Exosomes are attractive as nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and their ability to penetrate physiological barriers that are impermeable to ...synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs, into cell-secreted exosomes leads to low yields. Here we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared with bulk electroporation and other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and a more than 10
-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic phosphatase and tensin homologue (PTEN)-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced inhibition of tumour growth and increased survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.
Hepatitis E virus (HEV), a small, non-enveloped RNA virus in the family Hepeviridae, is associated with endemic and epidemic acute viral hepatitis in developing countries. Our 3.5-Å structure of a ...HEV-like particle (VLP) shows that each capsid protein contains 3 linear domains that form distinct structural elements: S, the continuous capsid; P1, 3-fold protrusions; and P2, 2-fold spikes. The S domain adopts a jelly-roll fold commonly observed in small RNA viruses. The P1 and P2 domains both adopt β-barrel folds. Each domain possesses a potential polysaccharide-binding site that may function in cell-receptor binding. Sugar binding to P1 at the capsid protein interface may lead to capsid disassembly and cell entry. Structural modeling indicates that native T = 3 capsid contains flat dimers, with less curvature than those of T = 1 VLP. Our findings significantly advance the understanding of HEV molecular biology and have application to the development of vaccines and antiviral medications.
Gold nanoparticles are promising drug delivery vehicles for nucleic acids, small molecules, and proteins, allowing various modifications on the particle surface. However, the instability and low ...bioavailability of gold nanoparticles compromise their clinical application. Here, we functionalized gold nanoparticles with CPP fragments (CALNNPFVYLI, CALRRRRRRRR) through sulfhydryl PEG to increase their stability and bioavailability. The resulting gold nanoparticles were characterized with transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-visible spectrometry and X-ray photoelectron spectroscopy (XPS), and the stability in biological solutions was evaluated. Comparing to PEGylated gold nanoparticles, CPP (CALNNPFVYLI, CALRRRRRRRR)-modified gold nanoparticles showed 46 folds increase in cellular uptake in A549 and B16 cell lines, as evidenced by the inductively coupled plasma atomic emission spectroscopy (ICP-AES). The interactions between gold nanoparticles and liposomes indicated CPP-modified gold nanoparticles bind to cell membrane more effectively than PEGylated gold nanoparticles. Surface plasmon resonance (SPR) was used to measure interactions between nanoparticles and the membrane. TEM and uptake inhibitor experiments indicated that the cellular entry of gold nanoparticles was mediated by clathrin and macropinocytosis. Other energy independent endocytosis pathways were also identified. Our work revealed a new strategy to modify gold nanoparticles with CPP and illustrated the cellular uptake pathway of CPP-modified gold nanoparticles.
The matrix 1 (M1) protein of Influenza A virus plays many critical roles throughout the virus life cycle. The oligomerization of M1 is essential for the formation of the viral matrix layer during the ...assembly and budding process.
In the present study, we report that M1 can oligomerize in vitro, and that the oligomerization is pH-dependent. The N-terminal domain of M1 alone exists as multiple-order oligomers at pH 7.4, and the C-terminal domain alone forms an exclusively stable dimer. As a result, intact M1 can display different forms of oligomers and dimer is the smallest oligomerization state, at neutral pH. At pH 5.0, oligomers of the N-terminal domain completely dissociate into monomers, while the C-terminal domain remains in dimeric form. As a result, oligomers of intact M1 dissociate into a stable dimer at acidic pH.
Oligomerization of M1 involves both the N- and C-terminal domains. The N-terminal domain determines the pH-dependent oligomerization characteristic, and C-terminal domain forms a stable dimer, which contributes to the dimerization of M1. The present study will help to unveil the mechanisms of influenza A virus assembly and uncoating process.
Due to the elimination of wear and lubrication system and effective solution to the vibration control issue, magnetic suspended dual-rotor system (MSDS) offers the possibility to significantly ...enhance the performance of aero-engines. However, research on MSDS vibration characteristics under fixed-point rubbing with the consideration of nonlinear support characteristics of active magnetic bearing (AMB) has rarely been addressed. In this research, an improved EF model of AMB is proposed by equivalent magnetic circuit method and verified by finite element model. Then, the Lankarani-Nikravesh model is employed to describe the impact force during rubbing process. On this basis, the rubbing dynamic model is established by finite element method and verified by comparing with the experimental results in literature. Finally, the MSDS vibration characteristics are explored, and some interesting phenomena are found. Fixed-point rubbing will cause abundant frequency components equally spaced around the combined frequency components of rotor rotational speeds at different frequency intervals. Due to the stimulation of the AMB nonlinear EF, the fractional frequency component of MSDS second-order critical speed emerges in a certain speed range and implies potential instability of the system, and the rotational speed corresponding to the initial appearance of this frequency component will decrease when fixed-point rubbing occurs. With the decrease of proportional coefficient and the increase of differential coefficient, the maximum normal impact force in the rubbing process decreases.
Zao5241 is an elite soybean Glycine max (L.) Merr. line and backbone parent. In this study, we employed iTRAQ to analyze the proteomes and protein expression profiles of Zao5241 during leaf ...development. We identified 1,245 proteins in all experiments, of which only 45 had been previously annotated. Among overlapping proteins between three biological replicates, 598 proteins with 2 unique peptides identified were reliably quantified. The protein datasets were classified into 36 GO functional terms, and the photosynthesis term was most significantly enriched. A total of 113 proteins were defined as being differentially expressed during leaf development; 41 proteins were found to be differently expressed between two and four week old leaves, and 84 proteins were found to be differently expressed between two and six week old leaves, respectively. Cluster analysis of the data revealed dynamic proteomes. Proteins annotated as electron carrier activity were greatly enriched in the peak expression profiles, and photosynthesis proteins were negatively modulated along the whole time course. This dataset will serve as the foundation for a systems biology approach to understanding photosynthetic development.
Cryo-electron microscopy (cryo-EM) plays an important role in determining the structure of proteins, viruses, and even the whole cell. It can capture dynamic structural changes of large protein ...complexes, which other methods such as X-ray crystallography and nuclear magnetic resonance analysis find difficult. The signal-to-noise ratio of cryo-EM images is low and the contrast is very weak, and therefore, the images are very noisy and require filtering. In this paper, a filtering method based on non-local means and Zernike moments is proposed. The method takes into account the rotational symmetry of some biological molecules to enhance the signal-to-noise ratio of cryo-EM images. The method may be useful in cryo-EM image processing such as the automatic selection of particles, orientation determination, and the building of initial models.
•Simultaneous reduction of V(V) and Cr(VI) by Shewanella loihica PV-4 is realized.•Enhancement of Cr(VI) bioreduction is observed with the suppressed V(V) reduction.•Both membrane components and ...cytoplasmic fractions contribute to these reductions.•Most Cr(VI) is reduced extracellularly, V(V) tends to be reduced intracellularly.
Toxic vanadium (V) and chromium (VI) often co-exist in wastewater from vanadium ore smelting and their reductions by bacterial strain Shewanella loihica PV-4 is realized simultaneously. After 27-d operation, 71.3% of V(V) and 91.2% of Cr(VI) were removed respectively, with citrate as organic carbon source. Enhancement of Cr(VI) bioreduction was observed with the suppressed V(V) reduction. V(IV) and Cr(III), the main reduction products, precipitated inside the organisms and attached on cell surfaces. Both membrane components containing cytochrome c and cytoplasmic fractions containing soluble proteins as well as NADH may contribute to these microbial reductions. Most Cr(VI) were reduced extracellularly and V(V) tended to be reduced through intracellular process, as revealed by mapping the microbial surface and a line scan across the cell, performed by scanning transmission electron microscopy. This study provides an efficient alternative for controlling combined pollution caused by these two metals based on microbial technology.
The ryanodine receptors (RyRs) are high-conductance intracellular Ca(2+) channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest ...known ion channels, with a homotetrameric organization and approximately 5,000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryomicroscopy. Three previously uncharacterized domains, named central, handle and helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative-charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity-filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities.
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