Search

Background & Aims: Hepatitis B surface antigen (HBsAg) drives hepatocarcinogenesis. Factors and mechanisms involved in this progression remain poorly defined, hindering the development of effective therapeutic strategies. Therefore, the mechanisms involved in the HBsAg-induced transformation of normal liver into hepatocellular carcinoma (HCC) were investigated. Methods: Hemizygous Tg(Alb1HBV)44Bri/J mice were examined for HBsAg-induced carcinogenic events. Gene set-enrichment analysis identified significant signatures in HBsAg-transgenic mice that correlated with endoplasmic reticulum (ER) stress, unfolded protein response, autophagy and proliferation. These events were investigated by western blotting, immunohistochemical and immunocytochemical staining in 2-, 8- and 12-month-old HBsAg-transgenic mice. The results were verified in HBsAg-overexpressing Hepa1-6 cells and validated in human HBV-related HCC samples. Results: Increased BiP expression in HBsAg-transgenic mice indicated induction of the unfolded protein response. In addition, early-phase autophagy was enhanced (increased BECN1 and LC3B) and late-phase autophagy blocked (increased p62) in HBsAg-transgenic mice. Finally, HBsAg altered lysosomal acidification via ATF4- and ATF6-mediated downregulation of lysosome-associated membrane protein 2 (LAMP2) expression. In patients, HBV-related HCC and adjacent tissues showed increased BiP, p62 and downregulated LAMP2 compared to uninfected controls. In vitro, the use of ER stress inhibitors reversed the HBsAg-related suppression of LAMP2. Furthermore, HBsAg promoted hepatocellular proliferation as indicated by Ki67, cleaved caspase-3 and AFP staining in paraffin-embedded liver sections from HBsAg-transgenic mice. These results were further verified by colony formation assays in HBsAg-expressing Hepa1-6 cells. Interestingly, inhibition of ER stress in HBsAg-overexpressing Hepa1-6 cells suppressed HBsAg-mediated cell proliferation. Conclusions: These data showed that HBsAg directly induces ER stress, impairs autophagy and promotes proliferation, thereby driving hepatocarcinogenesis. In addition, this study expanded the understanding of HBsAg-mediated intracellular events in carcinogenesis. Impact and implications: Factors and mechanisms involved in hepatocarcinogenesis driven by hepatitis B surface antigen (HBsAg) are poorly defined, hindering the development of effective therapeutic strategies. This study showed that HBsAg-induced endoplasmic reticulum stress suppressed LAMP2, thereby mediating autophagic injury. The present data suggest that restoring LAMP2 function in chronic HBV infection may have both antiviral and anti-cancer effects. This study has provided insights into the role of HBsAg-mediated intracellular events in carcinogenesis and thereby has relevance for future drug development.

BackgroundAs the leading cause of chronic kidney disease, diabetic kidney disease (DKD) is an enormous burden for all healthcare systems around the world. However, its early diagnosis has no effective methods.MethodsFirst, gene expression data in GEO database were extracted, and the differential genes of diabetic tubulopathy were obtained. Immune-related genesets were generated by WGCNA and immune cell infiltration analyses. Then, differentially expressed immune-related cuproptosis genes (DEICGs) were derived by the intersection of differential genes and genes related to cuproptosis and immune. To investigate the functions of DEICGs, volcano plots and GO term enrichment analysis was performed. Machine learning and protein-protein interaction (PPI) network analysis helped to finally screen out hub genes. The diagnostic efficacy of them was evaluated by GSEA analysis, receiver operating characteristic (ROC) curve, single-cell RNA sequencing and the Nephroseq website. The expression of hub genes at the animal level by STZ -induced and db/db DKD mouse models was further verified.ResultsFinally, three hub genes, including FSTL1, CX3CR1 and AGR2 that were up-regulated in both the test set GSE30122 and the validation set GSE30529, were screened. The areas under the curve (AUCs) of ROC curves of hub genes were 0.911, 0.935 and 0.922, respectively, and 0.946 when taking as a whole. Correlation analysis showed that the expression level of three hub genes demonstrated their negative relationship with GFR, while those of FSTL1 displayed a positive correlation with the level of serum creatinine. GSEA was enriched in inflammatory and immune-related pathways. Single-nucleus RNA sequencing indicated the main distribution of FSTL1 in podocyte and mesangial cells, the high expression of CX3CR1 in leukocytes and the main localization of AGR2 in the loop of Henle. In mouse models, all three hub genes were increased in both STZ-induced and db/db DKD models.ConclusionMachine learning was combined with WGCNA, immune cell infiltration and PPI analyses to identify three hub genes associated with cuproptosis, immunity and diabetic nephropathy, which all have great potential as diagnostic markers for DKD and even predict disease progression.

Ascorbic acid (AsA) is an essential nutritional component and powerful antioxidant in vegetables, and in plants, AsA levels are regulated by light. AsA levels in the leaves of Chinese chive (Allium tuberosum Rottler ex Spr), a popular vegetable, are poorly understood. Thus, this study was performed to assess the influence of light on AsA biosynthesis in chive and select related genes (AtuGGP1 and AtuGME1); in addition, bioinformatic analyses and gene expression level assays were performed. The biological information obtained for AtuGGP1 and AtuGME1 was analysed with several tools, including NCBI, DNAMAN, and MEGA11. After different light treatments were performed, the Chive AsA content and AtuGGP1 and AtuGME1 expression levels were determined. These results suggest that 1) compared with natural light, continuous darkness inhibited AsA synthesis in chives. 2) The amino acid sequences of AtuGGP1 and AtuGME1 are very similar to those of other plants. 3) The trends observed for the expression levels of AtuGGP1 and AtuGME1 were consistent with the AsA content observed in chives. Hence, we speculated that light controls AsA biosynthesis in chives by regulating AtuGGP1 and AtuGME1 expression. This study provided impactful and informative evidence regarding the functions of GGP and GME in chives.

Plant protection drone spraying technology is widely used to prevent and control crop diseases and pests due to its advantages of being unaffected by crop growth patterns and terrain restrictions, high operational efficiency, and low labor requirements. The operational parameters of plant protection drones significantly impact the distribution of spray droplets, thereby affecting pesticide utilization. In this study, a field experiment was conducted to determine the working modes of two representative plant protection drones and an electric backpack sprayer as a control to explore the characteristics of droplet deposition with different spray volumes in the citrus canopy. The results showed that the spraying volume significantly affected the number of droplets and the spray coverage. The number of droplets and the spray coverage area on the leaf surface were significantly increased by increasing the spray volume from 60 L/ha to 120 L/ha in plant protection drones. Particularly for the DJI T30, the mid-lower canopy showed a spray coverage increase of 52.5%. The droplet density demonstrated the most significant variations in the lower inner canopy, ranging from 18.7 droplets/cm2 to 41.7 droplets/cm2 by XAG V40. From the deposition distribution on fruit trees, the plant protection drones exhibit good penetration ability, as the droplets can achieve a relatively even distribution in different canopy layers of citrus trees. The droplet distribution uniformity inside the canopy is similar for XAG V40 and DJI T30, with a variation coefficient of approximately 50%-100%. Compared to the plant protection drones, the knapsack electric sprayer is suitable for pest and disease control in the mid-lower canopy, but they face challenges of insufficient deposition capability in the upper canopy and overall poor spray uniformity. The distribution of deposition determined in this study provides data support for the selection of spraying agents for fruit trees by plant protection drones and for the control of different pests and diseases.

The precise identification of corn and weeds plays an important role in precise spraying. This paper proposed a lightweight model based on the improved yolov5s and built a precision spraying robot. Firstly, we used a data augmentation method based on category balance and agronomic characteristics to solve the data imbalance problem. Then, compared with yolov5s, yolov5l, yolov5m, and yolov5x, we found that yolov5s has both real-time and accuracy and is easier to deploy the model on edge devices. Through the feature map visualization experiment, we found that the feature extraction network can’t pay close attention to the important feature of the target and suppress the feature of the noise. Therefore, we added the attention mechanism. In order to improve the real-time performance of the model, we designed the C3-Ghost-bottleneck module. Finally, we built a precision spraying robot. Compared with the original model, the value of map@0.5 is increased by 3.2%, the model file is reduced by 3.6 MB, the AP value for corn is increased from 93.2% to 96.3%, and the AP value for weeds is increased from 85.6% to 88.9%. Finally, the precision spraying experiment of weeds was carried out. The recognition accuracy of weeds is 83%, the probability of the spraying robot correctly identifying weeds and accurately spraying is 81%, and the detection speed is 30ms/f. The experimental results verify the feasibility of precision spraying weeding and the effectiveness of the improved model, which can provide a reference for the engineering application of precision weeding.

Environmental stressors such as salinity have pronounced impacts on the growth, productivity, nutrition, and flavor of horticultural crops, though yield loss sometimes is inevitable. In this study, the salinity influences were evaluated using hydroponic Chinese chive (Allium tuberosum) treated with different concentrations of sodium chloride. The results demonstrated that lower salinity could stimulate plant growth and yield. Accordingly, the contents of soluble sugar, ascorbic acid, and soluble protein in leaf tissues increased, following the decrease of the nitrate content, under mild salinity (6.25 or 12.5 mM NaCl). However, a higher level of salinity (25 or 50 mM NaCl) resulted in growth inhibition, yield reduction, and leaf quality deterioration of hydroponic chive plants. Intriguingly, the chive flavor was boosted by the salinity, as evidenced by pungency analysis of salinity-treated leaf tissues. UPLC-MS/MS analysis reveals that mild salinity promoted the accumulation of glutamic acid, serine, glycine, and proline in leaf tissues, and thereby enhanced the umami and sweet flavors of Chinese chive upon salinity stress. Considering the balance between yield and flavor, mild salinity could conduce to hydroponic Chinese chive cultivation. Transcriptome analysis revealed that enhanced pungency could be ascribed to a salt stress-inducible gene, AtuFMO1, associated with the biosynthesis of S-alk(en)yl cysteine sulphoxides (CSOs). Furthermore, correlation analysis suggested that two transcription factors, AtubHLH and AtuB3, were potential regulators of AtuFMO1 expressions under salinity. Thus, these results revealed the molecular mechanism underlying mild salinity-induced CSO biosynthesis, as well as a practical possibility for producing high-quality Chinese chive hydroponically.

Abstract Chinese chive (Allium tuberosum) is widely cultivated around the world for its unique flavor, nutrient, and medicinal values, yet its molecular mechanism on flavor formation and other metabolic pathways remains intangible. The elucidation of these complex processes begins with investigating the expression of the genes of interest, however the appropriate reference genes (RGs) for normalizing the gene expression are still unavailable in A. tuberosum. To fill this lacuna, transcriptome-wide screening was undertaken to identify the most stable genes according to the analysis of their FPKM values. The expression stability of the RGs was further evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The comprehensive analysis showed that GLY1 and SKP1, instead of two traditionally used RGs (eIF1α and ACT2), were the most stable genes across diverse A. tuberosum tissues, indicating the necessity to carefully validate the stability of RGs prior to their use for normalizations. As indicated by geNorm, the normalizations with at least two RGs could give more accurate results. qRT-PCR experiments were conducted with randomly selected genes, demonstrating that normalization with a combination of GLY1 and SKP1 resulted in reliable normalization results. Our finding represents the first attempt toward establishing a standardized qRT-PCR analysis in this economically important vegetable.

Functional maturation of liver sinusoidal endothelial cells (LSECs) plays an important role in intrahepatic T‐cell activation and control of viral infections. Natural killer (NK) cells have been reported to prompt the maturation of antigen‐presenting cells (APCs), especially for dendritic cells (DCs), but the interaction between NK cells and LSECs is elusive. Here, we investigated whether and how NK cells are involved in regulating LSEC maturation and if this has a role in controlling hepatitis B virus (HBV) infection in a mouse model. A chronic HBV replication mouse model was established by hydrodynamic injection (HI) of 6 µg adeno‐associated virus plasmid (pAAV)/HBV 1.2. The nucleotide‐binding oligomerization domain‐containing protein 1 (NOD1) ligand diaminopemelic acid (DAP) was imported into liver by HI at day 14 after plasmid injection. We found that HI of DAP recruited conventional NK cells (cNK) into the liver and promoted tumor necrosis factor alpha (TNF‐α) and interferon‐gamma (IFN‐γ) production of NK cells in a chemokine (C‐X‐C motif) receptor 3 (CXCR3)‐dependent manner. Importantly, the maturation of LSECs and the anti‐HBV effects of DAP were impaired in CXCR3−/− mice; this possibly was associated with the decreased number of intrahepatic cNK cells. Consistently, depleting cNK cells but not liver‐resident NK cells also impaired the maturation and antigen‐presenting function of LSECs, which reduced intrahepatic HBV‐specific T‐cell responses and thus inhibited HBV clearance both in wild‐type and in Rag1−/− mice. Moreover, TNF‐α or IFN‐γ stimulation as well as coculture with intrahepatic NK cells partly promoted LSEC phenotypic and functional maturation in vitro. Conclusion: NOD1‐triggered NK cell activation may lead to the enhancement of intrahepatic T‐cell responses by promoting maturation of LSECs through soluble cytokines and cell–cell contact, thereby controlling HBV replication and expression.

A better understanding of longitudinal changes in antibody responses in COVID-19 patients is needed. Here the authors analyze anti-spike and anti-nucleocapsid antibody responses to Sars-CoV-2 over a course of 6 months in a large cohort of patients with COVID-19, showing that IgM is mostly not detectable after 3 months, whereas IgG responses contract, yet remain at high levels at 6 months.

The susceptibility of mice to hepatitis B virus (HBV) infection depends on their genetic background. The gut microbiota modulates the antiviral immune response in the liver and plays a protective role against HBV infection. However, whether HBV infection outcomes depend on the gut microbiota remains unclear. In this study, we assessed the gut microbiota composition in naïve BALB/c and C57BL/6 mice using 16S rRNA gene sequencing. The gut microbiota in BALB/c mice was depleted using broad-spectrum antibiotics (ABX) and then reconstituted with fecal microbiota from naïve BALB/c or C57BL/6 mice to evaluate the effect of fecal microbiota transplantation (FMT) on the outcomes of and immune response to HBV infection. We found that HBV infection outcomes and the gut microbiota composition differed between BALB/c and C57BL/6 mice. Commensal bacteria from the fecal microbiota selectively colonized the guts of ABX-treated BALB/c mice. Mice receiving fecal microbiota from BALB/c or C57BL/6 mice displayed different HBV infection outcomes. The fecal microbiota from C57BL/6 mice induced immune tolerance in the liver and prolonged HBV infection. In conclusion, HBV infection outcomes in mice are determined by the host genetic background and gut microbiota composition. Reconstitution of the gut microbiota by FMT can alter the susceptibility to HBV infection in mice.

Flea distribution in northern region of Xinjiang Uygur Autonomous Region (XUAR) and fluctuations of the annual fleas index in Alataw Pass were investigated. During a 4-year (2015–2018) study, 5789 fleas were collected directly from 15 mammals at eight counties in northern XUAR of northwestern China. Nineteen flea species, belonging to sixteen genera and seven families, were further confirmed by four genetic markers (18S rDNA, 28S rDNA, COI and COII) after morphological observation. Pulex irritans and Paraceras crispum parasitizing Asian badgers (Meles leucurus) were recorded for the first time. In addition, the fluctuations of the annual fleas index in Alataw Pass were surveyed. Xenopsylla gerbilli minax, Xenopsylla conformis conformis and Nosopsyllus laeviceps laeviceps were highly detected in the warm season while Paradoxopsyllus repandus, Ctenophthalmus dolichus dolichus and Coptopsylla lamellifer ardua were only found in the cold season. These findings extend our knowledge of flea species, distribution and annual fluctuations especially in China-Kazakhstan border. Keywords: Flea distribution, XUAR, Fluctuations, The annual fleas index, Wild mammals

While the immunogenicity of inactivated vaccines against coronavirus disease 2019 (COVID‐19) has been characterized in several well-conducted clinical trials, real-world evidence concerning immune responses against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) raised by such vaccines is currently missing. Here, we comprehensively characterized various parameters of SARS-CoV-2-specific cellular and humoral immune responses induced by inactivated COVID-19 vaccines in 126 individuals under real-world conditions. After two doses of vaccination, S-receptor binding domain IgG (S-RBD IgG) and neutralizing antibody (NAb) were detected in 87.06% (74/85) and 78.82% (67/85) of individuals, respectively. Female participants developed higher concentrations of S-RBD IgG and NAb compared to male vaccinees. Interestingly, a longer dosing interval between the first and second vaccination resulted in a better long-term SARS-CoV-2 S-RBD IgG response. The frequencies of CD4+ T cells that produce effector cytokines (IFN-γ, IL-2, and TNF-α) in response to stimulation with peptide pools corresponding to the SARS-CoV-2 spike (S), nucleocapsid (N) or membrane (M) protein were significantly higher in individuals received two doses of vaccine than those received one dose of vaccine and unvaccinated individuals. S, N, or M-specific CD4+ and CD8+ T cell responses were detectable in 95.83% (69/72) and 54.16% (39/72) of double-vaccinated individuals, respectively. The longitudinal analysis demonstrated that CD4+ T cell responses recognizing S, N, and M waned quickly after a single vaccine dose, but were boosted and became more sustained following a second dose. Overall, we provide a comprehensive characterization of immune responses induced by inactivated COVID-19 vaccines in real-world settings, suggesting that both humoral and cellular SARS-CoV-2-specific immunity are elicited in the majority of individuals after two doses of inactivated COVID-19 vaccines.

Major advances have been made in understanding the dynamics of humoral immunity briefly after the acute coronavirus disease 2019 (COVID-19). However, knowledge concerning long-term kinetics of antibody responses in convalescent patients is limited. During a one-year period post symptom onset, we longitudinally collected 162 samples from 76 patients and quantified IgM and IgG antibodies recognizing the nucleocapsid (N) protein or the receptor binding domain (RBD) of the spike protein (S). After one year, approximately 90% of recovered patients still had detectable SARS-CoV-2-specific IgG antibodies recognizing N and RBD-S. Intriguingly, neutralizing activity was only detectable in ~43% of patients. When neutralization tests against the E484K-mutated variant of concern (VOC) B.1.351 (initially identified in South Africa) were performed among patients who neutralize the original virus, the capacity to neutralize was even further diminished to 22.6% of donors. Despite declining N- and S-specific IgG titers, a considerable fraction of recovered patients had detectable neutralizing activity one year after infection. However, neutralizing capacities, in particular against an E484K-mutated VOC were only detectable in a minority of patients one year after symptomatic COVID-19. Our findings shed light on the kinetics of long-term immune responses after natural SARS-CoV-2 infection and argue for vaccinations of individuals who experienced a natural infection to protect against emerging VOC.

Systemic sclerosis (SSc) is an immune-mediated systemic autoimmune disease with unknown etiology, which has high morbidity and mortality. Current treatments to dispose of this disorder are limited. And there are still no ideal animal models that can fully replicate the four basic pathophysiological features of SSc, including vascular lesions, fibrosis, inflammation, and autoimmunity, let alone animal models specifically designed to study gastrointestinal lesions. It’s essential to seek and establish appropriate animal models to explore the role of gut microbiota in the pathogenesis of SSc. In this study, we found similar gut microbiota aberration in patients of SSc and bleomycin (BLM)-induced mice model through 16S rRNA gene sequencing. In terms of phylum-level differences, the relative abundance of Bacteroidetes was significantly decreased and Firmicutes increased in the SSc patients and the mice. Notably, the genera of Lactobacillus, commonly used as a probiotic additive, was also elevated in SSc patients and BLM mice, which was consistent with a few of studies. Therefore, the model can likely mimic the pathological changes of gut microbiota in patients with SSc, which may offer an important potential platform for the in-depth understanding of gut microbiota aberration in patients with SSc and to devise potential disease-modifying treatments.

Wuhan was the very first city hit by SARS-CoV-2. Accordingly, the patients who experienced the longest phase of convalescence following COVID-19 reside here.

Commensal gut microbiota protects the immune defense of extra-intestinal organs. Gut microbiota depletion by antibiotics can impair host antiviral immune responses and alter hepatitis B virus (HBV) infection outcomes. However, how gut microbiota modulates antiviral immune response in the liver remains unclear. Here, mice were treated with broad-spectrum antibiotics to deplete gut microbiota. Gut integrity was evaluated, and translocation of live commensal gut bacteria and their components into the liver was investigated. An HBV infection model was established to evaluate impairment of antiviral immune response in the liver after gut microbiota depletion. We found that gut microbiota depletion was associated with impairment of colon epithelial integrity, and live commensal gut microbiota could translocate to the liver. Further, T cell antiviral function in the liver was impaired, partially relying on enhanced PD-1 expression, and HBV immune clearance was hampered. In conclusion, gut microbiota depletion by antibiotics can impair gut barrier function and suppress T cell antiviral immune response in the liver.

Background: The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. Methods: Peripheral blood samples were longitudinally collected from 40 confirmed COVID-19 patients and examined for lymphocyte subsets by flow cytometry and cytokine profiles by specific immunoassays. Findings: Of the 40 COVID-19 patients enrolled, 13 severe cases showed significant and sustained decreases in lymphocyte counts [0·6 (0·6-0·8)] but increases in neutrophil counts [4·7 (3·6-5·8)] than 27 mild cases [1.1 (0·8-1·4); 2·0 (1·5-2·9)]. Further analysis demonstrated significant decreases in the counts of T cells, especially CD8+ T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases. Moreover, the neutrophil-to-lymphocyte ratio (NLR) (AUC=0·93) and neutrophil-to-CD8+ T cell ratio (N8R) (AUC =0·94) were identified as powerful prognostic factors affecting the prognosis for severe COVID-19. Interpretation: The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R and NLR may serve as a useful prognostic factor for early identification of severe COVID-19 cases. Funding: The National Natural Science Foundation of China, the National Science and Technology Major Project, the Health Commission of Hubei Province, Huazhong University of Science and Technology, and the Medical Faculty of the University of Duisburg-Essen and Stiftung Universitaetsmedizin, Hospital Essen, Germany.

Abstract Background Rickettsia species belonging to the spotted fever group (SFG) cause infections in humans, domestic animals and wildlife. At least ten SFG Rickettsia species are known to occur in China. However, the distribution of rickettsiae in ticks and fleas in the border region of northwestern China have not been systematically studied to date. Results A total of 982 ticks (Rhipicephalus turanicus, Dermacentor marginatus, D. nuttalli and Haemaphysalis punctata) and 5052 fleas (18 flea species from 14 species of wild mammals) were collected in ten and five counties, respectively, of Xinjiang Uygur Autonomous Region (northwestern China). Tick and flea species were identified according to morphological and molecular characteristics. Seven sets of primers for amplifying the 17-kDa antigen gene (17-kDa), citrate synthase gene (gltA), 16S rRNA gene (rrs), outer membrane protein A and B genes (ompA, ompB), surface cell antigen 1 gene (sca1) and PS120-protein encoding gene (gene D) were used to identify the species of rickettsiae. Nine Rickettsia species have been detected, seven of them in ticks: R. aeschlimannii, R. conorii, R. raoultii, Rickettsia sibirica, R. slovaca, R. massiliae and “Candidatus R. barbariae”. In addition, R. bellii and two genotypes of a rickettsia endosymbiont (phylogenetically in an ancestral position to R. bellii) have been detected from flea pools. Conclusions This study provides molecular evidence for the occurrence of several SFG rickettsiae in Rhipicephalus turanicus, Dermacentor nuttalli and D. marginatus. Furthermore, R. bellii and two ancestral rickettsia endosymbionts are present in fleas infesting wild rodents in the border regions of northwestern China. These data extend our knowledge on the diversity of rickettsiae in Central Asia.

Abstract Background Chinese woodchucks (M. himalayana) were recently found to be susceptible to woodchuck hepatitis virus (WHV) infection. In this study, we aimed to determine the susceptibility to WHV infection of M. himalayana from different areas and their association with the animal genetic background exemplified by cytochrome B and MHC-DRB molecules. Methods Animals from four different areas in Qinghai province were inoculated with WHV59 strains. The virological markers including WHV surface antigen (WHsAg), WHV core antibody (WHcAb), and WHV DNA in serum were measured by ELISA and Real-time PCR, respectively. The sequences of cytochrome B gene and MHC-DRB molecules were obtained and sorted with Clustalx software. The nucleotide variation sites were identified using MEGA5 software. Results The animals from four different areas had different susceptibility to WHV infection. Animals from TR and TD areas had a high level of long-lasting viremia, while those from GD and WL areas had a low level of transient viremia after WHV inoculation. All of the animals belong to the same subspecies M. himalayana robusta identified by cytochrome B gene sequences. Based on their nucleotide variation pattern, 8 alleles of cytochrome B gene were identified, and 7 MHC-DRB alleles were identified. Allele A of cytochrome B and Allele Mamo-DRB1*02 of MHC-DRB was found to be frequent in animals from TR and TD areas, while Allele H of cytochrome B and Allele Mamo-DRB1*07 of MHC-DRB was predominant in animals from GD and WL areas. Conclusion Chinese woodchucks from different areas differed in their susceptibility to WHV infection, though they belong to the same subspecies M. himalayana robusta. The genetic background exemplified by cytochrome B and MHC-DRB differed in Chinese woodchucks with different susceptibility to WHV infection.

When Saccharomyces cerevisiae divides, a structure composed of different septin proteins arranged according to a certain rule is formed at the cell division site. The structure undergoes multiple remodeling stages during the cell cycle, thus guiding the yeast cells to complete the entire division process. Although the higher-order structure of septins can be determined using electron microscopy, the septin’s dynamic processes are poorly understood because of limitations in living cell super-resolution imaging technology. Herein, we describe a high lateral resolution and temporal resolution technique, known as fast fluctuation-enhanced structured illumination microscopy (fFE-SIM), which more than doubles the SIM resolution at a frame rate of 38 Hz in living cells. This allows a highly dynamic and sparse septin structure to be observed in Saccharomyces cerevisiae.

Upconversion nanoparticles, which do not suffer from the photophysical artifacts that limit fluorescent molecules, offer an exciting opportunity for biological super-resolution imaging. Here, Zhan et al. develop an efficient STED mechanism using optimized lanthanide upconversion nanoparticles, enabling cytoskeleton nanoscopic imaging.

Gut microbiota composition is known to be associated with the progression of hepatitis B virus (HBV)-related liver cirrhosis in humans, outcome of HBV infection in mice, and seroconversion of HBV e-antigen in nucleot(s)ide analog-treated patients. The dynamic alteration of the gut microbiota following HBV infection is still unknown. In this study, a hydrodynamic injection mouse model mimicking acute or chronic HBV infection in humans with comparable virological and immunological features was used. The composition of gut microbiota in the control mice and mice with acute or chronic HBV infection was analyzed at different time points using the Illumina MiSeq platform. The expression of immune molecules in the colon was detected by real-time polymerase chain reaction. We found that the changes in gut microbiota composition, including the total operational taxonomic unit (OTU) count and Shannon-Weaver index, were significantly delayed in mice with HBV infection. Furthermore, the ratio of Bacteroidetes and Firmicutes was stable in the control mice, whereas remarkable dynamic patterns were observed in mice with HBV infection. Interestingly, the dynamic changes in Lactobacillus and Bifidobacterium were found to differ in acute or chronic HBV infection. In addition, the expression of IFN-γ and PD-L1 in the colon was found to be up-regulated early in mice with acute HBV infection, whereas the expression of PD-L1 in the colon of mice with chronic HBV infection was up-regulated later. These data indicate that HBV infection could hamper the development of the gut microbiota community and dynamically change the gut Firmicutes/Bacteroidetes ratio. These data improve our understanding of the relationship between gut microbiota and HBV infection.

Lanthanide-doped upconversion nanoparticles (UCNPs) are promising bioimaging nanoprobes due to their excellent photostability. As one of the most commonly used lanthanide activators, Tm3+ ions have perfect ladder-type electron configuration and can be directly excited by bio-friendly near-infrared-II (NIR-II) wavelengths. Here, the emission characteristics of Tm3+-doped nanoparticles under laser excitations of different near-infrared-II wavelengths were systematically investigated. The 1064 nm, 1150 nm, and 1208 nm lasers are proposed to be three excitation strategies with different response spectra of Tm3+ ions. In particular, we found that 1150 nm laser excitation enables intense three-photon 475 nm emission, which is nearly 100 times stronger than that excited by 1064 nm excitation. We further optimized the luminescence brightness after investigating the luminescence quenching mechanism of bare NaYF4: Tm (1.75%) core. After growing an inert shell, a ten-fold increase of emission intensity was achieved. Combining the advantages of NIR-II wavelength and the higher-order nonlinear excitation, a promising facile excitation strategy was developed for the application of thulium-doped upconversion nanoparticles in nanoparticles imaging and cancer cell microscopic imaging.

Plant protection drone spraying technology is widely used to prevent and control crop diseases and pests due to its advantages of being unaffected by crop growth patterns and terrain restrictions, high operational efficiency, and low labor requirements. The operational parameters of plant protection drones significantly impact the distribution of spray droplets, thereby affecting pesticide utilization. In this study, a field experiment was conducted to determine the working modes of two representative plant protection drones and an electric backpack sprayer as a control to explore the characteristics of droplet deposition with different spray volumes in the citrus canopy. The results showed that the spraying volume significantly affected the number of droplets and the spray coverage. The number of droplets and the spray coverage area on the leaf surface were significantly increased by increasing the spray volume from 60 L/ha to 120 L/ha in plant protection drones. Particularly for the DJI T30, the mid-lower canopy showed a spray coverage increase of 52.5%. The droplet density demonstrated the most significant variations in the lower inner canopy, ranging from 18.7 droplets/cm2 to 41.7 droplets/cm² by XAG V40. From the deposition distribution on fruit trees, the plant protection drones exhibit good penetration ability, as the droplets can achieve a relatively even distribution in different canopy layers of citrus trees. The droplet distribution uniformity inside the canopy is similar for XAG V40 and DJI T30, with a variation coefficient of approximately 50%-100%. Compared to the plant protection drones, the knapsack electric sprayer is suitable for pest and disease control in the midlower canopy, but they face challenges of insufficient deposition capability in the upper canopy and overall poor spray uniformity. The distribution of deposition determined in this study provides data support for the selection of spraying agents for fruit trees by plant protection drones and for the control of different pests and diseases. [ABSTRACT FROM AUTHOR]

Th17 responses have been shown to play crucial roles in the pathogenesis of hepatitis B virus (HBV)-associated acute-on-chronic liver failure (ACLF). The mechanism underlying the enhanced Th17 responses in these patients remains largely unclear. Here we investigated toll-like receptors (TLRs) expression in peripheral T cells and their roles in Th17 cell differentiation and disease aggravation in ACLF patients. 18 healthy subjects (HS), 20 chronic HBV-infected (CHB) patients, and 26 ACLF patients were enrolled and examined for TLRs expression in peripheral blood mononuclear cells (PBMCs). The correlations of T cell TLR2 expression with the antigen non-specific Th17 responses and disease aggravation, as well as the Th17 response to TLR2 ligand stimulation were evaluated in ACLF patients. Compared to HS and CHB patients, ACLF patients showed a distinct TLRs expression pattern in PBMCs. Significantly increased TLR2 expression in T cells was observed in ACLF patients. The TLR2 expression in CD4+ T cells was correlated with the Th17 responses and the clinical markers for disease aggravation in ACLF patients. Moreover, TLR2 ligands stimulation promoted Th17 cell differentiation and response in PBMCs of ACLF patients. These findings implicate that TLR2 signaling plays critical roles in Th17 cell differentiation and disease aggravation of HBV-related ACLF.

Regulatory T cells (Tregs) play an important role in counter-regulating effector T cell responses in many infectious diseases. However, they can also contribute to the development of T cell dysfunction and pathogen persistence in chronic infections. Tregs have been reported to suppress virus-specific T cell responses in hepatitis B virus (HBV) infection of human patients as well as in HBV animal models. However, the phenotype and expansion of Tregs has so far only been investigated in other infections, but not in HBV. We therefore performed hydrodynamic injections of HBV plasmids into mice and analyzed the Treg response in the spleen and liver. Absolute Treg numbers significantly increased in the liver but not the spleen after HBV injection. The cells were natural Tregs that surprisingly did not show any activation or proliferation in response to the infection. However, they were able to suppress effector T cell responses, as selective depletion of Tregs significantly increased HBV-specific CD8+ T cell responses and accelerated viral antigen clearance. The data implies that natural Tregs infiltrate the liver in HBV infection without further activation or expansion but are still able to interfere with T cell mediated viral clearance.

The Eastern woodchuck (Marmota monax) is a classical animal model for studying hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) in humans. Recently, we found that Marmota himalayana, an Asian animal species closely related to Marmota monax, is susceptible to woodchuck hepatitis virus (WHV) infection and can be used as a new mammalian model for HBV infection. However, the lack of genomic sequence information of both Marmota models strongly limited their application breadth and depth. To address this major obstacle of the Marmota models, we utilized Illumina RNA-Seq technology to sequence the cDNA libraries of liver and spleen samples of two Marmota monax and four Marmota himalayana. In total, over 13 billion nucleotide bases were sequenced and approximately 1.5 billion clean reads were obtained. Following assembly, 106,496 consensus sequences of Marmota monax and 78,483 consensus sequences of Marmota himalayana were detected. For functional annotation, in total 73,603 Unigenes of Marmota monax and 78,483 Unigenes of Marmota himalayana were identified using different databases (NR, NT, Swiss-Prot, KEGG, COG, GO). The Unigenes were aligned by blastx to protein databases to decide the coding DNA sequences (CDS) and in total 41,247 CDS of Marmota monax and 34,033 CDS of Marmota himalayana were predicted. The single nucleotide polymorphisms (SNPs) and the simple sequence repeats (SSRs) were also analyzed for all Unigenes obtained. Moreover, a large-scale transcriptome comparison was performed and revealed a high similarity in transcriptome sequences between the two marmota species. Our study provides an extensive amount of novel sequence information for Marmota monax and Marmota himalayana. This information may serve as a valuable genomics resource for further molecular, developmental and comparative evolutionary studies, as well as for the identification and characterization of functional genes that are involved in WHV infection and HCC development in the woodchuck model.

The microRNA171 (miR171) family is widely distributed and highly conserved in a range of species and plays critical roles in regulating plant growth and development through repressing expression of () transcription factors. However, information on the evolutionary conservation and functional diversification of the miRNA171 family members remains scanty. We reconstructed the phylogenetic relationships among miR171 precursor and mature sequences so as to investigate the extent and degree of evolutionary conservation of miR171 in (L.) Heynh. (ath), grape ( L.) (vvi), poplar ( Torr. & A.Gray ex Hook.) (ptc), and rice ( L.) (osa). Despite strong conservation of over 80%, some mature miR171 sequences, such as , and and , -, and -, have undergone critical sequence variation, leading to functional diversification, since they target non gene transcript(s). Phylogenetic analyses revealed a combination of old ancestral relationships and recent lineage-specific diversification in the miR171 family within the four model plants. The -regulatory motifs on the upstream promoter sequences of genes were highly divergent and shared some similar elements, indicating their possible contribution to the functional variation observed within the miR171 family. This study will buttress our understanding of the functional differentiation of miRNAs and the relationships of miRNA–target pairs based on the evolutionary history of genes.

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.

Hepatitis B virus (HBV) reactivation and recurrence are common in patients under immunosuppression and can be controlled by hepatitis B immunoglobulin, antivirals, and hepatitis B vaccine. However, the detailed analysis of HBV infection under immunosuppression is essential for the prophylaxis and therapy for HBV reactivation and recurrence. In this study, HBV replication and T cell responses were analyzed in a HBV-transfected mouse model under immunosuppressive therapy. During the treatment, HBV replication was at a high level in mice treated with dexamethasone, cyclosporine, and cyclophosphamide, whereas was terminated in mice treated with mycophenolate mofetil. After the withdrawal, HBV replication was at low or high levels in the dexamethasone-treated mice or in both cyclosporine- and cyclophosphamide-treated mice. The early withdrawal of cyclosporine allowed the recovery of suppressed T cell responses and led to subsequent HBV clearance, while the adoptive immune transfer to the mice with HBV persistence led to HBV suppression. Taken together, long-term HBV persistence under immunosuppression depends on the immunosuppressive drugs used and on the treatment duration and is mediated by the suppressed intrahepatic CD8 T cell response. These data may be helpful for individualized immunosuppressive therapy in patients with high risk of HBV reactivation and recurrence, and the mouse system is suitable for studying HBV reactivation and recurrence under immunosuppression.

Interferon alpha (IFN-α) is commonly used for the treatment of chronic hepatitis B (CHB) patients. Many factors including viral genetics may determine the outcome of IFN-α therapy. In this study, we tested whether the expression of IFN-α directly in the liver inhibits HBV gene expression and replication using a HBV hydrodynamic injection (HI) mouse model. Two replication-competent clones from different HBV isolates that belonging to HBV genotype A and B based on a pAAV vector (pAAV-HBV-A and pAAV-HBV-B) were compared for their susceptibility to IFN-α. HBV clones were injected into mice either alone or in combination with a murine (m) IFN-α expression plasmid (pmIFN-α). HBsAg and HBeAg concentrations and HBV DNA levels in mice differed after injection of these two HBV clones. Co-application of pmIFN-α together with the two distinct isolates resulted in markedly different kinetics of decline of HBsAg, HBeAg, and HBV DNA levels in the mice. Immunohistochemical staining of liver sections with anti-HBc showed that mIFN-α application completely inhibited the expression of HBcAg in mice inoculated with pAAV-HBV-B, whereas the expression of HBcAg was only reduced in mice with pAAV-HBV-A. Consistently, mice injected with pAAV-HBV-B and pmIFN-α showed higher expression levels of the IFN-stimulated genes (ISGs) ISG15, OAS, PKR as well as proinflammatory cytokine IL-6 in the liver. In addition, expression levels of anti-inflammatory cytokine IL-10 was down-regulated significantly in liver of the mice injected with pAAV-HBV-B and pmIFN-α. Our data demonstrate that IFN-α exerts antiviral activity in HBV mouse model, but different HBV isolates may have diverse susceptibility to IFN-α.

BACKGROUND: The programmed cell death 1 (PD-1)/programmed death-1 ligand 1 (PD-L1) system may play a role in the negative regulation of T cell functions in hepatitis B virus (HBV) infection. Thus, it is important to study its role in the widely used animal model for HBV infection of woodchucks with woodchuck hepatitis virus (WHV). METHODS: Woodchuck PD-L1 (wPD-L1) and -L2 (wPD-L2) were cloned and characterized. The levels of wPD-L1 expression in primary woodchuck hepatocytes (PWH), peripheral blood mononuclear cells (PBMCs), and liver tissue of naive and WHV-infected woodchucks were examined by real time reverse transcription (RT)-PCR and flow cytometry. Using antibodies against wPD-L1 and -L2, the effect of blocking PD-1/PD-L1/PD-L2 interaction on the proliferation and degranulation of woodchuck PBMCs was examined. PRINCIPAL FINDINGS: Both wPD-L1 and -L2 showed a high homology to their counterparts of other mammalian species and humans. WPD-L1 expression in PWH and PBMCs of naive animals was low but could be stimulated by Toll-like receptor (TLR) ligands and interferons (IFN). WPD-L1 expression in liver tissue was significantly higher than that measured in PWHs and was slightly elevated during acute and chronic WHV infection. However, wPD-1 and wPD-L1 expression on PBMCs was strongly up-regulated during acute and chronic infection. In vitro blockade with antibodies against wPD-L1 and -L2 partially enhanced proliferation and degranulation of PBMCs from WHV-infected woodchucks. CONCLUSIONS: Our results demonstrated that wPD-1/wPD-L1 expression in hepatocytes and PBMCs can be induced by different inflammatory stimuli and is up-regulated mainly on PBMCs during WHV infection. A blockade of the woodchuck PD-1/PD-L pathway could partially enhance T cell functions in WHV infection.

BACKGROUND: The human asialoglycoprotein receptor (ASGPR) is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR) composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.

Cystic echinococcosis (CE) caused by E. granulosus is a serious helminthic zoonosis in humans, livestock and wildlife. Xinjiang is one of high endemic province for CE in China. A total of 55 sheep and cattle livers containing echinococcal cysts were collected from slaughterhouses in Changji and Yining City, northern region of Xinjiang. PCR was employed for cloning 2 gene fragments, 12S rRNA and CO1 for analysis of phylogenetic diversity of E. granulosus. The results showed that all the samples collected were identified as G1 genotype of E. granulosus. Interestingly, YL5 and CJ75 strains were the older branches compared to those strains from France, Argentina, Australia. CO1 gene fragment showed 20 new genotype haploids and 5 new genotype haplogroups (H1-H5) by the analysis of Network 5.0 software, and the YLY17 strain was identified as the most ancestral haplotype. The major haplotypes, such as CJ75 and YL5 strains, showed identical to the isolates from Middle East. The international and domestic trade of livestock might contribute to the dispersal of different haplotypes for E. granulosus evolution. [ABSTRACT FROM AUTHOR]

miR156 regulates the expression of its target SPL (PROMOTER BINDING-LIKE) genes during flower and fruit development, diverse developmental stage transitions, especially from vegetative to reproductive growth phases, by cleaving the target mRNA SPL of one plant-specific transcription factor. However, systematic reports on grapevine have yet to be presented. Here, the precise sequence of miR156 (vvi-miR156b/c/d) in grapevine "Takatsuma" was cloned with a previously cloned grapevine SPL (Vv-SPL9). Expression profiles in 18 grapevine tissues were identified through stem-loop RT-PCR. The interaction mode between vvi-miR156b/c/d and Vv-SPL9 was further validated by detecting the cleavage site and cleavage products of 3'- and 5'-ends via an integrated approach of 5'-RLM-RACE (RNA ligase-mediated 5'-rapid amplification of cDNA ends), 3'-PPM-RACE (poly(A) polymerase-mediated 3'-rapid amplification of cDNA ends), and qRT-PCR (real time reverse transcriptase-polymerase chain reaction). The variation in their cleavage roles in the whole growth stage of grapevine was also systematically investigated. Results showed that vvi-miR156b/c/d exhibited typical temporal-spatialspecific expression levels. The expression levels were higher in vegetative organs, such as leaf, than in reproductive organs, such as tendrils, flowers, and berries. A significant variation was observed during vegetative-to-reproductive transition. The expression patterns of Vv-SPL9 showed the opposite trends with those of vvi-miR156b. We confirmed that the cleavage site was at the 10th site of vvimiR156b/ c/d complementary to Vv-SPL9 in "Takatsuma" grapevine. We also identified the temporal- spatial variation of the cleavage products. This variation can indicate the regulatory function of miR156 on SPL in grapevines. Our findings provide further insights into the functions of vvi-miR156b/c/d and its target Vv-SPL9, and also help enrich our knowledge of small RNA-mediated regulation in grapevine. [ABSTRACT FROM AUTHOR]

Polyphenol oxidases catalyzing the oxygen-dependent oxidation of phenols to quinones are ubiquitous among angiosperms. They are key enzymes playing a significant role during the synthesis of lignin. The inhibition of the synthesis of lignin in grapevine can cause seedless grapevine berry development. In this study, grapevine PPO (Vv-PPO) was predicted as the target gene of Vv-miR058 by bioinformatics analysis, and it was further cloned and its homologous conservation in various plants was analyzed. The expression profiles of miR058 and its target Vv-PPO were detected by qRT-PCR in peel, pulp and seeds of three grapevine cultivars and Vv-PPO was expressed in an opposite variation way with Vv-miR058 where both of them could be detected, suggesting that Vv-miR058 can play an important role by regulating the expression of Vv-PPO. In addition, the potential target gene Vv-PPO for Vv-miR058 was verified by RLM-RACE. This result would be helpful in theoretical basis for further research and seedless grapevine berry production. [ABSTRACT FROM AUTHOR]

Background: It has been suggested that different hepatitis B virus (HBV) genotypes may have distinct virological characteristics that correlate with clinical outcomes during antiviral therapy and the natural course of infection. Hydrodynamic injection (HI) of HBV in the mouse model is a useful tool for study of HBV replication in vivo. However, only HBV genotype A has been used for studies with HI. Methods: We constructed 3 replication-competent clones containing 1.1, 1.2 and 1.3 fold overlength of a HBV genotype B genome and tested them both in vitro and in vivo. Moreover, A HBV genotype B clone based on the pAAV-MCS vector was constructed with the 1.3 fold HBV genome, resulting in the plasmid pAAV-HBV1.3B and tested by HI in C57BL/6 mice. Application of siRNA against HBx gene was tested in HBV genotype B HI mouse model. Results: The 1.3 fold HBV clone showed higher replication and gene expression than the 1.1 and 1.2 fold HBV clones. Compared with pAAV-HBV1.2 (genotype A), the mice HI with pAAV-HBV1.3B showed higher HBsAg and HBeAg expression as well as HBV DNA replication level but a higher clearance rate. Application of two plasmids pSB-HBxi285 and pSR-HBxi285 expressing a small/short interfering RNA (siRNA) to the HBx gene in HBV genotype B HI mouse model, leading to an inhibition of HBV gene expression and replication. However, HBV gene expression may resume in some mice despite an initial delay, suggesting that transient suppression of HBV replication by siRNA may be insufficient to prevent viral spread, particularly if the gene silencing is not highly effective. Conclusions: Taken together, the HI mouse model with a HBV genotype B genome was successfully established and showed different characteristics in vivo compared with the genotype A genome. The effectiveness of gene silencing against HBx gene determines whether HBV replication may be sustainably inhibited by siRNA in vivo. [ABSTRACT FROM AUTHOR]

The transgenic Bacillus thuringiensis (Bt) rice, Oryza sativa L., lines T2A-1 and T1c-19 expressing Cry2A* and Cry1C* from 'Minhui 63' (MH63) were evaluated for resistance to newly hatched and third-instar larvae of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), by using detached leaf laboratory bioassays. Both T2A-1 and T1c-19 rice showed high C. medinalis resistance; however, the lethal time (LT) 50 of larvae fed with T2A-1 rice was significantly longer than that of larvae fed with T1c-19 rice, implying T1c-19 rice was more toxic to C. medinalis larvae. Larval mortality after 4 d on nitrogen-free MH63 was 25.5% compared with 2.4% mortality on high nitrogen fertilizer (250 kg N/ha) plants. Larval mortality on high nitrogen T2A-1 plants declined by 20% compared with nitrogen-free plants. However, resistance in T1c-19 plants was unaffected by nitrogen fertilizer. C. medinalis moths preferred MH63 at both the seedling and grain milk stages for oviposition but not the T1c-19 and T2A-1 Bt rice lines. [ABSTRACT FROM AUTHOR]

It is often difficult to implement complex microscopy systems without spherical aberration. Herein, we developed a novel, robust, three-dimensional (3D), bifocal plane, single-particle tracking technique, based on a dual-objective fluorescent reflection system with spherical aberration (DOFR–SA). It can simultaneously image a pair of focused and defocused planes containing fluorescent particles with a single camera instead of splitting photons into two channels. Based on the 3D DOFR–SA, the desired position accuracy along the z-axis was achieved without compromising the precisions of the (x, y ) positions, even with limited number of photons from a single molecule. Accordingly, this method was applied to fluorescent particle tracking in biofluids and living cells with high-spatial and temporal precisions. [ABSTRACT FROM AUTHOR]

It remains controversial how interferon (IFN) response contributes to hepatitis B virus (HBV) control and pathogenesis. A previous study identified that hydrodynamic injection (HI) of type I IFN (IFN-I) inducer polyinosinic-poly(C) [poly(I·C)] leads to HBV clearance in a chronic HBV mouse model. However, recent studies have suggested that premature IFN-I activation in the liver may facilitate HBV persistence. In the present study, we investigated how the early IFN-I response induces an immunosuppressive signaling cascade and thus causes HBV persistence. We performed HI of the plasmid adeno-associated virus (pAAV)/HBV1.2 into adult BALB/c mice to establish an adult acute HBV replication model. Activation of the IFN-I signaling pathway following poly(I·C) stimulation or murine cytomegalovirus (MCMV) infection resulted in subsequent HBV persistence. HI of poly(I·C) with the pAAV/HBV1.2 plasmid resulted in not only the production of IFN-I and the anti-inflammatory cytokine interleukin-10 (IL-10) but also the expansion of intrahepatic regulatory T cells (Tregs), Kupffer cells (KCs), and myeloid-derived suppressor cells (MDSCs), all of which impaired the T cell response. However, when poly(I·C) was injected at day 14 after the HBV plasmid injection, it significantly enhanced HBV-specific T cell responses. In addition, interferon-alpha/beta receptor (IFNAR) blockade rescued T cell response by downregulating IL-10 expression and decreasing Treg and KC expansion. Consistently, Treg depletion or IL-10 blockade also controlled HBV replication. [ABSTRACT FROM AUTHOR]

Functional maturation of liver sinusoidal endothelial cells (LSECs) induced by a NOD1 ligand (diaminopimelic acid [DAP]) during viral infection has not been well defined. Thus, we investigated the role of DAP-stimulated LSEC maturation during hepatitis B virus (HBV) infection and its potential mechanism in a hydrodynamic injection (HI) mouse model. Primary LSECs were isolated from wild-type C57BL/6 mice and stimulated with DAP in vitro and in vivo and assessed for the expression of surface markers as well as for their ability to promote T cell responses via flow cytometry. The effects of LSEC maturation on HBV replication and expression and the role of LSECs in the regulation of other immune cells were also investigated. Pretreatment of LSECs with DAP induced T cell activation in vitro. HI-administered DAP induced LSEC maturation and subsequently enhanced T cell responses, which was accompanied by an increased production of intrahepatic cytokines, chemokines, and T cell markers in the liver. The HI of DAP significantly reduced the HBsAg and HBV DNA levels in the mice. Importantly, the DAP-induced anti-HBV effect was impaired in the LSEC-depleted mice, which indicated that LSEC activation and T cell recruitment into the liver were essential for the antiviral function mediated by DAP application. Taken together, the results showed that the Ag-presenting ability of LSECs was enhanced by DAP application, which resulted in enhanced T cell responses and inhibited HBV replication in a mouse model. The Journal of Immunology, 2018, 200: 3170-3179. [ABSTRACT FROM AUTHOR]

We have previously shown that poly(I:C) activates murine hepatic cells to produce interferon (IFN) and suppresses hepatitis B virus (HBV) replication in vitro. Therefore, we addressed whether poly(I:C) is able to induce the clearance of HBV in vivo. The chronic HBV replication mouse model was established by the hydrodynamic injection (HI) of pAAV-HBV1.2 into the tail veins of wild-type and IFN-α/βR-, IFN-γ-, and CXCR3-deficient C57BL/6 mice. Fourteen days post-HI of pAAV-HBV1.2, mice were administered poly(I:C) by intraperitoneal injection, intramuscular injection, or HI. Only treatment of poly(I:C) by HI led to HBV clearance in wild-type C57BL/6 mice. Serum HBsAg disappeared within 40 days postinfection (dpi) in mice that received poly(I:C) by HI, and this was accompanied by the appearance of anti-HBs antibodies. HBV-specific T-cell and antibody responses were significantly enhanced by HI of poly(I:C). HBV replication intermediates and HBcAg-positive hepatocytes were eliminated in the liver. HI of poly(I:C) induced the production of IFNs in mice and enhanced the levels of cytokines, IFN-stimulated genes, and T-cell markers in the liver. Importantly, poly(I:C)-induced HBV clearance was impaired in IFN-α/βR-, IFN-γ-, and CXCR3-deficient mice, indicating that the induction of type I IFN and the stimulation and recruitment of T cells into the liver are essential for HBV clearance in this model. Taken together, the application of poly(I:C) by HI into the liver enhances innate and adaptive immune responses and leads to HBV clearance in an HBV mouse model, implicating the potential of intrahepatic Toll-like receptor 3 (TLR3) activation for the treatment of chronic hepatitis B patients. [ABSTRACT FROM AUTHOR]