Four-Color SERS Monitoring of Size-dependent Nanoparticle Delivery in the Same Tumor.

The physicochemical properties of nanoparticles (NPs) significantly influence their deposition at the disease site, ultimately impacting the overall therapeutic efficacy; however, precisely assessing the effects of various factors on NP accumulation within a single cell/tumor tissue is challenging due to the lack of appropriate labeling techniques. Surface-enhanced Raman spectroscopy (SERS) tag is a powerful encoding method that has recently been intensively employed for immunodetection of biomarkers. Herein, we introduce a multiplexed SERS tracking approach for systematic investigation of size-dependent accumulation and distribution of NPs within the same tumor. Four-sized (34, 60, 108, and 147 nm) NPs encoded with different SERS "colors" were fabricated, mixed, and incubated with monolayer tumor cells, multicellular tumor spheroids, or injected into mouse models bearing xenograft solid tumors in a single dose. Multicolor SERS detection of the specimens revealed that NP accumulation in tumor cells, tumor spheroids, and solid tumors was in the order of 34 nm > 60 nm > 108 nm > 147 nm, 60 nm > 34 nm > 108 nm > 147 nm, and 34 nm > 147 nm > 108 nm > 60 nm, respectively. Inductively coupled plasma mass spectroscopy determination performed in parallel samples were in alignment with the four-color SERS probing results, demonstrating the effectiveness of this multiplexed evaluation assay. Furthermore, in combination with fluorescence labeling of specific biomolecules, this method can be applied for the colocalization of different NPs in various pathological structures and provide additional information for analysis of the possible mechanisms.

2, 2-dimethylthiazolidine hydrochloride protects against experimental contrast-induced acute kidney injury via inhibition of tubular ferroptosis.

Contrast-induced acute kidney injury (CI-AKI) has become the third leading cause of AKI acquired in hospital, lacking of effective interventions. In the study, we identified the renal beneficial role of 2, 2-dimethylthiazolidine hydrochloride (DMTD), a safer compound which is readily hydrolyzed to cysteamine, in the rodent model of CI-AKI. Our data showed that administration of DMTD attenuated the impaired renal function and tubular injury induced by the contrast agent. Levels of MDA, 4-hydroxynonenal, ferrous iron and morphological signs showed that contrast agent induced ferroptosis, which could be inhibited in the DMTD group. In vitro, DMTD suppressed ferroptosis induced by ioversol in the cultured tubular cells. Treatment of DMTD upregulated glutathione (GSH) and glutathione peroxidase 4 (GPX4). Moreover, we found that DMTD promoted the ubiquitin-mediated proteasomal degradation of Keap1, and thus increased the activity of nuclear factor erythroid 2-related factor 2 (Nrf2). Mechanistically, increase of the ubiquitylation degradation of Keap1 mediates the upregulated effect of DMTD on Nrf2. Consequently, activated Nrf2/Slc7a11 results in the increase of GSH and GPX4, and therefore leads to the inhibition of ferroptosis. Herein, we imply DMTD as a potential therapeutic agent for the treatment of CI-AKI.

Combined legumain- and integrin-targeted nanobubbles for molecular ultrasound imaging of breast cancer.

Molecular ultrasound imaging is a promising strategy for non-invasive and precise cancer diagnosis. Previously reported ultrasound contrast agents (UCAs) are mostly microbubbles or nanobubbles (NBs) larger than 200 nm, leading to less efficient tumor delivery. Here we synthesized NBs with a small size (~49 nm) and modified the NB surface with alanine-alanine-asparagine (NB-A) or arginine-glycine-aspartic acid peptide (NB-R) for concurrent active targeting towards legumain in tumor cells and integrin in tumor neovasculature. In vitro, the NB-A and NB-R presented echogenicity comparable with SonoVue MBs and showed specific binding with tumors cells and endothelial cells, respectively. In vivo, the combined NB-A/NB-R accumulated in tumor tissues selectively and provided ultrasound signals with prolonged duration and that were significantly stronger than non-targeted NBs, single-targeted NBs and SonoVue MBs. Overall, the dual targeted NBs served as efficient UCAs for specific imaging of breast cancer, and hold great potential for general cancer diagnosis/monitoring in the future.

Legumain, an asparaginyl endopeptidase, mediates the effect of M2 macrophages on attenuating renal interstitial fibrosis in obstructive nephropathy.

Two distinct macrophage phenotypes contribute to kidney injury and repair during the progression of renal interstitial fibrosis; proinflammatory (M1) and antiinflammatory (M2) macrophages. Legumain, an asparaginyl endopeptidase of the cysteine protease family, is overexpressed in macrophages in some pathological conditions. However, the macrophage subtype and function of macrophage-derived legumain remains unclear. To resolve this we tested whether M2 macrophages contribute to the accumulation of legumain in the unilateral ureteral obstruction model. Legumain-null mice exhibited more severe fibrotic lesions after obstruction compared with wild-type control. In vitro, IL4-stimulated M2 polarization led to the overexpression and secretion of legumain. The levels of fibronectin and collagen I/III, major components of the extracellular matrix, were reduced in the conditioned medium of TGF-ß1-stimulated tubular epithelial cells or fibroblasts after treatment with legumain or conditioned medium from IL4-stimulated macrophages. Administration of the legumain inhibitor RR-11a exacerbated fibrotic lesions following obstruction. Therapeutically, adoptive transfer of legumain-overexpressing macrophages or IL4-stimulated macrophages ameliorated the deposition of collagen and fibronectin induced by ureteral obstruction, either in the wild-type mice or in lgmn-/- mice. Thus, M2 macrophages overexpress and secret legumain and legumain mediates the anti-fibrotic effect of M2 macrophages in obstructive nephropathy.

Overexpression of Oct4 suppresses the metastatic potential of breast cancer cells via Rnd1 downregulation.

Although Oct4 is known as a critical transcription factor involved in maintaining "stemness", its role in tumor metastasis is still controversial. Herein, we overexpressed and silenced Oct4 expression in two breast cancer cell lines, MDA-MB-231 and 4T1, separately. Our data showed that ectopic overexpression of Oct4 suppressed cell migration and invasion in vitro and the formation of metastatic lung nodules in vivo. Conversely, Oct4 downregulation increased the metastatic potential of breast cancer cells both in vitro and in vivo. Furthermore, we identified Rnd1 as the downstream target of Oct4 by ribonucleic acid sequencing (RNA-seq) analysis, which was significantly downregulated upon Oct4 overexpression. Chromatin immunoprecipitation assays revealed the binding of Oct4 to the promoter region of Rnd1 by ectopic overexpression of Oct4. Dual luciferase assays indicated that Oct4 overexpression suppressed transcriptional activity of the Rnd1 promoter. Moreover, overexpression of Rnd1 partially rescued the inhibitory effects of Oct4 on the migration and invasion of breast cancer cells. Overexpression of Rnd1 counteracted the influence of Oct4 on the formation of cell adhesion and lamellipodia, which implied a potential underlying mechanism involving Rnd1. In addition, we also found that overexpression of Oct4 led to an elevation of E-cadherin expression, even in 4T1 cells that possess a relatively high basal level of E-cadherin. Rnd1 overexpression impaired the promoting effects of Oct4 on E-cadherin expression in MDA-MB-231 cells. These results suggest that Oct4 affects the metastatic potential of breast cancer cells through Rnd1-mediated effects that influence cell motility and E-cadherin expression.

Atypical PKC, regulated by Rho GTPases and Mek/Erk, phosphorylates Ezrin during eight-cell embryocompaction.

Phosphorylation of Ezrin T567 plays an important role in eight-cell embryo compaction. Yet, it is not clear how Ezrin phosphorylation is regulated during embryo compaction. Here, we demonstrated that inhibition of Mek/Erk or protein kinase C (PKC) signaling reduced the phosphorylation level of Ezrin T567 in eight-cell compacted embryos. Interestingly, the Rho GTPase inhibitor C3-transferase caused basolateral enrichment of atypical PKC (aPKC), as well as basolateral shift of phosphorylated Ezrin, suggesting aPKC may be a key regulator of Ezrin phosphorylation. Moreover, inhibition of PKC, but not Mek/Erk or Rho GTPases, affected the maintenance of Ezrin phosphorylation in compacted embryos. We further identified that aPKC is indeed required for Ezrin phosphorylation in eight-cell embryos. Taken together, Rho GTPases facilitate the apical distribution of aPKC and Ezrin. Subsequently, aPKC and Mek/Erk work together to promote Ezrin phosphorylation at the apical region, which in turn mediates the apical enrichment of filamentous actin, stabilizing the polarized apical region and allowing embryo compaction. Our data also suggested that aPKC might be the Ezrin kinase during eight-cell embryo compaction.

Self-Referenced Au Nanoparticles-Coated Glass Wafers for In Situ SERS Monitoring of Cell Secretion.

Surface-enhanced Raman spectroscopy (SERS) is a powerful technique for discrimination of bimolecules in complex systems. However, its practical applications face challenges such as complicated manufacturing procedures and limited scalability of SERS substrates, as well as poor reproducibility during detection which compromises the reliability of SERS-based analysis. In this study, we developed a convenient method for simultaneous fabrication of massive SERS substrates with an internal standard to eliminate the substrate-to-substrate differences. We first synthesized Au@CN@Au nanoparticles (NPs) which contain embedded internal standard molecules with a single characteristic peak in the Raman-silent region, and then deposited the NPs on 6 mm glass wafers in a 96-well plate simply by centrifugation for 3 min. The one-time obtained 96 SERS substrates have excellent intrasubstrate uniformity and intersubstrate repeatability for SERS detection by using the internal standard (relative standard deviation = 10.47%), and were able to detect both charged and neutral molecules (crystal violet and triphenylphosphine) at a concentration of 10-9 M. Importantly, cells can be directly cultured on glass wafers in the 96-well plate, enabling real time monitoring of the secretes and metabolism change in response to external stimulation. We found that the release of nucleic acids, amino acids and lipids by MDA-MB-231 cells significantly increased under hypoxic conditions. Overall, our approach enables fast and large-scale production of Au@CN@Au NPs-coated glass wafers as SERS substrates, which are homogeneous and highly sensitive for monitoring trace changes of biomolecules.

"Triple-Punch" Strategy Exosome-Mimetic Nanovesicles for Triple Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) is the most malignant breast cancer, with high rates of relapse and metastasis. Because of the nonspecific targeting of chemotherapy and insurmountable aggressiveness, TNBC therapy lacks an effective strategy. Exosomes have been reported as an efficient drug delivery system (DDS). CD82 is a tumor metastasis inhibitory molecule that is enriched in exosomes. Aptamer AS1411 specifically targets TNBC cells due to its high expression of nucleolin. We generated a "triple-punch" cell membrane-derived exosome-mimetic nanovesicle system that integrated with CD82 overexpression, AS1411 conjugation, and doxorubicin (DOX) delivery. CD82 enrichment effectively inhibits the migration of TNBC cells. AS1411 conjugation specifically targets TNBC cells. DOX loading effectively inhibits proliferation and induces apoptosis of TNBC cells. Our results demonstrate a system of exosome-mimetic nanovesicles with "triple-punch" that may facilitate TNBC therapeutics.

LATS1 inhibitor and zinc supplement synergistically ameliorates contrast-induced acute kidney injury: Induction of Metallothionein-1 and suppression of tubular ferroptosis.

Contrast-induced acute kidney injury (CI-AKI) is a prevalent cause of renal dysfunction among hospitalized patients, yet the precise pathogenesis and effective therapeutic strategies remain elusive. In this study, we investigated the role of tubular ferroptosis in both experimental CI-AKI models and in primary tubular epithelial cells (PTECs) treated with ioversol. Using whole exome sequencing, we identified metallothioneins (MTs) as being among the most significantly downregulated genes following ioversol exposure. Our findings reveal that overexpression of Mt1 mitigates, whereas suppression of Mt-1 exacerbates, ioversol-induced tubular ferroptosis. Interestingly, the level of MTF1 (metal regulatory transcription factor 1), a principal regulator of Mt1, was found to increase in response to ioversol treatment. We further elucidated that ioversol activates LATS1 (Large tumor suppressor homolog 1), a kinase that promotes the phosphorylation and nuclear translocation of MTF1, thereby inhibiting its transcriptional activity for Mt1. Both genetic and pharmacological inhibition of LATS1 reversed the ioversol-induced suppression of Mt-1. From a therapeutic perspective, the LATS1 inhibitor TDI-011536, in combination with zinc acetate, was administered to a rodent model of CI-AKI. Our data indicate that this combination synergistically upregulates Mt1 expression and provides protection against contrast media-induced tubular ferroptosis. In summary, our study demonstrates that the reduction of Mt-1 contributes to tubular ferroptosis associated with CI-AKI. We show that contrast media activate LATS1, which in turn suppresses the transcriptional activity of MTF1 for Mt1. Herein, the combination of zinc acetate and a LATS1 inhibitor emerges as a potential therapeutic approach for the treatment of CI-AKI.

Kukoamine A alleviates nephrolithiasis by inhibiting endogenous oxalate synthesis via the IL-6/JAK/STAT3/DAO signaling pathway.

BACKGROUND: The recurrent nature and socioeconomic burden of nephrolithiasis demand effective treatments. Delineating the crosstalk between inflammatory processes and the endogenous oxalate metabolism pathway, which underpins nephrolithiasis pathogenesis, is essential for advancing treatment strategies. PURPOSE: We aim to screen therapeutic Chinese herbal remedies and their bioactive constituents for kidney stone treatment using a fruit fly model, followed by efficacy and mechanism validation in a rodent nephrolithiasis model as well as in vitro human cell culture model. STUDY DESIGN AND METHODS: We developed a fruit fly model to screen for efficient traditional Chinese medicine herbs and their active compounds for kidney stone treatment. Candidate active compounds from efficient herbs were separated and identified by solid-phase chromatography coupled with LC-MS analysis. Fruit fly genetic tools were employed to manipulate the expression of related genes to explore the therapeutic mechanisms of the Lycii Cortex and kukoamine A (KuA). To confirm the therapeutic effects and mechanisms of KuA for mammalian nephrolithiasis, mouse model of glyoxylate-induced kidney stone and human renal tubular cells were used. The therapeutic role of kukoamine A in nephrolithiasis was evaluated by assessing tubular injury, crystal deposition, and adhesion. The level of expression and phosphorylation in cells and mice was assessed using RT-qPCR and western blot. RESULTS: Our findings indicate that Lycii Cortex potently inhibits calcium oxalate stone formation via activation of the JNK/Upd3/JAK/STAT signaling cascade, resulting in diminished endogenous oxalate synthesis by downregulating D-amino acid oxidase (DAO) gene expression, predominantly in fruit fly Malpighian tube stellate cells. KuA was identified as the principal bioactive constituent mediating these effects. Both mouse models and human cell assays confirmed KuA's efficacy in preventing calcium oxalate nephrolithiasis in mammals, through hepatic JAK/STAT3 pathway activation and upregulation of IL-6, culminating in reduced urinary crystal deposition. CONCLUSION: Our research underscores the potential of kukoamine A as a lead compound in treating nephrolithiasis and reveals the interplay between the IL-6/JAK/STAT3 inflammatory pathway and endogenous oxalate metabolism in nephrolithiasis pathogenesis. Additionally, it highlights the utility of the fruit fly model as a powerful tool for deciphering the therapeutic mechanisms of traditional Chinese herbs.

KDM6B regulates M2 polarization of macrophages by modulating the stability of nuclear ß-catenin.

Accumulating evidences suggest that the epigenetic regulation plays a pivotal role in establishing phenotype and function of tumor associated macrophages (TAMs). KDM6B is an epigenetic enzyme responsible for the H3K27me3 and reported to influence macrophage polarization. However, the underlying mechanism remains to be determined. Here, we demonstrated that inhibition of KDM6B in TAMs increased M2 polarization induced by coculture of breast cancer cells. Furthermore, we identified that KDM6B downregulation activated ß-catenin/c-Myc signaling, and thus promoted the M2-like phenotype. KDM6B accelerated the intranuclear ubiquitination degradation of ß-catenin, which depended on its demethylase activity. Therapeutically, our data showed that activated vitamin D analog paricalcitol upregulated the expression of KDM6B and decreased the M2 polarization, consequently protected against tumor progress in the xenograft mouse model of breast cancer. Taken together, our data reveal that epigenetic regulator KDM6B prevents M2 polarization via promoting the intranuclear degradation of ß-catenin. Active vitamin D analog induces KDM6B and suppresses tumor progress, suggesting a novel therapeutic potential of epigenetic modulation for the tumor treatment.

Ultrasensitive iodide detection in biofluids based on hot electron-induced reduction of p-Nitrothiophenol on Au@Ag core-shell nanoparticles.

Surveillance of iodine intake is important because either inadequate or excessive amount of iodine may lead to thyroid malfunctions. Herein, we report a method for fast iodide quantification based on a plasmonic hot electron-driven chemical reaction, which occurs on Au@Ag core-shell nanoparticles (NPs) coated with p-nitrothiophenol (PNTP) molecules. Upon resonant light illumination, hot electron-hole pairs are generated in the NPs. The hot holes capture iodide ions (I-) and form AgI which decomposes under light; while the hot electrons are shifted to the electron orbital (LUMO) of PNTP and trigger its reduction to p-aminothiophenol (PATP). By measuring characteristic surface-enhanced Raman spectroscopic (SERS) peaks of PNTP and PATP, the concentration of I- in water can be quantitatively determined, with a linear response in the 0.5-20 µM range and a detection limit of 0.30 µM. The Au@Ag nanosensor was then applied for I- detection in various biofluids including urine, serum and saliva, exhibiting superior detection sensitivity and high selectivity. This sensing assay requires a small sample volume of ∼10 µL and completes the entire detection process in ∼2 min, and therefore holds significant potential for application in point-of-care settings.

PDIA4 confers resistance to ferroptosis via induction of ATF4/SLC7A11 in renal cell carcinoma.

The prognosis of renal cell carcinoma (RCC) remains poor due to metastases and resistance to chemotherapy. Salinomycin (Sal) exhibits the potential of antitumor, while the underlying mechanism is not completely clear. Here, we found that Sal induced ferroptosis in RCCs and identified Protein Disulfide Isomerase Family A Member 4 (PDIA4) as a mediator of Sal's effect on ferroptosis. Sal suppressed PDIA4 by increasing its autophagic degradation. Downregulation of PDIA4 increased the sensitivity to ferroptosis, while ectopic overexpression of PDIA4 conferred ferroptosis resistance to RCCs. Our data showed that downregulation of PDIA4 suppressed activating transcription factor 4 (ATF4) and its downstream protein SLC7A11 (solute carrier family 7 member 11), thereby aggravating ferroptosis. In vivo, the administration of Sal promoted ferroptosis and suppressed tumor progress in the xenograft mouse model of RCC. Bioinformatical analyses based on clinical tumor samples and database indicated a positive correlation exists between PDIA4 and PERK/ATF4/SLC7A11 signaling pathway, as well as the malignant prognosis of RCCs. Together, our findings reveal that PDIA4 promotes ferroptosis resistance in RCCs. Treatment of Sal sensitizes RCC to ferroptosis via suppressing PDIA4, suggesting the potential therapeutical application in RCCs.

New Insights into the Mechanisms of Chaperon-Mediated Autophagy and Implications for Kidney Diseases.

Chaperone-mediated autophagy (CMA) is a separate type of lysosomal proteolysis, characterized by its selectivity of substrate proteins and direct translocation into lysosomes. Recent studies have declared the involvement of CMA in a variety of physiologic and pathologic situations involving the kidney, and it has emerged as a potential target for the treatment of kidney diseases. The role of CMA in kidney diseases is context-dependent and appears reciprocally with macroautophagy. Among the renal resident cells, the proximal tubule exhibits a high basal level of CMA activity, and restoration of CMA alleviates the aging-related tubular alternations. The level of CMA is up-regulated under conditions of oxidative stress, such as in acute kidney injury, while it is declined in chronic kidney disease and aging-related kidney diseases, leading to the accumulation of oxidized substrates. Suppressed CMA leads to the kidney hypertrophy in diabetes mellitus, and the increase of CMA contributes to the progress and chemoresistance in renal cell carcinoma. With the progress on the understanding of the cellular functions and uncovering the clinical scenario, the application of targeting CMA in the treatment of kidney diseases is expected.

YAP1-TEAD1 mediates the perineural invasion of prostate cancer cells induced by cancer-associated fibroblasts.

Perineural invasion (PNI) driven by the tumor microenvironment (TME) has emerged as a key pattern of metastasis of prostate cancer (PCa), while its underlying mechanism is still elusive. Here, we identified increased CAFs and YAP1 expression levels in patients with metastatic PCa. In the cultured PCa cell line LNCaP, co-culture with cancer-associated fibroblasts (CAFs) could upregulate YAP1 protein expression. Either ectopic overexpression of YAP1 or co-culture with CAFs could promote the infiltration of LNCaPs towards dorsal root ganglia (DRG). This effect could be blocked using an YAP1 inhibitor. In vivo, overexpression of YAP1 could increase PNI in a mouse model of sciatic nerve tumor invasion. Mechanistically, TEAD1 binds to the NGF promotor and YAP1/TEAD1 activates its transcription and consequently increases NGF secretion. In turn, PCa cells treated with CM from CAFs or stable YAP1 overexpression can stimulate DRG to secrete CCL2. The epithelial-to-mesenchymal transition (EMT) of PCa cells is thus activated via CCL2/CCR2. Overall, our data demonstrate that CAFs can activate YAP1/TEAD1 signaling and increase the secretion of NGF, therefore promoting PCa PNI. In addition, EMT induced by PNI suggests a feedback loop is present between neurons and PCa cells.

Gallic acid ameliorates calcium oxalate crystal-induced renal injury via upregulation of Nrf2/HO-1 in the mouse model of stone formation.

BACKGROUND: High prevalence and reoccurrence rate of nephrolithiasis bring about serious socioeconomic and healthcare burden, necessitating the need of effective therapeutic agents. Previous study revealed that gallic acid (GAL) alters the nucleation pathway of calcium oxalate (CaOx). On the other hand, it appears protective role against oxidative injury. Whether GAL could protect against crystal-induced lesion in vivo, and its underlying mechanism is yet unsolved. PURPOSE: This study aims to investigate the protective effects of GAL on the crystal-induced renal injury and its underlying mechanism in the mouse model of stone formation induced by glyoxylic acid. STUDY DESIGN AND METHODS: The mouse model of stone formation was established via successive intraperitoneal injection of glyoxylate. Proximal tubular epithelial cell line HK-2 treated with calcium oxalate monohydrate (COM) was used as in vitro model. The protective role of GAL on nephrolithiasis was tested by determination of tubular injury, crystal deposition and adhesion, levels of inflammatory cytokines, macrophage infiltration and the redox status of kidney. In vitro, effect of GAL on the ROS level and oxidative tubular injury induced by COM were detected, as well as major antioxidant pathway Nrf2/HO-1. RESULTS: Administration of GAL alleviates the renal deposition and adhesion of CaOx stone. Meanwhile, GAL ameliorates the inflammation and renal tubular injury. Level of intracellular ROS, osteopontin and CD44 are reduced, either in the mouse model of stone formation or in the COM-treated HK-2 cells after treatment of GAL. Mechanistically, GAL activates Nrf2/HO-1 pathway in HK-2 cells. Silencing Nrf2 abrogates the protective effect of GAL on the oxidative injury and adhesion of COM in HK-2 cells. CONCLUSION: Taken together, our study demonstrates the protective effect of GAL on the deposition of kidney stone and consequent tubular injury. Induction of the antioxidant pathway Nrf2/HO-1 was found to decrease the level of ROS and oxidative injury, thus implying that GAL could be a potential therapeutic agent for the treatment of nephrolithiasis.

Asparagine endopeptidase-targeted Ultrasound-responsive Nanobubbles Alleviate Tau Cleavage and Amyloid-ß Deposition in an Alzheimer's Disease Model.

Inhibition of asparagine endopeptidase (AEP) has been implied to be effective for treating tau- and amyloid-beta-mediated neurodegenerative diseases, although a method for targeted intracerebral delivery of AEP inhibitors has not yet been achieved. Here, we fabricated ultrasound-responsive nanobubbles (NBs) to load AEP inhibitor RR-11a, and modified the NB surface with either AEP recognizable peptide AAN or pro-transendothelial transversal motif RGD, i.e. NB(11a)-A and NB(11a)-R, for AEP-targeted treatment of Alzheimer's disease (AD). The developed NBs were uniform, small in size (50.1 ± 1.5 nm), with strong echogenicity and high drug loading efficiency (∼91.97%). When intravenously co-injected in the APP/PS1 mouse model, NB(11a)-R could adhere to endothelial cells and enhance transient opening of the blood-brain barrier (BBB) upon focused ultrasound oscillations, allowing the rest NBs/localized released RR-11a molecules to enter the brain, and then NB(11a)-A could selectively bind with the impaired neurons and deposit RR-11a molecules at the AD lesion. As a result, co-administration of NB(11a)-A and NB(11a)-R significantly promoted accumulation of RR-11a in the mouse brain, and substantially alleviated both tau cleavage and amyloid plaques deposition in the hippocampus. Most strikingly, the cognitive ability of the AD model mice was dramatically improved, achieving a level close to the normal mice. Overall, this unique AEP-targeted nanobubble design provides an efficient intracerebral drug delivery strategy and significantly enhances treatment efficacy of AD. STATEMENT OF SIGNIFICANCE: Asparagine endopeptidase (AEP) is an innovative therapeutic target simultaneously involved in Aß and tau-mediated Alzheimer's disease (AD) pathology, but targeted delivery of AEP inhibitors has not been achieved yet. Here we developed an efficient strategy to deliver AEP inhibitor RR-11a towards impaired neurons. We fabricated RR-11a-loaded ultrasound-responsive nanobubbles (NBs) and modified the NB surface with RGD peptide to promote BBB crossing upon focused ultrasound oscillations, or with AAN peptide to increase binding of NBs on the neurons. Our results indicated that, co-administration of the NB(11a)-A and NB(11a)-R significantly enhanced accumulation of RR-11a molecules at the AD lesion, alleviated both tau cleavage and amyloid plaques deposition in the hippocampus, and consequently restored cognitive function of the AD model mice.

Loss of legumain induces premature senescence and mediates aging-related renal fibrosis.

Aging is an independent risk factor for acute kidney injury and subsequent chronic kidney diseases, while the underlying mechanism is still elusive. Here, we found that renal tubules highly express a conserved lysosomal endopeptidase, legumain, which is significantly downregulated with the growing of age. Tubule-specific legumain-knockout mice exhibit spontaneous renal interstitial fibrosis from the 3rd month. In the tubule-specific legumain-knockout mice and the cultured legumain-knockdown HK-2 cells, legumain deficiency induces the activation of tubular senescence and thus increases the secretion of profibrotic senescence-associated cytokines, which in turn accelerates the activation of fibroblasts. Blockage of senescence mitigates the fibrotic lesion caused by legumain deficiency. Mechanistically, we found that silencing down of legumain leads to the elevated lysosome pH value, enlargement of lysosome size, and increase of lysosomal voltage dependent membrane channel proteins. Either legumain downregulation or aging alone induces the activation of nuclear transcription factors EB (TFEB) while it fails to further upregulate in the elderly legumain-knockdown tubules, accompanied with impaired mitophagy and increased mitochondrial ROS (mtROS) accumulation. Therapeutically, supplementation of exosomal legumain ameliorated fibronectin and collagen I production in an in vitro coculture system of tubular cells and fibroblasts. Altogether, our data demonstrate that loss of legumain in combined with aging dysregulates lysosomal homeostasis, although either aging or legumain deficiency alone induces lysosome adaptation via stimulating lysosomal biogenesis. Consequently, impaired mitophagy leads to mtROS accumulation and therefore activates tubular senescence and boosts the interstitial fibrosis.

Size-Dependent Penetration of Gold Nanoprobes into Fixed Cells.

Nanoprobes have been increasingly applied in the biomedical field due to their superior optical, electronic, or magnetic properties. Among the many aspects involved in the interaction between nanoprobes and biospecimens, size plays an essential role. Although the influence of size on their internalization behavior and distribution in live cells has been extensively studied, how does the size affect penetration of nanoprobes into fixed cells remains unknown. We investigate here the influence of size on the penetration behavior of gold nanoprobes into fixed mammalian cells by dark-field microscopy and surface-enhanced Raman scattering (SERS) microspectroscopy. We show that 14, 20, and 29 nm nanoprobes can readily enter into methanol-fixed MCF-7 cells, while 42 and 55 nm nanoprobes cannot cross the cell membrane. For 4% paraformaldehyde-fixed cells, even 14 nm nanoprobes can hardly get into the cells, but after treatment with permeabilization reagents, 14 and 20 nm nanoprobes are permitted to enter the cells. These findings provide important implications in future design of nanoprobes for cellular immunostaining.

Cancer-derived exosomal miR-138-5p modulates polarization of tumor-associated macrophages through inhibition of KDM6B.

Rationale: Differential activation of macrophages correlates closely with tumor progression, and the epigenetic factor lysine demethylase 6B (KDM6B, previously named JMJD3) mediates the regulation of macrophage polarization through an unknown mechanism. Methods: We developed a suspension coculture system comprising breast cancer cells and macrophages and used RT-qPCR and western blotting to measure KDM6B expression. Bioinformatics and luciferase reporter assays were used to identify candidate microRNAs of cancer cells responsible for the downregulation of KDM6B. To determine if exosomes mediated the transfer of miR-138-5p between cancer cells to macrophages, we treated macrophages with exosomes collected from the conditioned medium of cancer cells. The effects of exosomal miR-138-5p on macrophage polarization were measured using RT-qPCR, flow cytometry, and chromatin immunoprecipitation assays. We employed a mouse model of breast cancer, metastatic to the lung, to evaluate the effects on tumor metastasis of macrophages treated with miR-138-5p-enriched exosomes. To develop a diagnostic evaluation index, the levels of exosomal miR-138-5p in samples from patients with breast cancer were compared to those of controls. Results: Coculture of breast cancer cells led to downregulation of KDM6B expression in macrophages. Cancer cell-derived exosomal miR-138-5p inhibited M1 polarization and promoted M2 polarization through inhibition of KDM6B expression in macrophages. Macrophages treated with exosomal miR-138-5p promoted lung metastasis, and the level of circulating exosomal miR-138-5p positively correlated with the progression of breast cancer. Conclusion: Our data suggest that miR-138-5p was delivered from breast cancer cells to tumor-associated macrophages via exosomes to downregulate KDM6B expression, inhibit M1 polarization, and stimulate M2 polarization. Therefore, exosomal miR-138-5p represents a promising prognostic marker and target for the treatment of breast cancer.