An M2e-based multiple antigenic peptide vaccine protects mice from lethal challenge with divergent H5N1 influenza viruses

<p>Abstract</p> <p>Background</p> <p>A growing concern has raised regarding the pandemic potential of the highly pathogenic avian influenza (HPAI) H5N1 viruses. Consequently, there is an urgent need to develop an effective and safe vaccine against the divergent H5N1 influenza viruses. In the present study, we designed a tetra-branched multiple antigenic peptide (MAP)-based vaccine, designated M2e-MAP, which contains the sequence overlapping the highly conserved extracellular domain of matrix protein 2 (M2e) of a HPAI H5N1 virus, and investigated its immune responses and cross-protection against different clades of H5N1 viruses.</p> <p>Results</p> <p>Our results showed that M2e-MAP vaccine induced strong M2e-specific IgG antibody responses following 3-dose immunization of mice with M2e-MAP in the presence of Freunds' or aluminium (alum) adjuvant. M2e-MAP vaccination limited viral replication and attenuated histopathological damage in the challenged mouse lungs. The M2e-MAP-based vaccine protected immunized mice against both clade1: VN/1194 and clade2.3.4: SZ/406H H5N1 virus challenge, being able to counteract weight lost and elevate survival rate following lethal challenge of H5N1 viruses.</p> <p>Conclusions</p> <p>These results suggest that M2e-MAP presenting M2e of H5N1 virus has a great potential to be developed into an effective subunit vaccine for the prevention of infection by a broad spectrum of HPAI H5N1 viruses.</p

Effects of urease and nitrification inhibitors on nitrous oxide emissions and nitrifying/denitrifying microbial communities in a rainfed maize soil: A 6-year field observation

Application of the inhibitor-amended nitrogen fertilizers is a recommended method for reducing agricultural nitrous oxide (N2O) emission. However, the potential impacts of these inhibitors on soil environments still needs to be evaluated using long-term experiments. Through a 6-year field observation, the effects of combined application of a nitrification inhibitor (dicyandiamide, DCD) and a urease inhibitor (hydroquinone, HQ) on N2O emission, as well as soil ammonia oxidizers and denitrifiers in a maize (Zea mays L.) field in Northeast China were investigated. The results showed that annual soil N2O emissions were 0.466, 1.021 and 0.874 kg N2O-N ha(-1) for N0 fertilizer treatment (CK), Urea treatment (U) and Urea + DCD + HQ treatment (UDH), respectively. A significant linear correlation was found between the N2O accumulation in the first month after fertilizer application and the short-term precipitation (i.e., a period from 10 days before to 20 days after fertilizer application). The N2O emissions in the freeze-thaw period accounted for up to 42.5% of the year-round N2O emissions. The remarkable fluctuations of annual N2O emissions were observed (their coefficients of variation were 68.3%, 77.7% and 71.2% for CK, U and UDH treatments, respectively); these fluctuations were mainly attributed to the precipitation. The averaged N2O emission factors (EF) (0.308% and 0.227% for U and UDH treatments, respectively) were far less than the default mean EF of 1% proposed by (IPCC, 2006). An averaged N2O mitigation of 26.4% was fulfilled by UDH application. The results of quantitative PCR for soil nitrification and denitrification gene copy numbers measurement showed that UDH treatment significantly decreased the ammonia oxidation bacteria (AOB) amoA gene copy numbers by 74% on the 10th day after UDH application. No significant effects of combined application of DCD and HQ on microbial denitrification functional gene abundance were observed

Climate and soil parameters are more important than denitrifier abundances in controlling potential denitrification rates in Chinese grassland soils

Denitrification is an important process that influences nitrogen (N) loss and the production of greenhouse gas in grassland soils. However, the relative contributions of abiotic and biotic factors to soil denitrification potential at the regional and sub-regional scales in grassland ecosystems remain elusive. In this study, soil samples were collected from 21 sites at three steppes of China, including the Inner Mongolia Plateau (IMP), the Xinjiang Autonomous Region (XAR) and the Tibetan Plateau (TP) grasslands. Results showed that the key factors controlling the denitrification potential were regional and scale-dependent. At the sub-regional scales, soil pH, aridity index (AI) and total organic carbon (TOC) explained the highest variances on denitrification potential in the IMP, XAR and TP steppe, respectively. At the regional scale, the mean annual precipitation (MAP) was the most important environmental driver for the denitrification potential. Partial least squares (PLS) path modeling revealed that the MAP might regulate denitrification potential directly and indirectly by its effects on the plant and soil properties. Overall, these results help to improve our understandings on the prediction of the denitrification potential under global changes and revealed that the denitrification potential at various scales could be regulated by the multiple interactions of abiotic and biotic factors. (C) 2019 Published by Elsevier B.V

Stair-Step Pattern of Soil Bacterial Diversity Mainly Driven by pH and Vegetation Types Along the Elevational Gradients of Gongga Mountain, China

Ecological understandings of soil bacterial community succession and assembly mechanism along elevational gradients in mountains remain not well understood. Here, by employing the high-throughput sequencing technique, we systematically examined soil bacterial diversity patterns, the driving factors, and community assembly mechanisms along the elevational gradients of 1800–4100 m on Gongga Mountain in China. Soil bacterial diversity showed an extraordinary stair-step pattern along the elevational gradients. There was an abrupt decrease of bacterial diversity between 2600 and 2800 m, while no significant change at either lower (1800–2600 m) or higher (2800–4100 m) elevations, which coincided with the variation in soil pH. In addition, the community structure differed significantly between the lower and higher elevations, which could be primarily attributed to shifts in soil pH and vegetation types. Although there was no direct effect of MAP and MAT on bacterial community structure, our partial least squares path modeling analysis indicated that bacterial communities were indirectly influenced by climate via the effect on vegetation and the derived effect on soil properties. As for bacterial community assembly mechanisms, the null model analysis suggested that environmental filtering played an overwhelming role in the assembly of bacterial communities in this region. In addition, variation partition analysis indicated that, at lower elevations, environmental attributes explained much larger fraction of the β-deviation than spatial attributes, while spatial attributes increased their contributions at higher elevations. Our results highlight the importance of environmental filtering, as well as elevation-related spatial attributes in structuring soil bacterial communities in mountain ecosystems

Theoretical Study on Thermal Release of Helium-3 in Lunar Ilmenite

The in-situ utilization of lunar helium-3 resource is crucial to manned lunar landings and lunar base construction. Ilmenite was selected as the representative mineral which preserves most of the helium-3 in lunar soil. The implantation of helium-3 ions into ilmenite was simulated to figure out the concentration profile of helium-3 trapped in lunar ilmenite. Based on the obtained concentration profile, the thermal release model for molecular dynamics was established to investigate the diffusion and release of helium-3 in ilmenite. The optimal heating temperature, the diffusion coefficient, and the release rate of helium-3 were analyzed. The heating time of helium-3 in lunar ilmenite under actual lunar conditions was also studied using similitude analysis. The results show that after the implantation of helium-3 into lunar ilmenite, it is mainly trapped in vacancies and interstitials of ilmenite crystal and the corresponding concentration profile follows a Gaussian distribution. As the heating temperature rises, the cumulative amounts of released helium-3 increase rapidly at first and then tend to stabilize. The optimal heating temperature of helium-3 is about 1000 K and the corresponding cumulative release amount is about 74%. The diffusion coefficient and activation energy of helium-3 increase with the temperature. When the energy of helium-3 is higher than the binding energy of the ilmenite lattice, the helium-3 is released rapidly on the microscale. Furthermore, when the heating temperature increases, the heating time for thermal release of helium-3 under actual lunar conditions decreases. For the optimal heating temperature of 1000 K, the thermal release time of helium-3 is about 1 s. The research could provide a theoretical basis for in-situ helium-3 resources utilization on the moon

Stair-Step Pattern of Soil Bacterial Diversity Mainly Driven by pH and Vegetation Types Along the Elevational Gradients of Gongga Mountain, China

Ecological understandings of soil bacterial community succession and assembly mechanism along elevational gradients in mountains remain not well understood. Here, by employing the high-throughput sequencing technique, we systematically examined soil bacterial diversity patterns, the driving factors, and community assembly mechanisms along the elevational gradients of 1800-4100 m on Gongga Mountain in China. Soil bacterial diversity showed an extraordinary stair-step pattern along the elevational gradients. There was an abrupt decrease of bacterial diversity between 2600 and 2800 m, while no significant change at either lower (1800-2600 m) or higher (2800-4100 m) elevations, which coincided with the variation in soil pH. In addition, the community structure differed significantly between the lower and higher elevations, which could be primarily attributed to shifts in soil pH and vegetation types. Although there was no direct effect of MAP and MAT on bacterial community structure, our partial least squares path modeling analysis indicated that bacterial communities were indirectly influenced by climate via the effect on vegetation and the derived effect on soil properties. As for bacterial community assembly mechanisms, the null model analysis suggested that environmental filtering played an overwhelming role in the assembly of bacterial communities in this region. In addition, variation partition analysis indicated that, at lower elevations, environmental attributes explained much larger fraction of the beta-deviation than spatial attributes, while spatial attributes increased their contributions at higher elevations. Our results highlight the importance of environmental filtering, as well as elevation-related spatial attributes in structuring soil bacterial communities in mountain ecosystems

Data from: Differential impacts of nitrogen addition on rhizosphere and bulk-soil carbon sequestration in an alpine shrubland

1. Study site: The Field Research Station of Alpine Shrubland Ecosystem is located in Songpan County, Sichuan Province, China. This region belongs to a typical alpine climate. The community composition of plants was dominated by temperate and cold-temperate vegetation at our study region. The dominant shrub species was Sibiraea angustata, followed by Salix oritrepha, Spiraea alpine, and Potentilla fruticosa (Wang et al., 2017). The average coverage, basal diameter and height of Sibiraea angustata are 53%, 0.62 cm and 1.03 m, respectively. Our previous study showed that the aboveground and belowground biomasses of Sibiraea angustata were 3.71 kg m-2 and 4.39 kg m-2, respectively, accounting for 96% and 97% of the aboveground and belowground biomass (Wang et al., 2017). These results suggest that both the aboveground- and belowground-biomass of Sibiraea angustata have an overwhelming superiority within this shrub community. 2. The manipulated experiment of nitrogen addition: An experiment of nitrogen addition was initiated within this shrubland ecosystem in May 2012. A randomized block design with three replicated blocks of three treatments was established before the experiment; these treatments included the control (no N fertilizer), low-N addition (50 kg N ha-1 year-1), and HN (100 kg N ha-1 year-1). Each block included three plots in 5 m × 5 m, and each plot was surrounded by 10-m-wide buffer strips. According to the average increase of bulk N deposition across China (approximately 8 kg N ha-1 yr-1; Liu et al., 2013) and the level of N fertilizer experiments performed in neighbor sites on the QTP (10 and 350 kg N ha-1 year-1; Fu et al., 2017), we chose the doses of the N additions in our study equal to be nearly 3- and 5-times the value of the background N deposition. An ammonium nitrate (NH4NO3) solution has been sprayed onto the forest floor in 6 equal doses in the first week of every month from May to October (i.e., the growing season) since May 2012. In each N-addition event, the fertilizer was weighed, dissolved in 20 L of water, and sprayed evenly using backpack sprayers. The control plots received an equal volume of water. 3. Soil sampling protocol: We sampled the upper 15 cm of mineral soil in July 2017. After organic horizon removal, five replicate cores from each plot using a 6-cm diameter soil corer to ensure that fine roots would have a sufficient mass of adhering rhizosphere soil. The soil cores were stored on ice transported to the laboratory. The living roots of Sibiraea angustata in each core were empirically identified by features such as shape, color, and elasticity. The soil adhering to the roots was carefully separated from the roots using fine forceps; this fraction was operationally defined as rhizosphere soil, and non-adhering soil was considered to be bulk soil (Phillips et al., 2011). All soil samples were passed through a 2-mm mesh sieve and were divided into two parts: one part was used to determine the soil physicochemical properties and the soil carbon density fractionation; another part was stored at -20 °C for later analyses of microbial biomass carbon and enzymatic activity. 4. Basic physicochemical properties: For soil bulk density (SBD) determination, a bulk-density corer with 5-cm diameter stainless steel rings as an inner sleeve was manually inserted into the upper 15-cm depth of the mineral soil (three corers for each plot) (Davidson et al., 2004). The SOC was determined by dichromate oxidation and titration with ferrous ammonium sulfate (Walkley and Black, 1934). The DOC was extracted with 0.5 M K2SO4 and determined using a TOC analyzer (Vario TOC, Elementar Corp., UK). The total N concentration (SON) in soil was determined on a TOC analyzer (Vario TOC, Elementar Corp., UK). Soil NH4+ and NO3- were extracted by 2 M KCl (soil:solution = 1:5) and then determined on a continuous flow injection analyzer (SEAL Analytical, Germany). The dissolved inorganic N (DIN) was the sum of NH4+ and NO3-. The soil organic N concentration (SON) in soil was the difference between TSN and DIN. Available phosphorus (Av.P) was extracted with Bray-I solution (0.03 M NH4F - 0.025 M HCl) (Bray and Kurtz, 1945) and was determined by molybdenum antimony colorimetry. Soil pH was measured in slurry with a soil-to-water ratio of 1:2.5 using a pH meter (Mettler-Toledo Instruments Co., Ltd., Shanghai, China). 5. Soil carbon fractionations: The SOC fractions were separated using a density fraction method (McLauchlan and Hobbie, 2004). Briefly, a 15 g of air-dried rhizosphere or bulk soil (passed through 2mm-mesh sieve) was weighed and placed in a 100 mL centrifuge tube and dispersed in 50 mL of NaI (with a density of 1.8 g cm−3). The tubes were centrifuged at 3000 rpm for 20 min. The suspended materials (FLF) were decanted into a vacuum filter unit with 0.40 µm polycarbonate filter. This process was repeated 2-3 times until no floating material remained. The materials remaining at the bottom (HF) of the centrifuge tube were then rinsed into the vacuum filter unit. All samples on the filter paper were washed with 75 mL 0.01 mol L−1 CaCl2, followed by at least 75 mL of distilled water. The light and heavy materials were dried at 60°C for 48 h and weighed. All samples were passed through a 0.25 mm mesh sieve and analyzed for SOC as previously described. 6. Microbial gene abundance: For microbial gene abundance, DNA was extracted from 0.25 g of soil using the MoBio Power Soil DNA isolation kit (Mobio Laboratories, CA, USA). DNA quality and concentration were measured using a nanodrop spectrophotometer (NanoDrop, DE, USA) and electrophoresis in agarose gels (1% w/v in TAE), then stored at -20 °C prior to amplification. Quantitative PCR (qPCR) was used to quantify the gene copy numbers of bacterial 16S rRNA and fungal ITS using the primer pairs 515F/909R and ITS7F/ITS4R, respectively (Li et al., 2014; Schulz et al., 2018). Each 10-µL reaction contained 5 µL of SybrGreen (2×) PCR Master Mix (Bio-Rad, USA), 0.5 µL of each primer (10 pM), 2 µL of DNA templates and 2.5 µL of sterilized water. Bacterial 16S rRNA and fungal ITS conditions were 5 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 30 s at 55 °C, and 30 s at 72°C, with a final extension cycle of 8 min at 72°C. The qPCR standards for quantification were prepared from PCR products of target genes from environmental DNA with each primer set using the method described by Kou et al. (2017). Four replicates were performed for each sample. The amplification efficiencies of the 16S rDNA gene and ITS gene were 90% and 92%, respectively, with R2 values higher than 0.99, and no signals were observed in the negative controls. 7. Carbon-acquisition enzyme activities: Two grams of sieved soil was suspended in 125 mL of 50 mM sodium acetate buffer (pH = 5.0) in a homogenizer for 1 min to form slurry. Black 96-well microplates were used for fluorometric analysis. The microplates were assigned to six parts, including the sample assay, sample control, quench control, reference standard, negative control, and blank wells. First, 200 μL of buffer was pipetted into the blank, reference standard and negative control wells. Second, 50 μL of buffer was pipetted into the blank and sample control wells. Third, 200 μL of the soil slurry was pipetted into the sample assay, sample control, quench control wells, and then 50 μL of 10 μM 4-methylumbelliferyl (MUB) was pipetted into the reference standard and quench standard wells. Finally, 50 μL of 200 μM fluorogenic substrate (4-methyl-umbelliferyl β-D-glucopyranoside) were pipetted into into the negative control and sample assay wells. Plates were incubated for 4 h in the dark (25°C) and then scanned on a Varioskan Flash multiplate reader (Thermo Scientific, USA) at 365 nm excitation and 450 nm emission wavelengths. The unit for BG activity was expressed as μmol MU g-1dry soil (dry weight) h-1. Phenol oxidase and peroxidase activities were measured spectrophotometrically using L-3, 4-dihydroxyphenylalanine (L-DOPA, Sigma, St. Louis, USA) as the substrate. A total of 200 μL soil suspension (see above) and 50 μL of 25 mM DOPA were added to each sample well. The wells of peroxidase assays additionally received 10 μL of a 0.3% H2O2 solution. The microplates were incubated in the dark at 20°C for up to 8 h. Absorption was measured at 450 nm and expressed in units of μmol DOPA g−1 h−1. The background absorbance of DOPA was measured, and an extinction coefficient was calculated using a standard curve of DOPA degraded with mushroom tyrosinase.,1. Due to complex root-soil interactions, the responses of carbon (C) dynamics in the rhizosphere to elevated nitrogen (N) deposition may be different from those in bulk soil. However, the potentially different response of C dynamics in the rhizosphere and bulk soils and their contributions to soil C sequestration under N deposition is still not elucidated. 2. We conducted an N addition experiment in an alpine shrubland dominated by Sibiraea angustata located on the eastern Qinghai-Tibet Plateau (QTP). We measured the soil organic C (SOC) contents and density fractions in the rhizosphere and bulk soils in the top 15 cm of mineral soil and then employed a numerical model based on the rhizosphere extent to evaluate how the rhizosphere modulates soil C sequestration under N addition. We also measured the microbial gene abundance and C-acquisition enzyme activities to assess microbial community responses to N addition. 3. The results showed that nitrogen addition had opposite effects on the rhizosphere and bulk-soil C stocks. Specifically, N addition decreased the rhizosphere SOC content through increasing bacterial abundance, β-glucosidase activity, and thus accelerating the loss of free light fraction C (FLF-C). However, N addition increased the bulk-soil C content, which was corresponding with the reduced oxidase activities and the accelerated accumulation of heavy fraction C (HF-C) under N addition. Numerical model analysis showed that the decrease induced by N addition in rhizosphere SOC stock ranged from 0.11 to 3.01 kg C m-2 as root exudation diffusion distance extended from 0.5 mm to 2 mm, while the corresponding increase in the bulk-soil C stock ranged from 1.91 to 4.08 kg C m-2. By synthesizing the dynamics of the SOC stocks in these two soil compartments under N addition, the SOC stock at the ecosystem level exhibited an increase in range of 0.73-2.44 kg C m-2. 4. Synthesis Our results suggest that alpine shrublands on the eastern QTP have great potential for soil C sequestration under N deposition, and the magnitude of the sequestration would depend closely on the responses of rhizosphere microbial C processes and the rhizosphere extent. Our results highlight the importance of integrating rhizosphere processes into land surface models to accurately predict ecosystem functions in the background of elevated N deposition

Effects of Different Soils on the Biomass and Photosynthesis of Rumex nepalensis in Subalpine Region of Southwestern China

The performance of Rumex nepalensis, an important medicinal herb, varies significantly among subalpine grasslands, shrublands and forest ecosystems in southwestern China. Plant&ndash;soil feedback is receiving increasing interest as an important driver influencing plant growth and population dynamics. However, the feedback effects of soils from different ecosystems on R. nepalensis remain poorly understood. A greenhouse experiment was carried out to identify the effects of different soil sources on the photosynthesis and biomass of R. nepalensis. R. nepalensis was grown in soils collected from the rooting zones of R. nepalensis (a grassland soil, RS treatment), Hippophae rhamnoides (a shrub soil, HS treatment), and Picea asperata (a forest soil, PS treatment). The chlorophyll contents, net photosynthetic rates, and biomasses of R. nepalensis differed significantly among the three soils and followed the order of RS &gt; HS &gt; PS. After soil sterilization, these plant parameters followed the order of RS &gt; PS &gt; HS. The total biomass was 16.5 times higher in sterilized PS than in unsterilized PS, indicating that the existence of soil microbes in P. asperata forest ecosystems could strongly inhibit R. nepalensis growth. The root to shoot biomass ratio of R. nepalensis was the highest in the sterilized PS but the lowest in the unsterilized PS, which showed that soil microbes in PS could change the biomass allocation. Constrained redundancy analysis and path analysis suggested that soil microbes could impact the growth of R. nepalensis via the activities of soil extracellular enzymes (e.g., &beta;-1,4-N-acetylglucosaminidase (NAG)) in live soils. The soil total soluble nitrogen concentration might be the main soil factor regulating R. nepalensis performance in sterilized soils. Our findings underline the importance of the soil microbes and nitrogen to R. nepalensis performance in natural ecosystems and will help to better predict plant population dynamics

Effects of Different Soils on the Biomass and Photosynthesis of <i>Rumex nepalensis</i> in Subalpine Region of Southwestern China

The performance of Rumex nepalensis, an important medicinal herb, varies significantly among subalpine grasslands, shrublands and forest ecosystems in southwestern China. Plant–soil feedback is receiving increasing interest as an important driver influencing plant growth and population dynamics. However, the feedback effects of soils from different ecosystems on R. nepalensis remain poorly understood. A greenhouse experiment was carried out to identify the effects of different soil sources on the photosynthesis and biomass of R. nepalensis. R. nepalensis was grown in soils collected from the rooting zones of R. nepalensis (a grassland soil, RS treatment), Hippophae rhamnoides (a shrub soil, HS treatment), and Picea asperata (a forest soil, PS treatment). The chlorophyll contents, net photosynthetic rates, and biomasses of R. nepalensis differed significantly among the three soils and followed the order of RS > HS > PS. After soil sterilization, these plant parameters followed the order of RS > PS > HS. The total biomass was 16.5 times higher in sterilized PS than in unsterilized PS, indicating that the existence of soil microbes in P. asperata forest ecosystems could strongly inhibit R. nepalensis growth. The root to shoot biomass ratio of R. nepalensis was the highest in the sterilized PS but the lowest in the unsterilized PS, which showed that soil microbes in PS could change the biomass allocation. Constrained redundancy analysis and path analysis suggested that soil microbes could impact the growth of R. nepalensis via the activities of soil extracellular enzymes (e.g., β-1,4-N-acetylglucosaminidase (NAG)) in live soils. The soil total soluble nitrogen concentration might be the main soil factor regulating R. nepalensis performance in sterilized soils. Our findings underline the importance of the soil microbes and nitrogen to R. nepalensis performance in natural ecosystems and will help to better predict plant population dynamics