Critical role of G3BP1 in bovine parainfluenza virus type 3 (BPIV3)-inhibition of stress granules formation and viral replication.

Background: It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Methods: Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. Results: We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. Conclusion: This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.

Designing novel monolayer and multilayer h-CSe crystals with tunable photoelectric properties.

Using the first-principles method, a new structure of monolayer h-CSe was predicted, exhibiting good dynamical and thermal stability. The geometrical, electronic and optical properties of monolayer h-CSe are examined at the HSE level. Furthermore, the influences of the in-plane strain and layer number on the electric properties of the two dimensional h-CSe material are studied. The results indicate that it possesses an indirect band gap, which exhibits a rich variety of behaviors depending on the small in-plane biaxial strain. The band gap of monolayer h-CSe could be easily tuned in the energy range from 0.82 eV to 2.61 eV under small in-plane biaxial strain (from -3% to 3%). Also, a band gap transition between direct and indirect types is not found. The band gap of the h-CSe materials decreases with the increase of their layer number. In addition, it was found that these h-CSe materials show excellent optical properties, including strong light harvesting ability for the ultra-violet light range of the solar spectrum. The results obtained here indicate that monolayer h-CSe may have significant potential applications in future nanoelectronic fields.

Age-related differences in physiological and metabolic responses of Pleione aurita (Orchidaceae) pseudobulbs to drought stress and recovery.

The pseudobulb is a storage organ for water and nutrients that plays a crucial role in the growth and survival of epiphytic orchids. However, the role of water and metabolites in pseudobulb during adaptation to environmental stress are rarely detected through control experiments. In the present study, water-related physiological traits and metabolite changes in the pseudobulbs at the flowering stage and full leaf expansion stage for Pleione aurita were investigated after drought stress and recovery treatments. We found that the composition of non-structural carbohydrates (starch vs. soluble sugar) varied over the lifetime of pseudobulbs, and older pseudobulbs stored more water, whereas younger pseudobulbs stored more dry matter. When plants were subjected to drought stress and subsequent recovery, multiple metabolites in the pseudobulbs including non-structural carbohydrates, flavonoids, phenolic acids, as well as amino acids and their derivatives responded positively to these water level fluctuations. For those metabolites that differently accumulated in both stress and recovery processes, old pseudobulbs contained a higher number of these key metabolites than did the connected younger pseudobulbs. In addition, young and old pseudobulbs use different metabolic pathways to both respond and recover to drought. These results indicate that orchid pseudobulbs cope with water level fluctuations by mobilizing metabolite reserves and that pseudobulbs of different ages exhibit different physiological and metabolic responses to drought stress. These findings broadens our understanding of the role pseudobulbs play in the survival of orchids growing in epiphytic habitats.

Orphan gene PpARDT positively involved in drought tolerance potentially by enhancing ABA response in Physcomitrium (Physcomitrella) patens.

Almost all genomes have orphan genes, the majority of which are not functionally annotated. There is growing evidence showed that orphan genes may play important roles in the environmental stress response of Physcomitrium patens. We identified PpARDT (ABA-responsive drought tolerance) as a moss-specific and ABA-responsive orphan gene in P. patens. PpARDT is mainly expressed during the gametophytic stage of the life cycle, and the expression was induced by different abiotic stresses. A PpARDT knockout (Ppardt) mutant showed reduced dehydration-rehydration tolerance, and the phenotype could be rescued by exogenous ABA. Meanwhile, transgenic Arabidopsis lines exhibiting heterologous expression of PpARDT were more sensitive to exogenous ABA than wild-type (Col-0) plants and showed enhanced drought tolerance. These indicate that PpARDT confers drought tolerance among land plants potentially by enhancing ABA response. Further, we identified genes encoding abscisic acid receptor PYR/PYL family proteins, and ADP-ribosylation factors (Arf) as hub genes associated with the Ppardt phenotype. Given the lineage-specific characteristics of PpARDT, our results provide insights into the roles of orphan gene in shaping lineage-specific adaptation possibly by recruiting common pre-existed pathway components.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum.

The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in Cymbidium lowianum (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

Regulation of the Development in Physcomitrium (Physcomitrella) patens implicates the functional differentiation of plant RNase H1s.

R-loops, consisting of a DNA:RNA hybrid and a single-stranded DNA (ssDNA), form naturally as functional chromosome structures and are crucial in many vital biological processes. However, disrupted R-loop homeostasis will threat to the integrity and stability of genome. As the endonuclease, RNase H1 can efficiently recognize and remove excess R-loops to protect organisms from DNA damage induced by R-loop over-accumulation. Here, we investigated the function of RNase H1 in Physcomitrium (Physcomitrella) patens to illustrate its important role in the evolution of plants. We found that PpRNH1A dysfunction seriously affected shoot growth and branch formation in P. patens, revealing a noticeable functional difference between PpRNH1A and AtRNH1A of Arabidopsis. Furthermore, auxin signaling was significantly affected at the transcriptional level in PpRNH1A mutant plants, as a result of the accumulation of R-loops at several auxin-related genes. This study provides evidence that PpRNH1A regulates the development of P. patens by controlling R-loop formation at specific loci to modulate the transcription of auxin-related genes. It also highlights the interspecific functional differences between early land plants and vascular plants, despite crucial and conserved role of RNase H1 played in maintaining R-loop homeostasis.

An update on the function and regulation of methylerythritol phosphate and mevalonate pathways and their evolutionary dynamics.

Isoprenoids are among the largest and most chemically diverse classes of organic compounds in nature and are involved in the processes of photosynthesis, respiration, growth, development, and plant responses to stress. The basic building block units for isoprenoid synthesis-isopentenyl diphosphate and its isomer dimethylallyl diphosphate-are generated by the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways. Here, we summarize recent advances on the roles of the MEP and MVA pathways in plant growth, development and stress responses, and attempt to define the underlying gene networks that orchestrate the MEP and MVA pathways in response to developmental or environmental cues. Through phylogenomic analysis, we also provide a new perspective on the evolution of the plant isoprenoid pathway. We conclude that the presence of the MVA pathway in plants may be associated with the transition from aquatic to subaerial and terrestrial environments, as lineages for its core components are absent in green algae. The emergence of the MVA pathway has acted as a key evolutionary event in plants that facilitated land colonization and subsequent embryo development, as well as adaptation to new and varied environments.

Comprehensive analysis of the Ppatg3 mutant reveals that autophagy plays important roles in gametophore senescence in Physcomitrella patens.

BACKGROUND: Autophagy is an evolutionarily conserved system for the degradation of intracellular components in eukaryotic organisms. Autophagy plays essential roles in preventing premature senescence and extending the longevity of vascular plants. However, the mechanisms and physiological roles of autophagy in preventing senescence in basal land plants are still obscure. RESULTS: Here, we investigated the functional roles of the autophagy-related gene PpATG3 from Physcomitrella patens and demonstrated that its deletion prevents autophagy. In addition, Ppatg3 mutant showed premature gametophore senescence and reduced protonema formation compared to wild-type (WT) plants under normal growth conditions. The abundance of nitrogen (N) but not carbon (C) differed significantly between Ppatg3 mutant and WT plants, as did relative fatty acid levels. In vivo protein localization indicated that PpATG3 localizes to the cytoplasm, and in vitro Y2H assays confirmed that PpATG3 interacts with PpATG7 and PpATG12. Plastoglobuli (PGs) accumulated in Ppatg3, indicating that the process that degrades damaged chloroplasts in senescent gametophore cells was impaired in this mutant. RNA-Seq uncovered a detailed, comprehensive set of regulatory pathways that were affected by the autophagy mutation. CONCLUSIONS: The autophagy-related gene PpATG3 is essential for autophagosome formation in P. patens. Our findings provide evidence that autophagy functions in N utilization, fatty acid metabolism and damaged chloroplast degradation under non-stress conditions. We identified differentially expressed genes in Ppatg3 involved in numerous biosynthetic and metabolic pathways, such as chlorophyll biosynthesis, lipid metabolism, reactive oxygen species removal and the recycling of unnecessary proteins that might have led to the premature senescence of this mutant due to defective autophagy. Our study provides new insights into the role of autophagy in preventing senescence to increase longevity in basal land plants.

Functional redundancy and divergence of ß-carbonic anhydrases in Physcomitrella patens.

MAIN CONCLUSION: ß-carbonic anhydrases, which function in regulating plant growth, C/N status, and stomata number, showed functional redundancy and divergence in Physcomitrella patens. Carbonic anhydrases (CAs) catalyze the interconversion of CO2 and HCO3-. Plants have three evolutionarily unrelated CA families: α-, ß-, and γ-CAs. ßCAs are abundant in plants and are involved in CO2 assimilation, stress responses, and stomata formation. Recent studies of ßCAs have mainly examined C3 or C4 plants, whereas their functions in non-vascular plants are mostly unknown. In this study, phylogenetic analysis revealed that the evolution of ßCAs were conserved between subaerial green algae and bryophytes after terrestrialization event, and ßCAs from some cyanobacteria might begin evolving for the adaptation of terrestrial environment/habitat. In addition, we investigated the physiological roles of ßCAs in the basal land plant Physcomitrella patens. High PpßCA expression levels in different tissues suggest that PpßCAs play important roles in development in P. patens. Plants treated with 1-10 mM NaHCO3 had higher fresh and dry weight, PpßCA expression, total CA activity, and photosynthetic yield (Fv/Fm) compared with water-treated plants. However, treatment with 10 mM NaHCO3 influenced the C/N status. Further study of six Ppßca single-gene mutants revealed that PpßCAs have functional redundancy and divergence in regulating the C/N ratio of plants and stomatal formation. This study provides new insight into the physiological roles of ßCAs in basal land plants.

Genetic editing of the virulence gene of Escherichia coli using the CRISPR system.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is an emerging gene-editing technology that is widely used in prokaryotes and eukaryotes. It can realize the specific manipulation of the genome efficiently and accurately. CRISPR/Cas9 coupled λ-Red recombination technology was used to perform genome editing in different genes. For finding an efficient method to edit the virulence genes of enterotoxigenic E. coli (ETEC), the two-plasmid system was used. The coding sequence (CDS) region of the estA, eltI, estB, eltIIc1, and faeG locus were deleted. The coding region of estB was substituted with estA. Gene recombination efficiency ranged from 0 to 77.78% when the length of the homology arm was from 50 to 300 bp. Within this range, the longer the homology arm, the higher the efficiency of genetic recombination. The results showed that this system can target virulence genes located in plasmids and on chromosomes of ETEC strains. A single base mutation was performed by two-step gene fragment replacement. This study lays the foundation for research on virulence factors and genetic engineering of vaccines for ETEC.

Genome-Wide Analysis of the MYB Transcription Factor Superfamily in Physcomitrella patens.

MYB transcription factors (TFs) are one of the largest TF families in plants to regulate numerous biological processes. However, our knowledge of the MYB family in Physcomitrella patens is limited. We identified 116 MYB genes in the P. patens genome, which were classified into the R2R3-MYB, R1R2R3-MYB, 4R-MYB, and MYB-related subfamilies. Most R2R3 genes contain 3 exons and 2 introns, whereas R1R2R3 MYB genes contain 10 exons and 9 introns. N3R-MYB (novel 3RMYB) and NR-MYBs (novel RMYBs) with complicated gene structures appear to be novel MYB proteins. In addition, we found that the diversity of the MYB domain was mainly contributed by domain shuffling and gene duplication. RNA-seq analysis suggested that MYBs exhibited differential expression to heat and might play important roles in heat stress responses, whereas CCA1-like MYB genes might confer greater flexibility to the circadian clock. Some R2R3-MYB and CCA1-like MYB genes are preferentially expressed in the archegonium and during the transition from the chloronema to caulonema stage, suggesting their roles in development. Compared with that of algae, the numbers of MYBs have significantly increased, thus our study lays the foundation for further exploring the potential roles of MYBs in the transition from aquatic to terrestrial environments.

A CRISPR/LbCas12a-based method for highly efficient multiplex gene editing in Physcomitrella patens.

Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non-vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co-delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss-of-function mutants of multiple genes from different gene families.

Sulfur cycling in freshwater sediments: A cryptic driving force of iron deposition and phosphorus mobilization.

Sulfur cycling in freshwater ecosystems has been previously considered minor, and the direct evidence of its impacts on iron and phosphorus cycles in freshwater sediments remains unclear. In this study, mesocosms with amended acetate and various sulfate concentrations (1.5-3.0 mmol L-1) were set up to investigate sulfur cycling and its influences on iron-rich freshwater sediments. Acetate addition induced hypoxia and provided substrates, which stimulated the sulfur cycling with evidence of SO42- decline, ΣS2-, S0 increase and corresponding variations of sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria. Meanwhile, the growth of iron-reducing bacteria (IRB) was suppressed, and lower Fe(II) release was correspondingly related to larger SRB abundance at higher sulfate level, indicating that microbial iron reduction might be blocked by SRB activities. However, continuous dissolution of Fe(III) oxides and generation of iron sulfides were observed, suggesting that sulfide-mediated chemical iron reduction (SCIR) became the dominant iron-reducing pathway, and Fe(II) was buried as iron sulfides instead of released to water column, which resulted in a transition of iron cycling into unidirectional SCIR. Consequently, continuous dissolution of Fe(III) oxides led to significant increase of PO43- concentration in the water column and sediment pore-water, revealing the phosphorus mobility in sediments derived from the SCIR process. To note, sustained accumulation of iron sulfides was observed even without ΣS2- presence, suggesting that ΣS2- precipitation occurred prior to diffusion. Thus, ΣS2--missing sulfur cycling seemed "cryptic" in this study. To highlight, the transition of the iron-reducing pathway and resulting PO43- release can be induced even under current sulfate level of Lake Taihu, and elevated sulfate levels could significantly intensify SCIR and phosphorus mineralization. Thus, the stimulated iron deposition and the resulting phosphorus release derived from the sulfur cycling should be paid more attention to in the treatment of eutrophic freshwater ecosystems.

Histone Acetylation Modifications Affect Tissue-Dependent Expression of Poplar Homologs of C4 Photosynthetic Enzyme Genes.

Histone modifications play important roles in regulating the expression of C4 photosynthetic genes. Given that all enzymes required for the C4 photosynthesis pathway are present in C3 plants, it has been hypothesized that this expression regulatory mechanism has been conserved. However, the relationship between histone modification and the expression of homologs of C4 photosynthetic enzyme genes has not been well determined in C3 plants. In the present study, we cloned nine hybrid poplar (Populus simonii × Populus nigra) homologs of maize (Zea mays) C4 photosynthetic enzyme genes, carbonic anhydrase (CA), pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxykinase (PCK), and phosphoenolpyruvate carboxylase (PEPC), and investigated the correlation between the expression levels of these genes and the levels of promoter histone acetylation modifications in four vegetative tissues. We found that poplar homologs of C4 homologous genes had tissue-dependent expression patterns that were mostly well-correlated with the level of histone acetylation modification (H3K9ac and H4K5ac) determined by chromatin immunoprecipitation assays. Treatment with the histone deacetylase inhibitor trichostatin A further confirmed the role of histone acetylation in the regulation of the nine target genes. Collectively, these results suggest that both H3K9ac and H4K5ac positively regulate the tissue-dependent expression pattern of the PsnCAs, PsnPPDKs, PsnPCKs, and PsnPEPCs genes and that this regulatory mechanism seems to be conserved among the C3 and C4 species. Our findings provide new insight that will aid efforts to modify the expression pattern of these homologs of C4 genes to engineer C4 plants from C3 plants.

Phylogenic and phosphorylation regulation difference of phosphoenolpyruvate carboxykinase of C3 and C4 plants.

In C4 plants, phosphoenolpyruvate carboxykinase (PEPCK) plays a key role in the C4 cycle. PEPCK is also involved in gluconeogenesis and is conserved in both lower and higher organisms, including in animals and plants. A phylogenic tree constructed from PEPCK sequences from bacteria to higher plants indicates that the C4 Poaceae PEPCKs are conserved and have diverged from the PEPCKs of C3 plants. The maximum enzymatic activities of wild-type and phosphorylation mimic PEPCK proteins indicate that there is a significant difference between C3 and C4 plant PEPCKs. The conserved PEPCK phosphorylation sites are regulated differently in C3 and C4 plants. These results suggest that the functions of PEPCK have been conserved, but that sequences have diverged and regulation of PEPCK is important in C4 plants, but not in herbaceous and, in particular, woody C3 plants.

ERα inhibited myocardin-induced differentiation in uterine fibroids.

Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smooth muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids.

Analysis of gene expression and histone modification between C4 and non-C4 homologous genes of PPDK and PCK in maize.

More efficient photosynthesis has allowed C4 plants to adapt to more diverse ecosystems (such as hot and arid conditions) than C3 plants. To better understand C4 photosynthesis, we investigated the expression patterns of C4 genes (C4PPDK and PCK1) and their non-C4 homologous genes (CyPPDK1, CyPPDK2, and PCK2) in the different organs of maize (Zea mays). Both C4 genes and non-C4 genes showed organ-dependent expression patterns. The mRNA levels of C4 genes were more abundant in leaf organ than in seeds at 25 days after pollination (DAP), while non-C4 genes were mainly expressed in developing seeds. Further, acetylation of histone H3 lysine 9 (H3K9ac) positively correlates with mRNA levels of C4 genes (C4PPDK and PCK1) in roots, stems, leaves, and seeds at 25 DAP, acetylation of histone H4 lysine 5 (H4K5ac) in the promoter regions of both C4 (C4PPDK and PCK1) and non-C4 genes (CyPPDK1, CyPPDK2, and PCK2) correlated well with their transcripts abundance in stems. In photosynthetic organs (stems and leaves), dimethylation of histone H3 lysine 9 (H3K9me2) negatively correlated with mRNA levels of both C4 and non-C4 genes. Taken together, our data suggest that histone modification was involved in the transcription regulation of both C4 genes and non-C4 genes, which might provide a clue of the functional evolution of C4 genes.

Human cytomegalovirus immediate early protein 2 enhances myocardin-mediated survival of rat aortic smooth muscle cells.

Human cytomegalovirus (HCMV) may increase the incidence of restenosis and predispose to atherosclerosis. The lesions of restenosis and atherosclerosis often contain smooth muscle cells (SMCs) with high rates of proliferation and apoptosis. One of the immediate early (IE) gene products of HCMV-IE2 affects transcriptional activities of some cellular factors in SMCs, including myocardin. In this study, we studied the effects of IE2 and myocardin on PI3K pathway inducer wortmannin induced apoptosis in rat aortic SMCs. We show that the transcriptional activity of myocardin on Mcl-1 promoter is enhanced by co-expression of HCMV IE2 in rat aortic SMCs; and the expressions of mRNA and protein of antiapoptotic genes-Mcl-1 and Bcl-2 are upregulated by IE2 alone and co-transfection of myocardin and IE2, but decreased by myocardin-specific shRNA in rat aortic SMCs. We further demonstrate that co-expression of myocardin and HCMV IE2 declines apoptotic cell numbers and caspase-3 activities induced by serum starvation plus wortmannin in rat aortic SMCs. The results suggest that HCMV IE2 enhances myocardin-mediated survival of rat aortic SMCs under serum deprivation and PI3-kinase inhibition, partly via activation of Mcl-1's antiapoptosis effect. Our study connects HCMV IE2 to myocardin-induced transcriptional program for rat aortic SMCs survival and proliferation, involving in HCMV related restenosis and atherosclerosis.

Pathogenesis of a genotype C strain of bovine parainfluenza virus type 3 infection in albino guinea pigs.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important of the known viral respiratory tract agents of both young and adult cattle and widespread among cattle around the world. Up to present, three genotypes A, B and C of BPIV3 have been described on the basis of genetic and phylogenetic analysis and only limited studies on the pathogenesis of the genotype A of BPIV3 infection in calves and laboratory animals have been performed. The report about experimental infections of the genotypes B and C of BPIV3 in laboratory animals and calves was scant. Therefore, an experimental infection of guinea pigs with the Chinese BPIV3 strain SD0835 of the genotype C was performed. Sixteen guinea pigs were intranasally inoculated with the suspension of SD0835, while eight control guinea pigs were also intranasally inoculated with the same volume of supernatant from uninfected MDBK cells. The virus-inoculated guinea pigs displayed a few observable clinical signs that were related to the respiratory tract disease and two of the sixteen experimentally infected guinea pigs died at 2 and 3 days post inoculation (PI), respectively, and apparent gross pneumonic lesions were observed at necropsy. The gross pneumonic lesions in guinea pigs inoculated with SD0835 consisted of dark red, slightly depressed, irregular areas of consolidation in the lung lobes from the second to 9th day of infection at necropsy, and almost complete consolidation and atelectasis of the lung lobes were seen at 7 days PI. Histopathological changes including alveoli septa thickening and focal cellulose pneumonia were also observed in the lungs of guinea pigs experimentally infected with SD0835. Viral replication was detectable by virus isolation and titration, real-time RT-PCR and immunohistochemistry (IHC) staining in the respiratory tissues of guinea pigs as early as 24h after intranasal inoculation with SD0835. The results of virus isolation and titration showed that guinea pigs were permissive for SD0835 replication and exhibited a higher virus replication level in both lungs and tracheas. As well, the results of IHC staining implicated that the lungs and tracheas were the major tissues in which SD0835 replicated. Virus-specific serum neutralizing antibodies against BPIV3 were detected in virus-inoculated guinea pigs. The aforementioned results indicated that BPIV3 strain SD0835 of the genotype C was pathogenic to guinea pigs and could cause a few observable clinical signs, and gross and histologic lesions in virus-inoculated guinea pigs. Thus guinea pig is an ideal laboratory animal infection model for BPIV3 and would cast more light on the genotype C of BPIV3 infection process, in vivo tropism and pathogenesis or serve as a useful system for monitoring the pathogenesis of SD0835 and other BPIV3 isolates.

Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes.

BACKGROUND: The N-terminal protein processing mechanism (NPM) including N-terminal Met excision (NME) and N-terminal acetylation (N(α)-acetylation) represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To reveal the NPM in poplar, we investigated the N(α)-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α)-acetylated) proteins. Most proteins (47, >81%) are subjected to N(α)-acetylation following the N-terminal removal of Met, indicating that N(α)-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α)-acetylation (NPM) to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs) and N-terminal acetyltransferase (Nat) enzymes in poplar. The N(α)-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. CONCLUSIONS/SIGNIFICANCE: This study represents the first extensive investigation of N(α)-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α)-acetylation of proteins in poplar.