Heterologous expression and characterization of two delta glutathione S-transferases genes involved in imidacloprid metabolism in Grapholita molesta.

Glutathione S-transferases (GSTs) are multifunctional enzymes, and insect GSTs play a pivotal role in the metabolism of insecticides. Grapholita molesta is a worldwide pest that causes substantial economic losses to the fruit industry. However, it remains unclear how imidacloprid, a commonly used insecticide in orchards, is metabolized by G. molesta. In the present study, the synergist diethyl maleate (DEM), which inhibits the GST activity, exhibited a 22-fold synergistic ratio against imidacloprid. Two new GST genes, GmGSTD2 (OR096251) and GmGSTD3 (OR096252), were identified and successfully cloned, showing the highest expression in the Malpighian tubes. Knockdown of GmGSTD2 and GmGSTD3 by RNA interference, increased the mortality of G. molesta from 28% to 47% following imidacloprid treatment. Both recombinant GmGSTD2 and GmGSTD3 proteins exhibited 1-chloro-2,4-dinitrobenzene (CDNB) activity and could be inhibited by imidacloprid in vitro, with maximum inhibition was 60% for GmGSTD2 and 80% for GmGSTD3. These results suggested that GSTs participate in the metabolism of imidacloprid with GmGSTD2 and GmGSTD3 playing key roles in this process.

Various functions of detoxification enzymes against insecticides in Nilaparvata lugens selected by toxicity assays and RNAi methods.

The brown planthopper (BPH), Nilaparvata lugens is a devastating agricultural pest of rice, and they have developed resistance to many pesticides. In this study, we assessed the response of BPH nymphs to nitenpyram, imidacloprid, and etofenprox using contact and dietary bioassays, and investigated the underlying functional diversities of BPH glutathione-S-transferase (GST), carboxylesterase (CarE) and cytochrome P450 monooxygenase (P450) against these insecticides. Both contact and ingestion toxicity of nitenpyram to BPH were significantly higher than either imidacloprid or etofenprox. Under the LC50 concentration of each insecticide, they triggered a distinct response for GST, CarE, and P450 activities, and each insecticide induced at least one detoxification enzyme activity. These insecticides almost inhibited the expression of all tested GST, CarE, and P450 genes in contact bioassays but induced the transcriptional levels of these genes in dietary bioassays. Silencing of NlGSTD2 expression had the greatest effect on BPH sensitivity to nitenpyram in contact test and imidacloprid in dietary test. The sensitivities of BPH to insecticide increased the most in the contact test was etofenprox after silencing of NlCE, while the dietary test was nitenpyram. Knockdown of NlCYP408A1 resulted in BPH sensitivities to insecticide increasing the most in the contact test was nitenpyram, while the dietary test was imidacloprid. Taken together, these findings reveal that NlGSTD2, NlCE, and NlCYP408A1 play an indispensable role in the detoxification of the contact and ingestion toxicities of different types of insecticides to BPH, which is of great significance for the development of new strategies for the sucking pest control.

Variations in Mating and Reproduction in Oriental Fruit Moth Caused by Adult Physiological State in Laboratory Tests.

Grapholita molesta (Busck) is a pest of rosaceous fruit plants worldwide. Due to a combination of monandry and promiscuity in G. molesta, the age and mating history of both sexes significantly affected the mating and reproductive success. In this study, the interactions of different ages (3, 5, or 7 days) and mating history (unmated or mated) in each sex on the mating selection, reproductive system, and offspring production were investigated in the laboratory. The results showed that these differences mainly occurred in young females or males, associated with unmated or mated state. Especially, the 3-day-old unmated females were preferred by the 7-day-old males but discriminated against by the 3- or 5-day-old unmated males, whereas the 3-day-old mated males were preferred by the 3-day-old mated or 7-day-old females but discriminated against by the 3- or 5-day-old unmated females. The lengths of the ovarian ducts were affected by age in the unmated females, with the greatest length being found at 7 days old. The size of testes varied with age in the unmated males, being the largest at 3 days old. At 3 days old, the testes size of the unmated males was larger than that of the mated males. The pairing of 5-day-old unmated females × 3-day-old mated males maximized the successful matings. The least productive pairing was 7-day-old unmated females × 5-day-old mated males. The pairing of 5-day-old mated males × 3-day-old mated females had the lowest number of matings and the highest number of offspring. The pairing of 3-day-old mated females × 3-day-old mated males had a high rate of mating success and the most offspring. These results revealed the different roles between females and males because of physiological states in terms of the reproductive biology in G. molesta.

Complete Chloroplast Genome of Alternanthera sessilis and Comparative Analysis with Its Congeneric Invasive Weed Alternanthera philoxeroides.

Alternanthera sessilis is considered the closest relative to the invasive weed Alternanthera philoxeroides in China, making it an important native species for studying the invasive mechanisms and adaptations of A. philoxeroides. Chloroplasts play a crucial role in a plant's environmental adaptation, with their genomes being pivotal in the evolution and adaptation of both invasive and related species. However, the chloroplast genome of A. sessilis has remained unknown until now. In this study, we sequenced and assembled the complete chloroplast genome of A. sessilis using high-throughput sequencing. The A. sessilis chloroplast genome is 151,935 base pairs long, comprising two inverted repeat regions, a large single copy region, and a small single copy region. This chloroplast genome contains 128 genes, including 8 rRNA-coding genes, 37 tRNA-coding genes, 4 pseudogenes, and 83 protein-coding genes. When compared to the chloroplast genome of the invasive weed A. philoxeroides and other Amaranthaceae species, we observed significant variations in the ccsA, ycf1, and ycf2 regions in the A. sessilis chloroplast genome. Moreover, two genes, ccsA and accD, were found to be undergoing rapid evolution due to positive selection pressure. The phylogenetic trees were constructed for the Amaranthaceae family, estimating the time of independent species formation between A. philoxeroides and A. sessilis to be approximately 3.5186-8.8242 million years ago. These findings provide a foundation for understanding the population variation within invasive species among the Alternanthera genus.

Multifaceted functions of Drp1 in hypoxia/ischemia-induced mitochondrial quality imbalance: from regulatory mechanism to targeted therapeutic strategy.

Hypoxic-ischemic injury is a common pathological dysfunction in clinical settings. Mitochondria are sensitive organelles that are readily damaged following ischemia and hypoxia. Dynamin-related protein 1 (Drp1) regulates mitochondrial quality and cellular functions via its oligomeric changes and multiple modifications, which plays a role in mediating the induction of multiple organ damage during hypoxic-ischemic injury. However, there is active controversy and gaps in knowledge regarding the modification, protein interaction, and functions of Drp1, which both hinder and promote development of Drp1 as a novel therapeutic target. Here, we summarize recent findings on the oligomeric changes, modification types, and protein interactions of Drp1 in various hypoxic-ischemic diseases, as well as the Drp1-mediated regulation of mitochondrial quality and cell functions following ischemia and hypoxia. Additionally, potential clinical translation prospects for targeting Drp1 are discussed. This review provides new ideas and targets for proactive interventions on multiple organ damage induced by various hypoxic-ischemic diseases.

The brown planthopper NlDHRS11 is involved in the detoxification of rice secondary compounds.

BACKGROUND: The brown planthopper (Nilaparvata lugens, BPH) is the most destructive serious pest in rice production. Resistant varieties are effective means to defend against BPH, but the impact of the ingestion of resistant rice on BPH transcriptional regulation is still unclear. Here, we explore the molecular basis of the regulation by BPH feeding on resistant rice. RESULTS: BPH nymphs preferentially selected susceptible rice TN1 at 24 h after release in a choice test. Feeding on resistant rice IR56 under nonselective conditions increased mortality, decreased growth rate, and prolonged the molting time of BPH. Transcriptomic sequencing revealed 38 dysregulated genes, including 31 down-regulated and seven up-regulated genes in BPH feeding on resistant rice for 7 days compared with feeding on susceptible rice TN1. These genes were mainly involved in the pathways of growth and development, metabolism, energy synthesis, and transport. Finally, we showed that the toxicities of rice defensive compounds to BPH were dose-dependent, and silencing of the BPH gene dehydrogenase/reductase SDR family member 11 (NlDHRS11) increased sensibility to the rice secondary compounds ferulic acid and resorcinol. CONCLUSION: The adaption of BPH feeding on resistant rice is orchestrated by dynamically regulating gene expressions, and NlDHRS11 is a gene involved in the detoxification of plant defensive chemicals. The current work provides new insights into the interaction between insects and plants, and will help to develop novel BPH control strategies. © 2023 Society of Chemical Industry.

Morphological Characterstics of the Sensilla in a Monophagous Insect: Agasicles hygrophila Selman and Vogt (Coleoptera: Chrysomelidae, Halticinae).

Agasicles hygrophila Selman and Vogt (Coleoptera: Chrysomelidae) is the key natural enemy of Alternanthera philoxeroides (Mart.) Griseb, an invasive weed worldwide. To understand the morphology of A. hygrophila and further explore the specific host localization mechanism, scanning electron microscopy was used to observe and study the morphological characteristics of sensilla on the head appendages, tarsi, and external genital segments of A. hygrophila. Twelve types and forty-six subtypes of sensilla were observed. These contain various types of head appendices, including sensilla chaetica, sensilla trichodea, sensilla basiconca, sensilla coeloconica, sensilla styloconica, Böhm bristles, sensilla campaniform, sensilla terminal, sensilla dome, sensilla digit-like, sensilla aperture, and many subtypes. A new type of sensor was reported for the first time, which may be related to host plant recognition. This sensor was located on the distal segment of the maxillary palps of A. hygrophila and was named as sensilla petal-shaped based on its morphological characteristics. Sensilla chaetica, sensilla trichodea, and sensilla basiconca are also found on the tarsi and external genital segments. In addition, sensilla basiconica 4, sensilla coeloconica 1 and 2, sensilla styloconica 2, Böhm bristles 2, and sensilla campaniform 1 were only found in females. On the contrary, sensilla styloconica 3, sensilla coeloconica 3, and sensilla dome were only found in males. Numbers and sizes of the sensilla were also different between males and females. The potential functions related to structure were discussed in comparison with previous investigations on beetles and other monophagous insects. Our results provide a microscopic morphological basis for further research on the localization and recognition mechanism of A. hygrophila and its obligate host.

Identification, Pathogenicity, and Culture Conditions of a New Isolate of Cordyceps javanica (Hypocreales: Cordycipitaceae) From Soil.

This study decribes a highly effective insecticidal isolate of Cordyceps javanica (Frieder. & Bally) (Hypocreales: Cordycipitaceae) named IJ-tg19, which was isolated from soil. Spray bioassays were performed with IJ-tg19 on Myzus persicae (Sulzer) (Hemiptera: Aphididae) adults, third-instar nymphs of Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), and third-instar larvae of Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) to determine the pathogenicity of the isolate. The corrected mortality rates for all three pests were 100% when the conidia concentration was 1 × 106 conidia/ml, the lowest concentration in this study, and the median survival times (MST) were 4, 4, and 3 d. The MST shortens with increasing conidia concentration. The effects of laboratory culture conditions on the sporulation and growth of the isolate were also studied. This isolate had the greatest conidia production and fastest growth rate on malt extract agar medium at 25°C. The amount of conidia produced had positive correlation to light duration, with the highest production at 24 hr light. The growth of mycelium can adapt to a moderately alkaline environment, but the optimum conidial production occurred at the pH of 7. Our finding and research will be useful in biocontrol programs that are considering using the new isolate of C. javanica against greenhouse pests.

Development and Fecundity of Oriental Fruit Moth (Lepidoptera: Tortricidae) Reared on Various Concentrations of Amygdalin.

Grapholita molesta (Busck) (Lepidoptera: Tortricidae), Oriental fruit moth (OFM), attacks fruits and shoots of the economically important trees in Rosaceae. Amygdalin is a cyanogenic glucoside of rosaceous plants that may be related to the seasonal patterns of infestation in many pests. The amygdalin concentration of fruits and shoots of peach, pear, and apple varies over the growing season. However, the relationship between the amygdalin concentration and G. molesta performance has not been reported. Here, we measured the performance (feeding, growth, development, and fecundity) of G. molesta larvae (as subsequent adults) reared on artificial diets with six amygdalin concentrations (0, 3, 6, 12, 24, and 48 mg/g), and we then calculated the population parameters. We found that these different concentrations of amygdalin affected the developmental time and fecundity, except for the proportion of larvae feeding on the diet and the survival rates of larvae and pupae. When compared with the control diet without amygdalin, diets with 3 or 6 mg/g (low and moderate concentrations) of amygdalin shortened developmental times and increased the number of eggs laid by females; however, a diet with 12 mg/g (moderate concentration) of amygdalin only increased the number of eggs laid by females and did not affect the larval and pupal developmental rate. A diet with 48 mg/g (high concentration) of amygdalin prolonged developmental times and reduced the number of eggs laid by females when compared with the control diet without amygdalin. Furthermore, the intrinsic rate of increase (rm) for insects reared on diets with 3 or 6 mg/g (low and moderate concentrations) of amygdalin versus the control diet without amygdalin showed a slightly improved population growth. However, this increase in the rm value did not persist over ten successive generations of rearing on the same diet. We concluded that the diet with 6 mg of amygdalin per g of diet can enhance the performance and population growth of G. molesta, but the effects of amygdalin are concentration-dependent.

Oxidative-Damaged Mitochondria Activate GABARAPL1-Induced NLRP3 Inflammasomes in an Autophagic-Exosome Manner after Acute Myocardial Ischemia.

Objective: This study is aimed at identifying the potential diagnostic markers for circulating endothelial cells (CECs) for acute myocardial ischemia (AMI) and exploring the regulatory mechanisms of the selected biomarker in mitochondrial oxidative damage and vascular inflammation in AMI pathology. Methods: Utilizing the Gene Expression Omnibus dataset GSE66360, we scanned for differentially expressed genes (DEGs) in 49 AMI patients and 50 healthy subjects. To discover possible biomarkers, LASSO regression and support vector machine recursive feature elimination examinations were conducted. Using the GSE60993 and GSE123342 datasets and AMI rat models, the expression levels and diagnostic accuracy of the biomarkers in AMI were thoroughly verified. CIBERSORT was employed to evaluate the compositional patterns of 22 distinct immunological cell percentages in AMI according to combined cohorts. The oxidative-damaged mitochondria were detected by confocal microscopy observation of MitoTracker, ROS-DCFH-DA, and mCherry-GFP-LC3B. Results: In total, 122 genes were identified. The identified DEGs primarily contributed in arteriosclerosis, arteriosclerotic cardiovascular disorders, bacterial infectious disorder, coronary artery disease, and myocardial infarction. Nine features (NR4A2, GABARAPL1 (GEC1), CLEC4D, ITLN1, SNORD89, ZFP36, CH25H, CCR2, and EFEMP1) of the DEGs were shared by two algorithms, and GABARAPL1 (GEC1) was identified and verified as a diagnostic mitochondrial biomarker for AMI. Confocal results showed that there existed mitochondrial damage and oxidative stress in cardiac CMECs after AMI, and the blocked autophagy flux could be released by exosome burst in cardiac CMECs and blood CECs. Immune cell infiltration testing declared that elevated GEC1 expression in blood CECs was linked to the rise of monocytes and neutrophils. Functional tests revealed that high GEC1 expression in CMECs and CECs could activate the vascular inflammatory response by stimulating NLRP3 inflammasome production after AMI. Conclusion: Oxidative-damaged mitochondria in cardiac CMECs activate GEC1-mediated autophagosomes but block autophagy flux after AMI. The exfoliated cardiac CMECs evolve into abnormal blood CECs, and the undegraded GEC1 autophagosomes produce a large number of NLRP3 inflammasomes by exosome burst, stimulating the increase in monocytes and neutrophils and ultimately triggering vascular inflammation after AMI. Therefore, GEC1 in blood CECs is a highly specific diagnostic mitochondrial biomarker for AMI.

Identification of MMP9 as a Novel Biomarker to Mitochondrial Metabolism Disorder and Oxidative Stress in Calcific Aortic Valve Stenosis.

Calcific aortic valve stenosis (CAVS) is the most common heart valve disorder among humans. To date, no effective method has been identified to prevent this disease. Herein, we aimed to identify novel diagnostic and mitochondria-related biomarkers of CAVS, based on two machine learning algorithms. We further explored their association with infiltrating immune cells and studied their potential function in CAVS. The GSE12644, GSE51472, and GSE83453 expression profiles were downloaded from the Gene Expression Omnibus (GEO) repository. The GSE12644 and GSE51472 datasets were integrated to identify differentially expressed genes (DEGs). GSE12644 contains 10 normal and 10 CAVS samples, whereas GSE51472 contains 5 normal and 10 CAVS samples. GO and KEGG assays of DEGs were conducted, and the correlation between matrix metalloproteinase 9 (MMP9) expression and immune cell infiltration was explored, using CIBERSORT. The LASSO regression model and SVM-RFE analysis were used to identify diagnostic genes. The expression of MMP9 in CAVS and non-CAVS samples was measured using RT-PCR, western blotting and immunohistochemistry. A series of functional experiments were performed to explore the potential role of MMP9 in mitochondrial metabolism and oxidative stress during CAVS progression. Twenty-two DEGs were identified, of which six genes (SCG2, PPBP, TREM1, CCL19, WIF1, and MMP9) were ultimately distinguished as diagnostic genes in CAVS. Of these, MMP9 was indicated as a mitochondria-related gene, the expression and diagnostic value of which were further confirmed in the GSE83453 dataset. Correlation analysis revealed a positive correlation between MMP9 and infiltrating immune cells. In our cohort, MMP9 expression was distinctly increased in CAVS samples, and its inhibition attenuated the calcification of valve interstitial cells (VICs) by suppressing mitochondrial damage and oxidative stress. Taken together, our findings suggest MMP9 as a novel mitochondrial dysfunction biomarker and therapeutic target for CAVS.

Toxicity of crude toxin protein produced by Cordyceps fumosorosea IF-1106 against Myzus persicae (Sulze).

The entomopathogenic fungus Cordyceps fumosorosea IF-1106 is a potential biocontrol agent with high pathogenicity to the aphid Myzus persicae. We extracted the crude toxin protein from a liquid culture broth of an isolated C. fumosorosea strain using the ammonium sulfate precipitation method, and its toxicity to Myzus persicae was measured by injection, oral exposure, and topical exposure. The crude toxin protein of C. fumosorosea had insecticidal activity against M. persicae. Body cavity injection and oral exposure had significantly higher insecticidal activity against adults than contact sprays. The highest cumulative corrected mortality of adults after injection was 81.85 ± 13.45 %, and the highest cumulative corrected mortality of adults after ingestion was 85.45 ± 11.88 %. The proportion of plasmatocytes in adult blood lymphocytes reached the highest at 3 days after injection and feeding, and the proportion of granulocytes was the highest at 2 days after injection and feeding. These data confirmed the toxicity of the crude toxin protein of C. fumosorosea toxin to M. persicae and helped clarify the pathogenic mechanism of the strain. Population management of M. persicae may be possible by using a natural toxic compound produced by C. fumosorosea that is selective to this pest species.

SARS-CoV-2 Achieves Immune Escape by Destroying Mitochondrial Quality: Comprehensive Analysis of the Cellular Landscapes of Lung and Blood Specimens From Patients With COVID-19.

Mitochondria get caught in the crossfire of coronavirus disease 2019 (COVID-19) and antiviral immunity. The mitochondria-mediated antiviral immunity represents the host's first line of defense against viral infection, and the mitochondria are important targets of COVID-19. However, the specific manifestations of mitochondrial damage in patients with COVID-19 have not been systematically clarified. This study comprehensively analyzed one single-cell RNA-sequencing dataset of lung tissue and two bulk RNA-sequencing datasets of blood from COVID-19 patients. We found significant changes in mitochondrion-related gene expression, mitochondrial functions, and related metabolic pathways in patients with COVID-19. SARS-CoV-2 first infected the host alveolar epithelial cells, which may have induced excessive mitochondrial fission, inhibited mitochondrial degradation, and destroyed the mitochondrial calcium uniporter (MCU). The type II alveolar epithelial cell count decreased and the transformation from type II to type I alveolar epithelial cells was blocked, which exacerbated viral immune escape and replication in COVID-19 patients. Subsequently, alveolar macrophages phagocytized the infected alveolar epithelial cells, which decreased mitochondrial respiratory capacity and activated the ROS-HIF1A pathway in macrophages, thereby aggravating the pro-inflammatory reaction in the lungs. Infected macrophages released large amounts of interferon into the blood, activating mitochondrial IFI27 expression and destroying energy metabolism in immune cells. The plasma differentiation of B cells and lung-blood interaction of regulatory T cells (Tregs) was exacerbated, resulting in a cytokine storm and excessive inflammation. Thus, our findings systematically explain immune escape and excessive inflammation seen during COVID-19 from the perspective of mitochondrial quality imbalance.

Activated Drp1 regulates p62-mediated autophagic flux and aggravates inflammation in cerebral ischemia-reperfusion via the ROS-RIP1/RIP3-exosome axis.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive. METHODS: The 150 male C57 mice underwent middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h, Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1 (Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine (NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA (mtDNA) copy number, intracellular and mitochondrial reactive oxygen species (ROS), autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/LC3 I, TNF-α, IL-1ß, etc., were detected under normal or Drp1 interference conditions. RESULTS: The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI (P < 0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44% and 88% by Drp1 short hairpin RNA (shRNA) (P < 0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC (P < 0.05). RIP1/RIP3 inhibitor Necrostatin-1 (Nec-1) restored 75% to a low LC3 II/LC3 I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation (P < 0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes (P > 0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4 - 5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1 (P < 0.05). Furthermore, TNF-α and IL-1ß increased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen-glucose deprivation/reoxygenation (OGD/R) conditions (P < 0.05). CONCLUSIONS: CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation, triggering a vicious cycle.

[Identification and expression analysis of WRKY gene family in eukaryotic algae].

WRKY is a superfamily of plant-specific transcription factors, playing a critical regulatory role in multiple biological processes such as plant growth and development, metabolism, and responses to biotic and abiotic stresses. Although WRKY genes have been characterized in a variety of higher plants, little is known about them in eukaryotic algae, which are close to higher plants in evolution. To fully characterize algal WRKY family members, we carried out multiple sequence alignment, phylogenetic analysis, and conserved domain prediction to identify the WRKY genes in the genomes of 30 algal species. A total of 24 WRKY members were identified in Chlorophyta, whereas no WRKY member was detected in Rhodophyta, Glaucophyta, or Bacillariophyta. The 24 WRKY members were classified into Ⅰ, Ⅱa, Ⅱb and R groups, with a conserved heptapeptide domain WRKYGQ(E/A/H/N)K and a zinc finger motif C-X4-5-C-X22-23-H-X-H. Haematococcus pluvialis, a high producer of natural astaxanthin, contained two WRKY members (HaeWRKY-1 and HaeWRKY-2). Furthermore, the coding sequences of HaeWRKY-1 and HaeWRKY-2 genes were cloned and then inserted into prokaryotic expression vector. The recombinant vectors were induced to express in Escherichia coli BL21(DE3) cells and the fusion proteins were purified by Ni-NTA affinity chromatography. HaeWRKY-1 had significantly higher expression level than HaeWRKY-2 in H. pluvialis cultured under normal conditions. High light stress significantly up-regulated the expression of HaeWRKY-1 while down-regulated that of HaeWRKY-2. The promoters of HaeWRKY genes contained multiple cis-elements responsive to light, ethylene, ABA, and stresses. Particularly, the promoter of HaeWRKY-2 contained no W-box specific for WRKY binding. However, the W-box was detected in the promoters of HaeWRKY-1 and the key enzyme genes HaeBKT (ß-carotene ketolase) and HaePSY (phytoene synthase) responsible for astaxanthin biosynthesis. Considering these findings and the research progress in the related fields, we hypothesized that the low expression of HaeWRKY-2 under high light stress may lead to the up-regulation of HaeWRKY-1 expression. HaeWRKY-1 may then up-regulate the expression of the key genes (HaeBKT, HaePSY, etc.) for astaxanthin biosynthesis, consequently promoting astaxanthin enrichment in algal cells. The findings provide new insights into further analysis of the regulatory mechanism of astaxanthin biosynthesis and high light stress response of H. pluvialis.

Identification and Characterization of CYP6 Family Genes from the Oriental Fruit Moth (Grapholita molesta) and Their Responses to Insecticides.

Cytochrome P450 (CYP) monooxygenases comprise a superfamily of proteins that detoxify xenobiotics and plant secondary metabolites in insects. The CYP6 family is unique to the class Insecta, and its members participate in the metabolism of exogenous substances. In this study, we sequenced and characterized the full-length cDNAs of eight CYP6 family genes from Grapholita molesta (Busck), a global pest of pome fruits. P450 genes with the exception of CYP6AN35, which was most highly expressed in adults, consistently showed high expression in third- or fourth-instar larvae. The analysis of different tissues of adults showed that most of these genes were predominantly expressed in the midgut, Malpighian tubules, and/or fat body. The expression of these eight CYP6 genes was differentially affected by three representative insecticides: malathion (organophosphate), deltamethrin (pyrethroid), and chlorantraniliprole (carbamate). All eight CYP6 genes responded to malathion treatment. Only three CYP6 genes were highly expressed in deltamethrin-treated individuals. Chlorantraniliprole treatment exerted weak effects on gene expression. Interestingly, CYP6AN35 was a highly expression level in the adult head and its expression was induced by all three insecticides. CYP6AN35 may be a key gene in the metabolism of insecticides. This study provides a fundamental understanding of the functions of the CYP6 gene family in insecticide metabolism in G. molesta.

Hypoxia-primed monocytes/macrophages enhance postinfarction myocardial repair.

Background: Oxygen supplementation in myocardial infarction (MI) remains controversial. Inflammation is widely believed to play a central role in myocardial repair. A better understanding of these processes may lead to the design of novel strategies complementary to MI treatment. Methods: To investigate the role of hypoxia in inflammation and myocardial repair after acute MI, we placed MI mice in a tolerable mild hypoxia (10% O2) chamber for 7 days and then transferred the mice to ambient air for another 3 weeks. Results: We found that the cumulative survival rate of the MI mice under hypoxia was significantly higher than that under oxygen supplementation. Hypoxia promoted postinfarction myocardial repair. Importantly, we found that hypoxia modulated the phenotypic transition of blood monocytes from pro-inflammatory to pro-reparative in a timely manner, leading to the subsequent discontinuation of inflammation in myocardial tissues and promotion of myocardial repair post-MI. Specifically, cultured bone marrow-derived macrophages (BMDMs) primed by hypoxia in vitro exhibited improved reparative capacities and differed from M1 and M2 macrophages through the AMPKα2 signaling pathway. The deletion of AMPKα2 in monocytes/macrophages prevented the phenotypic transition induced by hypoxia and could not promote myocardial repair after MI when transplanted into the myocardium. Conclusions: Taken together, our work demonstrates that hypoxia promotes postinfarction myocardial repair by modulating the blood monocyte/macrophage phenotypic transition from pro-inflammatory to pro-reparative in a timely manner through the AMPKα2 signaling pathway. Hypoxia priming might be an attractive translational strategy for MI treatment by amplifying immune cells during early inflammation and subsequent resolution and repair.

A nomogram prediction model for sternal incision problems.

Presently, the incidence and mortality rates of sternal incision problems (SIPs) after thoracotomy remain high, and no effective preventive measures are available. The data on 23 182 patients at Xinqiao Hospital, Army Medical University treated with median sternotomy from 1 August 2009 to 31 July 2019 were retrospectively reviewed. A prediction model of SIPs after median thoracotomy was established using R software and then validated using the bootstrap method. Next, the validity and accuracy of the model were tested and evaluated. In total, 15 426 cases met the requirements of the present study, among which 309 cases were diagnosed with SIPs, with an incidence rate of 2%. The body mass index (BMI), intensive care unit (ICU) time, diabetes mellitus, and revision for bleeding were identified as independent risk factors for postoperative SIPs. The nomogram model achieved good discrimination (73.9%) and accuracy (70.2%) in predicting the risk of SIPs after median thoracotomy. Receiver operating characteristic curve analysis showed that the area under curve of the model was 0.705 (95% confidence interval [CI]: 0.746-0.803); the Hosmer-Lemeshow test showed that χ2  = 6.987 and P = 0.538, and the fitting degree of the calibration curve was good. Additionally, the clinical decision curve showed that the net benefit of the model was greater than 0, and the clinical application value was high. The nomogram based on BMI, ICU time, diabetes mellitus, and revision for bleeding can predict the individualised risk of SIPs after median sternotomy, showing good discrimination and accuracy, and has high clinical application value. It also provides significant guidance for screening high-risk populations and developing intervention strategies.

Identification and Evaluation of Reference Genes for Quantitative PCR Normalization in Alligator Weed Flea Beetle (Coleoptera: Chrysomelidae).

Stably expressed reference genes are critical internal standards for the quantification of gene transcription levels using quantitative real-time PCR. Housekeeping genes are commonly used as reference genes but their expressions were variable depending on experimental conditions in many insect species studied. Here we report the identification and evaluation of 10 housekeeping genes in alligator weed flea beetle, Agasicles hygrophila Selman & Vogt (Coleoptera: Chrysomelidae), a biocontrol agent of alligator weed. The 10 housekeeping genes are: beta-actin (Actin), ribosomal protein L13A (PRL13a), succinate dehydrogenase complex subunit A (SDHA), ribosomal protein S20 (RPS20), ribosomal protein S13 (RPS13), glyceraldehyde phosphate dehydrogenase (GAPDH), TATA-box-binding protein (TBP), ribosomal protein L32 (RPL32), tubulin alpha-1 chain (TUBULIN), and elongation factor-1 alpha (ELF). Five programs, geNorm, NormFinder, BestKeeper, ΔCt method, and RefFinder, were used to evaluate the expression stability of the 10 genes among various A. hygrophila body parts and with different nutrient types (starvation, diet types). The expression stability analysis showed that RPS32 and RPL13a were reliable reference genes for the study of gene transcription in different body parts; Actin and RPL13a were optimal reference genes for different nutrient types. The selections of reference genes were validated using a CarE gene (GeneBank No: KX353552). The results of this study provide useful bases for studies of gene expression in various aspects relating to A. hygrophila.

Enhancing growth and oil accumulation of a palmitoleic acid-rich Scenedesmus obliquus in mixotrophic cultivation with acetate and its potential for ammonium-containing wastewater purification and biodiesel production.

A palmitoleic acid-rich Scenedesmus obliquus strain SXND-02 was isolated from ammonium-containing wastewater. Biomass and lipid production were examined for this microalgal strain in photoautotrophic, heterotrophic, and mixotrophic cultivations, respectively, in order to extend its application in wastewater purification coupled with production of valued bio-products. Among the tested conditions, the microalga had better growth and higher lipid accumulation in mixotrophy. NH4Cl inhibited the microalgal growth in photoautotrophic cultivation. However, NaAc alleviated this inhibition in both heterotrophy and mixotrophy. Using 7 g L-1 NaAc and 0.5 g L-1 NH4Cl as carbon and nitrogen sources significantly increased the algal biomass and lipid yields under mixotrophic cultivation, with the highest levels up to 1.0 g L-1 and 59.88%, respectively. Fatty acid profiling indicated that palmitoleic acid was 23% in the S. obliquus SXND-02 under mixotrophic condition, which was about 21-fold higher than that in the control S. obliquus. Furthermore, this microalgal strain was tested in the chicken farm wastewater (CFW) containing high ammonium. Compared with other treatments, the S. obliquus SXND-02 cultivated in the 1/2 CFW + NaAc medium produced larger amounts of biomass (2.18 g L-1) and lipids (50.22%), and simultaneously higher removal rates of total nitrogen (TN) (80%), total ammonium nitrogen (TAN) (68%), total phosphate (TP) (82%), biological oxygen demand (BOD) (86%) and chemical oxygen demand (COD) (89%) from wastewater. The present data indicate that this excellent microalga can be used in mixotrophic cultivation for wastewater purification coupled with commercial production of valued biomass and high-quality algal oils.