Analysis of SARS-CoV-2 Mutations after Nirmatrelvir Treatment in a Lung Cancer Xenograft Mouse Model.

Paxlovid is the first approved oral treatment for coronavirus disease 2019 and includes nirmatrelvir, a protease inhibitor targeting the main protease (Mpro) of SARS-CoV-2, as one of the key components. While some specific mutations emerged in Mpro were revealed to significantly reduce viral susceptibility to nirmatrelvir in vitro, there is no report regarding resistance to nirmatrelvir in patients and animal models for SARS-CoV-2 infection yet. We recently developed xenograft tumors derived from Calu-3 cells in immunodeficient mice and demonstrated extended replication of SARS-CoV-2 in the tumors. In this study, we investigated the effect of nirmatrelvir administration on SARS-CoV-2 replication. Treatment with nirmatrelvir after virus infection significantly reduced the replication of the parental SARS-CoV-2 and SARS-CoV-2 Omicron at 5 days post-infection (dpi). However, the virus titers were completely recovered at the time points of 15 and 30 dpi. The virus genomes in the tumors at 30 dpi were analyzed to investigate whether nirmatrelvir-resistant mutant viruses had emerged during the extended replication of SARS-CoV-2. Various mutations in several genes including ORF1ab, ORF3a, ORF7a, ORF7b, ORF8, and N occurred in the SARS-CoV-2 genome; however, no mutations were induced in the Mpro sequence by a single round of nirmatrelvir treatment, and none were observed even after two rounds of treatment. The parental SARS-CoV-2 and its sublineage isolates showed similar IC50 values of nirmatrelvir in Vero E6 cells. Therefore, it is probable that inducing viral resistance to nirmatrelvir in vivo is challenging differently from in vitro passage.

Target-based discovery of a broad-spectrum flukicide.

Diseases caused by parasitic flatworms impart a considerable healthcare burden worldwide. Many of these diseases-for example, the parasitic blood fluke infection schistosomiasis-are treated with the drug praziquantel (PZQ). However, PZQ is ineffective against disease caused by liver flukes from the genus Fasciola because of a single amino acid change within the target of PZQ, a transient receptor potential ion channel in the melastatin family (TRPMPZQ), in Fasciola species. Here, we identify benzamidoquinazolinone analogs that are active against Fasciola TRPMPZQ. Structure-activity studies define an optimized ligand (BZQ) that caused protracted paralysis and tegumental damage to these liver flukes. BZQ also retained activity against Schistosoma mansoni comparable to PZQ and was active against TRPMPZQ orthologs in all profiled species of parasitic fluke. This broad-spectrum activity manifests as BZQ adopts a pose within the binding pocket of TRPMPZQ that is dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPMPZQ and a first-in-class, broad-spectrum flukicide.

Cellular Metabolism of Substance P Produces Neurokinin-1 Receptor Peptide Agonists with diminished cyclic AMP Signaling.

Substance P (SP) is released from sensory nerves in arteries and heart. It activates neurokinin-1 receptors (NK1R) causing vasodilation, immune modulation, and adverse cardiac remodeling. The hypothesis was tested that SP and SP metabolites activate different second messenger signaling pathways. Macrophages, endothelial cells and fibroblasts metabolized SP to N- and C-terminal metabolites to varying extents. SP 5-11 was the most abundant metabolite followed by SP 1-4, SP 7-11, SP 6-11, SP 3-11 and SP 8-11. In NK1R expressing HEK293 cells, SP and some C-terminal SP metabolites stimulate the NK1R promoting dissociation of several Ga proteins including Gas and Gaq from their bg subunits. SP increases intracellular calcium concentrations ([Ca]i) and cyclic AMP (cAMP) accumulation with similar -log EC50 values of 8.5±0.3 and 7.8±0.1 M, respectively. N-Terminal metabolism of SP by up to 5 amino acids and C-terminal deamidation of SP produce peptides that retain activity to increase [Ca]i but not to increase cAMP. C-Terminal metabolism results in loss of both activities. Thus, [Ca]i and cAMP signaling are differentially affected by SP metabolism. To assess the role of N-terminal metabolism, SP and SP 6-11 were compared on cAMP-mediated activities in NK1R expressing 3T3 fibroblasts. SP inhibits NFkB activity, cell proliferation and wound healing and stimulates collagen production. SP 6-11 had little or no activity. COX-2 expression is increased by SP but not SP 6-11. Thus, metabolism may select the cellular response to SP by inhibiting or re-directing the second messenger signaling pathway activated by the NK1R.

Analysis of SARS-CoV-2 omicron mutations that emerged during long-term replication in a lung cancer xenograft mouse model.

SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection.

A mouse xenograft long-term replication yields a SARS-CoV-2 Delta mutant with increased lethality.

We recently established a long-term SARS-CoV-2 infection model using lung-cancer xenograft mice and identified mutations that arose in the SARS-CoV-2 genome during long-term propagation. Here, we applied our model to the SARS-CoV-2 Delta variant, which has increased transmissibility and immune escape compared with ancestral SARS-CoV-2. We observed limited mutations in SARS-CoV-2 Delta during long-term propagation, including two predominant mutations: R682W in the spike protein and L330W in the nucleocapsid protein. We analyzed two representative isolates, Delta-10 and Delta-12, with both predominant mutations and some additional mutations. Delta-10 and Delta-12 showed lower replication capacity compared with SARS-CoV-2 Delta in cultured cells; however, Delta-12 was more lethal in K18-hACE2 mice compared with SARS-CoV-2 Delta and Delta-10. Mice infected with Delta-12 had higher viral titers, more severe histopathology in the lungs, higher chemokine expression, increased astrocyte and microglia activation, and extensive neutrophil infiltration in the brain. Brain tissue hemorrhage and mild vacuolation were also observed, suggesting that the high lethality of Delta-12 was associated with lung and brain pathology. Our long-term infection model can provide mutant viruses derived from SARS-CoV-2 Delta and knowledge about the possible contributions of emergent mutations to the properties of new variants.

Butein Increases Resistance to Oxidative Stress and Lifespan with Positive Effects on the Risk of Age-Related Diseases in Caenorhabditis elegans.

Butein is a flavonoid found in many plants, including dahlia, butea, and coreopsis, and has both antioxidant and sirtuin-activating activities. In light of the postulated role of free radicals in aging, we examined the effects of butein on aging and on genetic or nutritional models of age-related diseases in Caenorhabditis elegans. Butein showed radical scavenging activity and increased resistance to oxidative stress in Caenorhabditis elegans. The mean lifespan of Caenorhabditis elegans was significantly increased by butein, from 22.7 days in the untreated control to 25.0 days in the butein-treated group. However, the lifespan-extending effect of butein was accompanied by reduced production of progeny as a trade-off. Moreover, the age-related decline in motility was delayed by butein supplementation. Genetic analysis showed that the lifespan-extending effect of butein required the autophagic protein BEC-1 and the transcription factor DAF-16 to regulate stress response and aging. At the genetic level, expression of the DAF-16 downstream target genes hsp-16.2 and sod-3 was induced in butein-treated worms. Butein additionally exhibited a preventive effect in models of age-related diseases. In an Alzheimer's disease model, butein treatment significantly delayed the paralysis caused by accumulation of amyloid-beta in muscle, which requires SKN-1, not DAF-16. In a high-glucose-diet model of diabetes mellitus, butein markedly improved survival, requiring both SKN-1 and DAF-16. In a Parkinson's disease model, dopaminergic neurodegeneration was completely inhibited by butein supplementation and the accumulation of α-synuclein was significantly reduced. These findings suggest the use of butein as a novel nutraceutical compound for aging and age-related diseases.

The anthelmintic meclonazepam activates a schistosome transient receptor potential channel.

Parasitic flatworms cause various clinical and veterinary infections that impart a huge burden worldwide. The most clinically impactful infection is schistosomiasis, a neglected tropical disease caused by parasitic blood flukes. Schistosomiasis is treated with praziquantel (PZQ), an old drug introduced over 40 years ago. New drugs are urgently needed, as while PZQ is broadly effective it suffers from several limitations including poor efficacy against juvenile worms, which may prevent it from being completely curative. An old compound that retains efficacy against juvenile worms is the benzodiazepine meclonazepam (MCLZ). However, host side effects caused by benzodiazepines preclude development of MCLZ as a drug and MCLZ lacks an identified parasite target to catalyze rational drug design for engineering out human host activity. Here, we identify a transient receptor potential ion channel of the melastatin subfamily, named TRPMMCLZ, as a parasite target of MCLZ. MCLZ potently activates Schistosoma mansoni TRPMMCLZ through engagement of a binding pocket within the voltage-sensor-like domain of the ion channel to cause worm paralysis, tissue depolarization, and surface damage. TRPMMCLZ reproduces all known features of MCLZ action on schistosomes, including a lower activity versus Schistosoma japonicum, which is explained by a polymorphism within this voltage-sensor-like domain-binding pocket. TRPMMCLZ is distinct from the TRP channel targeted by PZQ (TRPMPZQ), with both anthelmintic chemotypes targeting unique parasite TRPM paralogs. This advances TRPMMCLZ as a novel druggable target that could circumvent any target-based resistance emerging in response to current mass drug administration campaigns centered on PZQ.

Contribution of HSP90 Cleavage to the Cytotoxic Effect of Suberoylanilide Hydroxamic Acid In Vivo and the Involvement of TXNIP in HSP90 Cleavage.

Heat shock protein (HSP) 90 is expressed in most living organisms, and several client proteins of HSP90 are necessary for cancer cell survival and growth. Previously, we found that HSP90 was cleaved by histone deacetylase (HDAC) inhibitors and proteasome inhibitors, and the cleavage of HSP90 contributes to their cytotoxicity in K562 leukemia cells. In this study, we first established mouse xenograft models with K562 cells expressing the wild-type or cleavage-resistant mutant HSP90ß and found that the suppression of tumor growth by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was interrupted by the mutation inhibiting the HSP90 cleavage in vivo. Next, we investigated the possible function of thioredoxin interacting protein (TXNIP) in the HSP90 cleavage induced by SAHA. TXNIP is a negative regulator for thioredoxin, an antioxidant protein. SAHA transcriptionally induced the expression of TXNIP in K562 cells. HSP90 cleavage was induced by SAHA also in the thymocytes of normal mice and suppressed by an anti-oxidant and pan-caspase inhibitor. When the thymocytes from the TXNIP knockout mice and their wild-type littermate control mice were treated with SAHA, the HSP90 cleavage was detected in the thymocytes of the littermate controls but suppressed in those of the TXNIP knockout mice suggesting the requirement of TXNIP for HSP90 cleavage. We additionally found that HSP90 cleavage was induced by actinomycin D, ß-mercaptoethanol, and p38 MAPK inhibitor PD169316 suggesting its prevalence. Taken together, we suggest that HSP90 cleavage occurs also in vivo and contributes to the anti-cancer activity of various drugs in a TXNIP-dependent manner.

Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage.

Flavonols are the major class of flavonoids of green Chinese cabbage (Brassica rapa subsp. pekinensis). The B. rapa genome harbors seven flavonol synthase genes (BrFLSs), but they have not been functionally characterized. Here, transcriptome analysis showed four BrFLSs mainly expressed in Chinese cabbage. Among them, only BrFLS1 showed major FLS activity and additional flavanone 3ß-hydroxylase (F3H) activity, while BrFLS2 and BrFLS3.1 exhibited only marginal F3H activities. We generated BrFLS1-knockout (BrFLS1-KO) Chinese cabbages using CRISPR/Cas9-mediated genome editing and obtained transgene-free homozygous plants without off-target mutation in the T1 generation, which were further advanced to the T2 generation showing normal phenotype. UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T2 plants, while dihydroflavonol glycosides accumulated concomitantly to levels corresponding to the reduced levels of flavonols. Quantitative PCR analysis revealed that the early steps of phenylpropanoid and flavonoid biosynthetic pathway were upregulated in the BrFLS1-KO plants. In accordance, total phenolic contents were slightly enhanced in the BrFLS1-KO plants, which suggests a negative role of flavonols in phenylpropanoid and flavonoid biosynthesis in Chinese cabbage. Phenotypic surveys revealed that the BrFLS1-KO Chinese cabbages showed normal head formation and reproductive phenotypes, but subtle morphological changes in their heads were observed. In addition, their seedlings were susceptible to osmotic stress compared to the controls, suggesting that flavonols play a positive role for osmotic stress tolerance in B.rapa seedling. In this study, we showed that CRISPR/Cas9-mediated BrFLS1-KO successfully generated a valuable breeding resource of Chinese cabbage with distinctive metabolic traits and that CRISPR/Cas9 can be efficiently applied in functional Chinese cabbage breeding.

Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546.

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24-36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes' integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum.

The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure-Activity Relationships at TRPMPZQ Orthologs.

The anthelmintic drug praziquantel remains a key clinical therapy for treating various diseases caused by parasitic flatworms. The parasite target of praziquantel has remained undefined despite longstanding usage in the clinic, although a candidate ion channel target, named TRPMPZQ, has recently been identified. Intriguingly, certain praziquantel derivatives show different activities against different parasites: for example, some praziquantel analogs are considerably more active against cestodes than against schistosomes. Here we interrogate whether the different activities of praziquantel analogs against different parasites are also reflected by unique structure-activity relationships at the TRPMPZQ channels found in these different organisms. To do this, several praziquantel analogs were synthesized and functionally profiled against schistosome and cestode TRPMPZQ channels. Data demonstrate that structure-activity relationships are closely mirrored between parasites and their TRPMPZQ orthologs, providing further support for TRPMPZQ as the therapeutically relevant target of praziquantel.

SARS-CoV-2 infection induces expression and secretion of lipocalin-2 and regulates iron in a human lung cancer xenograft model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to various clinical symptoms including anemia. Lipocalin-2 has various biological functions, including defense against bacterial infections through iron sequestration, and it serves as a biomarker for kidney injury. In a human protein array, we observed increased lipocalin-2 expression due to parental SARS-CoV-2 infection in the Calu-3 human lung cancer cell line. The secretion of lipocalin-2 was also elevated in response to parental SARS-CoV-2 infection, and the SARS-CoV-2 Alpha, Beta, and Delta variants similarly induced this phenomenon. In a Calu-3 implanted mouse xenograft model, parental SARSCoV- 2 and Delta variant induced lipocalin-2 expression and secretion. Additionally, the iron concentration increased in the Calu-3 tumor tissues and decreased in the serum due to infection. In conclusion, SARS-CoV-2 infection induces the production and secretion of lipocalin-2, potentially resulting in a decrease in iron concentration in serum. Because the concentration of iron ions in the blood is associated with anemia, this phenomenon could contribute to developing anemia in COVID-19 patients. [BMB Reports 2023; 56(12): 669-674].

Analysis of spike protein variants evolved in a novel in vivo long-term replication model for SARS-CoV-2.

Introduction: The spectrum of SARS-CoV-2 mutations have increased over time, resulting in the emergence of several variants of concern. Persistent infection is assumed to be involved in the evolution of the variants. Calu-3 human lung cancer cells persistently grow without apoptosis and release low virus titers after infection. Methods: We established a novel in vivo long-term replication model using xenografts of Calu-3 human lung cancer cells in immunodeficient mice. Virus replication in the tumor was monitored for 30 days and occurrence of mutations in the viral genome was determined by whole-genome deep sequencing. Viral isolates with mutations were selected after plaque forming assays and their properties were determined in cells and in K18-hACE2 mice. Results: After infection with parental SARS-CoV-2, viruses were found in the tumor tissues for up to 30 days and acquired various mutations, predominantly in the spike (S) protein, some of which increased while others fluctuated for 30 days. Three viral isolates with different combination of mutations produced higher virus titers than the parental virus in Calu-3 cells without cytopathic effects. In K18-hACE2 mice, the variants were less lethal than the parental virus. Infection with each variant induced production of cross-reactive antibodies to the receptor binding domain of parental SARS-CoV-2 S protein and provided protective immunity against subsequent challenge with parental virus. Discussion: These results suggest that most of the SARS-CoV-2 variants acquired mutations promoting host adaptation in the Calu-3 xenograft mice. This model can be used in the future to further study SARS-CoV-2 variants upon long-term replication in vivo.

Target-based discovery of a broad spectrum flukicide.

Diseases caused by parasitic flatworms impart a considerable healthcare burden worldwide. Many of these diseases - for example, the parasitic blood fluke infection, schistosomiasis - are treated with the drug praziquantel (PZQ). However, PZQ is ineffective against disease caused by liver flukes from the genus Fasciola. This is due to a single amino acid change within the target of PZQ, a transient receptor potential ion channel (TRPMPZQ), in Fasciola species. Here we identify benzamidoquinazolinone analogs that are active against Fasciola TRPMPZQ. Structure-activity studies define an optimized ligand (BZQ) that caused protracted paralysis and damage to the protective tegument of these liver flukes. BZQ also retained activity against Schistosoma mansoni comparable to PZQ and was active against TRPMPZQ orthologs in all profiled species of parasitic fluke. This broad spectrum activity was manifest as BZQ adopts a pose within the binding pocket of TRPMPZQ dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPMPZQ and a first-in-class, broad spectrum flukicide.

Isolation of Latilactobacillus curvatus with Enhanced Nitric Oxide Synthesis from Korean Traditional Fermented Food and Investigation of Its Probiotic Properties.

Nitric oxide (NO) is a free radical associated with physiological functions such as blood pressure regulation, cardiovascular health, mitochondrial production, calcium transport, oxidative stress, and skeletal muscle repair. This study aimed to isolate Latilactobacillus curvatus strains with enhanced NO production from the traditional Korean fermented food, jangajji, and evaluate their probiotic properties for industrial purposes. When cells were co-cultured with various bacterial stimulants, NO production generally increased, and NO synthesis was observed in the range of 20-40 mg/mL. The selected strains of Lat. curvatus were resistant to acid and bile conditions and with variable effectiveness (1-14%) in adhering to Caco-2 cells. Most bacterial strains can inhibit the growth of various pathogens. In addition, they are capable of reducing cholesterol levels via assimilation of cholesterol at 10-50%. Among the selected NO synthases from Lat. curvatus strains, the strain JBCC38 showed the highest capacity to scavenge ABTS (30.1%) and DPPH radicals (39.4%). Moreover, these strains exhibited immunomodulatory properties. The production of TNF-α and IL-6 in the macrophages treated with various bacterial stimulants was induced in all the selected strains.

Probiotic Properties of Pediococcus pentosaceus JBCC 106 and Its Lactic Acid Fermentation on Broccoli Juice.

To understand the biological roles of Pediococcus pentosaceus strains as probiotics isolated from the traditional Korean fermented food, Jangajji, Pediococcus pentosaceus was selected based on its high cinnamoyl esterase (CE) and antioxidant activities. The acid and bile stability, intestinal adhesion, antagonistic activity against human pathogens, cholesterol-lowering effects, and immune system stimulation without inflammatory effects were evaluated. Nitric oxide (NO) levels were measured in co-culture with various bacterial stimulants. Fermentation ability was measured by using a broccoli matrix and the sulforaphane levels were measured. Resistance to acidic and bilious conditions and 8% adherence to Caco-2 cells were observed. Cholesterol levels were lowered by 51% by assimilation. Moreover, these strains exhibited immunomodulatory properties with induction of macrophage TNF-α and IL-6 and had microstatic effects on various pathogens. Co-culture with various bacterial stimulants resulted in increased NO production. Fermentation activity was increased with the strains, and higher sulforaphane levels were observed. Therefore, in the future, the applicability of the selected strain to broccoli matrix-based fermented functional foods should be confirmed.

Sniffer worm, C. elegans, as a toxicity evaluation model organism with sensing and locomotion abilities.

Additive manufacturing, or 3D printing, has revolutionized the way we create objects. However, its layer-by-layer process may lead to an increased incidence of local defects compared to traditional casting-based methods. Factors such as light intensity, depth of light penetration, component inhomogeneity, and fluctuations in nozzle temperature all contribute to defect formations. These defective regions can become sources of toxic component leakage, but pinpointing their locations in 3D printed materials remains a challenge. Traditional toxicological assessments rely on the extraction and subsequent exposure of living organisms to these harmful agents, thus only offering a passive detection approach. Therefore, the development of an active system to both identify and locate sources of toxicity is essential in the realm of 3D printing technologies. Herein, we introduce the use of the nematode model organism, Caenorhabditis elegans (C. elegans), for toxicity evaluation. C. elegans exhibits distinctive 'sensing' and 'locomotion' capabilities that enable it to actively navigate toward safe zones while steering clear of hazardous areas. This active behavior sets C. elegans apart from other aquatic and animal models, making it an exceptional choice for immediate and precise identification and localization of toxicity sources in 3D printed materials.

PROTEIN PHOSPHATASE 2C08, a Negative Regulator of Abscisic Acid Signaling, Promotes Internode Elongation in Rice.

Clade A protein phosphatase 2Cs (PP2CAs) negatively regulate abscisic acid (ABA) signaling. Here, we investigated the functions of OsPP2CAs and their crosstalk with ABA and gibberellic acid (GA) signaling pathways in rice (Oryza sativa). Among the nine OsPP2CAs, OsPP2C08 had the highest amino acid sequence similarity with OsPP2C51, which positively regulates GA signaling in rice seed germination. However, OsPP2C08 was expressed in different tissues (internodes, sheaths, and flowers) compared to OsPP2C51, which was specifically expressed in seeds, and showed much stronger induction under abiotic stress than OsPP2C51. Transgenic rice lines overexpressing OsPP2C08 (OsPP2C08-OX) had a typical ABA-insensitive phenotype in a post-germination assay, indicating that OsPP2C08, as with other OsPP2CAs, negatively regulates ABA signaling. Furthermore, OsPP2C08-OX lines had longer stems than wild-type (WT) plants due to longer internodes, especially between the second and third nodes. Internode cells were also longer in OsPP2C08-OX lines than in the WT. As GA positively regulates plant growth, these results suggest that OsPP2C08 might positively regulate GA biosynthesis. Indeed, the expression levels of GA biosynthetic genes including gibberellin 20-oxidase (OsGA20ox4) and Ent-kaurenoic acid oxidase (OsKAO) were increased in OsPP2C08-OX lines, and we observed that GIBBERELLIN 2-OXIDASE 4 (OsGA2ox4), encoding an oxidase that catalyzes the 2-beta-hydroxylation of several biologically active GAs, was repressed in the OsPP2C08-OX lines based on a transcriptome deep sequencing and RT-qPCR analysis. Furthermore, we compared the accumulation of SLENDER RICE 1 (SLR1), a DELLA protein involved in GA signaling, in OsPP2C08-OX and WT plants, and observed lower levels of SLR1 in the OsPP2C08-OX lines than in the WT. Taken together, our results reveal that OsPP2C08 negatively regulates ABA signaling and positively regulates GA signaling in rice. Our study provides valuable insight into the molecular mechanisms underlying the crosstalk between GA and ABA signaling in rice.

LY6D is crucial for lipid accumulation and inflammation in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a serious metabolic disorder characterized by excess fat accumulation in the liver. Over the past decade, NAFLD prevalence and incidence have risen globally. There are currently no effective licensed drugs for its treatment. Thus, further study is required to identify new targets for NAFLD prevention and treatment. In this study, we fed C57BL6/J mice one of three diets, a standard chow diet, high-sucrose diet, or high-fat diet, and then characterized them. The mice fed a high-sucrose diet had more severely compacted macrovesicular and microvesicular lipid droplets than those in the other groups. Mouse liver transcriptome analysis identified lymphocyte antigen 6 family member D (Ly6d) as a key regulator of hepatic steatosis and the inflammatory response. Data from the Genotype-Tissue Expression project database showed that individuals with high liver Ly6d expression had more severe NAFLD histology than those with low liver Ly6d expression. In AML12 mouse hepatocytes, Ly6d overexpression increased lipid accumulation, while Ly6d knockdown decreased lipid accumulation. Inhibition of Ly6d ameliorated hepatic steatosis in a diet-induced NAFLD mouse model. Western blot analysis showed that Ly6d phosphorylated and activated ATP citrate lyase, which is a key enzyme in de novo lipogenesis. In addition, RNA- and ATAC-sequencing analyses revealed that Ly6d drives NAFLD progression by causing genetic and epigenetic changes. In conclusion, Ly6d is responsible for the regulation of lipid metabolism, and inhibiting Ly6d can prevent diet-induced steatosis in the liver. These findings highlight Ly6d as a novel therapeutic target for NAFLD.

Translation of paired fundus photographs to fluorescein angiographs with energy-based cycle-consistent adversarial networks.

Fluorescein angiography is a crucial examination in ophthalmology to identify retinal and choroidal pathologies. However, this examination modality is invasive and inconvenient, requiring intravenous injection of a fluorescent dye. In order to provide a more convenient option for high-risk patients, we propose a deep-learning-based method to translate fundus photography into fluorescein angiography using Energy-based Cycle-consistent Adversarial Networks (CycleEBGAN) We propose a deep-learning-based method to translate fundus photography into fluorescein angiography using CycleEBGAN. We collected fundus photographs and fluorescein angiographs taken at Changwon Gyeongsang National University Hospital between January 2016 and June 2021 and paired late-phase fluorescein angiographs and fundus photographs taken on the same day. We developed CycleEBGAN, a combination of cycle-consistent adversarial networks (CycleGAN) and Energy-based Generative Adversarial Networks (EBGAN), to translate the paired images. The simulated images were then interpreted by 2 retinal specialists to determine their clinical consistency with fluorescein angiography. A retrospective study. A total of 2605 image pairs were obtained, with 2555 used as the training set and the remaining 50 used as the test set. Both CycleGAN and CycleEBGAN effectively translated fundus photographs into fluorescein angiographs. However, CycleEBGAN showed superior results to CycleGAN in translating subtle abnormal features. We propose CycleEBGAN as a method for generating fluorescein angiography using cheap and convenient fundus photography. Synthetic fluorescein angiography with CycleEBGAN was more accurate than fundus photography, making it a helpful option for high-risk patients requiring fluorescein angiography, such as diabetic retinopathy patients with nephropathy.