Expression of a single-chain monellin (MNEI) mutant with enhanced stability in transgenic mice milk.

Monellin is a sweet protein that may be used as a safe and healthy sweetener. However, due to its low stability, the application of monellin is currently very limited. Here, we describe a wild-type, a double-sites mutant (E2N/E23A) and a triple-sites mutant (N14A/E23Q/S76Y) of single-chain monellin (MNEI) expressed in transgenic mice milk. Based on enzyme-linked immunoassay (ELISA), Western blot, and sweetness intensity testing, their sweetness and stability were compared. After boiling for 2 min at different pH conditions (2.5, 5.1, 6.8, and 8.2), N14A/E23Q/S76Y-MNEI showed significantly higher sweetness and stability than the wild-type and E2N/E23A-MNEI. These results suggest that N14A/E23Q/S76Y-MNEI shows remarkable potential as a sweetener in the future.

Inactivation of Zika virus in plasma and derivatives by four different methods.

Zika virus (ZIKV) is an emerging arbovirus with increasing prevalence in recent years. To reduce the risk of ZIKV transmission by transfusion, some mitigation strategies were recommended based on pathogen reduction technologies for blood products. In this study, we aimed to study the efficacy of several common pathogen reduction methods in the inactivation of ZIKV. The fresh frozen plasma and derivatives were spiked with a high titer of ZIKV or Sindbis virus (SINV). Viral titers and ZIKV RNA were measured before and after the inactivation treatment by methylene blue (MB), solvent/detergent (S/D), pasteurization, and low pH. The mean ZIKV infectivity titers in plasma and derivatives were 7.08 ± 0.14, 5.17 ± 0.14, 7.08 ± 0.14, and 5.80 ± 0.14 log10 TCID50 /mL, respectively before MB, S/D, pasteurization, and low pH inactivation. We found no detectable ZIKV RNA after five successive passages of inoculation on host cells, indicating there is no infectivity after inactivation. Similar inactivation results were observed for SINV. In conclusion, we achieved robust ZIKV inactivation through the four inactivation procedures in several blood products. These findings suggest that the pathogen reduction technologies commonly applied in plasma and derivatives have the capacity to mitigate the risk of ZIKV transmission by transfusion.

Six New Phragmalin Limonoids from the Stems of Chukrasia tabularis A. Juss.

Six new phragmalin limonoids, named moluccensin Z1 (1), moluccensin Z2 (2), carapanolide Y (3), tabulalin N (4), chukvelutilide A1 (5), and velutinasin J (6), as well as two known compounds, chukvelutilide A (7) and velutinasin D (8) were isolated from the stems of Chukrasia tabularis A. Juss. The structures of the new compounds 1⁻6 were confirmed by spectroscopic methods, including IR and HRESIMS, as well as 1D and 2D NMR, and by comparisons with the data of known analogues. All compounds were tested for α-glucosidase and acetylcholinesterase inhibitory activities. However, none of the compounds was active against α-glucosidase and acetylcholinesterase in vitro.

Spaceflight environment-induced variation in root yield and active constituents of Salvia miltiorrhiza.

Salvia miltiorrhiza is a significant source of bioactive compounds providing human health effects. Here, we surveyed root yield and the active constituents' divergences of second generation S. miltiorrhiza (SP2) responding to a spaceflight environment. High-performance liquid chromatography was conducted for the comprehensive constituents' characterizations of 28 SP2 lines (224 individuals) and the ground control (eight individuals). The results showed that the mean fresh and dry weight of roots ranged from 116 to 172 g and 25 to 119 g, respectively, in SP2 lines. In addition, the mean contents of four tanshinone compounds (tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I) of 28 SP2 lines varied from 0.32 to 1.04 mg ·â€Šg(-1), 0.47 to 2.39 mg ·â€Šg(-1), 0.25 to 1.60 mg ·â€Šg(-1), and 0.53 to 1.67 mg ·â€Šg(-1), respectively. Except for salvianolic acid B, which varied drastically from 72 % to 201 % of the ground control treatment, the other six phenolic acid contents of the 28 SP2 lines all increased after spaceflight. Principal component analysis was performed to obtain an overview of the distribution of all samples, and score plots clearly separated the SP2 accessions from ground controls. Moreover, a positive relationship was observed between tanshinone I and tanshinone IIA (r = 0.790, p < 0.01), and rosmarinic acid was positively correlated with salvianolic acid B (r = 0.728, p < 0.01). In conclusion, this study demonstrated that a spaceflight environment induced SP2 accessions remarkably in the variation of root yield and active constituent content.

Comparative Analysis of Transcriptomes of Diploid and Tetraploid Miscanthus lutarioriparius under Drought Stress.

Miscanthus lutarioriparius is a species of bioenergy crop unique to China. It is widely distributed in the south of China with high resistance to drought and salt stress. To date, the molecular mechanism of the adaption to drought stress in M. lutarioriparius is little known. In this study, RNA-seq technology was employed to analyze the transcriptome changes of diploid and tetraploid M. lutarioriparius after drought treatment. It was found that the number of differentially expressed genes in diploid M. lutarioriparius was much higher than tetraploid, whereas the tetraploid M. lutarioriparius may require fewer transcriptional changes. While the transcriptional changes in drought-tolerant tetraploid M. lutarioriparius are less than that of diploid, more known drought-tolerant pathways were significantly enriched than drought-sensitive diploid M. lutarioriparius. In addition, many drought-tolerance-related genes were constitutively and highly expressed in tetraploid under either normal condition or drought stress. These results together demonstrated that drought-tolerant tetraploid M. lutarioriparius, on the one hand, may preadapt to drought by constitutively overexpressing a series of drought-tolerant genes and, on the other hand, may adapt to drought by actively inducing other drought-tolerant-related pathways. Overall, this study could deepen our understanding of the molecular mechanism of drought-tolerance in bioenergy plants.

Expression of a triple mutational des-pGlu brazzein in transgenic mouse milk.

Brazzein has excellent potential for use as a sweetener because of its high level of sweetening potency and stability against extreme temperature and pH. It is extracted from the tropical and difficult-to-cultivate African plant Pentadiplandra brazzeana, which hampers its commercial viability. Here we report the mammary-specific expression of wildtype or triple mutational (H31R/E36D/E41A) des-pGlu brazzeins in the milk of transgenic mice. Using enzyme-linked immunoassay (ELISA), western blot, and sweetness intensity testing, we confirmed that the triple mutation made the des-pGlu brazzein molecule 10,000 times sweeter than sucrose in a weight base, even after 10 min of incubation at 100 °C; in addition, the triple mutant was also significantly sweeter than the wildtype des-pGlu brazzein. This study provides new insights for producing brazzein or brazzein-sweetened milk from animals for use in food and healthcare applications.

Characterization and Fine Mapping of qRPR1-3 and qRPR3-1, Two Major QTLs for Rind Penetrometer Resistance in Maize.

Stalk strength is one of the most important traits in maize, which affects stalk lodging resistance and, consequently, maize harvestable yield. Rind penetrometer resistance (RPR) as an effective and reliable measurement for evaluating maize stalk strength is positively correlated with stalk lodging resistance. In this study, one F2 and three F2:3 populations derived from the cross of inbred lines 3705I (the low RPR line) and LH277 (the high RPR line) were constructed for mapping quantitative trait loci (QTL), conferring RPR in maize. Fourteen RPR QTLs were identified in four environments and explained the phenotypic variation of RPR from 4.14 to 15.89%. By using a sequential fine-mapping strategy based on the progeny test, two major QTLs, qRPR1-3 and qRPR3-1, were narrowed down to 4-Mb and 550-kb genomic interval, respectively. The quantitative real-time PCR (qRT-PCR) assay was adopted to identify 12 candidate genes responsible for QTL qRPR3-1. These findings should facilitate the identification of the polymorphism loci underlying QTL qRPR3-1 and molecular breeding for RPR in maize.

The reference genome of Miscanthus floridulus illuminates the evolution of Saccharinae.

Miscanthus, a member of the Saccharinae subtribe that includes sorghum and sugarcane, has been widely studied as a feedstock for cellulosic biofuel production. Here, we report the sequencing and assembly of the Miscanthus floridulus genome by the integration of PacBio sequencing and Hi-C mapping, resulting in a chromosome-scale, high-quality reference genome of the genus Miscanthus. Comparisons among Saccharinae genomes suggest that Sorghum split first from the common ancestor of Saccharum and Miscanthus, which subsequently diverged from each other, with two successive whole-genome duplication events occurring independently in the Saccharum genus and one whole-genome duplication occurring in the Miscanthus genus. Fusion of two chromosomes occurred during rediploidization in M. floridulus and no significant subgenome dominance was observed. A survey of cellulose synthases (CesA) in M. floridulus revealed quite high expression of most CesA genes in growing stems, which is in agreement with the high cellulose content of this species. Resequencing and comparisons of 75 Miscanthus accessions suggest that M. lutarioriparius is genetically close to M. sacchariflorus and that M. floridulus is more distantly related to other species and is more genetically diverse. This study provides a valuable genomic resource for molecular breeding and improvement of Miscanthus and Saccharinae crops.

Interferon stimulated gene 15 promotes Zika virus replication through regulating Jak/STAT and ISGylation pathways.

Zika virus is an emergent arbovirus that has caused a public health emergency in South America. Zika virus infection is known to cause microcephaly and other congenital defects and Guillain-Barré syndrome. Unfortunately no direct antiviral treatments are available at present. IFN-stimulated gene 15 (ISG15) is one of the most upregulated host genes following type I interferon treatment or virus infections. ISG15 has been shown to have antiviral effect on a wide variety of viruses although pro-HCV replication was observed. However, the effect of ISG15 on ZIKV infection is not well defined. In this study, we try to clarify the effect of ISG15 on ZIKV replication and to further dissect the underlying mechanism. Our results indicated that ZIKV infection led to the increased expression of ISG15 in A549, 2fTGH, U5A cells. Overexpression of ISG15 stimulated ZIKV replication although ISG15 did not affect the viral entry. Further studies showed that this proviral effect was mediated through Jak/STAT signaling pathway and was ISGylation-dependent. Taken together, our work demonstrates that ISG15 is an important host factor exploited by ZIKV to facilitate its replication and might serve as a potential target for the development of novel antiviral agents.

Natural Variation of Lignocellulosic Components in Miscanthus Biomass in China.

Lignocellulose content is an important factor affecting the conversion efficiency of biomass energy plants. In this study, 179 Miscanthus accessions in China were used to determine the content of lignocellulose components in stems via acid hydrolysis and high-performance liquid chromatography. Results showed that the average lignocellulose content of wild Miscanthus germplasm resources was 80.27 ± 6.51%, and the average content of cellulose, hemicellulose, lignin, extracts, and total ash was 38.38 ± 3.52, 24.23 ± 4.21, 17.66 ± 1.56, 14.50 ± 5.60, and 2.53 ± 0.59%, respectively. The average lignocellulose content of M. sinensis, M. floridulus, M. nudipes, M. sacchariflorus, M. lutarioriparius, and the hybrids was 77.94 ± 6.06, 75.16 ± 4.98, 75.68 ± 3.02, 83.71 ± 4.78, 81.50 ± 5.23, and 74.72 ± 7.13%, respectively. In all the tested materials, the highest cellulose content was 48.52%, and the lowest was 29.79%. Hemicellulose had the maximum content of 34.23% and a minimum content of 15.71%. The highest lignin content was 23.75%, and the lowest was 13.01%. The lignocellulosic components of different ploidy materials were compared. The content of lignocellulosic components of diploid M. sacchariflorus was higher than that of tetraploid M. sacchariflorus, and the content of lignocellulosic components of diploid M. lutarioriparius was lower than that of tetraploid M. lutarioriparius. Analysis of the relationship between the changes in lignocellulosic components and geographical locations of Miscanthus showed that the holocellulose and hemicellulose content was significantly positive correlated with the latitude of the original growth location. Results indicated that the lignocellulosic components of Miscanthus resources in China are rich in genetic diversity.

Homoisoflavonoids and the Antioxidant Activity of Ophiopogon japonicus Root.

The root of Ophiopogon japonicus has been used as a traditional Chinese medicine and also a functional food ingredient for a long time in China. In the present study, 17 different homoisoflavonoid compounds were identified in the root extract of O. japonicus by HPLC-DAD and LCMS/MS analyses. The antioxidant activity of the of chloroform/methanol (1:1, v/v), methanol and 70% ethanol extracts, and two major isolated homoisoflavonoid compounds (methylophiopogonanone A and methylophiopogonanone B) from O. japonicus root were investigated by various in-vitro assays. Methylophiopogonanone B showed the highest antioxidant ability according to four antioxidant methods. Among the extracts, the chloroform/methanol extract which contained high amounts of homoisoflavonoids was found to exhibit the strongest antioxidant activity. The results showed that O. japonicus root can be regarded as a potential source of homoisoflavonoids and natural antioxidant.

Pulmonary immune cells and inflammatory cytokine dysregulation are associated with mortality of IL-1R1 -/-mice infected with influenza virus (H1N1).

Respirovirus infection can cause viral pneumonia and acute lung injury (ALI). The interleukin-1 (IL-1) family consists of proinflammatory cytokines that play essential roles in regulating immune and inflammatory responses in vivo. IL-1 signaling is associated with protection against respiratory influenza virus infection by mediation of the pulmonary anti-viral immune response and inflammation. We analyzed the infiltration lung immune leukocytes and cytokines that contribute to inflammatory lung pathology and mortality of fatal H1N1 virus-infected IL-1 receptor 1 (IL-1R1) deficient mice. Results showed that early innate immune cells and cytokine/chemokine dysregulation were observed with significantly decreased neutrophil infiltration and IL-6, TNF-α, G-CSF, KC, and MIP-2 cytokine levels in the bronchoalveolar lavage fluid of infected IL-1R1 -/- mice in comparison with that of wild type infected mice. The adaptive immune response against the H1N1 virus in IL-1R1 -/- mice was impaired with downregulated anti-viral Th1 cell, CD8+ cell, and antibody functions, which contributes to attenuated viral clearance. Histological analysis revealed reduced lung inflammation during early infection but severe lung pathology in late infection in IL-1R1 -/- mice compared with that in WT infected mice. Moreover, the infected IL-1R1 -/- mice showed markedly reduced neutrophil generation in bone marrow and neutrophil recruitment to the inflamed lung. Together, these results suggest that IL-1 signaling is associated with pulmonary anti-influenza immune response and inflammatory lung injury, particularly via the influence on neutrophil mobilization and inflammatory cytokine/chemokine production.

Insulin receptor substrate-4 interacts with ubiquitin-specific protease 18 to activate the Jak/STAT signaling pathway.

Ubiquitin-specific protease 18 (USP18) as a negative regulator of the Jak/STAT signaling pathway plays an important role in the host innate immune response. USP18 has been shown to bind to the type I interferon receptor subunit 2 (IFNAR2) to down-regulate the Jak/STAT signaling. In this study, we showed that insulin receptor substrate (IRS)-4 functioned as a novel USP18-binding protein. Co-precipitation assays revealed that two regions (amino acids 335-400 and 1094-1257) of IRS4 were related to bind to the C- terminal region of USP18. IRS4 binding to USP18 diminished the inhibitory effect of USP18 on Jak/STAT signaling. IRS4 over-expression enhanced while IRS4 knock-down suppressed the Jak/STAT signaling in the presence of IFN-a stimulation. As such, IRS4 increased IFN-a-mediated anti-HCV activity. Mechanistically, IRS4 promoted the IFN-a-induced Jak/STAT signaling by interact with USP18. These results suggested that IRS4 binds to USP18 to diminish the blunting effect of USP18 on IFN-a-induced Jak/STAT signaling. Our findings indicated that IRS4 is a novel USP18-binding protein that can be used to boost the host innate immunity to control HCV, and potentially other viruses that are sensitive to IFN-a.

Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD.

Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus.