An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic,Anionic, Non‐Viral Transfection Method

Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular‐matrix‐derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size‐ and concentration‐dependent gene silencing in a CD44‐positive human osteosarcoma cell line (MG‐63) and in human mesenchymal stromal cells (hMSCs). This native HA‐based siRNA transfection represents the first report on an anionic, non‐viral delivery method that resulted in approximately 60 % gene knockdown in both cell types tested, which correlated with a reduction in translation levels.     

Hyaluronic acid‐dependent protection against alkali‐burned human corneal cells

Hyaluronic acid (HA) is a high‐molecular‐weight glycosaminoglycan and extracellular matrix component that promotes cell proliferation. This study aimed to evaluate the effects of HA on alkali‐injured human corneal epithelial cells in vitro, and to elucidate the mechanisms by which HA mediates corneal cell protection. A human corneal epithelial cell line (HCE‐2) was treated with sodium hydroxide before incubation with low‐molecular‐weight HA (LMW‐HA, 127 kDa) or high‐molecular‐weight HA (HMW‐HA, 1525 kDa). A global proteomic analysis was then performed. Our data indicated that HA treatment protects corneal epithelial cells from alkali injury, and that the molecular weight of HA is a crucial factor in determining its effects. Only HMW‐HA reduced NaOH‐induced cytotoxic effects in corneal cells significantly and increased their migratory and wound healing ability. Results from 2D‐DIGE and MALDI‐TOF/TOF MS analyses indicated that HMW‐HA modulates biosynthetic pathways, cell migration, cell outgrowth, and protein degradation to stimulate wound healing and prevent cell death. To our knowledge, our study is the first to report the possible mechanisms by which HMW‐HA promotes repair in alkali‐injured human corneal epithelial cells.     

Drawing with Iron on a Gel Containing a Supramolecular Siderophore

Guanosine‐5′‐hydroxamic acid ( 3 ) forms hydrogels when mixed with guanosine ( 1 ) and KCl. The 5′‐hydroxamic acid (HA) unit is pH‐responsive and also chelates Fe³⁺. When gels are prepared under basic conditions, the 5′‐HA groups are deprotonated and the anionic hydrogel binds cationic thiazole orange (TO), signaled by enhanced fluorescence. The HA nucleoside 3 , when immobilized in the G‐quartet gel, acts as a supramolecular siderophore to form red complexes with Fe³⁺. We patterned the hydrogel's surface with FeCl₃, by hand and by using a 3D printer. Patterns form instantly, are visible by eye, and can be erased using vitamin C. This hydrogel, combining self‐assembled G‐quartet and siderophore–Fe³⁺ motifs, is strong, can be molded into different shapes, and is stable on the bench or under salt water.     

Design of the First‐in‐Class,Highly Potent Irreversible Inhibitor Targeting the Menin‐MLL Protein–Protein Interaction

The structure‐based design of M‐525 as the first‐in‐class, highly potent, irreversible small‐molecule inhibitor of the menin‐MLL interaction is presented. M‐525 targets cellular menin protein at sub‐nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL‐regulated gene expression in MLL leukemia cells. M‐525 demonstrates high cellular specificity over non‐MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co‐crystal structure of M‐525 in complex with menin firmly establish its mode of action. A single administration of M‐525 effectively suppresses MLL‐regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M‐525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.     

A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids

The use of noncanonical amino acids (ncAAs) to control the viability of an organism provides a strategy for the development of conditional “kill switches” for live vaccines or engineered human cells. We report an approach inspired by the posttranslational acetylation/deacetylation of lysine residues, in which a protein encoded by a gene with an in‐frame nonsense codon at an essential lysine can be expressed in its native state only upon genetic incorporation of N ‐ϵ ‐acetyl‐l ‐Lys (AcK), and subsequent enzymatic deacetylation in the host cell. We applied this strategy to two essential E. coli enzymes: the branched‐chain aminotransferase BCAT and the DNA replication initiator protein DnaA. We also devised a barnase‐based conditional suicide switch to further lower the escape frequency of the host cells. This strategy offers a number of attractive features for controlling host viability, including a single small‐molecule‐based kill switch, low escape frequency, and unaffected protein function.     

Potential‐Responsive Surfaces for Manipulation of Cell Adhesion,Release, and Differentiation

In living systems, interfacial molecular interactions control many biological processes. New stimuli‐responsive strategies are desired to provide versatile model systems that can regulate cell behavior in vitro. Described here are potential‐responsive surfaces that control cell adhesion and release as well as stem cell differentiation. Cell adhesion can be modulated dynamically by applying negative and positive potentials to surfaces functionalized with tailored monolayers. This process alters cell morphology and ultimately controls behavior and the fate of the cells. Cells can be detached from the electrode surface as intact clusters with different geometries using electrochemical potentials. Importantly, morphological changes during adhesion guide stem cell differentiation. The higher accessibility of the peptide under a positive applied potential causes phenotypic changes in the cells that are hallmarks of osteogenesis, whereas lower accessibility of the peptide promoted by negative potentials leads to adipogenesis.     

A Versatile Dynamic Mussel‐Inspired Biointerface: From Specific Cell Behavior Modulation to Selective Cell Isolation

Reported here is a novel dynamic biointerface based on reversible catechol‐boronate chemistry. Biomimetically designed peptides with a catechol‐containing sequence and a cell‐binding sequence at each end were initially obtained. The mussel‐inspired peptides were then reversibly bound to a phenylboronic acid (PBA) containing polymer‐grafted substrate through sugar‐responsive catechol‐boronate interactions. The resultant biointerface is thus capable of dynamic presentation of the bioactivity (i.e. the cell‐binding sequence) by virtue of changing sugar concentrations in the system (similar to human glycemic volatility). In addition, the sugar‐responsive biointerface enables not only dynamic modulation of stem cell adhesion behaviors but also selective isolation of tumor cells. Considering the highly biomimetic nature and biological stimuli‐responsiveness, this mussel‐inspired dynamic biointerface holds great promise in both fundamental cell biology research and advanced medical applications.     

Total Synthesis,Stereochemical Reassignment,and Biological Evaluation of (−)‐Lyngbyaloside B

(−)‐Lyngbyaloside B is a 14‐membered macrolide glycoside isolated from the marine cyanobacterium Lyngbya sp. as a cytotoxic substance by Moore and co‐workers. The first total synthesis of (−)‐lyngbyaloside B and the reassignment of its stereostructure is described. The synthesis features an Abiko–Masamune aldol reaction, a vinylogous Mukaiyama aldol reaction, and a macrocyclization involving an acyl ketene intermediate for the construction of the macrocyclic backbone, which contains an acylated tertiary alcohol. The antiproliferative activity of selected compounds against a small panel of human cancer cell lines is also reported.     

Disulfide‐Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA

Development of intracellular delivery methods for antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor‐ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10 minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide‐modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of antisense DNA and siRNA.     

The basic research on physiological property of functionalized hyaluronan—I. Effect of hyaluronan and sulfated hyaluronan on cell proliferation of human epidermal keratinocytes

Hyaluronan (HA) is one of the polysaccharides that is found widely in connective tissue of mammals, and it has no sulfate group and high molecular weight in comparison with other glycosaminoglycans. Glycosaminoglycans are deeply concerned with the manifestation of biofunctions not only by their physical properties but also by physiological ones. In this study, sulfated HA (S‐HA) with various degrees of sulfate substitution and high molecular weight will be synthesized in order to give HA new biological functions. Moreover, the effect of HA and S‐HA on cell proliferation of human epidermal keratinocytes in vitro will be discussed. HA did not affect lag phase, but growth rate (metabolic turnover) of the cell in a logarithmic growth phase which was controlled by the molecular weight of HA. S‐HA stimulated the cell proliferation in the low concentration region under 1 μg/ml. While it inhibited the cell proliferation in the high concentration region over 10 μg/ml. It strongly suppressed the cell proliferation in the logarithmic growth metaphase. These facts were considered to be caused by the change of the cell‐matrix and/or cell–cell interactions. Copyright © 2004 John Wiley & Sons, Ltd.     

High‐Throughput Isolation of Cell Protrusions with Single‐Cell Precision for Profiling Subcellular Gene Expression

Invading cancer cells extend cell protrusions, which guide cancer‐cell migration and invasion, eventually leading to metastasis. The formation and activity of cell protrusions involve the localization of molecules and organelles at the cell front; however, it is challenging to precisely isolate these subcellular structures at the single‐cell level for molecular analysis. Here, we describe a newly developed microfluidic platform capable of high‐throughput isolation of cell protrusions at single‐cell precision for profiling subcellular gene expression. Using this microfluidic platform, we demonstrate the efficient generation of uniform cell‐protrusion arrays (more than 5000 cells with protrusions) for a series of cell types. We show precise isolation of cell protrusions with high purity at single‐cell precision for subsequent RNA‐Seq analysis, which was further validated by RT‐qPCR and RNA FISH. Our highly controlled protrusion isolation method opens a new avenue for the study of subcellular functional mechanisms and signaling pathways in metastasis.     

High‐Frequency Mechanostimulation of Cell Adhesion

Cell adhesion is regulated by molecularly defined protein interactions and by mechanical forces, which can activate a dynamic restructuring of adhesion sites. Previous attempts to explore the response of cell adhesion to forces have been limited to applying mechanical stimuli that involve the cytoskeleton. In contrast, we here apply a new, oscillatory type of stimulus through push–pull azobenzenes. Push–pull azobenzenes perform a high‐frequency, molecular oscillation upon irradiation with visible light that has frequently been applied in polymer surface relief grating. We here use these oscillations to address single adhesion receptors. The effect of molecular oscillatory forces on cell adhesion has been analyzed using single‐cell force spectroscopy and gene expression studies. Our experiments demonstrate a reinforcement of cell adhesion as well as upregulated expression levels of adhesion‐associated genes as a result of the nanoscale “tickling” of integrins. This novel type of mechanical stimulus provides a previously unprecedented molecular control of cellular mechanosensing.     

The research on physiological property of functionalized hyaluronan: interaction between sulfated hyaluronan and plasma proteins

Hyaluronan (HA) is a natural polysaccharides which has no sulfated group but high molecular weight in comparison with other glycosaminoglycans (GAGs). Previously it has been cleared up that the cell function of human keratinocytes is affected by S‐HA (HA substituted with sulfated groups). Most biomedical materials contact with blood components, proteins, cells, etc. In this study, the interaction between S‐HA and blood components is discussed, that is, plasma proteins. And the application of S‐HA for new analytical and separation method of some proteins is pointed out. None of the proteins were adsorbed to HA. Fibronectin and fibinogen were adsorbed to S‐HA, but immunoglobulin‐G and insulin were not adsorbed to it as well as heparin. However, albumin could interact only with heparin, and it did not interact with S‐HA. Furthermore S‐HA adsorbed the plasma proteins that did not adsorb to heparin. It is clear that S‐HA showed different interaction with the plasma proteins in comparison with natural sulfated polysaccharides. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthetic Humic Acids Solubilize Otherwise Insoluble Phosphates to Improve Soil Fertility

Artificial humic acids (A‐HA) made from biomass in a hydrothermal process turn otherwise highly insoluble phosphates (e.g. iron phosphate as a model) into highly available phosphorus, which contributes to the fertility of soils and the coupled plant growth. A detailed electron microscopy study revealed etching of the primary iron phosphate crystals by the ‐COOH and phenolic groups of humic acids, but also illustrated the importance of the redox properties of humic matter on the nanoscale. The combined effects result in the formation of then bioavailable phosphate nanoparticles stabilized by humic matter. Typical agricultural chemical tests indicate that the content of total P and directly plant‐available P improved largely. Comparative pot planting experiments before and after treatment of phosphates with A‐HA demonstrate significantly enhanced plant growth, as quantified in higher aboveground and belowground plant biomass.     

Beyond the BET Family: Targeting CBP/p300 with 4‐Acyl Pyrroles

Bromodomain and extra‐terminal domain (BET) inhibitors are widely used both as chemical tools to study the biological role of their targets in living organisms and as candidates for drug development against several cancer variants and human disorders. However, non‐BET bromodomains such as those in p300 and CBP are less studied. XDM‐CBP is a highly potent and selective inhibitor for the bromodomains of CBP and p300 derived from a pan‐selective BET BRD‐binding fragment. Along with X‐ray crystal‐structure analysis and thermodynamic profiling, XDM‐CBP was used in screenings of several cancer cell lines in vitro to study its inhibitory potential on cancer cell proliferation. XDM‐CBP is demonstrated to be a potent and selective CBP/p300 inhibitor that acts on specific cancer cell lines, in particular malignant melanoma, breast cancer, and leukemia.     

Tetrazole‐Based Probes for Integrated Phenotypic Screening,Affinity‐Based Proteome Profiling,and Sensitive Detection of a Cancer Biomarker

Target‐identification phenotypic screening has been a powerful approach in drug discovery; however, it is hindered by difficulties in identifying the underlying cellular targets. To address this challenge, we have combined phenotypic screening of a fully functionalized small‐molecule library with competitive affinity‐based proteome profiling to map and functionally characterize the targets of screening hits. Using this approach, we identified ANXA2, PDIA3/4, FLAD1, and NOS2 as primary cellular targets of two bioactive molecules that inhibit cancer cell proliferation. We further demonstrated that a panel of probes can label and/or image annexin A2 (a cancer biomarker) from different cancer cell lines, thus providing opportunities for potential cancer diagnosis and therapy.     

Remote Regulation of Membrane Channel Activity by Site‐Specific Localization of Lanthanide‐Doped Upconversion Nanocrystals

The spatiotemporal regulation of light‐gated ion channels is a powerful tool to study physiological pathways and develop personalized theranostic modalities. So far, most existing light‐gated channels are limited by their action spectra in the ultraviolet (UV) or visible region. Simple and innovative strategies for the specific attachment of photoswitches on the cell surface without modifying or genetically encoding channel structures, and more importantly, that enable the remote activation of ion‐channel functions within near‐infrared (NIR) spectral window in living systems, remain a challenging concern. Herein, metabolic glycan biosynthesis is used to achieve site‐specific covalent attachment of near‐infrared‐light‐mediated lanthanide‐doped upconversion nanocrystals (UCNs) to the cell surface through copper‐free click cyclization. Upon irradiation with 808 nm light, the converted emission at 480 nm could activate a light‐gated ion channel, channelrhodopsins‐2 (ChR2), and thus remotely control the cation influx. This unique strategy provides valuable insights on the specific regulation membrane‐associated activities in vivo.     

An Epitope‐Imprinted Biointerface with Dynamic Bioactivity for Modulating Cell–Biomaterial Interactions

In this study, an epitope‐imprinting strategy was employed for the dynamic display of bioactive ligands on a material interface. An imprinted surface was initially designed to exhibit specific affinity towards a short peptide (i.e., the epitope). This surface was subsequently used to anchor an epitope‐tagged cell‐adhesive peptide ligand (RGD: Arg‐Gly‐Asp). Owing to reversible epitope‐binding affinity, ligand presentation and thereby cell adhesion could be controlled. As compared to current strategies for the fabrication of dynamic biointerfaces, for example, through reversible covalent or host–guest interactions, such a molecularly tunable dynamic system based on a surface‐imprinting process may unlock new applications in in situ cell biology, diagnostics, and regenerative medicine.     

Polymeric micelleplexes for improved photothermal endosomal escape and delivery of siRNA

Effective endosomal escape is required for practical application of nucleic acid therapeutics. In this study, we prepared siRNA micelleplexes with photothermally triggered endosomal escape and improved gene silencing activity in vitro. The micelleplexes were prepared from cholesterol‐modified and CXCR4‐inhibiting poly(amido amine)s (PAMD‐Ch). Near‐infrared dye IR780 was encapsulated in cationic PMAD‐Ch micelles, which then were used to form IR780 micelleplexes with siRNA. The micelleplexes displayed improved gene silencing efficiency upon laser irradiation, which was attributed to enhanced endosomal escape because of the photothermal effects of the encapsulated IR780. The IR780 micelleplexes retained the CXCR4 antagonism and inhibition of cancer cell invasion of the parent PAMD. Overall, this study validates codelivery of IR780 in siRNA micelleplexes as promising photothermally controlled siRNA delivery approach.