A novel BSL-2 Lassa virus reverse genetics system modelling the complete viral life cycle.

Lassa virus (LASV), a risk-group 4 pathogen, must be handled in biosafety level-4 (BSL-4) conditions, thereby limiting its research and antiviral development. Here, we developed a novel LASV reverse genetics system which, to our knowledge, is the first to study the complete LASV life cycle under BSL-2 conditions. Viral particles can be produced efficiently when LASV minigenomic RNA harbouring minimal viral cis-elements and reporter genes is transfected into a helper cell line stably expressing viral NP, GP, Z and L proteins. The resulting defective virions, named LASVmg, can propagate only in the helper cell line, providing a BSL-2 model to study the complete LASV life cycle. Using this model, we found that a previously reported cellular receptor α-dystroglycan is dispensable for LASVmg infection. Furthermore, we showed that ribavirin can inhibit LASVmg infection by inducing viral mutations. This new BSL-2 system should facilitate studying the LASV life cycle and screening antivirals.

Coronavirus and the Cytoskeleton of Virus-Infected Cells.

The cytoskeleton, which includes actin filaments, microtubules, and intermediate filaments, is one of the most important networks in the cell and undertakes many fundamental life activities. Among them, actin filaments are mainly responsible for maintaining cell shape and mediating cell movement, microtubules are in charge of coordinating all cargo transport within the cell, and intermediate filaments are mainly thought to guard against external mechanical pressure. In addition to this, cytoskeleton networks are also found to play an essential role in multiple viral infections. Due to the COVID-19 epidemic, including SARS-CoV-2, SARS-CoV and MERS-CoV, so many variants have caused wide public concern, that any virus infection can potentially bring great harm to human beings and society. Therefore, it is of great importance to study coronavirus infection and develop antiviral drugs and vaccines. In this chapter, we summarize in detail how the cytoskeleton responds and participates in coronavirus infection by analyzing the possibility of the cytoskeleton and its related proteins as antiviral targets, thereby providing ideas for finding more effective treatments.

Multi-view stitching phase measuring deflectometry for freeform specular surface metrology.

Phase measuring deflectometry (PMD) offers notable advantages for precision inspection of specular elements. Nevertheless, if confronts challenges when measuring freeform specular surfaces due to the dispersion of reflection rays from surfaces with high local slopes. Here, we propose a multi-view stitching PMD. It utilizes distinct sensors combining with a screen to capture the appearance of each region. After precisely calibrating the entire system to correct the absolute depth of each region, the appearances of all regions are precisely stitched together, reconstructing the comprehensive appearance of the surface. Through experimental setup, we measured the 3D morphology of a spherical lens with a curvature radius of 155.04 mm and a peak-to-valley (PV) value of 2.9 mm, which yielded a measurement accuracy of 5.3 µm (relative error: 0.18 %). Furthermore, we successfully measured the appearance of a curved mobile phone screen with local slopes ranging from -46.1° to 51.3°, and freeform acrylic sheet with local slopes ranging from -6.7° to 7.7° and a PV value of 5.3 mm.

Actomyosin-dependent cell contractility orchestrates Zika virus infection.

Emerging pathogen infections, such as Zika virus (ZIKV), pose an increasing threat to human health, but the role of mechanobiological attributes of host cells during ZIKV infection is largely unknown. Here, we reveal that ZIKV infection leads to increased contractility of host cells. Importantly, we investigated whether host cell contractility contributes to ZIKV infection efficacy, from both the intracellular and extracellular perspective. By performing drug perturbation and gene editing experiments, we confirmed that disruption of contractile actomyosin compromises ZIKV infection efficiency, viral genome replication and viral particle production. By culturing on compliant matrix, we further demonstrate that a softer substrate, leading to less contractility of host cells, compromises ZIKV infection, which resembles the effects of disrupting intracellular actomyosin organization. Together, our work provides evidence to support a positive correlation between host cell contractility and ZIKV infection efficacy, thus unveiling an unprecedented layer of interplay between ZIKV and the host cell.

Discovery of a fused bicyclic derivative of 4-hydroxypyrrolidine and imidazolidinone as a new anti-HCV agent.

Hepatitis C virus (HCV) infection causes severe liver diseases and remains a major global public health concern. Current direct-acting antiviral (DAA)-based therapies that target viral proteins involving HCV genome replication are effective, however a minority of patients still fail to cure HCV, rendering a window to develop additional antivirals particularly targeting host functions involving in HCV infection. Here, we utilized the HCV infection cell culture system (HCVcc) to screen in-house compounds bearing host-interacting preferred scaffold for the antiviral activity. Compound HXL-10, a novel fused bicyclic derivative of pyrrolidine and imidazolidinone, was identified as a potent anti-HCV agent with a low cytotoxicity and high specificity. Mechanistic studies showed that HXL-10 neither displayed a virucidal effect nor inhibited HCV genomic RNA replication. Instead, HXL-10 might inhibit HCV assembly by targeting host functions. In summary, we developed a novel anti-HCV agent that may potentially offer additive benefits to the current anti-HCV DDA.

Panax quinquefolius saponin inhibits vascular smooth muscle cell calcification via activation of nuclear factor-erythroid 2-related factor 2.

BACKGROUND: Panax quinquefolius saponin (PQS) is the main active component of Panax quinquefolius. Emerging evidence suggests that PQS exerts beneficial effects against cardiovascular diseases. However, the role and mechanism of PQS in vascular calcification are not unclear. The present study investigated the effects of PQS on the calcification of vascular smooth muscle cell (VSMCs). METHODS: The present study used calcification medium containing 3 mM inorganic phosphate (Pi) to induce rat VSMCs calcification. We investigated the effects of PQS on VSMCs calcification using alizarin red staining and alkaline phosphatase (ALP) activity assays. The intracellular reactive oxygen species (ROS) levels and the transcriptional activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined. The mRNA and protein expression levels of Nrf2, the antioxidant gene heme oxygenase-1 (HO-1), osteogenic markers, including runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and Kelch-like ECH-associated protein 1 (Keap1) were also measured. RESULTS: Treatment with Pi significantly increased intracellular calcium deposition and ALP activity, which were suppressed by PQS in a concentration-dependent manner. During VSMCs calcification, PQS inhibited the mRNA and protein expression of Runx2 and BMP2. PQS treatment reduced intracellular ROS production and significantly upregulated Nrf2 transcriptional activity and the expression of Nrf2 and its target antioxidant gene HO-1. PQS suppressed the Pi-induced protein expression of Keap1, which is an endogenous inhibitor of Nrf2. Keap1 siRNA treatment induced Nrf2 expression and downregulated Runx2 expression in the presence of Pi and PQS. CONCLUSION: Taken together, these findings suggest that PQS could effectively inhibit VSMCs calcification by ameliorating oxidative stress and regulating osteogenic genes via the promotion of Nrf2 expression.

MPK6-mediated HY5 phosphorylation regulates light-induced anthocyanin accumulation in apple fruit.

Light is known to regulate anthocyanin pigment biosynthesis in plants on several levels, but the significance of protein phosphorylation in light-induced anthocyanin accumulation needs further investigation. In this study, we investigated the dynamics of the apple fruit phosphoproteome in response to light, using high-performance liquid chromatography-tandem mass spectrometry analysis. Among the differentially phosphorylated proteins, the bZIP (basic leucine zipper) transcription factor, HY5, which has been identified as an anthocyanin regulator, was rapidly activated by light treatment of the fruit. We hypothesized that phosphorylated MdHY5 may play a role in light-induced anthocyanin accumulation of apple fruit. Protein interaction and phosphorylation assays showed that mitogen-activated protein kinase MdMPK6 directly interacted with, and activated, MdHY5 via phosphorylation under light conditions, thereby increasing its stability. Consistent with this finding, the suppression of the mitogen-activated protein kinase genes MdMPK6 or MdHY5 resulted in an inhibition of anthocyanin accumulation, and further showed that light-induced anthocyanin accumulation is dependent on MdMPK6 kinase activity, and is required for maximum MdHY5 activity. Under light conditions, active MdMPK6 phosphorylated MdHY5 leading to accumulation of phospho-MdHY5, which enhanced the binding of MdHY5 to its target anthocyanin related genes in fruit. Our findings reveal an MdMPK6-MdHY5 phosphorylation pathway in light-induced anthocyanin accumulation, providing new insights into the regulation of light-induced anthocyanin biosynthesis in apple fruit at both the transcriptional and post-translational levels.

Spectroscopic depth profilometry of organic thin films upon inductively coupled plasma etching.

During the deposition and post-treatments of organic films, phase separation along the film-depth direction is a commonly observed phenomenon. Thus, film-depth profilometry of organic thin films and the corresponding scientific instruments are attracting extensive interest. Here, we propose spectroscopic film-depth profilometry of organic thin films upon inductively coupled plasma etching. Compared with capacitively coupled plasma, which usually generates inhomogeneous filamentous discharge, damaging films underneath the etched surface, inductively coupled plasma studied in this work refers to a so-called soft plasma source generated by a well-defined homogenous glow discharge. The absorption spectra of the etched films are monitored by using a spectrometer, from which the film-depth-dependent light absorption spectra are, thus, numerically obtained with a film-depth resolution better than 1 nm. This methodology is available not only for non-conjugated molecules but also for conjugated organic semiconductors, which are usually known as unstable materials for many ionic plasma sources. Organic films for solar cells and field-effect transistors are investigated as model materials to demonstrate the applications of this depth profilometry.

Roles of illumination on distribution of phosphorus in Chlorella vulgaris under mixotrophic cultivation.

Phosphorus (P) is a non-substitutable resource and global reserves of phosphate rock are limited. In this study, phosphorus recovery by Chlorella vulgaris, and the effects of different light intensities (2000 Lux, 5000 Lux, 8000 Lux, 12,000 Lux) on the phosphorus distribution in the soluble microbial product (SMP), extracellular polymeric substance (EPS) and intracellular polymeric substance (IPS) were analyzed. The results showed that the 5000 Lux was the optimum light intensity for P uptake and transformation by Chlorella vulgaris under mixotrophic cultivation. At the light intensity of 5000 Lux, the P uptake rate was 100% after 32 days of cultivation, and the concentration of intracellular organic phosphorus (OP) was 5.77 mg P/L. Moreover, EPS was the main P pool when inorganic phosphorus (IP) was depleted in bulk solution. Phosphorus recovery by microalgae is an important solution to treat P-containing wastewater.

ROS1 promotes low temperature-induced anthocyanin accumulation in apple by demethylating the promoter of anthocyanin-associated genes.

Low temperature can affect the growth and development of plants through changes in DNA demethylation patterns. Another known effect of low temperature is the accumulation of anthocyanin pigments. However, it is not known whether the two phenomena are linked, specifically, whether DNA demethylation participates in anthocyanin accumulation in response to low-temperature stress. The ROS1 gene is involved in plant DNA demethylation and influences methylation levels in response to low temperature stress. In this study, using RNA sequencing, we detected that the transcription levels of MdROS1 correlate with the anthocyanin content, as well as with those of anthocyanin biosynthesis-related genes in apple (Malus domestica), at low temperatures. Genomic bisulfite sequencing showed that the methylation levels of the promoters of the anthocyanin related genes MdCHS, MdCHI, MdF3'H, MdANS, MdUFGT, and MdMYB10 decreased in apple leaves after low-temperature treatment. Similar expression and methylation results were also found in apple fruit. Transiently silencing MdROS1 in the leaves and fruit of apple cultivars inhibited the accumulation of anthocyanins and led to decreased expression of anthocyanin biosynthetic genes, and the opposite results were detected in MdROS1-overexpressing leaves and fruit. A promoter binding assay showed that the conserved RRD-DME domains of MdROS1 directly bind to the promoters of MdF3'H and MdUFGT. Taken together, these results suggest that ROS1 affects the anthocyanin biosynthetic pathway by decreasing the methylation level of anthocyanin-related gene promoters, thereby increasing their expression and increasing anthocyanin accumulation.

Actin nucleator formins regulate the tension-buffering function of caveolin-1.

Both the mechanosensitive actin cytoskeleton and caveolae contribute to active processes such as cell migration, morphogenesis, and vesicular trafficking. Although distinct actin components are well studied, how they contribute to cytoplasmic caveolae, especially in the context of mechano-stress, has remained elusive. Here, we identify two actin-associated mobility stereotypes of caveolin-1 (CAV-1)-marked intracellular vesicles, which are characterized as 'dwelling' and 'go and dwelling'. In order to exploit the reason for their distinct dynamics, elongated actin-associated formin functions are perturbed. We find drastically decreased density, increased clustering, and compromised motility of cytoplasmic CAV-1 vesicles resulting from lacking actin nucleator formins by both chemical treatment and RNA silencing of formin genes. Furthermore, hypo-osmosis-stimulated diminishing of CAV-1 is dramatically intensified upon blocking formins. The clustering of CAV-1 vesicles when cells are cultured on soft substrate is also aggravated under formin inhibition condition. Together, we reveal that actin-associated formins are essential for maintaining the dynamic organization of cytoplasmic CAV-1 and importantly its sensitivity upon mechanical challenge. We conclude that tension-controlled actin formins act as a safety valve dampening excessive tension on CAV-1 and safeguarding CAV-1 against mechanical damage.

Lifting Triplet Energy and Bipolar Characteristics by Limiting the Rotation of the Peripheral Groups in Host Materials to Achieve High-Efficiency Blue OLED.

Bipolar host materials with high triplet energy are of great significance for highly efficient blue organic light-emitting diodes (OLEDs). In this work, three donor-acceptor-donor (D-A-D) type host materials with identical non-rigid diphenylsulfone center but differing in rotation degree of peripheral amino substituted derivatives from rotating freely diphenylamine (SODP) to rotating partially iminodibenzyl (SOId) and rotating restricted carbazole (SOCz) were designed and synthesized. It was demonstrated that the triplet energy (ET ) level of the materials promoted by limiting the rotation degree of the peripheral groups, which was 2.72 eV for SODP, 2.73 eV for SOId and 2.78 eV for SOCz, respectively. Besides, the results of the single-carrier devices indicate SOCz possess better bipolar characteristic. Using FIrpic as guest emitter, the blue OLED with SOCz as host material exhibited superior device performance with a low turn-on voltage of 3.3 V, a maximum current efficiency (CE) of 30.1 cd A-1 , a maximum power efficiency (PE) of 32.2 lm W-1 , and a maximum external quantum efficiency (EQE) of 14.0%. This work provides a beneficial guideline for realizing promising host materials in efficient blue OLEDs.

The RNA Directed DNA Methylation (RdDM) Pathway Regulates Anthocyanin Biosynthesis in Crabapple (Malus cv. spp.) Leaves by Methylating the McCOP1 Promoter.

The synthesis of anthocyanin pigments in plants is known to be regulated by multiple mechanisms, including epigenetic regulation; however, the contribution of the RNA-directed DNA methylation (RdDM) pathway is not well understood. Here, we used bisulfite sequencing and Real Time (RT)-quantitative (q) PCR to analyze the methylation level of the promoter of constitutively photomorphogenic 1 (McCOP1) from Malus cv. spp, a gene involved in regulating anthocyanin biosynthesis. The CHH methylation level of the McCOP1 promoter was negatively correlated with McCOP1 RNA expression, and inhibiting DNA methylation caused decreased methylation of the McCOP1 promoter and asymmetric cytosine CHH methylation. We observed that the McCOP1 promoter was a direct target of the RdDM pathway argonaute RISC component 4 (McAGO4) protein, which bound to a McCOP1 promoter GGTTCGG site. Bimolecular fluorescence complementation (BIFC) analysis showed that RNA-directed DNA methylation (McRDM1) interacted with McAGO4 and another RdDM protein, domains rearranged methyltransferase 2 (McDRM2), to regulate the CHH methylation of the McCOP1 promoter. Detection of CHH methylation and COP1 gene expression in the Arabidopsis thalianaatago4, atdrm2 and atrdm1 mutants showed that RDM1 is the effector of the RdDM pathway. This was confirmed by silencing McRDM1 in crabapple leaves or apple fruit, which resulted in a decrease in McCOP1 CHH methylation and an increase in McCOP1 transcript levels, as well as in anthocyanin accumulation. In conclusion, these results show that the RdDM pathway is involved in regulating anthocyanin accumulation through CHH methylation of the McCOP1 promoter.

Infrared spectroscopy depth profiling of organic thin films.

Organic thin films are widely used in organic electronics and coatings. Such films often feature film-depth dependent variations of composition and optoelectronic properties. State-of-the-art depth profiling methods such as mass spectroscopy and photoelectron spectroscopy rely on non-intrinsic species (vaporized ions, etching-induced surface defects), which are chemically and functionally different from the original materials. Here we introduce an easily-accessible and generally applicable depth profiling method: film-depth-dependent infrared (FDD-IR) spectroscopy profilometry based on directly measuring the intrinsic material after incremental surface-selective etching by a soft plasma, to study the material variations along the surface-normal direction. This depth profiling uses characteristic vibrational signatures of the involved compounds, and can be used for both conjugated and non-conjugated, neutral and ionic materials. A film-depth resolution of one nanometer is achieved. We demonstrate the application of this method for investigation of device-relevant thin films, including organic field-effect transistors and organic photovoltaic cells, as well as ionized dopant distributions in doped semiconductors.

Almost periodic solutions for a SVIR epidemic model with relapse.

This paper is devoted to a nonautonomous SVIR epidemic model with relapse, that is, the recurrence rate is considered in the model. The permanent of the system is proved, and the result on the existence and uniqueness of globally attractive almost periodic solution of this system is obtained by constructing a suitable Lyapunov function. Some analysis for the necessity of considering the recurrence rate in the model is also presented. Moreover, some examples and numerical simulations are given to show the feasibility of our main results. Through numerical simulation, we have obtained the influence of vaccination rate and recurrence rate on the spread of the disease. The conclusion is that in order to control the epidemic of infectious diseases, we should increase the vaccination rate while reducing the recurrence rate of the disease.

Glycometabolism regulates hepatitis C virus release.

HCV cell-culture system uses hepatoma-derived cell lines for efficient virus propagation. Tumor cells cultured in glucose undergo active aerobic glycolysis, but switch to oxidative phosphorylation for energy production when cultured in galactose. Here, we investigated whether modulation of glycolysis in hepatocytes affects HCV infection. We showed HCV release, but not entry, genome replication or virion assembly, is significantly blocked when cells are cultured in galactose, leading to accumulation of intracellular infectious virions within multivesicular body (MVB). Blockade of the MVB-lysosome fusion or treatment with pro-inflammatory cytokines promotes HCV release in galactose. Furthermore, we found this glycometabolic regulation of HCV release is mediated by MAPK-p38 phosphorylation. Finally, we showed HCV cell-to-cell transmission is not affected by glycometabolism, suggesting that HCV cell-to-supernatant release and cell-to-cell transmission are two mechanistically distinct pathways. In summary, we demonstrated glycometabolism regulates the efficiency and route of HCV release. We proposed HCV may exploit the metabolic state in hepatocytes to favor its spread through the cell-to-cell transmission in vivo to evade immune response.

High-fidelity structured illumination microscopy by point-spread-function engineering.

Structured illumination microscopy (SIM) has become a widely used tool for insight into biomedical challenges due to its rapid, long-term, and super-resolution (SR) imaging. However, artifacts that often appear in SIM images have long brought into question its fidelity, and might cause misinterpretation of biological structures. We present HiFi-SIM, a high-fidelity SIM reconstruction algorithm, by engineering the effective point spread function (PSF) into an ideal form. HiFi-SIM can effectively reduce commonly seen artifacts without loss of fine structures and improve the axial sectioning for samples with strong background. In particular, HiFi-SIM is not sensitive to the commonly used PSF and reconstruction parameters; hence, it lowers the requirements for dedicated PSF calibration and complicated parameter adjustment, thus promoting SIM as a daily imaging tool.

UNC93B1 curbs cytosolic DNA signaling by promoting STING degradation.

UNC93B1 is a trafficking chaperone of endosomal Toll-like receptors (TLRs) and plays an essential role in the TLR-mediated innate signaling. However, whether it is also involved in other innate immune sensing or cellular pathways remains largely unexplored. Here we investigated the role of UNC93B1 in cytosolic DNA-triggered cGAS-STING signaling in mouse and human cell lines. We showed that while UNC93B1 deficiency blunts the signal transduction by TLR3, it augments innate immune responses to cytosolic DNA stimulation and DNA virus infection. Mechanistic study reveals a distinct action of UNC93B1 upon STING, but not other parts along the cGAS-STING-TBK1 axis, through regulating the protein level of STING at both resting and cytosolic DNA-stimulated conditions. UNC93B1 can directly interact and traffic along with STING, and the disruption of this interaction causes accumulation of STING that subsequently leads to augmented signaling responses upon its activation. These findings reveal a new function of UNC93B1 in negatively regulating STING-mediated signaling responses.

Effects of bioactive components of Pu-erh tea on gut microbiomes and health: A review.

Pu-erh tea is a post-fermentation tea with unique flavor and multiple health benefits. Due to the various microorganisms involved in the post-fermentation process, Pu-erh tea contains highly complex components, which have rich interactions with the gut microbiomes (GMs). Because the structure and homeostasis of GMs are closely related to human wellness and the various diseases progress, the beneficial effects of Pu-erh tea on GMs have a great potential for application in health care. However, there is no systematic summary of the bioactive components of Pu-erh tea, and their effects on the GMs. Here, we review the current studies on the effects of Pu-erh tea and its bioactive components on the structure of GMs as well as on health improvement, and further discuss the relevant quality indicators. This "components - function - indicators" clue will hopefully stimulate the standardization of Pu-erh tea fermentation process and the development of its functional products.

The efficacy of different seed priming agents for promoting sorghum germination under salt stress.

Sorghum [Sorghum bicolor (L.) Moench] seed germination is sensitive to salinity, and seed priming is an effective method for alleviating the negative effects of salt stress on seed germination. However, few studies have compared the effects of different priming agents on sorghum germination under salt stress. In this study, we quantified the effects of priming with distilled water (HP), sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2), and polyethylene glycol (PEG) on sorghum seed germination under 150 mM NaCl stress. The germination potential, germination rate, germination index, vigor index, root length, shoot length, root fresh weight, shoot fresh weight, root dry weight, and shoot dry weight were significantly reduced by salt stress. Different priming treatments alleviated the germination inhibition caused by salt stress to varying degrees, and 50 mM CaCl2 was the most effective treatment. In addition, the mitigation effect of priming was stronger on root traits than on shoot traits. Mitigation efficacy was closely related to both the type of agent and the concentration of the solution. Principal component analysis showed that all concentrations of CaCl2 had higher scores and were clearly distinguished from other treatments based on their positive effects on all germination traits. The effects of the other agents varied with concentration. The priming treatments were divided into three categories based on their priming efficacy, and the 50, 100, and 150 mM CaCl2 treatments were placed in the first category. The 150 mM KCl, 10% PEG, HP, 150 mM NaCl, 30% PEG, and 50 mM KCl treatments were placed in the second category, and the 100 mM NaCl, 100 mM KCl, 20% PEG, and 50 mM NaCl treatments were least effective and were placed in the third category. Choosing appropriate priming agents and methods for future research and applications can ensure that crop seeds germinate healthily under saline conditions.