“Smart” Materials for Biosensing Devices: Cell-Mimicking Supramolecular Assemblies and Colorimetric Detection of Pathogenic Agents

Mimicking cell membrane and the biomolecular recognition associated with membranes represents a great technical challenge, yet it has opened doors to innovative diagnostic and therapeutic methods. Our work has focused on design and synthesis of a class of smart materials exploiting biological principals for use in biosensors: these materials are functional polymeric assemblies that mimic the cell membrane and conveniently report the presence of pathogens with a color change. Biologically active cell membrane components are incorporated into conjugated polymers with desirable optical properties and the binding of the target molecules onto the material triggers conformational and electronic shifts that are reflected in a chromatic change (a so-called biochromic shift) that is conveniently observed and recorded. Langmuir–Blodgett thin films and vesicle bilayers provide ideal configurations for precise delivery of the biological binding entity to the sensing interface, and for control of molecular orientation for effective biomolecular interaction. Polydiacetylenic membrane-mimicking materials containing cell surface receptor gangliosides and sialic acid residues, respectively were formulated into these architectures and used for colorimetric detection of bacterial toxins and influenza virus. One advantage of these biochromic conjugated polymer (BCP) sensors is that their molecular recognition and signal transduction functionalities are resident in a single functional unit, making them amenable to convenient microfabrication and use.     

Oligomerization and polymerization of the filovirus matrix protein VP40

The matrix protein VP40 from Ebola virus plays an important role in the assembly process of virus particles by interacting with cellular factors, cellular membranes, and the ribonuclearprotein particle complex. Here we show that the N-terminal domain of VP40 folds into a mixture of two different oligomeric states in vitro, namely hexameric and octameric ringlike structures, as detected by gel filtration chromatography, chemical cross-linking, and electron microscopy. Octamer formation depends largely on the interaction with nucleic acids, which in turn confers in vitro SDS resistance. Refolding experiments with a nucleic acid free N-terminal domain preparation reveal a mostly dimeric form of VP40, which is transformed into an SDS resistant octamer upon incubation with E. coli nucleic acids. In addition, we demonstrate that the N-terminal domain of Marburg virus VP40 also folds into ringlike structures, similar to Ebola virus VP40. Interestingly, Marburg virus VP40 rings reveal a high tendency to polymerize into rods composed of stacked rings. These results may suggest distinct roles for different oligomeric forms of VP40 in the filovirus life cycle.     

Neutral Polymeric Surfactants Derived from Dextran: A Study of Their Aqueous Solution Behavior

Summary: Amphiphilic polysaccharides are obtained by hydrophobic modification of a neutral bacterial polysaccharide, dextran. By reacting the polysaccharide with aliphatic epoxides (epoxyoctane and epoxydodecane) in dimethyl sulfoxide, a series of amphiphilic polymers is obtained which covers a large range of structural parameters (length of the polysaccharide, number and nature of hydrocarbon moieties). The solution behavior of dextran derivatives is first characterized by viscometric measurements in dilute and semi‐dilute domains. The effects of molecular parameters on polymer viscosity behavior are evidenced and discussed. Information on the state of aggregation of polymers is obtained by the use of static and dynamic light scattering. The presence of aggregates in the dilute domain is clearly evidenced and their structural characteristics are estimated (size, molecular weight and number of aggregation). The aggregates are shown to account for the viscometric results in the examined concentration range, relating their chemical parameters (hydrodynamic radius and molecular weight) to the macroscopic behavior of the solutions.

     

非细胞体系核重构过程的超微结构研究

用电镜观察Lambda DNA与非洲爪蟾卵提取物在非细胞体系内的核重构,发现Lambda DNA首先诱导形成类染色质结构,膜泡、核孔复合体围绕这一结构组装成双层核膜,同时类染色质也随着核膜的装配,表现出从致密凝聚到呈现松散均匀分布的变化。有工作表明,在由染色质诱导的核重构过程中,或者是膜泡先与染色质结合,然后有核孔出现,或者是核孔物质先与染色质作用,膜泡通过结合核孔物质形成双层核膜。我们观察由外源DNA诱导的核重构过程,则发现膜泡与核孔复合体先分别独立地与类染色质结构相互作用,然后核孔复合体再镶嵌到双层核膜中。     

Virus Hijacks Host Proteins and Machinery for Assembly and Budding,with HIV-1 as an Example

Viral assembly and budding are the final steps and key determinants of the virus life cycle and are regulated by virus–host interaction. Several viruses are known to use their late assembly (L) domains to hijack host machinery and cellular adaptors to be used for the requirement of virus replication. The L domains are highly conserved short sequences whose mutation or deletion may lead to the accumulation of immature virions at the plasma membrane. The L domains were firstly identified within retroviral Gag polyprotein and later detected in structural proteins of many other enveloped RNA viruses. Here, we used HIV-1 as an example to describe how the HIV-1 virus hijacks ESCRT membrane fission machinery to facilitate virion assembly and release. We also introduce galectin-3, a chimera type of the galectin family that is up-regulated by HIV-1 during infection and further used to promote HIV-1 assembly and budding via the stabilization of Alix–Gag interaction. It is worth further dissecting the details and finetuning the regulatory mechanism, as well as identifying novel candidates involved in this final step of replication cycle.     

Molecule-to-Material-to-Bio Nanoarchitectonics with Biomedical Fullerene Nanoparticles

Nanoarchitectonics integrates nanotechnology with various other fields, with the goal of creating functional material systems from nanoscale units such as atoms, molecules, and nanomaterials. The concept bears strong similarities to the processes and functions seen in biological systems. Therefore, it is natural for materials designed through nanoarchitectonics to truly shine in bio-related applications. In this review, we present an overview of recent work exemplifying how nanoarchitectonics relates to biology and how it is being applied in biomedical research. First, we present nanoscale interactions being studied in basic biology and how they parallel nanoarchitectonics concepts. Then, we overview the state-of-the-art in biomedical applications pursuant to the nanoarchitectonics framework. On this basis, we take a deep dive into a particular building-block material frequently seen in nanoarchitectonics approaches: fullerene. We take a closer look at recent research on fullerene nanoparticles, paying special attention to biomedical applications in biosensing, gene delivery, and radical scavenging. With these subjects, we aim to illustrate the power of nanomaterials and biomimetic nanoarchitectonics when applied to bio-related applications, and we offer some considerations for future perspectives.     

A ribavirin-induced ORF2 single-nucleotide variant produces defective hepatitis E virus particles with immune decoy function

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious in vitro and in vivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.

Despite its rising global prevalence, hepatitis E is a disease that is mostly overlooked. Every year, more than 44,000 people die as a result of ∼20 million infections worldwide (1). Healthy individuals usually display no or only mild symptoms of viral hepatitis, such as fever, nausea, vomiting, and abdominal pain (2), while patients with preexisting liver disease, pregnant women, and immunocompromised individuals suffer from liver cirrhosis and liver failure (3). Pregnant women additionally present with increased mortality rates of >25% (4). Despite those liver-associated problems, there are also extrahepatic manifestations, such as hematopoietic disease, neurological disorders, and renal injury (5–9). The underlying agent, hepatitis E virus (HEV), is classed within the species of Paslahevepirus balayani (10), formerly known as Orthohepevirus A, which includes isolates from human, swine, wild boar, rat, and other mammals. HEV is a quasienveloped virus existing as both enveloped and non-enveloped particles (11, 12). To date, eight distinct genotypes (GT) of this species of the single-stranded RNA virus have been described (13), which display similar genomic structures. The positive orientated HEV genome is organized in three main open reading frames (ORF1 to ORF3) with a total length of 7.2 kb. Nonstructural proteins forming the HEV replicase complex, such as the RNA-dependent RNA polymerase (RdRp), RNA helicase, or methyltransferase, are encoded by ORF1, while the viral capsid protein is encoded by ORF2. During the HEV replication cycle, HEV produces at least three forms of ORF2 protein: infectious ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c) protein (14). The ORF2i protein is the structural component of infectious particles that is likely derived from the assembly of the intracellular ORF2 (ORF2intra) protein form. In contrast, ORF2g and ORF2c protein are not associated with infectious virions, but secreted in large amounts and are the most abundant antigens detected in patient sera (14). ORF3 encodes for a functional ion channel required for assembly and release of infectious particles by interacting with a variety of host factors (15).In immunocompetent patients, acute hepatitis E usually does not involve antiviral therapy; however, chronically infected and immunocompromised patients often require clinical intervention to clear the infection. Antiviral therapies include pegylated interferon (16–18), successfully implemented for many virus infections, and sofosbuvir (19, 20), a direct acting antiviral against hepatitis C virus, both of which have not yet been systematically evaluated in the context of HEV therapy. Recent studies have investigated the antiviral potential of silvestrol (21), zinc salts (22), and other possible drug candidates in vitro [reviewed in detail by Kinast et al. (23)], but the findings remain to be clinically validated. Lacking specific treatment options, the broad antiviral ribavirin (RBV) (24) is frequently used off-label. However, RBV therapy is often discontinued due to adverse side effects and is only effective in ∼80% of patients, implying that 20% of treated patients remain viremic (25). RBV treatment is specifically contraindicated in pregnant women and can give rise to variants such as G1634R, as well as other amino acid substitutions within the ORF1-encoded polyprotein, potentially contributing to treatment failure and poor clinical long-term outcomes (26–28). In this context, we recently identified viral populations of HEV harboring variations in the capsid-encoding ORF2 region during RBV therapy. With the use of an efficient HEV cell-culture model system, we characterized the impact of these ORF2 variants in the HEV replication cycle.     

Near-Native Visualization of SARS-CoV-2 Induced Membrane Remodeling and Virion Morphogenesis

Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, leads to profound remodeling of cellular membranes, promoting viral replication and virion assembly. A full understanding of this drastic remodeling and the process of virion morphogenesis remains lacking. In this study, we applied room temperature transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography to visualize the SARS-CoV-2 replication factory in Vero cells, and present our results in comparison with published cryo-EM studies. We obtained cryo-EM-like clarity of the ultrastructure by employing high-pressure freezing, freeze substitution (HPF-FS) and embedding, allowing room temperature visualization of double-membrane vesicles (DMVs) in a near-native state. In addition, our data illustrate the consecutive stages of virion morphogenesis and reveal that SARS-CoV-2 ribonucleoprotein assembly and membrane curvature occur simultaneously. Finally, we show the tethering of virions to the plasma membrane in 3D, and that accumulations of virus particles lacking spike protein in large vesicles are most likely not a result of defective virion assembly at their membrane. In conclusion, this study puts forward a room-temperature EM technique providing near-native ultrastructural information about SARS-CoV-2 replication, adding to our understanding of the interaction of this pandemic virus with its host cell.