Probe-Free Identification of RNA Virus Variants with Point Mutations by Surface-Enhanced Raman Spectroscopy.

As observed in the COVID-19 pandemic, RNA viruses continue to rapidly evolve through mutations. In the absence of effective therapeutics, early detection of new severely pathogenic viruses and quarantine of infected people are critical for reducing the spread of the viral infections. However, conventional detection methods require a substantial amount of time to develop probes specific to new viruses, thereby impeding immediate response to the emergence of viral pathogens. In this study, we identified multiple types of viruses by obtaining the spectral fingerprint of their surface proteins with probe-free surface-enhanced Raman scattering (SERS). In addition, the SERS-based method can remarkably distinguish influenza virus variants with several surface protein point mutations from their parental strain. Principal component analysis (PCA) of the SERS spectra systematically captured the key Raman bands to distinguish the variants. Our results show that the combination of SERS and PCA can be a promising tool for rapid detection of newly emerging mutant viruses without a virus-specific probe.

Graphene Oxide-Supported Microwell Grids for Preparing Cryo-EM Samples with Controlled Ice Thickness.

Cryogenic-electron microscopy (cryo-EM) is the preferred method to determine 3D structures of proteins and to study diverse material systems that intrinsically have radiation or air sensitivity. Current cryo-EM sample preparation methods provide limited control over the sample quality, which limits the efficiency and high throughput of 3D structure analysis. This is partly because it is difficult to control the thickness of the vitreous ice that embeds specimens, in the range of nanoscale, depending on the size and type of materials of interest. Thus, there is a need for fine regulation of the thickness of vitreous ice to deliver consistent high signal-to-noise ratios for low-contrast biological specimens. Herein, an advanced silicon-chip-based device is developed which has a regular array of micropatterned holes with a graphene oxide (GO) window on freestanding silicon nitride (Six Ny ). Accurately regulated depths of micropatterned holes enable precise control of vitreous ice thickness. Furthermore, GO window with affinity for biomolecules can facilitate concentration of the sample molecules at a higher level. Incorporation of micropatterned chips with a GO window enhances cryo-EM imaging for various nanoscale biological samples including human immunodeficiency viral particles, groEL tetradecamers, apoferritin octahedral, aldolase homotetramer complexes, and tau filaments, as well as inorganic materials.

Engineering Genetic Systems for Treating Mitochondrial Diseases.

Mitochondria are intracellular energy generators involved in various cellular processes. Therefore, mitochondrial dysfunction often leads to multiple serious diseases, including neurodegenerative and cardiovascular diseases. A better understanding of the underlying mitochondrial dysfunctions of the molecular mechanism will provide important hints on how to mitigate the symptoms of mitochondrial diseases and eventually cure them. In this review, we first summarize the key parts of the genetic processes that control the physiology and functions of mitochondria and discuss how alterations of the processes cause mitochondrial diseases. We then list up the relevant core genetic components involved in these processes and explore the mutations of the components that link to the diseases. Lastly, we discuss recent attempts to apply multiple genetic methods to alleviate and further reverse the adverse effects of the core component mutations on the physiology and functions of mitochondria.

Alteration of gammaretroviral vector integration patterns by insertion of histone and leucine zipper into integrase.

Retroviral vectors show long-term gene expression in gene therapy through the integration of transgenes into the human cell genome. Murine leukemia virus (MLV), a well-studied gammaretrovirus, has been often used as a representative retroviral vector. However, frequent integrations of MLV-based vectors into transcriptional start sites (TSSs) could lead to the activation of oncogenes by enhancer effects of the genetic components within the vectors. Therefore, the MLV integration preference for TSSs limits its wider use in clinical applications. To reduce the integration preference of MLV-based vectors, we attempted to perturb the structure of the viral integrase that plays a key role in determining integration sites. For this goal, we inserted histones and leucine zippers, having DNA-binding property, into internal sites of MLV integrase. This integrase engineering yielded multiple mutant vectors that showed significantly different integration patterns compared with that of wild-type vector. Some mutant vectors did not prefer the key regulatory genomic domains of human cells, TSSs. Moreover, a couple of engineered vectors did not integrate into the genomic sites near the TSSs of oncogenes. Overall, this study suggests that structural perturbation of integrase is a simple way to develop safer MLV-based retroviral vectors for use in clinical applications.

Activation of fractalkine/CX3CR1 by vascular endothelial cells induces angiogenesis through VEGF-A/KDR and reverses hindlimb ischaemia.

AIMS: The present study investigated the detailed mechanism by which fractalkine (Fkn), a CX3C chemokine, induces angiogenesis and its functional implication in alleviating ischaemia in vivo. METHODS AND RESULTS: Fkn induced new vessel formation on the excised rat aorta and chick chorioallantoic membrane (CAM) through CX3CR1 activation. Immunoblotting analysis, promoter assay and electrophoretic mobility shift assay showed that Fkn upregulated hypoxia-inducible factor-1 alpha (HIF-1alpha) by cultured human aortic endothelial cells (ECs), which in turn induced mRNA and protein levels of vascular endothelial growth factor (VEGF)-A through a p42/44 mitogen-activated protein kinase pathway. In vivo Fkn-induced angiogenesis on CAM was completely blocked by functional inhibition of VEGF receptor 2 kinase insert domain-containing receptor (KDR) and Rho GTPase. C57/BL6 mice with CX3CR1(-/-) bone marrow-derived cells developed angiogenesis in the implanted Fkn-mixed Matrigel plug, suggesting CX3CR1 activation in vascular ECs is sufficient for Fkn-induced angiogenesis in vivo. The condition of rat hindlimb ischaemia, which rapidly stimulated mRNA expression of both Fkn and VEGF-A, was significantly alleviated by the injection of whole-length Fkn protein. CONCLUSION: Fkn-induced activation of CX3CR1 by ECs leads to in vivo angiogenesis through two sequential steps: the induction of HIF-1alpha and VEGF-A gene expression by CX3CR1 activation and the subsequent VEGF-A/KDR-induced angiogenesis. The potent induction of angiogenesis by Fkn can be used as a therapeutic strategy for alleviating peripheral ischaemia.

On-line sample cleanup and chiral separation of gemifloxacin in a urinary solution using chiral crown ether as a chiral selector in microchip electrophoresis.

In chiral capillary electrophoresis of primary amine enantiomers using (+)-18-crown-6-tetracarboxylic acid (18C6H4) as a chiral selector, the presence of alkaline metal ions in the sample solution as well as in the run buffer is undesirable due to their strong competitive binding with 18C6H4. A channel-coupled microchip electrophoresis device was designed to clean up alkaline metal ions from a sample matrix for the chiral analysis of amine. In the first channel, the metal ions in the sample were monitored by indirect detection using quinine as a chromophore and drained to the waste. In the second separation channel, gemifloxacin enantiomers, free of the alkaline metal ions, were successfully separated using only a small amount of the chiral selector (50 microM 18C6H4).