Insulin-like Growth Factor 2-Tagged Aptamer Chimeras (ITACs) Modular Assembly for Targeted and Efficient Degradation of Two Membrane Proteins.

Overexpression of pathogenic membrane proteins drives abnormal proliferation and invasion of tumor cells. Various strategies to durably knockdown membrane proteins with heterobifunctional degraders have been successfully developed, including LYTAC, KineTAC, and AbTAC. However, challenges including complicated synthetic procedures and the inability to simultaneously degrade multiple pathogenic proteins still exist. Herein, we developed insulin-like growth factor 2 (IGF2)-tagged aptamer chimeras (ITACs) that link the cell-surface lysosome-targeting receptor IGF2R and membrane proteins of interest (POIs) based on specific recognition of aptamers to the POIs and high-affinity binding of IGF2 to IGF2R. We demonstrated that ITACs exhibit robust degradation efficiency of various membrane proteins in multiple cell lines. Furthermore, systematic studies revealed that a moderate cell-surface IGF2R level is responsible for the excellent degradation performance of ITACs. Importantly, we further established a modular assembly strategy that allows assembly of one IGF2 with two aptamers with precise stoichiometry (dITACs), enabling cooperative and simultaneous degradation of two membrane proteins. This work provides an efficient and facile target membrane protein degradation platform and will shed light on the treatment of diseases related to the overexpression of membrane proteins.

Mass Spectrometry Imaging-Based Single-Cell Lipidomics Profiles Metabolic Signatures of Heart Failure.

Heart failure (HF), leading as one of the main causes of mortality, has become a serious public health issue with high prevalence around the world. Single cardiomyocyte (CM) metabolomics promises to revolutionize the understanding of HF pathogenesis since the metabolic remodeling in the human hearts plays a vital role in the disease progression. Unfortunately, current metabolic analysis is often limited by the dynamic features of metabolites and the critical needs for high-quality isolated CMs. Here, high-quality CMs were directly isolated from transgenic HF mice biopsies and further employed in the cellular metabolic analysis. The lipids landscape in individual CMs was profiled with a delayed extraction mode in time-of-flight secondary ion mass spectrometry. Specific metabolic signatures were identified to distinguish HF CMs from the control subjects, presenting as possible single-cell biomarkers. The spatial distributions of these signatures were imaged in single cells, and those were further found to be strongly associated with lipoprotein metabolism, transmembrane transport, and signal transduction. Taken together, we systematically studied the lipid metabolism of single CMs with a mass spectrometry imaging method, which directly benefited the identification of HF-associated signatures and a deeper understanding of HF-related metabolic pathways.

Logical Analysis of Multiple Single-Nucleotide-Polymorphisms with Programmable DNA Molecular Computation for Clinical Diagnostics.

Analyzing complex single-nucleotide-polymorphism (SNP) combinations in the genome is important for research and clinical applications, given that different SNP combinations can generate different phenotypic consequences. Recent works have shown that DNA-based molecular computing is powerful for simultaneously sensing and analyzing complex molecular information. Here, we designed a switching circuit-based DNA computational scheme that can integrate the sensing of multiple SNPs and simultaneously perform logical analysis of the detected SNP information to directly report clinical outcomes. As a demonstration, we successfully achieved automatic and accurate identification of 21 different blood group genotypes from 83 clinical blood samples with 100 % accuracy compared to sequencing data in a more rapid manner (3 hours). Our method enables a new mode of automatic and logical sensing and analyzing subtle molecular information for clinical diagnosis, as well as guiding personalized medication.

[Characteristics analysis for Chinese patent medicine containing Jujubea Fructus based on data mining].

Chinese patent medicine prescriptions containing Jujubea Fructus in 2015 edition of Chinese Pharmacopoeia and the Composition Principles of Chinese Patent Drug were collected, and the characteristics of Chinese patent medicine containing Jujubea Fructus were analyzed by using data mining technology. Statistical software Excel 2019, Clementine 12.0 and SPSS 21.0 were used to conduct statistical analysis of conforming Chinese patent medicine prescriptions by means of frequency statistics, association rule analysis and cluster analysis. Finally, a total of 185 Chinese patent medicine prescriptions containing Jujubea Fructus were included in this study, involving 402 Chinese medicines and 28 kinds of high frequency Chinese medicines, with Jujubea Fructus, Poria, Zingiberis Rhizoma Recens, Glycyrrhizae Radix et Rhizoma, and Codonopsis Radix as the top five. The deficiency-nourishing drugs were in the most common efficacy classification, mainly sweet, bitter and pungent, with most medicine properties of warm and gentle, main meridians of spleen lung and stomach, dosage forms of pills, granules and tablets, and main indications of splenic diseases. Fifteen drug combinations were obtained in association rule analysis. Eleven drug combinations were obtained by association rule analysis of Chinese patent medicine containing Jujubea Fructus in the treatment of splenic diseases, and the drugs were divided into two categories by cluster analysis. According to the above analysis, it is found that the Chinese patent medicine prescriptions containing Jujubea Fructus are mainly composed of deficiency-nourishing drugs, mostly compatible with drugs of sweet, bitter and pungent flavors, warm and gentle properties, and spleen, lung, and stomach meridians in the treatment of splenic diseases, with Sijunzi Decoction as the main drug. This study provides guidance for modern clinical application and development of Jujubea Fructus.

[Animal model analysis of diabetic lower extremity arterial disease based on clinical symptoms of traditional Chinese and Western medicine].

The evaluation standard of LEAD animal model was established according to the understanding of the etiology and pathogenesis of diabetic lower extremity vascular disease based on Chinese and Western medicine. The consistency between the existing LEAD animal model and the clinical characteristics of traditional Chinese and Western medicine was analyzed and evaluated. The advantages and disadvantages of the existing model were compared,the application scope of different models was considered,and the possible improvement methods of the existing model were proposed,so as to provide impetus for the improvement of LEAD animal model.We should reflect more characteristics of traditional Chinese medicine syndromes in the process of model improvement and development,making the LEAD animal model to get closer to clinical features of traditional Chinese and Western medicine.

Bispecific Aptamer Chimeras Enable Targeted Protein Degradation on Cell Membranes.

The ability to regulate membrane protein abundance offers great opportunities for developing therapeutic sites for various diseases. Herein, we describe a platform for the targeted degradation of membrane-associated proteins using bispecific aptamer chimeras that bind both the cell-surface lysosome-shuttling receptor (IGFIIR) and the targeted membrane-bound proteins of interest. We demonstrate that the aptamer chimeras can efficiently and quickly shuttle the therapeutically relevant membrane proteins of Met and PTK-7 to lysosomes and degrade them through the lysosomal protein degradation machinery. We anticipate that our method will provide a universal platform for the use of readily synthesized aptamer materials for biochemical research and potential therapeutics.

Comparative Evaluation of the Transdermal Permeation Effectiveness of Fu's Cupping Therapy on Eight Different Types of Model Drugs.

BACKGROUND: Overcoming the skin barrier to achieve the transdermal penetration of drugs across the Stratum Corneum (SC) remains a significant challenge. Our previous study showed that Fu's Cupping Therapy (FCT) contributes to the transdermal enhancement and percutaneous absorption rate of representative drugs and improves their clinical effects. This work evaluated the transdermal enhancement effect of FCT on drugs with different Molecular Weights (MW). METHODS: We investigated the enhancements in the transdermal penetration of eight types of model drugs through the skin of BALB/c-nu mice and Sprague Dawley rats using Franz diffusion devices. In addition, 3% azone, 5% azone, 3% peppermint oil, and 5% peppermint oil were used as penetration enhancers to study the transdermal behaviour of these drugs. RESULTS: Our results showed that the BALB/c-nu mouse skin was the best transdermal media, and the optimal time for FCT was 10 min. Compared with other penetration enhancers, FCT exerted a significantly improved effect on enhancing the percutaneous penetration of the selected log(P)- model drugs in addition to the two large MW drugs (ginsenoside Rg1 and notoginsenoside R1). Statistical analysis revealed that the relationship between the log(P) of various model drugs and the permeability coefficient [log(Pcm)] of the FCT group was log(Pcm)=0.080(log(P))2-0.136 (log(P))-0.282. CONCLUSION: FCT may be used as a novel method for enhancing physical penetration and thus effectively promoting the transdermal absorption of drugs and might lay a foundation for future research on drug transdermal technology.

Highly Specific, Single-step Cancer Cell Isolation with Multi-Aptamer-Mediated Proximity Ligation on Live Cell Membranes.

A single-step method for isolation of specific cells based on multiple surface markers will have unique advantages because of its scalability, efficacy, and mildness. Herein, we developed multi-aptamer-mediated proximity ligation method on live cell membranes that leverages a multi-receptor co-recognition design for enhanced specificity, as well as a robust in situ signal amplification design for improved sensitivity of cell isolation. We demonstrated the promising efficacy of our method on differentiating tumor cell subtypes in both cell mixtures and clinical samples. Owing to its simple and fast operation with excellent cell isolation sensitivity and accuracy, this approach will have broad applications in biological science, biomedical engineering, and personalized medicine.

Total glucosides of herbaceous peony (Paeonia lactiflora Pall.) flower attenuate adenine- and ethambutol-induced hyperuricaemia in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbaceous peony (Paeonia lactiflora Pall.) flower has been used widely in dietotherapy in China and other countries. It has good ethnopharmacological value in the treatment of various metabolic diseases. However, the molecular mechanisms by which it lowers serum uric acid are unknown. The development of pharmaceutical resources is very important. Here, we sought to elucidate the mode of action of herbaceous peony in terms of reducing uric acid levels. AIM OF THE STUDY: In the present research, the effects of the total glucosides of herbaceous peony flower were investigated in a rat hyperuricaemia model. Another aim of the study was to clarify the mechanism by which herbaceous peony flower (TGPF) lowers serum uric acid levels. MATERIALS AND METHODS: A hyperuricaemic rat model was induced via intragastric administration of 100 mg/kg adenine and 250 mg/kg ethambutol hydrochloride (EH) for 23 d. Then TongFengShu 600 mg/kg, allopurinol 42 mg/kg, or TGPF (50 mg/kg, 100 mg/kg, or 200 mg/kg) was administered 1 h after the adenine and EH treatments. RESULTS: TGPF improved weight loss and decreased serum UA, XOD, MCP-1, TNF-α, Cr, and BUN in the rats with hyperuricaemic nephropathy. TGPF downregulated renal URAT1 and GLUT9, upregulated renal OAT1, and ameliorated histopathological changes in the thymus, spleen, and kidney. CONCLUSION: TGPF is promising as a therapeutic agent against hyperuricaemia. It regulates the uric acid transporters and diminished serum uric acid levels, and alleviates renal pathology associated with hyperuricaemia.

Cancer diagnosis with DNA molecular computation.

Early and precise cancer diagnosis substantially improves patient survival. Recent work has revealed that the levels of multiple microRNAs in serum are informative as biomarkers for the diagnosis of cancers. Here, we designed a DNA molecular computation platform for the analysis of miRNA profiles in clinical serum samples. A computational classifier is first trained in silico using miRNA profiles from The Cancer Genome Atlas. This is followed by a computationally powerful but simple molecular implementation scheme using DNA, as well as an effective in situ amplification and transformation method for miRNA enrichment in serum without perturbing the original variety and quantity information. We successfully achieved rapid and accurate cancer diagnosis using clinical serum samples from 22 healthy people (8) and people with lung cancer (14) with an accuracy of 86.4%. We envision that this DNA computational platform will inspire more clinical applications towards inexpensive, non-invasive and rapid disease screening, classification and progress monitoring.

CdSe/ZnS quantum dots induced spermatogenesis dysfunction via autophagy activation.

Recent researches have demonstrated that many nanoparticles are harmful to spermatogenesis. However, the reported nanoparticles -elicited testicular pathologies have been mostly confined to hormone levels and sperm quality and quantity, the detail mechanism is still largely unknown and the strategies to reduce the toxicity of nanoparticles on testis are lacking. Here, we found that CdSe/ZnS quantum dots (QDs) exposure impair double-strand break (DSB) repair in spermatocyte, leading to the disruption of meiotic progression and thus cell apoptosis and decreased sperm production. Furthermore, we found that QDs exposure elevates the autophagy. Crucially, both in vitro and in vivo studies indicated that elevated autophagy could down-regulate the expression of the genes responsible for homologous recombination, which is the main pathway for DSB repair during meiosis, indicating that spermatogenesis impairment by CdSe/ZnS QDs is mediated by autophagy. Consequently, injection of autophagy inhibitor (3-MA) restore DSB repair in spermatocytes, resulting in prevention of spermatocyte apoptosis and recovery of spermatogenesis. Our studies strongly indicate that autophagy is key for eliciting the spermatogenesis dysfunction after nanoparticle exposure, and autophagy inhibition can be used as a potential clinical remedy for alleviating the male reproductive toxicity of nanoparticles.

N6-Methyladenosine Modulates Nonsense-Mediated mRNA Decay in Human Glioblastoma.

The N6-methyladenosine (m6A) modification influences various mRNA metabolic events and tumorigenesis, however, its functions in nonsense-mediated mRNA decay (NMD) and whether NMD detects induced carcinogenesis pathways remain undefined. Here, we showed that the m6A methyltransferase METTL3 sustained its oncogenic role by modulating NMD of splicing factors and alternative splicing isoform switches in glioblastoma (GBM). Methylated RNA immunoprecipitation-seq (MeRIP-seq) analyses showed that m6A modification peaks were enriched at metabolic pathway-related transcripts in glioma stem cells (GSC) compared with neural progenitor cells. In addition, the clinical aggressiveness of malignant gliomas was associated with elevated expression of METTL3. Furthermore, silencing METTL3 or overexpressing dominant-negative mutant METTL3 suppressed the growth and self-renewal of GSCs. Integrated transcriptome and MeRIP-seq analyses revealed that downregulating the expression of METTL3 decreased m6A modification levels of serine- and arginine-rich splicing factors (SRSF), which led to YTHDC1-dependent NMD of SRSF transcripts and decreased SRSF protein expression. Reduced expression of SRSFs led to larger changes in alternative splicing isoform switches. Importantly, the phenotypes mediated by METTL3 deficiency could be rescued by downregulating BCL-X or NCOR2 isoforms. Overall, these results establish a novel function of m6A in modulating NMD and uncover the mechanism by which METTL3 promotes GBM tumor growth and progression. SIGNIFICANCE: These findings establish the oncogenic role of m6A writer METTL3 in glioblastoma stem cells.

Inhibition of CaMKIIα Activity Enhances Antitumor Effect of Fullerene C60 Nanocrystals by Suppression of Autophagic Degradation.

Fullerene C60 nanocrystals (nano-C60) possess various attractive bioactivities, including autophagy induction and calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) activation. CaMKIIα is a multifunctional protein kinase involved in many cellular processes including tumor progression; however, the biological effects of CaMKIIα activity modulated by nano-C60 in tumors have not been reported, and the relationship between CaMKIIα activity and autophagic degradation remains unclear. Herein, nano-C60 is demonstrated to elicit reactive oxygen species (ROS)-dependent cytotoxicity and persistent activation of CaMKIIα in osteosarcoma (OS) cells. CaMKIIα activation, in turn, produces a protective effect against cytotoxicity from nano-C60 itself. Inhibition of CaMKIIα activity by either the chemical inhibitor KN-93 or CaMKIIα knockdown dramatically promotes the anti-OS effect of nano-C60. Moreover, inhibition of CaMKIIα activity causes lysosomal alkalinization and enlargement, and impairs the degradation function of lysosomes, leading to autophagosome accumulation. Importantly, excessive autophagosome accumulation and autophagic degradation blocking are shown to play an important role in KN-93-enhanced-OS cell death. The synergistic anti-OS efficacy of KN-93 and nano-C60 is further revealed in an OS-xenografted murine model. The results demonstrate that CaMKIIα inhibition, along with the suppression of autophagic degradation, presents a promising strategy for improving the antitumor efficacy of nano-C60.

Transcatheter hepatic arterial chemoembolization plus cinobufotalin injection adjuvant therapy for advanced hepatocellular carcinoma: a meta-analysis of 27 trials involving 2,079 patients.

OBJECTIVE: The aim of this study was to systematically investigate the safety and efficacy of the combination of transcatheter hepatic arterial chemoembolization (TACE) and cinobufotalin injection for advanced hepatocellular carcinoma (HC). METHODS: Clinical trials were searched from Web of Science, Cochrane Library, PubMed, Embase, Chinese Medical Citation Index (CMCI), China National Knowledge Infrastructure (CNKI), Chinese Scientific Journal Database (VIP), and Wanfang database. Outcome measures including therapeutic efficacy, quality of life, liver function, immune function, and adverse events were extracted and evaluated. RESULTS: After final assessment, 27 studies including 2,079 advanced HC patients were involved in this study. Compared with TACE alone, the combination of TACE with cinobufotalin injection adjuvant therapy significantly prolonged the patients' 1-, 1.5-, 2-, and 3-year overall survival (OS) rate (1-year OS, OR=2.84, 95% CI=2.20-3.67, P<0.00001; 1.5-year OS, OR=3.57, 95% CI=1.92-6.66, P<0.0001; 2-year OS, OR=3.17, 95% CI=2.36-4.25, P<0.00001; 3-year OS, OR=2.88, 95% CI=1.82-4.57, P<0.00001). The combined therapy also improved patients' overall response rate (ORR; OR=1.86, 95% CI=1.54-2.24, P<0.00001), disease control rate (DCR; OR=2.05, 95% CI=1.59-2.64, P<0.00001), and quality of life improved rate (QIR; OR=3.45, 95% CI=2.52-4.72, P<0.00001). Moreover, the immune function and liver function of HC patients were all significantly enhanced after the combined therapy of TACE and cinobufotalin injection (CD3+, P=0.001; CD4+, P=0.0006; CD4+/CD8+, P=0.03; natural killer [NK] cell, P=0.01; total bilirubin [TBIL], P=0.003; alanine aminotransferase [ALT], P<0.00001; aspartate aminotransferase [AST], P<0.00001). No serious adverse events occurred during cinobufotalin injection-mediated therapy. CONCLUSION: The combination of TACE and cinobufotalin injection adjuvant therapy is safe and more effective for end-stage HC treatment than TACE alone.

Pan-Cancer Analysis Reveals the Functional Importance of Protein Lysine Modification in Cancer Development.

Large-scale tumor genome sequencing projects have revealed a complex landscape of genomic mutations in multiple cancer types. A major goal of these projects is to characterize somatic mutations and discover cancer drivers, thereby providing important clues to uncover diagnostic or therapeutic targets for clinical treatment. However, distinguishing only a few somatic mutations from the majority of passenger mutations is still a major challenge facing the biological community. Fortunately, combining other functional features with mutations to predict cancer driver genes is an effective approach to solve the above problem. Protein lysine modifications are an important functional feature that regulates the development of cancer. Therefore, in this work, we have systematically analyzed somatic mutations on seven protein lysine modifications and identified several important drivers that are responsible for tumorigenesis. From published literature, we first collected more than 100,000 lysine modification sites for analysis. Another 1 million non-synonymous single nucleotide variants (SNVs) were then downloaded from TCGA and mapped to our collected lysine modification sites. To identify driver proteins that significantly altered lysine modifications, we further developed a hierarchical Bayesian model and applied the Markov Chain Monte Carlo (MCMC) method for testing. Strikingly, the coding sequences of 473 proteins were found to carry a higher mutation rate in lysine modification sites compared to other background regions. Hypergeometric tests also revealed that these gene products were enriched in known cancer drivers. Functional analysis suggested that mutations within the lysine modification regions possessed higher evolutionary conservation and deleteriousness. Furthermore, pathway enrichment showed that mutations on lysine modification sites mainly affected cancer related processes, such as cell cycle and RNA transport. Moreover, clinical studies also suggested that the driver proteins were significantly associated with patient survival, implying an opportunity to use lysine modifications as molecular markers in cancer diagnosis or treatment. By searching within protein-protein interaction networks using a random walk with restart (RWR) algorithm, we further identified a series of potential treatment agents and therapeutic targets for cancer related to lysine modifications. Collectively, this study reveals the functional importance of lysine modifications in cancer development and may benefit the discovery of novel mechanisms for cancer treatment.

m6ASNP: a tool for annotating genetic variants by m6A function.

Background: Large-scale genome sequencing projects have identified many genetic variants for diverse diseases. A major goal of these projects is to characterize these genetic variants to provide insight into their function and roles in diseases. N6-methyladenosine (m6A) is one of the most abundant RNA modifications in eukaryotes. Recent studies have revealed that aberrant m6A modifications are involved in many diseases. Findings: In this study, we present a user-friendly web server called "m6ASNP" that is dedicated to the identification of genetic variants that target m6A modification sites. A random forest model was implemented in m6ASNP to predict whether the methylation status of an m6A site is altered by the variants that surround the site. In m6ASNP, genetic variants in a standard variant call format (VCF) are accepted as the input data, and the output includes an interactive table that contains the genetic variants annotated by m6A function. In addition, statistical diagrams and a genome browser are provided to visualize the characteristics and to annotate the genetic variants. Conclusions: We believe that m6ASNP is a very convenient tool that can be used to boost further functional studies investigating genetic variants. The web server "m6ASNP" is implemented in JAVA and PHP and is freely available at [60].

m6AVar: a database of functional variants involved in m6A modification.

Identifying disease-causing variants among a large number of single nucleotide variants (SNVs) is still a major challenge. Recently, N6-methyladenosine (m6A) has become a research hotspot because of its critical roles in many fundamental biological processes and a variety of diseases. Therefore, it is important to evaluate the effect of variants on m6A modification, in order to gain a better understanding of them. Here, we report m6AVar (http://m6avar.renlab.org), a comprehensive database of m6A-associated variants that potentially influence m6A modification, which will help to interpret variants by m6A function. The m6A-associated variants were derived from three different m6A sources including miCLIP/PA-m6A-seq experiments (high confidence), MeRIP-Seq experiments (medium confidence) and transcriptome-wide predictions (low confidence). Currently, m6AVar contains 16 132 high, 71 321 medium and 326 915 low confidence level m6A-associated variants. We also integrated the RBP-binding regions, miRNA-targets and splicing sites associated with variants to help users investigate the effect of m6A-associated variants on post-transcriptional regulation. Because it integrates the data from genome-wide association studies (GWAS) and ClinVar, m6AVar is also a useful resource for investigating the relationship between the m6A-associated variants and disease. Overall, m6AVar will serve as a useful resource for annotating variants and identifying disease-causing variants.

Single-walled carbon nanotube: One specific inhibitor of cancer stem cells in osteosarcoma upon downregulation of the TGFß1 signaling.

Cancer stem cells (CSCs) are believed to have a critical role in tumorigenesis, metastasis, therapeutic resistance or recurrence. Therefore, strategies designed to specifically target and eliminate CSCs have become one of the most promising and desirable ways for tumor treatment. Osteosarcoma stem cells (OSCs), the CSCs in osteosarcoma (OS), are critically associated with OS progression. Here, we show that single-walled carbon nanotubes (SWCNTs), including unmodified SWCNT (SWCNT-Raw) and SWCNT-COOH, have the ability to specifically inhibit the process of TGFß1-induced OS cells dedifferentiation, prevent the stem cell phenotypes acquisition in OS cells and reduce the OSC viability under conditions which mimic the OS microenvironment. Concurrently, SWCNT treatment significantly down-regulates the expression of OSC markers in OS, and markedly reduces the tumor microvessel density and tumor growth. Furthermore, we found that SWCNT could suppress the TGFß1-induced activation of TGFß type I receptor and downstream signaling, which are key for the OSC formation and maintenance. Our results reveal an unexpected function of SWCNT in negative modulation of OSCs, and provide significant implications for the potential CSCs-targeted therapeutic applications of SWCNT.