NinaB and BCO Collaboratively Participate in the ß-Carotene Catabolism in Crustaceans: A Case Study on Chinese Mitten Crab Eriocheir sinensis.

Carotenoid cleavage oxygenases can cleave carotenoids into a range of biologically important products. Carotenoid isomerooxygenase (NinaB) and ß, ß-carotene 15, 15'-monooxygenase (BCO1) are two important oxygenases. In order to understand the roles that both oxygenases exert in crustaceans, we first investigated NinaB-like (EsNinaBl) and BCO1-like (EsBCO1l) within the genome of Chinese mitten crab (Eriocheir sinensis). Their functions were then deciphered through an analysis of their expression patterns, an in vitro ß-carotene degradation assay, and RNA interference. The results showed that both EsNinaBl and EsBCO1l contain an RPE65 domain and exhibit high levels of expression in the hepatopancreas. During the molting stage, EsNinaBl exhibited significant upregulation in stage C, whereas EsBCO1l showed significantly higher expression levels at stage AB. Moreover, dietary supplementation with ß-carotene resulted in a notable increase in the expression of EsNinaBl and EsBCO1l in the hepatopancreas. Further functional assays showed that the EsNinaBl expressed in E. coli underwent significant changes in its color, from orange to light; in addition, its ß-carotene cleavage was higher than that of EsBCO1l. After the knockdown of EsNinaBl or EsBCO1l in juvenile E. sinensis, the expression levels of both genes were significantly decreased in the hepatopancreas, accompanied by a notable increase in the redness (a*) values. Furthermore, a significant increase in the ß-carotene content was observed in the hepatopancreas when EsNinaBl-mRNA was suppressed, which suggests that EsNinaBl plays an important role in carotenoid cleavage, specifically ß-carotene. In conclusion, our findings suggest that EsNinaBl and EsBCO1l may exhibit functional co-expression and play a crucial role in carotenoid cleavage in crabs.

Tuning Rheological Behaviors of Supramolecular Aqueous Gels via Charge Transfer Interactions.

Rheological properties are critical for determining real applications of supramolecular gels in various fields. Correspondingly, the modulation of gel rheology will be very important for meeting real requirements. In this aspect, a few strategies were applied to tune the rheological behaviors of supramolecular gels, but some specific interactions like charge transfer (CT) interactions were less explored at the molecular level. Herein, we report a pyrene-containing derivative of diphenylalanine as a donor gelator and naphthalenediimide or 3,5-dinitrobenzene as matching acceptor molecules. It was found that the viscoelastic properties and strength of the original gel could be tuned through addition of different acceptor molecules to the original gel with changing the ratios of the selected acceptor molecules. As a result, storage modulus was continuously adjusted over a wide range from 190,000 to 50,000 Pa by CT interactions. Furthermore, the mechanism of the CT-induced change in rheological properties was understood and clarified through relevant techniques (e.g., UV-Vis, fluorescence, and FT-IR spectroscopy and TEM). The findings in this work would provide a novel strategy to modulate the rheological properties of supramolecular gels for adaption to broader fields of real applications.

Recent advances in the biodegradation of azo dyes.

As dye demand continues to rapidly increase in the food, pharmaceutical, cosmetic, paper, textile, and leather industries, an industrialization increase is occurring. Meanwhile, the degradation and removal of azo dyes have raised broad concern regarding the hazards posed by these dyes to the ecological environment and human health. Physicochemical treatments have been applied but are hindered by high energy and economic costs, high sludge production, and chemicals handling. Comparatively, the bioremediation technique is an eco-friendly, removal-efficient, and cost-competitive method to resolve the problem. This paper provides scientific and technical information about recent advances in the biodegradation of azo dyes. It expands the biodegradation efficiency, characteristics, and mechanisms of various microorganisms containing bacteria, fungi, microalgae, and microbial consortia, which have been reported to biodegrade azo dyes. In addition, information about physicochemical factors affecting dye biodegradation has been compiled. Furthermore, this paper also sketches the recent development and characteristics of advanced bioreactors.

Small nucleolar RNA Sf-15 regulates proliferation and apoptosis of Spodoptera frugiperda Sf9 cells.

BACKGROUND: Small nucleolar RNAs (snoRNAs) function in guiding 2'-O-methylation and pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In recent years, more and more snoRNAs have been found to play novel roles in mRNA regulation, such as pre-mRNA splicing or RNA editing. In our previous study, we found a silkworm C/D box snoRNA Bm-15 can interact with Notch receptor gene in vitro. To further study the function of Bm-15, we cloned its homolog Sf-15 from Spodoptera frugiperda and investigate the function of Sf-15 in Sf9 cells. RESULTS: We showed that knocking down of Sf-15 can inhibit the proliferation, then induce apoptosis of insect S. frugiperda Sf9 cells, but the results were reversed when Sf-15 was overexpressed. De novo sequencing of transcriptome of Sf9 cells showed that the expression of 21 apoptosis-related genes were increased upon Sf-15 repression. Further analysis showed that a Ca2+-induced cell death pathway gene Cn (PPP3C, the serine/threonine-protein phosphatase 2B catalytic subunit), was significantly increased upon Sf-15 depression but decreased when Sf-15 was overexpressed, which indicated that Cn might be a potential target of Sf-15. CONCLUSIONS: We conclude that C/D box snoRNA Sf-15 can participate in apoptosis through regulating the expression of Ca2+-induced cell death pathway gene Cn in Sf9 cells. This is the first time that we found snoRNAs exhibiting dual functions in insect, which reveals a novel layer of ncRNA modulation in cell growth and death.

High Response, Self-Powered Photodetector Based on the Monolayer MoS2/P-Si Heterojunction with Asymmetric Electrodes.

Here, a self-powered photodetector based on the monolayer MoS2/P-Si heterojunction with asymmetric electrodes was fabricated. The MoS2/p-Si heterojunction photodetector with asymmetric electrodes offers the advantages over the conventional heterojunction photodetector on optoelectronic applications in terms of strong built-in electric field and fast photogenerated carrier separation and transport. Significantly, the MoS2/P-Si heterojunction exhibited an obvious photovoltaic effect, which can be used as the self-powered photodetector operating without any bias voltage. At a voltage bias of 0 V, the photocurrent of the detector is 23 nA, and its photoresponse/recovery time is 84 ms/136 ms. When at bias, the detector shows a ratio of photocurrent to dark current up to 3120, high responsivity of 117 A W-1, and fast photoresponse/recovery time of 74 ms/115 ms. Our work illustrates the great potential of the MoS2/P-Si heterojunction device with asymmetric electrodes on photovoltaic applications.

MiR-149 Compromises the Reactions of Liver Cells to Fatty Acid via its Polymorphism and Increases Non-Alcoholic Fatty Liver Disease (NAFLD) Risk by Targeting Methylene Tetrahydrofolate Reductase (MTHFR).

BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is a worldwide health problem, and microRNA (miRNA) has been reported to be involved in NAFLD. The objective of our study was to explore the effect of polymorphism in miR-149 on the pathogenesis of NAFLD. MATERIAL AND METHODS Real-time PCR was performed to explore the effect of long-chain fatty acid (FFA) on the level of miR-149 and methylene tetrahydrofolate reductase (MTHFR). Then in-silicon analysis and luciferase assay were investigated to verify MTHFR was the target gene of miR-149. Finally, Western-blot analysis and real-time PCR were performed to confirm the control of MTHFR by miR-149. RESULTS In this study, we found that miR-149 was apparently upregulated in hepatocytes genotyped as TT treated with FFA; and MTHFR in hepatocytes genotyped as TT treated with FFA was evidently downregulated compared to control. Whereas, FFA had no obvious effect on MTHFR level in hepatocytes genotyped as CC. We searched an online miRNA database and found that miR-149 was a regulator of MTHFR expression, which was confirmed by luciferase assay. In hepatocytes genotyped as TT and treated with or without FFA, miR-149 mimic dose-dependently decreased the level of MTHFR, and miR-149 inhibitor dose-dependently increased the level of MTHFR. And in hepatocytes genotyped as CC treated with or without FFA exhibited a similar inhibition effect of miR-149 on expression of MTHFR. CONCLUSIONS The data suggested that the polymorphism in miR-149 played an important role in the development of NAFLD via altering the expression of miR-149 as well as its target, MTHFR.

Arsenic Metabolites, Including N-Acetyl-4-hydroxy-m-arsanilic Acid, in Chicken Litter from a Roxarsone-Feeding Study Involving 1600 Chickens.

The poultry industry has used organoarsenicals, such as 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, ROX), to prevent disease and to promote growth. Although previous studies have analyzed arsenic species in chicken litter after composting or after application to agricultural lands, it is not clear what arsenic species were excreted by chickens before biotransformation of arsenic species during composting. We describe here the identification and quantitation of arsenic species in chicken litter repeatedly collected on days 14, 24, 28, 30, and 35 of a Roxarsone-feeding study involving 1600 chickens of two strains. High performance liquid chromatography separation with simultaneous detection by both inductively coupled plasma mass spectrometry and electrospray ionization tandem mass spectrometry provided complementary information necessary for the identification and quantitation of arsenic species. A new metabolite, N-acetyl-4-hydroxy-m-arsanilic acid (N-AHAA), was identified, and it accounted for 3-12% of total arsenic. Speciation analyses of litter samples collected from ROX-fed chickens on days 14, 24, 28, 30, and 35 showed the presence of N-AHAA, 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), inorganic arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), and ROX. 3-AHPAA accounted for 3-19% of the total arsenic. Inorganic arsenicals (the sum of As(III) and As(V)) comprised 2-6% (mean 3.5%) of total arsenic. Our results on the detection of inorganic arsenicals, methylarsenicals, 3-AHPAA, and N-AHAA in the chicken litter support recent findings that ROX is actually metabolized by the chicken or its gut microbiome. The presence of the toxic metabolites in chicken litter is environmentally relevant as chicken litter is commonly used as fertilizer.

Design, synthesis and biological evaluation of novel arylpiperazine derivatives on human prostate cancer cell lines.

A series of novel arylpiperazine derivatives was synthesized. The in vitro cytotoxic activities of all synthesized compounds against three human prostate cancer cell lines (PC-3, LNCaP, and DU145) were evaluated by a CCK-8 assay. Compounds 10, 24 and 29 exhibited strong cytotoxic activities against LNCaP cells (IC50 <3µM). In addition, these compounds exhibited weak cytotoxic effects on human epithelial prostate normal cells RWPE-1. The structure-activity relationship (SAR) of these arylpiperazine derivatives was also discussed based on the obtained experimental data.

The comparison of morphology and transcriptome in the inner membrane reveals the potential mechanism of the heritable carapace color of the Chinese mitten crab Eriocheir sinensis.

Carapace color plays an important role in the communication, reproduction, and self-defense of crustaceans, which is also related to their economic value. Chinese mitten crab (Eriocheir sinensis) is an important aquaculture species in China, and there are different strains with heritable carapace colors, i.e. Green, White, and Red. However, there is a lack of research on the formation mechanism of carapace color of this species. This study was conducted to compare the histology and transcriptome in the inner membrane of three carapace color strains of E. sinensis. Histological comparisons revealed that the inner membrane of green and red carapace crabs contained more melanin, appearing in clusters, and had a higher presence of yellow or orange pigments. In contrast, the inner membrane of white carapace crabs had smaller and fewer melanin particles, as well as a lower presence of yellow or orange pigments. Observation under an electron microscope showed that the inner membrane of E. sinensis contained a large number of collagen fibers and various types of cells, including fibroblasts, melanocytes, and other tissue cells, which exhibited different levels of activity. Transcriptome analysis showed that the Green, Red, and White strains of E. sinensis had approximately 80.3 K, 81.6 K and 80.3 K expressed unigenes in their inner membranes, respectively. When comparing Green and Red crabs, there were 2, 850 upregulated genes and 2, 240 downregulated genes. In the comparison between Red and White crabs, there were 2, 853 upregulated genes and 2, 583 downregulated genes. Furthermore, there were 2, 336 upregulated genes and 2, 738 downregulated genes in the inner membranes between White and Green crabs. Among these genes, some members of the solute carriers family, which are involved in carotenoid transportation, showed differential expression among the three carapace color strains. Additionally, significant differences were observed in the expression of genes related to melanin synthesis, including wingless/integrate, tyrosinase, guanine nucleotide-binding protein inhibitory subunit, cell adhesion molecule, adenylyl cyclase, and creb-binding protein. there were no differences in the gene expression levels of the crustacyanin family. In conclusion, this study identified several candidate genes associated with carapace color in the inner membrane of E. sinensis, suggesting a close relationship between the heritable carapace colors and the transport of the carotenoids as well as the synthesis of melanin.

Evolution and expression analysis of carotenoid cleavage oxygenase gene family in Chinese mitten crab Eriocheir sinensis.

Carotenoid cleavage oxygenase (CCO) plays a pivotal role in various biological activities, including antioxidant and immune functions in animals. This paper investigates the evolution and expression of CCO genes based on three chordates and 27 arthropods. Aquatic animals exhibit a higher abundance of CCO genes. Despite this, research on CCO in crustaceans has been notably limited, with a complete absence of any previous studies on the CCO genes for the Chinese mitten crab (Eriocheir sinensis). In this study, six CCO genes were identified in the E. sinensis genome database. Results reveal that the evolution of the CCO gene family in Crustacea is primarily characterized by purifying selection, with a preference for employing similar codons. EsCCO1 and EsCCO3 were mainly expressed in the epidermal layer, and EsCCO4 was mainly expressed in the hindgut. Meanwhile, EsCCO5 and EsCCO6 were mainly expressed in the hepatopancreas and endometrium. A notable detail that different EsCCO genes demonstrate distinct expression patterns within various tissues of E. sinensis. The findings of this study offer fundamental insights that could serve as a basis for further exploration into the functions and regulatory mechanisms of CCO genes in crustacean species.

Comparing the Performance of Three Computational Methods for Estimating the Effective Reproduction Number.

The effective reproduction number (Rt) is one of the most important epidemiological parameters, providing suggestions for monitoring the development trend of diseases and also for adjusting the prevention and control policies. However, a few studies have focused on the performance of some common computational methods for Rt. The purpose of this article is to compare the performance of three computational methods for Rt: the time-dependent (TD) method, the new time-varying (NT) method, and the sequential Bayesian (SB) method. Four evaluation methods-accuracy, correlation coefficient, similarity based on trend, and dynamic time warping distance-were used to compare the effectiveness of three computational methods for Rt under different time lags and time windows. The results showed that the NT method was a better choice for real-time monitoring and analysis of the epidemic in the middle and late stages of the infectious disease. The TD method could reflect the change of the number of cases stably and accurately, and was more suitable for monitoring the change of Rt during the whole process of the epidemic outbreak. When the data were relatively stable, the SB method could also provide a reliable estimate for Rt, while the error would increase when the fluctuation in the number of cases increased. The results would provide suggestions for selecting appropriate Rt estimation methods and making policy adjustments more timely and effectively according to the change of Rt.

Linear Fuzzy Information-Granule-Based Fuzzy C-Means Algorithm for Clustering Time Series.

This article aims to design a trend-oriented-granulation-based fuzzy C -means (FCM) algorithm that can cluster a group of time series at an abstract (granular) level. To achieve a better trend-oriented granulation of a time series, l1 trend filtering is firstly carried out to result in segments which are then optimized by the proposed segment merging algorithm. By constructing a linear fuzzy information granule (LFIG) on each segment, a granular time series which well reflects the linear trend characteristic of the original time series is produced. With the novel designed distance that can well measure the trend similarity of two LFIGs, the distance between two granular time series is calculated by the modified dynamic time warping (DTW) algorithm. Based on this distance, the LFIG-based FCM algorithm is developed for clustering time series. In this algorithm, cluster prototypes are iteratively updated by the specifically designed granule splitting and merging algorithm, which allows the lengths of prototypes to change in the process of iteration. This overcomes the serious drawback of the existing approaches, where the lengths of prototypes cannot be changed. Experimental studies demonstrate the superior performance of the proposed algorithm in clustering time series with different shapes or trends.

Formaldehyde Oxidation of Ce0.8Zr0.2O2 Nanocatalysts for Room Temperature: Kinetics and Effect of pH Value.

Ce0.8Zr0.2O2 catalysts were prepared via the co-precipitation method under different pH conditions. The catalysts were characterized via TEM, XRD, XPS, BET, Raman, and FTIR. The oxidation performance of formaldehyde was tested. Precipitation pH affects the physicochemical properties and performance of the Ce0.8Zr0.2O2 catalyst. By controlling the precipitation pH at 10.5, the Ce0.8Zr0.2O2 catalyst with the largest specific surface area, the smallest grain size with the best formaldehyde removal rate (98.85%), abundant oxygen vacancies, and the best oxidation performance were obtained. Meanwhile, the kinetic parameters of the catalyst were experimentally investigated and the calculated activation energy was 12.6 kJ/mol and the number of reaction steps was 1.4 and 1.2.

Impacts of exposure to nanopolystyrene and/or chrysene at ambient concentrations on neurotoxicity in Siniperca chuatsi.

Health risks caused by widespread environmental pollutants such as nanopolystyrene (NP) and chrysene (CHR) in aquatic ecosystems have aroused considerable concern. The present study established juvenile Mandarin fish (Siniperca chuatsi) models of NP and/or CHR exposure at ambient concentrations for 21 days to systematically investigate the underlying neurotoxicity mechanisms. The results showed that single and combined exposure to NP and CHR not only reduced the density of small neuronal cells in the grey matter layer of the optic tectum, but also induced brain oxidative stress according to physiological parameters including CAT, GSH-Px, SOD, T-AOC, and MDA. The co-exposure alleviated the histopathological damage, compared to NP and CHR single exposure group. These results indicate that NP and/or CHR causes neurotoxicity in S. chuatsi, in accordance with decreased acetylcholinesterase activity and altered expression of several marker genes of nervous system functions and development including c-fos, shha, elavl3, and mbpa. Transcriptomics analysis was performed to further investigate the potential molecular mechanisms of neurotoxicity. We propose that single NP and co-exposure induced oxidative stress activates MMP, which degrades tight junction proteins according to decreased expression of claudin, JAM, caveolin and TJP, ultimately damaging the integrity of the blood-brain barrier in S. chuatsi. Remarkably, the co-exposure exacerbated the blood-brain barrier disruption. More importantly, single NP and co-exposure induced neuronal apoptosis mainly activates the expression of apoptosis-related genes through the death receptor apoptosis pathway, while CHR acted through both death receptor apoptosis and endoplasmic reticulum apoptosis pathways. Additionally, subchronic CHR exposure caused neuroinflammation, supported by activation of TNF/NF-κB and JAK-STAT signaling pathways via targeting-related genes, while the co-exposure greatly alleviated the neuroinflammation. Collectively, our findings illuminate the underlying neurotoxicity molecular mechanisms of NP and/or CHR exposure on aquatic organisms.

Geometrical model establishment and preoperative evaluation on A-T flap design: Finite element method-based computer-aided simulation on surgical operation processes.

Objective: A-T flap has been extensively applied to repair dermal soft tissue defects. The flap design completely depends on the experience of doctors. Herein, we explored the approach of analyzing the reasonability of A-T flap design and performed a simulation of operation processes by computer-aided technology. Afterward, the finite element analysis software (MSC.Marc/Mentat) was used to establish the simulation model, based on which the computer simulation of flap suturing and release state in A-T flap surgery was performed. Methods: A geometrical model of the A-T flap was established, and the length-width ratio of the flap, maximum suture distance, and suture area that could influence the postoperative suture effects of the flap were analyzed. The reasonable surgical planning for A-T flap design based on the crossing constraint relationship was achieved. The simulation model was established by the finite element analysis software (MSC.Marc/Mentat), based on which computer simulation of flap suture and release state of A-T flap in surgery processes were performed. The flap's stress and deformation distribution results confirmed the applicability of the A-T flap design method proposed in the present study. Results: When the apex angle of the A-T flap was 60 degrees, the suture area was the smallest, and the flap design had the highest practicability. Conclusion: Computer-assisted preoperative assessment, which has high clinical value, could provide a theoretical basis for A-T flap design in clinical practice.

Application of Principal Component Analysis (PCA) to the Evaluation and Screening of Multiactivity Fungi.

Continued innovation in screening methodologies remains important for the discovery of high-quality multiactive fungi, which have been of great significance to the development of new drugs. Mangrove-derived fungi, which are well recognized as prolific sources of natural products, are worth sustained attention and further study. In this study, 118 fungi, which mainly included Aspergillus spp. (34.62%) and Penicillium spp. (15.38%), were isolated from the mangrove ecosystem of the Maowei Sea, and 83.1% of the cultured fungi showed at least one bioactivity in four antibacterial and three antioxidant assays. To accurately evaluate the fungal bioactivities, the fungi with multiple bioactivities were successfully evaluated and screened by principal component analysis (PCA), and this analysis provided a dataset for comparing and selecting multibioactive fungi. Among the 118 mangrove-derived fungi tested in this study, Aspergillus spp. showed the best comprehensive activity. Fungi such as A. clavatonanicus, A. flavipes and A. citrinoterreus, which exhibited high comprehensive bioactivity as determined by the PCA, have great potential in the exploitation of natural products and the development of new drugs. This study demonstrated the first use of PCA as a time-saving, scientific method with a strong ability to evaluate and screen multiactive fungi, which indicated that this method can affect the discovery and development of new drugs.

Regularized Spectral Spike Response Model: A Neuron Model for Robust Parameter Reduction.

The modeling procedure of current biological neuron models is hindered by either hyperparameter optimization or overparameterization, which limits their application to a variety of biologically realistic tasks. This article proposes a novel neuron model called the Regularized Spectral Spike Response Model (RSSRM) to address these issues. The selection of hyperparameters is avoided by the model structure and fitting strategy, while the number of parameters is constrained by regularization techniques. Twenty firing simulation experiments indicate the superiority of RSSRM. In particular, after pruning more than 99% of its parameters, RSSRM with 100 parameters achieves an RMSE of 5.632 in membrane potential prediction, a VRD of 47.219, and an F1-score of 0.95 in spike train forecasting with correct timing (±1.4 ms), which are 25%, 99%, 55%, and 24% better than the average of other neuron models with the same number of parameters in RMSE, VRD, F1-score, and correct timing, respectively. Moreover, RSSRM with 100 parameters achieves a memory use of 10 KB and a runtime of 1 ms during inference, which is more efficient than the Izhikevich model.

Unique functional responses of fungal communities to various environments in the mangroves of the Maowei Sea in Guangxi, China.

Fungi are important compartments of microbial communities of mangroves. Their diversity might be influenced by their habitat environment. This study analyzed the distribution and function of fungal communities in the sediments and plant samples from mangrove ecosystem of the Maowei Sea area in Guangxi, China. The results showed that phytopathogenic fungi Cladosporium (17.00%) was mainly observed in the sediments from the protected zone, while endophytic fungi Alternaria (9.22%) and Acremonium (6.09%) were only observed in the sediments from wharf. The fungi in the sediments from village and park were mainly consisted of high-activity endophytes and fungi related to lignin-degrading, respectively. Acaulospora and Aspergillus with higher relative abundance discovered in plant tissues could help plant growth. Cirrenalia (37.66%) and Lignincola (26.73%) with high-activity for lignin-degrading were discovered in decayed leaves. The distribution and function of fungi were highly dependent on the environment settings, thus the fungi can be used as indicators for monitoring the environmental change of mangrove ecosystems.

Ethanol as an efficient cosubstrate for the biodegradation of azo dyes by Providencia rettgeri: Mechanistic analysis based on kinetics, pathways and genomics.

Azo dyes pose hazards to ecosystems and human health and the cosubstrate strategy has become the focus for the bioremediation of azo dyes. Herein, Brilliant Crocein (BC), a model pollutant, was biodegraded by Providencia rettgeri domesticated from activated sludge. Additional ethanol, as a cosubstrate, could accelerate P. rettgeri growth and BC biodegradation, as reflected by the Gompertz models. This phenomenon was attributed to the smaller metabolites and greater number of potential pathways observed under the synergistic effect of ethanol. Genomic analysis of P. rettgeri showed that functional genes related to azo bond cleavage, redox reactions, ring opening and hydrolysis played crucial roles in azo dye biodegradation. Furthermore, the mechanism proposed was that ethanol might stimulate the production of additional reducing power via the expression of related genes, leading to the cleavage of azo bonds and aromatic rings. However, biodegradation without ethanol could only partly cleave the azo bonds.

The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious.

Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infectioninduces autophagy that enhances virus replication and vesicle release to evade immune systemsurveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells andshowed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobilitygroup box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradientcentrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in thecytoplasm of the infected cells were detected in the purified double-membrane autophagosome byimmunogold labeling under transmission electron microscopy. In DENV infected cells, the levels ofcapsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramaticaccumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked bychloroquine, indicating that these proteins are not regulated by autophagic degradation machinery.We further demonstrated that purified autophagosomes were infectious when co-cultured withuninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negativestrandand full-length genomic RNAs, but no viral particles. It is possible that the infectivity ofthe autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the firstto report that DENV2-induced double-membrane autophagosomes containing viral proteins andfull-length RNAs are infectious and not undergoing autophagic degradation. Our novel findingwarrants further validation of whether these intracellular vesicles undergo exocytosis to becomeinfectious autophagic vesicles.