Influenza A virus activates cellular Tropomyosin receptor kinase A (TrkA) signaling to promote viral replication and lung inflammation.

Influenza A virus (IAV) infection causes acute respiratory disease with potential severe and deadly complications. Viral pathogenesis is not only due to the direct cytopathic effect of viral infections but also to the exacerbated host inflammatory responses. Influenza viral infection can activate various host signaling pathways that function to activate or inhibit viral replication. Our previous studies have shown that a receptor tyrosine kinase TrkA plays an important role in the replication of influenza viruses in vitro, but its biological roles and functional mechanisms in influenza viral infection have not been characterized. Here we show that IAV infection strongly activates TrkA in vitro and in vivo. Using a chemical-genetic approach to specifically control TrkA kinase activity through a small molecule compound 1NMPP1 in a TrkA knock-in (TrkA KI) mouse model, we show that 1NMPP1-mediated TrkA inhibition completely protected mice from a lethal IAV infection by significantly reducing viral loads and lung inflammation. Using primary lung cells isolated from the TrkA KI mice, we show that specific TrkA inhibition reduced IAV viral RNA synthesis in airway epithelial cells (AECs) but not in alveolar macrophages (AMs). Transcriptomic analysis confirmed the cell-type-specific role of TrkA in viral RNA synthesis, and identified distinct gene expression patterns under the TrkA regulation in IAV-infected AECs and AMs. Among the TrkA-activated targets are various proinflammatory cytokines and chemokines such as IL6, IL-1ß, IFNs, CCL-5, and CXCL9, supporting the role of TrkA in mediating lung inflammation. Indeed, while TrkA inhibitor 1NMPP1 administered after the peak of IAV replication had no effect on viral load, it was able to decrease lung inflammation and provided partial protection in mice. Taken together, our results have demonstrated for the first time an important biological role of TrkA signaling in IAV infection, identified its cell-type-specific contribution to viral replication, and revealed its functional mechanism in virus-induced lung inflammation. This study suggests TrkA as a novel host target for therapeutic development against influenza viral disease.

Effect of Deep Learning Image Reconstruction Algorithms on Radiomic Features of Pulmonary Nodules in Ultra-Low-Dose CT.

OBJECTIVE: The purpose of this study is to explore the impact of deep learning image reconstruction (DLIR) algorithm on the quantification of radiomic features in ultra-low-dose computed tomography (ULD-CT) compared with adaptive statistical iterative reconstruction-Veo (ASIR-V). METHODS: One hundred eighty-three patients with pulmonary nodules underwent standard-dose computed tomography (SDCT) (4.30 ± 0.36 mSv) and ULD-CT (UL-A, 0.57 ± 0.09 mSv or UL-B, 0.33 ± 0.04 mSv). SDCT was the reference standard using (ASIR-V) at 50% strength (50%ASIR-V). ULD-CT was reconstructed with 50%ASIR-V, DLIR at medium and high strength (DLIR-M, DLIR-H). Radiomics analysis extracted 102 features, and the intraclass correlation coefficient (ICC) quantified reproducibility between ULD-CT and SDCT reconstructed by 50%ASIR-V, DLIR-M, and DLIR-H for each feature. RESULTS: Among 102 radiomic features, the percentages of reproducibility of 50%ASIR-V, DLIR-M, and DLIR-H were 48.04% (49/102), 49.02% (50/102), and 52.94% (54/102), respectively. Shape and first order features demonstrated high reproducibility across different reconstruction algorithms and radiation doses, with mean ICC values exceeding 0.75. In texture features, DLIR-M and DLIR-H showed improved mean ICC values for pure ground glass nodules (pGGNs) from 0.69 ± 0.23 to 0.75 ± 0.18 and 0.81 ± 0.12, respectively, compared with 50%ASIR-V. Similarly, the mean ICC values for solid nodules (SNs) increased from 0.60 ± 0.19 to 0.66 ± 0.14 and 0.69 ± 0.13, respectively. Additionally, the mean ICC values of texture features for pGGNs and SNs in both ULD-CT groups decreased with reduced radiation dose. CONCLUSIONS: DLIR can improve the reproducibility of radiomic features at ultra-low doses compared with ASIR-V. In addition, pGGNs showed better reproducibility at ultra-low doses than SNs.

Enhancing diagnostic performance and image quality in coronary CT angiography: Impact of SnapShot Freeze 2 algorithm across varied heart rates in stent patients.

PURPOSE: To investigate the enhancement of image quality achieved through the utilization of SnapShot Freeze 2 (SSF2), a comparison was made against the results obtained from the original SnapShot Freeze algorithm (SSF) and standard motion correction (STND) in stent patients undergoing coronary CT angiography (CCTA) across the entire range of heart rates. MATERIALS AND METHODS: A total of 118 patients who underwent CCTA, were retrospectively included in this study. Images of these patients were reconstructed using three different algorithms: SSF2, SSF, and STND. Objective assessments include signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), diameters of stents and artifact index (AI). The image quality was subjectively evaluated by two readers. RESULTS: Compared with SSF and STND, SSF2 had similar or even higher quality in the parameters (AI, SNR, CNR, inner diameters) of coronary artery, stent, myocardium, MV (mitral valve), TV (tricuspid valve), AV (aorta valve), and PV (pulmonary valve), and aortic root (AO). Besides the above structures, SSF2 also demonstrated comparable or even higher subjective scores in atrial septum (AS), ventricular septum (VS), and pulmonary artery root (PA). Furthermore, the enhancement in image quality with SSF2 was significantly greater in the high heart rate group compared to the low heart rate group. Moreover, the improvement in both high and low heart rate groups was better in the SSF2 group compared to the SSF and STND group. Besides, when using the three algorithms, an effect of heart rate variability on stent image quality was not detected. CONCLUSION: Compared to SSF and STND, SSF2 can enhance the image quality of whole-heart structures and mitigate artifacts of coronary stents. Furthermore, SSF2 has demonstrated a significant improvement in the image quality for patients with a heart rate equal to or higher than 85 bpm.

Alkyl-Doping Enables Significant Suppression of Conformational Relaxation and Intermolecular Nonradiative Decay for Improved Near-Infrared Fluorescence Imaging.

Despite various efforts to optimize the near-infrared (NIR) performance of perylene diimide (PDI) derivatives for bio-imaging, convenient and efficient strategies to amplify the fluorescence of PDI derivatives in biological environment and the intrinsic mechanism studies are still lacking. Herein, we propose an alkyl-doping strategy to amplify the fluorescence of PDI derivative-based nanoparticles for improved NIR fluorescence imaging. The developed PDI derivative, OPE-PDI, shows much brighter in n-Hexane (HE) compared with that in other organic media, and the excited state dynamics investigation experimentally elucidates the solvent effect-induced suppression of intermolecular energy transfer and intramolecular nonradiative decay as the underlying mechanism for the fluorescence improvement. Theoretical calculations reveal the lowest reorganization energies of OPE-PDI in HE among various solvents, indicating the effectively suppressed conformational relaxation to support the strongest radiative decay. Inspired by this, an alkyl atmosphere mimicking HE is constructed by incorporating the octadecane into OPE-PDI-based nanoparticles, permitting up to 3-fold fluorescence improvement compared with the counterpart nanoparticles. Owing to the merits of high brightness, anti-photobleaching, and low biotoxicity for the optimal nanoparticles, they have been employed for probing and long-term monitoring of tumor. This work highlights a facile strategy for the fluorescence enhancement of PDI derivative-based nanoparticles.

Evaluating the Biological Role of Lassa Viral Z Protein-Mediated RIG-I Inhibition Using a Replication-Competent Trisegmented Pichinde Virus System in an Inducible RIG-IN Expression Cell Line.

Lassa virus (LASV) is a mammarenavirus that can cause lethal Lassa fever disease with no FDA-approved vaccine and limited treatment options. Fatal LASV infections are associated with innate immune suppression. We have previously shown that the small matrix Z protein of LASV, but not of a nonpathogenic arenavirus Pichinde virus (PICV), can inhibit the cellular RIG-I-like receptors (RLRs), but its biological significance has not been evaluated in an infectious virus due to the multiple essential functions of the Z protein required for the viral life cycle. In this study, we developed a stable HeLa cell line (HeLa-iRIGN) that could be rapidly and robustly induced by doxycycline (Dox) treatment to express RIG-I N-terminal effector, with concomitant production of type I interferons (IFN-Is). We also generated recombinant tri-segmented PICVs, rP18tri-LZ, and rP18tri-PZ, which encode LASV Z and PICV Z, respectively, as an extra mScarlet fusion protein that is nonessential for the viral life cycle. Upon infection, rP18tri-LZ consistently expressed viral genes at a higher level than rP18tri-PZ. rP18tri-LZ also showed a higher level of a viral infection than rP18tri-PZ did in HeLa-iRIGN cells, especially upon Dox induction. The heterologous Z gene did not alter viral growth in Vero and A549 cells by growth curve analysis, while LASV Z strongly increased and prolonged viral gene expression, especially in IFN-competent A549 cells. Our study provides important insights into the biological role of LASV Z-mediated RIG-I inhibition and implicates LASV Z as a potential virulence factor. IMPORTANCE Lassa virus (LASV) can cause lethal hemorrhagic fever disease in humans but other arenaviruses, such as Pichinde virus (PICV), do not cause obvious disease. We have previously shown that the Z protein of LASV but not of PICV can inhibit RIG-I, a cytosolic innate immune receptor. In this study, we developed a stable HeLa cell line that can be induced to express the RIG-I N-terminal effector domain, which allows for timely control of RIG-I activation. We also generated recombinant PICVs encoding LASV Z or PICV Z as an extra gene that is nonessential for the viral life cycle. Compared to PICV Z, LASV Z could increase viral gene expression and viral infection in an infectious arenavirus system, especially when RIG-I signaling is activated. Our study presented a convenient cell system to characterize RIG-I signaling and its antagonists and revealed LASV Z as a possible virulence factor and a potential antiviral target.

Progress of proteomics-driven precision medicine: From a glycosylation view.

Currently, cancer is one of the leading causes of death worldwide, partially owing to the lack of early diagnosis methods and effective therapies. With the rapid development of various omics, the precision medicine strategy becomes a promising way to increase the survival rates by considering individual differences. Glycosylation is one of the most essential protein post-translational modifications and plays important roles in a variety of biological processes. Therefore, it is highly possible to acquire understanding of the molecular mechanisms as well as discover novel potential markers for diagnosis and prognosis based on glycoproteomics research. This review summarizes the recent glycoproteomics studies about N-glycosylation of several cancer types, mainly in the past 5 years. We also highlight corresponding mass spectrometry-based analytical methods to give a brief overview on the main techniques applied in glycoproteomics.

Regulation of Eosinophil Recruitment and Allergic Airway Inflammation by Tropomyosin Receptor Kinase A.

Eosinophilia is a hallmark of allergic airway inflammation (AAI). Identifying key molecules and specific signaling pathways that regulate eosinophilic inflammation is critical for development of novel therapeutics. Tropomycin receptor kinase A (TrkA) is the high-affinity receptor for nerve growth factor. AAI is associated with increased expression of TrkA by eosinophils; however, the functional role of TrkA in regulating eosinophil recruitment and contributing to AAI is poorly understood. This study identifies, to our knowledge, a novel mechanism of eotaxin-mediated activation of TrkA and its role in regulating eosinophil recruitment by using a chemical-genetic approach to specifically inhibit TrkA kinase activity with 1-NM-PP1 in TrkAF592A-knock-in (TrkA-KI) eosinophils. Blockade of TrkA by 1-NM-PP1 enhanced eosinophil spreading on VCAM-1 but inhibited eotaxin-1 (CCL11)-mediated eosinophil migration, calcium flux, cell polarization, and ERK1/2 activation, suggesting that TrkA is an important player in the signaling pathway activated by eotaxin-1 during eosinophil migration. Further, blockade of matrix metalloprotease with BB-94 inhibited eotaxin-1-induced TrkA activation and eosinophil migration, additively with 1-NM-PP1, indicating a role for matrix metalloproteases in TrkA activation. TrkA inhibition in Alternaria alternata-challenged TrkA-KI mice markedly inhibited eosinophilia and attenuated various features of AAI. These findings are indicative of a distinctive eotaxin-mediated TrkA-dependent signaling pathway, which, in addition to other TrkA-activating mediators, contributes to eosinophil recruitment during AAI and suggests that targeting the TrkA signaling pathway to inhibit eosinophil recruitment may serve as a therapeutic strategy for management of eosinophilic inflammation in allergic airway disease, including asthma.

Absolute decrease in regulatory T cells and low-dose interleukin-2 therapy: restoring and expanding regulatory T cells to treat systemic sclerosis: a 24-week study.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular lesions, immunological alterations and tissue fibrosis. There is some evidence of an imbalance between T-cell subsets in this disease. Interleukin (IL)-2 is a cytokine that can regulate the activity of immune cells and there is evidence that low-dose IL-2 therapy can be used to treat immune diseases. AIM: To investigate the changes of peripheral lymphocyte subsets, especially T helper (Th)17 and regulatory T (Treg) cells and the effects of low-dose IL-2 therapy in patients with SSc. METHODS: In total, 66 patients with SSc and 49 sex- and age-matched healthy controls (HCs), were enrolled. The absolute numbers of peripheral lymphocyte subsets in these individuals were determined by flow cytometry. The 66 patients, were divided into 2 groups: 23 (the IL-2 group) were treated with low-dose (5.0 × 105 IU) IL-2 by subcutaneous injection daily for 5 days combined with conventional therapy, while the remaining 23 patients received conventional therapy only. RESULTS: Compared with HCs, the absolute numbers of peripheral T, CD4+ T, CD8+ T, natural killer and Treg cells were significantly lower in patients with SSc, with the most dramatic difference seen in both the absolute number and percentage of Treg cells in these patients, including new (previously untreated) cases, resulting in an imbalance (elevated ratio) between Th17 and Treg cells. At Week 24 after commencement of IL-2 treatment, Treg cells were markedly increased and tended to restore the balance of Th17 to Treg cells compared with baseline. Erythrocyte sedimentation rate, C-reactive protein, modified Rodnan Skin Score and visual analogue scale score were significantly decreased in both the IL-2 and non-IL-2 groups, indicating disease improvement. Notably, compared with those in the non-IL-2 group, patients treated with IL-2 had greater improvement. CONCLUSION: Our study showed that the absolute numbers of peripheral Treg cells together with total T, CD4+ T, CD8+ T and NK is significantly decreased, leading to an imbalance of Th17 to Treg cells in patients with SSc, and that low-dose IL-2 treatment could restore the balance of the two immune cells and reduce disease activity without obvious adverse effects.

Chaotic Mapping-Based Anti-Sorting Radio Frequency Stealth Signals and Compressed Sensing-Based Echo Signal Processing Technology.

Radio frequency (RF) stealth anti-sorting technology can improve the battlefield survival rate of radar and is one of the research hotspots in the radar field. In this study, the signal design principle of anti-sequential difference histogram (SDIF) sorting was explored for the main sorting algorithm of the SDIF. Furthermore, we designed a piecewise linear chaotic system with interval number parameterization based on random disturbance and proposed a method to modulate the repetition period of widely spaced signal pulses using a chaotic system. Then, considering the difficulty of the traditional signal processing method to measure the velocity of the highly random anti-sorting signals designed in this paper, we used compressed sensing (CS) technology to process the echoes of the signals to solve the velocity and distance of the detection targets. Finally, simulation verification was performed from the correctness of the signal design principle, the performance of the chaotic system, the anti-sorting performance of the designed signals and the recovery and reconstruction performance of the signals by CS. The results show that: (a) the signal design principle presented in this paper can guide the signal design correctly; (b) the performance of the piecewise linear chaotic system with interval number parameterization is better than that of the classical one-dimensional chaotic system; (c) the anti-sorting signal modulated by the chaotic system can achieve anti-SDIF sorting, and the anti-sorting signals designed in this paper can be processed to obtain the velocity and distance of the targets.

Design of Multi-Parameter Compound Modulated RF Stealth Anti-Sorting Signals Based on Hyperchaotic Interleaving Feedback.

Radio frequency (RF) stealth anti-sorting technology is a research hotspot in the radar field. In this study, the signal design principles of anti-cluster and anti-SDIF sorting were investigated for processes of clustering pre-sorting and sequence-difference-histogram main sorting. Then, in accordance with the signal design principle, a 2D interleaving feedback hyperchaotic system based on the cosine-exponential was designed. A method to modulate the pulse repetition interval (PRI) of the signal parameters and carrier frequency with wide intervals through the hyperchaotic system was developed. Finally, we verified the correctness of the signal design principle, the performance of the hyperchaotic system, and the anti-sorting performance of the designed signal using simulations. The results showed that the signal design principle could guide the signal design. The hyperchaotic system outperformed the classical 1D and 2D chaotic systems and the classical 3D Lorenz systems in terms of randomness and complexity. Anti-cluster sorting and anti-SDIF sorting could be realized by anti-sorting signals modulated by a hyperchaotic system, with the anti-SDIF sorting performance being better than that of the PRI random jitter signal.

Inhibition of Arenaviruses by Combinations of Orally Available Approved Drugs.

Neglected diseases caused by arenaviruses such as Lassa virus (LASV) and filoviruses like Ebola virus (EBOV) primarily afflict resource-limited countries, where antiviral drug development is often minimal. Previous studies have shown that many approved drugs developed for other clinical indications inhibit EBOV and LASV and that combinations of these drugs provide synergistic suppression of EBOV, often by blocking discrete steps in virus entry. We hypothesize that repurposing of combinations of orally administered approved drugs provides effective suppression of arenaviruses. In this report, we demonstrate that arbidol, an approved influenza antiviral previously shown to inhibit EBOV, LASV, and many other viruses, inhibits murine leukemia virus (MLV) reporter viruses pseudotyped with the fusion glycoproteins (GPs) of other arenaviruses (Junin virus [JUNV], lymphocytic choriomeningitis virus [LCMV], and Pichinde virus [PICV]). Arbidol and other approved drugs, including aripiprazole, amodiaquine, sertraline, and niclosamide, also inhibit infection of cells by infectious PICV, and arbidol, sertraline, and niclosamide inhibit infectious LASV. Combining arbidol with aripiprazole or sertraline results in the synergistic suppression of LASV and JUNV GP-bearing pseudoviruses. This proof-of-concept study shows that arenavirus infection in vitro can be synergistically inhibited by combinations of approved drugs. This approach may lead to a proactive strategy with which to prepare for and control known and new arenavirus outbreaks.

Biological Characterization of Conserved Residues within the Cytoplasmic Tail of the Pichinde Arenaviral Glycoprotein Subunit 2 (GP2).

Several mammarenaviruses can cause deadly hemorrhagic fever infections in humans, with limited preventative and therapeutic measures available. Arenavirus cell entry is mediated by the viral glycoprotein (GP) complex, which consists of the stable signal peptide (SSP), the receptor-binding subunit GP1, and the transmembrane subunit GP2. The GP2 cytoplasmic tail (CT) is relatively conserved among arenaviruses and is known to interact with the SSP to regulate GP processing and membrane fusion, but its biological role in the context of an infectious virus has not been fully characterized. Using a Pichinde virus (PICV) GP expression vector and a PICV reverse genetics system, we systematically characterized the functional roles of 12 conserved residues within the GP2 CT in GP processing, trafficking, assembly, and fusion, as well as in viral replication. Except for P478A and K505A R508A, alanine substitutions at conserved residues abolished GP processing and membrane fusion in plasmid-transfected cells. Six invariant H and C residues and W503 are essential for viral replication, as evidenced by the fact that their mutant viruses could not be rescued. Both P480A and R482A mutant viruses were rescued, grew similarly to wild-type (WT) virus, and produced evidently processed GP1 and GP2 subunits in virus-infected cells, despite the fact that the same mutations abolished GP processing and membrane fusion in a plasmid-based protein expression system, illustrating the importance of using an infectious-virus system for analyzing viral glycoprotein function. In summary, our results demonstrate an essential biological role of the GP2 CT in arenavirus replication and suggest it as a potential novel target for developing antivirals and/or attenuated viral vaccine candidates.IMPORTANCE Several arenaviruses, such as Lassa virus (LASV), can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, for which no FDA-approved vaccines or therapeutics are available. Viral entry is mediated by the arenavirus GP complex, which consists of the stable signal peptide (SSP), the receptor-binding subunit GP1, and the transmembrane subunit GP2. The cytoplasmic tail (CT) of GP2 is highly conserved among arenaviruses, but its functional role in viral replication is not completely understood. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we show that the GP2 CT contains certain conserved residues that are essential for virus replication, implicating it as a potentially good target for developing antivirals and live-attenuated viral vaccines against deadly arenavirus pathogens.

Intervertebral disc degeneration associated with vertebral marrow fat, assessed using quantitative magnetic resonance imaging.

OBJECTIVE: To investigate the potential clinical application of quantitative MRI in assessing the correlation between lumbar vertebrae bone marrow fat deposition and intervertebral disc degeneration. MATERIALS AND METHODS: A total of 104 chronic lower-back pain volunteers underwent 3.0-T MRI with T2-weighted imaging, T2 mapping, and iterative decomposition of water and fat with echo asymmetry and least squares estimation (IDEAL-IQ) between August 2018 and June 2019. Each disc was assessed with T2 value by T2 mapping, and the L1-S1 vertebral bone marrow fat fraction was assessed by IDEAL-IQ. The differences and relationship between T2 value and the adjacent vertebral bone marrow fat fraction values within the five Pfirrmann groups, five age groups, and five lumbar levels were statistically analyzed. RESULTS: The vertebral bone marrow fat fraction had a significant negative correlation with T2 values of nucleus pulposus' T2 values (p < 0.001). However, the significant negative correlation was only found between T2 values of nucleus pulposus and adjacent vertebral bone marrow fat in Pfirrmann II-III, L1/2-L5/S1 level, and 40-49 years' age groups. Pfirrmann grades of the intervertebral disc were positively correlated with adjacent vertebrae bone marrow fat fraction (p < 0.05). CONCLUSION: Lumbar bone marrow fat deposition significantly increases during the early stages of intervertebral disc degeneration. Quantitative measurements of bone marrow fat deposition and water content of intervertebral discs have a predictive value and are an important supplement to the qualitative traditional classification strategies for the early stages of intervertebral disc degeneration.

Combination of Azithromycin and Gentamicin for Efficient Treatment of Pseudomonas aeruginosa Infections.

BACKGROUND: Trans-translation is a ribosome rescue system that plays an important role in bacterial tolerance to environmental stresses. It is absent in animals, making it a potential treatment target. However, its role in antibiotic tolerance in Pseudomonas aeruginosa remains unknown. METHODS: The role and activity of trans-translation during antibiotic treatment were examined with a trans-translation-deficient strain and a genetically modified trans-translation component gene, respectively. In vitro assays and murine infection models were used to examine the effects of suppression of trans-translation. RESULTS: We found that the trans-translation system plays an essential role in P. aeruginosa tolerance to azithromycin and multiple aminoglycoside antibiotics. We further demonstrated that gentamicin could suppress the azithromycin-induced activation of trans-translation. Compared with each antibiotic individually, gentamicin and azithromycin combined increased the killing efficacy against planktonic and biofilm-associated P. aeruginosa cells, including a reference strain PA14 and its isogenic carbapenem-resistance oprD mutant, the mucoid strain FRD1, and multiple clinical isolates. Furthermore, the gentamicin-azithromycin resulted in improved bacterial clearance in murine acute pneumonia, biofilm implant, and cutaneous abscess infection models. CONCLUSIONS: Combination treatment with gentamicin and azithromycin is a promising strategy in combating P. aeruginosa infections.

Arenaviral Nucleoproteins Suppress PACT-Induced Augmentation of RIG-I Function To Inhibit Type I Interferon Production.

RIG-I is a major cytoplasmic sensor of viral pathogen-associated molecular pattern (PAMP) RNA and induces type I interferon (IFN) production upon viral infection. A double-stranded RNA (dsRNA)-binding protein, PACT, plays an important role in potentiating RIG-I function. We have shown previously that arenaviral nucleoproteins (NPs) suppress type I IFN production via their RNase activity to degrade PAMP RNA. We report here that NPs of arenaviruses block the PACT-induced enhancement of RIG-I function to mediate type I IFN production and that this inhibition is dependent on the RNase function of NPs, which is different from that of a known mechanism of other viral proteins to abolish the interaction between PACT and RIG-I. To understand the biological roles of PACT and RIG-I in authentic arenavirus infection, we analyze growth kinetics of recombinant Pichinde virus (PICV), a prototypical arenavirus, in RIG-I knockout (KO) and PACT KO mouse embryonic fibroblast (MEF) cells. Wild-type (WT) PICV grew at higher titers in both KO MEF lines than in normal MEFs, suggesting the important roles of these cellular proteins in restricting virus replication. PICV carrying the NP RNase catalytically inactive mutation could not grow in normal MEFs but could replicate to some extent in both KO MEF lines. The level of virus growth was inversely correlated with the amount of type I IFNs produced. These results suggest that PACT plays an important role in potentiating RIG-I function to produce type I IFNs in order to restrict arenavirus replication and that viral NP RNase activity is essential for optimal viral replication by suppressing PACT-induced RIG-I activation.IMPORTANCE We report here a new role of the nucleoproteins of arenaviruses that can block type I IFN production via their specific inhibition of the cellular protein sensors of virus infection (RIG-I and PACT). Our results suggest that PACT plays an important role in potentiating RIG-I function to produce type I IFNs in order to restrict arenavirus replication. This new knowledge can be exploited for the development of novel antiviral treatments and/or vaccines against some arenaviruses that can cause severe and lethal hemorrhagic fever diseases in humans.

The bifurcation of periodic orbits and equilibrium points in the linked restricted three-body problem with parameter ω.

This paper is devoted to the bifurcation of periodic orbits and libration points in the linked restricted three-body problem (LR3BP). Inherited from the classic circular restricted three-body problem (CR3BP), it retains most of the dynamical structure of CR3BP, while its dynamical flow is dominated by angular velocity ω and Jacobi energy C. Thus, for the first time, the influence of the angular velocity in the three-body problem is discussed in this paper based on ω-motivated and C-motivated bifurcation. The existence and collision of equilibrium points in the LR3BP are investigated analytically. The dynamic bifurcation of the LR3BP under angular velocity variation is obtained based on three typical kinds of periodic orbits, i.e., planar and vertical Lyapunov orbits and Halo orbits. More bifurcation points are supplemented to Doedel's results in the CR3BP for a global sketch of bifurcation families. For the first time, a new bifurcation phenomenon is discovered that as ω approaches to 1.4, two period-doubling bifurcation points along the Halo family merge together. It suggests that the number and the topological type of bifurcation points themselves can be altered when the system parameter varies in LR3BP. Thus, it is named as "bifurcation of bifurcation" or "secondary bifurcation" in this paper. At selected values of ω, the phase space structures of equilibrium points L2 and L3 are revealed by Lie series method numerically, presenting the center manifolds on the Poincaré section and detecting three patterns of evolution for center manifolds in LR3BP.

Development and application of a real-time polymerase chain reaction assay for detection of a novel gut bacteriophage (crAssphage).

crAssphage is a novel and by far the most abundant bacteriophage in human gut. This bacteriophage might modulate gut microbiota balance so as to be involved in some diseases like obesity, diabetes, metabolic disorders, hypertension, and cancer. Therefore, a rapid and reliable detection and quantification method for crAssphage is essential for studying its molecular epidemiology and pathogenicity in human diseases. The primers-probes set for the quantitative real-time PCR assay was designed based on the DNA polymerase gene (ORF00018) of crAssphage. The sensitivity and specificity, as well as comparison testing with the conventional PCR and sequencing were evaluated. The assay could specifically detect crAssphage, and no cross-reactions with other gut microbes were observed. The detection limit was 15.6 copies/µL of clinical samples (46.8 copies/reaction). When using clinical samples, the assay showed higher ability to detect samples with low viral DNA copies and had an agreement of 93.33% when compared with the conventional PCR amplification and sequencing. The established real-time PCR assay is a sensitive, specific, and repeatable method for quantitatively detecting crAssphage, and thus is a very useful tool for investigating the molecular epidemiology, dynamics, and pathogenicity of crAssphage in human diseases.

Comparison of rRNA-based and DNA-based nucleic acid amplifications for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Ureaplasma urealyticum in urogenital swabs.

BACKGROUND: Nucleic acid amplification tests (NAAT) are well-accepted in diagnosis and surveillance of sexually infectious pathogens worldwide. However, performance differences between a RNA-based NAAT and DNA-based NAAT are rarely reported. This study compares the performances of the RNA-based SAT (simultaneous amplification and testing) assay and the DNA-based quantitative real-time polymerase chain reaction (qPCR) assay. METHODS: A total of 123 urogenital swabs were collected from outpatients with suspected genital infections in our hospital. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) in these swabs were simultaneously tested by SAT and qPCR. Any swabs were positive in the qPCR assay were further verified by following cloning and sequencing. All statistical analysis was performed using the SPSS software. RESULTS: When the concentrations of CT, NG, or UU were more than 1 × 103 copies/ml, 100% agreements between SAT and qPCR were observed regardless of the pathogen. No discrepancy was found. However, the sensitivity of SAT is significantly higher than qPCR in samples with concentration less than 1 × 103 copies/ml. When tested by SAT and qPCR, 57.14 and 28.57% were positive for CT, 46.15% and 0 were positive for NG, 80% and 0 were positive for UU, respectively. CONCLUSIONS: The SAT assay has better agreements and higher sensitivities when compared with the qPCR assay, and thus could be a better choice for screening, diagnosis, and surveillance of sexually transmitted diseases, especially for CT and NG.

Characterization of the Glycoprotein Stable Signal Peptide in Mediating Pichinde Virus Replication and Virulence.

Arenaviruses can cause lethal hemorrhagic fevers in humans with few preventative and therapeutic measures. The arenaviral glycoprotein stable signal peptide (SSP) is unique among signal peptides in that it is an integral component of the mature glycoprotein complex (GPC) and plays important roles not only in GPC expression and processing but also in the membrane fusion process during viral entry. Using the Pichinde virus (PICV) reverse genetics system, we analyzed the effects of alanine substitutions at many conserved residues within the SSP on viral replication in cell culture and in a guinea pig infection model. Our data showed that the K33A, F49A, and C57A mutations abolished GPC-mediated cell entry and therefore could not allow for the generation of viable recombinant viruses, demonstrating that these residues are essential for the PICV life cycle. The G2A mutation caused a marked reduction of cell entry at the membrane fusion step, and while this mutant virus was viable, it was significantly attenuated in vitro and in vivo The N20A mutation also reduced membrane fusion activity and viral virulence in guinea pigs, but it did not significantly affect cell entry or viral growth in cell culture. Two other mutations (N37A and R55A) did not affect membrane fusion or viral growth in vitro but significantly reduced viral virulence in vivo Taken together, our data suggest that the GPC SSP plays an essential role in mediating viral entry and also contributes to viral virulence in vivo IMPORTANCE: Several arenaviruses, such as Lassa fever virus, can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, and no FDA-approved vaccines or therapies are currently available. Viral entry into cells is mediated by arenavirus GPC that consists of an SSP, the receptor-binding GP1, and transmembrane GP2 protein subunits. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we have shown for the first time in the context of virus infections of cell culture and of guinea pigs that the SSP plays an essential role in mediating the membrane fusion step as well as in other yet-to-be-determined processes during viral infection. Our study provides important insights into the biological roles of GPC SSP and implicates it as a good target for the development of antivirals against deadly human arenavirus pathogens.

Clonal replacement of epidemic KPC-producing Klebsiella pneumoniae in a hospital in China.

BACKGROUND: Klebsiella pneumoniae is a frequent nosocomial pathogen causing difficult-to-treat infections worldwide. The prevalence of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-KP) is increasing in China. The aim of this study was to investigate the molecular epidemiology of KPC-KP in a nosocomial outbreak. METHODS: Fifty-four KPC-KP isolates were consecutively collected between November 2013 and August 2014 during a KPC-KP outbreak in a tertiary care hospital in Beijing, China. Antimicrobial susceptibility was determined by agar dilution. Carbapenemase, extended-spectrum ß-lactamase, 16S rRNA methylase, AmpC ß-lactamase, and plasmid-mediated quinolone resistance determinants were detected by PCR amplification. The genetic relatedness of isolates was analyzed by pulsed-field gel electrophoresis and multi-locus sequence typing. RESULTS: All isolates belonged to ST11 except one isolate which was identified as a new sequence type (ST2040). PFGE profile of genomic DNA revealed seven clusters, of which cluster A and C dominated the KPC-KP outbreak and cluster A was replaced by cluster C during the outbreak. PFGE of genomic DNA, S1-PFGE of plasmids, replicon typing, and drug resistant characteristics showed that clonal spread occurred during the outbreak. When compared with isolates within cluster A, all isolates in cluster C harbored rmtB and showed higher level of resistance to cefepime, amikacin, tobramycin, and tigecycline. CONCLUSION: We reported a nosocomial outbreak of KPC-KP with clonal replacement and a new sequence type (ST2040) of KP. High degree of awareness and surveillance of KPC-KP should be given to avoid potential outbreaks, especially in ICU wards.