry of LDL-DHA into the malignant TIB-75 cells, DHA is rapidly oxidized precipitating global and lysosomal lipid peroxidation along with increased lysosomal permeability. This leakage of lysosomal contents and lipid peroxidation products trigger subsequent mitochondrial dysfunction and nuclear injury. The cascade of LDL-DHA mediated lipid peroxidation and organelle damage was partially reversed by the administration of the antioxidant, N-acetylcysteine, or the iron-chelator, deferoxamine. LDL-DHA treatment in the normal TIB-73 cells was well tolerated and did not elicit any cell or organelle injury. CONCLUSION: These studies have shown that LDL-DHA is selectively cytotoxic to liver cancer cells and that increased levels of ROS and iron catalyzed reactions promote the peroxidation of DHA which lead to organelle dysfunction and ultimately the demise of the cancer cell. GENERAL SIGNIFICANCE: LDL-DHA selectively disrupts lysosomal, mitochondrial and nuclear function in cancer cells as a novel pathway for eliminating cancer cells.

Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity.

NLRP3 inflammasome activation occurs in response to hazardous particle exposures and is critical for the development of particle-induced lung disease. Mechanisms of Lysosome Membrane Permeabilization (LMP), a central pathway for activation of the NLRP3 inflammasome by inhaled particles, are not fully understood. We demonstrate that the lysosomal vATPases inhibitor Bafilomycin A1 blocked LMP in vitro and ex vivo in primary murine macrophages following exposure to silica, multi-walled carbon nanotubes, and titanium nanobelts. Bafilomycin A1 treatment of particle-exposed macrophages also resulted in decreased active cathepsin L in the cytosol, a surrogate measure for leaked cathepsin B, which was associated with less NLRP3 inflammasome activity. Silica-induced LMP was partially dependent upon lysosomal cathepsins B and L, whereas nanoparticle-induced LMP occurred independent of cathepsin activity. Furthermore, inhibition of lysosomal cathepsin activity with CA-074-Me decreased the release of High Mobility Group Box 1. Together, these data support the notion that lysosome acidification is a prerequisite for particle-induced LMP, and the resultant leak of lysosome cathepsins is a primary regulator of ongoing NLRP3 inflammasome activity and release of HMGB1.

The Parkinson-associated human P5B-ATPase ATP13A2 protects against the iron-induced cytotoxicity.

P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1-P5. The ion transported by the P5-ATPases is not known. Five genes named ATP13A1-ATP13A5 that belong to the P5-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor-Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA). Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage.

Candida albicans secreted aspartic proteases 4-6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism.

Systemic infection by the pathogenic yeast Candida albicans produces high mortality in immune-compromised people. Such infection starts with the penetration of the organism at the mucosal surfaces, facilitated by the secreted aspartic proteases (Saps) 4, 5, and 6. The functional mechanism of these virulence factors is unclear. We discovered that Saps 4-6 each contains amino acid motifs RGD/KGD to bind integrins on epithelial cell A549 and are internalized to endosomes and lysosomes. These processes are inhibited by RGD-containing peptides or by substituting RGD motifs of these Saps. The internalization of Saps 4-6 results in partial permeabilization of lysosomal membranes, measured by the redistribution of the lysosomal tropic dye acridine orange to the cytosol, and the triggering of apoptosis via caspase activation. Sap 2 and mutated Saps 4-6 contain no RGD motif, are ineffective in these processes, and a proteolytic inhibitor abolished Sap 4 activity in lysosome permeabilization. Same results were also seen for human tongue keratinocyte SCC-15 cells. Mucosal lesions from this fundamental new mechanism may permit C. albicans to enter the body and may be used to attack cells in immune defense during systemic infections. RGD-motif may also be incorporated in Sap inhibitors for Candidiasis drugs targeting to lysosomes.

Magnetic modulation of lysosomes for cancer therapy.

Lysosomes play a central role in biochemical signal transduction and oxidative stress in cells. Inducing lysosome membrane penetration (LMP) to cause lysosomal-dependent cell death (LCD) in tumor cells is an effective strategy for cancer therapy. Chemical drugs can destroy the stability of lysosomes by neutralizing protons within the lysosomes or enhancing the fragility of the lysosomal membranes. However, there remain several unsolved problems of traditional drugs in LMP induction due to insufficient lysosomal targeting, fast metabolism, and toxicity in normal cells. With the development of nanotechnology, magnetic nanoparticles have been demonstrated to target lysosomes naturally, providing a versatile tool for lysosomal modulation. Combined with excellent tissue penetration and spatiotemporal manipulability of magnetic fields, magnetic modulation of lysosomes progresses rapidly in inducing LMP and LCD for cancer therapy. This review comprehensively discussed the strategies of magnetic modulation of lysosomes for cancer therapy. The intrinsic mechanisms of LMP-induced LCD were first introduced. Then, the modulation of lysosomes by diverse physical outputs of magnetic fields was emphatically discussed. Looking forward, this review will shed the light on the prospect of magnetic modulation of lysosomes, inspiring future research of magnetic modulation strategy in cancer therapy. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Radiotherapy-activated NBTXR3 nanoparticles promote ferroptosis through induction of lysosomal membrane permeabilization.

PURPOSE: Radiotherapy-activated NBTXR3 (NBTXR3 + RT) has demonstrated superior efficacy in cancer cell destruction and tumor growth control, compared to radiotherapy (RT), in preclinical and clinical settings. Previous studies highlighted the immunomodulatory properties of NBTXR3 + RT, such as modification of tumor cell immunogenicity/adjuvanticity, producing an effective local tumor control and abscopal effect, related to an enhanced antitumor immune response. Furthermore, NBTXR3 + RT has shown potential in restoring anti-PD1 efficacy in a refractory tumor model. However, the early events leading to these results, such as NBTXR3 endocytosis, intracellular trafficking and primary biological responses induced by NBTXR3 + RT remain poorly understood. METHODS: We analyzed by transmission electron microscopy endocytosis and intracellular localization of NBTXR3 nanoparticles after endocytosis in various cell lines, in vitro and in vivo. A kinetic of NBTXR3 endocytosis and its impact on lysosomes was conducted using LysoTracker staining, and a RNAseq analysis was performed. We investigated the ability of NBTXR3 + RT to induce lysosomal membrane permeabilization (LMP) and ferroptosis by analyzing lipid peroxidation. Additionally, we evaluated the recapture by cancer cells of NBTXR3 released from dead cells. RESULTS: NBTXR3 nanoparticles were rapidly internalized by cells mainly through macropinocytosis and in a less extend by clathrin-dependent endocytosis. NBTXR3-containing endosomes were then fused with lysosomes. The day following NBTXR3 addition, we measured a significant increase in LysoTracker lysosome labeling intensity, in vitro as in vivo. Following RT, a significant lysosomal membrane permeabilization (LMP) was measured exclusively in cells treated with NBTXR3 + RT, while RT had no effect. The day post-irradiation, a significant increase in lipid peroxidation, a biomarker of ferroptosis, was measured with NBTXR3 + RT compared to RT. Moreover, we demonstrated that NBTXR3 nanoparticles released from dead cells can be recaptured by cancer cells. CONCLUSIONS: Our findings provide novel insights into the early and specific biological effects induced by NBTXR3 + RT, especially LMP, not induced by RT in our models. The subsequent significant increase in lipid peroxidation partially explains the enhanced cancer cell killing capacity of NBTXR3 + RT compared to RT, potentially by promoting ferroptosis. This study improves our understanding of the cellular mechanisms underlying NBTXR3 + RT and highlights its potential as an agnostic therapeutic strategy for solid cancers treatment.

Emerging roles of lysosome homeostasis (repair, lysophagy and biogenesis) in cancer progression and therapy.

In the era of personalized therapy, precise targeting of subcellular organelles holds great promise for cancer modality. Taking into consideration that lysosome represents the intersection site in numerous endosomal trafficking pathways and their modulation in cancer growth, progression, and resistance against cancer therapies, the lysosome is proposed as an attractive therapeutic target for cancer treatment. Based on the recent advances, the current review provides a comprehensive understanding of molecular mechanisms of lysosome homeostasis under 3R responses: Repair, Removal (lysophagy) and Regeneration of lysosomes. These arms of 3R responses have distinct role in lysosome homeostasis although their interdependency along with switching between the pathways still remain elusive. Recent advances underpinning the crucial role of (1) ESCRT complex dependent/independent repair of lysosome, (2) various Galectins-based sensing and ubiquitination in lysophagy and (3) TFEB/TFE proteins in lysosome regeneration/biogenesis of lysosome are outlined. Later, we also emphasised how these recent advancements may aid in development of phytochemicals and pharmacological agents for targeting lysosomes for efficient cancer therapy. Some of these lysosome targeting agents, which are now at various stages of clinical trials and patents, are also highlighted in this review.

G-quadruplex ligands as potent regulators of lysosomes.

Guanine-quadruplex structures (G4) are unusual nucleic acid conformations formed by guanine-rich DNA and RNA sequences and known to control gene expression mechanisms, from transcription to protein synthesis. So far, a number of molecules that recognize G4 have been developed for potential therapeutic applications in human pathologies, including cancer and infectious diseases. These molecules are called G4 ligands. When the biological effects of G4 ligands are studied, the analysis is often limited to nucleic acid targets. However, recent evidence indicates that G4 ligands may target other cellular components and compartments such as lysosomes and mitochondria. Here, we summarize our current knowledge of the regulation of lysosome by G4 ligands, underlying their potential functional impact on lysosome biology and autophagic flux, as well as on the transcriptional regulation of lysosomal genes. We outline the consequences of these effects on cell fate decisions and we systematically analyzed G4-prone sequences within the promoter of 435 lysosome-related genes. Finally, we propose some hypotheses about the mechanisms involved in the regulation of lysosomes by G4 ligands.

Calcium signaling in lysosome-dependent cell death.

Calcium is a crucial messenger of intracellular and extracellular signals, regulating a great variety of cellular processes such as cell death, proliferation, and metabolism. Inside the cell, calcium signaling is one of the main interorganelle communication mediators, with central functional roles at the endoplasmic reticulum (ER), mitochondria, Golgi complex, and lysosomes. Lysosomal function is highly dependent on lumenal calcium and most of the lysosomal membrane-localised ion channels regulate several lysosomal functions and properties such as lumenal pH. One of these functions configures a specific type of cell death involving lysosomes, named lysosome-dependent cell death (LDCD), which contributes to maintenance of tissue homeostasis, development and pathology when deregulated. Here, we cover the fundamental aspects of LDCD with a special focus on recent advances in calcium signaling in LDCD.

Lysosomes in retinal health and disease.

Lysosomes play crucial roles in various cellular processes - including endocytosis, phagocytosis, and autophagy - which are vital for maintaining retinal health. Moreover, these organelles serve as environmental sensors and act as central hubs for multiple signaling pathways. Through communication with other cellular components, such as mitochondria, lysosomes orchestrate the cytoprotective response essential for preserving cellular homeostasis. This coordination is particularly critical in the retina, given its high metabolic rate and susceptibility to photo-oxidative stress. Consequently, impaired lysosomal function and dysregulated communication between lysosomes and other organelles contribute significantly to the pathobiology of major retinal degenerative diseases. This review explores the pivotal role of lysosomes in retinal cells and their involvement in retinal degenerative diseases.

Annexin A7 mediates lysosome repair independently of ESCRT-III.

Lysosomes are crucial organelles essential for various cellular processes, and any damage to them can severely compromise cell viability. This study uncovers a previously unrecognized function of the calcium- and phospholipid-binding protein Annexin A7 in lysosome repair, which operates independently of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Our research reveals that Annexin A7 plays a role in repairing damaged lysosomes, different from its role in repairing the plasma membrane, where it facilitates repair through the recruitment of ESCRT-III components. Notably, our findings strongly suggest that Annexin A7, like the ESCRT machinery, is dispensable for membrane contact site formation within the newly discovered phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway. Instead, we speculate that Annexin A7 is recruited to damaged lysosomes and promotes repair through its membrane curvature and cross-linking capabilities. Our findings provide new insights into the diverse mechanisms underlying lysosomal membrane repair and highlight the multifunctional role of Annexin A7 in membrane repair.

Functions and mechanisms of cytosolic phospholipase A2 in central nervous system trauma.

Central nervous system (CNS) trauma, including traumatic brain injury and spinal cord injury, has a high rate of disability and mortality, and effective treatment is currently lacking. Previous studies have revealed that neural inflammation plays a vital role in CNS trauma. As the initial enzyme in neuroinflammation, cytosolic phospholipase A2 (cPLA2) can hydrolyze membranous phosphatides at the sn-2 position in a preferential way to release lysophospholipids and ω3-polyunsaturated fatty acid dominated by arachidonic acid, thereby inducing secondary injuries. Although there is substantial fresh knowledge pertaining to cPLA2, in-depth comprehension of how cPLA2 participates in CNS trauma and the potential methods to ameliorate the clinical results after CNS trauma are still insufficient. The present review summarizes the latest understanding of how cPLA2 participates in CNS trauma, highlighting novel findings pertaining to how cPLA2 activation initiates the potential mechanisms specifically, neuroinflammation, lysosome membrane functions, and autophagy activity, that damage the CNS after trauma. Moreover, we focused on testing a variety of drugs capable of inhibiting cPLA2 or the upstream pathway, and we explored how those agents might be utilized as treatments to improve the results following CNS trauma. This review aimed to effectively understand the mechanism of cPLA2 activation and its role in the pathophysiological processes of CNS trauma and provide clarification and a new referential framework for future research.

Diagnostic utility of serum and urine biomarkers in idiopathic membranous nephropathy: a systematic review and meta-analysis.

BACKGROUND: Membranous nephropathy is an autoimmune nephropathy that is one of the most common pathological types of nephrotic syndrome. It is important to find and apply specific biomarkers for the noninvasive diagnosis of idiopathic membranous nephropathy (IMN). However, there are limited data about their diagnostic value. Therefore, an overall meta-analysis helps to identify effective biomarkers for the clinical diagnosis of IMN. METHODS: A systematic literature search was carried out in PubMed, Embase, Cochrane and Web of Science from inception until December 31, 2020. Two researchers searched for studies that met the inclusion criteria. The results of the joint study were expressed in terms of sensitivity and specificity. RESULTS: The meta-analysis included 24 studies with biomarkers for the clinical diagnosis of IMN, including antibody against phospholipase A2 receptor (PLA2R-AB), antibody against thrombospondin type I domain-containing 7A (THSD7A-AB), lysosome membrane protein-2 (LIMP-2) and circular RNAs. The diagnostic efficiency of PLA2R-AB for IMN had a combined sensitivity of 60% and a combined specificity of 100%. The diagnostic efficiency of THSD7A-AB for IMN had a combined sensitivity of 3% and a combined specificity of 99%. The diagnostic efficiency of urinary LIMP-2 for IMN was 100%, and the specificity was 100%. The diagnostic efficiency of exosomal circRNAs for IMN was 100%, and the specificity was 100%. CONCLUSIONS: This meta-analysis shows that PLA2R-AB and THSD7A-AB are of important diagnostic value for IMN. More studies are needed in the future to reveal the diagnostic value of LIMP-2 and circRNAs for IMN.

Pol-miR-150 regulates anti-bacterial and viral infection in Japanese flounder (Paralichthys olivaceus) via the lysosomal protein LMP2L.

MiR-150 is a microRNA (miRNA) present in a number of teleost species, but its target and regulation mechanism are unknown. Similarly, lysosome membrane protein 2-like (LMP2L) is a gene identified in fish but with unknown function. In this study, we examined the regulation mechanism and function of flounder miR-150 (named pol-miR-150) and its target gene LMP2L (named PoLMP2L) in association with bacterial and viral infection. We found that pol-miR-150 expression was not only modulated by the bacterial pathogen Streptococcus iniae but also by the viral pathogen megalocytivirus. Pol-miR-150 targeted PoLMP2L by binding to the 3'-untranslated region (3'-UTR) of PoLMP2L and inhibited PoLMP2L expression in vitro and in vivo. PoLMP2L is a member of the CD36 superfamily of scavenger receptors and homologous to but phylogenetically distinct from lysosomal integral membrane protein type 2 (LIMP2). PoLMP2L was localized mainly in the lysosomes and expressed in multiple organs of flounder. In vivo knockdown and overexpression of PoLMP2L enhanced and suppressed, respectively, S. iniae dissemination in flounder tissues, whereas in vivo knockdown and overexpression of pol-miR-150 produced the opposite effects on S. iniae dissemination. In addition, pol-miR-150 knockdown also significantly inhibited the replication of megalocytivirus. The results of this study revealed the regulation mechanism and immune functions of fish miR-150 and LMP2L, and indicated that LMP2L and miR-150 play an important role in the antimicrobial immunity of fish.

Paclitaxel-loaded polypeptide-polyacrylamide nanomicelles overcome drug-resistance by enhancing lysosomal membrane permeability and inducing apoptosis.

The aim of the current project was to investigate the in vitro properties of Paclitaxel (PTX)-loaded pHPMA5kD -pHis5kD -pLeu3kD nanomicelles (NMs) on multidrug resistance cell line. Circular dichroism analysis was done to investigate the effect of pH on the secondary structure of the copolymer. Cytotoxicity assay together with fluorescence imaging and flow cytometry were performed to get an insight about toxicity and cellular uptake mechanism of NMs. Acridine orange assay, rhodamine 123 (Rh123) accumulation assay, and apoptosis analysis were conducted for further investigation. It was found that the secondary structure of the copolymer changed in response to pH, PTX-loaded NMs had higher cytotoxicity on both drug-sensitive (MES-SA and MCF-7) and multidrug resistant cells (MES-SA/DX5) compared to free PTX, and interestinly free copolymer inhibited the growth of MES-SA/DX5 cells while it was nontoxic on drug-sensitive cells. Moreover, the copolymer was able to induce lysosome membrane permeation and increase Rh123 accumulation inside cells indicating inhibition of the P-gp efflux pumps. Finally, apoptosis was strongly induced in MES-SA/DX5 cells upon treatment with PTX-loaded NMs. It can be concluded that the designed hybrid copolymer is a good candidate for in vivo assay and developing a powerful system against multidrug resistance tumors.