Effects of baculovirus infection on intestinal microflora of BmNPV resistant and susceptible strain silkworm.

Bombyx mori L. (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV) is a serious pathogen causing huge economic losses to sericulture. There is growing evidence that the gut microbiota of silkworms plays a critical role in shaping host responses and interactions with viral infection. However, little is known about the differences in the composition and diversity of intestinal microflora, especially with respect to silkworm strain differences and BmNPV infection-induced changes. Here, we aim to explore the differences between BmNPV-resistant strain A35 and susceptible strain P50 silkworm and the impact of BmNPV infection on intestinal microflora in different strains. The 16S rDNA sequencing analysis revealed that the fecal microbial populations were distinct between A35 and P50 and were significantly changed post BmNPV infection in both strains. Further analysis showed that the BmNPV-resistant strain silkworm possessed higher bacterial diversity than the susceptible strain, and BmNPV infection reduced the diversity of intestinal flora assessed by feces in both silkworm strains. In response to BmNPV infection, the abundance of Muribaculaceae increased in P50 and decreased in A35, while the abundance of Enterobacteriaceae decreased in P50 and increased in A35. These results indicated that BmNPV infection had various effects on the abundance of fecal microflora in different silkworm strains. Our findings not only broadened the understanding of host-pathogen interactions but also provided theoretical help for the breeding of resistant strains and healthy rearing of silkworms based on symbiotic bacteria.

BmNPV p35 regulates apoptosis in Bombyx mori via a novel target of interaction with the BmVDAC2-BmRACK1 complex.

Voltage-dependent anion channel 2 (VDAC2) is an important channel protein that plays a crucial role in the host response to viral infection. The receptor for activated C kinase 1 (RACK1) is also a key host factor involved in viral replication. Our previous research revealed that Bombyx mori VDAC2 (BmVDAC2) and B. mori RACK1 (BmRACK1) may interact with Bombyx mori nucleopolyhedrovirus (BmNPV), though the specific molecular mechanism remains unclear. In this study, the interaction between BmVDAC2 and BmRACK1 in the mitochondria was determined by various methods. We found that BmNPV p35 interacts directly with BmVDAC2 rather than BmRACK1. BmNPV infection significantly reduced the expression of BmVDAC2, and activated the mitochondrial apoptosis pathway. Overexpression of BmVDAC2 in BmN cells inhibited BmNPV-induced cytochrome c (cyto c) release, decrease in mitochondrial membrane potential as well as apoptosis. Additionally, the inhibition of cyto c release by BmVDAC2 requires the involvement of BmRACK1 and protein kinase C. Interestingly, overexpression of p35 inhibited cyto c release during mitochondrial apoptosis in a RACK1 and VDAC2-dependent manner. Even the mutant p35, which loses Caspase inhibitory activity, could still bind to VDAC2 and inhibit cyto c release. In summary, our results indicated that BmNPV p35 interacts with the VDAC2-RACK1 complex to regulate apoptosis by inhibiting cyto c release. These findings confirm the interaction between BmVDAC2 and BmRACK1, the interaction between p35 and the VDAC2-RACK1 complex, and a novel target that BmNPV p35 regulates apoptosis in Bombyx mori via interaction with the BmVDAC2-BmRACK1 complex. The result provide an initial exploration of the function of this interaction in the BmNPV-induced mitochondrial apoptosis pathway.

Bmo-miR-6498-5p suppresses Bombyx mori nucleopolyhedrovirus infection by down-regulating BmPLPP2 to modulate pyridoxal phosphate content in B. mori.

The RNA interference pathway mediated by microRNAs (miRNAs) is one of the methods to defend against viruses in insects. Recent studies showed that miRNAs participate in viral infection by binding to target genes to regulate their expression. Here, we found that the Bombyx mori miRNA, miR-6498-5p was down-regulated, whereas its predicted target gene pyridoxal phosphate phosphatase PHOSPHO2 (BmPLPP2) was up-regulated upon Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Both in vivo and in vitro experiments showed that miR-6498-5p targets BmPLPP2 and suppresses its expression. Furthermore, we found miR-6498-5p inhibits BmNPV genomic DNA (gDNA) replication, whereas BmPLPP2 promotes BmNPV gDNA replication. As a pyridoxal phosphate (PLP) phosphatase (PLPP), the overexpression of BmPLPP2 results in a reduction of PLP content, whereas the knockdown of BmPLPP2 leads to an increase in PLP content. In addition, exogenous PLP suppresses the replication of BmNPV gDNA; in contrast, the PLP inhibitor 4-deoxypyridoxine facilitates BmNPV gDNA replication. Taken together, we concluded that miR-6498-5p has a potential anti-BmNPV role by down-regulating BmPLPP2 to modulate PLP content, but BmNPV induces miR-6498-5p down-regulation to promote its proliferation. Our findings provide valuable insights into the role of host miRNA in B. mori-BmNPV interaction. Furthermore, the identification of the antiviral molecule PLP offers a novel perspective on strategies for preventing and managing viral infection in sericulture.

Connections between body composition and dysregulation of islet α- and ß-cells in type 2 diabetes.

BACKGROUND: Accompanying islet α- and ß-cell dysregulation in type 2 diabetes (T2D) at the microscopic scale, alterations in body composition at the macroscopic scale may affect the pathogenesis of T2D. However, the connections between body composition and islet α-cell and ß-cell functions in T2D have not been thoroughly explored. METHODS: For this cross-sectional study, we recruited a total of 729 Chinese Han patients with T2D in a consecutive manner. Dual-energy X-ray absorptiometry (DXA) was used to measure body composition, which included total bone-free mass, total fat and lean mass, trunk fat and lean mass and limb fat and lean mass. Every patient underwent an oral glucose tolerance test to simultaneously detect glucose, C-peptide and glucagon. The indices of islet α-cell function included fasting glucagon levels and the area under the curve of glucagon after a challenge (AUCglucagon), while the indices of ß-cell function included the insulin sensitivity index derived from C-peptide (ISIC-peptide) and the area under the curve of C-peptide after a challenge (AUCC-peptide). RESULTS: Among all patients, fat mass, especially trunk fat mass, was significantly correlated with ISIC-peptide and AUCC-peptide levels (r = - 0.330 and 0.317, respectively, p < 0.001), while lean mass, especially limb lean mass, was significantly correlated with fasting glucagon and AUCglucagon levels (r = - 0.196 and - 0.214, respectively, p < 0.001). Moreover, after adjusting for other relevant variables via multivariate linear regression analysis, increased trunk fat mass was independently associated with decreased ISIC-peptide (ß = - 0.247, t = - 3.628, p < 0.001, partial R2 = 10.9%) and increased AUCC-peptide (ß = 0.229, t = 3.581, p < 0.001, partial R2 = 8.2%), while decreased limb lean mass was independently associated with increased fasting glucagon (ß = - 0.226, t = - 2.127, p = 0.034, partial R2 = 3.8%) and increased AUCglucagon (ß = - 0.218, t = - 2.050, p = 0.041, partial R2 = 2.3%). Additionally, when separate analyses were performed with the same concept for both sexes, we found that increased trunk fat mass was still independently associated with decreased ISIC-peptide and increased AUCC-peptide, while decreased limb lean mass was still independently associated with increased fasting glucagon and AUCglucagon. CONCLUSIONS: Increased trunk fat mass may partly account for decreased insulin sensitivity and increased insulin secretion, while decreased limb lean mass may be connected to increased fasting glucagon and postprandial glucagon secretion.

Bmo-miR-3351 modulates glutathione content and inhibits BmNPV proliferation by targeting BmGSTe6 in Bombyx mori.

MicroRNAs (miRNAs) are small non-coding RNAs that play pivotal roles in the host response to invading pathogens. Among these pathogens, Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the main causes of substantial economic losses in sericulture, and there are relatively few studies on the specific functions of miRNAs in the B. mori-BmNPV interaction. Therefore, we conducted transcriptome sequencing to identify differentially expressed (DE) messenger RNAs (mRNAs) and miRNAs in the midgut of 2 B. mori strains (BmNPV-susceptible strain P50 and BmNPV-resistant strain A35) after BmNPV infection. Through correlation analysis of the miRNA and mRNA data, we identified a comprehensive set of 21 miRNAs and 37 predicted target mRNAs. Notably, miR-3351, which has high expression in A35, exhibited remarkable efficacy in suppressing BmNPV proliferation. Additionally, we confirmed that miR-3351 binds to the 3' untranslated region (3' UTR) of B. mori glutathione S-transferase epsilon 6 (BmGSTe6), resulting in its downregulation. Conversely, BmGSTe6 displayed an opposite expression pattern to miR-3351, effectively promoting BmNPV proliferation. Notably, BmGSTe6 levels were positively correlated with glutathione S-transferase activity, consequently influencing intracellular glutathione content in the infected samples. Furthermore, our investigation revealed the protective role of glutathione against BmNPV infection in BmN cells. In summary, miR-3351 modulates glutathione content by downregulating BmGSTe6 to inhibit BmNPV proliferation in B. mori. Our findings enriched the research on the role of B. mori miRNAs in the defense against BmNPV infection, and suggests that the antiviral molecule, glutathione, offers a novel perspective on preventing viral infection in sericulture.

Regulation of Bombyx mori ferritin heavy-chain homolog on ROS induces multiple effects on BmNPV replication.

Ferritin is an iron-binding protein composed of light-chain and heavy-chain homologs with a molecular weight of about 500 kDa. Free iron ions significantly affect reactive oxygen species (ROS) accumulation. Previous research has shown that Bombyx mori nucleopolyhedrosis virus (BmNPV) can increase ROS accumulation, activate autophagy, induce apoptosis, and upregulate the expression of B. mori ferritin heavy-chain homolog (BmFerHCH). However, the mechanism of mutual regulation between BmFerHCH and ROS-mediated autophagy and apoptosis induced by BmNPV remains unclear. In this study, we found that BmNPV induced the time-dependent accumulation of ROS in BmN cells, thereby promoting BmFerHCH expression. Interestingly, in BmFerHCH-overexpressed cells, BmNPV replication was inhibited in the first 18 h after infection but stimulated after 24 h. Further research on H2O2 or antioxidant-treated cells indicated that ROS-induced autophagy slightly increased in the early infection stage and increased BmNPV replication, while in the late stage, a large accumulation of ROS induced apoptosis and inhibited BmNPV replication. In this process, BmFerHCH inhibits BmNPV-induced ROS accumulation by chelating Fe2+. Taken together, BmFerHCH regulates ROS-mediated autophagy and apoptosis to achieve its various effects on BmNPV replication. These findings will help elucidate BmNPV-induced autophagy and apoptosis mediated by ROS and BmFerHCH, as well as the mutually fighting relationship between viruses and hosts.

RNA methyltransferase BmMettl3 and BmMettl14 in silkworm (Bombyx mori) and the regulation of silkworm embryonic development.

N6-methyladenosine (m6A) is a ubiquitous reversible epigenetic RNA modification that plays an important role in regulating many biological processes, especially embryonic development. However, regulation of m6A methylation during silkworm embryonic development and diapause remains to be investigated. In this study, we analyzed the phylogeny of subunits of methyltransferases BmMettl3 and BmMettl14, and detected the expression patterns of BmMettl3 and BmMettl14 in different tissues and at different developmental stages in silkworm. To investigate the function of m6A on the development of silkworm embryo, we analyzed the m6A/A ratio in diapause and diapause termination eggs. The results showed that BmMettl3 and BmMettl14 were highly expressed in gonads and eggs. Moreover, the expression of BmMettl3 and BmMettl14 and the m6A/A ratio were significantly increased in diapause termination eggs compared with diapause eggs in the early stage of silkworm embryonic development. Furthermore, in BmN cell cycle experiments, the percentage of cells in the S phase increased when lacking BmMettl3 or BmMettl14. This work contributes to understanding the role of m6A methylation during insect embryogenesis and gametogenesis. It also provides a research orientation to further analyze the role of m6A methylation in diapause initiation and termination during insect embryonic development.

Reactive oxygen species-mediated phosphorylation of JNK is involved in the regulation of BmFerHCH on Bombyx mori nucleopolyhedrovirus proliferation.

c-Jun N-terminal kinase (JNK) phosphorylation is widely observed during virus infection, modulating various aspects of the virus-host interaction. In our previous research, we have proved that B. mori ferritin heavy-chain homolog (BmFerHCH), an inhibitor of reactive oxygen species (ROS), facilitates B. mori nucleopolyhedrovirus (BmNPV) proliferation. However, one question remains: Which downstream signaling pathways does BmFerHCH regulate by inhibiting ROS? Here, we first determined that silencing BmFerHCH inhibits BmNPV proliferation, and this inhibition depends on ROS. Then, we substantiated that BmNPV infection activates the JNK signaling pathway. Interestingly, the JNK phosphorylation during BmNPV infection is activated by ROS. Further, we found that the enhanced nuclear translocation of phospho-JNK induced by BmNPV infection was dramatically reduced by pretreatment with the antioxidant N-acetylcysteine (NAC), whereas there was more detectable phospho-JNK in the cytoplasm. Next, we investigated how changes in BmFerHCH expression affect JNK phosphorylation. BmFerHCH overexpression suppressed the phosphorylation of JNK and nuclear translocation of phospho-JNK during BmNPV infection, whereas BmFerHCH knockdown facilitated phosphorylation of JNK and nuclear translocation of phospho-JNK. By measuring the viral load, we found the inhibitory effect of BmFerHCH knockdown on BmNPV infection depends on phosphorylated JNK. In addition, the JNK signaling pathway was involved in BmNPV-triggered apoptosis. Hence, we hypothesize that ROS-mediated JNK phosphorylation is involved in the regulation of BmFerHCH on BmNPV proliferation. These results elucidate the molecular mechanisms and signaling pathways of BmFerHCH-mediated response to BmNPV infection.

[Vertical Distribution Characteristics of Boundary Layer Volatile Organic Compounds in Autumn in the Mixed Industrial and Rural Areas over the Northern Suburb of Nanjing].

Based on the sounding data of VOCs in the lower troposphere (0-1000 m) in the northern suburb of Nanjing in the autumn of 2020, the vertical profile distribution, diurnal variation, and photochemical reactivity of VOCs in this area were analyzed. The results showed that the volume fraction of VOCs decreased with the increase in height (72.1×10-9±28.1×10-9-56.4×10-9±24.8×10-9). Alkanes at all heights accounted for the largest proportion (68%-75%), followed by aromatics (10%-12%), halohydrocarbons (10%-11%), alkenes (3%-7%), and acetylene (2%). The diurnal variation of the boundary layer had a great influence on the VOCs profile. The lower boundary layer in the morning and evening caused the volume fraction of VOCs to accumulate near the ground and lower in the upper layer. The vertical distribution of VOCs was more uniform in the afternoon. In the morning, the volume fraction proportion of alkenes (alkanes) with strong (weak) photochemical reactivity decreased (increased) with the increase in height, indicating that the photochemical aging of VOCs in the upper layer was significant. In the afternoon, the vertical distribution of VOCs volume fraction and OFP in the lower troposphere were more uniform. Affected by the surrounding air masses with different sources, the volume fraction and component proportion of VOCs at each height were significantly different. The alkanes in rural air masses were vertically evenly distributed, and the proportion increased gradually with the height. The vertical negative gradient of VOCs volume fraction in the urban air mass was the largest, the volume fraction of VOCs near the ground was high, and it was rich in aromatics. The proportion of aromatics increased with the increase in VOCs volume fraction between 200-400 m height of industrial air mass. The near-surface VOCs volume fraction of the highway traffic air mass was high, and alkanes accounted for the largest proportion.

Trehalose hydrolysis and transport-related genes promote Bombyx mori nucleopolyhedrovirus proliferation through the phosphoinositide 3-kinase-Akt signalling pathway in BmN cell.

The reprogramming of host physiology has been considered an essential process for baculovirus propagation. Trehalose, the main sugar in insect blood, plays a crucial role as an instant energy source. Although the trehalose level is modulated following infection with Bombyx mori nucleopolyhedrovirus (BmNPV), the mechanism of trehalose metabolism in response to BmNPV infection is still unclear. In this study, we demonstrated that the trehalose level tended to be lower in BmNPV-infected hemolymph and higher in the midgut. The omics analysis revealed that two trehalose transporters, BmTret1-1 and BmTret1-2, and trehalase, BmTRE1 and BmTRE2, were differentially expressed in the midgut after BmNPV infection. BmTret1-1 and BmTret1-2 had the ability to transport trehalose into the cell and promoted cellular absorption of trehalose. Furthermore, the functions of BmTret1-1, BmTret1-2, BmTRE1 and BmTRE2 in BmNPV infection were analyzed. These genes were upregulated in the midgut after BmNPV infection. Virus amplification analysis revealed that these genes could promote BmNPV proliferation in BmN cells. In addition, these genes could promote the expression of BmPI3K, BmPDK1 and BmAkt and inhibit the expression of BmFoxO in the phosphoinositide 3-kinase (PI3K)-Akt signalling pathway. Similarly, the increased trehalose level in BmN cells could promote the expression of BmPI3K, BmPDK1 and BmAkt and inhibit the expression of BmFoxO. Taken together, BmNPV infection promote the expression of trehalose hydrolysis and transport-related genes. These changes affect the PI3K-Akt signalling pathway to facilitate BmNPV proliferation. These findings help clarify the relationship between trehalose metabolism and BmNPV infection.

Bombyx mori ferritin heavy-chain homolog facilitates BmNPV proliferation by inhibiting reactive oxygen species-mediated apoptosis.

Ferritin heavy-chain homolog (FerHCH), an iron-binding protein, plays an important role in the host defense against oxidative stress and pathogen infections. In our previous research, Bombyx mori native ferritin had an interaction with B. mori nucleopolyhedrovirus (BmNPV). However, the underlying molecular mechanism of single ferritin homolog responses to BmNPV infection remains unclear. In this study, we found that BmNPV titer and B. mori FerHCH (BmFerHCH) expression were positively correlated with the ferric iron concentration. We performed RNA interference (RNAi) and overexpression experiments to investigate the effects of BmFerHCH on BmNPV proliferation. BmFerHCH knockdown suppressed BmNPV proliferation in vivo and in vitro, whereas BmFerHCH overexpression facilitated BmNPV proliferation. In addition, the oxidative stress level was increased significantly in BmN cells after budded virus infection, while BmFerHCH could neutralize the increased ROS production induced by BmNPV. Of note, we found that ROS was involved in BmNPV-induced apoptosis. Through inhibiting ROS, apoptosis was suppressed by BmFerHCH, whereas BmFerHCH knockdown facilitated apoptosis. Therefore, we hypothesize that BmFerHCH-mediated inhibition of virus-induced apoptosis depends on suppressing ROS accumulation and, thereby, facilitates virus replication. These results suggest that BmFerHCH plays an important role in facilitating BmNPV proliferation and modulating BmFerHCH is potential strategy for studying host-pathogen interactions.

Increased serum cystatin C levels and responses of pancreatic α- and ß-cells in type 2 diabetes.

Background: Increased serum cystatin C (CysC) can predict the onset of type 2 diabetes (T2D). Meanwhile, impaired pancreatic α- and ß-cell functions get involved in the pathophysiological processes of T2D. So this study was to explore the relationships between serum CysC levels and pancreatic α- and ß-cell functions in T2D. Methods: In this cross-sectional observational study, a total of 2634 patients with T2D were consecutively recruited. Each recruited patient received a serum CysC test and oral glucose tolerance test for synchronous detection of serum C-peptide and plasma glucagon. As components of pancreatic ß-cell function, insulin secretion and sensitivity indices were evaluated by C-peptide area under the curve (AUC-CP) and C-peptide-substituted Matsuda's index (Matsuda-CP), respectively. Fasting glucagon (F-GLA) and post-challenge glucagon calculated by glucagon area under the curve (AUC-GLA) were used to assess pancreatic α-cell function. These skewed indices and were further natural log-transformed (ln). Results: With quartiles of serum CysC levels ascending, AUC-CP, F-GLA and AUC-GLA were increased, while Matsuda-CP was decreased (P for trend <0.001). Moreover, serum CysC levels were positively related to lnAUC-CP, lnF-GLA and lnAUC-GLA (r= 0.241, 0.131 and 0.208, respectively, P < 0.001), and inversely related to lnMatsuda-CP (r= -0.195, P < 0.001). Furthermore, after controlling for other relevant variables via multivariable linear regression analysis, serum CysC levels were identified to account for lnAUC-CP (ß= 0.178, t= 10.518, P < 0.001), lnMatsuda-CP (ß= -0.137, t= -7.118, P < 0.001), lnF-GLA (ß= 0.049, t= 2.263, P = 0.024) and lnAUC-GLA (ß= 0.121, t= 5.730, P < 0.001). Conclusions: Increased serum CysC levels may be partly responsible for increased insulin secretion from ß-cells, decreased systemic insulin sensitivity, and elevated fasting and postprandial glucagon secretion from α-cells in T2D.

Two new sesquiterpenoid lactone derivatives from Lindera aggregata.

Two new sesquiterpenoid lactone derivatives, linderin A (1) and linderin B (2) comprising a sesquiterpenoid lactone and a methyl geranylhomogentisate moiety together with six known compounds were isolated from the roots of Lindera aggregata. Their chemical structures were elucidated using extensive spectroscopic analysis including 1 D, 2 D NMR, and HR-ESI-MS data and compared with previously reported data. The absolute configurations of 1 and 2 were assigned based on the electronic circular dichroism calculation. Compound 2 showed moderate anticoagulant activity.

[Spatiotemporal variations of wet-bulb temperature and its impact factors of Nanjing urban neighborhood]. / å—äº¬åŸŽå¸‚è¡—åŒºæ¹¿çƒæ¸©åº¦æ—¶ç©ºå˜åŒ–ç‰¹å¾åŠå ¶å½±å“å› å­.

Global climate change and local urban heat islands enhance urban heat stress. Studies focused at the urban neighborhood scale are limited. Wet-bulb temperature represents the combined effects of both temperature and humidity, and therefore can more accurately reflect human thermal comfort. In this study, air temperature, relative humidity and geographic information of different times, seasons, and sky conditions of the Nanjing Jiangbei New Area were obtained based on mobile measurements. The spatiotemporal variation of wet-bulb temperature at the urban neighborhood scale and the effects of sky conditions, land cover and urban morphology (sky view factor, SVF) were further analyzed. The results showed that: 1) the spatiotemporal variations of wet-bulb temperature at the Nanjing urban neighborhood scale were consistent with that of air temperature. Compared with vapor pressure, air temperature played a dominant role. The extremely high values of wet-bulb temperature in this area were mostly caused by the synergy between air temperature and vapor pressure. 2) The correlation between SVF and wet-bulb temperature was significantly positive in the daytime and negative at night. An increase in the vegetation fraction could reduce wet-bulb temperature, while impervious surfaces had the opposite effect. The wet-bulb temperature significantly decreased and its spatial distribution was much more homogeneous under overcast sky conditions. 3) The horizontal scale effect showed diurnal and seasonal differences and was more sensitive to sky conditions during nighttime than during daytime. Compared with vegetation, the horizontal effect of impervious surfaces was much larger in winter than in the other two seasons. The horizontal scale effects of vege-tation and impervious surfaces on wet-bulb temperature were similar to those of air temperature. These results could provide effective scientific support and a theoretical basis for improving and optimizing the thermal environment of urban neighborhoods, as well as alleviating urban heat stress.

[Characteristics of Atmospheric Particulate Matter Pollution and the Unique Wind and Underlying Surface Impact in the Twain-Hu Basin in Winter].

To understand the recent characteristics of atmospheric environmental changes in the Twain-Hu(Hunan-Hubei) Basin, including the middle reaches of the Yangtze River, this paper uses near-surface PM2.5 and PM10 observational data for the Twain-Hu Basin in the winters of 2015 to 2019, combined with wind-speed, topography, the normalized difference vegetation index(NDVI), and other datasets. The results show that:① PM2.5 pollution occurred frequently in the winters of 2015-2019 in the Twain-Hu Basin, and Xiangyang and Jingmen in the western part of the basin, experience PM2.5 pollution on an average of 62 and 61 days in winter(PM2.5>75 µg·m-3). And the heavy pollution days in Xiangyang reached 19 more days(PM2.5>150 µg·m-3), indicating that the Twain-Hu Basin is an air pollution center in the middle reaches of the Yangtze River Basin; ② Spatially, pollution in the Twain-Hu Basin is heavier in the northwest than in the southeast, and in the urban agglomeration, which is mainly related to the regional transport of air pollutants by the winter monsoon and the high levels of emissions from urban areas; ③ A "U-shaped" nonlinear relationship was observed between near-surface wind speeds and PM2.5 and PM10 concentrations. The inflection points of PM2.5 and PM10 concentrations were 153 and 210 µg·m-3, respectively. This implies that the accumulation of local atmospheric particulate matter in the Twain-Hu Basin dominates light/medium pollution, while the regional transport of air pollutants dominates period of severe pollution; and ④ PM2.5 and PM10 in winter were significantly negatively correlated with terrain height and the NDVI, which reflects the atmospheric environmental effects of topography and urbanization.

Bombyx mori ß-1,3-Glucan Recognition Protein 4 (BmßGRP4) Could Inhibit the Proliferation of B. mori Nucleopolyhedrovirus through Promoting Apoptosis.

ß-1,3-glucan recognition proteins (ßGRPs) as pattern recognition receptors (PRRs) play an important role in recognizing various pathogens and trigger complicated signaling pathways in insects. In this study, we identified a Bombyx mori ß-1,3-glucan recognition protein gene named BmßGRP4, which showed differential expression, from a previous transcriptome database. The full-length cDNA sequence was 1244 bp, containing an open reading frame (ORF) of 1128 bp encoding 375 amino acids. BmßGRP4 was strongly expressed in the larval stages and highly expressed in the midgut of B. mori larvae in particular. After BmNPV infection, the expression of BmßGRP4 was reduced significantly in the midgut. Furthermore, a significant increase in the copy number of BmNPV was observed after the knockdown of BmßGRP4 in 5th instar larvae, while the overexpression of BmßGRP4 suppressed the proliferation of BmNPV in BmN cells. Subsequently, the expression analysis of several apoptosis-related genes and observation of the apoptosis morphology demonstrated that overexpression of BmßGRP4 facilitated apoptosis induced by BmNPV in BmN cells. Moreover, BmßGRP4 positively regulated the phosphatase and tensin homolog gene (BmPTEN), while expression of the inhibitor of apoptosis gene (BmIAP) was negatively regulated by BmßGRP4. Hence, we hypothesize that BmNPV infection might suppress BmPTEN and facilitate BmIAP to inhibit cell apoptosis by downregulating the expression of BmßGRP4 to escape host antiviral defense. Taken together, these results show that BmßGRP4 may play a role in B. mori response to BmNPV infection and lay a foundation for studying its functions.

The digestive proteinase trypsin, alkaline A contributes to anti-BmNPV activity in silkworm (Bombyx mori).

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious pathogenic microorganism that causes tremendous loss to sericulture. Previous studies have found that some proteins of serine protease family in the digestive juice of B. mori larvae have anti-BmNPV activity. In our previous publication about proteome analysis of the digestive juice of B. mori larvae, the digestive enzyme trypsin, alkaline A (BmTA) was filtered as a differentially expressed protein possibly involved in BmNPV resistance. Here, the biological characteristics and anti-BmNPV functions of BmTA were comprehensively analysed. The cDNA sequence of BmTA had an ORF of 768 nucleotides encoding 255 amino acid residues. Domain architecture analysis showed that BmTA contained a signal peptide and a typical Tryp_SPc domain. Quantitative real-time PCR analysis showed that BmTA was highly expressed in the larval stages and specifically expressed in the midgut of B. mori larvae. The expression level of BmTA in BmNPV resistant strain A35 was higher than that in susceptible strain P50. After BmNPV infection, the expression of BmTA increased in both strains from 24 to 72 h. Virus amplification analysis showed that the relative levels of VP39 in B. mori larvae and BmN cells infected with the appropriate concentration of recombinant-BmTA-treated BmNPV were significantly lower than in the control groups. Moreover, overexpression of BmTA in BmN cells significantly inhibited the amplification of BmNPV. Taken together, the results of this study indicated that BmTA possessed anti-BmNPV activity in B. mori, which broadens the horizon for virus-resistant breeding of silkworms.

1 H NMR-based metabonomic evaluation of the pesticides camptothecin and matrine against larvae of Spodoptera litura.

BACKGROUND: Camptothecin (CPT) and matrine (MAT) have potential as botanical pesticides against several pest species. However, the mechanisms of metabolic and physiological changes in pests induced by CPT and MAT are unknown. In this study, a toxicological test, an NMR-based metabolomic study, an enzymatic test, and an RT quantitative PCR (RT-qPCR) experiment were all conducted to examine the effect of CPT and MAT on Spodoptera litura. RESULTS: CPT (0.5-1%) exerted high toxicity against larvae of S. litura and caused growth stagnation and high mortality of larvae. A variety of metabolites were significantly influenced by 0.5% CPT, including several energy-related metabolites such as trehalose, lactate, succinate, citrate, malate, and fumarate. In contrast, MAT showed low toxicity against larvae and induced almost no changes in hemolymph metabolites of S. litura. Enzymatic tests showed that trehalase activity was significantly decreased in larvae after feeding with 0.5% CPT. RT-qPCR showed that the transcription levels of alanine aminotransferase, malate dehydrogenase, and isocitrate dehydrogenase were decreased while lactate dehydrogenase was increased in the 0.5% CPT-treated group. CONCLUSIONS: These data indicate that one of the important mechanisms of CPT against S. litura larvae is via the inhibition of trehalose hydrolysis and glycolysis. Our findings also suggest that CPT exhibits a stronger toxicological effect than MAT against S. litura, which provides basic information for the application of CPT in the control of S. litura or other lepidoptera pests.

Identification and Functional Analysis of BmNPV-Interacting Proteins From Bombyx mori (Lepidoptera) Larval Midgut Based on Subcellular Protein Levels.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. However, the molecular mechanism of silkworm resistance to BmNPV and the interactions of this virus with the host during infection remain largely unclear. To explore the virus-binding proteins of silkworms, the midgut subcellular component proteins that may interact with BmNPV were analyzed in vitro based on one- and two-dimensional electrophoresis and far-western blotting combined with mass spectrometry (MS). A total of 24 proteins were determined to be specifically bound to budded viruses (BVs) in two subcellular fractions (mitochondria and microsomes). These proteins were involved in viral transportation, energy metabolism, apoptosis and viral propagation, and they responded to BmNPV infection with different expression profiles in different resistant strains. In particular, almost all the identified proteins were downregulated in the A35 strain following BmNPV infection. Interestingly, there were no virus-binding proteins identified in the cytosolic fraction of the silkworm midgut. Two candidate proteins, RACK1 and VDAC2, interacted with BVs, as determined with far-western blotting and reverse far-western blotting. We speculated that the proteins interacting with the virus could either enhance or inhibit the infection of the virus. The data provide comprehensive useful information for further research on the interaction of the host with BmNPV.

A Novel Digestive Proteinase Lipase Member H-A in Bombyx mori Contributes to Digestive Juice Antiviral Activity Against B. mori Nucleopolyhedrovirus.

Previous studies have revealed that some proteins in Bombyx mori larvae digestive juice show antiviral activity. Here, based on the label-free proteomics data, BmLipase member H-A (BmLHA) was identified as being involved in the response to BmNPV infection in B. mori larvae digestive juice. In the present study, a gene encoding the BmLHA protein in B. mori was characterized. The protein has an open reading fragment of 999 bp, encoding a predicted 332 amino acid residue-protein with a molecular weight of approximately 35.9 kDa. The phylogenetic analysis revealed that BmLHA shares a close genetic distance with Papilio xuthus Lipase member H-A. BmLHA was highly expressed in the middle part of the B. mori gut, and the expression level increased with instar rising in larvae. There was higher expression of BmLHA in A35 than in P50 strains, and it was upregulated in both A35 and P50 strains, following BmNPV infection. The expression level of VP39 decreased significantly in appropriate recombinant-BmLHA-treated groups compared with the PBS-treated group in B. mori larvae and BmN cells. Meanwhile, overexpression of BmLHA significantly reduced the infectivity of BmNPV in BmN cells. These results indicated that BmLHA did not have digestive function but had anti-BmNPV activity. Taken together, our work provides valuable data for the clarification of the molecular characterization BmLHA and supplements research on proteins of anti-BmNPV activity in B. mori.