Cross-Layer Contrastive Learning of Latent Semantics for Facial Expression Recognition.

Convolutional neural networks (CNNs) have achieved significant improvement for the task of facial expression recognition. However, current training still suffers from the inconsistent learning intensities among different layers, i.e., the feature representations in the shallow layers are not sufficiently learned compared with those in deep layers. To this end, this work proposes a contrastive learning framework to align the feature semantics of shallow and deep layers, followed by an attention module for representing the multi-scale features in the weight-adaptive manner. The proposed algorithm has three main merits. First, the learning intensity, defined as the magnitude of the backpropagation gradient, of the features on the shallow layer is enhanced by cross-layer contrastive learning. Second, the latent semantics in the shallow-layer and deep-layer features are explored and aligned in the contrastive learning, and thus the fine-grained characteristics of expressions can be taken into account for the feature representation learning. Third, by integrating the multi-scale features from multiple layers with an attention module, our algorithm achieved the state-of-the-art performances, i.e. 92.21%, 89.50%, 62.82%, on three in-the-wild expression databases, i.e. RAF-DB, FERPlus, SFEW, and the second best performance, i.e. 65.29% on AffectNet dataset. Our codes will be made publicly available.

Proprotein convertase cleavage of Ictalurid herpesvirus 1 spike-like protein ORF46 is modulated by N-glycosylation.

Viral spike proteins undergo a special maturation process that enables host cell receptor recognition, membrane fusion, and viral entry, facilitating effective virus infection. Here, we investigated the protease cleavage features of ORF46, a spike-like protein in Ictalurid herpesvirus 1 (IcHV-1) sharing similarity with spikes of Nidovirales members. We noted that during cleavage, full-length ORF46 is cleaved into ∼55-kDa and ∼100-kDa subunits. Moreover, truncation or site-directed mutagenesis at the recognition sites of proprotein convertases (PCs) abolishes this spike cleavage, highlighting the crucial role of Arg506/Arg507 and Arg668/Arg671 for the cleavage modification. ORF46 cleavage was suppressed by specific N-glycosylation inhibitors or mutation of its specific N-glycosylation sites (N192, etc.), suggesting that glycoprotein ORF46 cleavage is modulated by N-glycosylation. Notably, PCs and N-glycosylation inhibitors exhibited potent antiviral effects in host cells. Our findings, therefore, suggested that PCs cleavage of ORF46, modulated by N-glycosylation, is a potent antiviral target for fish herpesviruses.

Kaempferol is a novel antiviral agent against channel catfish virus infection through blocking viral attachment and penetration in vitro.

Channel catfish virus (CCV, Ictalurid herpesvirus 1) is the causative pathogen of channel catfish virus disease, which has caused high mortality and substantial economic losses in the catfish aquaculture industry. Due to the lack of licensed prophylactic vaccines and therapeutic drugs, the prevention and control of CCV infection seem to remain stagnant. Active compounds from medicinal plants offer eligible sources of pharmaceuticals and lead drugs to fight against endemic and pandemic diseases and exhibit excellent effect against viral infection. In this study, we evaluated the antiviral ability of 12 natural compounds against CCV with cell models in vitro and found kaempferol exhibited the strongest inhibitory compound against CCV infection among all the tested compounds. Correspondingly, kaempferol decreased transcription levels of viral genes and the synthesis of viral proteins, as well as reduced proliferation and release of viral progeny, the severity of the CPE induced by CCV in a dose-dependent manner, based on quantitative real-time PCR (RT-qPCR), western blotting, viral cytopathic effects (CPE) and viral titer assessment. Moreover, time-of-drug-addition assays, virus attachment, and penetration assays revealed that kaempferol exerted anti-CCV activity probably by blocking attachment and internalization of the viral entry process. Altogether, the present results indicated that kaempferol may be a promising candidate antiviral agent against CCV infection, which shed light on the development of a novel and potent treatment for fish herpesvirus infection.

Heparan sulfate is the attachment factor associated with channel catfish virus infection on host cells.

Channel catfish virus (CCV; family Alloherpesviridae) infects channel catfish, causing great harm to aquaculture fisheries and economic development. Attachment is the first step in viral infection and relies on the interaction of virions with components of the extracellular matrix (ECM). The present study aimed to explored the role of the main three ECM components in CCV attachment. Western blotting and quantitative real-time PCR analysis showed that neither collagen nor hyaluronic acid treatments had significant effects on CCV attachment. When exogenous heparin was used as a competitive inhibitor, the adhesion of heparin sodium salt to CCV was dose-dependent. When the concentration of heparin sodium salt was 10 mg/mL, the inhibitory effect on CCV infection of channel catfish ovary (CCO/BB) cells was more than 90%. Heparinase I could significantly prevent CCV attachment by digesting heparan sulfate on the cell surface, and both heparin sodium salt and heparinase I could dose-dependently reduce CCV titers, suggesting that heparin plays an important role in CCV attachment. In addition, the binding experiments between heparin-agarose beads and virions showed that CCV virions could specifically bind to heparin in a dose-dependent manner. The above results suggested that heparan sulfate might be an attachment factor involved in CCV infection of CCO/BB cells. These results increase our understand of the attachment mechanism of CCV and lay the foundation for further research on antiviral drugs.

Time to trimethoprim/sulfamethoxazole initiation among patients with rheumatic disease complicated by Pneumocystis jirovecii pneumonia: impact on 90-day mortality.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) is a life-threatening disease with increasing prevalence in patients with rheumatic disease. Trimethoprim/sulfamethoxazole (TMP/SMX) is an effective treatment for patients with rheumatic disease hospitalized for PJP. This study aimed to describe the 90-day mortality of patients with rheumatic disease complicated by PJP and investigate whether the administration of TMP/SMX after 7 days from initial symptoms correlates with 90-day mortality. METHODS: We enrolled consecutive patients with rheumatic disease complicated with PJP in our center from August 2018 to August 2021. The participants were classified into two groups according to when TMP/SMX was initiated: early (within the first 7 days) and late (after 7 days). The primary outcome was 90-day PJP-related mortality. Multivariate cox regression and Kaplan-Meier survival analyses were conducted to identify the risk factors for mortality and examine differences in survival between early and late use of TMP/SMX. RESULTS: Thirty-seven patients with rheumatic disease (median age 50.1 years, 24.3% male) complicated by PJP were enrolled in our study, and 15 (40.5%) patients died at or before 90 days of follow-up. The most common comorbidity was systemic lupus erythematosus (14, 37.8%), followed by inflammatory myopathy (11, 27.9%). Patients in the early group were less likely to require mechanical ventilation (8/27, 29.6% vs. 9/10, 90.0%, P = 0.002), lower doses glucocorticoids (43.2 mg/d vs. 72.2 mg/d, P = 0.039) and had lower mortality (7/27, 25.9% vs. 8/10, 80.0%, P = 0.006) than those in the late group. In the Kaplan-Meier analysis, the survivor probability of the early group was notably higher than that of the late group (P = 0.007). Multivariate cox regression analysis showed that initiation of TMP/SMX after 7 days from admission (hazard ratio [HR]: 5.9, 95% confidence interval [CI]: 1.1-30.4; P = 0.034) and a higher level of lactate dehydrogenase (LDH; HR: 6.0, 95% CI: 1.1-31.8; P = 0.035) were associated with 90-day mortality in patients with rheumatic disease complicated by PJP. CONCLUSION: Patients with rheumatic disease complicated by PJP had poor prognoses, with mortality rates as high as 40.5%. TMP/SMX initiation after 7 days from initial symptoms and a higher level of serum LDH were significantly associated with increased 90-day mortality.

Simultaneous Homogeneous Fluorescence Detection of AFP and GPC3 in Hepatocellular Carcinoma Clinical Samples Assisted by Enzyme-Free Catalytic Hairpin Assembly.

Simultaneous sensitive and cost-effective detection of multiple tumor markers has shown great potential for cancer diagnostics. Herein, we reported a simple enzyme-free parallel catalytic hairpin assembly (CHA) amplification strategy with N-methyl mesoporphyrin IX (NMM) and quantum dots (QDs) as signal reporters for the homogeneous fluorescent simultaneous detection of alpha-fetoprotein (AFP) and glypican-3 (GPC3). Upon selective binding, the released single-stranded DNA (ssDNA) from the two-aptamer double-stranded DNA (dsDNA) probes triggers CHA amplification, further releasing the G-quadruplex sequence and Ag+ from the C-Ag+-C structures at the same time. Then, NMM and CdTe QDs selectively recognize G-quadruplex and Ag+, respectively. Under optimized conditions, limits of detections (LODs) as low as 3 fg/mL for AFP and 0.25 fg/mL for GPC3 were achieved using fluorescence readout. Using color- and distance-based visual readouts, an LOD of 1 fg/mL for GPC3 was reached. This method was applied to quantitatively analyze AFP and GPC3 in 41 clinical serum samples of hepatocellular carcinoma (HCC) patients. The quantitative test results for AFP and GPC3 were consistent with those obtained using the electrochemiluminescence immunoassay (ECL-IA) clinical kit and correlated with radiological and pathological findings. The results of clinical tests demonstrated the potential of GPC3 as a tumor biomarker, and we propose a cut-off value of 2 ng/mL GPC3 for HCC.

Calibration and standardization of extracellular vesicle measurements by flow cytometry for translational prostate cancer research.

Extracellular vesicles (EVs) are microscopic particles released naturally in biofluids by all cell types. Since EVs inherits genomic and proteomic patterns from the cell of origin, they are emerging as promising liquid biomarkers for human diseases. Flow cytometry is a popular method that is able to detect, characterize and determine the concentration of EVs with minimal sample preparation. However, the limited awareness of the scientific community to utilize standardization and calibration methods of flow cytometers is an important roadblock for data reproducibility and inter-laboratory comparison. A significant collaborative effort by the Extracellular Vesicle Flow Cytometry Working Group has led to the development of guidelines and best practices for using flow cytometry and reporting data in a way to improve rigor and reproducibility in EV research. At first look, standardization and calibration of flow cytometry for EV detection may seem burdensome and technically challenging for non-academic laboratories with limited technical training and knowledge in EV flow cytometry. In this study, we build on prior research efforts and provide a systematic approach to evaluate the performance of a high sensitivity flow cytometer (herein Apogee A60-Micro Plus) and fine-tune settings to improve detection sensitivity for EVs. We performed calibration of our flow cytometer to generate data with comparable units (nanometers, MESF). Finally, we applied our optimized protocol to measure the concentrations of prostate-derived EVs in healthy individuals and prostate cancer patients. In conclusion, our proof-of-feasibility study can serve as a scientific and technical framework for other groups motivated in using flow cytometry for EV research.

Nanoparticulates reduce tumor cell migration through affinity interactions with extracellular migrasomes and retraction fibers.

Nano-tumor interactions are fundamental for cancer nanotherapy, and the cross-talk of nanomedicines with the extracellular matrix (ECM) is increasingly considered essential. Here, we specifically investigate the nano-ECM interactivity using drug-free nanoparticulates (NPs) and highly metastatic cancer cells as models. We discover with surprise that NPs closely bind to specific types of ECM components, namely, retraction fibers (RFs) and migrasomes, which are located at the rear of tumor cells during their migration. This interaction is observed to alter cell morphology, limit cell motion range and change cell adhesion. Importantly, NPs are demonstrated to inhibit tumor cell removal in vitro, and their anti-metastasis potential is preliminarily confirmed in vivo. Mechanically, the NPs are found to coat and form a rigid shell on the surface of migrasomes and retraction fibers via interaction with lipid raft/caveolae substructures. In this way, NPs block the recognition, endocytosis and elimination of migrasomes by their surrounding tumor cells. Thereby, NPs interfere with the cell-ECM interaction and reduce the promotion effect of migrasomes on cell movement. Additionally, NPs trigger alteration of the expression of proteins related to cell-cell adhesion and cytoskeleton organization, which also restricts cell migration. In summary, all the findings here provide a potential target for anti-tumor metastasis nanomedicines.

A new ALK inhibitor overcomes resistance to first- and second-generation inhibitors in NSCLC.

More than 60% of nonsmall cell lung cancer (NSCLC) patients show a positive response to the first ALK inhibitor, crizotinib, which has been used as the standard treatment for newly diagnosed patients with ALK rearrangement. However, most patients inevitably develop crizotinib resistance due to acquired secondary mutations in the ALK kinase domain, such as the gatekeeper mutation L1196M and the most refractory mutation, G1202R. Here, we develop XMU-MP-5 as a new-generation ALK inhibitor to overcome crizotinib resistance mutations, including L1196M and G1202R. XMU-MP-5 blocks ALK signaling pathways and inhibits the proliferation of cells harboring either wild-type or mutant EML4-ALK in vitro and suppresses tumor growth in xenograft mouse models in vivo. Structural analysis provides insights into the mode of action of XMU-MP-5. In addition, XMU-MP-5 induces significant regression of lung tumors in two genetically engineered mouse (GEM) models, further demonstrating its pharmacological efficacy and potential for clinical application. These preclinical data support XMU-MP-5 as a novel selective ALK inhibitor with high potency and selectivity. XMU-MP-5 holds great promise as a new therapeutic against clinically relevant secondary ALK mutations.

Tumor Associated Macrophages and TAMs-Based Anti-Tumor Nanomedicines.

As an important part of tumor microenvironment, tumor associated macrophages (TAMs) play a vital role in the occurrence, development, invasion, and metastasis of many malignant tumors and can significantly promote the formation of tumor blood vessels and lymphatic vessels, hence TAMs are greatly associated with poor prognosis. The research on nanomedicine has achieved huge progress, and nano-drugs have been widely utilized to treat various diseases through different mechanisms. Therefore, developing nano-drugs that are based on TAMs-associated anti-tumor mechanisms to effectively suppress tumor growth is expected to be a promising research filed. This paper introduces relevant information about TAMs in terms of their origin, and their roles in tumor genesis, development and metastasis. Furthermore, TAMs-related anti-tumor nano-drugs are summarized. Specifically, a wide range of nano-drugs targeting at TAMs are introduced, and categorized according to their therapeutic mechanisms toward tumors. Additionally, various nano delivery platforms using TAMs as cell carriers which aim at inhibiting tumor growth are reviewed. These two parts elucidate that the exploration of nanomedicine is essential to the study on TAMs-related anti-tumor strategies. This review is also intended to provide novel ideas for in-depth investigation on anti-tumor molecular mechanisms and nano-drug delivery systems based on TAMs.

Unsupervised fNIRS feature extraction with CAE and ESN autoencoder for driver cognitive load classification.

Objective. Understanding the cognitive load of drivers is crucial for road safety. Brain sensing has the potential to provide an objective measure of driver cognitive load. We aim to develop an advanced machine learning framework for classifying driver cognitive load using functional near-infrared spectroscopy (fNIRS).Approach. We conducted a study using fNIRS in a driving simulator with theN-back task used as a secondary task to impart structured cognitive load on drivers. To classify different driver cognitive load levels, we examined the application of convolutional autoencoder (CAE) and Echo State Network (ESN) autoencoder for extracting features from fNIRS.Main results. By using CAE, the accuracies for classifying two and four levels of driver cognitive load with the 30 s window were 73.25% and 47.21%, respectively. The proposed ESN autoencoder achieved state-of-art classification results for group-level models without window selection, with accuracies of 80.61% and 52.45% for classifying two and four levels of driver cognitive load.Significance. This work builds a foundation for using fNIRS to measure driver cognitive load in real-world applications. Also, the results suggest that the proposed ESN autoencoder can effectively extract temporal information from fNIRS data and can be useful for other fNIRS data classification tasks.

A combined "eat me/don't eat me" strategy based on extracellular vesicles for anticancer nanomedicine.

A long-term and huge challenge in nanomedicine is the substantial uptake and rapid clearance mediated by the mononuclear phagocyte system (MPS), which enormously hinders the development of nanodrugs. Inspired by the natural merits of extracellular vesicles, we therefore developed a combined "eat me/don't eat me" strategy in an effort to achieve MPS escape and efficient drug delivery. Methodologically, cationized mannan-modified extracellular vesicles derived from DC2.4 cells were administered to saturate the MPS (eat me strategy). Then, nanocarriers fused to CD47-enriched exosomes originated from human serum were administered to evade phagocytosis by MPS (don't eat me strategy). The nanocarriers were also loaded with antitumor drugs and functionalized with a novel homing peptide to promote the tumour tissue accumulation and cancer cell uptake (eat me strategy). The concept was proven in vitro as evidenced by the reduced endocytosis of macrophages and enhanced uptake by tumour cells, whereas prolonged circulation time and increased tumour accumulation were demonstrated in vivo. Specially, the strategy induced a 123.53% increase in tumour distribution compared to conventional nanocarrier. The study both shed light on the challenge overcoming of phagocytic evasion and provided a strategy for significantly improving therapeutic outcomes, potentially permitting active drug delivery via targeted nanomedicines.

Proteomic analysis of intracellular protein corona of nanoparticles elucidates nano-trafficking network and nano-bio interactions.

The merits of nanomedicines are significantly impacted by the surrounding biological environment. Similar to the protein corona generated on the surface of nanoparticles in the circulation system, the intracellular protein corona (IPC) might be formed on nanoparticles when transported inside the cells. However, little is known currently about the formation of IPC and its possible biological influence. Methods: Caco-2 cells, a classical epithelial cell line, were cultured in Transwell plates to form a monolayer. Gold nanoparticles (AuNPs) were prepared as the model nanomedicine due to their excellent stability. Here we focused on identifying IPC formed on the surface of AuNPs during cell transport. The nanoparticles in the basolateral side of the Caco-2 monolayer were collected and analyzed by multiple techniques to verify IPC formation. High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics was utilized to analyze the composition of IPC proteins. In particular, we established a dual-filtration strategy to exclude various interference in IPC identification. Based on the subcellular localization of specific IPC proteins, we elicited the nano-trafficking network of AuNPs. The transport pathways of AuNPs identified by proteomic analysis were also verified by various conventional technologies. Finally, we explored the influence of IPC on the uptake and stress response of endothelium. Results: The existence of IPC was demonstrated on the surface of AuNPs, in which 227 proteins were identified. Among them, 40 proteins were finally ascertained as the specific IPC proteins. The subcellular location analysis indicated that these "specific" IPC proteins could back-track the transport pathways of nanoparticles in the epithelial cell monolayer. According to the subcellular distribution of IPC proteins and co-localization, we discovered a new pathway of nanoparticles from endosomes to secretory vesicles which was dominant during the transcytosis. After employing conventional imageology and pharmacology strategies to verify the result of proteomic analysis, we mapped a comprehensive intracellular transport network. Our study also revealed the merits of IPC analysis, which could readily elucidate the molecular mechanisms of transcytosis. Besides, the IPC proteins increased the uptake and stress response of endothelium, which was likely mediated by extracellular matrix and mitochondrion-related IPC proteins. Conclusion: The comprehensive proteomic analysis of IPC enabled tracing of transport pathways in epithelial cells as well as revealing the biological impact of nanoparticles on endothelium.

A non-covalent inhibitor XMU-MP-3 overrides ibrutinib-resistant BtkC481S mutation in B-cell malignancies.

BACKGROUND AND PURPOSE: Bruton's tyrosine kinase (BTK) plays a key role in B-cell receptor signalling by regulating cell proliferation and survival in various B-cell malignancies. Covalent low-MW BTK kinase inhibitors have shown impressive clinical efficacy in B-cell malignancies. However, the mutant BtkC481S poses a major challenge in the management of B-cell malignancies by disrupting the formation of the covalent bond between BTK and irreversible inhibitors, such as ibrutinib. The present studies were designed to develop novel BTK inhibitors targeting ibrutinib-resistant BtkC481S mutation. EXPERIMENTAL APPROACH: BTK-Ba/F3, BTK(C481S)-Ba/F3 cells, and human malignant B-cells JeKo-1, Ramos, and NALM-6 were used to evaluate cellular potency of BTK inhibitors. The in vitro pharmacological efficacy and compound selectivity were assayed via cell viability, colony formation, and BTK-mediated signalling. A tumour xenograft model with BTK-Ba/F3, Ramos and BTK(C481S)-Ba/F3 cells in Nu/nu BALB/c mice was used to assess in vivo efficacy of XMU-MP-3. KEY RESULTS: XMU-MP-3 is one of a group of low MW compounds that are potent non-covalent BTK inhibitors. XMU-MP-3 inhibited both BTK and the acquired mutant BTKC481S, in vitro and in vivo. Further computational modelling, site-directed mutagenesis analysis, and structure-activity relationships studies indicated that XMU-MP-3 displayed a typical Type-II inhibitor binding mode. CONCLUSION AND IMPLICATIONS: XMU-MP-3 directly targets the BTK signalling pathway in B-cell lymphoma. These findings establish XMU-MP-3 as a novel inhibitor of BTK, which could serve as both a tool compound and a lead for further drug development in BTK relevant B-cell malignancies, especially those with the acquired ibrutinib-resistant C481S mutation.

Small GTPases: Structure, biological function and its interaction with nanoparticles.

Small GTPase is a kind of GTP-binding protein commonly found in eukaryotic cells. It plays an important role in cytoskeletal reorganization, cell polarity, cell cycle progression, gene expression and many other significant events in cells, such as the interaction with foreign particles. Therefore, it is of great scientific significance to understand the biological properties of small GTPases as well as the GTPase-nano interplay, since more and more nanomedicine are supposed to be used in biomedical field. However, there is no review in this aspect. This review summarizes the small GTPases in terms of the structure, biological function and its interaction with nanoparticles. We briefly introduced the various nanoparticles such as gold/silver nanoparticles, SWCNT, polymeric micelles and other nano delivery systems that interacted with different GTPases. These current nanoparticles exhibited different pharmacological effect modes and various target design concepts in the small GTPases study. This will help to elucidate the conclusion that the therapeutic strategy targeting small GTPases might be a new research direction. It is believed that the in-depth study on the functional mechanism of GTPases can provide insights for the design and study of nanomedicines.

Rho GTPases in A549 and Caco-2 cells dominating the endocytic pathways of nanocarbons with different morphologies.

INTRODUCTION: Endocytosis of nanomaterials is the first step of nano-bio interaction and current regulation is mostly by nanomaterials but seldom by intracellular signaling proteins. MATERIALS AND METHODS: Herein, we synthesized tubular nanocarbon (oxMWCNT) and lamellar-like nanocarbon (oxGRAPHENE) and formulated their aqueous dispersion. A549 and Caco-2 cells were selected as the models of tumor and intestinal epithelial cells, respectively. After knocking down three members of Rho GTPases (Cdc42, Rac1, RhoA) in these two cell lines, their silencing effects on the uptake pathways of nanomaterials with different morphologies were investigated. RESULTS: An unexpected finding was that the knock-down led to opposite uptake trends in different types of cells. The endocytosis of carbon nanomaterials increased in Caco-2 cells when Rho GTPases were inactivated, while that in A549 cells decreased. For nanomaterials with different shapes, the involved GTPase member of Rho family, or regulating protein molecule, was different. Concretely, Cdc42 and Rac1 were involved in oxMWCNT endocytosis, while all three GTPases participated in oxGRAPHENE internalization. More interestingly, such difference induced different uptake pathways, namely, the cellular uptake of oxMWCNT was clathrin-mediated and oxGRAPHENE was caveolin-modulated, both with the involvement of dynamin. CONCLUSION: In conclusion, this study provides new insights for the potential intervention in nano-bio interplay.

Two-degrees-of-freedom piezo-driven fast steering mirror with cross-axis decoupling capability.

Because mechanical cross coupling between its axes would lead to degradation of the scanning precision of a piezo-driven fast steering mirror (PFSM), a two-degrees-of-freedom (2-DoF) PFSM with a cross-axis decoupling capability, in which 2-DoF flexure hinges are used, is proposed in this work. The overall structure of the proposed PFSM is first introduced and then both static and dynamic models are established analytically; in addition, the decoupling mechanism is described in detail and the low dynamic cross coupling ratios that occur between the two DoFs are shown. Because of the decoupling property of the PFSM, the 2-DoF motion is treated as a combination of two independent one-degree-of-freedom (1-DoF) motions and two independent proportional-integral-derivative controllers are thus used separately in the control of the two DoFs. Based on this control strategy, experiments involving both 1-DoF trajectory tracking and 2-DoF trajectory tracking are implemented. The test results show that the proposed PFSM can achieve the tilt range of ±7 mrad for both axes with the low coupling ratios that are less than 2% (-34 dB), and the bandwidths of both axes are higher than 810 Hz; in addition, the maximal tracking full scale range errors for 1-DoF trajectory tracking and 2-DoF trajectory tracking are less than 0.2% and 1%, respectively, where the larger error of 2-DoF trajectory tracking is mainly caused by the remaining cross coupling between axes.

Intestinal Mucin Induces More Endocytosis but Less Transcytosis of Nanoparticles across Enterocytes by Triggering Nanoclustering and Strengthening the Retrograde Pathway.

Mucus, which is secreted by the goblet cells of enterocytes, constitutes the first obstacle encountered for the intestinal absorption of nanomedicines. For decades, mucus has simply been regarded as a physical barrier that hinders the permeation and absorption of drugs, because of its high viscosity and reticular structure, whereas the interaction of mucus ingredients with nanomedicines is usually neglected. It is unclear whether glycoproteins, as the main components of mucus, interact with nanomedicines. We also do not know how the potential interaction affects the subsequent transportation of nanomedicines through the intestinal epithelium. In this study, mucin as the key element of mucus was investigated to characterize the interaction of nanomedicines with mucus. PEG-modified gold nanoparticles (PGNPs) were fabricated as model nanoparticles. Mucin was found to adhere to the nanoparticle surface to form a corona structure and induce the clustering of PGNPs by joining particles together, demonstrating the interaction between mucin and PGNPs. In addition, two intestinal epithelia, Caco-2 (non- mucus secretion) and HT-29 (high mucus secretion), were compared to evaluate the influence of mucin on the cellular interaction of PGNPs. Amazingly, mucin altered the trafficking characteristic of PGNPs in intestinal epithelium. Both in vitro and in vivo investigations demonstrated more nanoparticles being internalized by cells due to the mucin coverage. However, mucin induced a significant reduction in the transcytosis of PGNPs across epithelial monolayers. The mechanism exploration further revealed that the "more endocytosis but less transcytosis (MELT)" effect was mainly attributed to the strengthened retrograde pathway in which more PGNPs were transported to Golgi apparatus and exocytosed back to the apical but not the basolateral side of the epithelial monolayers. The "MELT" effect endowed mucin with duality in the nanoparticle transportation. Therefore, the rational regulation based on the "MELT" effect will provide new insight into overcoming the mucus obstacle as a barrier and enhancing the oral absorption rate of nanomedicines.

Mycoepoxydiene suppresses HeLa cell growth by inhibiting glycolysis and the pentose phosphate pathway.

Upregulation of glycolysis and the pentose phosphate pathway (PPP) is a major characteristic of the metabolic reprogramming of cancer and provides cancer cells with energy and vital metabolites to support their rapid proliferation. Targeting glycolysis and the PPP has emerged as a promising antitumor therapeutic strategy. Marine natural products are attractive sources for anticancer therapeutics, as evidenced by the antitumor drug Yondelis. Mycoepoxydiene (MED) is a natural product isolated from a marine fungus that has shown promising inhibitory efficacy against HeLa cells in vitro. We used a proteomic approach with two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry to explore the cellular targets of MED and to unravel the molecular mechanisms underlying the antitumor activity of MED in HeLa cells. Our proteomic data showed that triosephosphate isomerase (TPI) and 6-phosphogluconolactonase (PGLS), which participate in glycolysis and the PPP, respectively, were significantly downregulated by MED treatment. Functional studies revealed that the expression levels of several other enzymes involved in glycolysis and the PPP, including hexokinase 2 (HK2), phosphofructokinase 1 (PFKM), aldolase A (ALDOA), enolase 1 (ENO1), lactate dehydrogenase A (LDHA), and glucose-6-phosphate dehydrogenase (G6PD), were also reduced in a dose-dependent manner. Moreover, the LDHA and G6PD enzymatic activities in HeLa cells were inhibited by MED, and overexpression of these downregulated enzymes rescued HeLa cells from the growth inhibition induced by MED. Our data suggest that MED suppresses HeLa cell growth by inhibiting glycolysis and the PPP, which provides a mechanistic basis for the development of new therapeutics against cervical cancer.