Nitrogen-mediated volatilisation of defensive metabolites in tomato confers resistance to herbivores.

Plants synthesise a vast array of volatile organic compounds (VOCs), which serve as chemical defence and communication agents in their interactions with insect herbivores. Although nitrogen (N) is a critical resource in the production of plant metabolites, its regulatory effects on defensive VOCs remain largely unknown. Here, we investigated the effect of N content in tomato (Solanum lycopersicum) on the tobacco cutworm (Spodoptera litura), a notorious agricultural pest, using biochemical and molecular experiments in combination with insect behavioural and performance analyses. We observed that on tomato leaves with different N contents, S. litura showed distinct feeding preference and growth and developmental performance. Particularly, metabolomics profiling revealed that limited N availability conferred resistance upon tomato plants to S. litura is likely associated with the biosynthesis and emission of the volatile metabolite α-humulene as a repellent. Moreover, exogenous application of α-humulene on tomato leaves elicited a significant repellent response against herbivores. Thus, our findings unravel the key factors involved in N-mediated plant defence against insect herbivores and pave the way for innovation of N management to improve the plant defence responses to facilitate pest control strategies within agroecosystems.

Influence of ganglioside combined with methylprednisolone sodium succinate on efficacy and neurological function in patients with acute myelitis.

BACKGROUND: Acute myelitis (AM) can lead to sudden sensory, motor and autonomic nervous dysfunction, which negatively affects their daily activities and quality of life, so it is necessary to explore optimization from a therapeutic perspective to curb the progression of the disease. AIM: To investigate the effect of ganglioside (GM) combined with methylprednisolone sodium succinate (MPSS) on the curative effect and neurological function of patients with AM. METHODS: First, we selected 108 AM patients visited between September 2019 and September 2022 and grouped them based on treatment modality, with 52 patients receiving gamma globulin (GG) + MPSS and 56 patients receiving GM + MPSS, assigned to the control group (Con) and observation group (Obs), respectively. The therapeutic effect, neurological function (sensory and motor function scores), adverse events (AEs), recovery (time to sphincter function recovery, time to limb muscle strength recovery above grade 2, and time to ambulation), inflammatory factors (IFs) [interleukin (IL)-6, C-reactive protein (CRP), and tumor necrosis factor (TNF)-α] and other data of the two groups were collected for evaluation and comparison. RESULTS: The Obs had: (1) A significantly higher response rate of treatment than the Con; (2) Higher scores of sensory and motor functions after treatment that were higher than the baseline (before treatment) and higher than the Con levels; (3) Lower incidence rates of skin rash, gastrointestinal discomfort, dyslipidemia, osteoporosis and other AEs; (4) Faster posttreatment recovery of sphincter function, limb muscle strength and ambulation; and (5) Markedly lower posttreatment IL-6, CRP and TNF-α levels than the baseline and the Con levels. CONCLUSION: From the above, it can be seen that GM + MPSS is highly effective in treating AM, with a favorable safety profile comparable to that of GG + MPSS. It can significantly improve patients' neurological function, speed up their recovery and inhibit serum IFs.

A novel mRNA rabies vaccine as a promising candidate for rabies post-exposure prophylaxis protects animals from different rabies viruses.

Rabies, caused by the rabies virus (RABV), is the most fatal zoonotic disease. It is a neglected tropical disease which remains a major public health problem, causing approximately 59,000 deaths worldwide annually. Despite the existence of effective vaccines, the high incidence of human rabies is mainly linked to tedious vaccine immunisation procedures and the overall high cost of post-exposure prophylaxis. Therefore, it is necessary to develop an effective vaccine that has a simple procedure and is affordable to prevent rabies infection in humans. RABV belongs to the genus Lyssavirus and family Rhabdoviridae. Previous phylogenetic analyses have identified seven major clades of RABV in China (China I-VII), confirmed by analysing nucleotide sequences from both the G and N proteins. This study evaluated the immunogenicity and protective capacity of SYS6008, an mRNA rabies vaccine expressing rabies virus glycoprotein, in mice and cynomolgus macaques. We demonstrated that SYS6008 induced sufficient levels of rabies neutralising antibody (RVNA) in mice. In addition, SYS6008 elicited strong and durable RVNA responses in vaccinated cynomolgus macaques. In the pre-exposure prophylaxis murine model, one or two injections of SYS6008 at 1/10 or 1/30 of dosage provided protection against a challenge with a 30-fold LD50 of rabies virus (China I and II clades). We also demonstrated that in the post-exposure prophylaxis murine model, which was exposed to lethal rabies virus (China I-VII clades) before vaccination, one or two injections of SYS6008 at both 1/10 and 1/30 dosages provided better protection against rabies virus challenge than the immunization by five injections of commercial vaccines at the same dosage. In addition, we proved that SYS6008-induced RVNAs could neutralise RABV from the China I-VII clades. Finally, 1/10 of the dosage of SYS6008 was able to stimulate significant RABV-G specificity in the T cell response. Furthermore, we found that SYS6008 induced high cellular immunity, including RABV-G-specific T cell responses and memory B cells. Our results imply that the SYS6008 rabies vaccine, with a much simpler vaccination procedure, better immunogenicity, and enhanced protective capacity, could be a candidate vaccine for post-exposure prophylaxis of rabies infections.

Net Water Uptake Calculated in Standardized and Blindly Outlined Regions of the Middle Cerebral Artery Territory Predicts the Development of Malignant Edema in Patients With Acute Large Hemispheric Infarction.

Background and purpose: Previous studies have demonstrated that Net Water Uptake (NWU) is associated with the development of malignant edema (ME). The current study aimed to investigate whether NWU calculated in standardized and blindly outlined regions of the middle cerebral artery can predict the development of ME. Methods: We retrospectively included 119 patients suffering from large hemispheric infarction within onset of 24 h. The region of the middle cerebral artery territory was blindly outlined in a standard manner to calculate NWU. Patients were divided into two groups according to the occurrence of ME, which is defined as space-occupying infarct requiring decompressive craniotomy or death due to cerebral hernia in 7 days from onset. The clinical characteristics were analyzed, and the receiver operating characteristic curve (ROC curve) was used to assess the predictive ability of NWU and other factors for ME. Results: Multivariable analysis showed that NWU was an independent predictor of ME (OR 1.168, 95% CI 1.041-1.310). According to the ROC curve, NWU≥8.127% identified ME with good predictive power (AUC 0.734, sensitivity 0.656, specificity 0.862). Conclusions: NWU calculated in standardized and blindly outlined regions of the middle cerebral artery territory is also a good predictor for the development of ME in patients with large hemispheric infarction.

Optimization, Test and Diagnostics of Miniaturized Hall Thrusters.

Miniaturized spacecraft and satellites require smart, highly efficient and durable low-thrust thrusters, capable of extended, reliable operation without attendance and adjustment. Thermochemical thrusters which utilize thermodynamic properties of gases as a means of acceleration have physical limitations on their exhaust gas velocity, resulting in low efficiency. Moreover, these engines demonstrate extremely low efficiency at small thrusts and may be unsuitable for continuously operating systems which provide real-time adaptive control of the spacecraft orientation, velocity and position. In contrast, electric propulsion systems which use electromagnetic fields to accelerate ionized gases (i.e., plasmas) do not have any physical limitation in terms of exhaust velocity, allowing virtually any mass efficiency and specific impulse. Low-thrust Hall thrusters have a lifetime of several thousand hours. Their discharge voltage ranges between 100 and 300 V, operating at a nominal power of <1 kW. They vary from 20 to 100 mm in size. Large Hall thrusters can provide fractions of millinewton of thrust. Over the past few decades, there has been an increasing interest in small mass, low power, and high efficiency propulsion systems to drive satellites of 50-200 kg. In this work, we will demonstrate how to build, test, and optimize a small (30 mm) Hall thruster capable of propelling a small satellite weighing about 50 kg. We will show the thruster operating in a large space environment simulator, and describe how thrust is measured and electric parameters, including plasma characteristics, are collected and processed to assess key thruster parameters. We will also demonstrate how the thruster is optimized to make it one of the most efficient small thrusters ever built. We will also address challenges and opportunities presented by new thruster materials.

Interactions of ABA signaling core components (SlPYLs, SlPP2Cs, and SlSnRK2s) in tomato (Solanum lycopersicon).

Abscisic acid (ABA) regulates fruit development and ripening via its signaling. However, the exact role of ABA signaling core components in fruit have not yet been clarified. In this study, we investigated the potential interactions of tomato (Solanum lycopersicon) ABA signaling core components using yeast two-hybrid analysis, with or without ABA at different concentrations. The results showed that among 12 PYR/PYL/RCAR ABA receptors (SlPYLs), SlPYL1, SlPYL2, SlPYL4, SlPYL5, SlPYL 7, SlPYL8, SlPYL9, SlPYL10, SlPYL11, and SlPYL13 were ABA-dependent receptors, while SlPYL3 and SlPYL12 were ABA-independent receptors. Among five SlPP2Cs (type 2C protein phosphatases) and seven SlSnRK2s (subfamily 2 of SNF1-related kinases), all SlSnRK2s could interact with SlPP2C2, while SlSnRK2.8 also interacted with SlPP2C3. SlSnRK2.5 could interact with SlABF2/4 (ABA-responsive element binding factors). Expressions of SlPYL1, SlPYL2, SlPYL8, and SlPYL10 were upregulated under exogenous ABA but downregulated under nordihydroguaiaretic acid (NDGA) at the mature green stage of fruit ripening. The expressions of SlPP2C1, SlPP2C2, SlPP2C3, and SlPP2C5 were upregulated in ABA-treated fruit, but downregulated in NDGA-treated fruit at the mature green stage. The expressions of SlSnRK2.4, SlSnRK2.5, SlSnRK2.6, and SlSnRK2.7 were upregulated by ABA, but downregulated by NDGA. However, SlSnRK2.2 was down regulated by ABA. Expression of SlABF2/3/4 was enhanced by ABA but decreased by NDGA. Based on these results, we concluded that the majority of ABA receptor PYLs interact with SlPP2Cs in an ABA-dependent manner. SlPP2C2 and SlPP2C3 can interact with SlSnRK2s. SlSnRK2.5 could interact with SlABF2/4. Most ABA signaling core components respond to exogenous ABA.

Euphorbia fischeriana Steud inhibits malignant melanoma via modulation of the phosphoinositide-3-kinase/Akt signaling pathway.

Euphorbia fischeriana Steud, a traditional Chinese medicine, has been shown to inhibit the growth of various cancers by the induction of apoptosis and cell cycle arrest. The purpose of the present study was to investigate the association between the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and the inhibitory effect of Euphorbia fischeriana Steud on the growth and metastasis of melanoma B16 cells in vitro, and the underlying mechanisms. MTT assay results indicated that Euphorbia fischeriana Steud inhibited the growth of B16 cells in a time- and dose-dependent manner. Flow cytometric analysis revealed that Euphorbia fischeriana Steud markedly induced apoptosis of the B16 cells, with arrest at the G0/G1 phase of the cell cycle. In addition, in a Transwell assay Euphorbia fischeriana Steud significantly suppressed the migration of B16 cells. Western blot analysis revealed that the expression levels of phosphatase and tensin homolog (PTEN) were upregulated, and the phosphorylation of Akt was downregulated, which resulted in inhibition of the PI3K/Akt signaling pathway and the eventual suppression of its downstream targets, such as matrix metalloproteinase-2 mRNA, in B16 cells. The results demonstrated that Euphorbia fischeriana Steud inhibited the growth and migration of B16 cells, possibly via modulation of the PI3K/Akt signaling pathway and upregulation of PTEN expression levels, in addition to downregulation of p-Akt expression. The aforementioned findings suggest that Euphorbia fischeriana Steud may have broad therapeutic applications in the treatment of malignant melanoma.

Hepatocyte growth factor gene-modified bone marrow-derived mesenchymal stem cells transplantation promotes angiogenesis in a rat model of hindlimb ischemia.

Angiogenic gene therapy and cell-based therapy for peripheral arterial disease(PAD) have been studied intensively currently. This study aimed to investigate whether combining mesenchymal stem cells(MSCs) transplantation with ex vivo human hepatocyte growth factor(HGF) gene transfer was more therapeutically efficient than the MSCs therapy alone in a rat model of hindlimb ischemia. One week after establishing hindlimb ischemia models, Sprague-Dawley(SD) rats were randomized to receive HGF gene-modified MSCs transplantation(HGF-MSC group), untreated MSCs transplantation (MSC group), or PBS injection(PBS group), respectively. Three weeks after injection, angiogenesis was significantly induced by both MSCs and HGF-MSCs transplantation, and capillary density was the highest in the HGF-MSC group. The number of transplanted cell-derived endothelial cells was greater in HGF-MSC group than in MSC group after one week treatment. The expression of angiogenic cytokines such as HGF and VEGF in local ischemic muscles was more abundant in HGF-MSC group than in the other two groups. In vitro, the conditioned media obtained from HGF-MSCs cultures exerted proproliferative and promigratory effects on endothelial cells. It is concluded that HGF gene-modified MSCs transplantation therapy may induce more potent angiogenesis than the MSCs therapy alone. Engraftment of MSCs combined with angiogenic gene delivery may be a promising therapeutic strategy for the treatment of severe PAD.

Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids.

To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids.

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system.

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10(4) molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.

Double Candida antarctica lipase B co-display on Pichia pastoris cell surface based on a self-processing foot-and-mouth disease virus 2A peptide.

To develop a high efficiency Candida antarctica lipase B (CALB) yeast display system, we linked two CALB genes fused with Sacchromyces cerevisiae cell wall protein genes, the Sed1 and the 3'-terminal half of Sag1, separately by a 2A peptide of foot-and-mouth disease virus (FMDV) in a single open reading frame. The CALB copy number of recombinant strain KCSe2ACSa that harbored the ORF was identified, and the quantity of CALB displayed on the cell surface and the enzyme activity of the strain were measured. The results showed that the fusion of multiple genes linked by 2A peptide was translated into two independent proteins displayed on the cell surface of stain KCSe2ACSa. Judging from the data of immunolabeling assay, stain KCSe2ACSa displayed 94 % CALB-Sed1p compared with stain KCSe1 that harbored a single copy CALB-Sed1 and 64 % CALB-Sag1p compared with stain KCSa that harbored a single copy CALB-Sag1 on its surface. Besides, strain KCSe2ACSa possessed 170 % hydrolytic activity and 155 % synthetic activity compared with strain KCSe1 as well as 144 % hydrolytic activity and 121 % synthetic activity compared with strain KCSa. Strain KCSe2ACSa even owned 124 % hydrolytic activity compared with strain KCSe2 that harbored two copies CALB-Sed1. The heterogeneous glycosylphosphatidylinositol-anchored proteins co-displaying yeast system mediated by FMDV 2A peptide was shown to be an effective method for improving the efficiency of enzyme-displaying yeast biocatalysts.

High-throughput screening of B factor saturation mutated Rhizomucor miehei lipase thermostability based on synthetic reaction.

Conventional lipase screening methods are mostly based on hydrolytic activity, which may not always be the best method to assess the enzyme activity, especially for evaluating synthetic activity. Here we developed a high throughput and visual method to screen clones with high synthetic activity and used it to assess lipases thermostability. All mutants' lipase synthetic activity were identified through esterification of caprylic acid and ethanol with methyl red as the pH indicator adding in the substrates on according to the color change halo around the colony on culture plates since synthetic reaction was often accompanied with a rise in pH. After two rounds operation with the pH indicator screening method, we obtained a double mutant Asn120Lys/Lys131Phe from the Rhizomucor miehei lipase saturation mutated library based on amino acid residue B factors. The mutant's initial synthetic activity was a little higher than wild type and its thermostability in synthetic reaction was enhanced, which remained 63.1% residual activity after being heated at 70°C for 5h comparing to 51.0% of wild type. The double mutant with the two residue replacements balanced well between stability and activity. Yeast surface display technology and the pH indicator method, combined with colony screening were shown to facilitate high-throughput screening for lipase synthetic activity.

Genetic differentiation of relictual populations of Alsophila spinulosa in southern China inferred from cpDNA trnL-F noncoding sequences.

The genetic differentiation and phylogeographical pattern of 11 relictual populations of Alsophila spinulosa distributed across Hainan, Guangdong, and Guangxi in southern China were inferred from sequence variations of trnL-F noncoding regions of chloroplast DNA (cpDNA). The length of trnL-F noncoding sequences varied from 863 to 940 bp. The A + T content was 62.23-63.36%. Sequences were neutral in terms of evolution (Tajima's criterion D=-0.62417, P>0.10 and Fu and Li's test D*=-1.45455, P>0.10; F*=-1.32798, P>0.10). Thirty-four haplotypes were identified based on nucleotide variation. Relatively high levels of haplotype diversity (h=0.929) and nucleotide diversity (Dij=0.022263) were detected in A. spinulosa, probably associated with its long evolutionary history which allowed the accumulation of genetic variation within lineages. Both the minimum spanning network and the strict consensus tree of the most parsimonious trees generated for haplotypes demonstrated that the investigated populations of A. spinulosa were subdivided into two geographical groups: Hainan and Guangdong-Guangxi. An analysis of molecular variance (AMOVA) indicated that most of the genetic variation (87.48%, P<0.001) was partitioned among regions. Spatial structure measurements revealed that population genetic structure was not related to geographical distance. This research suggests that blocked gene flow by Qiongzhou strait and an inbreeding system might result in the geographical subdivision between Hainan and Guangdong-Guangxi (F(ST)=0.92, Nm=0.09). Within each region, the "star like" pattern of phylogeography of haplotypes implied a population expansion process during evolutionary history. Gene genealogies together with coalescent theory were useful tools for uncovering the phylogeography of A. spinulosa.