Mitochondrial Glycerol-3-Phosphate Dehydrogenase Restricts HBV Replication via the TRIM28-Mediated Degradation of HBx.

Hepatitis B virus (HBV) infection affects hepatic metabolism. Serum metabolomics studies have suggested that HBV possibly hijacks the glycerol-3-phosphate (G3P) shuttle. In this study, the two glycerol-3-phosphate dehydrogenases (GPD1 and GPD2) in the G3P shuttle were analyzed for determining their role in HBV replication and the findings revealed that GPD2 and not GPD1 inhibited HBV replication. The knockdown of GPD2 expression upregulated HBV replication, while GPD2 overexpression reduced HBV replication. Moreover, the overexpression of GPD2 significantly reduced HBV replication in hydrodynamic injection-based mouse models. Mechanistically, this inhibitory effect is related to the GPD2-mediated degradation of HBx protein by recruiting the E3 ubiquitin ligase TRIM28 and not to the alterations in G3P metabolism. In conclusion, this study revealed GPD2, a key enzyme in the G3P shuttle, as a host restriction factor in HBV replication. IMPORTANCE The glycerol-3-phosphate (G3P) shuttle is important for the delivery of cytosolic reducing equivalents into mitochondria for oxidative phosphorylation. The study analyzed two key components of the G3P shuttle and identified GPD2 as a restriction factor in HBV replication. The findings revealed a novel mechanism of GPD2-mediated inhibition of HBV replication via the recruitment of TRIM28 for degrading HBx, and the HBx-GPD2 interaction could be another potential therapeutic target for anti-HBV drug development.

DNA Repair Factor Poly(ADP-Ribose) Polymerase 1 Is a Proviral Factor in Hepatitis B Virus Covalently Closed Circular DNA Formation.

The biogenesis of covalently closed circular DNA (cccDNA) from relaxed circular DNA (rcDNA) is essential for chronic hepatitis B virus (HBV) infection. Different host DNA repair proteins are involved in the conversion of rcDNA to cccDNA. Here, we reported that the DNA repair factor poly(ADP-ribose) polymerase 1 (PARP1) is engaged in HBV cccDNA formation. PARP1 depletion remarkably impaired HBV replication and cccDNA synthesis. Inhibition of PARP1 poly (ADP-ribosylation) activity by olaparib suppressed cccDNA synthesis both in vitro and in vivo. Specifically, the early stage of cccDNA reservoir establishment was more sensitive to olaparib, suggesting that PARP1 participated in de novo cccDNA formation. Furthermore, PARP1 was activated by recognizing the rcDNA-like lesions directly and combined with other DNA repair proteins. The results presented proposed that the DNA damage-sensing protein PARP1 and poly(ADP-ribosylation) modification play a key role in cccDNA formation, which might be the target for developing the anti-HBV drug. IMPORTANCE The biogenesis and eradication of HBV cccDNA have been a research priority in recent years. In this study, we identified the DNA repair factor PARP1 as a host factor required for the HBV de novo cccDNA formation. HBV infection caused PARylation through PARP1 in Huh7-NTCP cells, primary human hepatocytes, and human-liver chimeric mice. We found that PARP1 could directly bind to the rcDNA lesions and was activated, PARylating other DNA repair proteins. We address the importance of PARP1-mediated PARylation in HBV cccDNA formation, which is a potential therapeutic target for chronic hepatitis B.

RNA-binding motif protein 24 inhibits HBV replication in vivo.

Despite the extensive use of effective vaccines and antiviral drugs, chronic hepatitis B virus (HBV) infection continues to pose a serious threat to global public health. Therapies with novel mechanisms of action against HBV are being explored for achieving a functional cure. In this study, five murine models of HBV replication were used to investigate the inhibitory effect of RNA binding motif protein 24 (RBM24) on HBV replication. The findings revealed that RBM24 serves as a host restriction factor and suppresses HBV replication in vivo. The transient overexpression of RBM24 in hydrodynamics-based mouse models of HBV replication driven by the CMV or HBV promoters suppressed HBV replication. Additionally, the ectopic expression of RBM24 decreased viral accumulation and the levels of HBV covalently closed circular DNA (cccDNA) in an rcccDNA mouse model. The liver-directed transduction of adeno-associated viruses (AAV)-RBM24 mediated the stable hepatic expression of RBM24 in pAAV-HBV1.2 and HBV/tg mouse models, and markedly reduced the levels of HBV cccDNA and other viral indicators. Altogether, these findings revealed that RBM24 inhibits the replication of HBV in vivo, and RBM24 may be a potential therapeutic target for combating HBV infections.

Effect of greenhouse gas emissions on the life cycle of biomass energy production and conversion under different straw recycling modes.

With the gradual growth of greenhouse gas (GHG) emissions during the agricultural cultivation cycle, GHG emissions specific to the production and conversion of biomass energy is becoming increasingly problematic. Current studies lack analysis of net GHG emissions generated during full life cycle of agricultural cultivation, straw use and bioenergy production. This study measures the global warming potential of biomass energy production and conversion processes under different agricultural cultivation cycle systems based on life cycle approach, accompanied by four straw treatment methods: fast pyrolysis, slow pyrolysis, flash pyrolysis and anaerobic fermentation. The demonstration of Heilongjiang Province showed that the net GHG emissions of rice and soybean over 52.39% and 101.57% higher than those of corn, respectively. The amount of standard coal saved by fast pyrolysis treatment, slow pyrolysis treatment and anaerobic fermentation treatment of straw was only 38.38%, 78.02% and 61.98% of that of flash pyrolysis treatment. The relationship between environmental pressure and economic growth was decoupled during 2011-2017 and coupled in 2017-2020. This study contributes to green production of biomass energy. The methodology in this paper can be used to account for and assess the carbon effect of the entire straw recycling chain in any region.

[Reducing the Incidence of Postoperative Hypothermia in Patients Undergoing Robotic-Assisted Mitral Valve Surgery].

BACKGROUND & PROBLEMS: Post-operation hypothermia tends to induce complications. Sixty percent of robotic-assisted mitral valve surgery patients experienced hypothermia while admitted to our intensive care unit (ICU), resulting in prolonged ICU stays and 57% (eight) of those patients with hypothermia also experiencing cardiac arrhythmia. The causes of hypothermia in our ICU included low temperature in the operating room, delayed initiation of blanket coverage after surgery, and lack of postoperative thermal blankets, insufficient cardiopulmonary bypass rewarming time, cold ICU beds, lack of in-service training for hypothermia, and lack of procedure auditing. PURPOSE: This intervention was designed to reduce the incidence of hypothermia in ICU patients undergoing robotic-assisted mitral valve surgery upon ICU admission from 60% to 36% and the one-hour hypothermia rate from 43.3% to 26%. RESOLUTIONS: We implemented several measures including increasing the room temperature, pre-heating the ICU bed, achieving team consensus regarding prolonging the rewarming time after cardiopulmonary bypass, establishing a blanket warming area for postoperative patient use, and holding in-service training to enhance the awareness of the nurses were implemented. RESULTS: The incidence of hypothermia in ICU patients receiving robotic-assisted mitral valve surgery upon ICU admission decreased from 60% to 19.4%, while the one-hour hypothermia rate decreased from 43.3% to 19.4%. CONCLUSIONS: Using systemic interprofessional collaboration, combined thermal care can be achieved to significantly reduce the incidence of postoperative hypothermia in patients undergoing robotic-assisted mitral valve surgeries resulting in higher patient care quality and shorter ICU stays. We recommend applying this combined method to improve the quality of perioperative care for long-duration and major surgical procedures that involve large postoperative wounds and for patients who may require wider exposure during their operation.

Simultaneous or Prior Activation of Intrahepatic Type I Interferon Signaling Leads to Hepatitis B Virus Persistence in a Mouse Model.

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. IMPORTANCE IFN-I plays a double-edged sword role during chronic HBV infection. Here, we identified that application of IFN-I at different time points causes contrast outcomes. Activation of the IFN-I pathway before HBV replication induces an immunosuppressive signaling cascade in the liver and consequently caused HBV persistence, while IFN-I activation post HBV infection enhances HBV-specific T cell responses and thus promotes HBV clearance. This result provided an important clue to the mechanism of HBV persistence in adult individuals.

Mucosal-Associated Invariant T Cell Dysregulation Correlates With Conjugated Bilirubin Level in Chronic HBV Infection.

BACKGROUND AND AIMS: Mucosal-associated invariant T (MAIT) cells are nonconventional T cells restricted to major histocompatibility complex class I-related protein 1 (MR1). They are highly abundant in human liver and activated by T-cell receptor (TCR)-dependent and TCR-independent mechanisms to exhibit rapid, innate-like effector responses. However, the roles of MAIT cells in chronic HBV infection are still open for study. This study aims to test their antiviral potential and investigate their dynamic changes and regulating factors during chronic HBV infection. APPROACH AND RESULTS: Blood samples from 257 chronic HBV-infected patients were enrolled, and nontumor liver specimens were collected from 58 HBV-infected HCC patients. Combining cell-culture experiments and human data, we showed that MAIT cells had strong cytotoxicity against HBV-transfected hepatocytes in an MR1-dependent way. However, circulating and hepatic MAIT cells in HBV-infected patients decreased significantly compared to controls. Correlation analysis suggested that MAIT cell frequency was associated with disease progression and inversely correlated with serum-conjugated bilirubin level. In particular, conjugated bilirubin not only directly promoted MAIT cell activation and apoptosis, but also impaired TCR-induced proliferation and expansion of MAIT cells, which could be partially rescued by IL-2 in the absence of conjugated bilirubin. Despite that MAIT cells from patients with high conjugated bilirubin levels showed decreased cytokine-producing capacity, the increased TCR-dependent antiviral cytokine production suggested MAIT cells as an important guardian of chronic HBV with high conjugated bilirubin. CONCLUSIONS: We reveal the MR1-dependent, anti-HBV potential of MAIT cells and identify conjugated bilirubin as a major factor dysregulating its frequency and function in chronic HBV-infected patients, suggesting a therapeutic target for MAIT-cell-based immunity against chronic HBV infection.

Development of enzyme-free single-step immunoassays for glycocholic acid based on palladium nanoparticle-mediated signal generation.

Palladium nanoparticles (PdNPs) are composed mainly of inert noble metals, and their outstanding properties have attracted wide attention. PdNPs are not only capable of mimicking the oxidase-like characteristics of natural bio-enzymes, but they also present a clear black band in the test zone. In this work, the synthesized PdNPs promoted a transformation of colorless tetramethylbenzidine (TMB) to a blue oxidation product of TMB, providing a Km value of 0.09 mM for TMB, and revealing the good catalytic performance of the synthesized PdNPs. For both signal generation and amplification, PdNPs effectively replaced natural bio-enzymes as a new labeling tag. Thus, the PdNP-based enzyme-free single-step immunoassays were successfully developed for efficient and sensitive detection of glycocholic acid (GCA). Under optimal conditions, a noticeable linear relationship was identified by the enzyme-linked immunosorbent assay (ELISA) over a range of 8-2390 ng/mL, while the visual limit of detection (vLOD) in the constructed lateral flow immunoassay (LFA) was 10 ng/mL for GCA. The recovery rate in spiked urine samples obtained by the ELISA ranged from 84.2 to 117.9%, which was consistent with the results in LFA. The present work demonstrates the potential of PdNPs as labeling matrices in enzyme-free single-step immunoassays.

RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element.

Encapsidation of pregenomic RNA (pgRNA) is a crucial step in hepatitis B virus (HBV) replication. Binding by viral polymerase (Pol) to the epsilon stem-loop (ε) on the 5'-terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5'-TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of ε via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase (RT) domain of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol-ε interaction, whereas RBM24 knockdown decreases the interaction. RBM24 was able to rescue binding between ε and mutant Pol lacking ε-binding activity, further showing that RBM24 mediates the interaction between Pol and ε by forming a Pol-RBM24-ε complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol-ε interaction and formation of a Pol-RBM24-ε complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/virions for reverse transcription and DNA synthesis.IMPORTANCE Hepatitis B virus (HBV) is a ubiquitous human pathogen, and HBV infection is a major global health burden. Chronic HBV infection is associated with the development of liver diseases, including fulminant hepatitis, hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. A currently approved vaccine can prevent HBV infection, and medications are able to reduce viral loads and prevent liver disease progression. However, current treatments rarely achieve a cure for chronic infection. Thus, it is important to gain insight into the mechanisms of HBV replication. In this study, we found that the host factor RBM24 is involved in pregenomic RNA (pgRNA) packaging and regulates HBV replication. These findings highlight a potential target for antiviral therapeutics of HBV infection.

Platinum nanoflowers with peroxidase-like property in a dual immunoassay for dehydroepiandrosterone.

Platinum nanoflowers (PtNFs) were utilized in a competitive enzyme-linked immunosorbent assay (ELISA) and in a lateral flow immunoassay (LFIA) for superior peroxidase-like activity and intense brown color, respectively. PtNFs were linked to the polyclonal antibody (pAb) to form the pAb-PtNFs probes for the dual immunoassay. Based on optimized pAb-PtNF probes, both enzyme-linked immunosorbent assay (PtNFs-ELISA) and lateral flow immunoassay (PtNFs-LFIA) perform very well. The absorbance at 450 nm decreases linearly in the DHEA concentration range 2.1 to 118.1 ng mL-1, and the limit of detection is 1.3 ng mL-1 and the IC50 value is 15.7 ng mL-1 of PtNFs-ELISA. The visual cut-off value of PtNFs-LFIA is 10.0 ng mL-1. The average recoveries from spiked samples range from 95.0 to 108.9% with a coefficient of variation below 12.2%. Excellent recoveries and correlation between the two methods were observed. Furthermore, the designed immunosensors exhibited good selectivity, confirming a broad development prospect in DHEA monitoring. Graphical Abstract.

Ultrasensitive detection of H. pylori in human feces based on immunomagnetic bead capture and fluorescent quantum dots.

Given that Helicobacter pylori (H. pylori) generally infects people in early childhood and that such persons when not treated with antibiotics remain infected for the rest of their lives, it is quite important to detect H. pylori in children, and convenient to do so using non-invasive methods. Stool antigen tests constitute such an effective non-invasive method. In the current work, a novel fecal test was developed to detect H. pylori based on immunomagnetic beads (IMBs) with monoclonal antibodies sensitively recognizing and capturing the H. pylori, coupled with a polyclonal antibody-conjugating quantum dot probe, and ultrasensitive detection was achieved by using a fluorescence spectrometer. The detection method took 120 min to perform, and showed a limit of detection of 102 CFU mL-1 and a linear range of 10 to 106 CFU mL-1 (R2 = 0.9962). Most importantly, this method can be effectively applied to real samples. This study provided a novel method for the non-invasive detection of the fecal antigen H. pylori.

Hyaluronic Acid Coated Albumin Nanoparticles for Targeted Peptide Delivery to the Retina.

Recent studies have shown that Connexin43 mimetic peptide (Cx43 MP) can prevent secondary damage following several retinal ischemic and inflammatory disorders by blocking the pathological opening of gap junction hemichannels. However, the poor stability of peptides and the presence of various intraocular barriers limit efficient retinal delivery in the clinical setting. The present study aimed to prolong the bioactivity of Cx43 MP and achieve targeted delivery to the retina by loading the peptide into hyaluronic acid (HA) coated human serum albumin nanoparticles (HSA NPs). Two different loading methods, adsorption and incorporation, were used with the peptide released slowly over a period of up to four months. Compared to uncoated particles, HA coated HSA NPs exhibited enhanced in vitro cellular uptake and ex vivo retinal penetration via HA-CD44 receptor mediated interactions. Furthermore, cell viability and Cx43 MP functionality assays showed that NPs protected Cx43 MP from degradation, sustained its release, and thus prolonged its bioactivity without reducing cell viability at concentrations used for Cx43 hemichannel blocking. Therefore, HA coated HSA NPs could have great potential for sustained and targeted delivery of Cx43 MP to treat various retinal inflammatory conditions.

Recycling of straw-biochar-biogas-electricity for sustainable food production pathways: Toward an integrated modeling approach.

As global greenhouse gas emissions increase and fossil energy sources decline dramatically, the energy transition is at the heart of many countries' development initiatives. As a biomass resource, straw plays a positive role in energy transformation and environmental improvement. However, there is still a challenge to explore the best options and models for straw production and utilization of green and efficient biomass energy in agricultural systems. This study establishes an economic-environmental-resource synergistic Straw Green recycling optimization model based on straw-electricity-biochar-biogas core (Straw Green recycling optimization model, SGROM). Firstly, we explore the effects of biochar return to the field on crop yield and greenhouse gas emission by Meta-analysis method, and on this basis, we construct SGROM to weigh the three objectives of economic-greenhouse gas emission-resource utilization, and explore the best allocation ratio between four utilization methods of straw: power generation, biochar preparation, biogas and derivatives preparation and sale, so as to obtain a straw recycling and efficient low-carbon utilization model. Exploring the response of straw green utilization patterns to crop market prices with the help of deep learning methods, SGROM has been applied to the main grain producing areas in the Sanjiang Plain of China, and the results of comparison with the traditional straw utilization (TSU) model show that the greenhouse gas emissions per unit of production value of SGROM are 19.66 % lower than that of TSU model, the electricity consumption is saved by 2.00 %, and the optimal ratios of straw for power generation, biogas and biochar production, and sale are 1.00 %, 10.75 %, 62.11 % and 26.14 %. The economic benefits and total greenhouse gas emissions of the integrated straw utilization mode are better than those of the single straw utilization mode, proving the superiority of SGROM in optimizing the straw utilization mode.

Weather-driven synergistic water-economy-environment regulation of farmland ecosystems.

Farmland ecosystems (FEs) constitute the most important source of food production, and water is one of the most important factors influencing FEs. The amount of water can affect the yield and thus the economic efficiency. Water migration can generate environmental effects through the migration of fertilizers. Interlinkages and constraints exist between the water, economy and environment, which require synergistic regulation. Meteorological elements influence the reference crop uptake amount and thus the water cycle processes and are key drivers of regulation at the water-economy-environment nexus. However, the weather-driven, synergistic water-economy-environment regulation of FEs has not been sufficiently researched. As such, this paper employed a dynamic Bayesian prediction of the reference evapotranspiration (ETo) and a quantitative characterization of the total nitrogen (TN) and total phosphorus (TP) contents in agricultural crops and soils via field monitoring and indoor experimental analysis. Consequently, multiobjective optimization modeling was conducted to weigh the mutual trade-offs and constraints between water, the economy and the environment. The proposed method was verified via an example involving the modern agricultural high-tech demonstration park in Harbin, Heilongjiang Province, China. The results indicated that (1) the effect of meteorological factors gradually decreased over time, but the prediction results were very accurate, and the higher the delay order of the dynamic Bayesian network (DBN) was, the more accurate the predictions; (2) ETo was significantly driven by meteorological elements, and the most important meteorological factor influencing ETo throughout the year was average temperature. When the average temperature was reduced by 10.0 %, ETo was reduced by 1.4 %, the required amount of irrigation water was reduced by 4.9 %, and the economic benefits of a single cube of water increased by 6.3 %; (3) resource-economy-environment multidimensional synergy enabled a 12.8 % reduction in agricultural ecosystem pollutant emissions, while the economic benefits per unit of water increased by 8.2 % and the system synergy increased by 23.2 %.

RBM24 inhibits the translation of SARS-CoV-2 polyproteins by targeting the 5'-untranslated region.

SARS-CoV-2 is a betacoronavirus with single-stranded positive-sense RNA, which is a serious global threat to human health. Understanding the molecular mechanism of viral replication is crucial for the development of antiviral drugs. The synthesis of viral polyproteins is a crucial step in viral progression. The synthesis of viral polyproteins in coronaviruses is regulated by the 5'-untranslated region (UTR); however, the detailed regulatory mechanism needs further investigation. The present study demonstrated that the RNA binding protein, RBM24, interacts with the RNA genome of SARS-CoV-2 via its RNA recognition submotifs (RNPs). The findings revealed that RBM24 recognizes and binds to the GUGUG element at stem-loop 4 (SL4) in the 5'-UTR of SARS-CoV-2. The interaction between RBM24 and 5'-UTR prevents 80S ribosome assembly, which in turn inhibits polyproteins translation and the replication of SARS-CoV-2. Notably, other RNA viruses, including SARS-CoV, MERS-CoV, Ebolavirus, rhinovirus, West Nile virus, Zika virus, Japanese encephalitis virus, yellow fever virus, hepatitis C virus, and human immunodeficiency virus-1 also contain one or several G(U/C/A)GUG sequences in the 5'-UTR, which is also targeted by RBM24. In conclusion, the present study demonstrated that RBM24 functions by interacting with the 5'-UTR of SARS-CoV-2 RNA, and elucidated that RBM24 could be a host restriction factor for SARS-CoV-2 and other RNA viruses.

Crop pattern optimization for the coordination between economy and environment considering hydrological uncertainty.

With the rapid growth of population and economy, shortage and mismatch of land and water resources have deepened the need for cropping pattern optimization. In the context of the sustainable development of agriculture, cropping pattern optimization should not only pursue economic benefits, but the consequent environmental effects also deserve equal attention. Meanwhile, climate change increases the complexity of balancing conflicts of economic-environmental system by cropping pattern optimization. Therefore, this paper builds a multi-objective programming model for Economic-Environmental Synergistic Optimization for Cropping Pattern under Climate Change (EESO-CP-CC) model, with the goals of economic benefit increment and environmental pollutants emission reduction. The EESO-CP-CC model couples a non-point source pollution input-output model, a one-dimensional water quality model and an economic benefit function into an integrated framework. Fuzzy method was used to solve the optimization model, and the stochastic uncertainty of water supply under climate change was quantified by the integration of Bayesian approach and interval linear regression. The model was applied to Jinxi Irrigation District (JXID) in Heilongjiang Province, northeast of China. Results show that by adjusting the acreage of rice, corn and soybean, the harmony degree of economy-society-environment system increased by 10.7% compared to the current situation, indicating that the model tends to achieve the best possible economic benefits while ensuring the environmental effects. Compared with actual cropping pattern, the pollutants emissions reduced by 24.7% and 3% from corn and soybean, respectively. However, this led to a decrease of economic benefit by 8% in exchange, showing the trade-off between environmental pollution reduction and economic benefits improvement. The output coefficients of nitrogen and phosphorus pollutants were optimized, with the optimal output reducing by 20% compared to the standard. Cropping pattern and water resources allocation vary with different climate change conditions, however, the amplitude of variation is modest, indicating that the model can cope well with the changing environment. The developed model can help achieve synergistic development of economic benefits and environmental effects, and thus promote sustainable development of irrigation areas, and improve the coping capacity of agricultural water and land under climate change, by cropping pattern optimization and planning.

Comparison of viral propagation and drug response among SARS-CoV-2 VOCs using replicons capable of recapitulating virion assembly and release.

Several variants of concern (VOCs) have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first isolated in January 2020. Due to mutations in the spike (S) protein, these VOCs have evolved to enhance viral infectivity and immune evasion. However, whether mutations of the other viral proteins lead to altered viral propagation and drug resistance remains obscure. The replicon is a noninfectious viral surrogate capable of recapitulating certain steps of the viral life cycle. Although several SARS-CoV-2 replicons have been developed, none of them were derived from emerging VOCs and could only recapitulate viral genome replication and subgenomic RNA (sgRNA) transcription. In this study, SARS-CoV-2 replicons derived from the WIV04 strain and two VOCs (the Beta and Delta variants) were prepared by removing the S gene from their genomes, while other structural genes remained untouched. These replicons not only recapitulate viral genome replication and sgRNA transcription but also support the assembly and release of viral-like particles, as manifested by electron microscopic assays. Thus, the S-deletion replicon could recapitulate virtually all the post-entry steps of the viral life cycle and provides a versatile tool for measuring viral intracellular propagation and screening novel antiviral drugs, including inhibitors of virion assembly and release. Through the quantification of replicon RNA released into the supernatant, we demonstrate that viral intracellular propagation and drug response to remdesivir have not yet substantially changed during the evolution of SARS-CoV-2 from the WIV04 strain to the Beta and Delta VOCs.

RNA-Binding motif protein 38 (RBM38) mediates HBV pgRNA packaging into the nucleocapsid.

The binding of HBV polymerase (Pol) and the epsilon stem loop (ε) on the 5' terminal region of pgRNA is required for pgRNA packaging and HBV replication. Previous research has demonstrated that RNA binding motif protein 24 (RBM24) is involved in pgRNA packaging by mediating the interaction between HBV polymerase (Pol) and the ε element. Here, we demonstrate that RBM38 interacts with ε, pol, RBM24 and HBV core which mediate pgRNA packaging. RBM38 directly binds to the lower bulge of ε via RNA recognition submotifs (RNPs) and interacts with HBV Pol in an RNA-independent manner. RBM38 interacts with RBM24 and forms heterogeneous oligomers, which mediate Pol-ε binding and the formation of the Pol-RBM38/RBM24-ε complex. More important, RBM38 also binds to the HBV core via the C-terminal region (ARD domain), which facilitates the combination of Pol-ε with the HBV core protein. In conclusion, RBM38 facilitates the Pol-ε interaction and mediates Pol-ε in combining with the HBV core, triggering pgRNA packaging for reverse transcription and DNA synthesis. This study provides new insights into pgRNA encapsidation.

Intravitreal homocysteine-thiolactone injection leads to the degeneration of multiple retinal cells, including photoreceptors.

PURPOSE: Hyperhomocysteinemia is known to cause degeneration of retinal ganglion cells, but its influence on photoreceptors remains largely unknown. In particular, the role of homocysteine-thiolactone (Hcy-T)--the physiologic metabolite of homocysteine that has been proven to be more cytotoxic than homocysteine itself--as a factor that causes retinopathy, has not been defined. This study aimed to investigate the toxic effects of excessive Hcy-T in a mouse model. METHODS: A total of 60 six-week-old female ICR mice were used in this study. The mice were divided into 3 experimental groups and 2 control groups. The mice in the experimental groups were subjected to intravitreal injections of Hcy-T to reach final estimated intravitreal concentrations at 5, 25, and 200 µM, respectively. Mice without injection (blank) and with 0.9 NaCl injections (sham injection) were used as controls. The mice with 200 µM Hcy-T were sacrificed at days 7, 15, 45, and 90 after injection and the mice with 5 or 25 µM Hcy-T were sacrificed at day 90, with the controls sacrificed at day 15 or 90 for comparison. Semi-quantitative dot-blot analysis was performed for confirmation of retinal homocysteinylation. The mouse retinas were evaluated microscopically, with the thickness of total and specific retinal layers determined. Immunohistochemical analysis was performed and the labeled cells were quantified to determine the effects of excessive Hcy-T on specific retinal cells. RESULTS: Dose-dependent retinal homocysteinylation after Hcy-T injection was confirmed. The homocysteinylation was localized in the outer and inner segments of photoreceptors and the ganglion cell layer (GCL). Retinal cell degenerations were found in the GCL, inner nuclear layer, and outer nuclear layer at day 90 after 200 µM Hcy-T injection. Significant thickness reduction was found in the total retina, outer nuclear layer, and the outer and inner segment layers. A trend of thickness reduction was also found in the GCL and inner nuclear layer, although this was not statistically significant. The rhodopsin⁺ photoreceptors and the calbindin⁺ horizontal cells were significantly reduced at day 15, and were nearly ablated at day 90 after 200 µM Hcy-T injection (p<0.001 for both day 15 and day 90), which was not seen in the sham injection controls. The Chx-10⁺ or the Islet-1⁺ bipolar cells and the Pax-6⁺ amacrine cells were severely misarranged at day 90, but no significant reduction was found for both cell types. The GFAP⁺ Müller cells were activated at day 15, but were not significantly increased at day 90 after the injection. CONCLUSIONS: Excessive retinal homocysteinylation by Hcy-T, a condition of hyperhomocysteinemia, could lead to degeneration of photoreceptors, which might lead to retinopathies associated with severe hyperhomocysteinemia or diabetes mellitus.

Enhanced performance of a surface plasmon resonance-based immunosensor for the detection of glycocholic acid.

The concentration of glycocholic acid (GCA) in urine and blood is an important biomarker for liver cancer. Monitoring of GCA depends to a large extent on the availability of appropriate analytical techniques. In this work, based on the immobilization of GCA-OVA onto the sensor chip surface, a label-free competitive inhibition immunoassay for the determination of GCA with the surface plasmon resonance (SPR) technique was developed. The proposed SPR immunosensor is simple to prepare, recyclable and exhibits excellent sensitivity to GCA (a linear range of 13.3-119.4 ng mL-1 and a limit of detection (LOD) of 2.5 ng mL-1), which was 14 times lower than that of the traditional immunoassay. Excellent recoveries and correlation between these two methods were observed (R2 = 0.995). Hence, it can be proved that the SPR immunosensor could be used to achieve rapid and sensitive quantitative detection of GCA in real urine samples and meet clinical needs.