Discovery of Widespread Host Protein Interactions with the Pre-replicated Genome of CHIKV Using VIR-CLASP.

The primary interactions between incoming viral RNA genomes and host proteins are crucial to infection and immunity. Until now, the ability to study these events was lacking. We developed viral cross-linking and solid-phase purification (VIR-CLASP) to characterize the earliest interactions between viral RNA and cellular proteins. We investigated the infection of human cells using Chikungunya virus (CHIKV) and influenza A virus and identified hundreds of direct RNA-protein interactions. Here, we explore the biological impact of three protein classes that bind CHIKV RNA within minutes of infection. We find CHIKV RNA binds and hijacks the lipid-modifying enzyme fatty acid synthase (FASN) for pro-viral activity. We show that CHIKV genomes are N6-methyladenosine modified, and YTHDF1 binds and suppresses CHIKV replication. Finally, we find that the innate immune DNA sensor IFI16 associates with CHIKV RNA, reducing viral replication and maturation. Our findings have direct applicability to the investigation of potentially all RNA viruses.

OASL1 inhibits translation of the type I interferon-regulating transcription factor IRF7.

The production of type I interferon is essential for viral clearance but is kept under tight control to avoid unnecessary tissue damage from hyperinflammatory responses. Here we found that OASL1 inhibited translation of IRF7, the master transcription factor for type I interferon, and thus negatively regulated the robust production of type I interferon during viral infection. OASL1 inhibited the translation of IRF7 mRNA by binding to the 5' untranslated region (UTR) of IRF7 and possibly by inhibiting scanning of the 43S preinitiation complex along the message. Oasl1-/- mice were resistant to viral infection because of the greater abundance of type I interferon, which suggests that OASL1 could be a potential therapeutic target for boosting the production of type I interferon during viral infection.

Inhibition of endocytic uptake of severe acute respiratory syndrome coronavirus 2 and endo-lysosomal acidification by diphenoxylate.

Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The observed 50% effective concentrations ranged between 1.4 and 4.9 µM against the original wild-type strain and its variants. Time-of-addition experiments indicated that diphenoxylate is an entry blocker targeting a host factor involved in viral infection. Fluorescence microscopic analysis visualized that diphenoxylate prevented SARS-CoV-2 particles from penetrating the cell membrane and also impaired endo-lysosomal acidification. Diphenoxylate exhibited a synergistic inhibitory effect on SARS-CoV-2 infection in human lung epithelial Calu-3 cells when combined with a transmembrane serine protease 2 (TMPRSS2) inhibitor, nafamostat. This synergy suggested that efficient antiviral activity is achieved by blocking both TMPRSS2-mediated early and endosome-mediated late SARS-CoV-2 entry pathways. The antiviral efficacy of diphenoxylate against SARS-CoV-2 was reproducible in a human tonsil organoids system. In a transgenic mouse model expressing the obligate SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, intranasal administration of diphenoxylate (10 mg/kg/day) significantly reduced the viral RNA copy number in the lungs by 70% on day 3. This study underscores that diphenoxylate represents a promising core scaffold, warranting further exploration for chemical modifications aimed at developing a new class of clinically effective antiviral drugs against SARS-CoV-2.

Cutting Edge: The Histone Methyltransferase G9a Is Required for Silencing of Helper T Lineage-Associated Genes in Proliferating CD8 T Cells.

Helper versus cytotoxic T lineage decision in the thymus has been studied as a model for silencing of alternative lineage genes. Although the transcription factor RUNX3 is required for the initiation of Cd4 silencing in developing CD8 T cells, it is unknown how silencing of Cd4 and other helper T lineage genes is maintained. We show that the histone methyltransferase G9a is necessary for silencing helper T lineage genes in proliferating mouse CD8 T cells. Despite normal initial Cd4 downregulation, G9a-deficient CD8 T cells derepress Cd4 and other helper lineage genes during repeated division in lymphopenia or in response to tumor Ag. However, G9a was dispensable for continued silencing of those genes in CD8 T cells that respond to infection by Listeria monocytogenes These results demonstrate that G9a facilitates maintenance of cellular identity of CD8 T cells during cell division, which is further reinforced by inflammatory signals.

Design of High Corrosion Resistant Ni Alloy Using Electronic State Calculation.

Ni alloys are used as the prominent industrial material for heat-resistance and corrosion-resistance. Out of them, Inconel has excellent properties for high-temperature heat-resistant equipment as an excellent material for equipment such as ultrahigh-temperature electric furnace and or boiler. However, Ni alloys used in various areas contain high-priced metals such as Ta, Nb, or Re and their use have been limited to industrial sector so far due to the price incompetence. In this study, the alloy Ni-28Cr-4Mo-2Ti was designed by electronic state calculation by replacing the expensive material of Ta and Nb in Inconel 625 with Ti. In order to compare the corrosion properties, they were intentionally corroded in a furnace at 500 °C for 3 months and potentiodynamic polarization method was used to check the corrosion rate. The validity of the results was also confirmed by using the values of atomic radius and electronegativity. As a result, it was confirmed that the newly designed alloy had superior corrosion resistance at higher temperature than Inconel 625.

Restriction of Nonpermissive RUNX3 Protein Expression in T Lymphocytes by the Kozak Sequence.

The transcription factor Runx3 promotes differentiation of naive CD4(+) T cells into type-1 effector T (TH1) cells at the expense of TH2. TH1 cells as well as CD8(+) T cells express a subset-specific Runx3 transcript from a distal promoter, which is necessary for high protein expression. However, all T cell subsets, including naive CD4(+) T cells and TH2 cells, express a distinct transcript of Runx3 that is derived from a proximal promoter and that produces functional protein in neurons. Therefore, accumulation of RUNX3 protein generated from the proximal transcript needs to be repressed at the posttranscriptional level to preserve CD4(+) T cell capability of differentiating into TH2 cells. In this article, we show that expression of RUNX3 protein from the proximal Runx3 transcript is blocked at the level of translational initiation in T cells. A coding sequence for the proximal Runx3 mRNA is preceded by a nonoptimal context sequence for translational initiation, known as the Kozak sequence, and thus generates protein at low efficiencies and with multiple alternative translational initiations. Editing the endogenous initiation context to an "optimal" Kozak sequence in a human T cell line resulted in enhanced translation of a single RUNX3 protein derived from the proximal transcript. Furthermore, RUNX3 protein represses transcription from the proximal promoter in T cells. These results suggest that nonpermissive expression of RUNX3 protein is restricted at the translational level, and that the repression is further enforced by a transcriptional regulation for maintenance of diverse developmental plasticity of T cells for different effector subsets.

3D-printed airway model as a platform for SARS-CoV-2 infection and antiviral drug testing.

We present a bioprinted three-layered airway model with a physiologically relevant microstructure for the study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection dynamics. This model exhibited clear cell-cell junctions and mucus secretion with an efficient expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Having infected air-exposed epithelial cells in the upper layer with a minimum multiplicity of infection of 0.01, the airway model showed a marked susceptibility to SARS-CoV-2 within one-day post-infection (dpi). Furthermore, the unique longevity allowed the observation of cytopathic effects and barrier degradation for 21 dpi. The in-depth transcriptomic analysis revealed dramatic changes in gene expression affecting the infection pathway, viral proliferation, and host immune response which are consistent with COVID-19 patient data. Finally, the treatment of antiviral agents, such as remdesivir and molnupiravir, through the culture medium underlying the endothelium resulted in a marked inhibition of viral replication within the epithelium. The bioprinted airway model can be used as a manufacturable physiological platform to study disease pathogeneses and drug efficacy.

Prediction of Neoadjuvant Chemoradiotherapy Response in Rectal Cancer Patients Using Harmonized Radiomics of Multcenter 18F-FDG-PET Image.

We developed machine and deep learning models to predict chemoradiotherapy in rectal cancer using 18F-FDG PET images and harmonized image features extracted from 18F-FDG PET/CT images. Patients diagnosed with pathologic T-stage III rectal cancer with a tumor size > 2 cm were treated with neoadjuvant chemoradiotherapy. Patients with rectal cancer were divided into an internal dataset (n = 116) and an external dataset obtained from a separate institution (n = 40), which were used in the model. AUC was calculated to select image features associated with radiochemotherapy response. In the external test, the machine-learning signature extracted from 18F-FDG PET image features achieved the highest accuracy and AUC value of 0.875 and 0.896. The harmonized first-order radiomics model had a higher efficiency with accuracy and an AUC of 0.771 than the second-order model in the external test. The deep learning model using the balanced dataset showed an accuracy of 0.867 in the internal test but an accuracy of 0.557 in the external test. Deep-learning models using 18F-FDG PET images must be harmonized to demonstrate reproducibility with external data. Harmonized 18F-FDG PET image features as an element of machine learning could help predict chemoradiotherapy responses in external tests with reproducibility.

Viral crosslinking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes.

The initial interactions between incoming, pre-replicated virion RNA and host protein factors are important in infection and immunity. Yet currently there are no methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA-host protein interactions. First, host cells are infected with 4-thiouridine (4SU)-labeled RNA viruses and irradiated with 365 nm light to crosslink 4SU-labeled viral genomes and interacting proteins from host or virus. The crosslinked RNA binding proteins (RBPs) are purified by solid-phase reversible immobilization (SPRI) beads with protein-denaturing buffers, and then identified by proteomics. With VIR-CLASP, only the incoming virion RNAs are labeled with 4SU, so crosslinking events specifically occur between proteins and pre-replicated virion RNA. Since solid-phase purification under protein-denaturing conditions, rather than sequence-specific nucleic acid purification, is used to pull-down total RNA and crosslinked RBPs, this method facilitates investigation of potentially all RNA viruses, regardless of RNA sequence. Preparation of 4SU-labeled virus takes ∼7 days and VIR-CLASP takes 1 day.

ELAVL1 primarily couples mRNA stability with the 3' UTRs of interferon-stimulated genes.

Upon pathogen detection, the innate immune system triggers signaling events leading to upregulation of pro-inflammatory and anti-microbial mRNA transcripts. RNA-binding proteins (RBPs) interact with these critical mRNAs and regulate their fates at the post-transcriptional level. One such RBP is ELAVL1. Although significant progress has been made in understanding how embryonic lethal vision-like protein 1 (ELAVL1) regulates mRNAs, its target repertoire and binding distribution within an immunological context remain poorly understood. We overlap four high-throughput approaches to define its context-dependent targets and determine its regulatory impact during immune activation. ELAVL1 transitions from binding overwhelmingly intronic sites to 3' UTR sites upon immune stimulation of cells, binding previously and newly expressed mRNAs. We find that ELAVL1 mediates the RNA stability of genes that regulate pathways essential to pathogen sensing and cytokine production. Our findings reveal the importance of examining RBP regulatory impact under dynamic transcriptomic events to understand their post-transcriptional regulatory roles within specific biological circuitries.

Identification and functional analysis of antifungal immune response genes in Drosophila.

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-kappaB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.

Small molecule inhibition of human cGAS reduces total cGAMP output and cytokine expression in cells.

The cGAS-STING pathway is a major mechanism that mammalian cells utilize to detect cytoplasmic dsDNA from incoming viruses, bacteria, or self. CYCLIC GMP-AMP SYNTHASE (cGAS) is the sensor protein that directly binds dsDNAs. cGAS synthesizes cyclic GMP-AMP (cGAMP), which binds to the adaptor STIMULATOR OF INTERFERON GENES (STING), activating an INTERFERON REGULATORY FACTOR 3 (IRF3)-mediated immune response. Constitutive activation can result in interferonopathies such as Aicardi-Goutieres Syndrome (AGS) or other lupus-like autoimmune disorders. While inhibitors targeting mouse or human cGAS have been reported, the identification of a small molecule that targets both homologs of cGAS has been challenging. Here, we show that RU.521 is capable of potently and selectively inhibiting mouse and human cGAS in cell lines and human primary cells. This inhibitory activity requires the presence of cGAS, but it cannot suppress an immune response in cells activated by RNA, Toll-like receptor ligands, cGAMP, or recombinant interferon. Importantly, when RU.521 is applied to cells, the production of dsDNA-induced intracellular cGAMP is suppressed in a dose-dependent manner. Our work validates the use of RU.521 for probing DNA-induced innate immune responses and underscores its potential as an ideal scaffold towards pre-clinical development, given its potency against human and mouse cGAS.

Dual roles for the ER membrane protein complex in flavivirus infection: viral entry and protein biogenesis.

Hundreds of cellular host factors are required to support dengue virus infection, but their identity and roles are incompletely characterized. Here, we identify human host dependency factors required for efficient dengue virus-2 (DENV2) infection of human cells. We focused on two, TTC35 and TMEM111, which we previously demonstrated to be required for yellow fever virus (YFV) infection and others subsequently showed were also required by other flaviviruses. These proteins are components of the human endoplasmic reticulum membrane protein complex (EMC), which has roles in ER-associated protein biogenesis and lipid metabolism. We report that DENV, YFV and Zika virus (ZIKV) infections were strikingly inhibited, while West Nile virus infection was unchanged, in cells that lack EMC subunit 4. Furthermore, targeted depletion of EMC subunits in live mosquitoes significantly reduced DENV2 propagation in vivo. Using a novel uncoating assay, which measures interactions between host RNA-binding proteins and incoming viral RNA, we show that EMC is required at or prior to virus uncoating. Importantly, we uncovered a second and important role for the EMC. The complex is required for viral protein accumulation in a cell line harboring a ZIKV replicon, indicating that EMC participates in the complex process of viral protein biogenesis.

Development of a disposable kit with fully automatic self-shielding reactor for [18F]FDG synthesis.

A self-shielding device for the synthesis of radiopharmaceuticals was developed and fabricated in this study. Radiation exposure was minimized by the self-shielding of the kit, installation of the disposable kit in the auxiliary chamber while in a shielded state, and discharge of the kit into a radioactive waste container upon completion of the synthesis process. The developed self-shielding synthesis kit was tested by synthesizing 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) in order to verify its performance.

[Roles of F-18 FDG PET or PET/CT for the evaluation of gastrointestinal malignancies].

18F-FDG PET scan is an useful functional whole body imaging modality that images various types of malignancies with relative high sensitivity and specificity in a reasonably rapid time. It depicts a lesion based on abnormal glucose metabolism whereas CT detects malignant process mostly based on altered anatomy. In patients with gastric cancers, PET scan detects only less than 50% of early cancers and 62-98% of advanced cancers. For initial T staging, anatomical imaging with a high spatial resolution is essential. There are a few studies on the prognostic significance of FDG uptake with inconsistent results. In spite of low sensitivity for lymph node staging, the specificity of CT and PET scan are very high, and the specificity of PET scan tends to be higher than that of CT. Detection of distant metastases on PET scan is dependent on tumor histology, degree of FDG uptake in primary tumors, sites of distant metastases, etc. There are only a few data available for the evaluation of recurrence detection and treatment responses using FDG PET scan. FDG PET scan has been used in the preoperative staging of colorectal cancer with some promising results. It seems to be the most useful in restaging recurrent tumors and selecting those patients who would benefit from surgery. PET scan has a potential value in assessing treatment responses after various combination of treatments in patients with colorectal cancer.

Production of α-particle emitting ²¹¹At using 45 MeV α-beam.

Among the α-particle emitting radionuclides, (211)At is considered to be a promising radionuclide for targeted cancer therapy due to its decay properties. The range of alpha particles produced by the decay of (211)At are less than 70 µm in water with a linear energy transfer between 100 and 130 keV µm(-1), which are about the maximum relative biological effectiveness for heavy ions. It is important to note that at the present time, only a few of cyclotrons routinely produce (211)At. The direct production method is based on the nuclear reactions (209)Bi(α,2n)(211)At. Production of the radionuclide (211)At was carried out using the MC-50 cyclotron at the Korea Institute of Radiological and Medical Sciences (KIRAMS). To ensure high beam current, the α-beam was extracted with an initial energy of 45 MeV, which was degraded to obtain the appropriate α-beam energy. The calculations of beam energy degradation were performed utilizing the MCNPX. Alumina-baked targets were prepared by heating the bismuth metal powder onto a circular cavity in a furnace. When using an E(α, av) of 29.17 MeV, the very small contribution of (210)At confirms the right choice of the irradiation energy to obtain a pure production of (211)At isotope.

Genome-wide profiling of in vivo LPS-responsive genes in splenic myeloid cells.

Lipopolysaccharide (LPS), the major causative agent of bacterial sepsis, has been used by many laboratories in genome-wide expression profiling of the LPS response. However, these studies have predominantly used in vitro cultured macrophages (Macs), which may not accurately reflect the LPS response of these innate immune cells in vivo. To overcome this limitation and to identify inflammatory genes in vivo, we have profiled genome-wide expression patterns in non-lymphoid, splenic myeloid cells extracted directly from LPS-treated mice. Genes encoding factors known to be involved in mediating or regulating inflammatory processes, such as cytokines and chemokines, as well as many genes whose immunological functions are not well known, were strongly induced by LPS after 3 h or 8 h of treatment. Most of the highly LPS-responsive genes that we randomly selected from the microarray data were independently confirmed by quantitative RT-PCR, implying that our microarray data are quite reliable. When our in vivo data were compared to previously reported microarray data for in vitro LPS-treated Macs, a significant proportion (∼20%) of the in vivo LPS-responsive genes defined in this study were specific to cells exposed to LPS in vivo, but a larger proportion of them (∼60%) were influenced by LPS in both in vitro and in vivo settings. This result indicates that our in vivo LPS-responsive gene set includes not only previously identified in vitro LPS-responsive genes but also novel LPS-responsive genes. Both types of genes would be a valuable resource in the future for understanding inflammatory responses in vivo.

Effect of bisphosphonates on anodized and heat-treated titanium surfaces: an animal experimental study.

BACKGROUND: Recent investigations reported that osseointegration of titanium implants can be significantly reinforced with a nanostructure treated with anodic oxidation and heat treatment. This experimental study investigates the effect of bisphosphonates on the nanotubular implant surface in rats. METHODS: Thirty-six titanium implants were divided into three groups: 1) machine-turned (MT), 2) anodized and heat-treated (AH), and 3) anodized and heat- and bisphosphonate-treated (AHB) groups. The 36 implants were randomly placed in both tibias of 18 male Wistar rats. After 2 and 4 weeks, the levels of osseointegration of the implants were evaluated by a removal torque test and microcomputerized tomography (µCT). Peri-implant bone tissue on the extracted region was examined for the expression of type I collagen and osteocalcin. RESULTS: The AHB group showed the highest removal torque at 2 and 4 weeks (13.92 ± 1.51 Ncm and 18.10 ± 2.15 Ncm, respectively) followed, in order, by the AH group (11.63 ± 1.58 Ncm at 2 weeks and 14.80 ± 2.34 Ncm at 4 weeks) and MT group (4.30 ± 0.76 Ncm at 2 weeks and 6.20 ± 1.33 Ncm at 4 weeks) with statistically significant differences between the MT and other two groups at both time points. µCT images also revealed a denser appearance around implants in the AHB group than in the other groups. Levels of type I collagen and osteocalcin expression were similar between the MT and AH groups; however, the values were significantly higher in the AHB group compared to the other groups, which were 220.85% ± 71.09% and 363.04% ± 100.21%, respectively (P <0.05). CONCLUSION: Within the limits of this experiment, it was concluded that surface loading with bisphosphonates significantly improved the degree of osseointegration of titanium implants with a nanostructure.

Effect of polymer molecular weight on the fiber morphology of electrospun mats.

In this work, different fractions of solvent-induced polymer degraded solution were mixed with freshly prepared solution of same polymer, and its effect on fiber morphology of electrospun mats was investigated. Nylon-6 solution in formic acid was allowed to degrade for 3 weeks and different fractions of it were mixed with freshly prepared nylon-6 solution to get the electrospun mats. FE-SEM images of the mats indicated that the a large amount of sub-nanofibers (<50 nm in diameter) in the form of spider-net like structures were achieved by tailoring the amount of solvent degraded polymer solution in the freshly prepared nylon-6 solution. Large quantity of these ultrafine sub-nanofibers present in electrospun nylon-6 mats could increase its hydrophilicity and mechanical strength. The decreased average pore diameter and increased BET surface area of the mat, caused by spider-net like structure, can make it as a potential candidate for air/water filtration.