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.

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.

Identification of Newly Emerging Influenza Viruses by Detecting the Virally Infected Cells Based on Surface Enhanced Raman Spectroscopy and Principal Component Analysis.

Rapid diagnosis and quarantine of influenza virus mutant-infected people is critical to contain the fatal viral infection spread because effective antiviral drugs are normally not available. Conventional methods, however, cannot be used for the diagnosis because these methods need predefined labels, likely also unavailable for just emerging viruses. Here, we propose label-free identification of cells infected with different influenza viruses based on surface-enhanced Raman spectroscopy (SERS) and principal component analysis (PCA). Viral envelope proteins that are displayed on the surface of cells after infection of influenza viruses were targeted for this identification. Cells that expressed the envelope proteins of A/WSN/33 H1N1 or A/California/04/2009 H1N1 influenza viruses produced distinct SERS signals. Cells that displayed combinations of the envelope proteins from these two viral variants, an indication of emergence of a new virus, also generated characteristic SERS patterns. However, the cell's own surface proteins often hindered the identification of virally infected cells by producing SERS peaks similar to viral ones. PCA of the obtained SERS patterns could effectively capture the virus-specific signal components from the jumbled SERS peaks. Our study demonstrates a potential of combination of SERS and PCA to identify newly emerging influenza viruses through sensing the cells infected with the viruses.

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins.

The recent advances in the single cell genome analysis are generating a considerable amount of novel insights into complex biological systems. However, there are still technical challenges because each cell has a single copy of DNA to be amplified in most single cell genome analytical methods. In this paper, we present a novel approach to directly visualize a genomic map on a large DNA molecule instantly stained with red and green DNA-binding fluorescent proteins without DNA amplification. For this visualization, we constructed a few types of fluorescent protein-fused DNA-binding proteins: H-NS (histone-like nucleoid-structuring protein), DNA-binding domain of BRCA1 (breast cancer 1), high mobility group-1 (HMG), and lysine tryptophan (KW) repeat motif. Because H-NS and HMG preferentially bind A/T-rich regions, we combined A/T specific binder (H-NS-mCherry and HMG-mCherry as red color) and a non-specific complementary DNA binder (BRCA1-eGFP and 2(KW)2-eGFP repeat as green color) to produce a sequence-specific two-color DNA physical map for efficient optical identification of single DNA molecules.

Attomolar detection of virus by liquid coplanar-gate graphene transistor on plastic.

The direct method of detecting a virus with extremely low concentration is recommended for the diagnosis of viral disease. In this study, coplanar-gate graphene field-effect transistors (GFETs) were built on flexible polyethylene terephthalate substrates for the attomolar detection of a virus. The GFETs exhibited a very low detection limit of 47.8 aM with relatively low source/drain voltage due to aqueous dielectric media which stabilizes viruses and antibodies for specific bonding. The antibody as a probe molecule was decorated on a graphene surface using 1-pyrenebutanoic acid succinimidyl ester that had previously been immobilized on a graphene surface. The Dirac point voltage shifted downward after dropping the virus solution, due to the electrostatic gating effect of graphene in the antigen (namely, virus)-antibody complex. The virus detection platform used in this study is expected to be beneficial for direct diagnosis in saline environments, since the performances of GFETs were not significantly affected by the presence of Na+ and Cl-. Furthermore, since our flexible and transparent virus sensors can be used in a wearable device, they provide a simple and fast method for diagnosing viruses.

Molecular-Level Interactions between Engineered Materials and Cells.

Various recent experimental observations indicate that growing cells on engineered materials can alter their physiology, function, and fate. This finding suggests that better molecular-level understanding of the interactions between cells and materials may guide the design and construction of sophisticated artificial substrates, potentially enabling control of cells for use in various biomedical applications. In this review, we introduce recent research results that shed light on molecular events and mechanisms involved in the interactions between cells and materials. We discuss the development of materials with distinct physical, chemical, and biological features, cellular sensing of the engineered materials, transfer of the sensing information to the cell nucleus, subsequent changes in physical and chemical states of genomic DNA, and finally the resulting cellular behavior changes. Ongoing efforts to advance materials engineering and the cell-material interface will eventually expand the cell-based applications in therapies and tissue regenerations.

Recent Advances in Mitochondria-Targeted Gene Delivery.

Mitochondria are the energy-producing organelles of cells. Mitochondrial dysfunctions link to various syndromes and diseases including myoclonic epilepsy and ragged-red fiber disease (MERRF), Leigh syndrome (LS), and Leber hereditary optic neuropathy (LHON). Primary mitochondrial diseases often result from mutations of mitochondrial genomes and nuclear genes that encode the mitochondrial components. However, complete intracellular correction of the mutated genetic parts relevant to mitochondrial structures and functions is technically challenging. Instead, there have been diverse attempts to provide corrected genetic materials with cells. In this review, we discuss recent novel physical, chemical and biological strategies, and methods to introduce genetic cargos into mitochondria of eukaryotic cells. Effective mitochondria-targeting gene delivery systems can reverse multiple mitochondrial disorders by enabling cells to produce functional mitochondrial components.

Reliable RT-qPCR-based titration of retroviral and lentiviral vectors via quantification of residual vector plasmid DNA in samples.

OBJECTIVES: To develop a method for reliable quantification of viral vectors, which is necessary for determining the optimal dose of vector particles in clinical trials to obtain the desired effects without severe unwanted immune responses. RESULTS: A significant level of vector plasmid remained in retroviral and lentiviral vector samples, which led to overestimation of viral titers when using the conventional RT-qPCR-based genomic titration method. To address this problem, we developed a new method in which the residual plasmid was quantified by an additional RT-qPCR step, and standard molecules and primer sets were optimized. The obtained counts were then used to correct the conventionally measured genomic titers of viral samples. While the conventional method produced significantly higher genomic titers for mutant retroviral vectors than for wild-type vectors, our method produced slightly higher or equivalent titers, corresponding with the general idea that mutation of viral components mostly results in reduced or, at best, retained titers. CONCLUSION: Subtraction of the number of residual vector plasmid molecules from the conventionally measured genomic titer can yield reliable quantification of retroviral and lentiviral vector samples, a prerequisite to advancing the safety of gene therapy applications.

Identification of Newly Emerging Influenza Viruses by Surface-Enhanced Raman Spectroscopy.

In this work, we demonstrate in situ virus identification based on surface-enhanced Raman scattering (SERS). We hypothesized that newly emerging influenza viruses possess surface proteins and lipids that can generate distinctive Raman signals. To test this hypothesis, SERS signals were measured from the surface of a noninfluenza virus, two different influenza viruses, and a genetically shuffled influenza virus. To ensure the safety for experimenters we constructed nonreplicating pseudotyped viruses that display main influenza virus surface components. Pseudotype with influenza virus components produced enhanced Raman peaks, on gold nanoparticles, that are easily distinguishable from those of pseudotype with a noninfluenza virus component, vesicular stomatitis virus G protein (VSVG). Furthermore, virus with the surface components of a newly emerging influenza strain, A/California/04/2009 (H1N1), generated Raman peaks different from those of viruses with components of the conventional laboratory-adapted influenza strain, A/WSN/33 (H1N1). Interestingly, the virus simultaneously displaying surface components of both influenza strains, a model mutant with genome reassortment, also produced a Raman signal pattern that is clearly distinguishable from those of each strain. This work highlights that SERS can provide a powerful label-free strategy to quickly identify newly emerging and potentially fatal influenza viruses.

Scanning Electron Microscopy Imaging of Large DNA Molecules Using a Metal-Free Electro-Stain Composed of DNA-Binding Proteins and Synthetic Polymers.

This paper presents the first scanning electron microscopy (SEM)-based DNA imaging in biological samples. This novel approach incorporates a metal-free electro-stain reagent, formulated by combining DNA-binding proteins and synthetic polymers to enhance the visibility of 2-nm-thick DNA under SEM. Notably, DNA molecules stain with proteins and polymers appear as dark lines under SEM. The resulting DNA images exhibit a thickness of 15.0±4.0 nm. As SEM is the primary platform, it integrates seamlessly with various chemically functionalized large surfaces with the aid of microfluidic devices. The approach allows high-resolution imaging of various DNA structures including linear, circular, single-stranded DNA and RNA, originating from nuclear and mitochondrial genomes. Furthermore, quantum dots are successfully visualized as bright labels that are sequence-specifically incorporated into DNA molecules, which highlights the potential for SEM-based optical DNA mapping. In conclusion, DNA imaging using SEM with the novel electro-stain offers electron microscopic resolution with the ease of optical microscopy.

Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties.

Retroviral vectors offer benefits of efficient delivery and stable gene expression; however, their clinical use raises the concerns of insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSS). We have shifted the integration preferences of retroviral vectors by generating a library of viral variants with a DNA-binding domain inserted at random positions throughout murine leukemia virus Gag-Pol, then selecting for variants that are viable and exhibit altered integration properties. We found seven permissive zinc finger domain (ZFD) insertion sites throughout Gag-Pol, including within p12, reverse transcriptase, and integrase. Comprehensive genome integration analysis showed that several ZFD insertions yielded retroviral vector variants with shifted integration patterns that did not favor TSS. Furthermore, integration site analysis revealed selective integration for numerous mutants. For example, two retroviral variants with a given ZFD at appropriate positions in Gag-Pol strikingly integrated primarily into four common sites out of 3.1 x 10(9) possible human genome locations (P = 4.6 x 10(-29)). Our findings demonstrate that insertion of DNA-binding motifs into multiple locations in Gag-Pol can make considerable progress toward engineering safer retroviral vectors that integrate into a significantly narrowed pool of sites on human genome and overcome the preference for TSS.

Counting DNA molecules on a microchannel surface for quantitative analysis.

Microscopic visualization of DNA molecules is a simple, intuitive, and powerful method. Nonetheless, DNA-molecule quantification methods that employ microscopic visualization have not been reported so far. In this study, a new quantitative approach is presented that enables the counting of individual DNA molecules that have been rendered visible by fluorescence microscopy. Toward this, a microfluidic device was employed that directed DNA molecules into microchannels and deposited the molecules onto a positively charged surface. This microfluidic device had a vertically tapered channel inlet structure that prevented the accumulation of excess DNA molecules in the channel inlet while creating a tapering flow, thereby ensuring the even distribution of the DNA molecules in the microchannels. The channel heights and the density of positive charges on the surface were optimized for analysis. The linearity of this method with respect to the determination of the concentration of DNA in solutions was subsequently determined. The limit of detection was 0.48 fg/µL, which corresponds to 64 molecules of 7.25 kbp DNA in 1 µL of sample. This quantitative approach was finally used to count two types of plasmids co-transformed in an E. coli cell; a measurement that is typically considered challenging with gel electrophoresis.

Genome sequence of Vibrio sp. strain EJY3, an agarolytic marine bacterium metabolizing 3,6-anhydro-L-galactose as a sole carbon source.

The metabolic fate of 3,6-anhydro-L-galactose (L-AHG) is unknown in the global marine carbon cycle. Vibrio sp. strain EJY3 is an agarolytic marine bacterium that can utilize L-AHG as a sole carbon source. To elucidate the metabolic pathways of L-AHG, we have sequenced the complete genome of Vibrio sp. strain EJY3.

Resistive switching memory properties of layer-by-layer assembled enzyme multilayers.

The properties of enzymes, which can cause reversible changes in currents through redox reactions in solution, are of fundamental and practical importance in bio-electrochemical applications. These redox properties of enzymes are often associated with their charge-trap sites. Here, we demonstrate that reversible changes in resistance in dried lysozyme (LYS) films can be generated by an externally applied voltage as a result of charge trap/release. Based on such changes, LYS can be used as resistive switching active material for nonvolatile memory devices. In this study, cationic LYS and anionic poly(styrene sulfonate) (PSS) layers were alternately deposited onto Pt-coated silicon substrates using a layer-by-layer assembly method. Then, top electrodes were deposited onto the top of LYS/PSS multilayers to complete the fabrication of the memory-like device. The LYS/PSS multilayer devices exhibited typical resistive switching characteristics with an ON/OFF current ratio above 10(2), a fast switching speed of 100 ns and stable performance. Furthermore, the insertion of insulating polyelectrolytes (PEs) between the respective LYS layers significantly enhanced the memory performance of the devices showing a high ON/OFF current ratio of ~10(6) and low levels of power consumption.

Combinatorial latency reactivation for HIV-1 subtypes and variants.

The eradication of HIV-1 will likely require novel clinical approaches to purge the reservoir of latently infected cells from a patient. We hypothesize that this therapy should target a wide range of latent integration sites, act effectively against viral variants that have acquired mutations in their promoter regions, and function across multiple HIV-1 subtypes. By using primary CD4(+) and Jurkat cell-based in vitro HIV-1 latency models, we observe that single-agent latency reactivation therapy is ineffective against most HIV-1 subtypes. However, we demonstrate that the combination of two clinically promising drugs-namely, prostratin and suberoylanilide hydroxamic acid (SAHA)-overcomes the limitations of single-agent approaches and can act synergistically for many HIV-1 subtypes, including A, B, C, D, and F. Finally, by identifying the proviral integration position of latent Jurkat cell clones, we demonstrate that this drug combination does not significantly enhance the expression of endogenous genes nearest to the proviral integration site, indicating that its effects may be selective.

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.

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.

Computational fitness landscape for all gene-order permutations of an RNA virus.

How does the growth of a virus depend on the linear arrangement of genes in its genome? Answering this question may enhance our basic understanding of virus evolution and advance applications of viruses as live attenuated vaccines, gene-therapy vectors, or anti-tumor therapeutics. We used a mathematical model for vesicular stomatitis virus (VSV), a prototype RNA virus that encodes five genes (N-P-M-G-L), to simulate the intracellular growth of all 120 possible gene-order variants. Simulated yields of virus infection varied by 6,000-fold and were found to be most sensitive to gene-order permutations that increased levels of the L gene transcript or reduced levels of the N gene transcript, the lowest and highest expressed genes of the wild-type virus, respectively. Effects of gene order on virus growth also depended upon the host-cell environment, reflecting different resources for protein synthesis and different cell susceptibilities to infection. Moreover, by computationally deleting intergenic attenuations, which define a key mechanism of transcriptional regulation in VSV, the variation in growth associated with the 120 gene-order variants was drastically narrowed from 6,000- to 20-fold, and many variants produced higher progeny yields than wild-type. These results suggest that regulation by intergenic attenuation preceded or co-evolved with the fixation of the wild type gene order in the evolution of VSV. In summary, our models have begun to reveal how gene functions, gene regulation, and genomic organization of viruses interact with their host environments to define processes of viral growth and evolution.

Cellular Response of Ventricular-Subventricular Neural Progenitor/Stem Cells to Neonatal Hypoxic-Ischemic Brain Injury and Their Enhanced Neurogenesis.

PURPOSE: To elucidate the brain's intrinsic response to injury, we tracked the response of neural stem/progenitor cells (NSPCs) located in ventricular-subventricular zone (V-SVZ) to hypoxic-ischemic brain injury (HI). We also evaluated whether transduction of V-SVZ NSPCs with neurogenic factor NeuroD1 could enhance their neurogenesis in HI. MATERIALS AND METHODS: Unilateral HI was induced in ICR neonatal mice. To label proliferative V-SVZ NSPCs in response to HI, bromodeoxyuridine (BrdU) and retroviral particles encoding LacZ or NeuroD1/GFP were injected. The cellular responses of NSPCs were analyzed by immunohistochemistry. RESULTS: Unilateral HI increased the number of BrdU+ newly-born cells in the V-SVZ ipsilateral to the lesion while injury reduced the number of newly-born cells reaching the ipsilateral olfactory bulb, which is the programmed destination of migratory V-SVZ NSPCs in the intact brain. These newly-born cells were directed from this pathway towards the lesions. HI significantly increased the number of newly-born cells in the cortex and striatum by the altered migration of V-SVZ cells. Many of these newly-born cells differentiated into active neurons and glia. LacZ-expressing V-SVZ NSPCs also showed extensive migration towards the non-neurogenic regions ipsilateral to the lesion, and expressed the neuronal marker NeuN. NeuroD1+/GFP+ V-SVZ NSPCs almost differentiated into neurons in the peri-infarct regions. CONCLUSION: HI promotes the establishment of a substantial number of new neurons in non-neurogenic regions, suggesting intrinsic repair mechanisms of the brain, by controlling the behavior of endogenous NSPCs. The activation of NeuroD1 expression may improve the therapeutic potential of endogenous NSPCs by increasing their neuronal differentiation in HI.

Comparative study on the catalytic activity of Fe-doped ZrO2 nanoparticles without significant toxicity through chemical treatment under various pH conditions.

Despite advances in the construction of catalysts based on metal oxide nanoparticles (MO NPs) for various industrial, biomedical, and daily-life applications, the biosafety concerns about these NPs still remain. Recently, the need to analyze and improve the safety of MO NPs along with attempts to enhance their catalytic performance has been strongly perceived. Here, we prepared multiple variants of Fe-doped zirconium oxide (Fe@ZrO2) NPs under different pH conditions; then, we assessed their toxicity and finally screened the variant that exhibited the best catalytic performance. To assess the NP toxicity, the prepared NPs were introduced into three types of human cells originally obtained from different body parts likely to be most affected by NPs (skin, lung, and kidney). Experimental results from conventional cellular toxicity assays including recently available live-cell imaging indicated that none of the variants exerted severe negative effects on the viability of the human cells and most NPs were intracellular localized outside of nucleus, by which severe genotoxicity is unexpected. In contrast, Fe@ZrO2 NPs synthesized under a basic condition (pH = 13.0), exhibited the highest catalytic activities for three different reactions; each was biochemical (L-cysteine oxidation) or photochemical one (4-chlorophenol degradation and OH radical formation with benzoic acid). This study demonstrates that catalytic Fe@ZrO2 NPs with enhanced activities and modest or insignificant toxicity can be effectively developed and further suggests a potential for the use of these particles in conventional chemical reactions as well as in recently emerging biomedical and daily-life nanotechnology applications.