Hepatitis B virus e antigen induces atypical metabolism and differentially regulates programmed cell deaths of macrophages.

Macrophages can undergo M1-like proinflammatory polarization with low oxidative phosphorylation (OXPHOS) and high glycolytic activities or M2-like anti-inflammatory polarization with the opposite metabolic activities. Here we show that M1-like macrophages induced by hepatitis B virus (HBV) display high OXPHOS and low glycolytic activities. This atypical metabolism induced by HBV attenuates the antiviral response of M1-like macrophages and is mediated by HBV e antigen (HBeAg), which induces death receptor 5 (DR5) via toll-like receptor 4 (TLR4) to induce death-associated protein 3 (DAP3). DAP3 then induces the expression of mitochondrial genes to promote OXPHOS. HBeAg also enhances the expression of glutaminases and increases the level of glutamate, which is converted to α-ketoglutarate, an important metabolic intermediate of the tricarboxylic acid cycle, to promote OXPHOS. The induction of DR5 by HBeAg leads to apoptosis of M1-like and M2-like macrophages, although HBeAg also induces pyroptosis of the former. These findings reveal novel activities of HBeAg, which can reprogram mitochondrial metabolism and trigger different programmed cell death responses of macrophages depending on their phenotypes to promote HBV persistence.

Endosomal egress and intercellular transmission of hepatic ApoE-containing lipoproteins and its exploitation by the hepatitis C virus.

Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, the ApoE-LPs transport pathway from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that newly synthesized ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and CD63-positive intraluminal vesicles (ILVs), which are precursors of extracellular vesicles (EVs), along the late endosomal trafficking route in a microtubule-dependent manner. A fraction of ApoE-LPs associated with CD63-positive EVs appears to be co-transmitted from cell to cell. Given the important role of ApoE in viral infections, we employed as well-studied model the hepatitis C virus (HCV) and found that the viral replicase component nonstructural protein 5A (NS5A) is enriched in ApoE-containing ILVs. Interaction between NS5A and ApoE is required for the efficient release of ILVs containing HCV RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders.

Effective breast cancer combination therapy targeting BACH1 and mitochondrial metabolism.

Mitochondrial metabolism is an attractive target for cancer therapy1,2. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC)1,3. Here we show that BTB and CNC homology1 (BACH1)4, a haem-binding transcription factor that is increased in expression in tumours from patients with TNBC, targets mitochondrial metabolism. BACH1 decreases glucose utilization in the tricarboxylic acid cycle and negatively regulates transcription of electron transport chain (ETC) genes. BACH1 depletion by shRNA or degradation by hemin sensitizes cells to ETC inhibitors such as metformin5,6, suppressing growth of both cell line and patient-derived tumour xenografts. Expression of a haem-resistant BACH1 mutant in cells that express a short hairpin RNA for BACH1 rescues the BACH1 phenotype and restores metformin resistance in hemin-treated cells and tumours7. Finally, BACH1 gene expression inversely correlates with ETC gene expression in tumours from patients with breast cancer and in other tumour types, which highlights the clinical relevance of our findings. This study demonstrates that mitochondrial metabolism can be exploited by targeting BACH1 to sensitize breast cancer and potentially other tumour tissues to mitochondrial inhibitors.

Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells.

Autophagy is required for benign hepatic tumors to progress into malignant hepatocellular carcinoma. However, the mechanism is unclear. Here, we report that mitophagy, the selective removal of mitochondria by autophagy, positively regulates hepatic cancer stem cells (CSCs) by suppressing the tumor suppressor p53. When mitophagy is enhanced, p53 co-localizes with mitochondria and is removed by a mitophagy-dependent manner. However, when mitophagy is inhibited, p53 is phosphorylated at serine-392 by PINK1, a kinase associated with mitophagy, on mitochondria and translocated into the nucleus, where it binds to the NANOG promoter to prevent OCT4 and SOX2 transcription factors from activating the expression of NANOG, a transcription factor critical for maintaining the stemness and the self-renewal ability of CSCs, resulting in the reduction of hepatic CSC populations. These results demonstrate that mitophagy controls the activities of p53 to maintain hepatic CSCs and provide an explanation as to why autophagy is required to promote hepatocarcinogenesis.

Raf Kinase Inhibitory Protein regulates the cAMP-dependent protein kinase signaling pathway through a positive feedback loop.

Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the ß-adrenergic receptor (ß-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes ß-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the ß-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.

A Hepatitis C virus genotype 1b post-transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo.

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines.

Fermentative aminopyrrolnitrin production by metabolically engineered Corynebacterium glutamicum.

Aminopyrrolnitrin (APRN), a natural halogenated phenylpyrrole derivative (HPD), has strong antifungal and antiparasitic activities. Additionally, it showed 2.8-fold increased photostability compared to pyrrolnitrin, a commercially available HPD with antimicrobial activity. For microbial production of APRN, we first engineered anthranilate phosphoribosyltransferase encoded by trpD from Corynebacterium glutamicum, resulting in a TrpDA162D mutation that exhibits feedback-resistant against L-tryptophan and higher substrate affinity compared to wild-type TrpD. Plasmid-borne expression of trpDA162D in C. glutamicum TP851 strain with two copies of trpDA162D in the genome led to the production of 3.1 g/L L-tryptophan in flask culture. Subsequent step for L-tryptophan chlorination into 7-chloro-L-tryptophan was achieved by introducing diverse sources of genes encoding tryptophan 7-halogenase (PrnA or RebH) and flavin reductase (Fre, PrnF, or RebF). The combined expression of prnA from Serratia grimesii or Serratia plymuthica with flavin reductase gene from Escherichia coli, Pseudomonas fluorescens, or Lechevalieria aerocolonigenes yielded higher production of 7-chloro-L-tryptophan in comparison to other sets of two-component systems. In the next step, production of putative monodechloroaminopyrrolnitrin (MDAP) from 7-chloro-L-tryptophan was achieved through the expression of prnB encoding MDAP synthase from S. plymuthica or P. fluorescens. Finally, an artificial APRN biosynthetic pathway was constructed by simultaneously expressing genes coding for tryptophan 7-halogenase, flavin reductase, MDAP synthase, and MDAP halogenase (PrnC) from different microbial sources within the L-tryptophan-producing TP851 strain. As prnC from S. grimesii or S. plymuthica was introduced into the host strain, which carried plasmids expressing prnA from S. plymuthica, fre from E. coli, and prnB from S. plymuthica, APN3639 and APN3638 accumulated 29.5 mg/L and 28.1 mg/L of APRN in the culture broth. This study represents the first report on the fermentative APRN production by metabolically engineered C. glutamicum.

One Health Threat of Treated Wastewater Discharge in Urban Ohio Rivers: Implications for Surface Water and Fish Gut Microbiome and Resistome.

Wastewater treatment plants (WWTPs) are thought to be a major disseminating source of antibiotic resistance (AR) to the environment, establishing a crucial connection between human and environmental resistome. The objectives of this study were to determine how wastewater effluents impact microbiome and resistome of freshwater and fish, and identify potential AR-carrying clinically relevant pathogens in these matrices. We analyzed wastewater influent and effluent from four WWTPs in three metropolitan areas of Ohio, USA via shotgun metagenomic sequencing. We also sequenced river water and fish guts from three reaches (upstream, at the WWTP outfall, and downstream). Notably, we observed a decline in microbiome diversity and AR gene abundance from wastewater to the receiving river. We also found significant differences by reach and trophic level (diet) in beta-diversity of the fish gut microbiomes. SourceTracker revealed that 0.443 and 0.248 more of the of the fish gut microbiome was sourced from wastewater effluent in fish from the outfall and downstream locations, respectively, compared to upstream fish. Additionally, AR bacteria of public health concern were annotated in effluent and river water samples, indicating potential concern for human exposure. In summary, our findings show the continued role of wastewater as a significant AR reservoir and underscores the considerable impact of wastewater discharge on aquatic wildlife, which highlights the One Health nature of this issue.

High Glass Transition Temperature Fluorinated Polymers Based on Transfer Learning with Small Experimental Data.

Machine learning can be used to predict the properties of polymers and explore vast chemical spaces. However, the limited number of available experimental datasets hinders the enhancement of the predictive performance of a model. This study proposes a machine learning approach that leverages transfer learning and ensemble modeling to efficiently predict the glass transition temperature (Tg) of fluorinated polymers and guide the design of high Tg copolymers. Initially, the quantum machine 9 (QM9) dataset is employed for model pretraining, thus providing robust molecular representations for the subsequent fine-tuning of a specialized copolymer dataset. Ensemble modeling is used to further enhance prediction robustness and reliability, effectively addressing the problems owing to the limited and unevenly distributed nature of the copolymer dataset. Finally, a fine-tuned ensemble model is used to navigate a vast chemical space comprising 61 monomers and identify promising candidates for high Tg fluorinated polymers. The model predicts 247 entries capable of achieving a Tg over 390 K, of which 14 are experimentally validated. This study demonstrates the potential of machine learning in material design and discovery, highlighting the effectiveness of transfer learning and ensemble modeling strategies for overcoming the challenges posed by small datasets in complex copolymer systems.

Association between long-term PM2.5 exposure and risk of Kawasaki disease in children: A nationwide longitudinal cohort study.

BACKGROUND: Based on previous studies suggesting air pollution as a potential risk factor for Kawasaki Disease (KD), we examined the association of long-term exposure to childhood fine particulate matter (PM2.5) with the risk of KD. METHODS: We used National Health Insurance Service-National Sample Cohort data from 2002 to 2019, which included beneficiaries aged 0 years at enrollment and followed-up until the onset of KD or age 5 years. The onset of KD was defined as the first hospital visit record with a primary diagnostic code of M30.3, based on the 10th revision of the International Classification of Diseases, and with an intravenous immunoglobulin (IVIG) prescription. We assigned PM2.5 concentrations to 226 districts, based on mean annual predictions from a machine learning-based ensemble prediction model. We performed Cox proportional-hazards modeling with time-varying exposures and confounders. RESULTS: We identified 134,634 individuals aged five or less at enrollment and, of these, 1220 individuals who had a KD onset and an IVIG prescription during study period. The average annual concentration of PM2.5 exposed to the entire cohort was 28.2 µg/m³ (Standard Deviation 2.9). For each 5 µg/m³ increase in annual PM2.5 concentration, the hazard ratio of KD was 1.21 (95% CI 1.05-1.39). CONCLUSIONS: In this nationwide, population-based, cohort study, long-term childhood exposure to PM2.5 was associated with an increased incidence of KD in children. The study highlights plausible mechanisms for the association between PM2.5 and KD, but further studies are needed to confirm our findings.

Regulatory mechanisms for specification and patterning of plant vascular tissues.

Plant vascular tissues, the conduits of water, nutrients, and small molecules, play important roles in plant growth and development. Vascular tissues have allowed plants to successfully adapt to various environmental conditions since they evolved 450 Mya. The majority of plant biomass, an important source of renewable energy, comes from the xylem of the vascular tissues. Efforts have been made to identify the underlying mechanisms of cell specification and patterning of plant vascular tissues and their proliferation. The formation of the plant vascular system is a complex process that integrates signaling and gene regulation at transcriptional and posttranscriptional levels. Recently, a wealth of molecular genetic studies and the advent of cell biology and genomic tools have enabled important progress toward understanding its underlying mechanisms. Here, we provide a comprehensive review of the cell and developmental processes of plant vascular tissue and resources recently available for studying them that will enable the discovery of new ways to develop sustainable energy using plant biomass.

The effect of nondamaging subthreshold laser therapy in patients with chronic central serous chorioretinopathy.

PURPOSE: To evaluate the efficacy of nondamaging subthreshold laser therapy in Korean patients with chronic central serous chorioretinopathy (cCSC). METHODS: This retrospective interventional case series included 31 patients (31 eyes) with cCSC who underwent nondamaging laser therapy using Endpoint Management (EpM) software. Since a barely visible burn of the test spot was defined as 100% pulse energy, 30% pulse energy with a 200-µm spot was titrated to treat the macular area based on EpM settings. A 30% pulse laser with a spacing of 0.25-beam diameter was applied to cover the macular area where hyperfluorescent leaks were observed on fluorescein angiography. Changes in central macular thickness (CMT), subretinal fluid (SRF) height, subfoveal choroidal thickness (SCT), and logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA) were measured at baseline and after 3 and 6 months. If the subretinal fluid persisted for 3 months, retreatment was performed. RESULTS: At 6 months post-treatment, the complete SRF resolution rate was 48.39% (15/31 eyes), and the partial SRF resolution rate was 12.90% (4/31 eyes). The change in mean BCVA (logMAR) was not significant (0.31 ± 0.29 at the baseline and 0.31 ± 0.40 at month 6) (p = 0.943). At the baseline, the mean CMT (µm) decreased from 350.74 ± 112.76 at baseline to 239.71 ± 130.25 at month 6 (p < 0.001), and the mean SRF height (µm) decreased from 193.16 ± 90.69 at baseline to 70.58 ± 100.00 at month 6 (p < 0.001). However, the change in SCT was not statistically significant (p = 0.516). In 15 patients who were retreated at month 3, the mean SRF height (µm) decreased significantly from 144.67 ± 74.01 at month 3 to 77.13 ± 63.77 at month 6 (p = 0.002). No side effects associated with laser therapy were observed during the 6-month follow-up. CONCLUSIONS: Nondamaging laser therapy with a modified macular treatment was effective in reducing CMT and SRF and showed favorable visual and anatomical outcomes in patients with cCSC.

Clinical safety of remdesivir therapy in COVID-19 patients with renal insufficiency.

Though remdesivir benefits COVID-19 patients, its use in those with renal dysfunction is currently limited due to concerns about possible toxic effects of accumulated sulfobutylether-ß-cyclodextrin (SBECD) on liver and kidney. We examined renal and hepatic function for a month in renally-impaired COVID-19 patients who were treated or not treated with remdesivir to assess the safety of the drug. A retrospective study was performed in adult COVID-19 patients with glomerular filtration rates of <30 ml/min/1.73 m2 at admission to a tertiary care hospital between November 2020 and March 2022. Data on serum creatinine and liver chemistry were collected serially. A total of 101 patients with impaired renal function were analyzed, comprising 64 remdesivir-treated patients and 37 who did not receive any antiviral agent. Although remdesivir-treated patients were more likely to be infected with the Omicron variant (79.7% vs. 48.6%), baseline characteristics did not differ significantly between the two groups. Among patients who initially did not require dialysis, 18.4% (7/38) of remdesivir-treated patients developed acute kidney injury (AKI) at days 4-6, compared with 51.7% (15/29) of non-remdesivir-treated patients. Liver injury severity worsened in 3.1% (2/64) of remdesivir-treated patients and 5.4% (2/37) of non-remdesivir-treated patients at days 4-6. In addition, there was no significant increase in AKI and liver injury over time in remdesivir-treated patients, and there were no cases of discontinuation of remdesivir due to adverse reactions. Concerns regarding the safety of SBECD should not lead to hasty withholding of remdesivir treatment in renally-impaired COVID-19 patients.

Two-year carcinogenicity study of a novel plasticizer, bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate (Eco-DEHCH), by oral diet in Han Wistar rats.

Plasticizers are necessary for the usability of various products, including food contact materials. Exposure to plasticizers is most commonly made through the oral route. Several plasticizers have been reported to have adverse effects on humans and the environment. Thus, the present study aimed to determine the long-term toxicity and carcinogenicity of a novel plasticizer called bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate (Eco-DEHCH), which is an ecofriendly and biologically less harmful replacer. Groups of 50 male and 50 female Han Wistar rats were fed Eco-DEHCH at daily doses of 1,600, 5,000, or 16,000 ppm in their diet for at least 104 weeks. The rats were regularly monitored for mortality, clinical signs, body weight, food consumption, food efficiency, and perceivable mass. All animals were subjected to complete necropsy and histopathological examination. The results indicate that the rats well tolerated chronic exposure to Eco-DEHCH at highest daily doses of 16,000 ppm, with was equivalent to 805.1 mg/kg/day in males and 1060.6 mg/kg/day in females and did not show signs of toxicity or carcinogenicity. In conclusion, Eco-DEHCH could be a safe and promising alternative plasticizer.

Toxicology and safety study of L-tryptophan and its impurities for use in broiler feed.

L-tryptophan has been utilized as a feed additive in animal nutrition to improve growth performance, as well as a dietary supplement to alleviate various emotional symptoms in humans. Despite its benefits, concerns regarding its safety arose following the outbreak of eosinophilia-myalgia syndrome (EMS) among individuals who consumed L-tryptophan. The causative material of EMS was determined to be not L-tryptophan itself, but rather L-tryptophan impurities resulting from a specific manufacturing process. To investigate the effect of L-tryptophan and its impurities on humans who consume meat products derived from animals that were fed L-tryptophan and its impurities, an animal study involving broiler chickens was conducted. The animals in test groups were fed diet containing 0.065%-0.073% of L-tryptophan for 27 days. This study aimed to observe the occurrence of toxicological or EMS-related symptoms and analyze the residues of L-tryptophan impurities in meat products. The results indicated that there was no evidence of adverse effects associated with the test substance in the investigated parameters. Furthermore, most of the consumed EMS-causing L-tryptophan impurities did not remain in the meat of broiler chickens. Thus, this study demonstrated the safety of L-tryptophan and some of its impurities as a feed additive.

Toxicology and safety study of L-tryptophan and its impurities for use in swine.

L-tryptophan, an essential amino acid for physiological processes, metabolism, development, and growth of organisms, is widely utilized in animal nutrition and human health as a feed additive and nutritional supplement, respectively. Despite its known benefits, safety concerns have arisen due to an eosinophilia-myalgia syndrome (EMS) outbreak linked to L-tryptophan consumed by humans. Extensive research has established that the EMS outbreak was caused by an L-tryptophan product that contained certain impurities. Therefore, safety validations are imperative to endorse the use of L-tryptophan as a supplement or a feed additive. This study was conducted in tertiary hybrid [(Landrace × Yorkshire) × Duroc] pigs to assess general toxicity and potential risks for EMS-related symptoms associated with L-tryptophan used as a feed additive. Our investigation elucidated the relationship between L-tryptophan and EMS in swine. No mortalities or clinical signs were observed in any animals during the administration period, and the test substance did not induce toxic effects. Hematological analysis and histopathological examination revealed no changes in EMS-related parameters, such as eosinophil counts, lung lesions, skin lesions, or muscle atrophy. Furthermore, no test substance-related changes occurred in other general toxicological parameters. Through analyzing the tissues and organs of swine, most of the L-tryptophan impurities that may cause EMS were not retained. Based on these findings, we concluded that incorporating L-tryptophan and its impurities into the diet does not induce EMS in swine. Consequently, L-tryptophan may be used as a feed additive throughout all growth stages of swine without safety concerns.

The social epistemology of eating disorders: How our gaps in understanding challenge patient care.

In this article, I argue that various epistemic challenges associated with eating disorders (EDs) can negatively affect the care of already marginalized patient groups with various EDs. I will first outline deficiencies in our understanding of EDs-in research, healthcare settings, and beyond. I will then illustrate with examples cases where discriminatory misconceptions about what EDs are, the presentation and treatment of EDs, and who gets EDs, instantiate obstacles for the treatment of various ED patient groups.

MicroRNA-200c coordinates HNF1 homeobox B and apolipoprotein O functions to modulate lipid homeostasis in alcoholic fatty liver disease.

Hepatic steatosis is an initial manifestation of alcoholic liver disease. An imbalance of hepatic lipid processes including fatty acid uptake, esterification, oxidation, and triglyceride secretion leads to alcoholic fatty liver (AFL). However, the precise molecular mechanisms underlying the pathogenesis of AFL remain elusive. Here, we show that mice deficient in microRNAs (miRs)-141 and -200c display resistance to the development of AFL. We found that miR-200c directly targets HNF1 homeobox B (Hnf1b), a transcriptional activator for microsomal triglyceride transfer protein (Mttp), as well as apolipoprotein O (ApoO), an integral component of the mitochondrial contact site and cristae organizing system complex. We show that expression of these miRs is significantly induced by chronic ethanol exposure, which is accompanied by reduced HNF1B and APOO levels. Furthermore, miR-141/200c deficiency normalizes ethanol-mediated impairment of triglyceride secretion, which can be attributed to the restored levels of HNF1B and MTTP, as well as phosphatidylcholine abundance. Moreover, we demonstrate that miR-141/200c deficiency restores ethanol-mediated inhibition of APOO expression and mitochondrial dysfunction, improving mitochondrial antioxidant defense capacity and fatty acid oxidation. Taken together, these results suggest that miR-200c contributes to the modulation of lipid homeostasis in AFL disease by cooperatively regulating Hnf1b and ApoO functions.

Oxidative stress response and programmed cell death guided by NAC013 modulate pithiness in radish taproots.

Radish, Raphanus sativus L., is an important root crop that is cultivated worldwide. Owing to its evolutionary proximity to Arabidopsis thaliana, radish can be used as a model root crop in research on the molecular basis of agronomic traits. Pithiness is a significant defect that reduces the production of radish with commercial value; however, traditional breeding to eliminate this trait has thus far been unsuccessful. Here, we performed transcriptomics and genotype-by-sequencing (GBS)-based quantitative trait locus (QTL) analyses of radish inbred lines to understand the molecular basis of pithiness in radish roots. The transcriptome data indicated that pithiness likely stems from the response to oxidative stress, leading to cell death of the xylem parenchyma during the root-thickening process. Subsequently, we narrowed down a list of candidates responsible for pithiness near a major QTL and found polymorphisms in a radish homologue of Arabidopsis ANAC013 (RsNAC013), an endoplasmic reticulum bound NAC transcription factor that is targeted to the nucleus to mediate the mitochondrial retrograde signal. We analysed the effects of polymorphisms in RsNAC013 using Arabidopsis transgenic lines overexpressing RsNAC013 alleles as well as in radish inbred lines bearing these alleles. This analysis indicated that non-synonymous variations within the coding sequence result in different levels of RsNAC013 activities, thereby providing a genetic condition for root pithiness. The elevated oxidative stress or hypoxia that activates RsNAC013 for mitochondrial signalling enhances this process. Collectively, this study serves as an exemplary case of translational research taking advantage of the extensive information available from a model organism.

Comparison of HAV and HCV infections in vivo and in vitro reveals distinct patterns of innate immune evasion and activation.

BACKGROUND & AIMS: Hepatitis A virus (HAV) infections are considered not to trigger innate immunity in vivo, in contrast to hepatitis C virus (HCV). This lack of induction has been imputed to strong interference by HAV proteases 3CD and 3ABC. We aimed to elucidate the mechanisms of immune activation and counteraction by HAV and HCV in vivo and in vitro. METHODS: Albumin-urokinase-type plasminogen activator/severe combined immunodeficiency (Alb/uPA-SCID) mice with humanised livers were infected with HAV and HCV. Hepatic cell culture models were used to assess HAV and HCV sensing by Toll-like receptor 3 and retinoic acid-inducible gene I/melanoma differentiation-associated protein 5 (RIG-I/MDA5), respectively. Cleavage of the adaptor proteins TIR-domain-containing adapter-inducing interferon-ß (TRIF) and mitochondrial antiviral-signalling protein (MAVS) was analysed by transient and stable expression of HAV and HCV proteases and virus infection. RESULTS: We detected similar levels of interferon-stimulated gene induction in hepatocytes of HAV- and HCV-infected mice with humanised liver. In cell culture, HAV induced interferon-stimulated genes exclusively upon MDA5 sensing and depended on LGP2 (laboratory of genetics and physiology 2). TRIF and MAVS were only partially cleaved by HAV 3ABC and 3CD, not sufficiently to abrogate signalling. In contrast, HCV NS3-4A efficiently degraded MAVS, as previously reported, whereas TRIF cleavage was not detected. CONCLUSIONS: HAV induces an innate immune response in hepatocytes via MDA5/LGP2, with limited control of both pathways by proteolytic cleavage. HCV activates Toll-like receptor 3 and lacks TRIF cleavage, suggesting that this pathway mainly contributes to HCV-induced antiviral responses in hepatocytes. Our results shed new light on the induction of innate immunity and counteraction by HAV and HCV. IMPACT AND IMPLICATIONS: Understanding the mechanisms that determine the differential outcomes of HAV and HCV infections is crucial for the development of effective therapies. Our study provides insights into the interplay between these viruses and the host innate immune response in vitro and in vivo, shedding light on previously controversial or only partially investigated aspects. This knowledge could tailor the development of new strategies to combat HCV persistence, as well as improve our understanding of the factors underlying successful HAV clearance.