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Soil salinization is a globally prevalent abiotic environmental stress. The imbalance of ions caused by high concentrations of sodium chloride results in a 40% reduction in soybean yield. Soybean, as an important crop for soil quality improvement, necessitates the identification of salt-tolerant varieties and germplasms to effectively utilize and enhance saline-alkali land. In this study, we assessed the salt tolerance of 435 soybean varieties and germplasms during the seedling stage. Among them, Qihuang34, You2104, Hongzhudou, Pamanheidou, and Osage exhibited grade 1 salt tolerance rates surpassing other tested materials. Furthermore, Hongzhudou and Qihuang34 demonstrated higher salt tolerance during germination and emergence stages based on their elevated rates of emergence, salt tolerance index, chlorophyll content, and shoot fresh weights. Overall findings provide valuable resources for molecular breeding efforts aimed at developing salt-tolerant soybean varieties suitable for cultivation in saline-alkali soils.

Abstract Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease that is present in all major soybean-producing regions. The limited availability of resistant germplasm has resulted in a scarcity of commercial soybean cultivars that are resistant to the disease. To date, only the Chinese soybean landrace SX6907 has demonstrated an immune response to ASR. In this study, we present the isolation and characterization of Rpp6907-7 and Rpp6907-4, a gene pair that confer broad-spectrum resistance to ASR. Rpp6907-7 and Rpp6907-4 encode atypic nucleotide-binding leucine-rich repeat (NLR) proteins that are found to be required for NLR-mediated immunity. Genetic analysis shows that only Rpp6907-7 confers resistance, while Rpp6907-4 regulates Rpp6907-7 signaling activity by acting as a repressor in the absence of recognized effectors. Our work highlights the potential value of using Rpp6907 in developing resistant soybean cultivars.

Summary: Host specificity plays important roles in expanding the host range of rhizobia, while the genetic information responsible for host specificity remains largely unexplored. In this report, the roots of four symbiotic systems with notable different symbiotic phenotypes and the control were studied at four different post-inoculation time points by RNA sequencning (RNA-seq). The differentially expressed genes (DEGs) were divided into “found only in soybean or Lotus,” “only expressed in soybean or Lotus,” and “expressed in both hosts” according to the comparative genomic analysis. The distributions of enriched function ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways vary significantly in different symbiotic systems. Host specific genes account for the majority of the DEGs involved in response to stimulus, associated with plant-pathogen interaction pathways, and encoding resistance (R) proteins, the symbiotic nitrogen fixation (SNF) proteins and the target proteins in the SNF-related modules. Our findings provided molecular candidates for better understanding the mechanisms of symbiotic host-specificity.

Soybean (Glycine max), the primary source of high-quality plant protein, plays a crucial role as a grain and oil crop in China. Harnessing the full potential of symbiotic nitrogen fixation in soybean production holds immense significance for agriculture and ecology alike. Zhongdou 63, a newly developed early-maturing summer soybean cultivar in 2021, exhibits remarkable traits such as high yield, superior quality, multi-resistance, and wide adaptability. In this study, eight distinct rhizobia strains from diverse regions were meticulously screened to identify highly effective strains specifically suited for Zhongdou 63. The aboveground biomass, plant height, chlorophyll content, root length, nodule number, and nodule dry weight of Zhongdou 63 were measured and the data were subjected to statistical analysis. The results demonstrated that Y63-1 is a predominant strain of Zhongdou 63. Subsequently, we conducted further investigations on the broad-spectrum nodulation characteristics of Y63-1. Ten representative soybean cultivars were individually inoculated with Y63-1 and subsequently analyzed for nodule numbers and nodule dry weight in their symbiotic systems with rhizobia. The findings revealed that Y63-1 effectively formed nodules with all ten soybean varieties tested. In summary, our current study identified highly efficient broad-spectrum Bradyrhizobium elkanii strain Y63-1 as the predominant strain in Zhongdou 63 and provided a theoretical foundation for enhancing yield potential not only in Zhongdou 63 but also in other varieties through inoculation with highly efficient rhizobia in production.

Surface soil nitrogen (SN) is the main source of plant nutrient uptake, and its dynamic change is related to forest productivity, ecosystem carbon and nitrogen cycles, and water conservation. However, few studies have investigated the factors influencing SN. The linear mixing effect and generalized additivity model were used to investigate the spatial patterns and influencing factors of SN by collecting and measuring the total nitrogen content of forest soil samples in the middle and lower reaches of the Xijiang River Basin (XRB). We found that the total nitrogen content in broad-leaved forest soils was significantly higher than that in coniferous forest soils. Forest SN was influenced by soil properties and diversity indicators, and the full model constructed by influencing factors was more applicable to broad-leaved forests. Soil properties were the main explanatory factor for SN (53.53% in the full model, 61.02% in broad-leaved forests), with sub-surface soil nitrogen (sub SN) being the largest explanatory quantity factor (48.52% in the full model, 58.04% in broad-leaved forests). Therefore, forest management in the XRB should avoid damaging the sub-surface soils to prevent nitrogen loss. In the context of global change, the further study of the effect of nitrogen deposition on SN is warranted.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most destructive foliar diseases that affect soybeans. Developing resistant cultivars is the most cost-effective, environmentally friendly, and easy strategy for controlling the disease. However, the current understanding of the mechanisms underlying soybean resistance to P. pachyrhizi remains limited, which poses a significant challenge in devising effective control strategies. In this study, comparative transcriptomic profiling using one resistant genotype and one susceptible genotype was performed under infected and control conditions to understand the regulatory network operating between soybean and P. pachyrhizi. RNA-Seq analysis identified a total of 6540 differentially expressed genes (DEGs), which were shared by all four genotypes. The DEGs are involved in defense responses, stress responses, stimulus responses, flavonoid metabolism, and biosynthesis after infection with P. pachyrhizi. A total of 25,377 genes were divided into 33 modules using weighted gene co-expression network analysis (WGCNA). Two modules were significantly associated with pathogen defense. The DEGs were mainly enriched in RNA processing, plant-type hypersensitive response, negative regulation of cell growth, and a programmed cell death process. In conclusion, these results will provide an important resource for mining resistant genes to P. pachyrhizi infection and valuable resources to potentially pyramid quantitative resistance loci for improving soybean germplasm.

Sucrose metabolism plays a critical role in development, stress response, and yield formation of plants. Sucrose phosphate synthase (SPS) is the key rate-limiting enzyme in the sucrose synthesis pathway. To date, genome-wide survey and comprehensive analysis of the SPS gene family in soybean (Glycine max) have yet to be performed. In this study, seven genes encoding SPS were identified in soybean genome. The structural characteristics, phylogenetics, tissue expression patterns, and cold stress response of these GmSPSs were investigated. A comparative phylogenetic analysis of SPS proteins in soybean, Medicago truncatula, Medicago sativa, Lotus japonicus, Arabidopsis, and rice revealed four families. GmSPSs were clustered into three families from A to C, and have undergone five segmental duplication events under purifying selection. All GmSPS genes had various expression patterns in different tissues, and family A members GmSPS13/17 were highly expressed in nodules. Remarkably, all GmSPS promoters contain multiple low-temperature-responsive elements such as potential binding sites of inducer of CBF expression 1 (ICE1), the central regulator in cold response. qRT-PCR proved that these GmSPS genes, especially GmSPS8/18, were induced by cold treatment in soybean leaves, and the expression pattern of GmICE1 under cold treatment was similar to that of GmSPS8/18. Further transient expression analysis in Nicotiana benthamiana and electrophoretic mobility shift assay (EMSA) indicated that GmSPS8 and GmSPS18 transcriptions were directly activated by GmICE1. Taken together, our findings may aid in future efforts to clarify the potential roles of GmSPS genes in response to cold stress in soybean.

Abstract Exosomes derived from tumor cells play a key role in tumor development. In the present study, we identified the bioactivity of exosomes released from WERI-Rb1 retinoblastoma cells in tumor angiogenesis, as well as the underlying mechanism, through biochemical methods and animal experiments. Our in vitro data showed that exosomes could be engulfed by human vesicle endothelial cells (HUVECs), significantly promote cell viability and induce an inflammatory response in HUVECs by increasing the expression of a series of related genes, such as IL-1, IL-6, IL-8, MCP-1, VCAM1, and ICAM1. Significant increases in migration and tube formation were also observed in the HUVECs incubated with exosomes. Moreover, experiments with a nude mouse xenotransplantation model showed that exosomes injected near tumors could be strongly absorbed by tumor cells. The numbers of endothelial cells and blood vessels were significantly increased in tumor tissues treated with exosomes compared to control tissues. Furthermore, to reveal the mechanism underlying exosome-mediated angiogenesis in retinoblastoma, we analyzed the levels of 12 microRNAs in the exosomes. Specifically, our data showed that miR-92a-3p was enriched in RB exosomes. Accordingly, miR-92a-3p was increased in the HUVECs incubated with these exosomes. After treatment with a miR-92a-3p inhibitor, the promoting effect of exosomes on the migration and tube formation of HUVECs was significantly abrogated. The expression of the angiogenesis-related genes mentioned above was markedly decreased in HUVECs. Similarly, treatment with a microRNA mimic also demonstrated that miR-92a-3p was involved in the angiogenesis of HUVECs. More importantly, bioinformatics analysis predicted that Krüppel-like factor 2 (KLF2), a member of the KLF family of zinc-finger transcription factors, might be an active target of miR-92a-3p. Notably, this prediction was confirmed both in vitro and in vivo. Thus, our work suggests that exosomal miR-92a-3p is involved in tumor angiogenesis and might be a promising therapeutic candidate for retinoblastoma.

Auxin has been reported to regulate plant growth and development, as well as to mediate plant adaption to abiotic stresses, including drought. AUX/LAX family displays auxin uptake functions and comprises four highly conserved genes AUX1 and LIKE AUX1 (LAX1, LAX2, and LAX3) in Arabidopsis. There are fifteen GmLAX family genes in the soybean genomes and several members were regulated by dehydration stress. In this study, the sequence differences and expression pattern of GmLAXs-I were analyzed under stress treatment between the soybean drought-tolerant Jindou 21 and drought-sensitive varieties Zhongdou 33. Five homologous genes of AUX1 were all responsive to PEG, salt, ABA and IAA stimuli. There were two SNPs in the promoter region of GmLAX4 gene, and this gene was differentially expressed in two cultivars. Moreover, our results showed YFP-GmLAXs are predominantly localized in plasma membrane. Taken together, our results suggest that GmLAXs are involved in abiotic response, which can provide theoretical and technical support for the genetic improvement of soybean drought tolerance.

Abstract Background Plant papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes and play important roles in root nodule symbiosis (RNS), while the whole-genome studies of PLCP family genes in legume are quite limited, and the roles of Glycine max PLCPs (GmPLCPs) in nodulation, nodule development and senescence are not fully understood. Results In the present study, we identified 97 GmPLCPs and performed a genome-wide survey to explore the expansion of soybean PLCP family genes and their relationships to RNS. Nineteen paralogous pairs of genomic segments, consisting of 77 GmPLCPs, formed by whole-genome duplication (WGD) events were identified, showing a high degree of complexity in duplication. Phylogenetic analysis among different species showed that the lineage differentiation of GmPLCPs occurred after family expansion, and large tandem repeat segment were specifically in soybean. The expression patterns of GmPLCPs in symbiosis-related tissues and nodules identified RNS-related GmPLCPs and provided insights into their putative symbiotic functions in soybean. The symbiotic function analyses showed that a RNS-related GmPLCP gene (Glyma.04G190700) really participate in nodulation and nodule development. Conclusions Our findings improved our understanding of the functional diversity of legume PLCP family genes, and provided insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.

Leaf-chewing insects are important pests that cause yield loss and reduce seed quality in soybeans (Glycine max). Breeding soybean varieties that are resistant to leaf-chewing insects can minimize the need for insecticide use and reduce yield loss. The marker gene for QTL-M, Glyma.07g110300 (LOC100775351) that encodes a UDP-glycosyltransferase (UGT) is the major determinant of resistance against leaf-chewing insects in soybean; it exhibits a loss of function in insect-resistant soybean germplasms. In this study, Agrobacterium-mediated transformation introduced the CRISPR/Cas9 expression vector into the soybean cultivar Tianlong No. 1 to generate Glyma.07g110300-gene mutants. We obtained two novel types of mutations, a 33-bp deletion and a single-bp insertion in the GmUGT coding region, which resulted in an enhanced resistance to Helicoverpa armigera and Spodoptera litura. Additionally, overexpressing GmUGT produced soybean varieties that were more sensitive to H. armigera and S. litura. Both mutant and overexpressing lines exhibited no obvious phenotypic changes. The difference in metabolites and gene expression suggested that GmUGT is involved in imparting resistance to leaf-chewing insects by altering the flavonoid content and expression patterns of genes related to flavonoid biosynthesis and defense. Furthermore, ectopic expression of the GmUGT gene in the ugt72b1 mutant of Arabidopsis substantially rescued the phenotype of H. armigera resistance in the atugt72b1 mutant. Our study presents a strategy for increasing resistance against leaf-chewing insects in soybean through CRISPR/Cas9-mediated targeted mutagenesis of the UGT genes.

Seed vigor plays an essential role in soybean production. Soybean seed is sensitive to environment and easy to get seed deterioration. Generally soybean seed vigor can be maintained for less than one year and have to be multiplied every year. Varieties with good seed vigor are essential for maintaining planting population and stable yield. In this research, 419 germplasms from three eco-regions, South China, Huanghuai region and Northeast China, were evaluated for seed vigor by assessing germination ratio, germination potential and germination index. About 34.8% of tested germplasm had germination ratio equal or more than 85%, suggested by the national soybean seed quality standard of China. Only 9 from 82 tested cultivars met the seed standard. Seed germination ratio distribution had obvious regional feature. Landraces performed better seed vigor than cultivars. Germplasms from South China had stronger seed vigor compared to those from Huanghuai and Northeast regions. Seed vigor has no significant relationship with maturity and 100-seed weight. Totally 21 germplasms with strong seed vigor, favor maturation and seed size were identified and can be used in future breeding program.

Legume nodule development and senescence directly affect nitrogen fixation efficiency and involve a programmed series of molecular events. These molecular events are carried out synchronously by legumes and rhizobia. The characteristics and molecular mechanisms of nitrogen fixation at soybean important developmental stages play critical roles in soybean cultivation and fertilizer application. Although the gene expression of soybean were analyzed in nodules at five important soybean developmental stages, information on the expression of rhizobial genes in these nodule samples is limited. In the present study, we investigated the expression of Bradyrhizobium diazoefficiens 113-2 genes in the nodule samples from five developmental stages of soybean (Branching stage, flowering stage, fruiting stage, pod stage and harvest stage). Similar gene expression patterns of B. diazoefficiens 113-2 were existed during optimal symbiotic functioning, while different expression patterns were found among early nodule development, nitrogen fixation progress and nodule senescence. Besides, we identified 164 important different expression genes (DEGs) associated with nodule development and senescence. These DEGs included those encoding nod, nif, fix proteins and T3SS secretion system-related proteins, as well as proteins involved in nitrogen metabolism, ABC transporters and two-component system pathways. Gene Ontology, KEGG pathway and homology analysis of the identified DEGs revealed that most of these DEGs are uncharacterized genes associated with nodule development and senescence, and they are not core genes among the rhizobia genomes. Our results provide new clues for the understanding of the genetic determinants of soil rhizobia in nodule development and senescence, and supply theoretical basis for the creation of high efficiency soybean cultivation technology.

Previous studies have indicated that Brca1 (Breast cancer suppressor gene 1) plays an important role in neural development and degenerative diseases. However, the bioactivity and regulatory mechanism of Brca1 expression in retinal neurocytes remain unclear. In the present study, our data indicated that Brca1 maintains the state of neuronal precursor cells. Brca1 silencing induces differentiation in 661W cells. Nestin, a marker of precursor cells, was significantly decreased in parallel with Brca1 silencing in 661W cells, whereas Map2 (Microtubule associated protein 2), a marker of differentiated neurons, was significantly increased. Neurite outgrowth was increased by ~4.0-fold in Brca1-silenced cells. Moreover, DNA affinity purification assays and ChIP assays demonstrated that Gata3 (GATA binding protein 3) regulates Brca1 transcription in 661W cells. Silencing or overexpressing Gata3 could significantly regulate the expression of Brca1 and affect its promoter inducibility. Furthermore, the expression of Gata3 generally occurred in parallel with that of Brca1 in developing mouse retinas. Both Gata3 and Brca1 are expressed in the neonatal mouse retina but are developmentally silenced with age. Exogenous Gata3 significantly inhibited neural activity by decreasing synaptophysin and neurite outgrowth. Thus, this study demonstrated that Brca1 is transcriptionally regulated by Gata3. Brca1/Gata3 silencing is involved in neuronal differentiation and maturation.

With global warming and regional decreases in precipitation, drought has become a problem worldwide. As the number of arid regions in the world is increasing, drought has become a major factor leading to significant crop yield reductions and food crises. Soybean is a crop that is relatively sensitive to drought. It is also a crop that requires more water during growth and development. The aim of this study was to identify the quantitative trait locus (QTL) that affects drought tolerance in soybean by using a recombinant inbred line (RIL) population from a cross between the drought-tolerant cultivar ‘Jindou21’ and the drought-sensitive cultivar ‘Zhongdou33’. Nine agronomic and physiological traits were identified under drought and well-watered conditions. Genetic maps were constructed with 923,420 polymorphic single nucleotide polymorphism (SNP) markers distributed on 20 chromosomes at an average genetic distance of 0.57 centimorgan (cM) between markers. A total of five QTLs with a logarithm of odds (LOD) value of 4.035–8.681 were identified on five chromosomes. Under well-watered conditions and drought-stress conditions, one QTL related to the main stem node number was located on chromosome 16, accounting for 17.177% of the phenotypic variation. Nine candidate genes for drought resistance were screened from this QTL, namely Glyma.16G036700, Glyma.16G036400, Glyma.16G036600, Glyma.16G036800, Glyma.13G312700, Glyma.13G312800, Glyma.16G042900, Glyma.16G043200, and Glyma.15G100700. These genes were annotated as NAC transport factor, GATA transport factor, and BTB/POZ-MATH proteins. This result can be used for molecular marker-assisted selection and provide a reference for breeding for drought tolerance in soybean.

Abstract Background The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants. Results In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean. Conclusions Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.

Retinal organoids generated from human embryonic stem cells or iPSCs recreate the key structural and functional features of mammalian retinal tissue in vitro. However, the differences in the development of retinal organoids and normal retina in vivo are not well defined. Thus, in the present study, we analyzed the development of retinal organoids and zebrafish retina after inhibition of CXCR4, a key role in neurogenesis and optic nerve development, with the antagonist AMD3100. Our data indicated that CXCR4 was mainly expressed in ganglion cells in retinal organoids and was rarely expressed in amacrine or photoreceptor cells. AMD3100 treatment reduced the retinal organoid generation ratio, impaired differentiation, and induced morphological changes. Ganglion cells, amacrine cells, and photoreceptors were decreased and abnormal locations were observed in organoids treated with AMD3100. Neuronal axon outgrowth was also damaged in retinal organoids. Similarly, a decrease of ganglion cells, amacrine cells, and photoreceptors and the distribution of neural outgrowth was induced by AMD3100 treatment in zebrafish retina. However, abnormal photoreceptor ensembles induced by AMD3100 treatment in the organoids were not detected in zebrafish retina. Therefore, our study suggests that although retinal organoids might provide a reliable model for reproducing a retinal developmental model, there is a difference between the organoids and the retina in vivo.

GATA binding protein 3 (Gata3), a zinc-finger transcription factor, plays an important role in neural development. However, its expression and bioactivity in the retina remain unclear. In the present study, our data indicated that Gata3 maintains the precursor state of 661W cells, and Gata3 silencing induces cell differentiation. The expression of Nestin, a marker of precursor cells, was significantly decreased in parallel, whereas the expression of Map2, a marker of differentiated neurons, was significantly increased following the decrease in Gata3. Neurite outgrowth was increased by 2.78-fold in Gata3-silenced cells. Moreover, Gata3 expression generally paralleled that of Nestin in developing mouse retinas. Both Gata3 and Nestin were expressed in the retina at postnatal day 1 and silenced in the adult mouse retina. Exogenous Gata3 significantly inhibited the neural activity of primary retinal neurocytes (postnatal day 1) by decreasing synaptophysin levels, neurite outgrowth, and cell viability. Furthermore, in vivo, exogenous Gata3 significantly induced apoptosis and the contraction of retinal outlay filaments and decreased the a- and b-waves in adult mouse intravitreal injected with AAV-Re-Gata3-T2A-GFP. Thus, Gata3 silencing promotes neuronal differentiation and neurite outgrowth. Its abnormal expression impedes neural activity in adult retinal neurocytes. This study provides new insights into Gata3 bioactivity in retinal neurocytes.

Ocular inflammation is a common pathological condition of a series of retinal degenerative diseases. Tetramethylpyrazine (TMP), a Chinese herbal extraction, is widely used in the treatment of several ocular diseases in Eastern countries. However, the exact mechanisms correlating the vision protective effects of TMP have not been elucidated. Thus, this study aimed to investigate TMP’s molecular targets in anti-inflammatory activity in endotoxin lipopolysaccharide (LPS)-induced retinal inflammation both in vitro and in vivo. The primary cultured retinal microglial cells were pretreated with TMP and then activated by LPS. We found pretreatment with TMP significantly inhibited LPS-induced upregulation of CD68, a marker of mononuclear microglia activation. The morphological changes induced by LPS were also inhibited by the TMP pretreatment. Moreover, Toll like receptor 4 (TLR4), phosphorylation of inhibitor of NF-κB alpha (p-IκB-α) and the translocation of nuclear factor kappa B p65 (NF-κB p65) were significantly downregulated in retinal microglial cells with TMP pretreatment, which indicated that TMP might suppress LPS-induced retinal microglial activation through TLR4/NF-κB signalling pathway. And these results were confirmed in vivo. Pretreatment with TMP inhibited microglial activation, migration and regeneration, especially in ganglion cell layer (GCL). In addition to the inhibition of TLR4, TMP significantly inhibited the translocation of NF-κB p-65 to the nucleus in vivo. The downstream genes of NF-κB, such as the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), were significantly downregulated by TMP pretreatment in the retina. Accordingly, the increased expression of cleaved caspase-3 and the decreased ratio of B-cell lymphoma-2 (Bcl-2) to Bcl-2 associated X Protein (Bax) were significantly attenuated by TMP. TUNEL assay also demonstrated that TMP exerted neuroprotective effects in the retina. Therefore, this study elucidated a novel mechanism that TMP inhibits retinal inflammation by inhibiting microglial activation via a TLR4/NF-κB signalling pathway.

Cone rod homeobox (Crx) plays a key role at the center of a regulatory network that coordinates many pathways in the retina. Its abnormal expression induces retinal disorders. However, the underlying regulatory mechanism of Crx expression is not well defined. Here, we present data that show that the levels of Crx mRNA were inconsistent with that of Crx protein in primary retinal neurocytes cultured in light conditions. Crx protein levels were significantly higher (2.56-fold) in cells cultured in the dark than in cells cultured in light, whereas Crx mRNA was not changed in either type of cell. Moreover, the expression of Crx protein showed a significant light intensity-dependent decrease. Consistently, Crx downstream genes rhodopsin and arrestin also decreased in retinal neurocytes upon light exposure. Furthermore, Crx promoter activity assay performed in primary retinal neurocytes further indicated that light exposure and darkness did not affect its inducibility. In addition, the inconsistency between Crx mRNA and protein expression after light exposure was not observed in 661w cells transfected with plasmid pcDNA3.1-Crx, suggesting that the inconsistency between Crx mRNA and protein induced by light was specific to the endogenous Crx. More importantly, this observation was confirmed in vivo in postnatal day 15 (P15) retinas but not in adult retinas, further implicating that the posttranscriptional regulation mechanism may be involved in Crx expression in the developing retina. Therefore, our study sheds light on the mechanism of Crx expression in postnatal rat retina.

Objective To use a mathematical model to simulate an influenza outbreak in a school in order to assess the effectiveness of isolation (Iso), antiviral therapeutics, antiviral prophylactics (P), vaccination prior to the outbreak, and school closure (for 1 [S1w], 2 or 3 weeks). Methods This study developed a susceptible–exposed–infectious/asymptomatic–recovered model to estimate the effectiveness of commonly used interventions for seasonal influenza outbreaks in school. Results The most effective single-intervention strategy was isolation with a total attack rate of 1.99% and an outbreak duration of 30 days. The additional effectiveness of antiviral therapeutics and prophylactics and vaccination (prior to the outbreak) strategies were not obvious. Although Iso+P, P+Iso+S1w, four-, and five-combined intervention strategies had commendable effectiveness, total attack rate decreased only slightly, and outbreak duration was shortened by 9 days maximum, compared with the single-intervention isolation strategy. School closure for 1, 2 or 3 weeks was futile or even counterproductive. Conclusion Isolation, as a single intervention, was the most effective in terms of reducing the total attack rate and the duration of the outbreak.

The LOR (LURP-one related) family genes encode proteins containing a conserved LOR domain. Several members of the LOR family genes are required for defense against Hyaloperonospora parasitica (Hpa) in Arabidopsis. However, there are few reports of LOR genes in response to abiotic stresses in plants. In this study, a genome-wide survey and expression levels in response to abiotic stresses of 36 LOR genes from Glycine max were conducted. The results indicated that the GmLOR gene family was divided into eight subgroups, distributed on 14 chromosomes. A majority of members contained three extremely conservative motifs. There were four pairs of tandem duplicated GmLORs and nineteen pairs of segmental duplicated genes identified, which led to the expansion of the number of GmLOR genes. The expansion patterns of the GmLOR family were mainly segmental duplication. A heatmap of soybean LOR family genes showed that 36 GmLOR genes exhibited various expression patterns in different tissues. The cis-acting elements in promoter regions of GmLORs include abiotic stress-responsive elements, such as dehydration-responsive elements and drought-inducible elements. Real-time quantitative PCR was used to detect the expression level of GmLOR genes, and most of them were expressed in the leaf or root except that GmLOR6 was induced by osmotic and salt stresses. Moreover, GmLOR4/10/14/19 were significantly upregulated after PEG and salt treatments, indicating important roles in the improvement of plant tolerance to abiotic stress. Overall, our study provides a foundation for future investigations of GmLOR gene functions in soybean.

In this study, we report three cases of sporadic Creutzfeldt–Jakob disease in China, two confirmed cases and one probable case. The aim of this study was to enrich the data regarding clinical and epidemiological features of this disease and to provide reference for the diagnosis, control, and prevention of sporadic Creutzfeldt–Jakob disease.

Plants have evolved sophisticated defense mechanisms against herbivores to help them adapt to the environment. Understanding the defense mechanisms in plants can help us control insects in a more effective manner. In this study, we found that compared with Tianlong 2 (a cultivated soybean with insect susceptibility), ED059 (a wild soybean line with insect resistance) contains sharper pubescence tips, as well as lower transcript levels of wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK), which are important mitogen-activated protein kinases involved in early defense response to herbivores. The observed lower transcript levels of WIPK and SIPK induced higher levels of jasmonic acid (JA), JA biosynthesis enzymes (AOC3) and some secondary metabolites in ED059. Functional analysis of the KTI1 gene via Agrobacterium-mediated transformation in Arabidopsis thaliana indicated that it plays an important role in herbivore defense in ED059. We further investigated the molecular response of third-instar Helicoverpa armigera (Hübner) larvae to Tianlong 2 and ED059. We found apoptotic cells only in the midguts of larvae that fed on ED059. Compared with larvae reared on the susceptible cultivar Tianlong 2, transcript levels of catalase (CAT) and glutathione S-transferase (GST) were up-regulated, whereas those of CAR, CHSB, and TRY were down-regulated in larvae that fed on the highly resistant variety ED059. We propose that these differences underlie the different herbivore defense responses of ED059 and Tianlong 2.

We report rabies virus transmission among solid organ transplantation recipients in Changsha, China, in 2016. Two recipients were confirmed to have rabies and died. Our findings suggest that more attention should be paid to the possibility of rabies virus transmission through organ transplantation for clinical and public health reasons.

Chlorophyll plays an essential role in photosynthetic light harvesting and energy transduction in green tissues of higher plants and is closely related to photosynthesis and crop yield. Identification of transcription factors (TFs) involved in regulating chlorophyll biosynthesis is still limited in soybean (Glycine max), and the previously identified GmGATA58 is suggested to potentially modulate chlorophyll and nitrogen metabolisms, but its complete function is still unknown. In this study, subcellular localization assay showed that GmGATA58 was localized in the nucleus. Histochemical GUS assay and qPCR assay indicated that GmGATA58 was mainly expressed in leaves and responded to nitrogen, light and phytohormone treatments. Overexpression of GmGATA58 in the Arabidopsis thaliana ortholog AtGATA21 (gnc) mutant complemented the greening defect, while overexpression in Arabidopsis wild-type led to increasing chlorophyll content in leaves through up-regulating the expression levels of the large of chlorophyll biosynthetic pathway genes, but suppressing plant growth and yield, although the net photosynthetic rate was slightly improved. Dual-luciferase reporter assay also supported that GmGATA58 activated the transcription activities of three promoters of key chlorophyll biosynthetic genes of soybean in transformed protoplast of Arabidopsis. It is concluded that GmGATA58 played an important role in regulating chlorophyll biosynthesis, but suppressed plant growth and yield in transgenic Arabidopsis.

Three cases of the avian influenza A (H9N2) virus have been documented in Changsha, which is a large city that has nine districts and a population of 7.04 million in central South China. Among these patients, one was a girl and two were boys. The ages of the patients were 9 months, 2 years, and 15 years. Two cases of H9N2 were detected in September, 2015 and one was detected in 2017. Two patients were children who had not reached the age for kindergarten and one was a student. These three cases were all mild and were detected in a sentinel hospital of the Chinese Influenza Surveillance System. We describe the clinical and epidemiological features of the youngest patient with H9N2 in 2017 and the surveillance results of the H9N2 virus in live poultry markets in Changsha. From January 2014 to December 2017, 4212 samples were collected in live poultry markets in Changsha, among which 25.81% (1087/4212) were H9N2-positive. Public health concerns should be addressed for emerging H9N2 virus infection, and more strategies should be performed before this virus mutates to be more transmissible and highly pathogenic.

In January 2016, two patients died of rabies after receiving kidney transplants from a common organ donor at a hospital in Changsha, Hunan, China. The medical records, epidemiological data of the organ donor, two kidney and a liver recipients were reviewed. Intravitam saliva samples of the two kidney recipients were tested for rabies virus (RABV) using real-time RT-PCR, and the nucleoprotein (N) gene was amplified and sequenced by Sanger sequencing. Whole genome sequences were analyzed using next-generation sequencing. The N genes of the two kidney recipients showed 100% nucleic acid identity. Phylogenetic analysis of the complete genome, N and glycoprotein (G) genes indicated that the RABV was homologous with dog isolates from the Hunan province and belong to the China I lineage, which is widespread in China. The organ donor was a 22-month-old boy who died from unknown acute progressive encephalitis. After undergoing sub-hypothermia hibernation therapy, rabies-associated symptoms were atypical, and rabies was neglected because serum RABV-specific antibodies were negative. An unknown wound on the forehead of the donor was found 2 months before the onset of symptoms. Based on the clinical, epidemiological, and molecular findings, we speculated that the RABV initially originated in the donor from a dog bite, and was then transmitted to the recipients by organ transplantation. An uncertain exposure history and misdiagnosis played important roles in the spread of the RABV. Rabies should be considered in patients with acute progressive encephalitis of unexplained etiology, especially in potential organ donors.

Root nodule symbiosis (RNS) is one of the most productive and economical systems for nitrogen fixation, and previous studies have shown that several nodule-specific C2H2-zinc finger proteins (ZFPs) play important roles in symbiosis establishment and nodule function. However, C2H2-ZFPs are the most widespread ZFPs in eukaryotes, and a great variation of structure and function exist among the family members. It remains largely unclear whether or not special types of C2H2-ZF genes participate in symbiosis, especially in soybean. In the present study, we performed a genome-wide survey of soybean C2H2-ZF genes, and 321 soybean C2H2-ZF genes were identified and classified into 11 clearly distinguishable subsets (Gm-t1-SF, Gm-t2-SF, Gm-1i-Q-SF, Gm-1i-M-SF, Gm-1i-Z-SF, Gm-1i-D-SF, Gm-2i-Q-SF, Gm-2i-M-SF, Gm-2i-Mix-SF, Gm-3i-SF, and Gm-4i-SF) based on the arrangements, numbers, and types of C2H2-ZF domains. Phylogenetic and gene ontology analyses were carried out to assess the conserved sequence and GO function among these subsets, and the results showed that the classification of soybean C2H2-ZFPs was reasonable. The expression profile of soybean C2H2-ZFPs in multiple tissues showed that nearly half of soybean C2H2-ZFPs within different subsets had expressions in nodules, including a clustering branch consisting of 11 Gm-1i-Q-SF genes specifically expressed in symbiotic-relative tissues. RNA-Seq was used to identify symbiosis-related soybean C2H2-ZFPs, and the expression pattern of the soybean C2H2-ZFPs in roots and nodules at different development stages showed that soybean C2H2-ZFPs mainly played roles in nodule development or nodule function rather than nodulation signal transduction, and nearly half of these genes had high expressions and/or different expression patterns during soybean nodule development, especially for the six clustering branches of genes consisting of different subsets of C2H2-ZFPs. Furthermore, the selected symbiosis-related soybean C2H2-ZFPs might function in legume-rhizobium symbiosis through regulating or interacting with other key proteins. Taken together, our findings provided useful information for the study on classification and conservative function of C2H2-ZFPs, and offered solid evidence for investigation of rhizobium symbiosis-related C2H2-ZFPs in soybean or other legumes.

Identifying a superior soybean variety with high defoliator resistance is important to avoid yield loss. Cotton bollworm (Helicoverpa armigera Hübner) is one of the major defoliators of soybean (Glycine max [L.] Merr.) worldwide. In this study, we evaluated the effect of H. armigera larvae on ED059, a wild soybean (Glycine soja Sieb. et Zucc.), and three cultivated soybean varieties: Tianlong 2, PI 535807, and PI 533604, in choice and no-choice assays. The percentage of ED059 leaflets consumed by H. armigera was lower than that of the three cultivated soybeans. Larvae that fed on ED059 exhibited low weight gain and high mortality rate. Waldbauer nutritional indices suggested that ED059 reduced the growth, consumption, and frass production of H. armigera larvae. Larvae that fed on ED059 showed lower efficiency of conversion of ingested and of digested food than those that fed on Tianlong 2 and PI 533604. However, they showed statistically similar consumption index and approximate digestibility compared with those fed on the three cultivated soybeans. Quantitative real-time PCR analysis revealed that 24 h after insect attack, ED059 had higher transcript levels of Kunitz trypsin inhibitor 3, Cysteine proteinase inhibitor 2, and Nerolidol synthase 1 but a lower transcript level of Pathogenesis-related protein 1 than Tianlong 2. The gene expression results were consistent with the presence of higher levels of jasmonic acid (JA) and transcript levels of the JA biosynthesis enzyme allene oxide cyclase 3 in ED059 than in Tianlong 2. Our findings indicate that ED059 is a superior soybean line with strong insect resistance that may be mediated via the JA pathway.

BACKGROUND:Influenza A (H1N1) outbreaks have become common at schools in China since 2009. However, the effects of common countermeasures for school influenza outbreak have not been quantified so far, including isolation, vaccination, antivirus and school closure. We conducted a mathematically modeling study to address this unsolved issue. METHODS:We collected data of all small-scale school outbreaks caused by influenza A that occurred in Changsha city between January 2009 and December 2013. Two outbreaks (one was in 2009 and the other one was in 2013) were used for simulating the effects of single and combined use of common measures, including isolation (Iso), therapeutics (T), prophylactics (P), vaccinating 70% of susceptible individuals prior to the outbreak (VP70), vaccinating 70% of susceptible individuals every day during the outbreak (VD70) and school closure of one week (S1w). A susceptible-exposed-infectious/asymptomatic-recovered (SEIR) model was developed to implement the simulations based on the natural history of influenza A. RESULTS:When no control measures are taken, the influenza is expected to spread quickly at school for the selected outbreak in 2013; the outbreak would last 56 days, and the total attack rate (TAR) would reach up to 46.32% (95% CI: 46.12-46.52). Of all single control measures, VP70 is most effective to control the epidemic (TAR = 8.68%), followed by VP50, VD70, VD50 and Iso. The use of VP70 with any other measure can reduce TAR to 3.37-14.04% and showed better effects than any other combination of two kinds of measures. The best two-measure combination is 'S1w+VP70' (TAR = 3.37%, DO = 41 days). All combinations of three kinds of measures were not satisfactory when Vp70 and VD70 were excluded. The most effective three-intervention combination was 'Iso+S1w+VP70' (with TAR = 3.23%). When VP70 or VD70 is included, the combinations of four or five kinds of interventions are very effective, reducing TAR to lower than 5%. But the TAR of combination of 'T+P+Iso+S1w' is 23.20%. Similar simulation results were observed for the selected outbreak in 2009. CONCLUSION:Vaccinating no less than 70% of individuals prior to the outbreak and isolation are recommended as single measures to control H1N1outbreak at school. The combination of VP70+S1w can achieve very good control for school outbreak.

Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.

Nodulation, nodule development and senescence directly affects nitrogen fixation efficiency, and previous studies have shown that inhibition of some cysteine proteases delay nodule senescence, so their nature inhibitors, cystatin genes, are very important in nodulation, nodule development and senescence. Although several cystatins are actively transcribed in soybean nodules, their exact roles and functional diversities in legume have not been well explored in genome-wide survey studies. In this report, we performed a genome-wide survey of cystatin family genes to explore their relationship to nodulation and nodule development in soybean and identified 20 cystatin genes that encode peptides with 97~245 amino acid residues, different isoelectric points (pI) and structure characteristics, and various putative plant regulatory elements in 3000 bp putative promoter fragments upstream of the 20 soybean cystatins in response to different abiotic/biotic stresses, hormone signals and symbiosis signals. The expression profiles of these cystatin genes in soybean symbiosis with rhizobium strain Bradyrhizobium japonicum strain 113-2 revealed that 7 cystatin family genes play different roles in nodulation as well as nodule development and senescence. However, these genes were not root nodule symbiosis (RNS) - specific and did not encode special clade cystatin protein with structures related to nodulation and nodule development. Besides, only two of these soybean cystatins were not upregulated in symbiosis after ABA treatment. The functional analysis showed that a candidate gene Glyma.15G227500 (GmCYS16) was likely to play a positive role in soybean nodulation. Besides, evolutionary relationships analysis divided the cystatin genes from Arabidopsis thaliana, Nicotiana tabacum, rice, barley and four legume plants into three groups. Interestingly, Group A cystatins are special in legume plants, but only include one of the above-mentioned 7 cystatin genes related to nodulation and nodule development. Overall, our results provide useful information or clues for our understanding of the functional diversity of legume cystatin family proteins in soybean nodulation and nodule development and for finding nodule-specific cysteine proteases in soybean.

We collected 2768 Influenza-like illness emergency public health incidents from April 1, 2005 to November 30, 2013reported in the Emergency Public Reporting System. After screening by strict inclusion and exclusion criteria, there were 613 outbreaks analyzed with susceptible-exposed-infectious/asymptomatic-removed model in order to estimate the proportion of asymptomatic individuals (p) and the effective reproduction number (Rt). The relation between Rt and viral subtypes, regions, outbreak sites, populations, and seasons were analyzed. The mean values of p of different subtypes ranged from 0.09 to 0.15, but could be as high as up to 0.94. Different subtypes, provinces, regions, and sites of outbreak had statistically significantly different Rt. In particular, the southern region also manifested different Rt by affected population size and seasonality. Our results provide China and also the rest of the world a reference to understand characteristics of transmission and develop prevention and control strategies.

During the early stage of a pandemic, isolation is the most effective means of controlling transmission. However, the effectiveness of age-specific isolation policies is not clear; especially little information is available concerning their effectiveness in China. Epidemiological and serological survey data in the city of Changsha were employed to estimate key model parameters. The average infectious period (date of recovery-date of symptom onset) of influenza A (H1N1) was 5.2 days. Of all infected persons, 45.93% were asymptomatic. The basic reproduction number of the influenza A (H1N1) pandemic was 1.82. Based on the natural history of influenza A (H1N1), we built an extended susceptible-exposed-infectious/asymptomatic-removed model, taking age groups: 0-5, 6-14, 15-24, 25-59, and ≥60 years into consideration for isolation. Without interventions, the total attack rates (TARs) in each age group were 42.73%, 41.95%, 20.51%, 45.03%, and 37.49%, respectively. Although the isolation of 25-59 years-old persons was the most effective, the TAR of individuals of aged 0-5 and 6-14 could not be reduced. Paradoxically, isolating individuals ≥60 year olds was not predicted to be an effective way of reducing the TAR in this group but isolating the age-group 25-59 did, which implies inter-age-group transmission from the latter to the former is significant. Isolating multiple age groups increased effectiveness. The most effective combined isolation target groups were of 6-14 + 25-59 year olds, 6-14 + 15-24 + 25-59 year olds, and 0-5 + 6-14 + 25-59 + ≥60 year olds. The last of these isolation schemas reduced the TAR of the total population from 39.64% to 0.006%, which was exceptionally close to the effectiveness of isolating all five age groups (TAR = 0.004%).

Nitrogen is an important macronutrient required for plant growth, and is a limiting factor for crop productivity. Improving the nitrogen use efficiency (NUE) is therefore crucial. At present, the NUE mechanism is unclear and information on the genes associated with NUE in soybeans is lacking. cystathionine beta synthase (CBS) domain-containing proteins (CDCPs) may be implicated in abiotic stress tolerance in plants. We identified and classified a CBS domain–containing protein superfamily in soybean. A candidate gene for NUE, GmCBS21, was identified. GmCBS21 gene characteristics, the temporal expression pattern of the GmCBS21 gene, and the phenotype of GmCBS21 overexpression in transgenic Arabidopsis thaliana under low nitrogen stress were analyzed. The phenotypes suggested that the transgenic Arabidopsis thaliana seedlings performed better under the nitrogen-deficient condition. GmCBS21-overexpressing transgenic plants exhibit higher low nitrogen stress tolerance than WT plants, and this suggests its role in low nitrogen stress tolerance in plants. We conclude that GmCBS21 may serve as an excellent candidate for breeding crops with enhanced NUE and better yield.