The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.

Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca2+ influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.

Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic.

Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe 'coincidence-detecting' sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.

The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing.

The currently widely used CRISPR-Cas9 genome editing technology enables the editing of target genes (knock-out or knock-in) with high accuracy and efficiency. Guided by the small guide RNA, the Cas9 nuclease induces a DNA double-strand break at the targeted genomic locus. The DNA double-strand break can be repaired by the homology-directed repair pathway in the presence of a repair template. With the repair template containing the coding sequence of a fluorescent tag, the targeted gene can be inserted with the sequence of a fluorescent tag at the designed position. The genome editing mediated labeling of endogenous proteins with fluorescent tags avoids the potential artifacts caused by gene overexpression and substantially improves the reproductivity of imaging experiments. This protocol focuses on creating mammalian cell lines with endogenous proteins tagged with fluorescent proteins or self-labeling protein tags using CRISPR-Cas9 genome editing.

A Low-Cost, 3D-Printed Biosensor for Rapid Detection of Escherichia coli.

Detection of bacterial pathogens is significant in the fields of food safety, medicine, and public health, just to name a few. If bacterial pathogens are not properly identified and treated promptly, they can lead to morbidity and mortality, also possibly contribute to antimicrobial resistance. Current bacterial detection methodologies rely solely on laboratory-based techniques, which are limited by long turnaround detection times, expensive costs, and risks of inadequate accuracy; also, the work requires trained specialists. Here, we describe a cost-effective and portable 3D-printed electrochemical biosensor that facilitates rapid detection of certain Escherichia coli (E. coli) strains (DH5α, BL21, TOP10, and JM109) within 15 min using 500 µL of sample, and costs only USD 2.50 per test. The sensor displayed an excellent limit of detection (LOD) of 53 cfu, limit of quantification (LOQ) of 270 cfu, and showed cross-reactivity with strains BL21 and JM109 due to shared epitopes. This advantageous diagnostic device is a strong candidate for frequent testing at point of care; it also has application in various fields and industries where pathogen detection is of interest.

Dynamic Passivation in Perovskite Quantum Dots for Specific Ammonia Detection at Room Temperature.

Perovskite structured CsPbX3 (X = Cl, Br, or I) quantum dots (QDs) have attracted considerable interest in the past few years due to their excellent optoelectronic properties. Surface passivation is one of the main pathways to optimize the optoelectrical performance of perovskite QDs, in which the amino group plays an important role for the corresponding interaction between lead and halide. In this work, it is found that ammonia gas could dramatically increase photoluminescence of purified QDs and effectively passivate surface defects of perovskite QDs introduced during purification, which is a reversible process. This phenomenon makes perovskite QDs a kind of ideal candidate for detection of ammonia gas at room temperature. This QD film sensor displays specific recognition behavior toward ammonia gas due to its significant fluorescence enhancement, while depressed luminescence in case of other gases. The sensor, in turn-on mode, shows a wide detection range from 25 to 350 ppm with a limit of detection as low as 8.85 ppm. Meanwhile, a fast response time of ≈10 s is achieved, and the recovery time is ≈30 s. The fully reversible, high sensitivity and selectivity characteristics make CsPbBr3 QDs ideal active materials for room-temperature ammonia sensing.

Synthesis of Highly Active Sub-Nanometer Pt@Rh Core-Shell Nanocatalyst via a Photochemical Route: Porous Titania Nanoplates as a Superior Photoactive Support.

Sub-nanometer Pt@Rh nanoparticles highly dispersed on MIL-125-derived porous TiO2 nanoplates are successfully prepared for the first time by a photochemical route, where the porous TiO2 nanoplates with a relatively high specific surface area play a dual role as both effective photoreductant and catalyst support. The resulting Pt@Rh/p-TiO2 can be utilized as a highly active catalyst.

Self-Assembly of Tunable Heterometallic Ln-Ru Coordination Polymers with Near-Infrared Luminescence and Magnetocaloric Effect.

A series of heterometallic lanthanide (Ln)-Ru coordination polymers, denoted Gd-1, Yb-2, and Nd-3, were prepared by solvothermal reaction of a carboxylate derivative of [Ru(bpy)3 ]2+ (Rubpy, bpy=2,2'-bipyridine), oxalic acid, and Ln(OAc)3 by using the metalloligand strategy. Single-crystal X-ray diffraction indicated that the resulting isostructural heterometallic complexes have 1D butterfly-shaped Ln-Ru-based coordination chains but show slight differences in the coordination environments of the Ln centers. The introduced Ru(bpy) metalloligands could act as good light-harvesting antennas to effectively sensitize near-infrared (NIR) luminescence by energy transfer from the triplet metal-to ligand charge transfer state of Rubpy units to Ln (Yb or Nd) under the excitation in the visible-light region. Additionally, dopant-concentration-dependent behavior of the Ru-based emission and sensitized NIR emission was demonstrated in Gd-1. Finally, the magnetocaloric effect of Gd-1 was studied. The preparation of such heterometallic coordination polymers offers a versatile platform to investigate dimensionally controlled properties.

A Multifunctional Mn(II) Phosphonate for Rapid Separation of Methyl Orange and Electron-Transfer Photochromism.

A Mn(II) phosphonate of the general formula [Mn(H2 L)2 (H2 O)2 (H2 bibp)] adopts a layered motif with protonated H2 bibp(2+) cations embedded in the channels (H4 L=thiophene-2-phosphonic acid; bibp=4,4'-bis(1-imidazolyl)biphenyl). The title compound exhibits excellent adsorptive removal of methyl orange (MO) dye from aqueous solution. Its advantageous features include fast adsorption, high uptake capacity, selective removal, and reusability, which are of great significance for practical application in wastewater treatment. Meanwhile, the compound displays rapid photochromism upon irradiation with visible light at room temperature. Extensive research has demonstrated that such behavior is based on a ligand-to-ligand charge-transfer (LLCT) mechanism. The irradiated sample possesses an ultra-long-lived charge-separated state. Moreover, not only is the compound the first Mn-based photochromic MOF, but it is also one of the very few examples showing LLCT with non-photochromic components.

Photochromic Terbium Phosphonates with Photomodulated Luminescence and Metal Ion Sensitive Detection.

Rational selection and modification of rare earth metal centers and photoactive organic linkers enables designable multiphotofunctionality to come to fruition in new hybrid coordination polymer materials. By using a viologen-functionalized diphosphonate linker, two terbium phosphonate compounds (Tb-1 and Tb-2) have been constructed, which display reversible photochromic reactions in response to UV light and soft X-ray irradiation. In addition, the photo-induced electron-transfer reaction can modulate the luminescent emission to thus realize photoluminescence switching behavior. Furthermore, both terbium phosphonates can serve as highly sensitive sensors to probe Cu2+ in solution through their luminescence. Thus, they represent the first photochromic examples of lanthanide phosphonate-based materials with photomodulated luminescence and sensitive detection of metal ions.

Lanthanide metal-organic frameworks showing luminescence in the visible and near-infrared regions with potential for acetone sensing.

The luminescent properties of a family of lanthanide metal-organic frameworks LnL (Ln=Y, La-Yb, except Pm; L=4,4'-({2-[(4-carboxyphenoxy)methyl]-2-methylpropane-1,3-diyl}bis{oxy})dibenzoic acid) have been explored, and the energy-transfer process in the compounds has been carefully analyzed. The visible-emitting Tb(0.08)Gd(0.92)L and the near-infrared (NIR)-luminescent Yb(0.10)Gd(0.90)L show excellent optical performances and can be considered as fluorescent probes for acetone sensing based on luminescence quenching effects arising from host-guest interactions. Moreover, GdL exhibits a strong second harmonic generation (SHG) signal 6.1 times that of potassium dihydrogen phosphate (KDP) and an outstanding phase-matchable effect. These lanthanide compounds combining fluorescent and nonlinear optical (NLO) properties could meet further requirements as multifunctional materials.

Flexible diphosphonic acids for the isolation of uranyl hybrids with heterometallic U(VI)═O-Zn(II) cation-cation interactions.

A family of uranyl diphosphonates have been hydrothermally synthesized using various flexible diphosphonic acids and Zn(UO2)(OAc)4·7H2O in the presence of bipy or phen. Single-crystal X-ray analyses indicate that these compounds represent the first examples of uranyl phosphonates with heterometallic U(VI)═O-Zn(II) cation-cation interactions.

Syntheses and structures of a series of uranyl phosphonates and sulfonates: an insight into their correlations and discrepancies.

Six uranyl phosphonates and sulfonates have been hydrothermally synthesized, namely, (H2tib)[(UO2)3(PO3C6H5)4]·2H2O (UPhP-1), Zn(pi)2(UO2)(PO3C6H5)2 (UPhP-2), Zn(dib)(UO2)(PO3C6H5)2·2H2O (UPhP-3), (HTEA)[(UO2)(5-SP)] (USP-1), (Hdib)2[(UO2)2(OH)(O)(5-SP)] (USP-2), and Zn(phen)3(UO2)2(3-SP)2 (USP-3) (tib = 1,3,5-tri(1H-imidazol-1-yl)benzene, pi = 1-phenyl-1H-imidazole, dib = 1,4-di(1H-imidazol-1-yl)benzene, TEA = triethylamine, phen = 1,10-phenanthroline, 5-SP = 5-sulfoisophthalic acid, and 3-SP = 3-sulfoisophthalic acid). UPhP-1 has been determined to be a layered structure constructed by UO7 pentagonal bipyramids, UO6 octahedra, and phenylphosphonates. Protonated tib plays a role in balancing the negative charge and holding its structure together. UPhP-2 is made up of UO6 octahedra, ZnO2N2 tetrahedra and PO3C tetrahedra in phenylphosphonates, forming a 1D assembly, which is stabilized by chelate phen ligand. Further connection of such chainlike structure via dib yields a 2D layered architecture of UPhP-3. Although sulfonate group possesses similar tetrahedral structure as the phosphonate group, a unidentated coordination mode is only found in this work. UO7 pentagonal bipyramids are linked by 5-SP to form the layered assembly of USP-1. USP-2 also consists of the same sulfonate ligand, but features tetranulear uranyl clusters. Similarly, protonated TEA and dib molecules enable stabilization of their structures, respectively. Formed by dinuclear uranyl cluster and 3-SP ligand, USP-3 appears as a 1D arrangement, in which Zn(phen)3 acts as the counterion to compensate the negative charge. All of these compounds have been characterized by IR and photoluminescent spectroscopy. Their characteristic emissions have been attributed as transition properties of uranyl cations.

Synthesis, structures, and properties of uranyl hybrids constructed by a variety of mono- and polycarboxylic acids.

A series of uranyl-organic coordination polymers have been hydrothermally synthesized by using a variety of carboxylic ligands, 3,3'-((2-((3-carboxyphenoxy)methyl)-2-methylpropane-1,3-diyl)bis(oxy))dibenzoic acid (H3L(1)), 4,4'-(3-(4-carboxyphenethyl)-3-hydroxypentane-1,5-diyl)dibenzoic acid (H3L(2)), chelidamic acid (H2L(3)), and benzoic acid (HL(4)) in the presence of N-bearing coligands, including 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen), 1-([1,1'-biphenyl]-4-yl)-1H-imidazole (bpi), and 1,4-di(1H-imidazol-1-yl)benzene (dib). Compounds (UO2)(HL(1)) (1) and Zn(H2O)3(UO2)2(O)(OH)(L(2))·H2O (2) are constructed by semirigid ligands. The former is a one-dimensional ribbon-like structure with UO7 pentagonal bipyramids as the building unit, while the latter adopts a tetramer of UO7 pentagonal bipyramids to build a layered structure. Mononuclear UO7 pentagonal bipyramids are connected by L(3) groups to generate a two-dimensional arrangement of UO2(L(3))(H2dib)0.5 (3), in which the protonated dib molecules provide space filling and form π···π interactions with the layers. Compounds UO2(L(3))(phen) (4), UO2(L(3))2(Hbpi)2 (5), and UO2(L(4))2(bipy) (6) are molecular complexes, in which 4 and 6 are neutral, and 5 comprises protonated bpi as the counterion. The uranyl center in compound 4 is chelated by one phen and one L(3) group to form a UO5N2 pentagonal bipyramid, while in compound 5, two L(3) groups are coordinated to an uranyl center, producing a UO8 polyhedron. Compound 6 consists of a UO6N2 polyhedron of uranyl unit coordinated by one bipy and two benzoate groups. Compounds Zn(phen)3[(UO2)(C2O4)(L(4))]2 (7) and Zn(bpi)2(UO2)(O)(C2O4)0.5(L(4))·H2O (8) feature one-dimensional structures. In 7, UO7 pentagonal bipyramids are alternatively connected by oxalate groups to form the chain, in which unidentate benzoate groups are coordinated to the uranium atoms. Zn(phen)3 cations fill the void space of the chains to compensate the negative charge. Differently, the chain of 8 can be seen as the heterometallic tetramer of UO7 and ZnO2N2 polyhedra connected by oxalate groups, and then bpi and benzoate groups are coordinated to the chain. All of the compounds have been characterized by IR and photoluminescent spectroscopy, and compounds 2, 3, 5, 6, and 8 exhibit characteristic emissions of uranyl cations.

Syntheses and structures of uranyl ethylenediphosphonates: from layers to elliptical nanochannels.

A family of uranium diphosphonates have been hydrothermally synthesized through the reaction of ethylenediphosphonic acid (EDP, H4L) and uranyl nitrate/zinc uranyl acetate in the presence of organic templates, such as tetraethyl ammonium (NEt4(+)), 4,4'-bipyridine (bipy), and 1,10-phenanthroline (phen). The UO2(2+) in UO2(H2O)(H2L)(EDP-U1) is equatorially five-coordinated by four phosphonate groups and one aqua ligand, forming a pentagonal bipyramid. Each EDP ligand is doubly protonated and chelates three UO2(2+), resulting in a layered structure. Compounds (NEt4)2(UO2)3(HL)2(H2L)·4H2O (EDP-U2) and (H2bipy)UO2L (EDP-U3) have the same layered structure in which NEt4(+) and protonated bipy fill in the uranyl-phosphonate interlayers, respectively, and play a role to balance the negative charges. Different from that in EDP-U1, the UO2(2+) exists in the form of a UO6 tetragonal bipyramid and is surrounded by four different EDP ligands in EDP-U2 and EDP-U3. (Hphen)2(UO2)2(H2L)3 (EDP-U4) features a three-dimensional framework structure with large elliptical channels along the c axis (1.3 × 1.1 nm(2)). Monoprotonated phen molecules fill in these channels and hold together through strong π···π interactions. All of the four compounds have been characterized by IR and photoluminescent spectroscopy. Their characteristic emissions have been attributed as transition properties of uranyl cations. The ion-exchange study indicates that [Co(en)3](3+) could partially replace the protonated phen molecules.

Structure and dynamics of the EGFR/HER2 heterodimer.

HER2 belongs to the human epidermal growth factor receptor tyrosine kinase family. Its overexpression or hyperactivation is a leading cause for multiple types of cancers. HER2 functions mainly through dimerization with other family members, such as EGFR. However, the molecular details for heterodimer assembly have not been completely understood. Here, we report cryo-EM structures of the EGF- and epiregulin-bound EGFR/HER2 ectodomain complexes at resolutions of 3.3 Å and 4.5 Å, respectively. Together with the functional analyses, we demonstrate that only the dimerization arm of HER2, but not that of EGFR, is essential for their heterodimer formation and signal transduction. Moreover, we analyze the differential membrane dynamics and transient interactions of endogenous EGFR and HER2 molecules in genome-edited cells using single-molecule live-cell imaging. Furthermore, we show that the interaction with HER2 could allow EGFR to resist endocytosis. Together, this work deepens our understanding of the unique structural properties and dynamics of the EGFR/HER2 complex.

Comparative cytotoxicity study of water-soluble carbon nanoparticles on plant cells.

This study provides a parallel cytotoxicity study of water-soluble single-walled carbon nanotubes and fullerene C70(C(COOH)2)4-8 on Nicotiana tabacum BY-2 cells. After BY-2 cells were treated with single-walled carbon nanotubes or C70(C(COOH)2)4-8, the cells adopted a shrunken morphology and exhibited lower proliferation rate. Fluorescence imaging of the treated cells revealed increased reactive oxygen species generation, abnormal mitochondrial distribution, decreased mitochondrial activity, and impaired actin cytoskeleton arrangement. However, no obvious cells death was induced by either single-walled carbon nanotubes or C70(C(COOH)2)4-8. Compared with single-walled carbon nanotubes, C70(C(COOH)2)4-8 exerted more serious adverse effects on BY-2 cells.

Chinese Medicine Shensong Yangxin Capsule () Ameliorates Myocardial Microcirculation Dysfunction in Rabbits with Chronic Myocardial Infarction.

OBJECTIVE: To investigate the effect of Chinese compound Shensong Yangxin Capsule ( , SSYX) on myocardial microcirculation in myocardial-infarcted rabbits. METHODS: Myocardial infarction (MI) was established in rabbits by ligation of the left circumflex coronary. Thirty rabbits were randomly divided into the control group, the MI group (model), and the MI treated with SSYX group (MI+SSYX) by a random number table method. After 4 weeks of administration, low-energy real-time myocardial contrast echocardiography (RT-MCE) was conducted to assess the microcirculatory perfusion. Immunofluorescence double staining was used to detect the capillary density. The endothelial ultrastructure was observed with a transmission electron microscope. The mRNA expression levels of vascular endothelial growth factor (VEGF), endothelin 1 (ET-1), prostaglandin I2 (PGI2) and endothelial nitric oxide synthase (eNOS) were measured by real-time quantitative polymerase chain reaction (Real-time PCR). The plasmic levels of ET-1, thromboxane A2 (TXA2), nitric oxide (NO) and von willebrand factor (vWF) were examined with enzyme-linked immunosorbent assays (ELISA). RESULTS: SSYX significantly improved the myocardial blood volume, myocardial micro bubble velocity, and myocardial inflow according to the examination of RT-MCE, and it visibly ameliorated the capillary endothelial structure. Furthermore, compared with the MI group, the plasma levels of TXA2, ET-1 and vWF contents significantly decreased in the MI+SSYX group, and the ET-1 mRNA expression levels of myocardium in the border zone significantly decreased, and the VEGF, PGI2 and eNOS mRNA expression levels significantly increased (all P<0.05). CONCLUSIONS: SSYX has favorable advantages in ameliorating the impaired myocardial microcirculation following MI. The mechanisms of the effect are related to the ability of SSYX in balancing the endothelial-derived vasodilators and vasoconstrictors, and up-regulating the expression of VEGF and eNOS.

Inherited nuclear pore substructures template post-mitotic pore assembly.

Nuclear envelope assembly during late mitosis includes rapid formation of several thousand complete nuclear pore complexes (NPCs). This efficient use of NPC components (nucleoporins or "NUPs") is essential for ensuring immediate nucleocytoplasmic communication in each daughter cell. We show that octameric subassemblies of outer and inner nuclear pore rings remain intact in the mitotic endoplasmic reticulum (ER) after NPC disassembly during prophase. These "inherited" subassemblies then incorporate into NPCs during post-mitotic pore formation. We further show that the stable subassemblies persist through multiple rounds of cell division and the accompanying rounds of NPC mitotic disassembly and post-mitotic assembly. De novo formation of NPCs from newly synthesized NUPs during interphase will then have a distinct initiation mechanism. We postulate that a yet-to-be-identified modification marks and "immortalizes" one or more components of the specific octameric outer and inner ring subcomplexes that then template post-mitotic NPC assembly during subsequent cell cycles.

A Fluidics-Based Biosensor to Detect and Characterize Inhibition Patterns of Organophosphate to Acetylcholinesterase in Food Materials.

A chip-based electrochemical biosensor is developed herein for the detection of organophosphate (OP) in food materials. The principle of the sensing platform is based on the inhibition of dimethoate (DMT), a typical OP that specifically inhibits acetylcholinesterase (AChE) activity. Carbon nanotube-modified gold electrodes functionalized with polydiallyldimethylammonium chloride (PDDA) and oxidized nanocellulose (NC) were investigated for the sensing of OP, yielding high sensitivity. Compared with noncovalent adsorption and deposition in bovine serum albumin, bioconjugation with lysine side chain activation allowed the enzyme to be stable over three weeks at room temperature. The total amount of AChE was quantified, whose activity inhibition was highly linear with respect to DMT concentration. Increased incubation times and/or DMT concentration decreased current flow. The composite electrode showed a sensitivity 4.8-times higher than that of the bare gold electrode. The biosensor was challenged with organophosphate-spiked food samples and showed a limit of detection (LOD) of DMT at 4.1 nM, with a limit of quantification (LOQ) at 12.6 nM, in the linear range of 10 nM to 1000 nM. Such performance infers significant potential for the use of this system in the detection of organophosphates in real samples.

Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic.

Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 "uncoating ATPase." The J- and PTEN-like domain-containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin-mediated membrane scission has released the vesicle from the plasma membrane. These bursts contain a very small number of auxilins, and even four to six molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated pit assembly. We previously showed that clathrin-coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. The differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.