Single-Molecule Monitoring of Membrane Association of the Necroptosis Executioner MLKL with Discernible Anchoring and Insertion Dynamics.

The dynamics of membrane proteins that are well-folded in water and become functional after self-insertion into cell membranes is not well understood. Herein we report on single-molecule monitoring of membrane association dynamics of the necroptosis executioner MLKL. We observed that, upon landing, the N-terminal region (NTR) of MLKL anchors onto the surface with an oblique angle and then is immersed in the membrane. The anchoring end does not insert into the membrane, but the opposite end does. The protein is not static, switching slowly between water-exposed and membrane-embedded conformations. The results suggest a mechanism for the activation and function of MLKL in which exposure of H4 is critical for MLKL to adsorb on the membrane, and the brace helix H6 regulates MLKL rather than inhibits it. Our findings provide deeper insights into membrane association and function regulation of MLKL and would have impacts on biotechnological applications.

Mismatch sensing by nucleofilament deciphers mechanism of RecA-mediated homologous recombination.

Recombinases polymerize along single-stranded DNA (ssDNA) at the end of a broken DNA to form a helical nucleofilament with a periodicity of ∼18 bases. The filament catalyzes the search and checking for homologous sequences and promotes strand exchange with a donor duplex during homologous recombination (HR), the mechanism of which has remained mysterious since its discovery. Here, by inserting mismatched segments into donor duplexes and using single-molecule techniques to catch transient intermediates in HR, we found that, even though 3 base pairs (bp) is still the basic unit, both the homology checking and the strand exchange may proceed in multiple steps at a time, resulting in ∼9-bp large steps on average. More interestingly, the strand exchange is blocked remotely by the mismatched segment, terminating at positions ∼9 bp before the match-mismatch joint. The homology checking and the strand exchange are thus separated in space, with the strand exchange lagging behind. Our data suggest that the strand exchange progresses like a traveling wave in which the donor DNA is incorporated successively into the ssDNA-RecA filament to check homology in ∼9-bp steps in the frontier, followed by a hypothetical transitional segment and then the post-strand-exchanged duplex.

Subnanometer-Precision Measurements of Transmembrane Motions of Biomolecules in Plasma Membranes Using Quenchers in Extracellular Environment.

Characterization of biomolecular dynamics at cellular membranes lags far behind that in solutions because of challenges to measure transmembrane trafficking with subnanometer precision. Herein, by introducing nonfluorescent quenchers into extracellular environment of live cells, we adopted Förster resonance energy transfer from one donor to multiple quenchers to measure positional changes of biomolecules in plasma membranes. We demonstrated the method by monitoring flip-flops of individual lipids and by capturing transient states of the host defense peptide LL-37 in plasma membranes. The method was also applied to investigate the interaction of the necroptosis-associated protein MLKL with plasma membranes, showing a few distinct depths of MLKL insertion. Our method is especially powerful to quantitate the dynamics of proteins at the cytosolic leaflets of plasma membranes which are usually not accessible by conventional techniques. The method will find wide applications in the systematic analysis of fundamental cellular processes at plasma membranes.

Detection of tBid Oligomerization and Membrane Permeabilization by Graphene-Based Single-Molecule Surface-Induced Fluorescence Attenuation.

The permeabilization of organelle membranes by BCL-2 family proteins is a pivotal step during the regulation of apoptosis; the underlying mechanisms remain unclear. Based on the fluorescence attenuation by graphene oxide, we developed a single-molecule imaging method termed surface-induced fluorescence attenuation (smSIFA), which enabled us to track both vertical and lateral kinetics of singly labeled BCL-2 family protein tBid during membrane permeabilization. We found that tBid monomers lie shallowly on the lipid bilayer, where they self-assemble to form oligomers. During the initiation phase of self-assembly, the two central hydrophobic helices (α6 and α7) of tBid insert halfway into the phospholipid core, while the other helices remain on the surface. In oligomerized tBid clusters, α6 and α7 prefer to float up, and the other helices may sink to the bottom of the membrane and cause the formation of transient two-dimensional, micelle-like pore structures, which are responsible for the permeabilization of membranes and the induction of apoptosis. Our results shed light on the understanding of tBid-induced apoptosis, and this nanotechnology-based smSIFA approach could be used to dissect the kinetic interaction between membrane protein and lipid bilayer at the single-molecule level with subnanometer precision.

Watching Three-Dimensional Movements of Single Membrane Proteins in Lipid Bilayers.

It is challenging to assess protein-membrane interactions because of the lack of appropriate tools to detect position changes of single proteins in the ∼4 nm range of biological membranes. We developed an assay recently, termed surface-induced fluorescence attenuation (SIFA). It is able to track both vertical and lateral dynamic motion of singly labeled membrane proteins in supported lipid bilayers. Similar to the FRET (fluorescence resonance energy transfer) principle, SIFA takes advantage of the energy transfer from a fluorophore to a light-absorbing surface to determine the distance at 2-8 nm away from the surface. By labeling a protein with a proper fluorophore and using graphene oxide as a two-dimensional quencher, we showed that SIFA is capable of monitoring three-dimensional movements of the fluorophore-labeled protein not only inside but also above the lipid bilayer atop the graphene oxide. Our data show that SIFA is a well-suited method to study the interplay between proteins and membranes.

Free-Standing Single-Molecule Thick Crystals Consisting of Linear Long-Chain Polymers.

Organic two-dimensional (2D) crystals are fundamentally important for development of future devices. Despite that more than a half of man-made products contain polymers, 2D crystals consisting of long linear chains have yet to be explored. Here we report on the fabrication of 2D polyaniline (PANI) crystals via rational electrochemical polymerization followed by liquid-phase exfoliation. The 2D PANI is molecularly thin (∼0.8 nm) and composed of PANI chains with a number-average molecular weight of ∼31 000. The chains are parallel to each other with the benzene rings standing almost vertically to the surface, implying a face-to-face arrangement of the neighboring chains held together by abundant π-π interactions augmented with hydrogen bonds. The 2D PANI can be readily transferred to various solid surfaces and exhibit interesting electrical and optical properties, suggesting that they would be potentially useful in photoelectronic devices and other applications.

Curcumin suppresses soluble tau dimers and corrects molecular chaperone, synaptic, and behavioral deficits in aged human tau transgenic mice.

The mechanisms underlying Tau-related synaptic and cognitive deficits and the interrelationships between Tau species, their clearance pathways, and synaptic impairments remain poorly understood. To gain insight into these mechanisms, we examined these interrelationships in aged non-mutant genomic human Tau mice, with established Tau pathology and neuron loss. We also examined how these interrelationships changed with an intervention by feeding mice either a control diet or one containing the brain permeable beta-amyloid and Tau aggregate binding molecule curcumin. Transgene-dependent elevations in soluble and insoluble phospho-Tau monomer and soluble Tau dimers accompanied deficits in behavior, hippocampal excitatory synaptic markers, and molecular chaperones (heat shock proteins (HSPs)) involved in Tau degradation and microtubule stability. In human Tau mice but not control mice, HSP70, HSP70/HSP72, and HSP90 were reduced in membrane-enriched fractions but not in cytosolic fractions. The synaptic proteins PSD95 and NR2B were reduced in dendritic fields and redistributed into perikarya, corresponding to changes observed by immunoblot. Curcumin selectively suppressed levels of soluble Tau dimers, but not of insoluble and monomeric phospho-Tau, while correcting behavioral, synaptic, and HSP deficits. Treatment increased PSD95 co-immunoprecipitating with NR2B and, independent of transgene, increased HSPs implicated in Tau clearance. It elevated HSP90 and HSC70 without increasing HSP mRNAs; that is, without induction of the heat shock response. Instead curcumin differentially impacted HSP90 client kinases, reducing Fyn without reducing Akt. In summary, curcumin reduced soluble Tau and elevated HSPs involved in Tau clearance, showing that even after tangles have formed, Tau-dependent behavioral and synaptic deficits can be corrected.

Single-molecule reconstruction of eukaryotic factor-dependent transcription termination.

Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.

Dissecting the mechanism of atlastin-mediated homotypic membrane fusion at the single-molecule level.

Homotypic membrane fusion of the endoplasmic reticulum (ER) is mediated by dynamin-like GTPase atlastin (ATL). This fundamental process relies on GTP-dependent domain rearrangements in the N-terminal region of ATL (ATLcyto), including the GTPase domain and three-helix bundle (3HB). However, its conformational dynamics during the GTPase cycle remain elusive. Here, we combine single-molecule FRET imaging and molecular dynamics simulations to address this conundrum. Different from the prevailing model, ATLcyto can form a loose crossover dimer upon GTP binding, which is tightened by GTP hydrolysis for membrane fusion. Furthermore, the α-helical motif between the 3HB and transmembrane domain, which is embedded in the surface of the lipid bilayer and self-associates in the crossover dimer, is required for ATL function. To recycle the proteins, Pi release, which disassembles the dimer, activates frequent relative movements between the GTPase domain and 3HB, and subsequent GDP dissociation alters the conformational preference of the ATLcyto monomer for entering the next reaction cycle. Finally, we found that two disease-causing mutations affect human ATL1 activity by destabilizing GTP binding-induced loose crossover dimer formation and the membrane-embedded helix, respectively. These results provide insights into ATL-mediated homotypic membrane fusion and the pathological mechanisms of related disease.

Interface engineering of semiconductor/dielectric heterojunctions toward functional organic thin-film transistors.

Interface modification is an effective and promising route for developing functional organic field-effect transistors (OFETs). In this context, however, researchers have not created a reliable method of functionalizing the interfaces existing in OFETs, although this has been crucial for the technological development of high-performance CMOS circuits. Here, we demonstrate a novel approach that enables us to reversibly photocontrol the carrier density at the interface by using photochromic spiropyran (SP) self-assembled monolayers (SAMs) sandwiched between active semiconductors and gate insulators. Reversible changes in dipole moment of SPs in SAMs triggered by lights with different wavelengths produce two distinct built-in electric fields on the OFET that can modulate the channel conductance and consequently threshold voltage values, thus leading to a low-cost noninvasive memory device. This concept of interface functionalization offers attractive new prospects for the development of organic electronic devices with tailored electronic and other properties.

Nanomolar LL-37 induces permeability of a biomimetic mitochondrial membrane.

LL-37, the only human host cathelicidin peptide, is proposed to be able to induce host cell apoptosis through mitochondrial membrane permeabilization (MMP). Detailed pathways of the LL-37-triggered MMP are however still disputed. It is generally believed that cationic peptides permeate a membrane mostly in conditions of micromolar peptide concentrations and negatively charged membranes, which are not usually satisfied in the mitochondrial circumstance. Herein, using a variety of single-molecule techniques, we show that nanomolar LL-37 specifically induces permeability of a phosphoethanolamine (PE)-rich biomimetic mitochondrial membrane in a protein-independent manner. The insertion dynamics of single LL-37 molecules exhibit different metastable states in bilayers composed of different lipids. Moreover, the PE lipids significantly facilitate adsorption and accumulation of LL-37 on the PE-rich bilayer, and produce deeper insertion of peptide oligomers, especially tetramers, into the bilayer. This work offers an alternative pathway of the LL-37-triggered MMP and apoptosis.

Correction: Nanomolar LL-37 induces permeability of a biomimetic mitochondrial membrane.

Correction for 'Nanomolar LL-37 induces permeability of a biomimetic mitochondrial membrane' by Xin Jiang et al., Nanoscale, 2022, https://doi.org/10.1039/d2nr05409d.

Exploring the environmental properties and resource utilization of construction waste in Beijing-Tianjin-Hebei region.

Beijing-Tianjin-Hebei region is a capital economic circle for the future. Promoting the coordinated development of its population, economy, resources and environment is a major national strategy. And as towns and cities continue to expand, the volume of construction waste is gradually expanding, posing a major challenge to the sustainable development of the construction industry. In order to solve this problem, this paper used portable X-ray fluorescence spectrometry to realize the on-site rapid monitoring of heavy metals in construction waste, and the correlation analysis result was R2 = 0.9908. The visualization of enrichment factor evaluation results was realized through ArcGIS. The Beijing-Tianjin-Hebei region is mainly polluted by heavy metal elements Cr, Zn, Pb and Hg, showing regional pollution characteristics, and the results of mercury morphology analysis show that all are inorganic mercury pollution, and methylmercury is not detected, and the cause can be traced to heavy industrial production in Tangshan City, which is consistent with industrial ecology. The results of leaching toxicity and cation anion analysis showed that the construction waste in Beijing-Tianjin-Hebei region had environmental risks to the surrounding surface water and groundwater. The resource treatment and disposal path were determined by means of XRD, ternary phase diagram and oxide composition analysis to avoid secondary pollution. This study explores the environmental properties and resource utilization pathways of construction waste in the Beijing-Tianjin-Hebei region, laying the foundation for research work on construction waste in the development of national urban agglomerations, effectively solving regional environmental pollution problems and promoting the sustainable development of the construction industry.

Electrical bistability in self-assembled hybrid multilayers of phospholipid and nanoparticles.

A novel kind of biomolecule-based electrical bistable device composed of phospholipid-CdTe nanoparticle multilayered films was demonstrated. The composite film was fabricated by a facile solution-cast method. X-ray reflectivity and transmission electron microscopy measurements showed the homogeneous distribution of nanoparticles within the lamellar lipid matrix with long-range ordering. Current-voltage scans on the Al/(lipid-nanoparticle composite film)/ITO/glass structures at room temperature exhibited an obvious current bistable phenomenon. Further investigation of such bionanoparticle composite film promises to show its importance for applications in future memory nanodevices with tailored performance.

Real-time imaging of structure and dynamics of transmembrane biomolecules by FRET-induced single-molecule fluorescence attenuation.

Tracking the transmembrane topology and conformational dynamics of membrane proteins is key to understand their functions. It is however challenging to monitor position changes of individual proteins in cell membranes with high sensitivity and high resolution. We review on three single-molecule fluorescence imaging methods - SIFA, LipoFRET and QueenFRET - recently developed in our lab for studying the dynamics of membrane proteins. They can be applied, progressively, to investigate membrane proteins in solid-supported lipid bilayers, artificial liposome membranes and live-cell plasma membranes. The techniques take advantage of the energy transfer from a fluorophore to a cloud of quenchers and are able to extract in real time positions and position changes of a single fluorophore-labeled protein in the direction normal to the membrane surface. The methods have sub-nanometer precision and have proved powerful to investigate biomolecules interacting with bio-membranes.

A unique red-emitting molecular rotor for high-fidelity visualizing and long-term tracking mitochondria.

Visualizing and tracking mitochondrial changes is the key to understand the processes of diseases related to mitochondria, which is meaningful to physiology, pathology, and pharmacology. So, a great deal of mitochondrial probes was designed and synthesized according to the principle that probes with a positive charge can target mitochondria through mitochondrial membrane potential (MMP). However, these traditional mitochondrial probes are not able to visualize and track mitochondrial changes, because their targeting abilities depend on high MMP. Once MMP decreases, they will leak from mitochondria. Herein, we designed and synthesized a red-emitting molecule rotor (SQ, sensitive to viscosity) that could visualize mitochondria with high-fidelity. The rotor was able to firmly immobilize in mitochondrial inner membrane through the cooperation of MMP and the high viscosity property of mitochondrial membrane, and it could still stain mitochondria with long-term regardless of MMP changes. Hence, the probe is able to real-time image and distinguish four kinds of mitochondria with high-fidelity in muscle tissues. In addition, SQ can monitor mitochondrial autophagy in real time. These results demonstrate that SQ is a powerful tool for high-fidelity visualizing and long-term tracking mitochondria in vitro and in vivo.

Structure and phase transformation of oligodeoxynucleotide/lipid lipoplexes on solid supports.

Lipoplexes are composed of lipids and nucleic acids and have an ordered multilamellar structure with a periodic 1D array of parallel nucleic acid strands in the lipid bilayers. We report a low-angle X-ray diffraction study on solid-supported lipoplex films composed of synthetic single-stranded oligodeoxynucleotides (ssODN) and lipids. The ssODN molecules distribute sparsely in the headgroup regions when the weight concentration of the ssODN in the lipoplex is low. The lipoplex separates into two phases, an ODN-poor phase and an ODN-rich phase, when the weight concentration of the ssODN is increased beyond a level at which the ssODN molecules contact each other so that some regions of the lipid bilayers must accommodate two layers of the ssODN. The phase separation is a result of the fusion of such regions to minimize the total interfacial energy of the system. The ssODN molecules distort the lipid bilayers in the ODN-poor phase. The local area per lipid molecule is increased by the distortions so that the interbilayer distance of the lipoplex film is smaller than that of the lipid film without the ssODN. The ODN-rich phase has a much larger interbilayer distance because two layers of ssODN are intercalated into each lipid bilayer. The ssODN molecules are tightly compacted, and the lipid bilayers are not distorted in the ODN-rich phase.

Effects of Cortex Phellodendri extract on post-weaning piglets diarrhoea.

The diarrhoea incidence rate is often high among weaning piglets. In light of the fact that Cortex phellodendri has long been used to treat diarrhoea in China, this study aimed to evaluate the effects of Cortex Phellodendri Extract (CPE) on diarrhoea in weaning piglets and the mechanism behind such effects. In the first trial, 36 diarrhoeal weaning piglets were randomly divided into three groups. The control group was injected with 20 mg oxytetracycline/kg BW, while the two treatment groups were orally administered with 10 mg and 20 mg CPE/kg BW respectively. In the second trial, 96 weaning piglets were randomly divided into two groups. The control group was fed basal diet, while 300 mg CPE/kg BW was added to the diet of the treatment group. The pathogenic bacteria were then isolated and identified from the diarrhoeal faecal samples. Cell adhesion and RT-PCR tests were used to investigate the effect of CPE on the adhesion of pathogenic bacteria to IPEC-J2 cells. 16S rDNA-based high-throughput sequencing was used to analyse faecal microflora. The results showed that CPE reduced the diarrhoea incidence rate (p < 0.05) and diarrhoea index (p < 0.05) compared to control group, and increased the richness and evenness of weaning piglets' gut microbiota. Escherichia coli (E. coil) was identified as the causative organism. Cell adhesion and RT-PCR tests suggested that CPE reduced the adhesion of E. coli to IPEC-J2 cells (p < 0.05) and the expression of fae and faeG gene (p < 0.05) responsible for encoding E. coli fimbriae protein.

GSK3 inhibitors show benefits in an Alzheimer's disease (AD) model of neurodegeneration but adverse effects in control animals.

The dysregulation of glycogen synthase kinase-3 (GSK3) has been implicated in Alzheimer disease (AD) pathogenesis and in Abeta-induced neurotoxicity, leading us to investigate it as a therapeutic target in an intracerebroventricular Abeta infusion model. Infusion of a specific GSK3 inhibitor SB216763 (SB) reduced a downstream target, phospho-glycogen synthase 39%, and increased glycogen levels 44%, suggesting effective inhibition of enzyme activity. Compared to vehicle, Abeta increased GSK3 activity, and was associated with elevations in levels of ptau, caspase-3, the tau kinase phospho-c-jun N-terminal kinase (pJNK), neuronal DNA fragmentation, and gliosis. Co-infusion of SB corrected all responses to Abeta infusion except the induction of gliosis and behavioral deficits in the Morris water maze. Nevertheless, SB alone was associated with induction of neurodegenerative markers and behavioral deficits. These data support a role for GSK3 hyperactivation in AD pathogenesis, but emphasize the importance of developing inhibitors that do not suppress constitutive activity.

A Novel Model of Mixed Vascular Dementia Incorporating Hypertension in a Rat Model of Alzheimer's Disease.

Alzheimer's disease (AD) and mixed dementia (MxD) comprise the majority of dementia cases in the growing global aging population. MxD describes the coexistence of AD pathology with vascular pathology, including cerebral small vessel disease (SVD). Cardiovascular disease increases risk for AD and MxD, but mechanistic synergisms between the coexisting pathologies affecting dementia risk, progression and the ultimate clinical manifestations remain elusive. To explore the additive or synergistic interactions between AD and chronic hypertension, we developed a rat model of MxD, produced by breeding APPswe/PS1ΔE9 transgenes into the stroke-prone spontaneously hypertensive rat (SHRSP) background, resulting in the SHRSP/FAD model and three control groups (FAD, SHRSP and non-hypertensive WKY rats, n = 8-11, both sexes, 16-18 months of age). After behavioral testing, rats were euthanized, and tissue assessed for vascular, neuroinflammatory and AD pathology. Hypertension was preserved in the SHRSP/FAD cross. Results showed that SHRSP increased FAD-dependent neuroinflammation (microglia and astrocytes) and tau pathology, but plaque pathology changes were subtle, including fewer plaques with compact cores and slightly reduced plaque burden. Evidence for vascular pathology included a change in the distribution of astrocytic end-foot protein aquaporin-4, normally distributed in microvessels, but in SHRSP/FAD rats largely dissociated from vessels, appearing disorganized or redistributed into neuropil. Other evidence of SVD-like pathology included increased collagen IV staining in cerebral vessels and PECAM1 levels. We identified a plasma biomarker in SHRSP/FAD rats that was the only group to show increased Aqp-4 in plasma exosomes. Evidence of neuron damage in SHRSP/FAD rats included increased caspase-cleaved actin, loss of myelin and reduced calbindin staining in neurons. Further, there were mitochondrial deficits specific to SHRSP/FAD, notably the loss of complex II, accompanying FAD-dependent loss of mitochondrial complex I. Cognitive deficits exhibited by FAD rats were not exacerbated by the introduction of the SHRSP phenotype, nor was the hyperactivity phenotype associated with SHRSP altered by the FAD transgene. This novel rat model of MxD, encompassing an amyloidogenic transgene with a hypertensive phenotype, exhibits several features associated with human vascular or "mixed" dementia and may be a useful tool in delineating the pathophysiology of MxD and development of therapeutics.