Red-grouper nervous necrosis virus B1 protein inhibits fish IFN response by targeting Ser5-phosphorylated RNA polymerase II to promote viral replication.

Nervous necrosis virus (NNV) could infect more than 200 fish species worldwide, with almost 100% mortality in affected larvae and juvenile fish. Among different genotypes of NNV, the red-grouper nervous necrosis virus (RGNNV) genotype is the most widely reported with the highest number of susceptible species. Interferon (IFN) is a crucial antiviral cytokine and RGNNV needs to develop some efficient strategies to resist host IFN-stimulated antiviral immune. Although considerable researches on RGNNV, whether RGNNV B1 protein participates in regulating the host's IFN response remains unknown. Here, we reported that B1 protein acted as a transcript inhibition factor to suppress fish IFN production. We firstly found that ectopic expression of B1 protein significantly decreased IFN and IFN-stimulated genes (ISGs) mRNA levels and IFNφ1 promoter activity induced by polyinosinic:polycytidylic acid [poly (I:C)]. Further studies showed that B1 protein inhibited the IFNφ1 promoter activity stimulated by the key RIG-I-like receptors (RLRs) factors, including MDA5, MAVS, TBK1, IRF3, and IRF7 and decreased their protein levels. Moreover, B1 protein significantly inhibited the activity of constitutively active cytomegalovirus (CMV) promoter, which suggested that B1 protein was a transcription inhibitor. Western blot indicated that B1 protein decreased the Ser5 phosphorylation of RNA polymerase II (RNAP II) C-terminal domain (CTD). Together, our data demonstrated that RGNNV B1 protein was a host transcript antagonist, which intervened RNAP II Ser5-phosphorylation, inhibiting host IFN response and facilitating RGNNV replication.

Development of a droplet digital PCR method for detection of porcine circovirus 4.

BACKGROUND: Porcine circovirus 4 (PCV4), a newly emerging virus that was first discovered in 2019, may pose a potential threat to the pig industry. Droplet digital PCR (ddPCR) is an absolute quantitative method that has high sensitivity and accuracy. In this study, we developed a novel ddPCR assay to detect PCV4. Furthermore, we evaluated the detection limit, sensitivity, specificity and reproducibility of the ddPCR and TaqMan real-time quantitative PCR (qPCR) and tested 160 clinical samples to compare the detection rate of the two methods. RESULTS: The detection limit for ddPCR was 0.54 copies/µL, 10.6 times greater sensitivity than qPCR. Both ddPCR and qPCR assays exhibited good linearity and repeatability, and the established ddPCR method was highly specific for PCV4. The results of clinical sample testing showed that the positivity rate of ddPCR (5.6%) was higher than that of qPCR (4.4%). CONCLUSIONS: This study successfully developed a sensitive, specific and repeatable ddPCR assay for PCV4 detection, which can be widely used in clinical diagnosis of PCV4 infections.

First report of powdery mildew caused by Erysiphe cruciferarum on spider flower (Tarenaya hassleriana) in China.

Spider flower (Tarenaya (Cleome) hassleriana (Chodat) Iltis, Cleomaceae) is an excellent ornamental landscape plant and has an extensive flowering period, and therefore, plays an important role in horticulture (Parma et al. 2022). In May 2020 and April 2021, severe powdery mildew symptoms were observed on spider flower plants in a public garden (22.35°N and 113.56°E) in Shenzhen, China. Approximately 60 % of the plants were infected, and the adaxial surface of diseased leaves were covered with irregular white patches, which developed on tender to old leaves. In severe infections, drying and premature defoliation of infected leaves were observed. Microscopic examinations of mycelia showed irregularly lobed hyphal appressoria. Conidiophores (n = 30) were straight, unbranched, 65.65-92.11 µm long, and consisted of two to three cells. Conidia were formed singly on the top of conidiophores, cylindrical to oblong, 32.15-42.60 × 14.88-18.43 µm (mean 38.26 × 16.89, n = 50), and without distinct fibrosin bodies. Chasmothecia were not observed. The internal transcribed spacer (ITS) region and 28S rDNA was amplified using the primer sets ITS1/ITS5 and NL1/NL4, respectively. The representative sequences of ITS and 28S rDNA (GenBank accession nos.: MW879365 for ITS and MW879435 for 28S rDNA) analyzed by BLASTN search and showed 100 % identity with the sequences from Erysiphe cruciferarum found in GenBank (accession nos.: LC009943 for ITS and MF192846 for 28S rDNA). Phylogenetic analyses were conducted for further confirmation by using the combined sequences of ITS and 28S rDNA and indicated that the isolate ZDH046 grouped in a clade with isolates of E. cruciferarum (Figure S2). Based on morphological and molecular characteristics, this fungus was identified as E. cruciferarum (Braun and Cook, 2012). Koch's postulates were confirmed by gently pressing conidia from diseased leaves onto 30 leaves of healthy spider flower plants. After incubating for 10 d in a greenhouse (25 ℃ and 75 % relative humidity), similar symptoms to the diseased plants appeared on all inoculated leaves, whereas control leaves remained symptomless. Powdery mildew caused by E. cruciferarum on T. hassleriana has so far only been reported from France (Ale-Agha et al. 2008), Germany (Jage et al. 2010), Italy (Garibaldi et al. 2009), and New Zealand (Pennycook 1989, E. polygoni). To our knowledge, this is the first report of E. cruciferarum causing powdery mildew on T. hassleriana in China. This finding expands the known host range of E. cruciferarum in China and indicates a potential threat to plantations of T. hassleriana in China.

First report of powdery mildew caused by Podosphaera fusca on Coreopsis tinctoria in China.

Coreopsis tinctoria is an annual herb and commonly cultivated in gardens due to its attractive flowers, its capitula also have been used as a traditional medicine in China, Asia, North America and Europe (Shen et al. 2021). In June 2023, severe powdery mildew infection was observed on C. tinctoria in a hillside near headwork of the middle route of the South to North Water Diversion Project (32°40'55''N, 111°41'59''E). Abundant irregular white spots were found on adaxial surface of the leaves and tender stems. Approximately 75% of the observed C. tinctoria plants showed these signs and symptoms. Generative hyphae were thin-walled, smooth or almost so, and 5 to 9 µm wide. Conidiophores were unbranched, straight, 80.5 to 162.5 × 9.3 to 12.9 µm (n=25), and produced one to three immature conidia. Foot-cells of conidiophores were cylindrical, 38.5 to 62.3 µm (n=20) long. Conidia were ellipsoid to ovoid, 25.1 to 31.9 × 15.2 to 19.5 µm (n=30). The morphological characteristics of asexual structures corresponded to Podosphaera sp. (Braun and Cook 2012). For further identification, genomic DNA was extracted directly from the mycelia and conidia using Chelex 100 (Sigma Aldrich, Shanghai, China). The internal transcribed spacer (ITS) regions and 28S large subunit (LSU) of ribosomal DNA from the specimen (CT2302) were amplified using the primers ITS1/ITS4 (expected amplicon size 566 bp) (White et al. 1990) and NL1/NL4 (expected amplicon size 618 bp) (Baten et al. 2014), respectively. The sequences of ITS (GenBank accession no. OR649304) and LSU (GenBank accession no. OR649305) showed 99.63% and 100% identity values to the Podosphaera fusca isolate HMNWAFU-CF2012074 in the NCBI database (KR048109 for ITS and KR048178 for LSU), respectively. Phylogenetic analyses based on the combined ITS and LSU sequences using MEGA 7.0 software indicated that CT2302 formed a monophyletic clade together with isolates of P. fusca. Therefore, this fungus was identified as P. fusca based on the morphological and molecular characteristics. Pathogenicity tests were performed by gently pressing the infected leaves onto 15 young leaves of five healthy plants and three noninoculated plants were used as controls. All plants were maintained in a greenhouse (25℃ and 70% relative humidity). Powdery mildew symptoms similar to those of originally diseased plants were observed on all inoculated leaves after 12 days, whereas no symptoms were observed on the control leaves. Powdery mildew caused by P. fusca (previously Sphaerotheca fusca) on C. tinctoria has been reported in Russia, Poland, Korea, Romania and Ukraine (Cho and Shin 2004; Rusanov and Bulgakow 2008). To our knowledge, this is the first report of P. fusca on C. tinctoria in China. The identification of P. fusca as the causal agent on C. tinctoria is critical to the prevention and control of this disease in the future.

Engineering of formate dehydrogenase for improving conversion potential of carbon dioxide to formate.

Formate dehydrogenase (FDH) is a D-2-hydroxy acid dehydrogenase, which can reversibly reduce CO2 to formate and thus act as non-photosynthetic CO2 reductase. In order to increase catalytic efficiency of formate dehydrogenase for CO2 reduction, two mutants V328I/F285W and V354G/F285W were obtained of which reduction activity was about two times more than the parent CbFDHM2, and the formate production from CO2 catalyzed by mutants were 2.9 and 2.7-fold higher than that of the parent CbFDHM2. The mutants had greater potential in CO2 reduction. The optimal temperature for V328I/F285W and V354G/F285W was 55 °C, and they showed increasement of relative activity under 45 °C to 55 °C compared with parent. The optimal pH for the mutants was 9.0, and they showed excellent stability in pH 4.0-11.5. The kcat/Km values of mutants were 1.75 times higher than that of the parent. Then the molecular basis for its improvement of biochemical characteristics were preliminarily elucidated by computer-aided methods. All of these results further established a solid foundation for molecular modification of formate dehydrogenase and CO2 reduction.

Identification and expression analysis of GARP superfamily genes in response to nitrogen and phosphorus stress in Spirodela polyrhiza.

BACKGROUND: GARP transcription factors perform critical roles in plant development and response to environmental stimulus, especially in the phosphorus (P) and nitrogen (N) sensing and uptake. Spirodela polyrhiza (giant duckweed) is widely used for phytoremediation and biomass production due to its rapid growth and efficient N and P removal capacities. However, there has not yet been a comprehensive analysis of the GRAP gene family in S. polyrhiza. RESULTS: We conducted a comprehensive study of GRAP superfamily genes in S. polyrhiza. First, we investigated 35 SpGARP genes which have been classified into three groups based on their gene structures, conserved motifs, and phylogenetic relationship. Then, we identified the duplication events, performed the synteny analysis, and calculated the Ka/Ks ratio in these SpGARP genes. The regulatory and co-expression networks of SpGARPs were further constructed using cis-acting element analysis and weighted correlation network analysis (WGCNA). Finally, the expression pattern of SpGARP genes were analyzed using RNA-seq data and qRT-PCR, and several NIGT1 transcription factors were found to be involved in both N and P starvation responses. CONCLUSIONS: The study provides insight into the evolution and function of GARP superfamily in S. polyrhiza, and lays the foundation for the further functional verification of SpGARP genes.

Molecular cloning, expression and functional analysis of STAT2 in orange-spotted grouper, Epinephelus coioides.

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signaling pathway. To better understand the functions of STAT2 in fish immune response, a STAT2 gene from orange-spotted grouper (Epinephelus coioides) (EcSTAT2) was cloned and characterized in this study. EcSTAT2 encoded a 802-amino acid peptide which shared 99.5% and 91.5% identity with giant grouper (Epinephelus lanceolatus) and leopard coral grouper (Plectropomus leopardus), respectively. Amino acid alignment analysis showed that EcSTAT2 contained five conserved domains, including N-terminal protein interaction domain, coiled coil domain (CCD), DNA binding domain (DBD), Src-homology 2 (SH2) domain, and C-terminal transactivation domain (TAD). Phylogenetic analysis indicated that EcSTAT2 clustered into fish STAT2 group and showed the nearest relationship to giant grouper STAT2. In healthy grouper, EcSTAT2 was distributed in all tissues tested, and the expression of EcSTAT2 was predominantly detected in spleen, kidney and gill. In vitro, EcSTAT2 expression was significantly increased in response to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization showed that EcSTAT2 was located in the cytoplasm in a punctate manner. EcSTAT2 overexpression significantly inhibited RGNNV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral genes and protein. Consistently, knockdown of EcSTAT2 using small interfering RNA (siRNA) promoted RGNNV replication. Furthermore, EcSTAT2 overexpression increased both interferon (IFN) and interferon stimulated genes (ISGs) expression. In addition, EcSTAT2 knockdown decreased the transcription levels of IFN and ISGs. Together, our data demonstrated that EcSTAT2 exerted antiviral activity against RGNNV through up-regulation of host interferon response.

Case report of enterobacter hormaechei in sheep with respiratory disease and death.

BACKGROUND: Enterobacter hormaechei is typically a opportunistic pathogenic bacterium in humans, and no pathological change of of Enterobacter hormaechei in diseased sheep has previously been documented. CASE PRESENTATION: Three free-range, four-month-old female sheep were ill with respiratory disease and died three days after receiving treatment with ceftiofur sodium. A frozen lung sample of one sheep was studied using bacterium isolation, and lung samples of the other two sheep were collected and analyzed by histopathological examination and bacterium isolation. The 16S rRNA gene sequences and biochemical characteristics of the isolates were analyzed. All results showed the isolated strain to be Enterobacter hormaechei. Phylogenetic analysis of the 16S rRNA sequence showed three representative strains were most closely related to the strains isolated from calf. Antimicrobial sensitivity tests indicated that no sensitivity to the ß-lactam antimicrobials involved in treatment of sheep respiratory disease in China. Detection of the genes responsible for ß-lactam resistance showed that all three isolates from sheep harbor blaSHV and blaKPC. Interstitial pneumonia, bronchial epithelial cells shedding, and massive mucous secretion were observed in the lung histopathological sections. Immunohistochemical staining showed that specific staining was mainly limited to the alveoli and alveolar septum. CONCLUSIONS: This appears to be the first report of pathological changes in lungs of sheep with respiratory disease and death associated with Enterobacter hormaechei.

Molecular detection of haemophilic pathogens reveals evidence of Candidatus Mycoplasma haemobos in dogs and parasitic ticks in central China.

BACKGROUND: In addition to Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum, a few hemoplasma species that mainly infect other livestock have been detected in dogs. 'Candidatus Mycoplasma haemobos' (Ca. M. haemobos) has been found in a variety of animals in China. The present study was aimed to investigate the occurrence of 'Ca. M. haemobos' infections in dogs and ticks collected from the Henan province, China. RESULTS: Overall, 55 dog blood samples and 378 ticks on skins were collected from anemic and healthy dogs, and these samples were subjected to PCR, sequence analysis, and identification. The results showed that Haemaphysalis longicornis (266) and Rhipicephalus (Boophilus) microplus (112) were the only two parasitic ticks on dogs. Molecular detection revealed that 163 M. haemocanis, 88 'Ca. M. haemobos' and 32 Anaplasma platys positive amplicons could be amplified from dogs, H. longicornis and R. (B.) microplus. In addition, co-infections (M. haemocanis + A. platys and 'Ca. M. haemobos'+ A. platys) could be also detected. CONCLUSIONS: To the best of our knowledge, this is the first molecular evidence of 'Ca. M. haemobos' natural infection in dogs and tick species identified as H. longicornis and R. (B.) microplus from China.

Transcriptome analysis reveals potential function of long non-coding RNAs in 20-hydroxyecdysone regulated autophagy in Bombyx mori.

BACKGROUND: 20-hydroxyecdysone (20E) plays important roles in insect molting and metamorphosis. 20E-induced autophagy has been detected during the larval-pupal transition in different insects. In Bombyx mori, autophagy is induced by 20E in the larval fat body. Long non-coding RNAs (lncRNAs) function in various biological processes in many organisms, including insects. Many lncRNAs have been reported to be potential for autophagy occurrence in mammals, but it has not been investigated in insects. RESULTS: RNA libraries from the fat body of B. mori dissected at 2 and 6 h post-injection with 20E were constructed and sequenced, and comprehensive analysis of lncRNAs and mRNAs was performed. A total of 1035 lncRNAs were identified, including 905 lincRNAs and 130 antisense lncRNAs. Compared with mRNAs, lncRNAs had longer transcript length and fewer exons. 132 lncRNAs were found differentially expressed at 2 h post injection, compared with 64 lncRNAs at 6 h post injection. Thirty differentially expressed lncRNAs were common at 2 and 6 h post-injection, and were hypothesized to be associated with the 20E response. Target gene analysis predicted 6493 lncRNA-mRNA cis pairs and 42,797 lncRNA-mRNA trans pairs. The expression profiles of LNC_000560 were highly consistent with its potential target genes, Atg4B, and RNAi of LNC_000560 significantly decreased the expression of LNC_000560 and Atg4B. These results indicated that LNC_000560 was potentially involved in the 20E-induced autophagy of the fat body by regulating Atg4B. CONCLUSIONS: This study provides the genome-wide identification and functional characterization of lncRNAs associated with 20E-induced autophagy in the fat body of B. mori. LNC_000560 and its potential target gene were identified to be related to 20-regulated autophagy in B. mori. These results will be helpful for further studying the regulatory mechanisms of lncRNAs in autophagy and other biological processes in this insect model.

A rapid reduction of Au(I→0) strategy for the colorimetric detection and discrimination of proteins.

A gold nanoparticle (AuNP)-based sensing strategy based on rapid reduction of Au(I→0) is proposed. As a proof-of-concept study, the proposed sensing principle is designed for simultaneous and colorimetric detection and discrimination of multiple proteins. In the presence of H2O2, the target proteins could reduce Au(I) (i.e. HAuCl2) to AuNPs with different sizes, shapes and dispersion/aggregation states, thus resulting in rapidly colorimetric identification of different proteins. The optical response (i.e. color) of AuNPs is found to be characteristic of a given protein. The color response patterns are characteristic for each protein and can be quantitatively differentiated by statistical techniques. The sensor array is capable of discriminating proteins at concentrations as low as 0.1 µg/mL with high accuracy. A linear relationship was observed between the total Euclidean distances and protein concentration, providing the potential for protein quantification using this sensor array. The limit of detection (LOD) for catalase (Cat) is 0.08 µg/mL. The good linear range (from 0 to 8 µg/mL) has been used for the quantitative assay of Cat. To show a potentially practical application, this method was used to detect and discriminate proteins in human urine and tear samples. Graphical abstract We report a facile gold nanoparticle (AuNP)-based sensing strategy, that is, "a rapid reduction of Au(I) to Au(0) nanoparticles with different sizes and shapes by analytes that having certain reducing capabilities, resulting in different colours." The proposed sensing principle is designed for simultaneous, colorimetric detection and discrimination of multiple proteins.

Rhodobacter xinxiangensis sp. nov., isolated from pakchoi-cultivated soil contaminated with heavy metal and its potential to reduce Cd and Pb accumulation in pakchoi (Brassica campestris L.).

A Gram-stain-negative, aerobic, and motile strain, TJ48T, was isolated from pakchoi-cultivated soil contaminated with Cd and Pb in Xinxiang (China). Cells of the strain were rod-shaped and colonies on LB agar were faint yellow. Strain TJ48T was positive for catalase and oxidase and the optimal condition for growth was 28 °C, with 1% (w/v) NaCl and at pH 7.0. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain TJ48T was closely related to the genus Rhodobacter and the closest relatives were Rhodobacter ovatus JA234T (97.4%, 16S rRNA gene sequence similarity) and Rhodobacter azotoformans KA25T (96.5%). The DNA G + C content of strain TJ48T was 64.7 mol%. Genome-to-genome distance calculations (GGDC) and ANIb values from genomic comparison between the genomes of strain TJ48T and the related reference species were less than 70% and 95%, respectively. The major cellular fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C17:0. The only isoprenoid quinone detected was Ubiquinone-10 (Q-10). The polar lipid profile contains diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipids, and three unidentified lipids. Strain TJ48T significantly increased the dry weight of roots (26.2-66.3%) and shoots (16.7-37.8%) of pakchoi and reduced the Cd (50.2-60.1%) and Pb (55.6-60.9%) contents in pakchoi shoots and roots. On the basis of the physiological, genotypic and genomic characteristics, the strain TJ48T represent a novel species of the genus Rhodobacter, and the name Rhodobacter xinxiangensis sp. nov. is proposed (type strain TJ48T = CCTCC AB2019120T = KCTC 72510T).

Environmental Factors and Microbial Diversity and Abundance Jointly Regulate Soil Nitrogen and Carbon Biogeochemical Processes in Tibetan Wetlands.

Wetlands have numerous critical ecological functions, some of which are regulated by several nitrogen (N) and carbon (C) biogeochemical processes, such as denitrification, organic matter decomposition, and methane emission. Until now, the underlying pathways of the effects of environmental and biological factors on wetland N and C cycling rates are still not fully understood. Here, we investigated soil potential/net nitrification, potential/unamended denitrification, methane production/oxidation rates in 36 riverine, lacustrine, and palustrine wetland sites on the Tibet Plateau. The results showed that all the measured N and C cycling rates did not differ significantly among the wetland types. Stepwise multiple regression analyses revealed that soil physicochemical properties (e.g., moisture, C and N concentration) explained a large amount of the variance in most of the N and C cycling rates. Microbial abundance and diversity were also important in controlling potential and unamended denitrification rates, respectively. Path analysis further revealed that soil moisture and N and C availability could impact wetland C and N processes both directly and indirectly. For instance, the indirect effect of soil moisture on methane production rates was mainly through the regulating the soil C content and methanogenic community structure. Our findings highlight that many N and C cycling processes in high-altitude and remote Tibetan wetlands are jointly regulated by soil environments and functional microorganisms. Soil properties affecting the N and C cycling rates in wetlands through altering their microbial diversity and abundance represent an important but previously underestimated indirect pathway.

Study of the immunogenicity of the VP2 protein of canine parvovirus produced using an improved Baculovirus expression system.

BACKGROUND: Canine parvovirus (CPV) is now recognized as a serious threat to the dog breeding industry worldwide. Currently used CPV vaccines all have their specific drawbacks, prompting a search for alternative safe and effective vaccination strategies such as subunit vaccine. VP2 protein is the major antigen targeted for developing CPV subunit vaccine, however, its production in baculovirus expression system remains challenging due to the insufficient yield. Therefore, our study aims to increase the VP2 protein production by using an improved baculovirus expression system and to evaluate the immunogenicity of the purified VP2 protein in mice. RESULTS: The results showed that high-level expression of the full length VP2 protein was achieved using our modified baculovirus expression system. The recombinant virus carrying two copies of VP2 gene showed the highest expression level, with a productivity of 186 mg/L, which is about 1.4-1.6 fold that of the recombinant viruses carrying only one copy. The purified protein reacted with Mouse anti-His tag monoclonal antibody and Rabbit anti-VP2 polyclonal antibody. BALB/c mice were intramuscularly immunized with purified VP2 protein twice at 2 week intervals. After vaccination, VP2 protein could induce the mice produce high level of hemagglutination inhibition antibodies. CONCLUSIONS: Full length CPV VP2 protein was expressed at high level and purified efficiently. Moreover, it stimulated mice to produce high level of antibodies with hemmaglutination inhibition properties. The VP2 protein expressed in this study could be used as a putative economic and efficient subunit vaccine against CPV infection.

First report of Enterobacter hormaechei with respiratory disease in calves.

BACKGROUND: Enterobacter hormaechei is commonly considered a causative pathogen for nosocomial infections and it does not usually cause diseases in animals. However, researchers have recently dissociated the pathogenic Enterobacter hormaechei from foxes and piglets. Here, the Enterobacter hormaechei was first found to be associated with respiratory disease in unweaned calves in China. CASE PRESENTATION: A 2-month-old calf was severely sick and diagnosed with respiratory infection by a rural veterinarian, and it died 5 days after treatment with penicillin G. The lung sample was then run through histopathological analysis and pathogen isolation. The sequence analysis and biochemical tests results showed the isolated bacterium strain to be Enterobacter hormaechei, and drug sensitivity tests showed resistance to all ß-lactam antimicrobials and sensitivity to quinolones. Thickened alveoli septum, inflammatory cell infiltration, and erythrocyte diapedesis around the pulmonary alveoli septum were visible in lung histopathological sections. One week later, at the same farm, another calf showed similar clinical signs, and the Enterobacter hormaechei strain was isolated from its nasal discharge; after a week of treatment with enrofloxacin, as suggested by the results of drug sensitivity tests, this calf fully recovered. CONCLUSIONS: To the best of our knowledge, this is the first case report of calves with respiratory disease that was associated with E. hormaechei, and multi-drug resistance was observed in isolates.

Heavy metal-immobilizing bacteria increase the biomass and reduce the Cd and Pb uptake by pakchoi (Brassica chinensis L.) in heavy metal-contaminated soil.

Microbial immobilization is a novel and environmentally friendly technology that uses microbes to reduce metal availability in soil and accumulation of heavy metals in plants. We used urea agar plates to isolate urease-producing bacteria from the rhizosphere soil of pakchoi in Cd- and Pb-contaminated farmland and investigated their effects on Cd and Pb accumulation in pakchoi and the underlying mechanisms. The results showed that two urease-producing bacteria, Bacillus megaterium N3 and Serratia liquefaciens H12, were identified by screening. They had higher ability to produce urease (57.5 ms cm-1 min-1 OD600-1 and 76.4 ms cm-1 min-1 OD600-1, respectively). The two strains allowed for the immobilization of Cd and Pb by extracellular adsorption, bioprecipitation, and increasing the pH (from 6.94 to 7.05-7.09), NH4+ content (69.1%-127%), and NH4+/NO3- ratio (from 1.37 to 1.67-2.11), thereby reducing the DTPA-extractable Cd (35.3%-58.8%) and Pb (37.8%-62.2%) contents in the pakchoi rhizosphere soils and the Cd (76.5%-79.7%) and Pb (76.3%-83.5%) contents in the leaves (edible tissue) of pakchoi. The strains were highly resistant to heavy metal toxicity; produced IAA, siderophores and abscisic acid; and increased the NH4+/NO3- ratio, which might be related to the two strains protectiing pakchoi against the toxic effect of Cd and Pb and increasing pakchoi biomass. Thus, the results were supposed to strain resources and a theoretical basis for the remediation of Cd- and Pb-contaminated farmlands for the safe production of vegetables.

Adsorption of Pb2+ onto freeze-dried microalgae and environmental risk assessment.

Dry microalgae Spirulina platensis shows a high capacity for heavy metal uptake, but there is a concern about dissolved organic carbon (DOC) residue, which is the precursor of disinfection by-products (DBPs). Vsp, a kind of Spirulina platensis powder prepared by vacuum freeze-drying, and Osp, a kind of Spirulina platensis powder prepared by the conventional oven drying-pulverization method, were subjected to assessments of their adsorption potential for Pb2+ and DOC residue. The adsorption mechanism of Pb2+ by the two adsorbents was studied by SEM, FT-IR, EDX and N2-BET. The effects of pH, adsorbent dosage, initial Pb2+ concentration and contact time on the biosorption process were investigated. The results showed that Pb2+ biosorption by Vsp and Osp were fit well by a pseudo-second-order kinetic model and the Langmuir model. The maximum amount of Pb2+ biosorption by Vsp was 253 mg/g, which was 33 mg/g greater than that of Osp. In comparison with Osp, Vsp reached adsorption saturation 8 h earlier and had a remarkable effect on the control of DOC residue in water. When both adsorption capacity and environmental risks were considered, it was determined that the dosage of 0.5 g/L Vsp for 2 h of contact time was the best method, with 85.89 mg/g of Pb2+ removal and 3.45 mg/L of DOC residue. In summary, Vsp is a highly efficient and environmentally friendly biosorbent that can be used for heavy metal removal from water.

Dynamic nano-Ag colloids cytotoxicity to and accumulation by Escherichia coli: Effects of Fe3+, ionic strength and humic acid.

Released Ag ions or/and Ag particles are believed to contribute to the cytotoxicity of Ag nanomaterials, and thus, the cytotoxicity and mechanism of Ag nanomaterials should be dynamic in water due to unfixed Ag particle:Ag+ ratios. Our recent research found that the cytotoxicity of PVP-Ag nanoparticles is attributable to Ag particles alone in 3 hr bioassays, and shifts to both Ag particles and released Ag+ in 48 hr bioassays. Herein, as a continued study, the cytotoxicity and accumulation of 50 and 100 nm Ag colloids in Escherichia coli were determined dynamically. The cytotoxicity and mechanisms of nano-Ag colloids are dynamic throughout exposure and are derived from both Ag ions and particles. Ag accumulation by E. coli is derived mainly from extracellular Ag particles during the initial 12 hr of exposure, and thereafter mainly from intracellular Ag ions. Fe3+ accelerates the oxidative dissolution of nano-Ag colloids, which results in decreasing amounts of Ag particles and particle-related toxicity. Na+ stabilizes nano-Ag colloids, thereby decreasing the bioavailability of Ag particles and particle-related toxicity. Humic acid (HA) binds Ag+ to form Ag+-HA, decreasing ion-related toxicity and binding to the E. coli surface, decreasing particle-related toxicity. HA in complex conditions showed a stronger relative contribution to toxicity and accumulation than Na+ or Fe3+. The results highlighted the cytotoxicity and mechanism of nano-Ag colloids are dynamic and affected by environmental factors, and therefore exposure duration and water chemistry should be seriously considered in environmental and health risk assessments.

Small nucleolar RNA Sf-15 regulates proliferation and apoptosis of Spodoptera frugiperda Sf9 cells.

BACKGROUND: Small nucleolar RNAs (snoRNAs) function in guiding 2'-O-methylation and pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In recent years, more and more snoRNAs have been found to play novel roles in mRNA regulation, such as pre-mRNA splicing or RNA editing. In our previous study, we found a silkworm C/D box snoRNA Bm-15 can interact with Notch receptor gene in vitro. To further study the function of Bm-15, we cloned its homolog Sf-15 from Spodoptera frugiperda and investigate the function of Sf-15 in Sf9 cells. RESULTS: We showed that knocking down of Sf-15 can inhibit the proliferation, then induce apoptosis of insect S. frugiperda Sf9 cells, but the results were reversed when Sf-15 was overexpressed. De novo sequencing of transcriptome of Sf9 cells showed that the expression of 21 apoptosis-related genes were increased upon Sf-15 repression. Further analysis showed that a Ca2+-induced cell death pathway gene Cn (PPP3C, the serine/threonine-protein phosphatase 2B catalytic subunit), was significantly increased upon Sf-15 depression but decreased when Sf-15 was overexpressed, which indicated that Cn might be a potential target of Sf-15. CONCLUSIONS: We conclude that C/D box snoRNA Sf-15 can participate in apoptosis through regulating the expression of Ca2+-induced cell death pathway gene Cn in Sf9 cells. This is the first time that we found snoRNAs exhibiting dual functions in insect, which reveals a novel layer of ncRNA modulation in cell growth and death.

Expression of dysregulated miRNA in vivo in DF-1 cells during the course of subgroup J avian leukosis virus infection.

Aberrant expression of microRNAs (miRNAs) is known to be involved in cancer progression caused by subgroup J avian leukosis virus (ALV-J) in liver tissues. To advance our understanding of the related pathological mechanisms and virus-host interactions, seven previously reported miRNAs were selected for a comparative analysis of miRNA expression between infected and uninfected DF-1 cells, including six miRNAs related to tumorigenesis (let-7b/7i, miR-221/222, miR-125b, miR-375 and miR-2127. The results showed that six of the seven miRNAs except gga-miR-375 were upregulated in cells infected with NX0101 (caused myeloma (ML)) and GD1109 (caused hemangioma (HE)) at 1 h post infection. On day 2 post-infection, all seven miRNAs were upregulated in infected DF-1 cells. On day 6 post-infection, gga-let-7b, gga-miR-125b, and gga-miR-375 were downregulated whereas gga-miR-221 and gga-miR-222 were upregulated in DF-1 cells infected with the two ALV-J strains of different phenotypes. However, expression of gga-let-7i was reduced in DF-1 cells infected with NX0101 and was increased in those infected with GD1109; gga-miR-2127 expression showed no significant difference between infected and uninfected cells. This study is the first to report the changes in the miRNA expression levels in DF-1 cells during the course of ALV-J infection, and suggests a relationship between its pathological mechanisms and miRNAs.