HIV-1 Integrase Assembles Multiple Species of Stable Synaptic Complex Intasomes That Are Active for Concerted DNA Integration In vitro.

Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis.

Structural dynamics of RAF1-HSP90-CDC37 and HSP90 complexes reveal asymmetric client interactions and key structural elements.

RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 µs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1's unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding.

Three-Dimensional Reconstruction of the Hepatitis C Virus Envelope Glycoprotein E1E2 Heterodimer by Electron Microscopic Analysis.

Despite the development of highly effective hepatitis C virus (HCV) treatments, an effective prophylactic vaccine is still lacking. HCV infection is mediated by its envelope glycoproteins, E1 and E2, during the entry process, with E2 binding to cell receptors and E1 mediating endosomal fusion. The structure of E1E2 has only been partially resolved by X-ray crystallography of the core domain of E2 protein (E2c) and its complex with various neutralizing antibodies. Structural understanding of the E1E2 heterodimer in its native form can advance the design of candidates for HCV vaccine development. Here, we analyze the structure of the recombinant HCV E1E2 heterodimer with the aid of well-defined monoclonal anti-E1 and E2 antibodies, as well as a small-molecule chlorcyclizine-diazirine-biotin that can target and cross-link the putative E1 fusion domain. Three-dimensional (3D) models were generated after extensive 2D classification analysis with negative-stain single-particle data sets. We modeled the available crystal structures of the E2c and Fabs into 3D volumes of E1E2-Fab complexes based on the shape and dimension of the domain density. The E1E2 heterodimer exists in monomeric form and consists of a main globular body, presumably depicting the E1 and E2 stem/transmembrane domain, and a protruding structure representing the E2c region, based on anti-E2 Fab binding. At low resolution, a model generated from negative-stain analysis revealed the unique binding and orientation of individual or double Fabs onto the E1 and E2 components of the complex. Cryo-electron microscopy (cryo-EM) of the double Fab complexes resulted in a refined structural model of the E1E2 heterodimer, presented here. IMPORTANCE Recombinant HCV E1E2 heterodimer is being developed as a vaccine candidate. Using electron microscopy, we demonstrated unique features of E1E2 in complex with various neutralizing antibodies and small molecule inhibitors that are important to understanding its antigenicity and induction of immune response.

Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine.

mRNA function is influenced by modifications that modulate canonical nucleobase behavior. We show that a single modification mediates distinct impacts on mRNA translation in a position-dependent manner. Although cytidine acetylation (ac4C) within protein-coding sequences stimulates translation, ac4C within 5' UTRs impacts protein synthesis at the level of initiation. 5' UTR acetylation promotes initiation at upstream sequences, competitively inhibiting annotated start codons. Acetylation further directly impedes initiation at optimal AUG contexts: ac4C within AUG-flanking Kozak sequences reduced initiation in base-resolved transcriptome-wide HeLa results and in vitro utilizing substrates with site-specific ac4C incorporation. Cryo-EM of mammalian 80S initiation complexes revealed that ac4C in the -1 position adjacent to an AUG start codon disrupts an interaction between C and hypermodified t6A at nucleotide 37 of the initiator tRNA. These findings demonstrate the impact of RNA modifications on nucleobase function at a molecular level and introduce mRNA acetylation as a factor regulating translation in a location-specific manner.

Cryo-EM structure of an active central apparatus.

Accurately regulated ciliary beating in time and space is critical for diverse cellular activities, which impact the survival and development of nearly all eukaryotic species. An essential beating regulator is the conserved central apparatus (CA) of motile cilia, composed of a pair of microtubules (C1 and C2) associated with hundreds of protein subunits per repeating unit. It is largely unclear how the CA plays its regulatory roles in ciliary motility. Here, we present high-resolution structures of Chlamydomonas reinhardtii CA by cryo-electron microscopy (cryo-EM) and its dynamic conformational behavior at multiple scales. The structures show how functionally related projection proteins of CA are clustered onto a spring-shaped scaffold of armadillo-repeat proteins, facilitated by elongated rachis-like proteins. The two halves of the CA are brought together by elastic chain-like bridge proteins to achieve coordinated activities. We captured an array of kinesin-like protein (KLP1) in two different stepping states, which are actively correlated with beating wave propagation of cilia. These findings establish a structural framework for understanding the role of the CA in cilia.

Structures of outer-arm dynein array on microtubule doublet reveal a motor coordination mechanism.

Thousands of outer-arm dyneins (OADs) are arrayed in the axoneme to drive a rhythmic ciliary beat. Coordination among multiple OADs is essential for generating mechanical forces to bend microtubule doublets (MTDs). Using electron microscopy, we determined high-resolution structures of Tetrahymena thermophila OAD arrays bound to MTDs in two different states. OAD preferentially binds to MTD protofilaments with a pattern resembling the native tracks for its distinct microtubule-binding domains. Upon MTD binding, free OADs are induced to adopt a stable parallel conformation, primed for array formation. Extensive tail-to-head (TTH) interactions between OADs are observed, which need to be broken for ATP turnover by the dynein motor. We propose that OADs in an array sequentially hydrolyze ATP to slide the MTDs. ATP hydrolysis in turn relaxes the TTH interfaces to effect free nucleotide cycles of downstream OADs. These findings lead to a model explaining how conformational changes in the axoneme produce coordinated action of dyneins.

Structural basis for strand-transfer inhibitor binding to HIV intasomes.

The HIV intasome is a large nucleoprotein assembly that mediates the integration of a DNA copy of the viral genome into host chromatin. Intasomes are targeted by the latest generation of antiretroviral drugs, integrase strand-transfer inhibitors (INSTIs). Challenges associated with lentiviral intasome biochemistry have hindered high-resolution structural studies of how INSTIs bind to their native drug target. Here, we present high-resolution cryo-electron microscopy structures of HIV intasomes bound to the latest generation of INSTIs. These structures highlight how small changes in the integrase active site can have notable implications for drug binding and design and provide mechanistic insights into why a leading INSTI retains efficacy against a broad spectrum of drug-resistant variants. The data have implications for expanding effective treatments available for HIV-infected individuals.

Dissipation behavior of chlorpyrifos residues and risk assessment in sugarcane fields.

As chlorpyrifos is used globally to control pests in sugarcane fields, analysis of its residues on food crops is essential to assess product safety for humans. In this study, chlorpyrifos content in sugarcane plants, soil and juice was determined using a gas chromatography with an electron capture detector. The limit of quantification was 0.01 mg/kg for plant and soil, and 0.01 mg/L for juice. The degradation and residual risk in sugarcane fields after applying chlorpyrifos to two sample sites (Changsha and Danzhou, China) were assessed. Chlorpyrifos concentrations in plants and soil decreased rapidly over time, reaching a degradation rate ranging from 98.82 to 99.25% on day 35. The half-life of chlorpyrifos in both plants and soil was only 5.97-6.12 days. Regardless of application dosage (standard or high) at a pre-harvest interval of 60 days, chlorpyrifos was undetectable in the harvested sugarcane. Risk assessment indicated that chlorpyrifos residue in sugarcane did not pose a health risk to humans.

Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome.

Like all retroviruses, HIV-1 irreversibly inserts a viral DNA (vDNA) copy of its RNA genome into host target DNA (tDNA). The intasome, a higher-order nucleoprotein complex composed of viral integrase (IN) and the ends of linear vDNA, mediates integration. Productive integration into host chromatin results in the formation of the strand transfer complex (STC) containing catalytically joined vDNA and tDNA. HIV-1 intasomes have been refractory to high-resolution structural studies. We used a soluble IN fusion protein to facilitate structural studies, through which we present a high-resolution cryo-electron microscopy (cryo-EM) structure of the core tetrameric HIV-1 STC and a higher-order form that adopts carboxyl-terminal domain rearrangements. The distinct STC structures highlight how HIV-1 can use the common retroviral intasome core architecture to accommodate different IN domain modules for assembly.

A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks.

We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E. coli BL21(DE3) cells in shaker flasks that reduces D2O usage tenfold and d7-glucose usage by 30 %. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD600 of up to 10. Inducing the cells with isopropyl ß-D-1-thiogalactopyranoside at an OD600 of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD600 between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of E. coli from H2O-based to D2O-based medium is also key for ensuring high levels of protein expression in D2O. We find that a simple three-step adaptation approach-Luria-Bertani (LB) medium in H2O to LB in D2O to modified-M9 medium in D2O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.

Optimal Shell Thickness of Metal@Insulator Nanoparticles for Net Enhancement of Photogenerated Polarons in P3HT Films.

Embedding metal nanoparticles in the active layer of organic solar cells has been explored as a route for improving charge carrier generation, with localized field enhancement as a proposed mechanism. However, embedded metal nanoparticles can also act as charge recombination sites. To suppress such recombination, the metal nanoparticles are commonly coated with a thin insulating shell. At the same time, this insulating shell limits the extent that the localized enhanced electric field influences charge generation in the organic medium. It is presumed that there is an optimal thickness which maximizes field enhancement effects while suppressing recombination. Atomic Layer Deposition (ALD) was used to deposit Al2O3 layers of different thicknesses onto silver nanoparticles (Ag NPs), in a thin film of P3HT. Photoinduced absorption (PIA) spectroscopy was used to study the dependence of the photogenerated P3HT(+) polaron population on the Al2O3 thickness. The optimal thickness was found to be 3-5 nm. This knowledge can be further applied in the design of metal nanoparticle-enhanced solar cells.

[Influence of combined ecological floating bed on nitrogen and phosphorus between overlying water and sediment].

A new type of combined ecological floating bed was developed on Swan Lake. It was combined with aquatic and hydrophilous plants and ancillary equipments that included floating controllers, water-cycling aerator systems and wave-making systems. Combined ecological floating bed changed the physical and chemical environment of Swan Lake and had an effect on the transportation and transformation of TN, NH4(+) -N and TP between overlying water and sediment during the experiment. The concentration change of TN, NH4(+) -N and TP and the effect of DO, Eh and pH on the nutrient in the overlying water and sediment were investigated. The results indicated that: in overlying water, the removal efficiencies of TN, NH4(+) -N and TP were 61.92%, 63.09% and 80.0%, respectively; the removal efficiencies of TN and NH4(+) -N were 23.79% and 37.04%, respectively; the concentration of TP increased by 43.71% in sediment during the experiment. The combined ecological floating bed influenced environmental factors such as DO, Eh and pH in overlying water in some degree. DO concentration rose from 8.7-8.9 mg x L(-1) to 9.3-10.4 mg x L(-1), and Eh concentration was from 163-178 mV to 191-198 mV. Both of them were higher than those in the contrast area. Phosphorus release was inhibited, and phosphorus adsorption was promoted through improving DO and Eh of the overlying water. The pH volatility was less and maintained between 7.51 and 8.32, and did not promote phosphorus release in sediment. TN, TP and NH4(+) -N in overlying water and TN and NH4(+) -N in sediment were significantly positively correlated with each other and with TP in sediment. The pH had no significant correlation with TN, TP and NH4(+) -N in overlying water and sediment. DO was significantly positively corrected with Eh in overlying water and was significantly negatively corrected with TP in sediment.

[Experimental studies on stability of flocs from cadmium pollution emergency treatment].

Taking the flocs from cadmium pollution emergency treatment of Longjiang River in Guangxi province as the research object, the stability of the flocs in the simulated static reservoirs and acidic floods was investigated based on the effects of disturbance and pH on the stability of the flocs. The results indicated that disturbance and pH had great effects on the stability of the flocs, and the concentrations of Cd2+ followed the order of pH 5.0 >> pH 6.0 > pH 7. 0 approximately pH 8.0 > pH 9.0 with the original pH of water. When the original pH of water was 5.0, the concentrations of Cd2+ in samples were 19-58 times higher than the national standard limit, and when the original pH of water were 6.0, 7.0, 8.0 and 9.0, respectively, the concentrations of Cd2+ in samples varied from below to 11 times higher than the national standard limit. The release of cadmium from the flocs was higher in the disturbed water, with the concentrations of Cd2+ in most samples higher than 5.0 microg x L(-1), and the highest was double of the national standard limit. In contrast, there was little release in the simulated static reservoirs, with the concentrations of Cd2+ in all samples below 5.0 microg x L(-1), which was lower than the national standard limit. Therefore, the flocs had good stability in the simulated static reservoirs. But it had poor stability in the simulated acidic floods, with higher release of cadmium, and the concentrations of Cd2+ in samples were 14-25 times higher than the national standard limit. Therefore, the monitoring of cadmium concentrations in the floods should be strengthened in the post project analysis for eco-environmental impact of Longjiang River.

A novel T-type overhangs improve the enzyme-free cloning of PCR products.

PCR product cloning is the foundational technology for almost all fields in the life sciences. Numerous innovative methods have been designed during the past few decades. Enzyme-free cloning is the only one that avoids post-amplification enzymatic treatments, making the technique reliable and cost effective. However, the complementary staggered overhangs used in enzyme-free cloning tend to result in self-ligation of the vector under some circumstances. Here, we describe a "T-type" enzyme-free cloning method: instead of designing the complementary staggered overhangs used in conventional enzyme-free cloning, we create "T-type" overhangs that reduce the possibility of self-ligation and are more convenient for multi-vector cloning. In this study, we systematically optimize "T-type" enzyme-free cloning, compare its cloning background with that in conventional enzyme-free cloning, and demonstrate a promising application of this technique in multi-vector cloning. Our method simplifies post-amplification procedures and greatly reduces cost, offering a competitive option for PCR product cloning.

Enhanced photocatalysis on TiO2 nanotube arrays modified with molecularly imprinted TiO2 thin film.

A molecularly imprinted TiO(2) film was constructed onto the surface of a TiO(2) nanotube (NT) array, getting a novel composite TiO(2) catalyst. Compared with unmodified TiO(2) NT and the non-imprinted film modified TiO(2) NT, the molecularly imprinted film modified TiO(2) NT not only exhibits a much higher adsorption capacity for the target contaminant but also shows an enhanced photocatalytic activity in degrading the target contaminant. Moreover, the molecularly imprinted inorganic semiconductor film is not degraded during photocatalysis, owing to its stable physicochemical properties which is superior to those of conventional molecularly imprinted organic polymer films.

General assembly method for linear metal nanoparticle chains embedded in nanotubes.

We demonstrate a flexible assembly method for producing linear metal nanoparticle chains embedded in nanotubes. The chain formation is based on the Rayleigh instability after annealing metal nanowires confined in nanotubes. Beginning with metal nanowires from arbitrary synthesis methods, atomic layer deposition (ALD) was applied to coat the wires first with a sacrificial layer then with a shell layer. Subsequently, the sacrificial layer was removed leading to confined wires in nanotubes with a free volume. Finally, embedded nanoparticle chains were produced inducing the Rayleigh instability by annealing the confined nanowires. This method is quite general not only for different metals but also for different shell materials. We are able to tune the particle spacing and diameter, the shape of the nanochains, the tube diameter and the shell thickness by ALD significantly.

Clomazone dissipation, adsorption and translocation in four paddy topsoils.

Laboratory experiments about the dissipation, adsorption and translocation in four paddy topsoils were conducted in this paper. From the results it can be concluded as follows: the dissipation rate of clomazone differed greatly in different paddy soil derived from different parent materials. The half-lives for clomazone degradation in paddy soils ranged from 5.7 to 22.0 d. The order of clomazone dissipation rate was reddish yellow paddy soil > alluvial sandy paddy soil > yellow clayey paddy soil > purple sandy paddy soil. Clomazone sorption quantity was significantly correlated with organic carbon (R2 = 0.62) and clay content(R2 = 0.67) in the tested paddy soils. Positive correlation was found between apparent Kd value and cation exchange content(CEC). The consequences for the adsorption of different soils were purple sandy paddy soil > yellow clayey paddy soil > reddish yellow paddy soil > alluvial sandy paddy soil. Under the simulated rainfall of 200 mm through four different unsaturated soil lysimeters over 24 h, clomazone was readily to be leached into lower surface soil and there was about 2.6%--4.2% of applied clomazone leached out of 20 cm cultivated soil layer. Translocation experiments showed that the order of clomazone leaching ability was: alluvial sandy paddy soil > reddish yellow paddy soil > yellow clayey paddy soil > purple sandy paddy soil. Simple regression results manifested that factors like CEC, organic carbon, clay, and adsorption rate constant had been negatively correlated with the percentage of clomazone loss from soil lysimeters.