Dynamic bidirectional regulation of NLRC3 and gammaherpesviruses during viral latency in B lymphocytes.

While most NOD-like receptors (NLRs) are predominately expressed by innate immune cells, NLRC3, an inhibitory NLR of immune signaling, exhibits the highest expression in lymphocytes. The role of NLRC3 or any NLRs in B lymphocytes is completely unknown. Gammaherpesviruses, including human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV-68), establish latent infection in B lymphocytes, which requires elevated NF-κB. This study shows that during latent EBV infection of human B cells, viral-encoded latent membrane protein 1 (LMP1) decreases NLRC3 transcript. LMP1-induced-NF-κB activation suppresses the promoter activity of NLRC3 via p65 binding to the promoter. Conversely, NLRC3 inhibits NF-κB activation by promoting the degradation of LMP1 in a proteasome-dependent manner. In vivo, MHV-68 infection reduces Nlrc3 transcripts in splenocytes, and Nlrc3-deficient mice show greater viral latency than controls. These results reveal a bidirectional regulatory circuit in B lymphocytes, where viral latent protein LMP1 reduces NLRC3 expression, while NLRC3 disrupts gammaherpesvirus latency, which is an important step for tumorigenesis.

Pipette-free field-deployable molecular diagnostic kit for bimodal visual detection of infectious RNA viruses.

Here, we developed a field-deployable molecular diagnostic kit for the detection of RNA viruses that operates in a pipette-free manner. The kit is composed of acrylic sticks, PCR tubes, and palm-sized three-dimensional(3D)-printed heaters operated by batteries. The kit performs RNA extraction, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), and visual detection in one kit. An acrylic stick was engraved with one shallow and one deep cylindrical chamber at each end for the insertion of an FTA card and ethidium homodimer-1 (EthD-1), respectively, to perform RNA extraction/purification and bimodal visual detection of the target amplicons. First, an intercalation of EthD-1 into the target DNA initially produces fluorescence upon UV illumination. Next, the addition of a strong oxidant, in this case sodium (meta) periodate (NaIO4 ), produces intense aggregates in the presence of EthD-1-intercalated DNA, realized by electrostatic interaction. In the absence of the target amplicon, no fluorescence or aggregates are observed. Using this kit, two major infectious viruses-severe fever with thrombocytopenia syndrome virus (SFTSV) and severe acute respiratory syndrome coronavirus (SARS-CoV-2)-were successfully detected in 1 h, and the limits of detection (LOD) were approximately 1 virus µL-1 for SFTSV and 103 copies µL-1 for SARS-CoV-2 RNA. The introduced kit is portable, end-user-friendly, and can be operated in a pipette-free manner, paving the way for simple and convenient virus detection in resource-limited settings.

A DNA vaccine against GII.4 human norovirus VP1 induces blocking antibody production and T cell responses.

Human noroviruses (HuNoVs) are highly contagious and a leading cause of epidemics of acute gastroenteritis worldwide. Among the various HuNoV genotypes, GII.4 is the most prevalent cause of outbreaks. However, no vaccines have been approved for HuNoVs to date. DNA vaccines are proposed to serve as an ideal platform against HuNoV since they can be easily produced and customized to express target proteins. In this study, we constructed a CMV/R vector expressing a major structural protein, VP1, of GII.4 HuNoV (CMV/R-GII.4 HuNoV VP1). Transfection of CMV/R-GII.4 HuNoV VP1 into human embryonic kidney 293T (HEK293T) cells resulted in successful expression of VP1 proteins in vitro. Intramuscular or intradermal immunization of mice with the CMV/R-GII.4 HuNoV VP1 construct elicited the production of blocking antibodies and activation of T cell responses against GII.4 HuNoV VP1. Our collective data support the utility of CMV/R-GII.4 HuNoV VP1 as a promising DNA vaccine candidate against GII.4 HuNoV.

A study on the fabrication of metal microneedle array electrodes for ECG detection based on low melting point Bi-In-Sn alloys.

This study describes the fabrication and characteristics of microneedle array electrodes (MAEs) using Bismuth-Indium-Tin (Bi-In-Sn) alloys. The MAEs consist of 57 pyramid-shaped needles measuring 340 µm wide and 800 µm high. The fabrication process involved micromolding the alloys in a vacuum environment. Physical tests demonstrated that Bi-In-Sn MAEs have good mechanical strength, indicating their suitability for successful skin penetration. The electrode-skin interface impedance test confirmed that Bi-In-Sn MAEs successfully penetrated the skin. Impedance measurements revealed the importance of insulating the microneedle electrodes for optimal electrical performance, and a UV-curable Polyurethane Acrylate coating was applied to enhance insulation. Electrocardiogram measurements using the Bi-In-Sn MAEs demonstrated performance comparable to that of traditional Ag/AgCl electrodes, which shows promise for accurate data collection. Overall, the study demonstrates successful, minimally-invasive skin insertion, improved electrical insulation, and potential applications of Bi-In-Sn microneedle array. These findings contribute to advancements in microneedle technology for biomedical applications.

A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V.

The Japanese encephalitis virus (JEV) is prevalent in Asian countries, including Korea, Japan, China, Vietnam, and India. JEV is transmitted to humans by Culex mosquitoes. Despite extensive research efforts, no approved antiviral agents are currently available, although JE can be prevented by vaccination. DNA endonuclease-targeted CRISPR trans reporter (DETECTR) is a newly emerging CRISPR-Cas12a-based molecular diagnostic method combined with isothermal nucleic acid amplification. In this study, DETECTR with reverse transcription-recombinase polymerase amplification (RT-RPA) was effectively utilized for JEV diagnosis and detected down to 10 RNA copies for JEV genotype I (GI) and 1 × 102 copies for both GIII and GV, achieving similar sensitivity to RT-PCR while displaying no cross-reaction with other viruses. A one-tube, one-temperature format of DETECTR was further developed, and its efficiency compared with that of conventional DETECTR.

Schizophrenia-associated NRXN1 deletions induce developmental-timing- and cell-type-specific vulnerabilities in human brain organoids.

De novo mutations and copy number deletions in NRXN1 (2p16.3) pose a significant risk for schizophrenia (SCZ). It is unclear how NRXN1 deletions impact cortical development in a cell type-specific manner and disease background modulates these phenotypes. Here, we leveraged human pluripotent stem cell-derived forebrain organoid models carrying NRXN1 heterozygous deletions in isogenic and SCZ patient genetic backgrounds and conducted single-cell transcriptomic analysis over the course of brain organoid development from 3 weeks to 3.5 months. Intriguingly, while both deletions similarly impacted molecular pathways associated with ubiquitin-proteasome system, alternative splicing, and synaptic signaling in maturing glutamatergic and GABAergic neurons, SCZ-NRXN1 deletions specifically perturbed developmental trajectories of early neural progenitors and accumulated disease-specific transcriptomic signatures. Using calcium imaging, we found that both deletions led to long-lasting changes in spontaneous and synchronous neuronal networks, implicating synaptic dysfunction. Our study reveals developmental-timing- and cell-type-dependent actions of NRXN1 deletions in unique genetic contexts.

Method to Generate Dorsal Forebrain Brain Organoids from Human Pluripotent Stem Cells.

Region-specific brain organoids, such as dorsal forebrain brain organoid, have become increasingly useful to model early brain development. Importantly, these organoids provide an avenue to investigate mechanisms underlying neurodevelopmental disorders, as they undergo developmental milestones resembling early neocortical formation. These milestones include the generation of neural precursors which transition into intermediate cell types and subsequently to neurons and astrocytes, as well as the fulfillment of key neuronal maturation events such as synapse formation and pruning. Here we describe how to generate free-floating dorsal forebrain brain organoids from human pluripotent stem cells (hPSCs). We also describe validation of the organoids via cryosectioning and immunostaining. Additionally, we include an optimized protocol that allows high-quality dissociation of the brain organoids to live single cells, a critical step for downstream single-cell assays.

Kaempferol Interferes with Varicella-Zoster Virus Replication in Human Foreskin Fibroblasts.

Kaempferol, a natural flavonoid abundantly found in plants, is known to have pharmacological properties, such as anti-inflammatory and anti-cancer effects. In this study, we investigated the antiviral effects of kaempferol against a varicella-zoster virus (VZV) clinical isolate in vitro. We found that kaempferol significantly inhibited VZV replication without exhibiting cytotoxicity. Kaempferol exerted its antiviral effect at a similar stage of the VZV life cycle as acyclovir, which inhibits VZV DNA replication. Taken together, our results suggest that kaempferol inhibits VZV infection by blocking the DNA replication stage in the viral life cycle.

A CRISPR-Cas12a-based diagnostic method for multiple genotypes of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) infection is commonly reported in countries of Northeast Asia including China, Japan and South Korea. The majority of the SFTS patients are elderly and the average fatality rate is more than 10%. A rapid and sensitive diagnostic method to monitor and prevent SFTSV transmission remains an urgent clinical challenge. In this study, we developed a molecular diagnostic technique for detection of SFTSV using the CRISPR-Cas12a system combined with reverse transcription recombinase polymerase amplification (RT-RPA). Using this method, we successfully diagnosed SFTSV infections with the reaction time of 50 min from blood plasma without cross-reactivity to other viruses, supporting its application for rapid and sensitive diagnosis of SFTS.

Probing the molecular and cellular pathological mechanisms of schizophrenia using human induced pluripotent stem cell models.

With recent advancements in psychiatric genomics, as a field, "stem cell-based disease modelers" were given the exciting yet daunting task of translating the extensive list of disease-associated risks into biologically and clinically relevant information in order to deliver therapeutically meaningful leads and insights. Despite their limitations, human induced pluripotent stem cell (iPSCs) based models have greatly aided our understanding of the molecular and cellular mechanisms underlying the complex etiology of brain disorders including schizophrenia (SCZ). In this review, we summarize the major findings from studies in the past decade which utilized iPSC models to investigate cell type-specific phenotypes relevant to idiopathic SCZ and disease penetrant alleles. Across cell type differences, several biological themes emerged, serving as potential neurodevelopmental mechanisms of SCZ, including oxidative stress and mitochondrial dysfunction, depletion of progenitor pools and insufficient differentiation potential of these progenitors, and structural and functional deficits of neurons and other supporting cells. Here, we discuss both the recent progress as well as challenges and improvements needed for future studies utilizing iPSCs as a model for SCZ and other neuropsychiatric disorders.

Aggressive Treatment Including Endonasal Surgical Sequestrectomy with Vascularized Nasoseptal Flap Can Improve Outcomes of Skull Base Osteoradionecrosis.

Objective Skull base osteoradionecrosis (SB-ORN) is a serious, potentially lethal complication of radiation therapy. We aimed to review the clinical characteristics and outcomes of SB-ORN according to the extent of treatment. Design Retrospective analysis design was used for this study. Setting The study was conducted in two tertiary care hospitals. Participants Patients included who had been clinically diagnosed with SB-ORN from January 2006 to 2017. Main Outcome Measures Clinical characteristics, including demographics, predisposing factors, presenting symptoms, radiological findings, treatment modalities, and treatment outcomes, were reviewed. Treatment was classified into conservative and aggressive types. Aggressive treatment included radical surgical removal of soft tissue and bony sequestrum with the placement of vascularized tissue. Treatment outcome was analyzed in terms of clinical control, survival, and carotid artery blow out. Results Fifteen patients (11 males and 4 females) were identified during the study period. Eight patients were managed conservatively, whereas seven patients were managed with aggressive treatment. The 2-year survival was 75% in the aggressive treatment group and 15% in the conservative group (log-rank, p = 0.049). The estimated 2-year blow out free rate was 46.7% for the conservative group and 100% for the aggressive group (log-rank, p = 0.100). Conclusion In patients with SB-ORN, aggressive management, including surgical removal of sequestrum and coverage with a pedicled flap, is associated with increased survival.

Discovery and Photoisomerization of New Pyrrolosesquiterpenoids Glaciapyrroles D and E, from Deep-Sea Sediment Streptomyces sp.

Two new pyrrolosesquiterpenes, glaciapyrroles D (1) and E (2) were discovered along with the previously reported glaciapyrrole A (3) from Streptomyces sp. GGS53 strain isolated from deep-sea sediment. This study elucidated the planar structures of 1 and 2 using nuclear magnetic resonance (NMR), mass spectrometry (MS), ultraviolet (UV), and infrared (IR) spectroscopic data. The absolute configurations of the glaciapyrroles were determined by Mosher's method, circular dichroism spectroscopy, and X-ray crystallography. Under 366 nm UV irradiation, the glaciapyrroles were systematically converted to the corresponding photoglaciapyrroles (4-6) via photoisomerization, resulting in the diversification of the glaciapyrrole family compounds. The transformation of the glaciapyrrole Z to E isomers occurred in a 1:1 ratio, based on virtual validation of the photoisomerization of these olefinic compounds by 1H-NMR spectroscopy and liquid chromatography/mass spectrometry (LC/MS) analysis. Finally, when encapsulated in poly(lactic-co-glycolic acid) nanoparticles, glaciapyrrole E and photoglaciapyrrole E displayed significant inhibitory activity against influenza A virus. This is the first report of antiviral effects from glaciapyrrole family compounds, whose biological functions have only been subjected to limited studies so far.

Coordinated regulation of interferon and inflammasome signaling pathways by SARS-CoV-2 proteins.

Type I and III interferons (IFNs) and the nucleotide-binding domain (NBD) leucine-rich repeat (LRR)-containing receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome play pivotal roles in the pathogenesis of SARS-CoV-2. While optimal IFN and inflammasome responses are essential for limiting SARS-CoV-2 infection, aberrant activation of these innate immune responses is associated with COVID-19 pathogenesis. In this review, we focus our discussion on recent findings on SARS-CoV-2-induced type I and III IFNs and NLRP3 inflammasome responses and the viral proteins regulating these mechanisms.

Antiviral Activities of Ethyl Pheophorbides a and b Isolated from Aster pseudoglehnii against Influenza Viruses.

Screening of the antiviral and virucidal activities of ethanol extracts from plants endemic to the Republic of Korea revealed the inhibitory activity of a 70% ethanol extract of the whole plant of A. pseudoglehnii (APE) against influenza virus infection. Two chlorophyll derivatives, ethyl pheophorbides a and b, isolated as active components of APE, exerted virucidal effects with no evident cytotoxicity. These compounds were effective only under conditions of direct incubation with the virus, and exerted no effects on the influenza A virus (IAV) surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Interestingly, virucidal activities of ethyl pheophorbides a and b were observed against enveloped but not non-enveloped viruses, suggesting that these compounds act by affecting the integrity of the viral membrane and reducing infectivity.

Microfluidic electrical cell lysis for high-throughput and continuous production of cell-free varicella-zoster virus.

Varicella-zoster virus (VZV), the causative agent of varicella and herpes zoster, is highly cell-associated and spreads via cell-to-cell contact in tissue culture. The lack of cell-free VZV hampers studies on VZV biology as well as antiviral and vaccine development. In the present study, a poly(methylmethacrylate) microfluidic device integrated with arrays of microelectrode was fabricated to continuously electrolyse VZV-infected cells to produce cell-free viruses. By designing multiple constrictions and microelectrode arrays, a high electric field is focused on the constricted region of the microchannel to disrupt large numbers of virus-infected cells with high-throughput on a microfluidic platform. Plaque assay and scanning electron microscopy were conducted to quantify and characterize cell-free VZV produced using the microfluidic continuous-flow electrical cell lysis device. The process of microfluidic electrical cell lysis followed by subsequent filtration and virus concentration process yielded a 1.4-2.1 × 104 plaque-forming units (PFUs) per mL of cell-free VZV from 7.0 × 106 VZV-infected human foreskin fibroblasts (HFF) cells. The high electric field formed inside a microfluidic channel combined with the continuous-flow of virus-infected cells within the microchannel enabled the rapid and efficient production of high-titer cell-free virus in large quantities with relatively low input of the voltage.

SARS-CoV-2 Nonstructural Proteins 1 and 13 Suppress Caspase-1 and the NLRP3 Inflammasome Activation.

Viral infection-induced activation of inflammasome complexes has both positive and negative effects on the host. Proper activation of inflammasome complexes induces down-stream effector mechanisms that inhibit viral replication and promote viral clearance, whereas dysregulated activation has detrimental effects on the host. Coronaviruses, including SARS-CoV and MERS-CoV, encode viroporins that activate the NLRP3 inflammasome, and the severity of coronavirus disease is associated with the inflammasome activation. Although the NLRP3 inflammasome activation is implicated in the pathogenesis of coronaviruses, these viruses must evade inflammasome-mediated antiviral immune responses to establish primary replication. Screening of a complementary DNA (cDNA) library encoding 28 SARS-CoV-2 open reading frames (ORFs) showed that two nonstructural proteins (NSPs), NSP1 and NSP13, inhibited caspase-1-mediated IL-1ß activation. NSP1 amino acid residues involved in host translation shutoff and NSP13 domains responsible for helicase activity were associated with caspase-1 inhibition. In THP-1 cells, both NSP1 and NSP13 significantly reduced NLRP3-inflammasome-induced caspase-1 activity and IL-1ß secretion. These findings indicate that SARS-CoV-2 NSP1 and NSP13 are potent antagonists of the NLRP3 inflammasome.

Antiviral Activities of Quercetin and Isoquercitrin Against Human Herpesviruses.

We previously reported that the ethyl acetate (EtOAc) fraction of a 70% ethanol extract of Elaeocarpus sylvestris (ESE) inhibits varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) replication in vitro. PGG (1,2,3,4,6-penta-O-galloyl-ß-D-glucose) is a major chemical constituent of the EtOAc fraction of ESE that inhibits VZV but not HCMV replication. In this study, we comprehensively screened the chemical compounds identified in the EtOAc fraction of ESE for potential antiviral properties. Among the examined compounds, quercetin and isoquercitrin displayed potent antiviral activities against both VZV and HCMV with no significant cytotoxic effects. Both compounds strongly suppressed the expression of VZV and HCMV immediate-early (IE) genes. Our collective results indicated that, in addition to PGG, quercetin and isoquercitrin are bioactive compounds in the EtOAc fraction of ESE that effectively inhibit human herpesvirus replication.

Human cytomegalovirus IE86 protein aa 136-289 mediates STING degradation and blocks the cGAS-STING pathway.

We previously reported that human cytomegalovirus (HCMV) 86 kDa immediate-early 2 gene product (IE86) promotes proteasome-dependent degradation of STING. In the present study, we determined the specific residues of IE86 responsible for STING degradation using a STING-firefly luciferase fusion protein expression system for quantitative meas-urement of STING protein levels. IE86 amino acids (aa) 136-289 were sufficient to promote STING degradation and further induced down-regulation of 2'3'-cyclic GMP-AMP (cGAMP)-mediated IFN-ß promoter activation. Interestingly, transactivation domains (TAD) of the IE86 protein located at the N- and C-termini were required for down-regulation of Toll/interleukin-1 receptor (TIR) domain-containing adaptor-inducing interferon ß (IFN-ß) (TRIF)-mediated IFN-ß-and p65/RelA-induced NF-κB-dependent promoter activation while amino acids (aa) 136-289 had no significant effects. Our collective data suggest that the IE86 protein utilizes the aa 136-289 region to promote STING degradation and inhibit the cGAS-STING pathway.

Clinical Characteristics Other Than Intralesional Hyperdensity May Increase the Preoperative Diagnostic Accuracy of Maxillary Sinus Fungal Ball.

OBJECTIVES: This study aimed to evaluate the clinical characteristics of maxillary sinus fungus ball (MFB) to increase the preoperative diagnostic accuracy. METHODS: A retrospective review of 247 patients who underwent endoscopic sinus surgery for unilateral maxillary sinusitis from January 2015 to December 2017 at a single institution was performed. Patients with pathologically proven MFB were compared to those with unilateral chronic maxillary sinusitis (CMS). Patient demographics and computed tomography (CT) findings were evaluated. The CT features were categorized as intralesional hyperdensity (calcification), the irregular lobulated protruding lesion (fuzzy appearance), maxillary sinus full haziness without mass effect, maxillary sinus full haziness with mass effect, and others. A regression tree analysis was performed. RESULTS: In total, 247 patients were analyzed; among them, 179 (72.5%) had MFB and 68 (27.5%) had CMS. MFB showed predominance in older individuals. Among the radiological features, intralesional hyperdensity was most commonly associated with MFB. The presence of a fuzzy appearance or full opacity with mass effect was also associated with MFB. The highest area under the curve was noted with the regression tree analysis based on the model, which included the presence of intralesional hyperdensity, demographic data (age), and presence of fuzzy appearance or maxillary sinus full haziness with mass effect in case of absence of intralesional hyperdensity (0.904). CONCLUSION: A simple algorithm to optimize the preoperative diagnosis of MFB was developed. Physicians should be aware of such findings in the management of patients presenting with unilateral CMS.

Spicatoside A derived from Liriope platyphylla root ethanol extract inhibits hepatitis E virus genotype 3 replication in vitro.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans worldwide. Although hepatitis E is self-limiting without chronic infection development, HEV infection often leads to severe liver diseases causing high mortality in pregnant women in addition to chronic hepatitis and cirrhosis in immunosuppressed patients. In this study, we investigated the effect of a Liriope platyphylla ethanol extract (LPE) on HEV replication. Interestingly, LPE suppressed replication of the genotype 3 HEV replicon. Sequential solvent fractionation revealed that the ethyl acetate (EA) fraction of LPE exerts the most potent inhibitory effects. With the aid of activity-guided fractionation and multi-step column chromatography, spicatoside A was subsequently isolated in the EA fraction of LPE and specifically shown to exert inhibitory effects on replication of the genotype 3 HEV replicon. In addition, spicatoside A interfered with replication of the HEV genotype 3 strain 47832c and expression of HEV ORF2 capsid proteins. Our findings clearly support the potential utility of spicatoside A as an effective anti-HEV agent.