Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability.

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Structure-based design of a single-chain triple-disulfide-stabilized fusion-glycoprotein trimer that elicits high-titer neutralizing responses against human metapneumovirus.

The Pneumoviridae family of viruses includes human metapneumovirus (HMPV) and respiratory syncytial virus (RSV). The closely related Paramyxoviridae family includes parainfluenza viruses (PIVs). These three viral pathogens cause acute respiratory tract infections with substantial disease burden in the young, the elderly, and the immune-compromised. While promising subunit vaccines are being developed with prefusion-stabilized forms of the fusion glycoproteins (Fs) of RSV and PIVs, for which neutralizing titers elicited by the prefusion (pre-F) conformation of F are much higher than for the postfusion (post-F) conformation, with HMPV, pre-F and post-F immunogens described thus far elicit similar neutralizing responses, and it has been unclear which conformation, pre-F or post-F, would be the most effective HMPV F-vaccine immunogen. Here, we investigate the impact of further stabilizing HMPV F in the pre-F state. We replaced the furin-cleavage site with a flexible linker, creating a single chain F that yielded increased amounts of pre-F stabilized trimers, enabling the generation and assessment of F trimers stabilized by multiple disulfide bonds. Introduced prolines could increase both expression yields and antigenic recognition by the pre-F specific antibody, MPE8. The cryo-EM structure of a triple disulfide-stabilized pre-F trimer with the variable region of antibody MPE8 at 3.25-Å resolution confirmed the formation of designed disulfides and provided structural details on the MPE8 interface. Immunogenicity assessments in naïve mice showed the triple disulfide-stabilized pre-F trimer could elicit high titer neutralization, >10-fold higher than elicited by post-F. Immunogenicity assessments in pre-exposed rhesus macaques showed the triple disulfide-stabilized pre-F could recall high neutralizing titers after a single immunization, with little discrimination in the recall response between pre-F and post-F immunogens. However, the triple disulfide-stabilized pre-F adsorbed HMPV-directed responses from commercially available pooled human immunoglobulin more fully than post-F. Collectively, these results suggest single-chain triple disulfide-stabilized pre-F trimers to be promising HMPV-vaccine antigens.

Diverse Murine Vaccinations Reveal Distinct Antibody Classes to Target Fusion Peptide and Variation in Peptide Length to Improve HIV Neutralization.

While neutralizing antibodies that target the HIV-1 fusion peptide have been elicited in mice by vaccination, antibodies reported thus far have been from only a single antibody class that could neutralize ~30% of HIV-1 strains. To explore the ability of the murine immune system to generate cross-clade neutralizing antibodies and to investigate how higher breadth and potency might be achieved, we tested 17 prime-boost regimens that utilized diverse fusion peptide-carrier conjugates and HIV-1 envelope trimers with different fusion peptides. We observed priming in mice with fusion peptide-carrier conjugates of variable peptide length to elicit higher neutralizing responses, a result we confirmed in guinea pigs. From vaccinated mice, we isolated 21 antibodies, belonging to 4 distinct classes of fusion peptide-directed antibodies capable of cross-clade neutralization. Top antibodies from each class collectively neutralized over 50% of a 208-strain panel. Structural analyses - both X-ray and cryo-EM - revealed each antibody class to recognize a distinct conformation of fusion peptide and to have a binding pocket capable of accommodating diverse fusion peptides. Murine vaccinations can thus elicit diverse neutralizing antibodies, and altering peptide length during prime can improve the elicitation of cross-clade responses targeting the fusion peptide site of HIV-1 vulnerability. IMPORTANCE The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence. We found that variation in peptide length during prime elicits enhanced neutralizing responses in mice and guinea pigs. We identified vaccine-elicited murine monoclonal antibodies from distinct classes capable of cross-clade neutralization and of diverse fusion peptide recognition. Our findings lend insight into improved immunogens and regimens for HIV-1 vaccine development.

Assessment of Crosslinkers between Peptide Antigen and Carrier Protein for Fusion Peptide-Directed Vaccines against HIV-1.

Conjugate-vaccine immunogens require three components: a carrier protein, an antigen, and a crosslinker, capable of coupling antigen to carrier protein, while preserving both T-cell responses from carrier protein and B-cell responses from antigen. We previously showed that the N-terminal eight residues of the HIV-1 fusion peptide (FP8) as an antigen could prime for broad cross-clade neutralizing responses, that recombinant heavy chain of tetanus toxin (rTTHC) as a carrier protein provided optimal responses, and that choice of crosslinker could impact both antigenicity and immunogenicity. Here, we delve more deeply into the impact of varying the linker between FP8 and rTTHC. In specific, we assessed the physical properties, the antigenicity, and the immunogenicity of conjugates for crosslinkers ranging in spacer-arm length from 1.5 to 95.2 Å, with varying hydrophobicity and crosslinking-functional groups. Conjugates coupled with different degrees of multimerization and peptide-to-rTTHC stoichiometry, but all were well recognized by HIV-fusion-peptide-directed antibodies VRC34.01, VRC34.05, PGT151, and ACS202 except for the conjugate with the longest linker (24-PEGylated SMCC; SM(PEG)24), which had lower affinity for ACS202, as did the conjugate with the shortest linker (succinimidyl iodoacetate; SIA), which also had the lowest peptide-to-rTTHC stoichiometry. Murine immunizations testing seven FP8-rTTHC conjugates elicited fusion-peptide-directed antibody responses, with SIA- and SM(PEG)24-linked conjugates eliciting lower responses than the other five conjugates. After boosting with prefusion-closed envelope trimers from strains BG505 clade A and consensus clade C, trimer-directed antibody-binding responses were lower for the SIA-linked conjugate; elicited neutralizing responses were similar, however, though statistically lower for the SM(PEG)24-linked conjugate, when tested against a strain especially sensitive to fusion-peptide-directed responses. Overall, correlation analyses revealed the immunogenicity of FP8-rTTHC conjugates to be negatively impacted by hydrophilicity and extremes of length or low peptide-carrier stoichiometry, but robust to other linker parameters, with several commonly used crosslinkers yielding statistically indistinguishable serological results.

Inter-Group Face Recognition Bias Was Modulated by the Group Status.

Previous studies have shown that social categorization can induce an own-group face recognition bias. However, similar and better other-group face recognition emerged recently. In this research, we aimed to examine whether competitive cues and group status accompanied by social categorization can modulate the inter-group face recognition bias. Moreover, we investigated how the group identification of individuals with different statuses affected the inter-group face recognition bias. The results indicated that an own-group face recognition bias emerged for targets with in-group labels compared to out-group labels. Moreover, when the group labels signaled competitive cues, the own-group face recognition bias was reversed. Furthermore, low-status and similar-status individuals exhibited out-group face recognition bias, but high-status individuals did not. In addition, the higher the in-group identification scores of participants from the low-status group, the stronger the out-group face recognition bias. These results suggested that competitive cues would reverse the own-group face recognition bias and the group status would play a modulating role in face recognition bias.

[Impacts of arbuscular mycorrhizal fungi (AMF)on growth, N bio-fixation, and phosphorus uptake of legume crop.] / ä¸›æžèŒæ ¹çœŸèŒå¯¹è±†ç§‘ä½œç‰©ç”Ÿé•¿å’Œç”Ÿç‰©å›ºæ°®åŠç£·ç´ å¸æ”¶çš„å½±å“.

To explore the effects of arbuscular mycorrhizal fungi (AMF) on the growth of legume crop, pot and field experiments with soybean were conducted. Treatments of inoculation (+AMF) and non-inoculation with AMF (-AMF) were set up for the pot experiment, and AMF mycelium non-limited and limited for the field experiment. Results of the pot experiment showed that inoculation with AMF significantly increased soybean aboveground biomass (16.5%) and root nodules number (131.4%), above-ground plant phosphorus and nitrogen concentrations and uptakes. In the field trial, the above-ground and root biomasses and root nodules number under AMF mycelium non-limited were significantly increased by 123.6%, 61.5%, and 212.5% compared with those under the limited condition, respectively. Plant phosphorus uptake, nitrogen concentration and uptake, and soil available nitrogen and phosphorus concentrations were significantly higher under AMF mycelium non-limited than the limited both in both shoot and root. Our findings provide theoretical reference for further understanding the relationship between legume crop and AMF, as well as the efficient utilization of phosphorus fertilizer.

Long-term organic and inorganic fertilization alters the diazotrophic abundance, community structure, and co-occurrence patterns in a vertisol.

Diazotrophs play a critical role in converting air-inactive nitrogen to bio-available nitrogen. Assessing the influences of different fertilization regimes on diazotrophs is essential for a better understanding of their maintenance of soil fertility and agricultural sustainability. In this study, we targeted the nifH gene to investigate the effects of different long-term fertilization on the diazotrophic community in a vertisol, using real-time quantitative polymerase chain reaction (PCR) and MiSeq sequencing. Five fertilization regimes were tested: no fertilizer (CK), chemical nitrogen, phosphorus, and potassium fertilizer (NPK), organic fertilizer (O), chemical NPK plus organic fertilizer with an equivalent application rate of nitrogen (NPKO), and chemical NPK plus organic fertilizer with a high application rate of nitrogen (HNPKO). Our results showed that fertilization significantly affected the diazotrophic activity, abundance and composition. NPK tended to reduce the activity, abundance, operational taxonomic units (OTU)-richness and alpha-diversity of the diazotrophs, while O had the opposite effect. The effects of inorganic and organic fertilization on the diazotrophs depended on the N application rate, showing that the diazotrophic activity, abundance, and alpha-diversity in NPKO were higher than that of HNPKO. For the diazotrophic community structure, CK, O, and NPKO were grouped and separated from NPK and HNPKO. The diazotrophic community structure strongly correlated with the soil pH, electrical conductivity (EC), total carbon content (TC), and total nitrogen content (TN), among which pH was the major factor shaping the diazotrophic community structure. Different network patterns were observed between the long-term organic and non-organic fertilizers, suggesting that the organic amendment resulted in a more complicated diazotrophic community than the non-organic amendments. Rhizobium was the most important hub connecting members in the community. These results indicated that organic amendments are beneficial to diazotrophic activity, abundance, OTU richness, alpha-diversity, and the diazotrophic communities' potential interactions, which may enhance biological nitrogen fixation in vertisols.

Assessment of Water and Nitrogen Use Efficiencies Through UAV-Based Multispectral Phenotyping in Winter Wheat.

Unmanned aerial vehicle (UAV) based remote sensing is a promising approach for non-destructive and high-throughput assessment of crop water and nitrogen (N) efficiencies. In this study, UAV was used to evaluate two field trials using four water (T0 = 0 mm, T1 = 80 mm, T2 = 120 mm, and T3 = 160 mm), and four N (T0 = 0, T1 = 120 kg ha-1, T2 = 180 kg ha-1, and T3 = 240 kg ha-1) treatments, respectively, conducted on three wheat genotypes at two locations. Ground-based destructive data of water and N indictors such as biomass and N contents were also measured to validate the aerial surveillance results. Multispectral traits including red normalized difference vegetation index (RNDVI), green normalized difference vegetation index (GNDVI), normalized difference red-edge index (NDRE), red-edge chlorophyll index (RECI) and normalized green red difference index (NGRDI) were recorded using UAV as reliable replacement of destructive measurements by showing high r values up to 0.90. NGRDI was identified as the most efficient non-destructive indicator through strong prediction values ranged from R 2 = 0.69 to 0.89 for water use efficiencies (WUE) calculated from biomass (WUE.BM), and R 2 = 0.80 to 0.86 from grain yield (WUE.GY). RNDVI was better in predicting the phenotypic variations for N use efficiency calculated from nitrogen contents of plant samples (NUE.NC) with high R 2 values ranging from 0.72 to 0.94, while NDRE was consistent in predicting both NUE.NC and NUE.GY by 0.73 to 0.84 with low root mean square errors. UAV-based remote sensing demonstrates that treatment T2 in both water 120 mm and N 180 kg ha-1 supply trials was most appropriate dosages for optimum uptake of water and N with high GY. Among three cultivars, Zhongmai 895 was highly efficient in WUE and NUE across the water and N treatments. Conclusively, UAV can be used to predict time-series WUE and NUE across the season for selection of elite genotypes, and to monitor crop efficiency under varying N and water dosages.

Higher yields and lower methane emissions with new rice cultivars.

Breeding high-yielding rice cultivars through increasing biomass is a key strategy to meet rising global food demands. Yet, increasing rice growth can stimulate methane (CH4 ) emissions, exacerbating global climate change, as rice cultivation is a major source of this powerful greenhouse gas. Here, we show in a series of experiments that high-yielding rice cultivars actually reduce CH4 emissions from typical paddy soils. Averaged across 33 rice cultivars, a biomass increase of 10% resulted in a 10.3% decrease in CH4 emissions in a soil with a high carbon (C) content. Compared to a low-yielding cultivar, a high-yielding cultivar significantly increased root porosity and the abundance of methane-consuming microorganisms, suggesting that the larger and more porous root systems of high-yielding cultivars facilitated CH4 oxidation by promoting O2 transport to soils. Our results were further supported by a meta-analysis, showing that high-yielding rice cultivars strongly decrease CH4 emissions from paddy soils with high organic C contents. Based on our results, increasing rice biomass by 10% could reduce annual CH4 emissions from Chinese rice agriculture by 7.1%. Our findings suggest that modern rice breeding strategies for high-yielding cultivars can substantially mitigate paddy CH4 emission in China and other rice growing regions.

Optimizing rice plant photosynthate allocation reduces N2O emissions from paddy fields.

Rice paddies are a major source of anthropogenic nitrous oxide (N2O) emissions, especially under alternate wetting-drying irrigation and high N input. Increasing photosynthate allocation to the grain in rice (Oryza sativa L.) has been identified as an effective strategy of genetic and agronomic innovation for yield enhancement; however, its impacts on N2O emissions are still unknown. We conducted three independent but complementary experiments (variety, mutant study, and spikelet clipping) to examine the impacts of rice plant photosynthate allocation on paddy N2O emissions. The three experiments showed that N2O fluxes were significantly and negatively correlated with the ratio of grain yield to total aboveground biomass, known as the harvest index (HI) in agronomy (P < 0.01). Biomass accumulation and N uptake after anthesis were significantly and positively correlated with HI (P < 0.05). Reducing photosynthate allocation to the grain by spikelet clipping significantly increased white root biomass and soil dissolved organic C and reduced plant N uptake, resulting in high soil denitrification potential (P < 0.05). Our findings demonstrate that optimizing photosynthate allocation to the grain can reduce paddy N2O emissions through decreasing belowground C input and increasing plant N uptake, suggesting the potential for genetic and agronomic efforts to produce more rice with less N2O emissions.

Climatic warming increases winter wheat yield but reduces grain nitrogen concentration in east China.

Climatic warming is often predicted to reduce wheat yield and grain quality in China. However, direct evidence is still lacking. We conducted a three-year experiment with a Free Air Temperature Increase (FATI) facility to examine the responses of winter wheat growth and plant N accumulation to a moderate temperature increase of 1.5°C predicted to prevail by 2050 in East China. Three warming treatments (AW: all-day warming; DW: daytime warming; NW: nighttime warming) were applied for an entire growth period. Consistent warming effects on wheat plant were recorded across the experimental years. An increase of ca. 1.5°C in daily, daytime and nighttime mean temperatures shortened the length of pre-anthesis period averagely by 12.7, 8.3 and 10.7 d (P<0.05), respectively, but had no significant impact on the length of the post-anthesis period. Warming did not significantly alter the aboveground biomass production, but the grain yield was 16.3, 18.1 and 19.6% (P<0.05) higher in the AW, DW and NW plots than the non-warmed plot, respectively. Warming also significantly increased plant N uptake and total biomass N accumulation. However, warming significantly reduced grain N concentrations while increased N concentrations in the leaves and stems. Together, our results demonstrate differential impacts of warming on the depositions of grain starch and protein, highlighting the needs to further understand the mechanisms that underlie warming impacts on plant C and N metabolism in wheat.

[Effects of tillage patterns on photosynthetic and chlorophyll fluorescence characteristics of maize in rainfed area of Northeast China].

In 2010-2011, a field experiment was conducted in Northeast China to evaluate the effects of different tillage patterns on the temperature and moisture in topsoil layer and the leaf photosynthesis and chlorophyll fluorescence of maize. The effects of tillage patterns on the soil temperature and moisture mainly manifested at sowing-jointing stage. In treatments flat planting with ridging at early jointing stage (PL) and flat planting without ridging (PP), the soil moisture content at the depth of 0-40 cm was significantly higher than that in treatment ridge planting (LL), with the increment being 5.6% and 5.2%, 4.6% and 7.3%, and 3.9% and 4.8% at emergency, seedling, and jointing stages, respectively. The minimum temperature at the soil depth 5 cm at seedling stage in PL and PP was 1.4 and 1.3 degrees C higher than that in LL, respectively. Due to the improvement of soil water and thermal conditions, the leaf photosynthetic rate (Pn) and transpiration rate (Tr) at jointing stage in PL and PP were significantly higher than those in LL, whereas the PS II potential activity (Fv/Fo) and PS II maximal photochemical efficiency (Fv/Fm) had no significant differences among the treatments, indicating that the stomatal factors such as stomata conductivity and stomata limitation were the main factors inducing the photosynthesis differences among the treatments. Furthermore, the Pn and Tr at grain filling stage in LL and PL were higher than those in PP, mainly due to the high water-logging risk in PP in strong rainfall season. Consequently, treatment PL could promote maize photosynthesis through improving soil water and thermal conditions, and further, increase maize grain yield.

[Effects of irrigation amount on water use characteristics and grain yield of wheat under different nitrogen application rates].

A field experiment was conducted to examine the effects of irrigation amount on the water consumption, flag leaf physiological characteristics, and grain yield of wheat under the nitrogen application rates 180 kg x hm(-2)(N180)) and 240 kg x hm(-2)(N240). Four irrigation regimes were designed, i.e., no irrigation during whole growth period (W0), irrigation with 60 mm water before sowing (W1), irrigation with 60 mm water before sowing and at jointing stage, respectively (W2), and irrigation with 60 mm water before sowing, at jointing stage, and at flowering stage, respectively (W3). In treatment W0, the water consumption amount below 100 cm soil layer was lower than that in other treatments; and in treatments W1 and W2, the water consumption amount in 100-200 cm and 0-200 cm soil layers was higher than that in treatment W3. The soil water consumption amount in 0-80 cm soil layer, the consumption percentage from flowering to maturing stage, and the total water consumption amount were all higher under N240 than under N180. At mid and late grain-filling stages, the relative water content (RWC) and water potential (psi w) of flag leaves were higher in treatments W2 and W3 than in treatments W0 and W1. The RWC and psi w at late grain-filling stage were higher in treatments N240W0 and N240 than in N180W0 and N180 W1, respectively, but had no significant differences between treatments N240W2 and N180W2, and N240W3 and N180W3. In this study, treatment N180W2 had the higher grain yield, water use efficiency (WUE), and nitrogen use efficiency, while over-irrigation increased the water consumption amount, and decreased the WUE, irrigation water use efficiency, and irrigation benefit.

[Effects of water-nitrogen interaction on soil water utilization by wheat and fructan content in wheat stem].

A field experiment was conducted to study the effects of water-nitrogen interaction on the flag leaf photosynthetic rate, penult stem fructan content, fertilizer N use efficiency, and soil water use efficiency of strong gluten wheat variety 'Jimai 20'. Three N application levels (N0, 0 kg x hm(-2); N1, 180 kg x hm(-2); and N2, 240 kg x hm(-2)) and four irrigation schedules (W0, no irrigation; W1, irrigation before sowing and at jointing and flowering stages; W2, irrigation before sowing, before wintering, and at jointing and flowering stages; W3, irrigation before sowing, before wintering, and at jointing, flowering, and grain-filling stages) were designed. The irrigation amount was 60 mm each time. Among the N treatments, treatment N1 had a higher flag leaf net photosynthetic rate and penult stem fructan content, and the highest grain yield, apparent N use efficiency, agronomic N use efficiency, and water use efficiency. Treatment N2 had a higher penult stem fructan content than treatments N0 and N1. No N application and applying excessive N did not benefit the increase of grain yield, fertilizer N use efficiency, and water use efficiency. Treatment W1 promoted the fructan accumulation in penult stem and accelerated the translocation of the accumulated fructan to grain, being beneficial to the increase of grain yield. Treatment N1W1 had the highest grain yield, and higher fertilizer N use efficiency and water use efficiency. Excessive irrigation and N application increased the flag leaf net photosynthetic rate and penult stem fructan content, but decreased the fertilizer N use efficiency and water use efficiency, with no significant effects on grain yield.