Dopamine inhibits group 2 innate lymphoid cell-driven allergic lung inflammation by dampening mitochondrial activity.

Neuronal signals have emerged as pivotal regulators of group 2 innate lymphoid cells (ILC2s) that regulate tissue homeostasis and allergic inflammation. The molecular pathways underlying the neuronal regulation of ILC2 responses in lungs remain to be fully elucidated. Here, we found that the abundance of neurotransmitter dopamine was negatively correlated with circulating ILC2 numbers and positively associated with pulmonary function in humans. Dopamine potently suppressed lung ILC2 responses in a DRD1-receptor-dependent manner. Genetic deletion of Drd1 or local ablation of dopaminergic neurons augmented ILC2 responses and allergic lung inflammation. Transcriptome and metabolic analyses revealed that dopamine impaired the mitochondrial oxidative phosphorylation (OXPHOS) pathway in ILC2s. Augmentation of OXPHOS activity with oltipraz antagonized the inhibitory effect of dopamine. Local administration of dopamine alleviated allergen-induced ILC2 responses and airway inflammation. These findings demonstrate that dopamine represents an inhibitory regulator of ILC2 responses in allergic airway inflammation.

The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency.

Newcastle disease virus (NDV) has been extensively studied as a promising oncolytic virus for killing tumor cells in vitro and in vivo in clinical trials. However, the viral components that regulate the oncolytic activity of NDV remain incompletely understood. In this study, we systematically compared the replication ability of different NDV genotypes in various tumor cells and identified NP protein determines the oncolytic activity of NDV. On the one hand, NDV strains with phenylalanine (F) at the 450th amino acid position of the NP protein (450th-F-NP) exhibit a loss of oncolytic activity. This phenotype is predominantly associated with genotype VII NDVs. In contrast, the NP protein with a leucine amino acid at this site in other genotypes (450th-L-NP) can facilitate the loading of viral mRNA onto ribosomes more effectively than 450th-F-NP. On the other hand, the NP protein from NDV strains that exhibit strong oncogenicity interacts with eIF4A1 within its 366-489 amino acid region, leading to the inhibition of cellular mRNA translation with a complex 5' UTR structure. Our study provide mechanistic insights into how highly oncolytic NDV strains selectively promote the translation of viral mRNA and will also facilitate the screening of oncolytic strains for oncolytic therapy.

Intervertebral Disk Degeneration and Bone Mineral Density: A Bidirectional Mendelian Randomization Study.

This study aimed to investigate the causal relationship between bone mineral density (BMD) and intervertebral disk degeneration (IVDD) using a two-sample bidirectional Mendelian randomization analysis. Summary-level data from the Genome-Wide Association Study (GWAS) were used. Instrumental variables (IVs) for IVDD were selected from the large-scale Genome-Wide Association Study (GWAS) (20,001 cases and 164,682 controls). Bone mineral density (BMD) at five different sites (heel (n = 426,824), total body (TB) (n = 56,284), forearm (FA) (n = 8143), femoral neck (FN) (n = 32,735), and lumbar spine (LS) (n = 28,498)) was used as a phenotype for OP. Bidirectional causality between IVDD and BMD was assessed using inverse variance weighting (IVW) and other methods. Related sensitivity analyses were performed. Myopia was also analyzed as a negative control result to ensure the validity of IVs. Heel bone mineral density (heel BMD), total body bone mineral density (TB-BMD), femoral neck bone mineral density (FN-BMD), and lumbar spine bone mineral density (LS-BMD) have a direct causal relationship on intervertebral disk degeneration (IVDD) [heel BMD-related analysis: beta = 0.06, p = 0.03; TB-BMD-related analysis: beta = 0.18, p = 8.72E-08; FN-BMD-related analysis: beta = 0.15, p = 4.89E-03; LS-BMD-related analysis: beta = 0.16, p = 1.43E-04]. There was no evidence of a significant causal effect of IVDD on BMD. In conclusion, our study found a significant positive causal effect of lower BMD on IVDD, and we identified significant causal effects of heel, TB-, FN-, and LS-BMD on IVDD, but there was no evidence of a significant causal effect of IVDD on BMD.

Adenosine Alleviates Necrotizing Enterocolitis by Enhancing the Immunosuppressive Function of Myeloid-Derived Suppressor Cells in Newborns.

Necrotizing enterocolitis (NEC) is a common disorder in premature infants that is characterized by hyperinflammation and severe necrosis in the intestine. The pathogenesis of NEC remains to be elucidated. In this study, we demonstrate that adenosine, a metabolite more abundant in infants than in adults, plays an important role in the prevention of NEC. Administration of adenosine or its analog, adenosine-5'-N-ethyluronamide (NECA), dramatically relieved the severity of NEC in neonatal mice. Meanwhile, adenosine treatment significantly enhanced the immunosuppressive function, antibacterial activity, and migration of myeloid-derived suppressor cells (MDSCs). However, depletion of MDSCs or inhibition of their migration using the CXCR2 inhibitor SB225002 almost completely abrogated the protective effect of adenosine on NEC. Mechanistic studies showed that MDSCs in newborns expressed abundant adenosine receptor A2B (A2BR) that elicits intracellular cAMP signaling and its downstream target NF-κB. Importantly, intestinal tissues from patients with NEC showed significantly lower infiltration of A2BR-positive MDSCs than those from healthy donors. These observations revealed that adenosine-induced MDSCs represent an essential immune axis for intestinal homeostasis in newborns.

Critical Role of Intestinal Microbiota in ATF3-Mediated Gut Immune Homeostasis.

Secretory Ig A (sIgA) plays an important role in the maintenance of intestinal homeostasis via cross-talk with gut microbiota. The defects in sIgA production could elicit dysbiosis of commensal microbiota and subsequently facilitate the development of inflammatory bowel disease. Our previous study revealed activating transcription factor 3 (ATF3) as an important regulator of follicular helper T (TFH) cells in gut. ATF3 deficiency in CD4+ T cells impaired the development of gut TFH cells, and therefore diminished sIgA production, which increased the susceptibility to murine colitis. However, the potential role of microbiota in ATF3-mediated gut homeostasis remains incompletely understood. In this study, we report that both Atf3-/- and CD4creAtf3fl/fl mice displayed profound dysbiosis of gut microbiota when compared with their littermate controls. The proinflammatory Prevotella taxa, especially Prevotella copri, were more abundant in ATF3-deficient mice when compared with littermate controls. This phenotype was obviously abrogated by adoptive transfer of either TFH cells or IgA+ B cells. Importantly, depletion of gut microbiota dramatically alleviated the severity of colitis in Atf3-/- mice, whereas transfer of microbiota from Atf3-/- mice to wild-type recipients increased their susceptibility to colitis. Collectively, these observations indicate the importance of IgA-microbiota interaction in ATF3-mediated gut homeostasis.

Multiplex one-step real-time PCR assay for rapid simultaneous detection of velogenic and mesogenic Newcastle disease virus and H5-subtype avian influenza virus.

H5 avian influenza virus (AIV) and velogenic Newcastle disease virus (v-NDV) are pathogens listed in the OIE Terrestrial Animal Health Code and are considered key pathogens to be eliminated in poultry production. Molecular techniques for rapid detection of H5 AIV and v-NDV are required to investigate their transmission characteristics and to guide prevention. Traditional virus isolation, using embryonated chicken eggs, is time-consuming and cannot be used as a rapid diagnostic technology. In this study, a multiplex real-time RT-PCR (RRT-PCR) detection method for six H5 AIV clades, three v-NDV subtypes, and one mesogenic NDV subtype was successfully established. The detection limit of our multiplex NDV and H5 AIV RRT-PCR was five copies per reaction for each pathogen, with good linearity and efficiency (y = -3.194x + 38.427 for H5 AIV and y = -3.32x + 38.042 for NDV). Multiplex PCR showed good intra- and inter-assay reproducibility, with coefficient of variance (CV) less than 1%. Furthermore, using the RRT-PCR method, H5 AIV and NDV detection rates in clinical samples were higher overall than those obtained using the traditional virus isolation method. Therefore, our method provides a promising technique for surveillance of various H5 AIV clades and multiple velogenic and mesogenic NDV subtypes in live-poultry markets.

Ultrasensitive and rapid count of Escherichia coli using magnetic nanoparticle probe under dark-field microscope.

BACKGROUND: Escherichia coli (E. coli) is one of the best-known zoonotic bacterial species, which pathogenic strain can cause infections in humans and animals. However, existing technologies or methods are deficient for quickly on-site identifying infection of E. coli before they breakout. Herein, we present an ultrasensitive and on-site method for counting E. coli using magnetic nanoparticle (MNP) probe under a dark-field in 30 min. RESULTS: The antibodies functionalized MNP, binding to E. coli to form a golden ring-like structure under a dark-field microscope, allowing for counting E. coli. This method via counting MNP-conjugated E. coli under dark-field microscope demonstrated the sensitivity of 6 CFU/µL for E. coli detection. Importantly, due to the advantages such as time-saving (only 30 min) and almost free of instrument (only require a portable microscope), our MNP-labeled dark-field counting strategy has the potential of being a universal tool for on-site quantifying a variety of pathogens with size ranges from a few hundreds of nanometers to a few micrometers. CONCLUSION: In summary, the MNP-labeled dark-field counting strategy is a rapid, simple, sensitive as well as low-cost assay strategy, which has the potential of being a universal tool for on-site quantification of micrometer-size pathogens like E. coli.

Characterization of the nuclear import pathway for BLM protein.

Numerous studies have shown that nuclear localization of BLM protein, a member of the RecQ helicases, mediated by nuclear localization signal (NLS) is critical for DNA recombination, replication and transcription, but the mechanism by which BLM protein is imported into the nucleus remains unknown. In this study, the nuclear import pathway for BLM was investigated. We found that nuclear import of BLM was inhibited by two dominant-negative mutants of importin ß1 and NTF2/E42K, which lacks the ability to bind Ran and RanGDP, respectively, but was not inhibited by the Ran/Q69L, which is deficient in GTP hydrolysis. Further studies revealed that nuclear import of BLM was reconstituted using importin ß1, RanGDP and NTF2 in digitonin-permeabilized HeLa cells. Moreover, BLM had direct binding to importin ß1 through its NLS domain with the 14-16 HEAT repeats of importin ß1. Furthermore, importin ß1, Ran or NTF2 depletion by siRNA disrupted the accumulation of BLM protein in the nucleus. These results showed that BLM enters the nucleus via the importin ß1, RanGDP and NTF2 dependent pathway, demonstrating for the first time the nuclear trafficking mechanism of a DNA helicase.

The T160A hemagglutinin substitution affects not only receptor binding property but also transmissibility of H5N1 clade 2.3.4 avian influenza virus in guinea pigs.

We generated and characterized site-directed HA mutants on the genetic backbone of H5N1 clade 2.3.4 virus preferentially binding to α-2,3 receptors in order to identify the key determinants in hemagglutinin rendering the dual affinity to both α-2,3 (avian-type) and α-2,6 (human-type) linked sialic acid receptors of the current clade 2.3.4.4 H5NX subtype avian influenza reassortants. The results show that the T160A substitution resulted in the loss of a glycosylation site at 158N and led not only to enhanced binding specificity for human-type receptors but also transmissibility among guinea pigs, which could be considered as an important molecular marker for assessing pandemic potential of H5 subtype avian influenza isolates.

[The nuclear import of Newcastle disease virus matrix protein depends on KPNB1 and Ran protein].

Objective: The aim of this study was to identify the transport proteins that mediates the nuclear import of Newcastle disease virus (NDV) matrix (M) protein. Methods: Chicken KPNA1 to KPNA6 gene and KPNB1 gene were cloned from DF-1 cells and then inserted into eukaryotic expression vectors. The constructed recombinant plasmids with a combination of grouping were transfected into HEK-293T cells to identify the transport proteins interacting with NDV M protein by co-immunoprecipitation (Co-IP) assay. Moreover, fluorescent co-localization assay was used to verify the transport proteins by co-expressing M and Ran protein mutant or M and its interactive protein deletant. Results: The recombinant proteins could normally express in plasmid-transfected HEK-293T cells. Indirect immunofluorescence detection showed that the recombinant proteins except for Myc-KPNA2 displayed the same nuclear localization as NDV M protein. The results of Co-IP revealed that M protein could interact with KPNA1 and KPNB1. Further fluorescent co-localization indicated that co-expression of M and DN-KPNA1 did not change the nuclear localization of M, whereas co-expression of M and DN-KPNB1 or M and Ran-Q69L disrupted the nuclear localization of M, demonstrating that the nuclear import of M protein was dependent on KPNB1 and Ran protein. Conclusion: KPNB1 and Ran protein jointly mediated the nuclear import of NDV M protein, showing that KPNB1 protein interacted with NDV M protein to form binary complex and then entered into the nucleus with the assistance of Ran protein.

A single R36Q mutation in the matrix protein of pigeon paramyxovirus type 1 reduces virus replication and shedding in pigeons.

Pigeon paramyxovirus type 1 (PPMV-1) is considered an antigenic and variant of avian paramyxovirus type 1 (APMV-1) that has adapted to pigeons as hosts. However, how this host-specific adaption of PPMV-1 is related to its biological characteristics is unknown. In this study, seven unique amino acids in PPMV-1 that are not present in other APMV-1 strains (n = 39 versus n = 106) were identified. R36 of the M protein was found to be not only a unique amino acid but also a positive-selection site. To investigate the role of R36 in host adaptation, a recombinant PPMV-1 with R36Q mutation was constructed. Our results indicated that the an R36Q mutation significantly attenuates pathogenicity in chickens, viral growth in both chicken embryo fibroblasts (CEFs) and pigeon embryo fibroblasts (PEFs), and virus replication and shedding in pigeons in comparison with the wild-type virus, suggesting that R36 is a key residue that evolved during the adaptation of PPMV-1 in pigeons.

Simultaneous mutation of G275A and P276A in the matrix protein of Newcastle disease virus decreases virus replication and budding.

The matrix (M) protein of Newcastle disease virus (NDV) is a highly conserved hydrophobic viral protein. In some paramyxoviruses (measles virus and Sendai virus), the paired glycine (G) near the C terminus of the M protein may form a turn that mediates the specific interaction with the cell membrane. Similar amino acids (glycine-proline [GP], at position 275-276) exist in the M protein of NDV. However, the role of these residues in the replication and pathogenicity of NDV is unknown. In this study, recombinant NDV with the sequence GP/AA or LGP/GGL in the M protein was generated to investigate the role of this conserved sequence. Budding experiments on the mutant viruses revealed that the GP/AA mutation reduced virus budding and virus replication in DF-1 cells; biological characterization revealed attenuated virulence and pathogenicity in chickens, indicating that the GP sequence plays a critical role in the life cycle of the virus.

Characterization of virulent Newcastle disease viruses from vaccinated chicken flocks in Eastern China.

BACKGROUND: Newcastle disease (ND) is one of the most contagious and devastating diseases to poultry in the world. The causative agents are virulent strains of Newcastle disease virus (NDV), which belong to the genus Avulavirus, sub-family Paramyxoviridae, family Paramyxovirinae. Knowing the genomic and antigenic characteristics of virulent NDVs might contribute to ND control in China. RESULTS: The results showed that all of the virulent strains belonged sub-genotype VIId shared the same cleavage site (112)RRQKR/F(117) in the fusion protein. At least 69 % (38 of 55) of the NDV strains possessed E347K variation in the hemagglutinin-neuraminidase protein. The cross-neutralization tests confirmed that the strains harboring 347 K showed lower antigenic relatedness with LaSota. Furthermore, the immune-challenge experiment indicated that LaSota could not provide complete protection against infection with the E347K variant NDVs as the vaccinated birds were still able to be infected and shed virulent challenge viruses. CONCLUSIONS: Currently, sub-genotype VIId NDVs are the prevalent virulent strains circulating among vaccinated chicken flocks in Eastern China. Our findings indicated that the E347K variation in HN gene would expand the antigenic difference with LaSota, which may be responsible for the increasing isolation rate of these strains from vaccinated chickens.

The M, F and HN genes of genotype VIId Newcastle disease virus are associated with the severe pathological changes in the spleen of chickens.

BACKGROUND: The strains of the genotype VIId Newcastle disease virus (NDV) induce more severe tissue damage in lymphoid organs than other virulent strains. The underlying molecular mechanisms are poorly understood. METHODS: Genotype IV NDV Herts/33 and genotype VIId NDV JS5/05 have a distinctive pathological profile in the spleen. These two strains of viruses were selected as parental viruses to generate a panel of chimeric viruses by replacing the M, F and HN genes of Herts/33 individually or in combination with the corresponding genes of JS5/05 using reverse genetic. Virulence and in vitro characteristics of the recombinant viruses were assessed. In addition, pathological changes, virus load, and transcriptional cytokine response in the spleen of chickens infected with these recombinant viruses were also analyzed. RESULTS: Pathogenicity test showed that all chimeric viruses are virulent. In vitro characterization revealed that gene replacement did not change growth kinetics and HN expression on cell surface of the recombinant viruses. However, replacement of the M, F and HN genes resulted in apparent changes in the fusion activity. Moreover, pathological studies revealed that only inclusion of the homologous M, F and HN genes of JS5/05 in Herts/33 backbone resulted in severe pathological changes characterized by extensive necrosis in the spleen, similar to that induced by JS5/05. In addition, this gene replacement significantly increased virus replication and the levels of transcriptional cytokine response, compared to Herts/33. Conversely, inclusion of the M, F and HN genes of Herts/33 into JS5/05 backbone resulted in Herts/33-specific pathological changes and significantly decreased virus load and the expression levels of cytokine genes, compared to JS5/05. CONCLUSIONS: The M, F and HN genes are related to the severe pathological changes in the spleen of chickens infected with genotype VIId NDV.

A single amino acid mutation, R42A, in the Newcastle disease virus matrix protein abrogates its nuclear localization and attenuates viral replication and pathogenicity.

The Newcastle disease virus (NDV) matrix (M) protein is a highly basic and nucleocytoplasmic shuttling viral protein. Previous study has demonstrated that the N-terminal 100 aa of NDV M protein are somewhat acidic overall, but the remainder of the polypeptide is strongly basic. In this study, we investigated the role of the N-terminal basic residues in the subcellular localization of M protein and in the replication and pathogenicity of NDV. We found that mutation of the basic residue arginine (R) to alanine (A) at position 42 disrupted M's nuclear localization. Moreover, a recombinant virus with R42A mutation in the M protein reduced viral replication in DF-1 cells and attenuated the virulence and pathogenicity of the virus in chickens. This is the first report to show that a basic residue mutation in the NDV M protein abrogates its nuclear localization and attenuates viral replication and pathogenicity.

Two amino acid substitutions in the haemagglutinin of the 2009 pandemic H1N1 virus decrease direct-contact transmission in guinea pigs.

The 2009 pandemic H1N1 influenza A virus spread across the globe and caused the first influenza pandemic of the 21st century. Many of the molecular factors that contributed to the airborne transmission of this pandemic virus have been determined; however, the direct-contact transmission of this virus remains poorly understood. In this study, we report that a combination of two mutations (N159D and Q226R) in the haemagglutinin (HA) protein of the representative 2009 H1N1 influenza virus A/California/04/2009 (CA04) caused a switch in receptor binding preference from the α2,6-sialoglycan to the α2,3-sialoglycan receptor, and decreased the binding intensities for both glycans. In conjunction with a significantly decreased replication efficiency in the nasal epithelium, this limited human receptor binding affinity resulted in inefficient direct-contact transmission of CA04 between guinea pigs. Our findings highlight the role of the HA gene in the transmission of the influenza virus.

Surveillance of avirulent Newcastle disease viruses at live bird markets in Eastern China during 2008-2012 reveals a new sub-genotype of class I virus.

BACKGROUND: The strains of Newcastle disease virus (NDV) can be divided into two distinct clades: class I and class II. At present, limited molecular epidemiological data are available for the class I virus at live bird markets (LBMs). Knowing the genomic and antigenic characteristics of class I NDVs might provide important insights into the evolution dynamics of these viruses. In this study class I NDVs isolated from LBMs in Eastern China between 2008 and 2012 were characterized. RESULTS: We characterized 34 class I NDVs genetically and 15 of the 34 NDVs pathologically which originated from geese, chickens and ducks at live bird markets. Based on the older classification system, twelve of fourteen strains isolated from 2008 to 2010 belonged to sub-genotype 3b. However, the rest 22 strains formed a separate novel cluster in genotype 3, which was designated as sub-genotype 3c. When based on the new classification system, sub-genotype 3b was classified into sub-genotype 1a and the sub-genotype 3c was classified into sub-genotype 1b. Over 62% (21/34) of the viruses were chicken-origin and only 13 isolates were waterfowl-origin. The Cross-neutralization reactions between CK/JS/05/11, CK/JS/06/12 and the vaccine strain LaSota showed significant antigenic differences between them. CONCLUSIONS: Currently, sub-genotype 3c (or 1b) NDVs are the most frequently isolated classI strains at LBMs in Eastern China., and the class I NDVs has transferred from waterfowls to chickens and circulated in chicken flocks extensively.

Mutations in the FPIV motif of Newcastle disease virus matrix protein attenuate virus replication and reduce virus budding.

The FPIV-like late domains identified in the matrix (M) proteins of parainfluenza virus 5 and mumps virus have been demonstrated to be critical for virus budding. In this study, we found that the same FPIV sequence motif is present in the N-terminus of the Newcastle disease virus (NDV) M protein. Mutagenesis experiments demonstrated that mutation of either phenylalanine (F) or proline (P) to alanine led to a more obvious decrease in viral virulence and replication and resulted in poor budding of the mutant viruses. Additionally, evidence for the involvement of cellular multivesicular body (MVB) proteins was obtained, since NDV production was inhibited upon expression of dominant-negative versions of the VPS4A-E228Q protein. Together, these results demonstrate that the FPIV motif, especially the residues F and P, within the NDV M protein, plays a critical role in NDV replication and budding, and this budding process likely involves the cellular MVB pathway.

Differences in transmissibility and pathogenicity of reassortants between H9N2 and 2009 pandemic H1N1 influenza A viruses from humans and swine.

Both H9N2 subtype avian influenza and 2009 pandemic H1N1 viruses (pH1N1) can infect humans and pigs, which provides the opportunity for virus reassortment, leading to the genesis of new strains with potential pandemic risk. In this study, we generated six reassortant H9 viruses in the background of three pH1N1 strains from different hosts (A/California/04/2009 [CA04], A/Swine/Jiangsu/48/2010 [JS48] and A/Swine/Jiangsu/285/2010 [JS285]) by replacing either the HA (H9N1-pH1N1) or both the HA and NA genes (H9N2-pH1N1) from an h9.4.2.5-lineage H9N2 subtype influenza virus, A/Swine/Taizhou/5/08 (TZ5). The reassortant H9 viruses replicated to higher titers in vitro and in vivo and gained both efficient transmissibility in guinea pigs and increased pathogenicity in mice compared with the parental H9N2 virus. In addition, differences in transmissibility and pathogenicity were observed among these reassortant H9 viruses. The H9N2-pH1N1viruses were transmitted more efficiently than the corresponding H9N1-pH1N1 viruses but showed significantly decreased pathogenicity. One of the reassortant H9 viruses that were generated, H9N-JS48, showed the highest virulence in mice and acquired respiratory droplet transmissibility between guinea pigs. These results indicate that coinfection of swine with H9N2 and pH1N1viruses may pose a threat for humans if reassortment occurs, emphasizing the importance of surveillance of these viruses in their natural hosts.