Transcriptome profiling and digital gene expression analysis of the skin of Dybowski's frog (Rana dybowskii) exposed to Aeromonas hydrophila.

Recently, populations of Rana dybowskii, an important amphibian species in Northeast China, have decreased, mainly owing to the disease caused by Aeromonas hydrophila. However, effective control methods have not yet been developed. In order to explore the immune responses of R. dybowskii upon exposure to A. hydrophila infection, Illumina high-throughput transcriptome sequencing and digital gene expression (DGE) technology were employed to investigate transcriptomic changes in the skin of R. dybowskii exposed to A. hydrophila. In this work, a total of 26,244,446 transcriptome sequencing reads were obtained and assembled into 109,089 unique unigenes using de novo assembly, and a total of 37,105 unigenes (34.0%) were functionally annotated against the non-redundant (Nr), Swiss-Prot, Cluster of Orthologous Groups of Proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases. Gene expression changes in the skin tissue of R. dybowskii exposed to A. hydrophila were investigated by a tag-based DGE system, and a total of 1435 significantly differentially expressed genes were identified, including 460 that were up-regulated and 975 that were down-regulated, indicating a large change in the host transcriptome profile exposed to A. hydrophila. Among these, 478 genes were associated with immune-relevant pathways, metabolic pathways, cellular components, growth, migration, and muscle and hormone signaling pathways. We confirmed the differential expression of 106 immune-relevant genes associated with innate and adaptive immune responses. Our data provide a fairly comprehensive molecular biology background for the deeper understanding of the amphibian immune system following A. hydrophila infection.

Directed chromosomal integration and expression of porcine rotavirus outer capsid protein VP4 in Lactobacillus casei ATCC393.

Using two-step plasmid integration in the presence of 5-fluorouracil (5-FU), we developed a stable and markerless Lactobacillus casei strain for vaccine antigen expression. The upp of L. casei, which encodes uracil phosphoribosyltransferase (UPRTase), was used as a counterselection marker. We employed the Δupp isogenic mutant, which is resistant to 5-FU, as host and a temperature-sensitive suicide plasmid bearing upp expression cassette as counterselectable integration vector. Extrachromosomal expression of UPRTase complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. The resultant genotype can either be wild type or recombinant. The efficacy of the system was demonstrated by insertion and expression of porcine rotavirus (PRV) VP4. To improve VP4 expression, we analyzed L. casei transcriptional profiles and selected the constitutive highly expressed enolase gene (eno). The VP4 inserted after the eno termination codon were screened in the presence of 5-FU. Using genomic PCR amplification, we confirmed that VP4 was successfully integrated and stably inherited for at least 50 generations. Western blot demonstrated that VP4 was steadily expressed in medium with different carbohydrates. RT-qPCR and ELISA analysis showed that VP4 expression from the chromosomal location was similar to that achieved by a plasmid expression system. Applying the recombinant strain to immunize BALB/c mice via oral administration revealed that the VP4-expressing L. casei could induce both specific local and systemic humoral immune responses in mice. Overall, the improved gene replacement system represents an efficient method for chromosome recombination in L. casei and provides a safe tool for vaccine production.

Immunogenicity in Swine of Orally Administered Recombinant Lactobacillus plantarum Expressing Classical Swine Fever Virus E2 Protein in Conjunction with Thymosin α-1 as an Adjuvant.

Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious disease that results in enormous economic losses in pig industries. The E2 protein is one of the main structural proteins of CSFV and is capable of inducing CSFV-neutralizing antibodies and cytotoxic T lymphocyte (CTL) activities in vivo. Thymosin α-1 (Tα1), an immune-modifier peptide, plays a very important role in the cellular immune response. In this study, genetically engineered Lactobacillus plantarum bacteria expressing CSFV E2 protein alone (L. plantarum/pYG-E2) and in combination with Tα1 (L. plantarum/pYG-E2-Tα1) were developed, and the immunogenicity of each as an oral vaccine to induce protective immunity against CSFV in pigs was evaluated. The results showed that recombinant L. plantarum/pYG-E2 and L. plantarum/pYG-E2-Tα1 were both able to effectively induce protective immune responses in pigs against CSFV infection by eliciting immunoglobulin A (IgA)-based mucosal, immunoglobulin G (IgG)-based humoral, and CTL-based cellular immune responses via oral vaccination. Significant differences (P < 0.05) in the levels of immune responses were observed between L. plantarum/pYG-E2-Tα1 and L. plantarum/pYG-E2, suggesting a better immunogenicity of L. plantarum/pYG-E2-Tα1 as a result of the Tα1 molecular adjuvant that can enhance immune responsiveness and augment specific lymphocyte functions. Our data suggest that the recombinant Lactobacillus microecological agent expressing CSFV E2 protein combined with Tα1 as an adjuvant provides a promising strategy for vaccine development against CSFV.

How to build a habitable planet: an interview with Charles H. Langmuir.

Earth is the only known habitable planet in the solar system. Understanding how Earth developed its unique habitability has been the frontier of Earth sciences and has become one of the main themes of current deep-space explorations. What are the decisive factors that led to a habitable planet? What is the role of solid Earth processes in the origin of life and in modulating the surface environment? Are Earth's habitability studies relevant to current challenges that human beings face? These questions have attracted the interest of both scientists and the public alike. NSR spoke to Prof. Charles H. Langmuir from Harvard University in the USA, who is a solid Earth geochemist who carries out research on diverse aspects of the plate tectonic geochemical cycle, including ocean ridges, convergent margins and intraplate volcanism. Prof. Langmuir is the author of the book How to Build a Habitable Planet (www.habitableplanet.org), one of the best Earth science books published in 2012.

The terrestrial end-Permian mass extinction in the paleotropics postdates the marine extinction.

The end-Permian mass extinction was the most severe ecological event during the Phanerozoic and has long been presumed contemporaneous across terrestrial and marine realms with global environmental deterioration triggered by the Siberian Traps Large Igneous Province. We present high-precision zircon U-Pb geochronology by the chemical abrasion-isotope dilution-thermal ionization mass spectrometry technique on tuffs from terrestrial to transitional coastal settings in Southwest China, which reveals a protracted collapse of the Cathaysian rainforest beginning after the onset of the end-Permian marine extinction. Integrated with high-resolution geochronology from coeval successions, our results suggest that the terrestrial extinction occurred diachronously with latitude, beginning at high latitudes during the late Changhsingian and progressing to the tropics by the early Induan, spanning a duration of nearly 1 million years. This latitudinal age gradient may have been related to variations in surface warming with more degraded environmental conditions at higher latitudes contributing to higher extinction rates.

New constraints on Cenozoic subduction between India and Tibet.

The type of lithosphere subducted between India and Tibet since the Paleocene remains controversial; it has been suggested to be either entirely continental, oceanic, or a mixture of the two. As the subduction history of this lost lithosphere strongly shaped Tibetan intraplate tectonism, we attempt to further constrain its nature and density structure with numerical models that aim to reproduce the observed history of magmatism and crustal thickening in addition to present-day plateau properties between 83°E and 88°E. By matching time-evolving geological patterns, here we show that Tibetan tectonism away from the Himalayan syntaxis is consistent with the initial indentation of a craton-like terrane at 55 ± 5 Ma, followed by a buoyant tectonic plate with a thin crust, e.g., a broad continental margin (Himalandia). This new geodynamic scenario can explain the seemingly contradictory observations that had led to competing hypotheses like the subduction of Greater India versus largely oceanic subduction prior to Indian indentation.

Induction of antimicrobial peptides from Rana dybowskii under Rana grylio virus stress, and bioactivity analysis.

The skin glands of Ranidae are a rich source of antimicrobial peptides. In this study, the genomic RNA of Rana dybowskii was extracted from its skin while under Rana grylio virus stress. Five new cDNA sequences encoding 5 mature peptides, Ranatuerin-2YJ (GLMDIFKVAVNKLLAAGMNKPRCKAAHC), Dybowskin-YJb (IIPLPLGYFAKKP), Dybowskin-YJa (IIPLPLGYFAKKKKKKDPVPLDQ), Temperin-YJa (VLPLLETCSMTCWENNQTFGK), and Temperin-YJb (VLPLVGNLLNDLLGK), were obtained by reverse transcription polymerase chain reaction with a pair of degenerate primers designed according to the conserved terminal sequences of cDNA encoding antimicrobial peptide precursors of genus Rana. The antimicrobial activities of the peptides were analyzed, and the results demonstrated that all these peptides showed a significant anti-Rana grylio virus activity, and the virus was gradually cleared with the increase in gene expression. Among the 5 peptides obtained in this work, Ranatuerin-2YJ also showed a broad-spectrum anti-Gram-positive bacteria and anti-Gram-negative bacteria activity with a minimal inhibitory concentration of 22.5 µg/mL and 7.64% hemolysis activity, both of which were significantly lower (p < 0.05) than that of the other peptides. Moreover, Ranatuerin-2YJ was widely distributed in the skin, liver, spleen, and blood of R. dybowskii, while the other 4 peptides could only be cloned from the skin, indicating that the Ranatuerin-2YJ in vivo plays an important role in the protection against pathogen invasion.

Boron isotopes in boninites document rapid changes in slab inputs during subduction initiation.

How subduction-related magmatism starts at convergent plate margins is still poorly understood. Here we show that boron isotope variations in early-formed boninites from the Izu-Bonin arc, combined with radiogenic isotopes and elemental ratios document rapid (~0.5 to 1 Myr) changes in the sources and makeup of slab inputs as subduction begins. Heterogeneous hornblende-granulite facies melts from ocean crust gabbros ± basalts fluxed early melting to generate low silica boninites. Hydrous fluids from slab sediments and basalts later fluxed the low silica boninites mantle source to produce high silica boninites. Our results suggest that initially the uppermost parts of the slab were accreted near the nascent trench, perhaps related to early low-angle subduction. The rapid changes in slab inputs recorded in the boninites entail a steepening subduction angle and cooling of the plate interface, allowing for subduction of slab sediment and basalt, and generating hydrous fluids at lower slab temperatures.

Cretaceous basin evolution in northeast Asia: tectonic responses to the paleo-Pacific plate subduction.

Cretaceous rift basin evolution was an important part of the tectonic history of northeast Asia in the late Mesozoic. Three types of rift basins are identified-active, passive and wide rift basins-and they developed in different regions. Passive rift basins in the eastern North China craton are thought to be the consequence of crustal stretching and passive asthenospheric upwelling. Wide rift basins in the eastern Central Asian orogen are assumed to originate from gravitational collapse of the thickened and heated orogenic crust. Active rift basins in the northern North China craton are attributed to uprising of asthenospheric materials along a lithospheric-scale tear fault. Slab tearing of the subducting paleo-Pacific plate is postulated and well explains the spatial distribution of different types of rift basins and the eastward shifting of magmatism in the northern North China craton. The Late Cretaceous witnessed a period of mild deformation and weak magmatism, which was possibly due to kinematic variation of the paleo-Pacific plate.

High-precision geochronological constraints on the duration of 'Dinosaur Pompeii' and the Yixian Formation.

High-precision U-Pb zircon ages of 125.755 ± 0.061 Ma and 124.122 ± 0.048 Ma, respectively, represent the onset and termination of Yixian Formation in the Jin-Yang basin, bracketing its duration to 1.633 ± 0.078 Myr.

Molybdenum isotopes unmask slab dehydration and melting beneath the Mariana arc.

How serpentinites in the forearc mantle and subducted lithosphere become involved in enriching the subarc mantle source of arc magmas is controversial. Here we report molybdenum isotopes for primitive submarine lavas and serpentinites from active volcanoes and serpentinite mud volcanoes in the Mariana arc. These data, in combination with radiogenic isotopes and elemental ratios, allow development of a model whereby shallow, partially serpentinized and subducted forearc mantle transfers fluid and melt from the subducted slab into the subarc mantle. These entrained forearc mantle fragments are further metasomatized by slab fluids/melts derived from the dehydration of serpentinites in the subducted lithospheric slab. Multistage breakdown of serpentinites in the subduction channel ultimately releases fluids/melts that trigger Mariana volcanic front volcanism. Serpentinites dragged down from the forearc mantle are likely exhausted at >200 km depth, after which slab-derived serpentinites are responsible for generating slab melts.

Felsic volcanism as a factor driving the end-Permian mass extinction.

The Siberian Traps large igneous province (STLIP) is commonly invoked as the primary driver of global environmental changes that triggered the end-Permian mass extinction (EPME). Here, we explore the contributions of coeval felsic volcanism to end-Permian environmental changes. We report evidence of extreme Cu enrichment in the EPME interval in South China. The enrichment is associated with an increase in the light Cu isotope, melt inclusions rich in copper and sulfides, and Hg concentration spikes. The Cu and Hg elemental and isotopic signatures can be linked to S-rich vapor produced by felsic volcanism. We use these previously unknown geochemical data to estimate volcanic SO2 injections and argue that this volcanism would have produced several degrees of rapid cooling before or coincident with the more protracted global warming. Large-scale eruptions near the South China block synchronous with the EPME strengthen the case that the STLIP may not have been the sole trigger.

Wet deposition of nitrogen and sulfur in Guangzhou, a subtropical area in South China.

With the aim of understanding the seasonal distribution of deposition fluxes of nitrogen (N) and sulfur (S) in South China, a main acid deposition region in China, precipitation samples were collected and analyzed from Guangzhou area, between March 2005 and February 2006. The estimated wet deposition of N (including ammonium nitrogen (NH(4)-N) and nitrate nitrogen (NO(3)-N)) and S (sulfate sulfur (SO(4)-S)) during the monitoring period were 40.47 kg N ha(-1) and 65.29 kg S ha(-1), respectively. The average deposition of NH(4)-N was ∼1.5 times of the NO(3)-N deposition, suggesting that the reduced and oxidized N depositions were comparable in the study area. The S and N depositions in the rainy season were greater than those in the dry season, showing great seasonal variation, which was consistent with both the distribution of precipitation and the period of fertilizer application for agriculture. The N and S wet deposition fluxes in Guangzhou were greater than those in Beijing and Zhengzhou, located in the northern China, but comparable to the level of Chongqing, located in the southwestern China, another major acid deposition region. The atmospheric N and S depositions in these cities from north to south were affected by both intensive agricultural and industrial activities.

Cloning, Prokaryotic Soluble Expression, and Analysis of Antiviral Activity of Two Novel Feline IFN-ω Proteins.

Cats are becoming more popular as household companions and pets, forming close relationships with humans. Although feline viral diseases can pose serious health hazards to pet cats, commercialized preventative vaccines are lacking. Interferons (IFNs), especially type I IFNs (IFN-α, IFN-ß, and interferon omega (IFN-ω)), have been explored as effective therapeutic drugs against viral diseases in cats. Nevertheless, there is limited knowledge regarding feline IFN-ω (feIFN-ω), compared to IFN-α and IFN-ß. In this study, we cloned the genes encoding feIFN-ωa and feIFN-ωb from cat spleen lymphocytes. Homology and phylogenetic tree analysis revealed that these two genes belonged to new subtypes of feIFN-ω. The recombinant feIFN-ωa and feIFN-ωb proteins were expressed in their soluble forms in Escherichia coli, followed by purification. Both proteins exhibited effective anti-vesicular stomatitis virus (VSV) activity in Vero, F81 (feline kidney cell), Madin-Darby bovine kidney (MDBK), Madin-Darby canine kidney (MDCK), and porcine kidney (PK-15) cells, showing broader cross-species antiviral activity than the INTERCAT IFN antiviral drug. Furthermore, the recombinant feIFN-ωa and feIFN-ωb proteins demonstrated antiviral activity against VSV, feline coronavirus (FCoV), canine parvovirus (CPV), bovine viral diarrhea virus (BVDV), and porcine epidemic diarrhea virus (PEDV), indicating better broad-spectrum antiviral activity than the INTERCAT IFN. The two novel feIFN-ω proteins (feIFN-ωa and feIFN-ωb) described in this study show promising potential to serve as effective therapeutic agents for treating viral infections in pet cats.

Immunogenicity in chickens with orally administered recombinant chicken-borne Lactobacillus saerimneri expressing FimA and OmpC antigen of O78 avian pathogenic Escherichia coli.

PURPOSE: Avian colibacillosis is responsible for economic losses to poultry producers worldwide. To combat this, we aimed to develop an effective oral vaccine for chicken against O78 avian pathogenic Escherichia coli (APEC) infection through a Lactobacillus delivery system. METHODOLOGY: Eight Lactobacillus strains isolated from the intestines of broiler chickens were evaluated based on their in vitro adherence ability to assess their potential as a delivery vector. Fimbrial subunit A (FimA) and outer-membrane protein C (OmpC) of APEC with and without fusion to dendritic cell-targeting peptide (DCpep) and microfold cell-targeting peptide (Co1) were displayed on the surface of Lactobacillus saerimneri M-11 and yielded vaccine groups (pPG-ompC-fimA/M-11 and pPG-ompC-fimA-Co1-DCpep/M-11, respectively). The colonization of the recombinant strains in vivo was assessed and the immunogenicity and protective efficacy of orally administered recombinant strains in chickens were evaluated. RESULTS: The colonization of the recombinant strains in vivo revealed no significant differences between the recombinant and wild-type strains. Chickens orally administered with vaccine groups showed significantly higher levels of OmpC/FimA-specific IgG in serum and mucosal IgA in cecum lavage, nasal lavage and stool compared to the pPG/M-11 group. After challenge with APEC CVCC1553, better protective efficacy was observed in chickens orally immunized with pPG-ompC-fimA/M-11 and pPG-ompC-fimA-Co1-DCpep/M-11, but no significant differences were observed between the two groups. CONCLUSIONS: Recombinant chicken-borne L. saerimneri M-11 showed good immunogenicity in chickens, suggesting that it may be a promising vaccine candidate against APEC infections. However, the activity of mammalian DCpep and Co1 was not significant in chickens.

[Co-expression of CSFV T cell epitope E290 peptide and PPV VP2 protein in Lactobacillus casei and determination of specific antibodies in immunized mice].

Lactobacillus casei strain 393 was selected as an antigen delivery vehicle for the development of oral vaccine to express recombinant classical swine fever virus (CSFV) T cell epitope E290 peptide and porcine parvovirus (PPV) VP2 protein. The recombinant genes encoding CSFV T cell epitope E290 peptide and PPV VP2 protein, respectively, were cloned into the secretion expression vector pPG, and then the pPG-VP2-E290 was electrotransformed into L. casei 393 giving rise to recombinant strain pPG-VP2-E290/L. casei 393. The recombinant L. casei 393 was induced by 2% lactose in MRS and about 70kDa protein was detected with SDS-PAGE in induced recombinant strain and culture supernatants. The result of Western blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein. The indirect ELISA test also indicated that the interest protein was expressed and secreted from the recombinant strain. Specific anti-PPV VP2 secret immunoglobulin A (sIgA) antibody was detected by indirect ELISA in the feces, anti-PPV VP2 and anti-CSFV E290 peptide immunoglobulin G (IgG) antibody was detected by indirect ELISA in the serum of immunized mice after intragastric administration. The results indicated that the mice immunized with recombinant strain pPG-VP2-E290/L. casei 393 could produce clear antibody level, which establish important material basement for the development of lactic acid bacteria oral vaccine of recombinant CSFV and PPV.

[Construction of recombinant Lactococcus lactis expressing porcine transmissible gastroenteritis spike glycoprotein and analysis of immunogenicity].

Transmissible gastroenteritis virus (TGEV), is an enteropathogenic coronavirus that causes a highly fatal acute diarrhea in newborn pigs. It's typically clinical manifestations consist of omitting, severe diarrhea, loss water and highly infectious disease. All kinds and ages of pigs can be infected. Particular, the mortality piglets under 3 weeks may reach 100% . The effective protection against TGEV requires the development of vaccines that are able to induce local mucosal immunization. Lactococcus lactis was selected as a bacterial carrier for the expression of TGEV spike glycoprotein. The gene of S glycoprotein was cloned into the Lactococcus lactis vectors pNZ8112. An approximately 2000 bps fragments of TGEV gene S that encompasses all the four major antigenic domains critical for neutralization was transformed into Lactococcus lactis NZ9000 by electroporation, resulting in the recombinant strain pNZ8112-Sa/NZ9000. The recombinant glycoprotein S was detected by SDS-PAGE and Western blot after induced by 1ng/mL nisin. The result indicated that the expressed product maintain the antigenicity of TGEV as expected. In order to detect the location of expressed protein, the yellow and green fluorescence of the recombinant strain pNZ8112-Sa/NZ9000 was detected by the IFA experiments, which indicated that the expressed recombinant protein was secreted and located on the surface of the bacterium cell. Oral immunization of BALB/c mice with recombinant strain that constitutively express the 66kDa fragment of the glycoprotein S, Specific anti-TGEV glycoprotein S secret immunoglobulin A (sIgA) antibodies were detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces after immunization. It was showed that the mice immunized with pNZ8112-Sa/NZ9000 recombinant strain had produced clear antibody level anti TGEV, and which had provided important substance foundation and explored the feasibility of Lactobacillus as oral vaccine.