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Although immunogenic cell death (ICD) induced by lysosomal membrane permeabilization (LMP) evidently enhance the effectiveness of antitumor immunity for triple-negative breast cancer (TNBC) with poor immunogenicity, their potential is increasingly restricted by the development of other death pathways and the repair of lysosomes by endoplasmic reticulum (ER) during LMP induction. Herein, a polydopamine nanocomposite with i-motif DNA modified and BNN6 loaded is prepared toward boosting LMP and immunotherapy of TNBC by synergy of spatially confined photoacoustic (PA) effects and nitric oxide. Combining the high-frequency pulsed laser (4000 kHz) with the intra-lysosomal assembly of nanocomposites produced spatially confined and significantly boosted PA effects (4.8-fold higher than the individually dispersed particles extracellular), suppressing damage to other cellular components and selectively reducing lysosomal integrity to 19.2 %. Simultaneously, the releasing of nitric oxide inhibited the repair of lysosomes by ER stress, causing exacerbated LMP. Consequently, efficient immune activation was achieved, including the abundant releasing of CRT/HMGB1 (5.93–6.8-fold), the increasing maturation of dendritic cells (3.41-fold), and the fostered recruitment of CD4+/CD8+ T cells (3.99–3.78-fold) in vivo. The study paves a new avenue for the rational design and synergy of confined energy conversion and responsive nanostructures to achieve the treatment of low immunogenicity tumors. A strategy of boosting lysosomal membrane permeabilization (LMP) and concomitantly preventing the repair was developed to address the immunotherapy challenge of triple-negative breast cancer. Spatially confined and significantly enhanced photoacoustic (PA) effects were achieved through DNA-guided pH-responsive assembly of polydopamine nanocomposites in lysosomes and application of a high-frequency pulsed laser. Efficient immunogenic cell death was guaranteed by selective and powerful damage of lysosomal membranes through the significant contrast of PA intensities for dispersed/assembled particles and nitric oxide release induced endoplasmic reticulum stress. The study paves a new avenue for the rational design and synergy of confined energy conversion and responsive nanostructures to achieve the treatment of low immunogenicity tumors. [Display omitted] [ABSTRACT FROM AUTHOR]

BACKGROUND: Cardiac hypertrophy and subsequent heart failure impose a considerable burden on public health worldwide. Impaired protein degradation, especially endo-lysosome-mediated degradation of membrane proteins, is associated with cardiac hypertrophy progression. CHMP4C (charged multivesicular body protein 4C), a critical constituent of multivesicular bodies, is involved in cellular trafficking and signaling. However, the specific role of CHMP4C in the progression of cardiac hypertrophy remains largely unknown. METHODS: Mouse models with CHMP4C knockout or cardiadc-specific overexpression were subjected to transverse aortic constriction surgery for 4 weeks. Cardiac morphology and function were assessed through histological staining and echocardiography. Confocal imaging and coimmunoprecipitation assays were performed to identify the direct target of CHMP4C. An EGFR (epidermal growth factor receptor) inhibitor was administrated to determine whether effects of CHMP4C on cardiac hypertrophy were EGFR dependent. RESULTS: CHMP4C was significantly upregulated in both pressure-overloaded mice and spontaneously hypertensive rats. Compared with wild-type mice, CHMP4C deficiency exacerbated transverse aortic constriction-induced cardiac hypertrophy, whereas CHMP4C overexpression in cardiomyocytes attenuated cardiac dysfunction. Mechanistically, the effect of CHMP4C on cardiac hypertrophy relied on the EGFR signaling pathway. Fluorescent staining and coimmunoprecipitation assays confirmed that CHMP4C interacts directly with EGFR and promotes lysosome-mediated degradation of activated EGFR, thus attenuating cardiac hypertrophy. Notably, an EGFR inhibitor canertinib counteracted the exacerbation of cardiac hypertrophy induced by CHMP4C knockdown in vitro and in vivo. CONCLUSIONS: CHMP4C represses cardiac hypertrophy by modulating lysosomal degradation of EGFR and is a potential therapeutic candidate for cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Intracellular organelles carry out many of their functions by engaging in extensive interorganellar communication through specialized membrane contact sites (MCSs) formed where two organelles tether to each other or to the plasma membrane (PM) without fusing. In recent years, these ubiquitous membrane structures have emerged as central signaling hubs that control a multitude of cellular pathways, ranging from lipid metabolism/transport to the exchange of metabolites and ions (i.e., Ca2+), and general organellar biogenesis. The functional crosstalk between juxtaposed membranes at MCSs relies on a defined composite of proteins and lipids that populate these microdomains in a dynamic fashion. This is particularly important in the nervous system, where alterations in the composition of MCSs have been shown to affect their functions and have been implicated in the pathogenesis of neurodegenerative diseases. In this review, we focus on the MCSs that are formed by the tethering of the endoplasmic reticulum (ER) to the mitochondria, the ER to the endo‐lysosomes and the mitochondria to the lysosomes. We highlight how glycosphingolipids that are aberrantly processed/degraded and accumulate ectopically in intracellular membranes and the PM change the topology of MCSs, disrupting signaling pathways that lead to neuronal demise and neurodegeneration. In particular, we focus on neurodegenerative lysosomal storage diseases linked to altered glycosphingolipid catabolism. [ABSTRACT FROM AUTHOR]

Intracellular pathogens such as Mycobacterium tuberculosis (Mtb) have evolved diverse strategies to counteract macrophage defence mechanisms including phagolysosomal biogenesis. Within macrophages, Mtb initially resides inside membrane‐bound phagosomes that interact with lysosomes and become acidified. The ability of Mtb to control and subvert the fusion between phagosomes and lysosomes plays a key role in the pathogenesis of tuberculosis. Therefore, understanding how pathogens interact with the endolysosomal network and cope with intracellular acidification is important to better understand the disease. Here, we describe in detail the use of fluorescence microscopy‐based approaches to investigate Mtb responses to acidic environments in cellulo. We report high‐content imaging modalities to probe Mtb sensing of external pH or visualise in real‐time Mtb intrabacterial pH within infected human macrophages. We discuss various methodologies with step‐by‐step analyses that enable robust image‐based quantifications. Finally, we highlight the advantages and limitations of these different approaches and discuss potential alternatives that can be applied to further investigate Mtb–host cell interactions. These methods can be adapted to study host–pathogen interactions in different biological systems and experimental settings. Altogether, these approaches represent a valuable tool to further broaden our understanding of the cellular and molecular mechanisms underlying intracellular pathogen survival. [ABSTRACT FROM AUTHOR]

As the final step in apoptosis, apoptotic cells (ACs) are swiftly removed by specialized phagocytes, such as macrophages, or nonprofessional phagocytes, such as epidermal cells. Genetic studies of model organisms such as Caenorhabditis elegans have helped to elucidate the mechanisms of AC clearance and the underlying causes of disorders related to the dysregulation of these pathways. C. elegans possesses six class B scavenger receptor homologs, but whether they affect apoptosis is unknown. Here, we show that only the loss of function of scav‐3, the C. elegans homolog of human lysosomal integral membrane protein‐2, resulted in a considerable accumulation of cell corpses, which was caused by a failure in degradation rather than engulfment. SCAV‐3 was found to be widely distributed and localized in lysosomes to maintain the integrity of the lysosomal membrane. Further study revealed that loss of scav‐3 had no effect on phagosome maturation or the recruitment of lysosomes to phagosomes carrying cell corpses. Moreover, we discovered that the hydrolytic enzymes contained in the lysosomes were reduced in phagosomes in scav‐3 mutants. Thus, hydrolases may leak from the damaged lysosome during phagolysosome formation due to the loss of scav‐3 function, which reduces lysosome digestion activity and thus directly contributes to the elimination of ACs. [ABSTRACT FROM AUTHOR]

Postoperative pain management would benefit significantly from an anesthetic that could take effect in an on-demand manner. An ultrasound would be an appropriate tool for such nanoplatform because it is widely used in clinical settings for ultrasound-guided anesthesia. Herein, we report a nanoplatform for postoperative on-demand pain management that can effectively enhance their analgesic time while providing ultrasonic imaging. Levobupivacaine and perfluoropentane were put into dendritic mesoporous silica and covered with red blood cell membranes to make the pain relief last longer in living organisms. The generated nanoplatform with gas-producing capability is ultrasonic responsive and can finely escape from the lysosomal in cells under ultrasound irradiation, maximizing the anesthetic effect with minimal toxicity. Using an incision pain model in vivo, levobupivacaine's sustained and controlled release gives pain reduction for approximately 3 days straight. The duration of pain relief is over 20 times greater than with a single injection of free levobupivacaine. Effective pain management was reached in vivo , and the pain reduction was enhanced by repeated ultrasonic irradiation. There was no detectable systemic or tissue injury under either of the treatments. Thus, our results suggest that nanoplatform with lysosomal escape capability can provide a practical ultrasound imaging-guided on-demand pain management strategy. On-demand pain management is essential to postoperative patients. However, the traditional on-demand pain management strategy is hampered by the limited tissue penetration depth of near-infrared stimuli and the lack of proper imaging guidance. The proposed research is significant because it provides a nanoplatform for deep penetrated ultrasound controlled pain management under clinical applicable ultrasound imaging guidance. Moreover, the nanoplatform with prolonged retention time and lysosomal escape capability can provide long-term pain alleviation. Therefore, our results suggest that nanoplatform with lysosomal escape capability can provide an effective strategy for ultrasound imaging-guided on-demand pain management. [Display omitted] [ABSTRACT FROM AUTHOR]

Intracellular protein delivery has been a major challenge due to various physiological barriers including low proteolytic stability and poor membrane permeability of the biologics. Nanoparticles were widely proposed to deliver cargo proteins into cells by endocytosis, however, the materials and complexes with proteins are often entrapped in endosomes and subject to lysosome degradation. In this study, we report a piperazine modified dendrimer for stabilizing the complexes via a combination of electrostatic interaction and hydrophobic interactions. The complexes show rapid cell internalization and the loaded proteins are released into the cytosols as early as half an hour post incubation. Mechanism study suggests that the complexes are endocytosed into cells via caveolae-based pathways, which could be inhibited by inhibitors such as genistein, filipin III, brefeldin A and nystatin. The phenylpiperazine-modified polymer enables the delivery of cargo proteins with reserved bioactivity and show high permeability in three-dimensional cell spheroids. The results prove the beneficial roles of phenylpiperazine ligands in polymer-mediated cytosolic protein delivery systems. We synthesized a list of piperazine and derivatives modified dendrimers as cytosolic protein delivery vectors via facile reactions. Phenylpiperazine modification enables the efficient protein binding through the combination of electrostatic, hydrogen bonding and hydrophobic interactions. Phenylpiperazine modified dendrimers were internalized into the cells via a caveolae-based endo/lysosome-independent path and could release the cargo proteins into the cytosols as early as half an hour post incubation. Phenylpiperazine modified dendrimers delivered cargo proteins with reserved bioactivity and showed high permeability in three-dimensional cell spheroids [Display omitted] [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 mediated genome editing have provided a promising strategy to correct multiple mutations of Duchenne muscular dystrophy (DMD). However, the delivery of CRISPR/Cas9 system into mammalian cell for DMD gene editing mainly relies on adeno associated virus (AAV)-mediated transport. Meanwhile, the protospacer adjacent motif (PAM) requirement of wild-typed Cas9 protein causing the target sites for exon splice acceptor site are restricted to limited regions. Here, we developed a biomineralized PAMLess Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo. This method described a facile synthesis of biomineralized NPs with high SpRY pDNA encapsulation efficiency. In vitro results show that the Bm-SpRY NPs have the obvious advantages of well biocompatibility and protecting SpRY pDNA from enzyme degradation and efficient delivery under high serum condition. Cell studies demonstrated that Bm-SpRY NPs enable rapid cellular uptake, endo-lysosomes escape and nucleus transport. Meanwhiles, the DMD gene editing via Bm-SpRY NPs pathway is transient process without genomic integration. We evaluated multiple target regions with different PAMs for the DMD exon 51 splice acceptor site through Bm-SpRY NPs method and found that the target region with TAG PAM has the highest editing efficiency and significant preferential mutation. In vivo results show that intramuscular injection of Bm-SpRY NPs enable DMD gene mutation in muscle tissue without tissue damage. This study may extend the advanced application of CRISPR system for DMD therapy. The gene editing technology of CRISPR/Cas9 provides an effective treatment strategy for the Duchenne muscular dystrophy (DMD) therapy. However, the delivery of CRISPR system in mammalian cell mainly relies on viral mediated transport and the NGG or NAG requirement of wild-typed Cas9 protein limits the target region in DMD gene. Here, the present study provides a biomineralized PAM Less Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo. This study may extend the application of CRISPR system for DMD gene therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Nanoparticle-anchored platelet systems hold great potential to act as drug carriers in post-surgical cancer treatment due to their intrinsic ability to target the bleeding sites. However, rational design is still needed to further improve its cargo release profiles to meet the cytosolic delivery of therapeutic proteins with intracellular targets. Herein, we developed a tumor microenvironment (TME)-responsive backpack-conjugated platelet system to enhance intracellular protein delivery, thereby significantly inhibiting tumor recurrence after surgery. Specifically, protein nanogels encapsulating GALA and Granzyme B (GrB) are conjugated on the platelet surface via an acid-sensitive benzoic-imine linker through a biorthogonal reaction (GALA-GNGs-P). Taking advantage of wound-tropism of platelets, GALA-GNGs-P could actively accumulate at the surgical trauma and release nanogels in response to acidic TME for promoting deep penetration. Following cellular uptake, the pore-forming peptide GALA helps nanogels escape from lysosome. Subsequently, high glutathione (GSH) concentration in tumor cytoplasm facilitates GrB release from NGs, leading to intense cell apoptosis. GALA-GNGs-P shows remarkable tumor-targeting capability, high cellular uptake, and outstanding lysosomal escaping ability, which can significantly inhibit tumor recurrence in mice models with incomplete tumor resection. Our findings indicate that platelets bioengineered with TME-responsive protein nanogels provide an option to intracellularly deliver therapeutic proteins for the post-surgical treatment of cancer. Platelet-based drug delivery systems (DDSs) have gained considerable achievements in post-surgical cancer treatment. However, there is no research exploring their potential in realizing the controllable release of cargoes in the acidic tumor microenvironment (TME). Herein, we developed a TME-responsive bioengineered platelet delivery platform (GALA-GNGs-P) for achieving controllable and effective protein intracellular delivery to overcome post-surgical tumor recurrence. Our surface-anchored nanogel-platelet system has the following advantages: (i) improving the loading efficiency of therapeutic proteins, (ii) affecting no physiological function of platelets, (iii) realizing on-demand cargo release in the acidic TME, and (iv) helping proteins escape from endosomal entrapment. Our findings further explored the prospect of cellular backpack system and realized the controllable release of cargoes in the acidic TME. [Display omitted] [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA)-based therapies have shown great potential in the repair of spinal cord injury (SCI). MicroRNA 21 (miR21) has been proven to have an essential protective effect on SCI. However, there are some challenges for miRNAs application due to their easy degradation and ineffective cell penetration. As natural vesicles, exosomes were considered ideal carriers for miRNAs delivery for their advantages of low immunogenicity, inherent stability and tissue/cell penetration. However, poor targeting and the low capacity of specific miRNAs impede their practical applications. This study aims to develop a type of genetically engineered miR21-loaded exosomes that can be entrapped in collagen-I (Col-I) scaffold to repair SCI. The collagen-binding domain (CBD)-fused lysosome-associated membrane glycoprotein 2b (Lamp2b) protein (CBD-LP) and miR21 were overexpressed in host HEK293T (293T) cells that were used to produce engineered miR21-loaded exosomes. The CBD peptide fused in Lamp2b on the exosome surface can stably tether exosomes to Col-I scaffold, facilitate the retention of miR21-loaded exosomes in lesion sites, promote the sustained release of miR21 to cells. Finally, a functionalized Col-I scaffold biomaterial enriched with miR21-loaded exosomes was developed and it could benefit the repair of SCI. MiRNA-based therapeutics have promising potential in spinal cord injury (SCI) repair. However, easy degradation and ineffective cell penetration impede miRNAs application. Exosomes are natural vehicles for miRNAs delivery but face the challenge of diffusion in vivo. Here, the collagen-binding domain (CBD)-fused Lamp2b and miR21 were overexpressed in HEK293T cells to produce miR21-loaded and CBD-modified exosomes (CBD-LP-miR21-EXOs). The CBD modified on the exosome surface can stably tether exosomes to collagen-I scaffold to form functionalized CBD-LP-miR21-EXO-Col scaffold that can facilitate the retention of miR21-loaded exosomes, promote the sustained release of miR21 to cells and finally benefit SCI repair. Furthermore, this type of functionalized collagen-I materials can be widely applied for other tissue injury repairs by enriching the CBD-LP-EXOs loaded with appropriate miRNAs. [Display omitted] [ABSTRACT FROM AUTHOR]

Deoxynivalenol (DON) is a secondary metabolite of Fusarium fungi and belonged to trichothecenes, and it widely presents in various food commodities. Previous studies have highlighted its potent toxicity, adversely affecting the growth, development, and reproductive in both humans and animals. However, the potential impact of DON on porcine oocyte organelles remains elusive. In present study, we delved into the toxic effects of DON on mitochondria, endoplasmic reticulum, Golgi during the porcine oocyte maturation. Our findings revealed that DON exposure significantly impeded granulosa cell diffusion and the expulsion of the first polar body. Additionally, mitochondrial fluorescence intensity and membrane potential underwent notable alterations under DON exposure. Notably, lysosomal fluorescence intensity decreased significantly, suggesting protein degradation and potential autophagy, which was further corroborated by the enhanced fluorescence intensity of LC3. Furthermore, endoplasmic reticulum fluorescence intensity declined, and DON exposure elevated endoplasmic reticulum stress levels, evident from the upregulated expression of GRP78. Concurrently, we observed disruption in the fusiform cortex distribution of the Golgi apparatus, characterized by reduced Golgi apparatus fluorescence intensity and GM130 expression. Collectively, our results indicate that DON exposure profoundly affects the fundamental functions of porcine oocyte organelles during meiosis and maturation. • DON impeded granulosa cell diffusion and porcine oocyte polar body extrusion. • DON altered mitochondria distribution and membrane potential of porcine oocytes. • DON disrupted lysosomal function and induced autophagy in porcine oocytes. • DON increased endoplasmic reticulum stress by GRP78 in porcine oocytes. • DON reduced GM130 and altered Golgi apparatus distribution of porcine oocytes. [ABSTRACT FROM AUTHOR]

Perfluorooctane sulfonate (PFOS) is known as a persistent organic pollutant. A significant correlation between PFOS and liver ferroptosis has been unveiled, but the precise mechanism needs to be elucidated. In prior research, we found that PFOS treatment provoked mitochondrial iron overload. In this study, we observed a gradual increase in lysosomal iron in L-O2 cells after exposure to PFOS for 0.5–24 h. In PFOS-exposed L-O2 cells, suppressing autophagy relieved the lysosomal iron overload. Inhibiting transient receptor potential mucolipin 1 (TRPML1), a calcium efflux channel on the lysosomal membrane, led to a further rise in lysosomal iron levels and decreased mitochondrial iron overload during PFOS treatment. Suppressing VDAC1, a subtype of voltage-dependent anion-selective channels (VDACs) on the outer mitochondrial membrane, had no impact on PFOS-triggered mitochondrial iron overload, whereas restraining VDAC2/3 relieved this condition. Although silencing VDAC2 relieved PFOS-induced mitochondrial iron overload, it had no effect on PFOS-triggered lysosomal iron overload. Silencing VDAC3 alleviated PFOS-mediated mitochondrial iron overload and led to an additional increase in lysosomal iron. Therefore, we regarded VDAC3 as the specific VDACs subtype that mediated the lysosomes-mitochondria iron transfer. Additionally, in the presence of PFOS, an enhanced association between TRPML1 and VDAC3 was found in mice liver tissue and L-O2 cells. Our research unveils a novel regulatory mechanism of autophagy on the iron homeostasis and the effect of TRPML1-VDAC3 interaction on lysosomes-mitochondria iron transfer, giving an explanation of PFOS-induced ferroptosis and shedding some light on the role of classic calcium channels in iron transmission. [Display omitted] • Autophagy induces lysosomal iron overload under PFOS exposure. • TRPML1 mediates PFOS-provoked iron transfer between lysosomes and mitochondria. • VDAC3, not VDAC1/2, mediates PFOS-induced lysosomes-mitochondria iron transfer. • TRPML1 interacts with VDAC3 under PFOS exposure. • Lysosomes-mitochondria iron transmission mediates the ferroptosis elicited by PFOS. [ABSTRACT FROM AUTHOR]

A central bearded dragon (Pogona vitticeps) presented with periorbital swelling and exophthalmos. A retrobulbar mass was detected, and enucleation with the mass was performed. Histologically, the mass was composed of a dense sheet and interlacing bundles of round to polygonal to short spindle-shaped cells with occasional bizarre mononuclear and multinucleated giant cells. Immunohistochemically, the neoplastic cells had various degrees of membranous and/or cytoplasmic granular reactivity to anti-ionized calcium-binding adapter molecule 1 and anti-CD204 antibodies. Ultrastructurally, the neoplastic cells had irregular nuclei and abundant cytoplasm with membrane-bound electron-dense lysosomes and endoplasmic reticula. These findings were consistent with a histiocytic sarcoma. The present study provided a detailed description of retrobulbar histiocytic sarcoma for the first time in a central bearded dragon. [ABSTRACT FROM AUTHOR]

Background: We have demonstrated that protein aggregation plays a pivotal role in the pathophysiology of preeclampsia and identified several aggregated proteins in the circulation of preeclampsia patients, the most prominent of which is the serum protein TTR (transthyretin). However, the mechanisms that underlie protein aggregation remain poorly addressed.Methods: We examined TTR aggregates in hypoxia/reoxygenation-exposed primary human trophoblasts (PHTs) and the preeclampsia placenta using complementary approaches, including a novel protein aggregate detection assay. Mechanistic analysis was performed in hypoxia/reoxygenation-exposed PHTs and Ttr transgenic mice overexpressing transgene-encoded wild-type human TTR or Ttr-/- mice. High-resolution ultrasound analysis was used to measure placental blood flow in pregnant mice.Results: TTR aggregation was inducible in PHTs and the TCL-1 trophoblast cell line by endoplasmic reticulum stress inducers or autophagy-lysosomal disruptors. PHTs exposed to hypoxia/reoxygenation showed increased intracellular BiP (binding immunoglobulin protein), phosphorylated IRE1α (inositol-requiring enzyme-1α), PDI (protein disulfide isomerase), and Ero-1, all markers of the unfolded protein response, and the apoptosis mediator caspase-3. Blockade of IRE1α inhibited hypoxia/reoxygenation-induced upregulation of Ero-1 in PHTs. Excessive unfolded protein response activation was observed in the early-onset preeclampsia placenta. Importantly, pregnant human TTR mice displayed aggregated TTR in the junctional zone of the placenta and severe preeclampsia-like features. High-resolution ultrasound analysis revealed low blood flow in uterine and umbilical arteries in human TTR mice compared with control mice. However, Ttr-/- mice did not show any pregnancy-associated abnormalities.Conclusions: These observations in the preeclampsia placenta, cultured trophoblasts, and Ttr transgenic mice indicate that TTR aggregation is an important causal contributor to preeclampsia pathophysiology. [ABSTRACT FROM AUTHOR]

Tollip is a multifunctional adaptor protein implicated in innate immunity, lysosomal trafficking/autophagy of protein aggregates and various signaling processes in mammalian models. To verify evolutionary conservation of these functions, we used CRISPR/Cas9 editing to construct a zebrafish line bearing a stable tollip knockout. In contrast to previously reported tollip morphants, Tollip-deficient fish display normal development until adulthood, are fertile, and have no apparent physiological defects. When challenged with lipopolysaccharide (LPS), inflammatory gene expression is unaffected. Moreover, Tollip deficiency does not aggravate swimming deficiency resulting from lysosomal dysfunction and proteotoxicity in a fish model of Gaucher disease. Thus, individual functions of Tollip may be organism-specific or manifest only upon certain conditions/challenges or disease backgrounds. [ABSTRACT FROM AUTHOR]