Cooperative assembly of a designer peptide and silk fibroin into hybrid nanofiber gels for neural regeneration after spinal cord injury.

Local reconstruction of a permissive environment with biomaterials is a promising strategy to treat spinal cord injury (SCI). We reported a hybrid hydrogel fabricated from a small functional self-assembling peptide (F-SAP) and large silk fibroin (SF). The diffusion of SF micelles into F-SAP solution was driven by the dynamic synergy between osmotic pressure and F-SAP/SF electrostatic interactions, resulting in the rearrangement of SF micelles and the formation of rod-like filaments with axes nearly perpendicular to F-SAP nanofibers. Spectroscopy analysis, including circular dichroism, Raman and fluorescence, indicated conformation changes of SF from random coil to ß sheet, which contributed to enhanced mechanical properties of the resultant hybrid hydrogel. Furthermore, the F-SAP/SF hybrid hydrogel coupled with controlled release of NT-3 provided a permissive environment for neural regeneration by providing nanofibrous substrates for regenerating axons, inflammatory modulation and remyelination, consequently resulting in improved locomotion and electrophysiological properties. This hydrogel could be used as a long-term stent in vivo for the treatment of SCI.

Genome organization in double-stranded DNA viruses observed by cryoET.

Double-stranded DNA (dsDNA) viruses package their genetic material into protein cages with diameters usually a few hundred times smaller than the length of their genome. Compressing the relatively stiff and highly negatively charged dsDNA into a small volume is energetically costly and mechanistically enigmatic. Multiple models of dsDNA packaging have been proposed based on various experimental evidence and simulation methods, but direct observation of any viral genome organization is lacking. Here, using cryoET and an improved data processing scheme that utilizes information from the encaging protein shell, we present 3D views of dsDNA genome inside individual viral particles at resolution that densities of neighboring DNA duplexes are readily separable. These cryoET observations reveal a "rod-and-coil" fold of the dsDNA that is conserved among herpes simplex virus type 1 (HSV-1) with a spherical capsid, bacteriophage T4 with a prolate capsid, and bacteriophage T7 with a proteinaceous core inside the capsid. Finally, inspired by the genome arrangement in partially packaged T4 particles, we propose a mechanism for the genome packaging process in dsDNA viruses.

Cryo-EM analysis of the HCoV-229E spike glycoprotein reveals dynamic prefusion conformational changes.

Coronaviruses spike (S) glycoproteins mediate viral entry into host cells by binding to host receptors. However, how the S1 subunit undergoes conformational changes for receptor recognition has not been elucidated in Alphacoronavirus. Here, we report the cryo-EM structures of the HCoV-229E S trimer in prefusion state with two conformations. The activated conformation may pose the potential exposure of the S1-RBDs by decreasing of the interaction area between the S1-RBDs and the surrounding S1-NTDs and S1-RBDs compared to the closed conformation. Furthermore, structural comparison of our structures with the previously reported HCoV-229E S structure showed that the S trimers trended to open the S2 subunit from the closed conformation to open conformation, which could promote the transition from pre- to postfusion. Our results provide insights into the mechanisms involved in S glycoprotein-mediated Alphacoronavirus entry and have implications for vaccine and therapeutic antibody design.

Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-Gi Complex Structures.

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.

Self-capping of nucleoprotein filaments protects the Newcastle disease virus genome.

Non-segmented negative-strand RNA viruses, such as measles, ebola and Newcastle disease viruses (NDV), encapsidate viral genomic RNAs into helical nucleocapsids, which serve as the template for viral replication and transcription. Here, the clam-shaped nucleocapsid structure, where the NDV viral genome is sequestered, was determined at 4.8 Å resolution by cryo-electron microscopy. The clam-shaped structure is composed of two single-turn spirals packed in a back-to-back mode. This tightly packed structure functions as a seed for the assembly of a nucleocapsid from both directions, facilitating the growth of double-headed filaments with two separate RNA strings inside. Disruption of this structure by mutations in its loop interface yielded a single-headed unfunctional filament.

Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells.

BACKGROUND & AIMS: As hepatitis B virus (HBV) spreads through the infected liver it is simultaneously secreted into the blood. HBV-susceptible in vitro infection models do not efficiently amplify viral progeny or support cell-to-cell spread. We sought to establish a cell culture system for the amplification of infectious HBV from clinical specimens. METHODS: An HBV-susceptible sodium-taurocholate cotransporting polypeptide-overexpressing HepG2 cell clone (HepG2-NTCPsec+) producing high titers of infectious progeny was selected. Secreted HBV progeny were characterized by native gel electrophoresis and electron microscopy. Comparative RNA-seq transcriptomics was performed to quantify the expression of host proviral and restriction factors. Viral spread routes were evaluated using HBV entry- or replication inhibitors, visualization of viral cell-to-cell spread in reporter cells, and nearest neighbor infection determination. Amplification kinetics of HBV genotypes B-D were analyzed. RESULTS: Infected HepG2-NTCPsec+ secreted high levels of large HBV surface protein-enveloped infectious HBV progeny with typical appearance under electron microscopy. RNA-seq transcriptomics revealed that HBV does not induce significant gene expression changes in HepG2-NTCPsec+, however, transcription factors favoring HBV amplification were more strongly expressed than in less permissive HepG2-NTCPsec-. Upon inoculation with HBV-containing patient sera, rates of infected cells increased from 10% initially to 70% by viral spread to adjacent cells, and viral progeny and antigens were efficiently secreted. HepG2-NTCPsec+ supported up to 1,300-fold net amplification of HBV genomes depending on the source of virus. Viral spread and amplification were abolished by entry and replication inhibitors; viral rebound was observed after inhibitor discontinuation. CONCLUSIONS: The novel HepG2-NTCPsec+ cells efficiently support the complete HBV life cycle, long-term viral spread and amplification of HBV derived from patients or cell culture, resembling relevant features of HBV-infected patients. LAY SUMMARY: Currently available laboratory systems are unable to reproduce the dynamics of hepatitis B virus (HBV) spread through the infected liver and release into the blood. We developed a slowly dividing liver-derived cell line which multiplies infectious viral particles upon inoculation with patient- or cell culture-derived HBV. This new infection model can improve therapy by measuring, in advance, the sensitivity of a patient's HBV strain to specific antiviral drugs.

A Chemical Strategy for Amphiphile Replacement in Membrane Protein Research.

Membrane mimics are indispensable tools in the structural and functional understanding of membrane proteins (MPs). Given stringent requirements of integral MP manipulations, amphiphile replacement is often required in sample preparation for various biophysical purposes. Current protocols generally rely on physical methodologies and rarely reach complete replacement. In comparison, we report herein a chemical alternative that facilitates the exhaustive exchange of membrane-mimicking systems for MP reconstitution. This method, named sacrifice-replacement strategy, was enabled by a class of chemically cleavable detergents (CCDs), derived from the disulfide incorporation in the traditional detergent n-dodecyl-ß-d-maltopyranoside. The representative CCD behaved well in both solubilizing the diverse α-helical human G protein-coupled receptors and refolding of the ß-barrel bacterial outer membrane protein X, and more importantly, it could also be readily degraded under mild conditions. By this means, the A2A adenosine receptor was successfully reconstituted into a series of commercial detergents for stabilization screening and nanodiscs for electron microscopy analysis. Featured by the simplicity and compatibility, this CCD-mediated strategy would later find more applications when being integrated in other biophysics studies.

Assembly Pathway Selection of Designer Self-Assembling Peptide and Fabrication of Hierarchical Scaffolds for Neural Regeneration.

The self-assembling peptide (SAP) RADA 16-I has been modified with various functional motifs to improve its performances in biomedical applications. Nevertheless, the assembly mechanisms of designer functional RADA 16-I SAPs (F-SAPs) have not been clearly illustrated. The main problem is the difficulty in preparing a completely molecular aqueous solution of F-SAP. In the current study, we demonstrated that different procedures for preparing the F-SAP solution could result in the formation of different conformations and consequently micro/macroscopic morphologies. F-SAP was molecularly dissolved in an appropriate solvent, such as hexafluoroisopropanol (HFIP), as evidenced by random coil conformation characterized by circular dichroism spectroscopy and morphologies under transmission electron microscopy. The monomers were induced into monolayers when the F-SAP solution in HFIP was adsorbed on mica as observed by atomic force microscopy. However, nanoscaled filaments containing ß-sheets dominated in the F-SAP aqueous solution, in which case water acted as a poor solvent of F-SAP. Furthermore, the results of molecular dynamics simulation implicated that water facilitated F-SAP aggregation, whereas HFIP inhibited it. The ß-sheet assemblies formed in water exhibited a high kinetic stability and did not disassemble rapidly after the addition of HFIP. Our study indicated that selecting the right assembly pathway of F-SAP required for targeted functions, for example, delivery of hydrophobic drugs in aqueous conditions, could be achieved by optimizing the preparation protocol in addition to molecular design. Moreover, hierarchical scaffolds mimicking the natural extracellular matrix could be fabricated by the direct electrospinning of F-SAP molecular solution in HFIP and biodegradable polymer for applications in neural regeneration by promoting neural differentiation, neurite outgrowth, and synapse formation.

Functional Self-Assembling Peptide Nanofiber Hydrogels Designed for Nerve Degeneration.

Self-assembling peptide (SAP) RADA16-I (Ac-(RADA)4-CONH2) has been suffering from a main drawback associated with low pH, which damages cells and host tissues upon direct exposure. In this study, we presented a strategy to prepare nanofiber hydrogels from two designer SAPs at neutral pH. RADA16-I was appended with functional motifs containing cell adhesion peptide RGD and neurite outgrowth peptide IKVAV. The two SAPs were specially designed to have opposite net charges at neutral pH, the combination of which created a nanofiber hydrogel (-IKVAV/-RGD) characterized by significantly higher G' than G″ in a viscoelasticity examination. Circular dichroism, Fourier transform infrared spectroscopy, and Raman measurements were performed to investigate the secondary structure of the designer SAPs, indicating that both the hydrophobic/hydrophilic properties and electrostatic interactions of the functional motifs play an important role in the self-assembling behavior of the designer SAPs. The neural progenitor cells (NPCs)/stem cells (NSCs) fully embedded in the 3D-IKVAV/-RGD nanofiber hydrogel survived, whereas those embedded within the RADA 16-I hydrogel hardly survived. Moreover, the -IKVAV/-RGD nanofiber hydrogel supported NPC/NSC neuron and astrocyte differentiation in a 3D environment without adding extra growth factors. Studies of three nerve injury models, including sciatic nerve defect, intracerebral hemorrhage, and spinal cord transection, indicated that the designer -IKVAV/-RGD nanofiber hydrogel provided a more permissive environment for nerve regeneration than the RADA 16-I hydrogel. Therefore, we reported a new mechanism that might be beneficial for the synthesis of SAPs for in vitro 3D cell culture and nerve regeneration.

Tumor necrosis factor-alpha antagonism protects from myocardial inflammation and fibrosis in experimental diabetic cardiomyopathy.

To investigate the effect of anti-cytokine-based therapy in the course of diabetic cardiomyopathy, we performed a study using an anti-TNF-alpha monoclonal antibody treatment (mab) in Sprague male Dawley (SD) rats with streptozotocin-induced diabetic cardiomyopathy. Five days after streptozotocin injection, rats were treated with the anti-TNF-alpha mAb C432A for 6 weeks.At the end of the study, left ventricular (LV) function was determined by a pressure-catheter. Intercellular adhesion molecule (ICAM)-1, vascular adhesion molecule (VCAM)-1, beta2-lymphocyte-integrins(+) (CD18(+), CD11a(+), CD11b(+)), ED1/CD68(+) and cytokine (TNF-alpha, interleukin (IL)-1beta)- expressing infiltrates, total collagen content and stainings of collagen I and III were quantified by digital image analysis. LV phosphorylated and total ERK protein levels were determined by Western Blot. TNFalpha-antagonism reduced ICAM-1- and VCAM-1 expression and leukocyte infiltration to levels of non-diabetics and decreased macrophage residence by 3.3-fold compared with untreated diabetics. In addition, anti-TNF-alpha mAb-treatment decreased diabetes-induced cardiac TNF-alpha and IL-1beta expression by 2.0-fold and 1.8- fold, respectively, and reduced the ratio of phosphorylated to total ERK by 2.7-fold. The reduction in intramyocardial inflammation was associated with a 5.4-fold and 3.6-fold reduction in cardiac collagen I and III content, respectively. This was reflected by a normalization of cardiac total collagen content to levels of non-diabetics and associated with an improved LV function. TNFalpha-antagonism attenuates the development of experimental diabetic cardiomyopathy associated with a reduction of intramyocardial inflammation and cardiac fibrosis.