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Results demonstrate that spontaneous self-assembly of the amyloid Abeta(sub)1-42 oligomers result in the formation of globular, neurotoxic Abeta-derived diffusible ligands, which are stable and contribute to Alzheimer's disease pathogenesis.

Insulin administration mimicking physiological insulin secretion behavior remains a challenge for the treatment of patients with type I diabetes. Herein, we report a novel glucose-responsive insulin-release nanosystem through an enzyme-polymer layer-by-layer coating strategy on silica vesicles loaded with insulin. With a choice of polyethylenimine with prioritized proton binding capability and glucose specific enzymes in the coating layer, the insulin-release threshold can be adjusted in a desired glucose concentration range 5-20 mM for the first time. In vitro tests show that the insulin release is switched "ON" in response to hyperglycemia (10, 20 mM) and "OFF" to normal glucose level (5 mM) repeatedly. Moreover, in vivo experiments in type I diabetes mice demonstrate that our nanosystem enables a fast glucose response insulin release and regulates the glycemia levels in a normal range up to 84 h with a single administration. In addition, when applied to healthy mice, the nanosystem maintains the blood glucose concentration in the normal range without causing hypoglycemia. Our nanosystem which mimics the physiological insulin secretion has the potential to be developed as a convenient and safe insulin delivery carrier for diabetes treatment. [ABSTRACT FROM AUTHOR]

Nucleotidepyrophosphatase/phosphodiesterase 1 (NPP1) belongs tothe family of ecto-nucleotidases, which control extracellular nucleotide,nucleoside, and (di)phosphate levels. To study the (patho)physiologicalroles of NPP1 potent and selective inhibitors with drug-like propertiesare required. Therefore, a compound library was screened for NPP1inhibitors using a colorimetric assay with p-nitrophenyl5′-thymidine monophosphate (p-Nph-5′-TMP)as an artificial substrate. This led to the discovery of 2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (5a) as ahit compound with a Kivalue of 217 nM.Subsequent structure–activity relationship studies led to thedevelopment of purine and imidazo[4,5-b]pyridineanalogues with high inhibitory potency (Kivalues of 5.00 nM and 29.6 nM, respectively) when assayed with p-Nph-5′-TMP as a substrate. Surprisingly, the compoundswere significantly less potent when tested versus ATP as a substrate,with Kivalues in the low micromolar range.A prototypic inhibitor was investigated for its mechanism of inhibitionand found to be competitive versus both substrates. [ABSTRACT FROM AUTHOR]

The ferroelectric photovoltaic effect has been extensively studied for possible applications in energy conversion and photo-electrics. The reversible spontaneous polarization gives rise to a switchable photovoltaic behavior. However, despite its long history, the origin of the ferroelectric photovoltaic effect still lacks a full understanding since multiple mechanisms such as bulk and Schottky-barrier-related interface effects are involved. Herein, we report a comprehensive study on the photovoltaic response of BiFeO 3 -based vertical heterostructures, using multiple strategies to clarify its origin. We found that, under white light illumination, polarization-modulated Schottky barrier at the interface is the dominating mechanism. By varying the top metal contacts, only the photovoltaic effect of the polarization downward state is strongly modulated, suggesting selective interface contribution in different polarization states. A Schottky-barrier-free device shows negligible photovoltaic effect, suggesting the lack of bulk photovoltaic effect in vertical heterostructures under white light illumination.

Blastobotrys adeninivorans plays an essential role in pile-fermenting of Pu-erh tea. Its ability to assimilate various carbon and nitrogen sources makes it available for application in a wide range of industry sectors. The genome of B. adeninivorans TMCC 70007 isolated from pile-fermented Pu-erh tea was sequenced and assembled. Proteomics analysis indicated that 4900 proteins in TMCC 70007 were expressed under various culture conditions. Proteogenomics mapping revealed 48 previously unknown genes and corrected 118 gene models predicted by GeneMark-ES. Ortho-proteogenomics analysis identified 17 previously unidentified genes in B. adeninivorans LS3, the first strain with a sequenced genome among the genus Blastobotrys as well. More importantly, five species specific genes were identified from TMCC 70007, which could serve as a barcode for strain typing and were applicable for fermentation process protection of this industrial species. The datasets generated from tea aqueous extract culture not only increased the proteome coverage and accuracy but also contributed to the identification of proteins related to polyphenols and caffeine, which were considered to change greatly during the microbial fermentation of Pu-erh tea. This study provides a proteome perspective on TMCC 70007, which was considered to be an important strain in the production of Pu-erh tea. The systematic proteogenomics analysis not only made a better annotation on the genome of B. adeninivorans TMCC 70007 as previous proteogenomics study but also provided solution for fermentation process protection on valuable industrial species with species specific genes uniquely identified from proteogenomics study.

As a hepadnavirus, hepatitis B virus (HBV) can cause damage to extrahepatic organs. The kidney is one of the organs that is more susceptible to damage. Research studies on HBV-associated glomerulonephritis (HBV-GN) have been going on for decades. However, the underlying molecular mechanism remains obscure. Here, we applied a tandem mass tag (TMT) isobaric labeling-based method to quantitatively profile the kidney proteome of HBV transgenic mice to illustrate the pathological mechanisms of HBV-GN. Weighted correlation network analysis, a clustering method for gene expression, is used to cluster proteins. Totally, we identified 127 proteins that were highly associated with HBV expression out of a total of 5169 quantified proteins. Among them, the downregulated solute carrier (SLC) family proteins are involved in the process of HBV-GN. We also found that IL1B was upregulated in the kidney tissue of HBV transgenic mice. These findings suggest that HBV disrupts the small molecule transport network of the kidney, which contributes to the occurrence of HBV-GN. The transporter, particularly SLC family 7 member 7 (SLC7A7), is involved in this process, which might serve as an intervention target for HBV-GN. All MS data have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the data set identifier PXD016450.

Polyubiquitination encompasses complex topologies through various linkage types to deliver diverse cellular signals, which has been recognized as a sophisticated ubiquitin code. Accurate comparison of polyubiquitination signals is critical for revealing the dynamic cellular ubiquitination-regulated events. Western blotting (WB) is the most widely used biochemical method to quantify proteins and posttranslational modifications under diverse physiological conditions. The accuracy and sensitivity of the WB mainly depend on the quality and specificity of the antibody. In this study, we found that the antiubiquitin antibodies exhibited different affinities to the eight linkage types of ubiquitin chains, with the highest sensitivity for the K63-linked chain, lower efficiency for M1 and K48, and very low affinity for the other types of chains. Herein, we introduced the tandem hybrid ubiquitin-binding domain (ThUBD)-based far-Western blotting (TUF-WB) to visualize the signal of synthetic ubiquitin chains or ubiquitinated conjugates on a solid membrane by utilizing the unbiased affinity of ThUBD to all types of ubiquitin linkages. As compared to antiubiquitin antibody detection, TUF-WB can accurately quantify the signal intensity to the mass amounts of all eight ubiquitin chains. Meanwhile, the sensitivity of this method in detecting complex ubiquitinated samples was 4-5-fold higher than those of antibodies. Consequently, TUF-WB allows accurate quantification of polyubiquitination signal on the membrane with great sensitivity and wider dynamic range.

Epitaxial strain can cause both lattice distortion and oxygen nonstoichiometry, effects that are strongly coupled at heterojunctions of complex nickelate oxides. Here we decouple these structural and chemical effects on the oxygen evolution reaction (OER) by using a set of coherently strained epitaxial NdNiO 3 films. We show that within the regime where oxygen vacancies (V O ) are negligible, compressive strain is favorable for the OER whereas tensile strain is unfavorable; the former induces orbital splitting, resulting in a higher occupancy in the d 3 z 2 - r 2 orbital and weaker Ni-O chemisorption. However, when the tensile strain is sufficiently large to promote V O formation, an increase in the OER is also observed. The partial reduction of Ni 3+ to Ni 2+ due to V O  makes the e g occupancy slightly larger than unity, which is thought to account for the increased OER activity. Our work highlights that epitaxial-strain-induced lattice distortion and V O generation can be individually or collectively exploited to tune OER activity, which is important for the predictive synthesis of high-performance electrocatalysts.

Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

The CRISPR/Cas9 system has made significant contributions to genome editing, gene regulation and chromatin studies in recent years. High-throughput and systematic investigations into the multiplexed biological systems require simultaneous expression and coordinated functioning of multiple sgRNAs. However, current cotransfection based sgRNA coexpression systems remain inefficient, and virus-based transfection approaches are relatively costly and labor intensive. Here we established a vector-independent method allowing multiple sgRNA expression cassettes to be assembled in series into a single plasmid. This synthetic biology-based strategy excels in its efficiency, controllability and scalability. Taking the flexibility advantage of this all-in-one sgRNA expressing system, we further explored its applications in single nonrepetitive genomic locus imaging as well as coordinated gene regulation in live cells. With its full potency, our method will facilitate the research in understanding genome structure, function and dynamics.

Identifying missing proteins (MPs) has been one of the critical missions of the Chromosome-Centric Human Proteome Project (C-HPP). Since 2012, over 30 research teams from 17 countries have been trying to search adequate and accurate evidence of MPs through various biochemical strategies. MPs mainly fall into the following classes: (1) low-molecular-weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), (4) nucleic acid-associated proteins, (5) low abundance, and (6) unexpressed genes. In this study, kidney cancer and adjacent tissues were used for phosphoproteomics research, and 8962 proteins were identified, including 6415 phosphoproteins, and 44 728 phosphosites, of which 10 266 were unreported previously. In total, 75 candidate detections were found, including 45 phoshoproteins. GO analysis for these 75 candidate detections revealed that these proteins mainly clustered as membrane proteins and took part in nephron and kidney development. After rigorous screening and manual check, 9 of them were verified with the synthesized peptides. Finally, only one missing protein was confirmed. All mass spectrometry data from this study have been deposited in the PRIDE with identifier PXD006482.

The recent development of reversibly switchable fluorescent proteins (RSFPs) has promoted reversible saturable optical fluorescence transitions (RESOLFT) nanoscopy as a general scheme for live-cell super-resolution imaging. However, continuous, long-term live-cell RESOLFT nanoscopy is still hindered mainly because of the unsatisfactory properties of existing RSFPs. In this work, we report GMars-Q, a monomeric RSFP with low residual off-state fluorescence and strong fatigue resistance attributed to a biphasic photobleaching process. We further demonstrate that GMars-Q is particularly suitable for long-term parallelized RESOLFT nanoscopy as it supports an order of magnitude longer imaging durations than existing RSFPs. The excellent photophysical properties of GMars-Q also suggest that it would be of general interest for other RESOLFT nanoscopic methods.

A one-dimensional (1D) organic-inorganic hybrid iodoplumbate crystal (1, C4H12NPbI3, TMAPbI3) can undergo two reversible phase transitions as the temperature decreases. Its dynamic phase-transition behaviors were carefully studied by dielectric measurements, thermal analysis, and variable-temperature crystallographic studies. These results indicate that the phase transitions possess a disorder-order feature with a noncentrosymmetrical intermediate phase structure. Due to the existence of the ordered motion and reorientation of the C4H12N(+) cation, 1 undergoes two phase transitions: the first one from space group P63/m at room temperature to Pm at 163 K with symmetry breaking, and the second one from space group Pm at 163 K to P61 at 142 K with partial symmetry restoration. Our results indicate that there is an existence of a transitional structure with a low symmetry space group during the disorder-order-type phase transitions, which can provide us valuable information to deeply understand the disorder-order phase transition in organic-inorganic hybrids.

Oxygen vacancy is intrinsically coupled with magnetic, electronic, and transport properties of transition-metal oxide materials and directly determines their multifunctionality. Here, we demonstrate reversible control of oxygen content by postannealing at temperature lower than 300 °C and realize the reversible metal-insulator transition in epitaxial NdNiO₃ films. Importantly, over 6 orders of magnitude in the resistance modulation and a large change in optical bandgap are demonstrated at room temperature without destroying the parent framework and changing the p-type conductive mechanism. Further study revealed that oxygen vacancies stabilized the insulating phase at room temperature is universal for perovskite nickelate films. Acting as electron donors, oxygen vacancies not only stabilize the insulating phase at room temperature, but also induce a large magnetization of ∼50 emu/cm³ due to the formation of strongly correlated Ni²⁺ t(2g)⁶e(g)² states. The bandgap opening is an order of magnitude larger than that of the thermally driven metal-insulator transition and continuously tunable. Potential application of the newly found insulating phase in photovoltaics has been demonstrated in the nickelate-based heterojunctions. Our discovery opens up new possibilities for strongly correlated perovskite nickelates.

Nanoscale spatiotemporal clustering of RNA polymerase II (Pol II) plays an important role in transcription regulation. However, dynamics of individual Pol II clusters in live-cell nuclei has not been measured directly, prohibiting in-depth understanding of their working mechanisms. In this work, we studied the dynamics of Pol II clustering using Bayesian nanoscopy in live mammalian cell nuclei. With 50 nm spatial resolution and 4 s temporal resolution, Bayesian nanoscopy allows direct observation of the assembly and disassembly dynamics of individual Pol II clusters. The results not only provide quantifications of Pol II clusters but also shed light on the understanding of cluster formation and regulation. Our study suggests that transcription factories form on-demand and recruit Pol II molecules in their pre-elongation phase. The assembly and disassembly of individual Pol II clusters take place asynchronously. Overall, the methods developed herein are also applicable to studying a wide realm of real-time nanometer-scale nuclear processes in live cells.

Cyclin G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Cocrystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV life cycle (i.e., viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer, and Parkinson's disease).

It is extremely important for emerging applications and still enormously challenging to develop multifunctional stimuli-responsive anisotropic polymeric micelles with integration of potentially targeted therapeutic and diagnostic function. Herein, we report a first example of fluorescent anisotropic micelles (FAMs) with Fe(3+), DTT, H2O2, and thermal responsive fluorescence and morphology. FAMs from direct aqueous self-assembly of amphiphilic diblock copolymer showed reversible "switch off/on" of aqua fluorescent emission and controllable structural change by sequential addition of Fe(3+) and DTT. In addition, the FAMs had reversible dual-thermal responsiveness of fluorescence and morphology. This micelle could serve as a promising candidate for all-in-one application of quantitative detecting, imaging, drug delivery, and targeted release.

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ∼1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.

A nanoscale optical biosensor based on localized surface plasmon resonance (LSPR) spectroscopy has been developed to monitor the interaction between the antigen, amyloid-beta derived diffusible ligands (ADDLs), and specific anti-ADDL antibodies. Using the sandwich assay format, this nanosensor provides quantitative binding information for both antigen and second antibody detection that permits the determination of ADDL concentration and offers the unique analysis of the aggregation mechanisms of this putative Alzheimer's disease pathogen at physiologically relevant monomer concentrations. Monitoring the LSPR-induced shifts from both ADDLs and a second polyclonal anti-ADDL antibody as a function of ADDL concentration reveals two ADDL epitopes that have binding constants to the specific anti-ADDL antibodies of 7.3 x 10(12) M(-1) and 9.5 x 10(8) M(-1). The analysis of human brain extract and cerebrospinal fluid samples from control and Alzheimer's disease patients reveals that the LSPR nanosensor provides new information relevant to the understanding and possible diagnosis of Alzheimer's disease.