Kilohertz droplet-on-demand serial femtosecond crystallography at the European XFEL station FXE.

X-ray Free Electron Lasers (XFELs) allow the collection of high-quality serial femtosecond crystallography data. The next generation of megahertz superconducting FELs promises to drastically reduce data collection times, enabling the capture of more structures with higher signal-to-noise ratios and facilitating more complex experiments. Currently, gas dynamic virtual nozzles (GDVNs) stand as the sole delivery method capable of best utilizing the repetition rate of megahertz sources for crystallography. However, their substantial sample consumption renders their use impractical for many protein targets in serial crystallography experiments. Here, we present a novel application of a droplet-on-demand injection method, which allowed operation at 47 kHz at the European XFEL (EuXFEL) by tailoring a multi-droplet injection scheme for each macro-pulse. We demonstrate a collection rate of 150 000 indexed patterns per hour. We show that the performance and effective data collection rate are comparable to GDVN, with a sample consumption reduction of two orders of magnitude. We present lysozyme crystallographic data using the Large Pixel Detector at the femtosecond x-ray experiment endstation. Significant improvement of the crystallographic statistics was made by correcting for a systematic drift of the photon energy in the EuXFEL macro-pulse train, which was characterized from indexing the individual frames in the pulse train. This is the highest resolution protein structure collected and reported at the EuXFEL at 1.38 Å resolution.

Epithelium-derived kallistatin promotes CD4+ T-cell chemotaxis to TH2-type inflammation in chronic rhinosinusitis.

BACKGROUND: The function of kallistatin in airway inflammation, particularly chronic rhinosinusitis with nasal polyps (CRSwNP), has not been elucidated. OBJECTIVE: We sought to investigate the role of kallistatin in airway inflammation. METHODS: Kallistatin and proinflammatory cytokine expression levels were detected in nasal polyps. For the in vivo studies, we constructed the kallistatin-overexpressing transgenic mice to elucidate the role of kallistatin in airway inflammation. Furthermore, the levels of plasma IgE and proinflammatory cytokines in the airways were evaluated in the kallistatin-/- rat in vivo model under a type 2 inflammatory background. Finally, the Notch signaling pathway was explored to understand the role of kallistatin in CRSwNP. RESULTS: We showed that the expression of kallistatin was significantly higher in nasal polyps than in the normal nasal mucosa and correlated with IL-4 expression. We also discovered that the nasal mucosa of kallistatin-overexpressing transgenic mice expressed higher levels of IL-4 expression, associating to TH2-type inflammation. Interestingly, we observed lower IL-4 levels in the nasal mucosa and lower total plasma IgE of the kallistatin-/- group treated with house dust mite allergen compared with the wild-type house dust mite group. Finally, we observed a significant increase in the expression of Jagged2 in the nasal epithelium cells transduced with adenovirus-kallistatin. This heightened expression correlated with increased secretion of IL-4, attributed to the augmented population of CD4+CD45+Notch1+ T cells. These findings collectively may contribute to the induction of TH2-type inflammation. CONCLUSIONS: Kallistatin was demonstrated to be involved in the CRSwNP pathogenesis by enhancing the TH2 inflammation, which was found to be associated with more expression of IL-4, potentially facilitated through Jagged2-Notch1 signaling in CD4+ T cells.

An Expeditious Neutralization Assay for Porcine Reproductive and Respiratory Syndrome Virus Based on a Recombinant Virus Expressing Green Fluorescent Protein.

Due to the extensive genetic and antigenic variation in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), as well as its rapid mutability and evolution, PRRS prevention and control can be challenging. An expeditious and sensitive neutralization assay for PRRSV is presented to monitor neutralizing antibodies (NAbs) in serum during vaccine research. Here, a PRRSV expressing eGFP was successfully rescued with reverse genetics based on the infectious clone HuN4-F112-eGFP which we constructed. The fluorescent protein expressions of the reporter viruses remained stable for at least five passages. Based on this reporter virus, the neutralization assay can be easily used to evaluate the level of NAbs by counting cells with green fluorescence. Compared with the classical CPE assay, the newly developed assay increases sensitivity by one- to four-fold at the early antibody response stage, thus saving 2 days of assay waiting time. By using this assay to unveil the dynamics of neutralizing antibodies against PRRSV, priming immunity through either a single virulent challenge or only vaccination could produce limited NAbs, but re-infection with PRRSV would induce a faster and stronger NAb response. Overall, the novel HuN4-F112-eGFP-based neutralization assay holds the potential to provide a highly efficient platform for evaluating the next generation of PRRS vaccines.

Transbronchial cryoablation in peripheral lung parenchyma with a novel thin cryoprobe and initial clinical testing.

BACKGROUND: Transbronchial cryoablation shows potential as a local therapy for inoperable peripheral lung cancer. However, its clinical application for peripheral pulmonary lesions has not been reported yet. METHODS: An improved cryoprobe with an 8-mm-long, 1.9-mm-wide cryotip was used. Initially, the safety and effectiveness of this cryoprobe were assessed in an in vivo porcine model. Transbronchial cryoablation with 2 or 3 freeze-thaw cycles (10 min or 15 min in each freezing time) was performed in 18 pigs under CT monitoring. Radiological and pathological examinations were performed to evaluate the extent of cryoablation. Subsequently, nine patients with stage IA peripheral lung cancer or metastases underwent transbronchial cryoablation with this cryoprobe under the guidance of navigation bronchoscopy and cone-beam CT. Technical success, safety and outcomes were assessed. RESULTS: 36 cryoablation procedures were performed successfully without any major complications in the porcine model. The extent of cryoablation increased with freezing time and the number of freeze-thaw cycles, which peaked at 24 hours and then gradually decreased. Pathological results showed a change from massive haemorrhage at 24 hours to fibrous hyperplasia with chronic inflammation after 4 weeks. In the clinical trial, 10 cryoablations were performed on 9 tumours with a technical success rate of 100%. One mild treatment-related complication occurred. Of the nine tumours, seven achieved complete ablation, while two exhibited incomplete ablation and subsequent local progression at 6 months. CONCLUSION: Our initial experience indicated that transbronchial cryoablation was a safe and feasible procedure for non-surgical peripheral stage IA lung cancer or pulmonary metastases. TRIAL REGISTRATION NUMBER: ChiCTR2200061544.

The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity.

African swine fever virus (ASFV) is a highly contagious and deadly virus that leads to high mortality rates in domestic swine populations. Although the envelope protein CD2v of ASFV has been implicated in immunomodulation, the molecular mechanisms underlying CD2v-mediated immunoregulation remain unclear. In this study, we generated a stable CD2v-expressing porcine macrophage (PAM-CD2v) line and investigated the CD2v-dependent transcriptomic landscape using RNA-seq. GO terms enrichment analysis and gene set enrichment analysis revealed that CD2v predominantly affected the organization and assembly process of the extracellular matrix. Wound healing and Transwell assays showed that CD2v inhibited swine macrophage migration. Further investigation revealed a significant decrease in the expression of transcription factor early growth response 1 (EGR1) through inhibiting the activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Notably, EGR1 knockout in swine macrophages restricted cell migration, whereas EGR1 overexpression in PAM-CD2v restored the ability of macrophage migration, suggesting that CD2v inhibits swine macrophage motility by downregulating EGR1 expression. Furthermore, we performed chromatin immunoprecipitation and sequencing for EGR1 and the histone mark H3K27 acetylation (H3K27ac), and we found that EGR1 co-localized with the activated histone modification H3K27ac neighboring the transcriptional start sites. Further analysis indicated that EGR1 and H3K27ac co-occupy the promoter regions of cell locomotion-related genes. Finally, by treating various derivatives of swine macrophages with lipopolysaccharides, we showed that depletion of EGR1 decreased the expression of inflammatory cytokines including TNFα, IL1α, IL1ß, IL6, and IL8, which play essential roles in inflammation and host immune response. Collectively, our results provide new insights into the immunomodulatory mechanism of ASFV CD2v.

Expression of ASFV p17 in CHO cells and identification of one novel epitope using a monoclonal antibody.

As a highly pathogenic large DNA virus, African swine fever virus (ASFV) has huge particles and numerous encoded proteins. At present, few of the existing studies on ASFV proteins have investigated the function of p17. Specific antibodies against p17 to promote the development of prevention techniques against African swine fever (ASF) are urgently needed. Herein, we successfully expressed ASFV p17 in CHO cells using a suspension culture system and generated a monoclonal antibody (mAb) against p17. The mAb recognized a novel linear epitope (8LLSHNLSTREGIK20) and exhibited specific reactivity, which was conducive to the identification of ectopically expressed p17, the recombinant porcine reproductive and respiratory syndrome virus expressing p17, and the ASFV-SY18. The epitope was conservative among genotype I and genotype II ASFV strains. Overall, the mAb against p17 revealed efficient detection and promising application prospects, making it a useful tool for future vaccine research on ASF. Determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of ASFV antigen protein.

XFEL Microcrystallography of Self-Assembling Silver n-Alkanethiolates.

New synthetic hybrid materials and their increasing complexity have placed growing demands on crystal growth for single-crystal X-ray diffraction analysis. Unfortunately, not all chemical systems are conducive to the isolation of single crystals for traditional characterization. Here, small-molecule serial femtosecond crystallography (smSFX) at atomic resolution (0.833 Å) is employed to characterize microcrystalline silver n-alkanethiolates with various alkyl chain lengths at X-ray free electron laser facilities, resolving long-standing controversies regarding the atomic connectivity and odd-even effects of layer stacking. smSFX provides high-quality crystal structures directly from the powder of the true unknowns, a capability that is particularly useful for systems having notoriously small or defective crystals. We present crystal structures of silver n-butanethiolate (C4), silver n-hexanethiolate (C6), and silver n-nonanethiolate (C9). We show that an odd-even effect originates from the orientation of the terminal methyl group and its role in packing efficiency. We also propose a secondary odd-even effect involving multiple mosaic blocks in the crystals containing even-numbered chains, identified by selected-area electron diffraction measurements. We conclude with a discussion of the merits of the synthetic preparation for the preparation of microdiffraction specimens and compare the long-range order in these crystals to that of self-assembled monolayers.

Locating cellular contents during cryoFIB milling using cellular secondary-electron imaging.

Cryo-electron tomography (cryoET) is a powerful technology that allows in-situ observation of the molecular structure of tissues and cells. Cryo-focused ion beam (cryoFIB) milling plays an important role in the preparation of high-quality thin lamellar samples for cryoET studies, thus, promoting the rapid development of cryoET in recent years. However, locating the regions of interest in a large cell or tissue during cryoFIB milling remains a major challenge limiting cryoET applications on arbitrary biological samples. Here, we report an on-the-fly localization method based on cellular secondary electron imaging (CSEI), which is derived from a basic imaging function of the cryoFIB instruments and enables high-contrast imaging of the cellular contents of frozen-hydrated biological samples. Moreover, CSEI does not require fluorescent labels and additional devices. The present study discusses the imaging principles and settings for optimizing CSEI. Tests on several commercially available cryoFIB instruments demonstrated that CSEI was feasible on mainstream instruments to observe all types of cellular contents and reliable under different milling conditions. We established a simple milling-localization workflow and tested it using the basal body of Chlamydomonas reinhardtii.

Watching Excited State Dynamics with Optical and X-ray Probes: The Excited State Dynamics of Aquocobalamin and Hydroxocobalamin.

Femtosecond time-resolved X-ray absorption (XANES) at the Co K-edge, X-ray emission (XES) in the Co Kß and valence-to-core regions, and broadband UV-vis transient absorption are combined to probe the femtosecond to picosecond sequential atomic and electronic dynamics following photoexcitation of two vitamin B12 compounds, hydroxocobalamin and aquocobalamin. Polarized XANES difference spectra allow identification of sequential structural evolution involving first the equatorial and then the axial ligands, with the latter showing rapid coherent bond elongation to the outer turning point of the excited state potential followed by recoil to a relaxed excited state structure. Time-resolved XES, especially in the valence-to-core region, along with polarized optical transient absorption suggests that the recoil results in the formation of a metal-centered excited state with a lifetime of 2-5 ps. This combination of methods provides a uniquely powerful tool to probe the electronic and structural dynamics of photoactive transition-metal complexes and will be applicable to a wide variety of systems.

Ultrafast Jahn-Teller Photoswitching in Cobalt Single-Ion Magnets.

Single-ion magnets (SIMs) constitute the ultimate size limit in the quest for miniaturizing magnetic materials. Several bottlenecks currently hindering breakthroughs in quantum information and communication technologies could be alleviated by new generations of SIMs displaying multifunctionality. Here, ultrafast optical absorption spectroscopy and X-ray emission spectroscopy are employed to track the photoinduced spin-state switching of the prototypical complex [Co(terpy)2 ]2+ (terpy = 2,2':6',2″-terpyridine) in solution phase. The combined measurements and their analysis supported by density functional theory (DFT), time-dependent-DFT (TD-DFT) and multireference quantum chemistry calculations reveal that the complex undergoes a spin-state transition from a tetragonally elongated doublet state to a tetragonally compressed quartet state on the femtosecond timescale, i.e., it sustains ultrafast Jahn-Teller (JT) photoswitching between two different spin multiplicities. Adding new Co-based complexes as possible contenders in the search for JT photoswitching SIMs will greatly widen the possibilities for implementing magnetic multifunctionality and eventually controlling ultrafast magnetization with optical photons.

Secondary Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (HP-PRRSV2) Infection Augments Inflammatory Responses, Clinical Outcomes, and Pathogen Load in Glaesserella-parasuis-Infected Piglets.

Glaesserella parasuis (Gps), Gram-negative bacteria, are a universal respiratory-disease-causing pathogen in swine that colonize the upper respiratory tract. Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (HP-PRRSV2HP-PRRSV2) and Gps coinfections are epidemics in China, but little is known about the influence of concurrent coinfection on disease severity and inflammatory responses. Herein, we studied the effects of secondary HP-PRRS infection on clinical symptoms, pathological changes, pathogen load, and inflammatory response of Gps coinfection in the upper respiratory tract of piglets. All coinfected piglets (HP-PRRSV2 + Gps) displayed fever and severe lesions in the lungs, while fever was present in only a few animals with a single infection (HP-PRRSV2 or Gps). Additionally, HP-PRRSV2 and Gps loading in nasal swabs and blood and lung tissue samples was significantly increased in the coinfected group. Necropsy data showed that coinfected piglets suffered from severe lung damage and had significantly higher antibody titers of HP-PRRSV2 or Gps than single-infected piglets. Moreover, the serum and lung concentrations of inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8) were also significantly higher in coinfected piglets than in those infected with HP-PRRSV2 or Gps alone. In conclusion, our results show that HP-PRRSV2 promotes the shedding and replication of Gps, and their coinfection in the upper respiratory tract aggravates the clinical symptoms and inflammatory responses, causing lung damage. Therefore, in the unavoidable situation of Gps infection in piglets, necessary measures must be made to prevent and control secondary infection with HP-PRRSV2, which can save huge economic losses to the pork industry.

CryoFIB milling large tissue samples for cryo-electron tomography.

Cryo-electron tomography (cryoET) is a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, localization and cutting out objects of interest from a large tissue sample are still difficult steps. In this study, we report a sample thinning strategy and workflow for tissue samples based on cryo-focused ion beam (cryoFIB) milling. This workflow provides a full solution for isolating objects of interest by starting from a millimeter-sized tissue sample and ending with hundred-nanometer-thin lamellae. The workflow involves sample fixation, pre-sectioning, a two-step milling strategy, and localization of the object of interest using cellular secondary electron imaging (CSEI). The milling strategy consists of two steps, a coarse milling step to improve the milling efficiency, followed by a fine milling step. The two-step milling creates a furrow-ridge structure with an additional conductive Pt layer to reduce the beam-induced charging issue. CSEI is highlighted in the workflow, which provides on-the-fly localization during cryoFIB milling. Tests of the complete workflow were conducted to demonstrate the high efficiency and high feasibility of the proposed method.

The rPRRSV-E2 strain exhibited a low level of potential risk for virulence reversion.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Classical Swine Fever Virus (CSFV) are two important pathogens, which cause serious impact on swine industry worldwide. In our previous research, rPRRSV-E2, the recombinant PRRSV expressing CSFV E2 protein, could provide sufficient protection against the lethal challenge of highly pathogenic PRRSV and CSFV, and could maintained genetically stable in vitro. Here, to evaluate the virulence reversion potential risk, rPRRSV-E2 had been continuously passaged in vivo, the stability of E2 expression and virulence of the passage viruses were analyzed. The results showed that no clinical symptoms or pathological changes could be found in the inoculated groups, and there were no significant differences of viraemia among the test groups. Sequencing and IFA analysis showed that the coding gene of exogenous CSFV E2 protein existed in the passaged viruses without any sequence mutations, deletions or insertions, and could expressed steadily. It could be concluded that the foreign CSFV E2 gene in the genome of rPRRSV-E2 could be maintained genetically stable in vivo, and rPRRSV-E2 strain had relatively low level of potential risk for virulence reversion.

PSMB1 Inhibits the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Recruiting NBR1 To Degrade Nonstructural Protein 12 by Autophagy.

The nonstructural proteins (Nsps) of porcine reproductive and respiratory syndrome virus (PRRSV) play essential roles in virus replication-a multistep process that requires the participation of host factors. It is of great significance for the development of antiviral drugs to characterize the host proteins that interact with PRRSV Nsps and their functions in PRRSV replication. Here, we determined that proteasome subunit ß type 1 (PSMB1) interacted with viral Nsp12 to inhibit PRRSV replication in target and permissive cells. PSMB1 could be downregulated by PRRSV infection through interaction with the transcription factor EBF1. Proteasome and autophagy inhibitor assays showed that PSMB1 was regulated by the autophagic pathway to degrade Nsp12. Cotransfection of PSMB1 and Nsp12 increased the level of intracellular autophagy; both molecules were colocated in lysosomes. We also found that the selective autophagy cargo receptor protein NBR1 and E3 ubiquitin ligase STUB1 interacted with PSMB1 and Nsp12, respectively, in the autophagic degradation of Nsp12. Furthermore, the degradation of Nsp12 by PSMB1 was mainly dependent on the ubiquitination of Nsp12 at lysine site 130. Our results indicate for the first time that PSMB1 is an anti-PRRSV host protein that inhibits the replication of PRRSV by degradation of Nsp12 through the selective autophagy pathway. IMPORTANCE PRRS is a major threat to the global pig industry and urgently requires an effective and sustainable control strategy. PRRSV Nsps have important roles in viral RNA synthesis, proteinase activity, induction of replication-associated membrane rearrangements, replicative endoribonuclease activity, determination of virulence, and regulation of host immune response. Research associated with PRRSV Nsps can provide vital guidance to modify the PRRSV genome through reverse genetics in the development of vaccines and diagnostics. The function of Nsp12, which generally plays essential roles in virus replication, remains unclear. We demonstrated that PSMB1 interacted with and degraded Nsp12 through an autophagic pathway to inhibit PRRSV replication. Our data confirmed a novel antiviral function of PSMB1 and allowed us to elaborate on the roles of Nsp12 in PRRSV pathogenesis. These findings suggest a valid and highly conserved candidate target for the development of novel therapies and more effective vaccines and demonstrate the complex cross talk between selective autophagy and PRRSV infection.

Kinetic Resolution of Azaarylethynyl Tertiary Alcohols by Chiral Brønsted Acid Catalysed Phosphine-Mediated Deoxygenation.

A chiral Brønsted acid catalysed phosphine-mediated deoxygenation protocol is reported. This metal-free method provides a precise kinetic resolution platform for azaarylethynyl tertiary alcohols, which are a broad category of biologically and synthetically important azaarene derivatives. In addition to providing an efficient method for the first asymmetric preparation of these tertiary alcohols, the strategy facilitates the construction of azaaryl-functionalized allenes with good to excellent enantioselectivities. The high selectivity factors (s up to 235), broad substrate scope, and ability to convert azaaryl compounds into both chiral tertiary alcohols and allenes robustly underscore the efficiency and promising utility of this method. The practicability is further validated by the successful synthesis of deuterated allenes with high ee values and substantial incorporation of deuterium using inexpensive D2 O as the deuterium source.

Nonstructural Protein 2 Is Critical to Infection Efficiency of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus on PAMs and Influence Virulence In Vivo.

Porcine reproductive and respiratory syndrome (PRRS) is an important viral disease, causing significant economic losses to the swine industry worldwide. Atypical cases caused by highly pathogenic PRRS virus (HP-PRRSV) emerged in 2006 in China. The vaccine strain HuN4-F112 has been developed from the wild-type HP-PRRSV HuN4 through repeated passages on MARC-145 cells. However, the mechanisms of attenuation have yet to be defined. Previous studies have shown that the vaccine strain HuN4-F112 could not effectively replicate in porcine alveolar macrophages (PAMs). In the present study, a series of chimeric and mutant PRRSVs were constructed to investigate regions associated with the virus attenuation. Firstly, the corresponding genome regions (ORF1a, ORF1b and ORFs 2-7) were exchanged between two infectious clones of HuN4 and HuN4-F112, and then the influence of small regions in ORF1a and ORF2-7 was evaluated, then influence of specific amino acids on NSP2 was tested. NSP2 was determined to be the key gene that regulated infection efficiency on PAMs, and amino acids at 893 and 979 of NSP2 were the key amino acids. The results of in vivo study indicated that NSP2 was not only important for infection efficiency in vitro, but also influenced the virulence, which was indicated by the results of survival rate, temperature, viremia, lung score and tissue score.

Identification and Characterization of Cell Lines HepG2, Hep3B217 and SNU387 as Models for Porcine Epidemic Diarrhea Coronavirus Infection.

Porcine epidemic diarrhea virus (PEDV), a member of the genera alphacoronavirus, causes acute watery diarrhea and dehydration in suckling piglets and results in enormous economic losses in the swine industry worldwide. Identification and characterization of different cell lines are not only invaluable for PEDV entry and replication studies but also important for the development of various types of biological pharmaceuticals against PEDV. In this study, we present an approach to identify suitable permissive cell lines for PEDV research. Human cell lines were screened for a high correlation coefficient with the established PEDV infection model Huh7 based on RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE). Experimentally testing permissiveness towards PEDV infection, three highly permissive human cell lines, HepG2, Hep3B217, and SNU387 were identified. The replication kinetics of PEDV in HepG2, Hep3B217, and SNU387 cells were similar to that in Vero and Huh7 cells. Additionally, the transcriptomes analysis showed robust induction of transcripts associated with the innate immune in response to PEDV infection in all three cell lines, including hundreds of inflammatory cytokine and interferon genes. Moreover, the expression of inflammatory cytokines and interferons were confirmed by qPCR assay. Our findings indicate that HepG2, Hep3B217, and SNU387 are suitable cell lines for PEDV replication and innate immune response studies.

How Safety Climate Impacts Safety Voice-Investigating the Mediating Role of Psychological Safety from a Social Cognitive Perspective.

Safety voice has become a popular research topic in the organizational safety field because it helps to prevent accidents. A good safety climate and psychological safety can motivate employees to actively express their ideas about safety, but the specific mechanisms of safety climate and psychological safety, on safety voice, are not yet clear. Based on the "environment-subject cognition-behavior" triadic interaction model of social cognitive theory, this paper explores the relationship between safety climate and safety voice, and the mediating role of psychological safety. We collected questionnaires and conducted data analysis of the valid questionnaires using analytical methods such as hierarchical regression, stepwise regression, and the bootstrap sampling method. We found that safety climate significantly and positively influenced safety voice, and psychological safety played a mediating role between safety climate and safety voice, which strengthened the positive relationship between them. From the research results, it was clear that to stimulate employees to express safety voice behavior, organizations should strive to create a good safety climate and pay attention to building employees' psychological safety. The findings of this paper provide useful insights for the management of employee safety voice behavior in enterprises.

A predictive model based on ground glass nodule features via high-resolution CT for identifying invasiveness of lung adenocarcinoma.

Objective: The morphology of ground-glass nodule (GGN) under high-resolution computed tomography (HRCT) has been suggested to indicate different histological subtypes of lung adenocarcinoma (LUAD); however, existing studies only include the limited number of GGN characteristics, which lacks a systematic model for predicting invasive LUAD. This study aimed to construct a predictive model based on GGN features under HRCT for LUAD. Methods: A total of 301 surgical LUAD patients with HRCT-confirmed GGN were enrolled, and their GGN-related features were assessed by 2 individual radiologists. The pathological diagnosis of the invasive LUAD was established by pathologic examination following surgery (including 171 invasive and 130 non-invasive LUAD patients). Results: GGN features including shorter distance from pleura, larger diameter, area and mean CT attenuation, more heterogeneous uniformity of density, irregular shape, coarse margin, not defined nodule-lung interface, spiculation, pleural indentation, air bronchogram, vacuole sign, vessel changes, lobulation were observed in invasive LUAD patients compared with non-invasive LUAD patients. After adjustment by multivariate logistic regression model, GGN diameter (OR = 1.490, 95% CI, 1.326-1.674), mean CT attenuation (OR = 1.007, 95% CI, 1.004-1.011) and heterogeneous uniformity of density (OR = 3.009, 95% CI, 1.485-6.094) were independent risk factors for invasive LUAD. In addition, a predictive model integrating these three independent GGN features was established (named as invasion of lung adenocarcinoma by GGN features (ILAG)), and receiver-operating characteristic curve illustrated that the ILAG model presented good predictive value for invasive LUAD (AUC: 0.919, 95% CI, 0.889-0.949). Conclusions: ILAG predictive model integrating GGN diameter, mean CT attenuation and heterogeneous uniformity of density via HRCT shows great potential for early estimation of LUAD invasiveness.

Porcine epidemic diarrhea virus strain FJzz1 infection induces type I/III IFNs production through RLRs and TLRs-mediated signaling.

Interferons (IFNs) including type I/III IFNs are the major components of the host innate immune response against porcine epidemic diarrhea virus (PEDV) infection, and several viral proteins have been identified to antagonize type I/III IFNs productions through diverse strategies. However, the modulation of PEDV infection upon the activation of the host's innate immune response has not been fully characterized. In this study, we observed that various IFN-stimulated genes (ISGs) were upregulated significantly in a time- and dose-dependent manner in LLC-PK1 cells infected with the PEDV G2 strain FJzz1. The transcriptions of IRF9 and STAT1 were increased markedly in the late stage of FJzz1 infection and the promotion of the phosphorylation and nuclear translocation of STAT1, implicating the activation of the JAK-STAT signaling pathway during FJzz1 infection. In addition, abundant type I/III IFNs were produced after FJzz1 infection. However, type I/III IFNs and ISGs decreased greatly in FJzz1-infected LLC-PK1 cells following the silencing of the RIG-I-like receptors (RLRs), including RIG-I and MDA5, and the Toll-like receptors (TLRs) adaptors, MyD88 and TRIF. Altogether, FJzz1 infection induces the production of type-I/III IFNs in LLC-PK1 cells, in which RLRs and TLRs signaling pathways are involved, followed by the activation of the JAK-STAT signaling cascade, triggering the production of numerous ISGs to exert antiviral effects of innate immunity.