DMF-scMT-seq linking methylome and transcriptome within single cells with digital microfluidics

Single-cell joint analysis of methylome and transcriptome reveals how the methylation regulates the transcriptional activity.However,traditional bench-top protocols for single-cell DNA methylation and RNA transcription co-detection are laborintensive,cost-ineffective and contaminant-prone.Herein,we establish the DMF-sc MT-seq,a highly-efficient and cost-effective method to simultaneously analyze single-cell DNA methylation and transcriptional activity based on digital microfluidics.DMFsc MT-seq automates the workflow of single-cell isolation,cellular hypotonic lysis,nucleic acid separation and methylome/transcriptome library construction in a contactless and addressable way.The system ensures high accuracy(R>0.85),high gene detection ability(14,697 genes per cell at 4 million sequencing depth),and high CpG coverage(677,198 CpG sites per cell at 1million sequencing depth).By using DMF-sc MT-seq,the relationship of DNA methylation and RNA transcription under different genomic contexts is resolved.We further apply DMF-sc MT-seq to study the dynamics of transcription regulation with methylation-inhibiting anti-tumor Decitabine,and identify the methylated promoter/gene body driven genes in response to Decitabine treatment.DMF-sc MT-seq facilitates the construction of the correlation of DNA methylation and transcriptional activity at the single-cell level in a flexible,sensitive and accurate way,which is anticipated to be a powerful tool in studying single-cell biological systems.

Nano-Medicine for Thrombosis:A Precise Diagnosis and Treatment Strategy

Thrombosis is a global health issue and one of the leading factors of death.However,its diagnosis has been limited to the late stages,and its therapeutic window is too narrow to provide reasonable and effective treatment.In addition,clinical thrombolytics suffer from a short half-life,allergic reactions,inactivation,and unwanted tissue hemorrhage.Nano-medicines have gained extensive attention in diagnosis,drug delivery,and photo/sound/magnetic-theranostics due to their convertible properties.Furthermore,diagnosis and treatment of thrombosis using nano-medicines have also been widely studied.This review summarizes the recent advances in this area,which revealed six types of nanoparticle approaches:(1)in vitro diagnostic kits using“synthetic biomarkers”;(2)in vivo imaging using nano-contrast agents;(3)targeted drug delivery systems using artificial nanoparticles;(4)microenvironment responsive drug delivery systems;(5)drug delivery systems using biological nanostructures;and(6)treatments with external irradiation.The investigations of nano-medicines are believed to be of great significance,and some of the advanced drug delivery systems show potential applications in clinical theranotics.

μ-opioid receptor agonist facilitates circulating tumor cell formation in bladder cancer via the MOR/AKT/Slug pathway:a comprehensive study including randomized controlled trial

Background:μ-opioid receptor agonists(MORAs)are indispensable for analgesia in bladder cancer(BC)patients,both during surgery and for chronic pain treatment.Whether MORAs affect BC progression and metastasis remains largely unknown.This study focused on the effects of MORAs on the formation of circulating tumor cells(CTCs)in BC and aimed to provide potential therapeutic targets,which would retain the pain-relieving effects of MORAs in BC patients without sacrificing their long-term prognosis.Methods:Different preclinical models were used to identify the effects of MORAs on the progression of BC.A novel immunocapture microfluidic chip was utilized to analyze whether MORAs affected the number of CTCs in mouse models and clinical BC patients.Bioinformatic analyses,total transcriptome sequencing,and molecular biology methods were then used to investigate the underlying mechanisms in these models and in BC cell lines.Results:Mouse models of hematogenous metastasis and in situ BC demonstrated that tumor metastasis was significantly increased after MORA treatment.A significant increase in the number of mesenchymal and/or epithelial CTCs was detected after MORA treatment in both the mouse models and clinical trial patients.Mechanistically,MORAs facilitated the formation of CTCs by activating the MOR/PI3K/AKT/Slug signaling pathway,hereby promoting the epithelialmesenchymal transition(EMT)of BC cells,as knockdown of MOR,Slug or blockade of PI3K inhibited the EMT process and CTC formation.Conclusion:MORAs promoted BC metastasis by facilitating CTC formation.The EMT-CTC axis could be targeted for preventive measures during MORA treatment to inhibit the associated tumormetastasis or recurrence in BC patients.

Assembly of peptide nanostructures with controllable sizes

Controlled peptide assembly offers significant promise to develop synthetic supramolecular nanostructures to display material and biological properties that mimic protein assemblies in nature.Despite the progress in forming peptide nanostructures of various morphology,there exists a distinct gap between natural and synthetic assembly systems in terms of size control.Constructing nanostructures with a narrow size distribution that can be tuned over a wide range of length-scales is essential for applications that require precise spacing between objects.This approach provides the opportunity to correlate materials and biological properties of interest with assembly size.In this review,we discuss representative endeavors over the past two decades for design of size-controllable peptide nanostructures using tunable building blocks.Other mechanisms for size control,e.g.,molecular frustration,template-directed peptide assembly,and multi-component peptide co-assembly,will also be discussed.We also demonstrate the applicable scopes of these strategies and suggest potential future avenues for scientific advances in this field.

A mini-review and perspective on multicyclic peptide mimics of antibodies

Affinity reagents are important tools in the biological sciences for understanding biological processes and for studying protein expression, localization and interactions. However, traditional affinity reagents such as antibodies(and their fragments) and non-immunoglobulin(non-Ig) scaffold binders, usually suffer from problems of poor cellular uptake efficiency, high production cost, and low structural stability. This leads to rapid development of small antibody-like affinity reagents such as scaffold-free cyclic and multicyclic peptides, which usually have 5-30 amino acid residues, thus lying between non-Ig scaffolds and small molecules in size. In this mini-review, we highlight the recent development in mono-and multi-cyclic peptide mimics of antibodies, including cyclic peptide affinity reagents that have been developed for use in antibody-like applications, novel synthetic strategies for multicyclic peptides, and promising peptide library screening platforms. We also provide a perspective on the future development in multicyclic peptide mimics of antibodies.

Cu^(2+)-Assisted Synthesis of Au@AgI Core/Shell Nanorods via In Situ Oxidation of Iodide:A Strategy for Colorimetric Iodide Sensing

Colorimetric nanoprobes are frequently employed to construct sensitive sensors via various strategies.In this work,a novel strategy for simple and ultrasensitive sensing of iodide(I_(−))was constructed based on Cu^(2+)-assisted generation of Au@AgI core/shell nanorods(NRs),by etching silver-coated gold nanorods(Ag@Au NRs).Ag@Au NRs were first prepared and characterized with UV-visible spectroscopy,transmission electron microscopy,high-angle annular dark-field scanning transmission electron microscopy,and energy-dispersive X-ray spectroscopy before and after iodide treatment.The mechanism of detection was based on the generation of AgI shells via etching Ag shells by I_(2),which was produced from the oxidation of I_(−)with Cu^(2+),resulting in the peak shift of localized surface plasmon resonance(LSPR)while the color changed from green,blue to light-violet.The red-shift of the longitudinal LSPR peak had a linear relationship with I_(−)concentration in the range of 0-50μmol/L with a correlation coefficient of 0.982.Furthermore,the proposed method was used for the quantitative detection of I_(−)in real samples and outstanding recoveries in the range of 91-106%with relative standard deviations lower than 2.5%were observed.

Interchain doubly-bridged α-helical peptides for the development of protein binders

Constrained peptide scaffolds that are tolerant to extensive sequence manipulation and amenable to bioactive peptide design are of great value to the development of novel protein binders and peptide therapeutics. In this work, we reported strategies for the design and synthesis of a kind of novel interchain doubly-bridged α-helical peptides, involving mutual stabilization of two peptide α-helices linked by two interchain bisthioether crosslinkers. By taking a MDM2-binding peptide with an α-helical tendency as a model, we demonstrated that α-helical dimers with significantly improved structural and proteolytic stability and nanomolar binding affinity to the target protein can be obtained. By modulating the surface charges on the dimeric peptides, we also obtained a dimeric peptide with enhanced cellpenetrating capability, which can efficiently penetrate into cancer cells and inhibit the intracellular MDM2-p53 interactions to promote cell apoptosis. Considering that many proteins take a surface α-helical segment as the binding motif to mediate their interactions with other proteins, we believe that our interchain doubly-bridged α-helical peptides would provide a promising scaffold for the development of novel high-affinity protein binders.

Integrated identification of growth pattern and taxon of bacterium in gut microbiota via confocal fluorescence imaging‐oriented single‐cell sequencing

Despite the fast progress in our understanding of the complex functions of gut microbiota,it is still challenging to directly investigate the in vivo microbial activities and processes on an individual cell basis.To gain knowledge of the indigenous growth/division patterns of the diverse mouse gut bacteria with a relatively high throughput,here,we propose an integrative strategy,which combines the use of fluorescent probe labeling,confocal imaging with single‐cell sorting,and sequencing.Mouse gut bacteria sequentially labeled by two fluorescent D‐amino acid probes in vivo were first imaged by confocal microscopy to visualize their growth patterns,which can be unveiled by the distribution of the two fluorescence signals on each bacterium.Bacterial cells of interest on the imaging slide were then sorted using a laser ejection equipment,and the collected cells were then sequenced individually to identify their taxa.Our strategy allows integrated acquirement of the growth pattern knowledge of a variety of gut bacteria and their genomic information on a single‐cell basis,which should also have great potential in studying many other complex bacterial systems.

Fabrication of Non-woven Fabric-Based SERS Substrate for Direct Detection of Pesticide Residues in Fruits

In surface-enhanced Raman scattering(SERS),flexible substrate plays an important role in target molecular collection from various shape surfaces and increases the analytical sensitivity.In this study,silver nanoparticles(Ag NPs)were deposited on a non-woven fabric used as an SERS substrate by self-assembly,in situ growing or the self-assembly/in situ growing combination method.4-Aminothiophenol was selected as a model molecular for the evaluation of the SERS performance using the substrates.The Ag NPs substrate prepared by self-assembly/in situ growing method presented the best Raman enhancement effect and its enhancement factor was estimated as high as 3.59106.The substrate was applied to the determination of four pesticide residues on the surfaces of fruit samples through wipe sampling,and the results revealed the good reproducibility of SERS responses and high detection sensitivity.The prepared flexible substrate was simple to fabricate and environmentally friendly.It could be expected to be a useful tool in rapid on-site test of pesticide residues on fruit surfaces because of its high sensitivity,convenience and non-destructive characteristics.

Crown ether-thiourea conjugates as ion transporters

Na^(+),Cl^(‒)and K^(+)are the most abundant electrolytes present in biological fluids that are essential to the regulation of pH homeostasis,membrane potential and cell volume in living organisms.Herein,we report synthetic crown ether-thiourea conjugates as a cation/anion symporter,which can transport both Na+and Cl^(–)across lipid bilayers with relatively high transport activity.Surprisingly,the ion transport activities were diminished when high concentrations of K+ions were present outside the vesicles.This unusual behavior resulted from the strong affinity of the transporters for K^(+)ions,which led to predominant partitioning of the transporters as the K^(+)complexes in the aqueous phase preventing the transporter incorporation into the membrane.Synthetic membrane transporters with Na^(+),Cl^(‒)and K^(+)transport capabilities may have potential biological and medicinal applications.

Nucleic acids analysis

Nucleic acids are natural biopolymers of nucleotides that store, encode, transmit and express genetic information, which play central roles in diverse cellular events and diseases in living things. The analysis of nucleic acids and nucleic acids-based analysis have been widely applied in biological studies, clinical diagnosis, environmental analysis, food safety and forensic analysis.During the past decades, the field of nucleic acids analysis has been rapidly advancing with many technological breakthroughs.In this review, we focus on the methods developed for analyzing nucleic acids, nucleic acids-based analysis, device for nucleic acids analysis, and applications of nucleic acids analysis. The representative strategies for the development of new nucleic acids analysis in this field are summarized, and key advantages and possible limitations are discussed. Finally, a brief perspective on existing challenges and further research development is provided.

Integrated microfluidic devices for in vitro diagnostics at point of care

Given the continuous and growing demand for point of care(POC)diagnostic tests,attention has been shifted toward integration and miniaturization of laboratory protocols into“sample-in-answer-out”devices.Microfluidic technologies have been considered an ideal solution to address the requirements of POC diagnostics since many laboratory functions can be miniaturized and incorporated onto a single integrated chip.In this review,we summarize the advances of integrated microfluidic devices for POC diagnostics in the last 3 years.Particularly,we summarize current materials used for microfluidic chip fabrication,discuss the innovation of versatile integrated microfluidic devices,especially the strategies for simplifying sample preparation in manual or self-driven systems,and new detection methods of microfluidic chips.In addition,we describe new integrated microfluidic devices for POC diagnostics of protein-targeted immunodiagnostics,nucleic acid molecular tests,and small molecule metabolites analysis.We also provide future perspectives and current challenges for clinical translation and commercialization of these microfluidic technologies.