Amorphous calcium carbonate (ACC) plays an important role in microbially induced calcium carbonate precipitation (MICP), which has great potential in broad applications such as building restoration, ...CO2 sequestration, and bioremediation of heavy metals, etc. However, our understanding of ACC is still limited. By combining microscopy of cell-laden microdroplets with confocal Raman microspectroscopy, we investigated the ACC dynamics during MICP. The results show that MICP inside droplets can be divided into three stages: liquid, gel-like ACC, and precipitated CaCO3 stages. In the liquid stage, the droplets are transparent. As the MICP process continues into the gel-like stage, the ACC structure appears and the droplets become opaque. Subsequently, dissolution of the gel-like structure is accompanied by growth of precipitated CaCO3 crystals. The size, morphology, and lifetime of the gel-like structures depend on the Ca2+ concentration. Using polystyrene colloids as tracers, we find that the colloids exhibit diffusive behavior in both the liquid and precipitated CaCO3 stages, while their motion becomes arrested in the gel-like ACC stage. These results provide direct evidence for the formation-dissolution process of the ACC-formed structure and its gel-like mechanical properties. Our work provides a detailed view of the time evolution of ACC and its mechanical properties at the microscale level, which has been lacking in previous studies.
Mannose-sensitive hemagglutinin (MSHA) pili and flagellum are critical for the surface attachment of
Vibrio cholerae
, the first step of
V. cholerae
colonization on host surfaces. However, the cell ...landing mechanism remains largely unknown, particularly in viscoelastic environments such as the mucus layers of intestines. Here, combining the cysteine-substitution-based labeling method with single-cell tracking techniques, we quantitatively characterized the landing of
V. cholerae
by directly observing both pili and flagellum of cells in a viscoelastic non-Newtonian solution consisting of 2% Luria-Bertani and 1% methylcellulose (LB+MC). The results show that MSHA pili are evenly distributed along the cell length and can stick to surfaces at any point along the filament. With such properties, MSHA pili are observed to act as a brake and anchor during cell landing which includes three phases: running, lingering, and attaching. Importantly, loss of MSHA pili results in a more dramatic increase in mean path length in LB+MC than in 2% LB only or in 20% Ficoll solutions, indicating that the role of MSHA pili during cell landing is more apparent in viscoelastic non-Newtonian fluids than viscous Newtonian ones. Our work provides a detailed picture of the landing dynamics of
V. cholerae
under viscoelastic conditions, which can provide insights into ways to better control
V. cholerae
infections in a real mucus-like environment.
African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by the ASF virus (ASFV). ASFV has evolved multiple strategies to escape host antiviral immune responses. Here, ...we reported that ASFV pB318L, a trans-geranylgeranyl-diphosphate synthase, reduced the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs). Mechanically, pB318L not only interacted with STING to reduce the translocation of STING from the endoplasmic reticulum to the Golgi apparatus but also interacted with IFN receptors to reduce the interaction of IFNAR1/TYK2 and IFNAR2/JAK1. Of note, ASFV with interruption of B318L gene (ASFV-intB318L) infected PAMs produces more IFN-I and ISGs than that in PAMs infected with its parental ASFV HLJ/18 at the late stage of infection. Consistently, the pathogenicity of ASFV-intB318L is attenuated in piglets compared with its parental virus. Taken together, our data reveal that B318L gene may partially affect ASFV pathogenicity by reducing the production of IFN-I and ISGs. This study provides a clue to design antiviral agents or live attenuated vaccines to prevent and control ASF.
Double emulsion (DE) droplets with controlled size and internal structure are a promising platform for biological analysis, chemical synthesis, and drug delivery systems. However, to further ..."democratize" their application, new methods that enable simple and precise spatial patterning of the surface wettability of droplet-generating microfluidic devices are still needed. Here, by leveraging the increase in hydrophilicity of polydimethylsiloxane (PDMS) due to the plasma-treatment used to permanently bond to glass, we developed a one-step method to selectively pattern the wettability of PDMS microfluidic devices for DE generation. Our results show that both Aquapel-treated and 1H,1H,2H,2H-Perfluorodecyltriethoxysilan (PFDTES)-treated devices are functionally showing the generality of our method. With the resulting microfluidic devices, both water-in-oil-in-water (w/o/w) and oil-in-water-in-oil (o/w/o) DE droplets can be produced. Using a PDMS mixture containing cross-linking agents, we formed PDMS microcapsules by solidifying the shell layer of water-in-PDMS-in-water DE droplets. We also characterize the morphological properties of the generated droplets/microcapsules. We anticipate the method developed in this work could be used in a broad range of applications of DE droplets.
African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease in domestic pigs and wild boars. Domestic pigs infected with virulent African swine fever virus (ASFV) isolates ...have a high mortality, approaching 100%. Identification of ASFV genes related to virulence/pathogenicity and deletion of them are considered to be key steps in the development of live attenuated vaccines, because the ability of ASFV to escape host innate immune responses is related to viral pathogenicity. However, the relationship between the host antiviral innate immune responses and the pathogenic genes of ASFV has not been fully understood. In this study, the ASFV H240R protein (pH240R), a capsid protein of ASFV, was found to inhibit type I interferon (IFN) production. Mechanistically, pH240R interacted with the N-terminal transmembrane domain of stimulator of interferon genes (STING) and inhibited its oligomerization and translocation from the endoplasmic reticulum to the Golgi apparatus. Additionally, pH240R inhibited the phosphorylation of interferon regulatory factor 3 (IRF3) and TANK binding kinase 1 (TBK1), leading to reduced production of type I IFN. Consistent with these results, infection with
-deficient ASFV (ASFV-ΔH240R) induced more type I IFN than infection with its parental strain, ASFV HLJ/18. We also found that pH240R may enhance viral replication via inhibition of type I IFN production and the antiviral effect of interferon alpha (IFN-α). Taken together, our findings provide a new explanation for the reduction of ASFV's replication ability by knockout of the
gene and a clue for the development of live attenuated ASFV vaccines.
African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and acute hemorrhagic viral disease with a high mortality, approaching 100% in domestic pigs. However, the relationship between viral pathogenicity and immune evasion of ASFV is not fully understood, which limits the development of safe and effective ASF vaccines, specifically, live attenuated vaccines. In this study, we found that pH240R, as a potent antagonist, inhibited type I IFN production by targeting STING and inhibiting its oligomerization and translocation from the endoplasmic reticulum to the Golgi apparatus. Furthermore, we also found that deletion of the
gene reduced viral pathogenicity by enhancing type I IFN production, which decreases ASFV replication. Taken together, our findings provide a clue for the development of an ASFV live attenuated vaccine via deleting the
gene.
Tomato (Solanum lycopersicum) is one of the most widely cultivated vegetable crop species in the world. Tomato late blight caused by Phytophthora infestans is a severe disease, which can cause ...serious losses in tomato production. In this study, tomato SlbZIP68 was identified as a transcription factor that can be induced by P. infestans, salicylic acid (SA) and jasmonic acid (JA). Knockout of SlbZIP68 via clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology revealed a significant decrease in tomato resistance to P. infestans. Furthermore, knockout of SlbZIP68 reduced the activity of defense enzymes and increased the accumulation of reactive oxygen species (ROS). Our findings also indicated that SlbZIP68 can activate the expression of the PR genes and enhance resistance to P. infestans. In addition, SlbZIP68 can bind to the PR3 and PR5 promoters and induce gene expression, as revealed by yeast one-hybrid (Y1H) and dual-luciferase (LUC) assays. These findings not only elucidate the mechanisms of response to P. infestans but also enable targeted breeding strategies for tomato resistance to P. infestans.
•To identify tomato SlbZIP68 as a transcription factor that can be induced by P. infestans, SA and JA.•Knockout of SlbZIP68 using CRISPR-Cas9 technology resulted in a significant decrease in tomato resistance to P. infestans.•SlbZIP68 was found to play a positive regulatory role in tomato resistance to late blight.•The mechanism model of slbzip68 regulating tomato late blight immunity was proposed from an innovative perspective.
African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by ASF virus (ASFV) infection. At present, there are still no safe and effective drugs and vaccines to prevent ...ASF. Mining the important proteins encoded by ASFV that affect the virulence and replication of ASFV is the key to developing effective vaccines and drugs. In this study, ASFV pH240R, a capsid protein of ASFV, was found to inhibit the type I interferon (IFN) signaling pathway. Mechanistically, pH240R interacted with IFNAR1 and IFNAR2 to disrupt the interaction of IFNAR1-TYK2 and IFNAR2-JAK1. Additionally, pH240R inhibited the phosphorylation of IFNAR1, TYK2, and JAK1 induced by IFN-α, resulting in the suppression of the nuclear import of STAT1 and STAT2 and the expression of IFN-stimulated genes (ISGs). Consistent with these results, H240R-deficient ASFV (ASFV-∆H240R) infection induced more ISGs in porcine alveolar macrophages compared with its parental ASFV HLJ/18. We also found that pH240R enhanced viral replication via inhibition of ISGs expression. Taken together, our results clarify that pH240R enhances ASFV replication by inhibiting the JAK-STAT signaling pathway, which highlights the possibility of pH240R as a potential drug target.IMPORTANCEThe innate immune response is the host's first line of defense against pathogen infection, which has been reported to affect the replication and virulence of African swine fever virus (ASFV) isolates. Identification of ASFV-encoded proteins that affect the virulence and replication of ASFV is the key step in developing more effective vaccines and drugs. In this study, we found that pH240R interacted with IFNAR1 and IFNAR2 by disrupting the interaction of IFNAR1-TYK2 and IFNAR2-JAK1, resulting in the suppression of the expression of interferon (IFN)-stimulated genes (ISGs). Consistent with these results, H240R-deficient ASFV (ASFV-∆H240R) infection induces more ISGs' expression compared with its parental ASFV HLJ/18. We also found that pH240R enhanced viral replication via inhibition of ISGs' expression. Taken together, our findings showed that pH240R enhances ASFV replication by inhibiting the IFN-JAK-STAT axis, which highlights the possibility of pH240R as a potential drug target.
African swine fever (ASF) is a highly contagious infectious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV), with a mortality rate of up to 100%. In order to ...replicate efficiently in macrophages and monocytes, ASFV has evolved multiple strategies to evade host antiviral responses. However, the underlying molecular mechanisms by which ASFV-encoded proteins execute immune evasion are not fully understood. In this study, we found that ASFV pH240R strongly inhibits transcription, maturation, and secretion of interleukin-1β (IL-1β). Importantly, pH240R not only targeted NF-κB signaling but also impaired NLRP3 inflammasome activation. In this mechanism, pH240R interacted with NF-kappa-B essential modulator (NEMO), a component of inhibitor of kappa B kinase (IKK) complex and subsequently reduced phosphorylation of IκBα and p65. In addition, pH240R bonded to NLRP3 to inhibit NLRP3 inflammasome activation, resulting in reduced IL-1β production. As expected, infection with
-deficient ASFV (ASFV-ΔH240R) induced more inflammatory cytokine expression both
and
than its parental ASFV HLJ/18 strain. Consistently,
deficiency reduced the viral pathogenicity in pigs compared with its parental strain. These findings reveal that the
gene is an essential virulence factor, and deletion of the
gene affects the pathogenicity of ASFV HLJ/18 by enhancing antiviral inflammatory responses, which provides insights for ASFV immune evasion mechanisms and development of attenuated live vaccines and drugs for prevention and control of ASF.
African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and acute hemorrhagic viral disease of domestic pigs, with a high mortality approaching 100%. ASFV has spread rapidly worldwide and caused huge economic losses and ecological consequences. However, the pathogenesis and immune evasion mechanisms of ASFV are not fully understood, which limits the development of safe and effective ASF attenuated live vaccines. Therefore, investigations are urgently needed to identify virulence factors that are responsible for escaping the host antiviral innate immune responses and provide a new target for development of ASFV live-attenuated vaccine. In this study, we determined that the
gene is an essential virulence factor, and its depletion affects the pathogenicity of ASFV by enhancing NLRP3-mediated inflammatory responses, which provides theoretical support for the development of an ASFV attenuated live vaccine.
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