Bidirectional TFDM 200†G coherent PON with a simplified receiver at the ONU side.

Time and frequency division multiplexing (TFDM) coherent passive optical networks (PONs) are considered as a promising candidate for future optical access networks due to the advantage of high sensitivity, high spectral efficiency, and flexibility. We propose a novel, to our knowledge, bidirectional TFDM 200-Gb/s coherent PON architecture based on the digital subcarrier multiplexing (DSCM) technology. A polarization-insensitive simplified coherent receiver is achieved at the ONU side by Alamouti coding and heterodyne detection. We experimentally demonstrate this bidirectional coherent PON with a 50-Gbaud Alamouti 16 quadrature amplitude modulation (QAM) signal transmitted downstream and a 25-Gbaud dual polarization (DP)-16QAM signal transmitted upstream. By using a single sideband modulated transmitter, only one laser source is required at the optical network unit (ONU) side. The power budgets can reach 30 and 33 dB for the downstream and upstream, respectively, after the 20-km transmission over a standard single-mode fiber (SSMF).

Identifying PE2 and PE5 Proteins from Existing Mass Spectrometry Data Using pFind.

The Chromosome-Centric Human Proteome Project (C-HPP) aims to identify all proteins encoded by the human genome. Currently, the human proteome still contains approximately 2000 PE2-PE5 proteins, referring to annotated coding genes that lack sufficient protein-level evidence. During the past 10 years, it has been increasingly difficult to identify PE2-PE5 proteins in C-HPP approaches due to the limited occurrence. Therefore, we proposed that reanalyzing massive MS data sets in repository with newly developed algorithms may increase the occurrence of the peptides of these proteins. In this study, we downloaded 1000 MS data sets via the ProteomeXchange database. Using pFind software, we identified peptides referring to 1788 PE2-PE5 proteins. Among them, 11 PE2 and 16 PE5 proteins were identified with at least 2 peptides, and 12 of them were identified using 2 peptides in a single data set, following the criteria of the HPP guidelines. We found translation evidence for 16 of the 11 PE2 and 16 PE5 proteins in our RNC-seq data, supporting their existence. The properties of the PE2 and PE5 proteins were similar to those of the PE1 proteins. Our approach demonstrated that mining PE2 and PE5 proteins in massive data repository is still worthy, and multidata set peptide identifications may support the presence of PE2 and PE5 proteins or at least prompt additional studies for validation. Extremely high throughput could be a solution to finding more PE2 and PE5 proteins.

LINC00982-encoded protein PRDM16-DT regulates CHEK2 splicing to suppress colorectal cancer metastasis and chemoresistance.

Metastasis is one of the key factors of treatment failure in late-stage colorectal cancer (CRC). Metastatic CRC frequently develops resistance to chemotherapeutic agents. This study aimed to identify the novel regulators from "hidden" proteins encoded by long noncoding RNAs (lncRNAs) involved in tumor metastasis and chemoresistance. Methods: CRISPR/Cas9 library functional screening was employed to identify the critical suppressor of cancer metastasis in highly invasive CRC models. Western blotting, immunofluorescence staining, invasion, migration, wound healing, WST-1, colony formation, gain- and loss-of-function experiments, in vivo experimental metastasis models, multiplex immunohistochemical staining, immunohistochemistry, qRT-PCR, and RT-PCR were used to assess the functional and clinical significance of FOXP3, PRDM16-DT, HNRNPA2B1, and L-CHEK2. RNA-sequencing, co-immunoprecipitation, qRT-PCR, RT-PCR, RNA affinity purification, RNA immunoprecipitation, MeRIP-quantitative PCR, fluorescence in situ hybridization, chromatin immunoprecipitation and luciferase reporter assay were performed to gain mechanistic insights into the role of PRDM16-DT in cancer metastasis and chemoresistance. An oxaliplatin-resistant CRC cell line was established by in vivo selection. WST-1, colony formation, invasion, migration, Biacore technology, gain- and loss-of-function experiments and an in vivo experimental metastasis model were used to determine the function and mechanism of cimicifugoside H-1 in CRC. Results: The novel protein PRDM16-DT, encoded by LINC00982, was identified as a cancer metastasis and chemoresistance suppressor. The down-regulated level of PRDM16-DT was positively associated with malignant phenotypes and poor prognosis of CRC patients. Transcriptionally regulated by FOXP3, PRDM16-DT directly interacted with HNRNPA2B1 and competitively decreased HNRNPA2B1 binding to exon 9 of CHEK2, resulting in the formation of long CHEK2 (L-CHEK2), subsequently promoting E-cadherin secretion. PRDM16-DT-induced E-cadherin secretion inhibited fibroblast activation, which in turn suppressed CRC metastasis by decreasing MMP9 secretion. Cimicifugoside H-1, a natural compound, can bind to LEU89, HIS91, and LEU92 of FOXP3 and significantly upregulated PRDM16-DT expression to repress CRC metastasis and reverse oxaliplatin resistance. Conclusions: lncRNA LINC00982 can express a new protein PRDM16-DT to function as a novel regulator in cancer metastasis and drug resistance of CRC. Cimicifugoside H-1 can act on the upstream of the PRDM16-DT signaling pathway to alleviate cancer chemoresistance.

PTPLAD1 Regulates PHB-Raf Interaction to Orchestrate Epithelial-Mesenchymal and Mitofusion-Fission Transitions in Colorectal Cancer.

Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. The poor prognosis of this malignancy is attributed mainly to the persistent activation of cancer signaling for metastasis. Here, we showed that protein tyrosine phosphatase-like A domain containing 1 (PTPLAD1) is down-regulated in highly metastatic CRC cells and negatively associated with poor survival of CRC patients. Systematic analysis reveals that epithelial-to-mesenchymal transition (EMT) and mitochondrial fusion-to-fission (MFT) transition are two critical features for CRC patients with low expression of PTPLAD1. PTPLAD1 overexpression suppresses the metastasis of CRC in vivo and in vitro by inhibiting the Raf/ERK signaling-mediated EMT and mitofission. Mechanically, PTPLAD1 binds with PHB via its middle fragment (141-178 amino acids) and induces dephosphorylation of PHB-Y259 to disrupt the interaction of PHB-Raf, resulting in the inactivation of Raf/ERK signaling. Our results unveil a novel mechanism in which Raf/ERK signaling activated in metastatic CRC induces EMT and mitochondrial fission simultaneously, which can be suppressed by PTPLAD1. This finding may provide a new paradigm for developing more effective treatment strategies for CRC.

YjgM is a crotonyltransferase critical for polymyxin resistance of Escherichia coli.

Lysine crotonylation has attracted widespread attention in recent years. However, little is known about bacterial crotonylation, particularly crotonyltransferase and decrotonylase, and its effects on antibiotic resistance. Our study demonstrates the ubiquitous presence of crotonylation in E. coli, which promotes bacterial resistance to polymyxin. We identify the crotonyltransferase YjgM and its regulatory pathways in E. coli with a focus on crotonylation. Further studies show that YjgM upregulates the crotonylation of the substrate protein PmrA, thereby boosting PmrA's affinity for binding to the promoter of eptA, which, in turn, promotes EptA expression and confers polymyxin resistance in E. coli. Additionally, we discover that PmrA's crucial crotonylation site and functional site is Lys 164. These significant discoveries highlight the role of crotonylation in bacterial drug resistance and offer a fresh perspective on creating antibacterial compounds.

Pan-Cancer Proteomics Analysis Reveals Wiskott-Aldrich Syndrome Protein as a Potential Regulator of Programmed Death-Ligand 1.

The programmed death-ligand 1 (PD-L1) is a key mediator of immunosuppression in the tumor microenvironment. The expression of PD-L1 in cancer cells is useful for the clinical determination of an immune checkpoint blockade (ICB). However, the regulatory mechanism of the PD-L1 abundance remains incompletely understood. Here, we integrated the proteomics of 52 patients with solid tumors and examined immune cell infiltration to reveal PD-L1-related regulatory modules. Wiskott-Aldrich syndrome protein (WASP) was identified as a potential regulator of PD-L1 transcription. In two independent cohorts containing 164 cancer patients, WASP expression was significantly associated with PD-L1. High WASP expression contributed to immunosuppressive cell composition, including cells positive for immune checkpoints (PD1, CTLA4, TIGIT, and TIM3), FoxP3+ Treg cells, and CD163+ tumor-associated macrophages. Overexpression of WASP increased, whereas knockdown of WASP decreased the protein level of PD-L1 in cancer cells without alteration of PD-L1 protein stability. The WASP-mediated cell migration and invasion were markedly attenuated by the silence of PD-L1. Collectively, our data suggest that WASP is a potential regulator of PD-L1 and the WASP/PD-L1 axis is responsible for cell migration and an immunosuppressive microenvironment.

Two synonymous single-nucleotide polymorphisms promoting fluoroquinolone resistance of Escherichia coli in the environment.

Single-nucleotide polymorphism (SNP) is one of the core mechanisms that respond to antibiotic resistance of Escherichia coli (E. coli), which is a major issue in environmental pollution. A specific type of SNPs, synonymous SNPs, have been generally considered as the "silent" SNPs since they do not change the encoded amino acid. However, the impact of synonymous SNPs on mRNA splicing, nucleo-cytoplasmic export, stability, and translation was gradually discovered in the last decades. Figuring out the mechanism of synonymous SNPs in regulating antibiotic resistance is critical to improve antimicrobial therapy strategies in clinics and biological treatment strategies of antibiotic-resistant E. coli-polluted materials. With our newly designed antibiotic resistant SNPs prediction algorithm, Multilocus Sequence Type based Identification for Phenotype-single nucleotide polymorphism Analysis (MIPHA), and in vivo validation, we identified 2 important synonymous SNPs 522 G>A and 972 C>T, located at hisD gene, which was previously predicted as a fluoroquinolone resistance-related gene without a detailed mechanism in the E. coli samples with environmental backgrounds. We first discovered that hisD causes gyrA mutation via the upregulation of sbmC and its downstream gene umuD. Moreover, those 2 synonymous SNPs of hisD cause its own translational slowdown and further reduce the expression levels of sbmC and its downstream gene umuD, making the fluoroquinolone resistance determining region of gyrA remains unmutated, ultimately causing the bacteria to lose their ability to resist drugs. This study provided valuable insight into the role of synonymous SNPs in mediating antibiotic resistance of bacteria and a new perspective for the treatment of environmental pollution caused by drug-resistant bacteria.

Repurposing cinacalcet suppresses multidrug-resistant Staphylococcus aureus by disruption of cell membrane and inhibits biofilm by targeting IcaR.

BACKGROUND: MDR Staphylococcus aureus infections, along with the severity of biofilm-associated infections, continue to threaten human health to a great extent. It necessitates the urgent development of novel antimicrobial and antibiofilm agents. OBJECTIVES: To reveal the mechanism and target of cinacalcet as an antibacterial and antimicrobial agent for S. aureus. METHODS: Screening of non-antibiotic drugs for antibacterial and antibiofilm properties was conducted using a small-molecule drug library. In vivo efficacy was assessed through animal models, and the antibacterial mechanism was studied using quantitative proteomics, biochemical assays, LiP-SMap, BLI detection and gene knockout techniques. RESULTS: Cinacalcet, an FDA-approved drug, demonstrated antibacterial and antibiofilm activity against S. aureus, with less observed development of bacterial resistance. Importantly, cinacalcet significantly improved survival in a pneumonia model and bacterial clearance in a biofilm infection model. Moreover, the antibacterial mechanism of cinacalcet mainly involves the destruction of membrane-targeted structures, alteration of energy metabolism, and production of reactive oxygen species (ROS). Cinacalcet was found to target IcaR, inhibiting biofilm formation through the negative regulation of IcaADBC. CONCLUSIONS: The findings suggest that cinacalcet has potential for repurposing as a therapeutic agent for MDR S. aureus infections and associated biofilms, warranting further investigation.

Unveiling a novel mechanism for competitive advantage of ciprofloxacin-resistant bacteria in the environment through bacterial membrane vesicles.

Ciprofloxacin (CIP) is a prevalent environmental contaminant that poses a high risk of antibiotic resistance. High concentrations of antibiotics can lead to the development of resistant bacteria with high fitness costs, which often face a competitive disadvantage. However, it is unclear whether low-cost resistant bacteria formed by exposure to sub-MIC CIP in the environment can evolve competitive mechanisms against sensitive Escherichia coli (SEN) other than stronger resistance to CIP. Our study exposed E. coli to sub-MIC CIP levels, resulting in the development of CIP-resistant E. coli (CIPr). In antibiotic-free co-culture assays, CIPr outcompeted SEN. This indicates that CIPr is very likely to continue to develop and spread in antibiotic-free environments such as drinking water and affect human health. Further mechanism investigation revealed that bacterial membrane vesicles (BMVs) in CIPr, functioning as substance delivery couriers, mediated a cleavage effect on SEN. Proteomic analysis identified Entericidin B (EcnB) within CIPr-BMVs as a key factor in this competitive interaction. RT-qPCR analysis showed that the transcription of its negative regulator ompR/envZ was down-regulated. Moreover, EcnB plays a crucial role in the development of CIP resistance, and some resistance-related proteins and pathways have also been discovered. Metabolomics analysis highlighted the ability of CIPr-BMVs to acidify SEN, increasing the lytic efficiency of EcnB through cationization. Overall, our study reveals the importance of BMVs in mediating bacterial resistance and competition, suggesting that regulating BMVs production may be a new strategy for controlling the spread of drug-resistant bacteria.

Super-tetragonal Sr4Al2O7 as a sacrificial layer for high-integrity freestanding oxide membranes.

Identifying a suitable water-soluble sacrificial layer is crucial to fabricating large-scale freestanding oxide membranes, which offer attractive functionalities and integrations with advanced semiconductor technologies. Here, we introduce a water-soluble sacrificial layer, "super-tetragonal" Sr4Al2O7 (SAOT). The low-symmetric crystal structure enables a superior capability to sustain epitaxial strain, allowing for broad tunability in lattice constants. The resultant structural coherency and defect-free interface in perovskite ABO3/SAOT heterostructures effectively restrain crack formation during the water release of freestanding oxide membranes. For a variety of nonferroelectric oxide membranes, the crack-free areas can span up to a millimeter in scale. This compelling feature, combined with the inherent high water solubility, makes SAOT a versatile and feasible sacrificial layer for producing high-quality freestanding oxide membranes, thereby boosting their potential for innovative device applications.

A Interacting Model: How TRIM21 Orchestrates with Proteins in Intracellular Immunity.

Tripartite motif-containing protein 21 (TRIM21), identified as both a cytosolic E3 ubiquitin ligase and FcR (Fragment crystallizable receptor), primarily interacts with proteins via its PRY/SPRY domains and promotes their proteasomal degradation to regulate intracellular immunity. But how TRIM21 involves in intracellular immunity still lacks systematical understanding. Herein, it is probed into the TRIM21-related literature and raises an interacting model about how TRIM21 orchestrates proteins in cytosol. In this novel model, TRIM21 generally interacts with miscellaneous protein in intracellular immunity in two ways: For one, TRIM21 solely plays as an E3, ubiquitylating a glut of proteins that contain specific interferon-regulatory factor, nuclear transcription factor kappaB, virus sensors and others, and involving inflammatory responses. For another, TRIM21 serves as both E3 and specific FcR that detects antibody-complexes and facilitates antibody destroying target proteins. Correspondingly delineated as Fc-independent signaling and Fc-dependent signaling in this review, how TRIM21's interactions contribute to intracellular immunity, expecting to provide a systematical understanding of this important protein and invest enlightenment for further research on the pathogenesis of related diseases and its prospective application is elaborated.

LAFEM: A Scoring Model to Evaluate Functional Landscape of Lysine Acetylome.

Protein lysine acetylation is a critical post-translational modification involved in a wide range of biological processes. To date, about 20,000 acetylation sites of Homo sapiens were identified through mass spectrometry-based proteomic technology, but more than 95% of them have unclear functional annotations because of the lack of existing prioritization strategy to assess the functional importance of the acetylation sites on large scale. Hence, we established a lysine acetylation functional evaluating model (LAFEM) by considering eight critical features surrounding lysine acetylation site to high-throughput estimate the functional importance of given acetylation sites. This was achieved by selecting one of the random forest models with the best performance in 10-fold cross-validation on undersampled training dataset. The global analysis demonstrated that the molecular environment of acetylation sites with high acetylation functional scores (AFSs) mainly had the features of larger solvent-accessible surface area, stronger hydrogen bonding-donating abilities, near motif and domain, higher homology, and disordered degree. Importantly, LAFEM performed well in validation dataset and acetylome, showing good accuracy to screen out fitness directly relevant acetylation sites and assisting to explain the core reason for the difference between biological models from the perspective of acetylome. We further used cellular experiments to confirm that, in nuclear casein kinase and cyclin-dependent kinase substrate 1, acetyl-K35 with higher AFS was more important than acetyl-K9 with lower AFS in the proliferation of A549 cells. LAFEM provides a prioritization strategy to large scale discover the fitness directly relevant acetylation sites, which constitutes an unprecedented resource for better understanding of functional acetylome.

214.7 Tbit/s S, C, and L-band transmission over 50†km SSMF based on silicon photonic integrated transceivers.

We experimentally demonstrate a 214.7 Tbit/s generalized mutual information (GMI) estimated throughput by ultra-wideband wavelength division multiplexing (WDM) transmission in standard single-mode fiber (SSMF). With 50-GHz grid, 396 transmission channels are used to deliver 49 GBaud probabilistically constellation-shaped (PCS) 256 quadrature amplitude modulation (QAM) and PCS-64QAM signals. Silicon photonic integrated transceiver is employed to complete electro-optic and optic-electro conversion of the modulated signals. S, C, and L-band rare-earth-doped amplifiers enable the 19.8 THz bandwidth WDM transmission without the assistance of distributed Raman amplification. The measured data rate shows great potential for Silicon photonic devices deployed in ultra-wideband WDM transmission.

Monitoring acoustic vibrations in optical fibers by estimating polarization matrix variation with the integration of coherent optical communication and sensing.

In this paper, we propose a novel architecture called as the Direct-Computation-Sensing Architecture (DCSA) to directly calculates the polarization state changes caused by optical fiber vibrations with training data, offering a more accurate and responsive method than that with adaptive filter-based sensing architectures. We detected the distinct fiber vibration induced by piezoelectric ceramics in an established experimental platform, and recovered a song melody played near the optical fiber buddle from the fiber's polarization changes. We locate the source of the vibration by comparing data from both ends of a bidirectional transmission setup. Lastly, we conducted field tests under conditions involving machine-induced vibrations and natural cable movements.

Histone deacetylase inhibitor pracinostat suppresses colorectal cancer by inducing CDK5-Drp1 signaling-mediated peripheral mitofission.

Divisions at the periphery and midzone of mitochondria are two fission signatures that determine the fate of mitochondria and cells. Pharmacological induction of excessively asymmetric mitofission-associated cell death (MFAD) by switching the scission position from the mitochondrial midzone to the periphery represents a promising strategy for anticancer therapy. By screening a series of pan-inhibitors, we identified pracinostat, a pan-histone deacetylase (HDAC) inhibitor, as a novel MFAD inducer, that exhibited a significant anticancer effect on colorectal cancer (CRC) in vivo and in vitro. Pracinostat increased the expression of cyclin-dependent kinase 5 (CDK5) and induced its acetylation at residue lysine 33, accelerating the formation of complex CDK5/CDK5 regulatory subunit 1 and dynamin-related protein 1 (Drp1)-mediated mitochondrial peripheral fission. CRC cells with high level of CDK5 (CDK5-high) displayed midzone mitochondrial division that was associated with oncogenic phenotype, but treatment with pracinostat led to a lethal increase in the already-elevated level of CDK5 in the CRC cells. Mechanistically, pracinostat switched the scission position from the mitochondrial midzone to the periphery by improving the binding of Drp1 from mitochondrial fission factor (MFF) to mitochondrial fission 1 protein (FIS1). Thus, our results revealed the anticancer mechanism of HDACi pracinostat in CRC via activating CDK5-Drp1 signaling to cause selective MFAD of those CDK5-high tumor cells, which implicates a new paradigm to develop potential therapeutic strategies for CRC treatment.

Loss of UCP2 causes mitochondrial fragmentation by OMA1-dependent proteolytic processing of OPA1 in podocytes.

Mitochondrial dysfunction plays an important role in the onset and progression of podocyte injury and proteinuria. However, the process by which the change in the podocyte mitochondria occurs is not well understood. Uncoupling protein 2 (UCP2) is a mitochondrial anion carrier protein, which is located in the mitochondrial inner membrane. Here, we reported that mice with podocyte-specific Ucp2 deficiency developed podocytopathy with proteinuria with aging. Furthermore, those mice exhibited increased proteinuria in experimental models evoked by Adriamycin. Our findings suggest that UCP2 mediates mitochondrial dysfunction by regulating mitochondrial dynamic balance. Ucp2-deleted podocytes exhibited increased mitochondrial fission and deficient in ATP production. Mechanistically, opacity protein 1 (OPA1), a key protein in fusion of mitochondrial inner membrane, was regulated by UCP2. Ucp2 deficiency promoted proteolysis of OPA1 by activation OMA1 which belongs to mitochondrial inner membrane zinc metalloprotease. Those finding demonstrate the role of UCP2 in mitochondrial dynamics in podocytes and provide new insights into pathogenesis associated with podocyte injury and proteinuria.

A multi-omics dataset of human transcriptome and proteome stable reference.

The development of high-throughput omics technology has greatly promoted the development of biomedicine. However, the poor reproducibility of omics techniques limits their application. It is necessary to use standard reference materials of complex RNAs or proteins to test and calibrate the accuracy and reproducibility of omics workflows. The transcriptome and proteome of most cell lines shift during culturing, which limits their applicability as standard samples. In this study, we demonstrated that the human hepatocellular cell line MHCC97H has a very stable transcriptome (r = 0.983~0.997) and proteome (r = 0.966~0.988 for data-dependent acquisition, r = 0.970~0.994 for data-independent acquisition) after 9 subculturing generations, which allows this steady standard sample to be consistently produced on an industrial scale in long term. Moreover, this stability was maintained across labs and platforms. In sum, our study provides omics standard reference material and reference datasets for transcriptomic and proteomics research. This helps to further standardize the workflow and data quality of omics techniques and thus promotes the application of omics technology in precision medicine.

Residue Lys219 of CpxR is critical in the regulation of the antibiotic resistance of Escherichia coli.

BACKGROUND: CpxR is a critical regulator in bacterial adaptation to various harmful stresses, and is known to regulate bacterial resistance to commonly used antibiotics, such as aminoglycosides, ß-lactams and polypeptides. However, the detailed study of functional residues of CpxR remains insufficient. OBJECTIVES: To investigate the contribution of Lys219 to CpxR's function in regulating antibiotic resistance of Escherichia coli. METHODS: We performed sequence alignment and conservative analysis of the CpxR protein and constructed mutant strains. We then performed electrophoretic mobility shift assay, real-time quantitative PCR assay, determination of reactive oxygen species (ROS) levels, molecular dynamics simulation, conformational analysis and circular dichroism. RESULTS: All mutant proteins (K219Q, K219A and K219R) lost the cpxP DNA-binding ability. Additionally, the three complemented strains eK219A, eK219Q, and eK219R exhibited lower resistance to copper toxicity and alkaline pH toxicity than eWT. Molecular dynamics analysis revealed that mutation of Lys219 leads to looser and more unstable conformation of CpxR, leading to its decreased binding affinity with downstream genes. Moreover, the Lys219 mutation resulted in the down-regulation of efflux pump genes (acrD, tolC, mdtB and mdtA), leading to the accumulation of antibiotics inside the cells and an increase in ROS production, which significantly reduces antibiotic resistance. CONCLUSIONS: The mutation of the key residue Lys219 causes a conformational change that results in the loss of regulatory ability of CpxR, which may potentially reduce to antibiotic resistance. Therefore, this study suggests that targeting the highly conserved sequence of CpxR could be a promising strategy for the development of new antibacterial drugs.

Genome-wide CRISPR/Cas9 screening identifies a targetable MEST-PURA interaction in cancer metastasis.

BACKGROUND: Metastasis is one of the most lethal hallmarks of esophageal squamous cell carcinoma (ESCC), yet the mechanisms remain unclear due to a lack of reliable experimental models and systematic identification of key drivers. There is urgent need to develop useful therapies for this lethal disease. METHODS: A genome-wide CRISPR/Cas9 screening, in combination with gene profiling of highly invasive and metastatic ESCC sublines, as well as PDX models, was performed to identify key regulators of cancer metastasis. The Gain- and loss-of-function experiments were taken to examine gene function. Protein interactome, RNA-seq, and whole genome methylation sequencing were used to investigate gene regulation and molecular mechanisms. Clinical significance was analyzed in tumor tissue microarray and TCGA databases. Homology modeling, modified ELISA, surface plasmon resonance and functional assays were performed to identify lead compound which targets MEST to suppress cancer metastasis. FINDINGS: High MEST expression was associated with poor patient survival and promoted cancer invasion and metastasis in ESCC. Mechanistically, MEST activates SRCIN1/RASAL1-ERK-snail signaling by interacting with PURA. miR-449a was identified as a direct regulator of MEST, and hypermethylation of its promoter led to MEST upregulation, whereas systemically delivered miR-449a mimic could suppress tumor metastasis without overt toxicity. Furthermore, molecular docking and computational screening in a small-molecule library of 1,500,000 compounds and functional assays showed that G699-0288 targets the MEST-PURA interaction and significantly inhibits cancer metastasis. INTERPRETATION: We identified the MEST-PURA-SRCIN1/RASAL1-ERK-snail signaling cascade as an important mechanism underlying cancer metastasis. Blockade of MEST-PURA interaction has therapeutic potential in management of cancer metastasis. FUNDING: This work was supported by National Key Research and Development Program of China (2021YFC2501000, 2021YFC2501900, 2017YFA0505100); National Natural Science Foundation of China (31961160727, 82073196, 81973339, 81803551); NSFC/RGC Joint Research Scheme (N_HKU727/19); Natural Science Foundation of Guangdong Province (2021A1515011158, 2021A0505030035); Key Laboratory of Guangdong Higher Education Institutes of China (2021KSYS009).

Polyphyllin D punctures hypertrophic lysosomes to reverse drug resistance of hepatocellular carcinoma by targeting acid sphingomyelinase.

Hypertrophic lysosomes are critical for tumor progression and drug resistance; however, effective and specific lysosome-targeting compounds for cancer therapy are lacking. Here we conducted a lysosomotropic pharmacophore-based in silico screen in a natural product library (2,212 compounds), and identified polyphyllin D (PD) as a novel lysosome-targeted compound. PD treatment was found to cause lysosomal damage, as evidenced by the blockade of autophagic flux, loss of lysophagy, and the release of lysosomal contents, thus exhibiting anticancer effects on hepatocellular carcinoma (HCC) cell both in vitro and in vivo. Closer mechanistic examination revealed that PD suppressed the activity of acid sphingomyelinase (SMPD1), a lysosomal phosphodieserase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine, by directly occupying its surface groove, with Trp148 in SMPD1 acting as a major binding residue; this suppression of SMPD1 activity irreversibly triggers lysosomal injury and initiates lysosome-dependent cell death. Furthermore, PD-enhanced lysosomal membrane permeabilization to release sorafenib, augmenting the anticancer effect of sorafenib both in vivo and in vitro. Overall, our study suggests that PD can potentially be further developed as a novel autophagy inhibitor, and a combination of PD with classical chemotherapeutic anticancer drugs could represent a novel therapeutic strategy for HCC intervention.