WRN inhibition leads to its chromatin-associated degradation via the PIAS4-RNF4-p97/VCP axis.

Synthetic lethality provides an attractive strategy for developing targeted cancer therapies. For example, cancer cells with high levels of microsatellite instability (MSI-H) are dependent on the Werner (WRN) helicase for survival. However, the mechanisms that regulate WRN spatiotemporal dynamics remain poorly understood. Here, we used single-molecule tracking (SMT) in combination with a WRN inhibitor to examine WRN dynamics within the nuclei of living cancer cells. WRN inhibition traps the helicase on chromatin, requiring p97/VCP for extraction and proteasomal degradation in a MSI-H dependent manner. Using a phenotypic screen, we identify the PIAS4-RNF4 axis as the pathway responsible for WRN degradation. Finally, we show that co-inhibition of WRN and SUMOylation has an additive toxic effect in MSI-H cells and confirm the in vivo activity of WRN inhibition using an MSI-H mouse xenograft model. This work elucidates a regulatory mechanism for WRN that may facilitate identification of new therapeutic modalities, and highlights the use of SMT as a tool for drug discovery and mechanism-of-action studies.

Targeted protein degradation systems to enhance Wnt signaling.

Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.

Ag-thiolate interactions to enable an ultrasensitive and stretchable MXene strain sensor with high temporospatial resolution.

High-sensitivity strain sensing elements with a wide strain range, fast response, high stability, and small sensing areas are desirable for constructing strain sensor arrays with high temporospatial resolution. However, current strain sensors rely on crack-based conductive materials having an inherent tradeoff between their sensing area and performance. Here, we present a molecular-level crack modulation strategy in which we use layer-by-layer assembly to introduce strong, dynamic, and reversible coordination bonds in an MXene and silver nanowire-matrixed conductive film. We use this approach to fabricate a crack-based stretchable strain sensor with a very small sensing area (0.25 mm2). It also exhibits an ultrawide working strain range (0.001-37%), high sensitivity (gauge factor ~500 at 0.001% and >150,000 at 35%), fast response time, low hysteresis, and excellent long-term stability. Based on this high-performance sensing element and facile assembly process, a stretchable strain sensor array with a device density of 100 sensors per cm2 is realized. We demonstrate the practical use of the high-density strain sensor array as a multichannel pulse sensing system for monitoring pulses in terms of their spatiotemporal resolution.

Nanocellulose based ultra-elastic and durable foams for smart packaging applications.

Foams with advanced sensing properties and excellent mechanical properties are promising candidates for smart packaging materials. However, the fabrication of ultra-elastic and durable foams is still challenging. Herein, we report a universal strategy to obtain ultra-elastic and durable foams by crosslinking cellulose nanofiber and MXene via strong covalent bonds and assembling the composites into anisotropic cellular structures. The obtained composite foam shows an excellent compressive strain of up to 90 % with height retention of 97.1 % and retains around 90.3 % of its original height even after 100,000 compressive cycles at 80 % strain. Their cushioning properties were systematically investigated, which are superior to that of wildly-used petroleum-based expanded polyethylene and expanded polystyrene. By employing the foam in a piezoelectric sensor, a smart cushioning packaging and pressure monitoring system is constructed to protect inner precision cargo and detect endured pressure during transportation for the first time.

Assembled one-dimensional nanowires for flexible electronic devices via printing and coating: Techniques, applications, and perspectives.

The rapid progress in flexible electronic devices has necessitated continual research into nanomaterials, structural design, and fabrication processes. One-dimensional nanowires, characterized by their distinct structures and exceptional properties, are considered essential components for various flexible electronic devices. Considerable attention has been directed toward the assembly of nanowires, which presents significant advantages. Printing and coating techniques can be used to assemble nanowires in a relatively simple, efficient, and cost-competitive manner and exhibit potential for scale-up production in the foreseeable future. This review aims to provide an overview of nanowire assembly using printing and coating techniques, such as bar coating, spray coating, dip coating, blade coating, 3D printing, and so forth. The application of assembled nanowires in flexible electronic devices is subsequently discussed. Finally, further discussion is presented on the potential and challenges of flexible electronic devices based on assembled nanowires via printing and coating.

Tailoring Silver Nanowire Nanocomposite Interfaces to Achieve Superior Stretchability, Durability, and Stability in Transparent Conductors.

Silver nanowires (AgNWs) have been considered as a promising candidate for transparent stretchable conductors (TSCs). However, the strong interface mismatch of stiff AgNWs and elastic substrates leads to the stress concentration at their interface and ultimately the low stretchability and poor durability of TSCs. Here, to address the interfacial mismatch of AgNWs-based TSCs we put forward a universal interface tailoring strategy that introduces the mercapto compound as the intermediate cross-linked layer. The mercapto compound strongly interacts with the AgNWs, forming a dense protective layer on their surface to improve their corrosion resistance, and reacts with the polymer substrate, forming a buffer layer to release the concentrated stress. As a result, the optimized TSCs showed superior stretchability (160%), exceptional durability (230 000 cycles), competent optoelectrical performance (18.0 ohm·sq-1 with a transmittance of 86.5%), and prominent stability. This work provides clear guidance and a strong impetus for the development of transparent stretchable electronics.

Chitosan and procyanidin composite films with high antioxidant activity and pH responsivity for cheese packaging.

Chitosan-procyanidin composite films (CS-PC films) with different mass ratios were prepared by solution casting method. Their structural, thermal, physical, and antioxidant properties, antibacterial activity and pH responsivity were determined. Compared with CS-control film, CS-PC films exhibited lower solubility and higher tensile strength. The antimicrobial properties against Escherichia coli and Aspergillus niger were improved by 20.0% and 30.4%, respectively. CS-PC films indicated good antioxidant activity through their DPPH and ABTS+ scavenging rates, which were 2.45 times higher than CS-control film. pH responsivity was represented by the outstanding changes in color, which were visible to the naked eye. Food packaging film with high antioxidant activity, bacteriostatic properties and pH responsivity was prepared by CS and PC. Compared with the initial properties of cheese, the characteristics of cheese packaged with CS-PC films were obviously better than those of the control groups.

Tissue-targeted R-spondin mimetics for liver regeneration.

R-spondin (RSPO) proteins amplify Wnt signaling and stimulate regeneration in a variety of tissues. To repair tissue in a tissue-specific manner, tissue-targeted RSPO mimetic molecules are desired. Here, we mutated RSPO (RSPO2 F105R/F109A) to eliminate LGR binding while preserving ZNRF3/RNF43 binding and targeted the mutated RSPO to a liver specific receptor, ASGR1. The resulting bi-specific molecule (αASGR1-RSPO2-RA) enhanced Wnt signaling effectively in vitro, and its activity was limited to ASGR1 expressing cells. Systemic administration of αASGR1-RSPO2-RA in mice specifically upregulated Wnt target genes and stimulated cell proliferation in liver but not intestine (which is more responsive to non-targeted RSPO2) in healthy mice, and improved liver function in diseased mice. These results not only suggest that a tissue-specific RSPO mimetic protein can stimulate regeneration in a cell-specific manner, but also provide a blueprint of how a tissue-specific molecule might be constructed for applications in a broader context.

Assessment of five satellite-derived LAI datasets for GPP estimations through ecosystem models.

Ecosystem models have been widely used for obtaining gross primary productivity (GPP) estimations at multiple scales. Leaf area index (LAI) is a critical variable in these models for describing the vegetation canopy structure and predicting vegetation-atmosphere interactions. However, the uncertainties in LAI datasets and the effects of their representation on simulated GPP remain unclear, especially over complex terrain. Here, five most popular datasets, namely the Long-term Global Mapping (GLOBMAP) LAI, Global LAnd Surface Satellite (GLASS) LAI, Geoland2 version 1 (GEOV1) LAI, Global Inventory Monitoring and Modeling System (GIMMS) LAI, and Moderate Resolution Imaging Spectroradiometer (MODIS) LAI, were selected to examine the influences of LAI representation on GPP estimations at 95 eddy covariance (EC) sites. The GPP estimations from the Boreal Ecosystem Productivity Simulator (BEPS) model and the Eddy Covariance Light Use Efficiency (EC-LUE) model were evaluated against EC GPP to assess the performances of LAI datasets. Results showed that MODIS LAI had stronger linear correlations with GLASS and GEOV1 than GIMMS and GLOMAP at the study sites. The GPP estimations from GLASS LAI had a better agreement with EC GPP than those from other four LAI datasets at forest sites, while the GPP estimations from GEOVI LAI matched best with EC GPP at grass sites. Additionally, the GPP estimations from GLASS and GEOVI LAI presented better performances than the other three LAI datasets at crop sites. Besides, the results also showed that complex terrain had larger discrepancies of LAI and GPP estimations, and flat terrain presented better performances of LAI datasets in GPP estimations. Moreover, the simulated GPP from BEPS was more sensitive to LAI than those from EC - LUE, suggesting that LAI datasets can also lead to different uncertainties in GPP estimations from different model structures. Our study highlights that the satellite-derived LAI datasets can cause uncertainties in GPP estimations through ecosystem models.

Measuring dynamics of eukaryotic transcription initiation: Challenges, insights and opportunities.

Transcription of protein-encoding genes in eukaryotic cells is a dynamically coordinated process. Many of the key transcription regulators contain functionally essential intrinsically disordered regions (IDRs), the dynamic nature of which creates extra challenges to traditional biochemical analyses. Recent advances in single-molecule fluorescence imaging technology have enabled direct visualization of these rapid, complex and dynamic molecular interactions in real time.

Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB-promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II-TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

Real-time quantification of single RNA translation dynamics in living cells.

Although messenger RNA (mRNA) translation is a fundamental biological process, it has never been imaged in real time in vivo with single-molecule precision. To achieve this, we developed nascent chain tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify protein synthesis dynamics at the single-mRNA level. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds. Polysomes contain ~1 ribosome every 200 to 900 nucleotides and are globular rather than elongated in shape. By developing multicolor probes, we showed that most polysomes act independently; however, a small fraction (~5%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics.

Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation.

Intrinsically disordered proteins/regions (IDPs/IDRs) are proteins or peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. In this study, we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.

[Clinical comparative study on the treatment characteristics of secretory otitis media between cleft and non-cleft palate patients].

OBJECTIVE: To discuss the treatment characteristics of secretory otitis media (SOM) in cleft palate children. METHODS: A total of 319 patients (524 ears) with SOM and cleft palate (3-14 years old) who accepted treatment were divided into experiment group A, group B, and group C according to effusion characteristics in the middle ear and tympanic pressure. Group A included 112 patients with serous effusion (198 ears). Group B included 162 patients with mucinous effusion (248 ears). Group C included 45 patients (78 ears) with negative pressure in the middle ear without effusion and an acoustic immittance. A total of 208 patients (246 ears) with SOM and tonsil and adenoid hypertrophy were divided into control group Al, group B1, and group Cl matched with the same effusion characteristics in the middle ear and tympanic pressure. Group A and Al accepted puncture in the tympanic cavity, group B and B1 accepted tympanostomy tubes, and group C and Cl accepted puncture in the tympanic cavity after palatoplasty, adenoidectomy, and tonsillectomy. All groups were treated with antibiotics and ear drops. Cure rate and recurrence rate between the experiment group and the control group were compared. RESULTS: The control group had a better cure rate [93.09% (229/246)] than the experiment group [77.29% (405/524)] 12 months after treatment. The experiment group had a higher recurrence rate [14.57% (59/405)] than the control group [3.93% (9/229)]. Statistical differences were observed between the two groups (P<0.05). SOM with cleft palate initially had a low cure rate, and thus it was treated repeatedly for many times. CONCLUSION: SOM with cleft palate is different from normal otitis media in terms of clinical manifestation, treatment, outcome, and prognosis. This case should be considered a special otitis media to be treated with special examination and therapy to obtain better results. Repeated puncture in the tympanic cavity and tympanostomy tubes for six months according to effusion characteristics are better treatment options for patients with SOM and cleft palate.

A general method to improve fluorophores for live-cell and single-molecule microscopy.

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.

Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.

Single-molecule tracking of the transcription cycle by sub-second RNA detection.

Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (∼10e7 M(-1)s(-1)), and allows deterministic detection of single nascent transcripts. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. DOI: http://dx.doi.org/10.7554/eLife.01775.001.

Dual functions of TAF7L in adipocyte differentiation.

The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood. Here we report that TAF7L, a paralogue of TFIID subunit TAF7, is enriched in adipocytes and white fat tissue (WAT) in mouse. Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well. Ectopic expression of TAF7L in myoblasts reprograms these muscle precursors into adipocytes upon induction. Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARγ at enhancers and TBP/Pol II at core promoters. In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARγ. These findings suggest that TAF7L plays an integral role in adipocyte gene expression by targeting enhancers as a cofactor for PPARγ and promoters as a component of the core transcriptional machinery.DOI:http://dx.doi.org/10.7554/eLife.00170.001.

Transcription initiation by human RNA polymerase II visualized at single-molecule resolution.

Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at single-molecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.