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Background: The quantification of mRNA expression in urinary sediments is a reliable biomarker for various diseases. However, few studies have investigated the clinical relevance of urinary mRNA levels in IgA nephropathy (IgAN). Thus, we investigated the expression of urinary mRNAs and their clinical significance in IgAN. Methods: Overall, 200 patients with biopsy-proven IgAN, 48 disease controls, and 76 healthy controls were enrolled. We identified the differential expression of mRNAs in renal tissue between patients with IgAN and normal subjects using the Gene Expression Omnibus dataset and selected candidate mRNAs. mRNA expression in the urinary sediment was measured using quantitative real-time polymerase chain reaction. Associations between urinary mRNA levels and clinicopathological parameters were analyzed and the predictive value of mRNAs for disease progression was evaluated. Results: The urinary expression of CCL2, CD14, DNMT1, FKBP5, Nephrin, and IL-6 was significantly upregulated in patients with IgAN compared with healthy controls. C3, FLOT1, and Podocin levels were significantly correlated with renal function, where C3, FLOT1, and TfR levels were significantly correlated with urinary protein excretion. During follow-up, 26 (13.0%) patients with IgAN experienced disease progression, defined as a greater than 50% reduction in the estimated glomerular filtration rate or progression to end-stage renal disease. Urinary mRNA levels of FLOT1 (HR 3.706, 95% CI 1.373–10.005, P = 0.010) were independently associated with an increased risk of disease progression. Conclusions: Our results suggest that urinary sediment mRNAs are a useful biomarker in IgAN patients. Further studies with larger sample sizes and longer follow-up durations are required. [ABSTRACT FROM AUTHOR]

BACKGROUND: Chronic stroke can impair cardiopulmonary function, mobility, and daily activities. This study assessed the impact of robot-assisted gait training (RAGT) on such impairments. OBJECTIVE: To investigate the effects of robot-assisted gait training on cardiopulmonary function, walking ability, lower extremity function and strength, activities of daily living (ADLs), and blood test results among individuals with chronic stroke. METHODS: A multicenter, prospective, single-blinded, randomized controlled trial with 22 chronic stroke participants compared RAGT against a control exercise regimen. RAGT involved three days weekly sessions of high-intensity interval training for 8 weeks (24 sessions) with a Morning Walk® device. The control group also performed home exercises. (24 sessions) Measures included VO2max, Functional Ambulatory Category, 2-minute walk test, 10-meter walk test, Motricity Index-Lower, Korean version of the Fugl-Meyer Assessment Scale, Modified Barthel Index, Berg Balance Scale, muscle strength, InBody body composition, and blood tests (cholesterol, lipid, glucose). RESULTS: RAGT significantly improved VO2max, gait, balance, and lower limb strength compared with controls, with significant improvements in 2-minute walk test, 10-meter walk test, Motricity Index-Lower, and Fugl-Meyer Assessment outcomes. No changes were seen in muscle mass or blood markers. CONCLUSION: RAGT enhances cardiopulmonary function and ambulatory capacity in chronic stroke patients, underscoring its potential in stroke rehabilitation. [ABSTRACT FROM AUTHOR]

SUMMARY: Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright‐red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5‐Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus‐induced gene silencing showed that ZEP silencing also results in bright‐red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations. Significance Statement: Despite health benefits, such as macular protection and antioxidant effects, most crops contain low levels of zeaxanthin. Orange pepper is an important source of zeaxanthin which is scarce in our diet. We demonstrated that a leaky mutation in zeaxanthin epoxidase leads to the development of bright‐red fruit, elevated zeaxanthin levels, and affects endogenous plant hormones in pepper. Our findings suggest a novel breeding strategy for developing red pepper cultivars with high levels of zeaxanthin. [ABSTRACT FROM AUTHOR]

Key message: Segmental introgression and advanced backcross lines were developed and validated as important tools for improving agronomically important traits in pepper, offering improved sensitivity in detecting quantitative trait loci for breeding. Segmental introgression lines (SILs) and advanced backcross lines (ABs) can accelerate genetics and genomics research and breeding in crop plants. This study presents the development of a complete collection of SILs and ABs in pepper using Capsicum annuum cv. ‘CM334’ as the recipient parent and Capsicum baccatum ‘PBC81’, which displays various agronomically important traits including powdery mildew and anthracnose resistance, as donor parent. Using embryo rescue to overcome abortion in interspecific crosses, and marker-assisted selection with genotyping-in-thousands by sequencing (GT-seq) to develop SILs and ABs containing different segments of the C. baccatum genome, we obtained 63 SILs and 44 ABs, covering 94.8% of the C. baccatum genome. We characterized them for traits including powdery mildew resistance, anthracnose resistance, anthocyanin accumulation, trichome density, plant architecture, and fruit morphology. We validated previously known loci for these traits and discovered new sources of variation and quantitative trait loci (QTLs). A total of 15 QTLs were identified, including four for anthracnose resistance with three novel loci, seven for plant architecture, and four for fruit morphology. This is the first complete collection of pepper SILs and ABs validated for agronomic traits and will enhance QTL detection and serve as valuable breeding resources. Further, these SILs and ABs will be useful for comparative genomics and to better understand the genetic mechanisms underlying important agronomic traits in pepper, ultimately leading to improved crop productivity and sustainability. [ABSTRACT FROM AUTHOR]

Background: Anthracnose is a fungal disease caused by Colletotrichum spp. that has a significant impact on worldwide pepper production. Colletotrichum scovillei is the most common pathogenic anthracnose-causing species in the Republic of Korea. Results: The resistances of 197 pepper (Capsicum chinense) accessions deposited in Korea's National Agrobiodiversity Center were evaluated for their response against the virulent pathogens Colletotrichum acutatum isolate 'KSCa-1' and C. scovillei isolate 'Hana') in the field and in vitro methods for three consecutive years (2018 to 2020). The severity of the disease was recorded and compared between inoculation methods. Six phenotypically resistant pepper accessions were selected based on three years of disease data. All of the selected resistant pepper accessions outperformed the control resistant pepper in terms of resistance (PI 594,137). A genome-wide association study (GWAS) was carried out to identify single nucleotide polymorphisms (SNPs) associated with anthracnose resistance. An association analysis was performed using 53,518 SNPs and the disease score of the 2020 field and in vitro experiment results. Both field and in vitro experiments revealed 25 and 32 significantly associated SNPs, respectively. These SNPs were found on all chromosomes except Ch06 and Ch07 in the field experiment, whereas in the in vitro experiment they were found on all chromosomes except Ch04 and Ch11. Conclusion: In this study, six resistant C. chinense accessions were selected. Additionally, in this study, significantly associated SNPs were found in a gene that codes for a protein kinase receptor, such as serine/threonine-protein kinase, and other genes that are known to be involved in disease resistance. This may strengthen the role of these genes in the development of anthracnose resistance in Capsicum spp. As a result, the SNPs discovered to be strongly linked in this study can be used to identify a potential marker for selecting pepper material resistant to anthracnose, which will assist in the development of resistant varieties. [ABSTRACT FROM AUTHOR]

The increasing use of rapidly fluctuating renewable energy sources, such as sunlight, has necessitated the use of supercapacitors, which are a type of energy storage system with high power. Chemically exfoliated graphene oxide (GO) is a representative starting material in the fabrication of supercapacitor electrodes based on reduced GO (rGO). However, the restacking of rGO sheets driven by π–π stacking interactions leads to a significant decrease in the electrochemically active surface area, leading to a loss of energy density. Here, to effectively inhibit restacking and construct a three-dimensional wrinkled structure of rGO (3DWG), we propose an agarose gel-templating method that uses agarose gel as a soft and removable template. The 3DWG, prepared via the sequential steps of gelation, freeze-drying, and calcination, exhibits a macroporous 3D structure and 5.5-fold higher specific capacitance than that of rGO restacked without the agarose template. Further, we demonstrate a "gel-stamping" method to fabricate thin-line patterned 3DWG, which involves the gelation of the GO–agarose gel within micrometer-sized channels of a customized polydimethylsiloxane (PDMS) mold. As an easy and low-cost manufacturing process, the proposed agarose gel templating method could provide a promising strategy for the 3D structuring of rGO. [ABSTRACT FROM AUTHOR]

Flower production provides the foundation for crop yield and increased profits. Capsicum annuum is a pepper species with a sympodial shoot structure with solitary flowers. By contrast, C. chinense produces multiple flowers per node. C. annuum accounts for 80% of pepper production worldwide. The identification of C. chinense genes that control multiple flowers and their transfer into C. annuum may open the way to increasing fruit yield. In this study, we dissected the genetic factors were dissected controlling the multiple-flower-per-node trait in Capsicum. 85 recombinant inbred lines (RILs) between the contrasting C. annuum 'TF68' and C. chinense 'Habanero' accessions were phenotyped and genotyped. Quantitative Trait Loci (QTL) analysis identified four novel QTLs on chromosomes 1, 2, 7, and 11 that accounted for 65% of the total phenotypic variation. Genome-wide association study was also performed on a panel of 276 genotyped and phenotyped C. annuum accessions, which revealed 28 regions significantly associated with the multiple-flower trait, of which three overlapped the identified QTLs. Five candidate genes involved in the development of the shoot and flower meristems were identified and these genes could cause multiple flowers per node in pepper. These results contribute to our understanding of multiple flower formation in Capsicum and will be useful to develop high-yielding cultivars. [ABSTRACT FROM AUTHOR]

Genotyping by sequencing (GBS) enables genotyping of multiple loci at low cost. However, the single nucleotide polymorphisms (SNPs) revealed by GBS tend to be randomly distributed between individuals, limiting their direct comparisons without applying the various filter options to obtain a comparable dataset of SNPs. Here, we developed a panel of a multiplex targeted sequencing method, genotyping-in-thousands by sequencing (GT-seq), to genotype SNPs in Capsicum spp. Previously developed Fluidigm® SNP markers were converted to GT-seq markers and combined with new GT-seq markers developed using SNP information obtained through GBS. We then optimized multiplex PCR conditions: we obtained the highest genotyping rate when the first PCR consisted of 25 cycles. In addition, we determined that 101 primer pairs performed best when amplifying target sequences of 79 bp. We minimized interference of multiplex PCR by primer dimer formation using the PrimerPooler program. Using our GT-seq pipeline on Illumina Miseq and Nextseq platforms, we genotyped up to 1,500 (Miseq) and 1,300 (Nextseq) samples for the optimum panel size of 100 loci. To allow the genotyping of Capsicum species, we designed 332 informative GT-seq markers from Fluidigm SNP markers and GBS-derived SNPs. This study illustrates the first application of GT-seq in crop plants. The GT-seq marker set developed here will be a useful tool for molecular breeding of peppers in the future. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) is the most prevalent and internal modification of eukaryotic mRNA. Multiple m6A methylation sites have been identified in the viral RNA genome and transcripts of DNA viruses in recent years. m6A modification is involved in all the phases of RNA metabolism, including RNA stability, splicing, nuclear exporting, RNA folding, translational modulation, and RNA degradation. Three protein groups, methyltransferases (m6A-writers), demethylases (m6A-erasers), and m6A-binding proteins (m6A-readers) regulate this dynamic reversible process. Here, we have reviewed the role of m6A modification dictating viral replication, morphogenesis, life cycle, and its contribution to disease progression. A better understanding of the m6A methylation process during viral pathogenesis is required to reveal novel approaches to combat the virus-associated diseases. [ABSTRACT FROM AUTHOR]

Pepper (Capsicum spp.) fruit-related traits are critical determinants of quality. These traits are controlled by quantitatively inherited genes for which marker-assisted selection (MAS) has proven insufficiently effective. Here, we evaluated the potential of genomic selection, in which genotype and phenotype data for a training population are used to predict phenotypes of a test population with only genotype data, for predicting fruit-related traits in pepper. We measured five fruit traits (fruit length, fruit shape, fruit width, fruit weight, and pericarp thickness) in 351 accessions from the pepper core collection, including 229 Capsicum annuum , 48 Capsicum baccatum , 48 Capsicum chinense , 25 Capsicum frutescens , and 1 Capsicum chacoense in 4 years at two different locations and genotyped these accessions using genotyping-by-sequencing. Among the whole core collection, considering its genetic distance and sexual incompatibility, we only included 302 C. annum complex (229 C. annuum , 48 C. chinense , and 25 C. frutescens) into further analysis. We used phenotypic and genotypic data to investigate genomic prediction models, marker density, and effects of population structure. Among 10 genomic prediction methods tested, Reproducing Kernel Hilbert Space (RKHS) produced the highest prediction accuracies (measured as correlation between predicted values and observed values) across the traits, with accuracies of 0.75, 0.73, 0.84, 0.83, and 0.82 for fruit length, fruit shape, fruit width, fruit weight, and pericarp thickness, respectively. Overall, prediction accuracies were positively correlated with the number of markers for fruit traits. We tested our genomic selection models in a separate population of recombinant inbred lines derived from two parental lines from the core collection. Despite the large difference in genetic diversity between the training population and the test population, we obtained moderate prediction accuracies of 0.32, 0.34, 0.50, and 0.48 for fruit length, fruit shape, fruit width, and fruit weight, respectively. This use of genomic selection for fruit-related traits demonstrates the potential use of core collections and genomic selection as tools for crop improvement. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to evaluate the possibility of attic cholesteatomas concealed within a tiny retraction of the pars flaccida (classification of Tos and Poulsen type I or II attic retraction) in patients with an intact pars tensa of the tympanic membrane. Methods: The clinical records of patients with a tiny retraction of the pars flaccida and an intact pars tensa of the tympanic membrane who presented to the ear clinic of a tertiary care medical center for the first time between March 2012 and February 2015 were retrospectively reviewed. All patients who had an abnormal pars flaccida of the tympanic membrane were recommended to undergo temporal bone computed tomography (CT) scans. In cases of a soft tissue density lesion within Prussak's space, an exploratory operation was recommended. Results: Among 1320 adult patients, 146 patients (n = 168 ears) who had a tiny attic retraction with a normal pars tensa in unilateral or bilateral ears underwent temporal bone CT scans, and 18 ears had a soft tissue density lesion within Prussak's space. Among the ears with a tiny retraction of the pars flaccida and a normal pars tensa, an attic cholesteatoma was suspected in 10.7% (n = 18 ears) of cases based on the CT scans. After exploratory operations, 2% of patients who underwent CT scans (3 out of 146 patients) and 23% of patients who had a soft tissue density lesion within Prussak's space on CT scans (3 out of 13 operations) had an attic cholesteatoma. Conclusion: All attic retractions which are even in cases of Tos type I or II should be examined closely using endoscopy, microscopy, and, if necessary, temporal bone CT scan. [ABSTRACT FROM AUTHOR]

Interferon (IFN) stimulates a whole repertoire of cellular genes, collectively referred to as ISGs (Interferon-stimulated genes). ISG20, a 3´-5´ exonuclease enzyme, has been previously shown to bind and degrade hepatitis B Virus (HBV) transcripts. Here, we show that the N6-methyladenosine (m6A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m6A reader protein, YTHDF2. Previously, we identified a unique m6A site within HBV transcripts and confirmed that methylation at nucleotide A1907 regulates HBV lifecycle. In this report, we now show that the methylation at A1907 is a critical regulator of IFN-α mediated decay of HBV RNA. We observed that the HBV RNAs become less sensitive to ISG20 mediated degradation when methyltransferase enzymes or m6A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20D94G, we further demonstrated that ISG20 forms a complex with m6A modified HBV RNA and YTHDF2 protein. Due to terminal redundancy, HBV genomic nucleotide A1907 position is acquired two times by pregenomic RNA (pgRNA) during transcription and therefore the sites of methylation are encoded within 5´ and 3´ epsilon stem loops. We generated HBV mutants that lack m6A site at either one (5´ or 3´) or both the termini (5´& 3´). Using these mutants, we demonstrated that m6A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m6A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m6A modification of RNA in IFN-α induced viral RNA degradation and proposes a new role of YTHDF2 protein as a cofactor required for IFN-α mediated viral RNA degradation. Author summary: Hepatitis B Virus (HBV) is a DNA virus but replicates through a transitional pregenomic RNA (pgRNA) synthesized by reverse transcription. Interferon stimulated antiviral RNase, ISG20 selectively binds to the lower epsilon stem loop of HBV RNA and causes their degradation. Surprisingly this ISG20 binding site is chemically modified by N6-methyladenosine addition to A1907 residue, which resides in the lower region of the epsilon stem loop. This single m6A site occurs twice in due to terminal redundancy of sequences in the pgRNA. We demonstrated herein that IFN-α-induced ISG20 can selectively degrade m6A modified HBV RNA. Using a combined strategy of silencing cellular methyltransferases, m6A binding protein YTHDF2 and the m6A sites mutants, we show that HBV transcripts are resistant to either IFN-α treatment or ectopically introduced ISG20 mediated degradation. YTHDF2 is an m6A binding protein which makes the HBV RNAs unstable. YTHDF2 protein forms a complex with IFN-α stimulated ISG20 and executes the nuclease digestion of the recruited m6A modified transcripts. Absence of cellular m6A machinery (methyltransferases or m6A reader proteins) makes the HBV RNA unresponsive to ISG20 mediated decay. This study provides molecular explanation of IFN-α mediated degradation of m6A modified HBV RNAs. [ABSTRACT FROM AUTHOR]

Subspecies of Brassica rapa are morphologically and genetically diverse, and include a variety of fresh vegetables grown worldwide. Among them, turnip (B. rapa subsp. rapa) produces a large bulbous taproot, and thus is primarily consumed as a root vegetable in Europe and Asia. In comparison to Chinese cabbage (B. rapa subsp. pekinensis), however, genetic analysis and breeding of turnip is hampered in practice due in part to scarcity of useful genetic resources. In this study, we produced a doubled haploid (DH) line of Ganghwa turnip, an heirloom specialty crop in Korea that is usually propagated by open pollination. Microspores were isolated from young flower buds of Ganghwa turnip, and shoots and roots were sequentially regenerated in vitro. Chromosome doubling was induced with the colchicine treatment, and verified by flow cytometry analysis. The G14 DH line displayed uniformity in overall morphology compared to heterogeneous commercial Ganghwa turnips. The whole genome of G14 was sequenced on an Illumina HiSeq 4000 platform, and the reads mapped onto the B. rapa reference genome identified 1,163,399 SNPs and 779,700 indels. Despite high similarity in overall genome sequence, turnips and Chinese cabbage have different compositions of transposable elements (TEs). In particular, long terminal repeat (LTR) retrotransposons are more enriched in turnips than in Chinese cabbage genomes, in which the gypsy elements are classified as major LTR sequences in the turnip genome. These findings suggest that subspecies-specific TE divergence is in part responsible for huge phenotypic variations observed within the same species. [ABSTRACT FROM AUTHOR]

Finding the same individual across cameras in disjoint views at different locations and times, which is known as person re-identification (re-id), is an important but difficult task in intelligent visual surveillance. However, to build a practical re-id system for large-scale and crowdsourced environments, the existing approaches are largely unsuitable because of their high model complexity. In this paper, we present a deep feature learning framework for automated large-scale person re-id with low computational cost and memory usage. The experimental results show that the proposed framework is comparable to the state-of-the-art methods while having low model complexity. [ABSTRACT FROM AUTHOR]

The Visual Internet of Things has received much attention in recent years due to its ability to get the object location via image information of the scene, attach the visual label to the object, and then return information of scene objects to the network. In particular, face recognition is one of the most suitable means to Visual IoT because face feature is inherent label for human being. However, current state-of-the-art face recognition methods based on huge deep neural networks are difficult to apply in the embedded platform for Visual IoT due to the lack of computational resources. To solve this problem, we present compact deep neural network-based face recognition method for Visual Internet of Things. The proposed method has a low model complexity to operate in an embedded environment while using deep neural networks, which is strong against posture and illumination changes. We show competitive accuracy and performance results for the LFW verification benchmark and the collected mobile face recognition dataset. Additionally, we demonstrate that the implementation of the proposed system can be run in real time on the Android-based mobile embedded platform. [ABSTRACT FROM AUTHOR]

The liver-specific microRNA miR-122, which has essential roles in liver development and metabolism, is a key proviral factor for hepatitis C virus (HCV). Despite its crucial role in the liver and HCV life cycle, little is known about the molecular mechanism of miR-122 expression regulation by HCV infection. Here, we show that the HCV core protein downregulates the abundance of miR-122 by promoting its destabilization via the inhibition of GLD-2, a non-canonical cytoplasmic poly(A) polymerase. The decrease in miR-122 expression resulted in the dysregulation of the known functions of miR-122, including its proviral activity for HCV. By high-throughput sequencing of small RNAs from human liver biopsies, we found that the 22-nucleotide (nt) prototype miR-122 is modified at its 3′ end by 3′-terminal non-templated and templated nucleotide additions. Remarkably, the proportion of miR-122 isomers bearing a single nucleotide tail of any ribonucleotide decreased in liver specimens from patients with HCV. We found that these single-nucleotide-tailed miR-122 isomers display increased miRNA activity and stability over the 22-nt prototype miR-122 and that the 3′-terminal extension is catalyzed by the unique terminal nucleotidyl transferase activity of GLD-2, which is capable of adding any single ribonucleotide without preference of adenylate to the miR-122 3′ end. The HCV core protein specifically inhibited GLD-2, and its interaction with GLD-2 in the cytoplasm was found to be responsible for miR-122 downregulation. Collectively, our results provide new insights into the regulatory role of the HCV core protein in controlling viral RNA abundance and miR-122 functions through miR-122 stability modulation. [ABSTRACT FROM AUTHOR]

In recent years, the convergence of IT space and physical space is increasingly studied. In the legacy IT-based systems, developments of services were focusing on just the cyber space. However, as ubiquitous computing environment is expanding into the real world, considerations about how to design and develop the systems for ensuring the interoperability between two spaces must be taken. For indeed converging IT/physical spaces and ensuring the ubiquity, a new model to efficiently identify a moving object needs to be established. Although the identifier information resulted from successful authentication procedure is used in the most security systems, each authentication method adopts a variety of identifiable information (II) specification. So in this paper, we suggest a scheme to access any ubiquitous service with single authentication at initial stage for efficiently identifying an object moving multiple convergence spaces by relaying the II along the movement. This is performed by enabling distributed smart cameras to deliver II of the identified moving object. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The hepatitis C virus (HCV) internal ribosome entry site (IRES) that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA)-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts) 277–343. Based on their antiviral activity, we mapped a druggable region (nts 313–343) where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5′ or 3′ direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens. [ABSTRACT FROM AUTHOR]

Background and Purpose: Hepatitis C virus (HCV) infection is responsible for various chronic inflammatory liver diseases. Here, we have identified a naturally occurring compound with anti-HCV activity and have elucidated its mode of antiviral action.Experimental Approach: Luciferase reporter and real-time RT-PCR assays were used to measure HCV replication. Western blot, fluorescence-labelled HCV replicons and infectious clones were employed to quantitate expression levels of viral proteins. Resistant HCV mutant mapping, in vitro NS3 protease, helicase, NS5B polymerase and drug affinity responsive target stability assays were also used to study the antiviral mechanism.Key Results: A resveratrol tetramer, vitisin B from grapevine root extract showed high potency against HCV replication (EC50 = 6 nM) with relatively low cytotoxicity (EC50 >10 μM). Combined treatment of vitisin B with an NS5B polymerase inhibitor (sofosbuvir) exhibited a synergistic or at least additive antiviral activity. Analysis of a number of vitisin B-resistant HCV variants suggested an NS3 helicase as its potential target. We confirmed a direct binding between vitisin B and a purified NS3 helicase in vitro. Vitisin B was a potent inhibitor of a HCV NS3 helicase (IC50 = 3 nM). In vivo, Finally, we observed a preferred tissue distribution of vitisin B in the liver after i.p. injection in rats, at clinically attainable concentrations. Conclusion and Implications Vitisin B is one of the most potent HCV helicase inhibitors identified so far. Vitisin B is thus a prime candidate to be developed as the first HCV drug derived from natural products. [ABSTRACT FROM AUTHOR]

Objectives Hepatitis C virus (HCV) infection causes chronic liver disease and is a major public health problem worldwide. The aim of this study was to evaluate the potential of Monascus pigment derivatives, which were derived from a microbial secondary metabolite synthesized from polyketides by Monascus spp., as HCV antiviral agents. Methods We performed an in vitro RNA-dependent RNA polymerase (RdRp) assay to screen for HCV RdRp inhibitors. The anti-HCV activity of RdRp inhibitors in HCV-replicating cells was evaluated by quantification of the RNA viral genome. Molecular docking analysis was performed to predict the binding sites of the selected RdRp inhibitors. Results We have identified a Monascus pigment and its derivatives as inhibitors of the HCV NS5B RdRp. A group of Monascus orange pigment (MOP) amino acid derivatives, in which the reactive oxygen moiety was changed to amino acids, significantly inhibited HCV replication. Further, combination of the MOP derivatives (Phe, Val or Leu conjugates) with interferon (IFN)-α inhibited HCV replication more than IFN-α treatment alone. Lastly, molecular docking studies indicate the inhibitors may bind to a thumb subdomain allosteric site of NS5B. The antiviral activity of the MOP derivatives was related to a modulation of the mevalonate pathway, since the mevalonate-induced increase in HCV replication was suppressed by the MOP compounds. Conclusions Our results identify amino acid derivatives of MOP as potential anti-HCV agents and suggest that their combination with IFN-α might offer an alternative strategy for the control of HCV replication. [ABSTRACT FROM PUBLISHER]

Background: Adjuvant Online (AOL) is web-accessible risk-assessment model that predicts the mortality and the benefits of adjuvant therapy (www.newadjuvantonline.com). AOL has never been validated for Asian colon cancer patients. Methods: Using the Yonsei Tumor Registry database, patients who were treated within the Yonsei University Health System between 1990 and 2005 for T1-4, N0-2, and M0 colon cancer were included in the calculations for survival. Observed and predicted 5-year overall survival was compared for each patient. Results: The median age of the study population of 1431 patients was 60 years (range, 15–87 years), and the median follow-up duration was 7.9 years (range, 0.06–19.8 years). The predicted 5-year overall survival rate (77.7%) and observed survival (79.5%) was not statistically different (95% Confidential interval, 76.3–81.5) in all patients. Predicted outcomes were within 95% confidential interval of observed survival in both stage II and III disease, including most demographic and pathologic subgroups. Moreover, AOL more accurately predicted OS for patients with stage II than stage III. Conclusions: AOL tended to offer reliable prediction for 5-year overall survival and could be used as a decision making tool for adjuvant treatment in Korean colon cancer patients whose prognosis is similar to other Asian patients. [ABSTRACT FROM AUTHOR]

Pulmonary manifestations of benign metastasizing leiomyoma (BML) usually include multiple well-defined, round, bilateral nodules. Low-grade endometrial stromal sarcoma (LG-ESS) is a rare uterine tumor. A 70-year-old woman visited the clinic complaining of acute cough and dyspnea in April 2017. Chest computed tomography (CT) revealed pneumothorax and multiple pulmonary nodules. She had a history of hysterectomy for uterine leiomyoma 23 years ago. Biopsy revealed that the pulmonary masses were consistent with BML. However, the patient had two subsequent episodes of acute, recurrent respiratory distress, accompanied by massive pleural effusions and hydropneumothorax over the next two years. A chest CT performed for acute dyspnea revealed large and multiple hydropneumothoraces. The size and distribution of pulmonary masses were aggravated along with cystic changes and bilateral pleural effusions. Given this aggressive feature, additional immunohistochemical findings and gynecologic pathologist review confirmed the correct diagnosis to be LG-ESS. After initiating anti-estrogen therapy, the patient achieved a partial response, without recurrence of symptoms, for 28 months. Metastatic LG-ESS responds well to anti-hormonal therapy. If the clinical pattern of a disease is different than expected, the possibility of a correction in the diagnosis should be considered. [ABSTRACT FROM AUTHOR]

Recent outbreaks of zoonotic coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused tremendous casualties and great economic shock. Although some repurposed drugs have shown potential therapeutic efficacy in clinical trials, specific therapeutic agents targeting coronaviruses have not yet been developed. During coronavirus replication, a replicase gene cluster, including RNA-dependent RNA polymerase (RdRp), is alternatively translated via a process called -1 programmed ribosomal frameshift (−1 PRF) by an RNA pseudoknot structure encoded in viral RNAs. The coronavirus frameshifting has been identified previously as a target for antiviral therapy. In this study, the frameshifting efficiencies of MERS-CoV, SARS-CoV and SARS-CoV-2 were determined using an in vitro −1 PRF assay system. Our group has searched approximately 9689 small molecules to identify potential −1 PRF inhibitors. Herein, we found that a novel compound, 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline (KCB261770), inhibits the frameshifting of MERS-CoV and effectively suppresses viral propagation in MERS-CoV-infected cells. The inhibitory effects of 87 derivatives of furo[2,3-b]quinolines were also examined showing less prominent inhibitory effect when compared to compound KCB261770. We demonstrated that KCB261770 inhibits the frameshifting without suppressing cap-dependent translation. Furthermore, this compound was able to inhibit the frameshifting, to some extent, of SARS-CoV and SARS-CoV-2. Therefore, the novel compound 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline may serve as a promising drug candidate to interfere with pan-coronavirus frameshifting. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19) are emerging zoonotic diseases caused by coronavirus (CoV) infections. The viral RNA-dependent RNA polymerase (RdRp) has been suggested as a valuable target for antiviral therapeutics because the sequence homology of CoV RdRp is highly conserved. We established a cell-based reporter assay for MERS-CoV RdRp activity to test viral polymerase inhibitors. The cell-based reporter system was composed of the bicistronic reporter construct and the MERS-CoV nsp12 plasmid construct. Among the tested nine viral polymerase inhibitors, ribavirin, sofosbuvir, favipiravir, lamivudine, zidovudine, valacyclovir, vidarabine, dasabuvir, and remdesivir, only remdesivir exhibited a dose-dependent inhibition. Meanwhile, the Z-factor and Z′-factor of this assay for screening inhibitors of MERS-CoV RdRp activity were 0.778 and 0.782, respectively. Ribavirin and favipiravir did not inhibit the MERS-CoV RdRp activity, and non-nucleoside HCV RdRp inhibitor, dasabuvir, partially inhibited MERS-CoV RdRp activity. Taken together, the cell-based reporter assay for MERS-CoV RdRp activity confirmed remdesivir as a direct inhibitor of MERS-CoV RdRp in cells. A cell-based MERS-CoV RdRp activity reporter assay is reliable and accurate for screening MERS-CoV RdRp-specific inhibitors. It may provide a valuable platform for developing antiviral drugs for emerging CoV infections. [ABSTRACT FROM AUTHOR]

The kinase C-related kinase 2 (PRK2), which phosphorylates hepatitis C virus (HCV) RNA polymerase, is a proviral factor enhancing HCV replication. Here, we report on the in vivo anti-HCV efficacy of HA1077, which inhibits viral genome replication by targeting PRK2 and displays viral entry inhibitory activity by targeting Rho-associated kinase. HA1077 showed synergistic antiviral activity selectively with nonstructural protein 5 A (NS5A) inhibitors including daclatasvir (DCV). HA1077 oral administration substantially reduced serum viral loads in mice bearing HCV genotype 2a-replicating Huh7 xenografts. When administered with DCV, HA1077 potentiated the antiviral efficacy of DCV and suppressed the generation of DCV resistance-associated variants (RAVs). By deep-sequencing analysis, we uncovered an unprecedented DCV-induced polymorphism at the poly-proline motif (PxxPxxP) of NS5A. Coadministration of HA1077 reduced such a polymorphism. Overall, our results demonstrate the potential therapeutic benefit of combination therapy with HA1077 plus DCV for HCV patients carrying emerging or pre-existing RAVs toward NS5A inhibitors. [ABSTRACT FROM AUTHOR]