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The circadian clock plays a critical role in the regulation of host immune defense. However, the mechanistic basis for this regulation is largely unknown. Herein, the core clock gene cryptochrome1 (cry1) knockout line in Bombyx mori, an invertebrate animal model, was constructed to obtain the silkworm with dysfunctional molecular clock, and the dynamic regulation of the circadian clock on the immune responsiveness within 24 h of Staphylococcus aureus infection was analyzed. We found that deletion of cry1 decreased viability of silkworms and significantly reduced resistance of larvae to S. aureus. Time series RNA-seq analysis identified thousands of rhythmically expressed genes, including immune response genes, in the larval immune tissue, fat bodies. Uninfected cry1 knockout silkworms exhibited expression patterns of rhythmically expressed genes similar to wild-type (WT) silkworms infected with S. aureus. However, cry1 knockout silkworms exhibited a seriously weakened response to S. aureus infection. The immune response peaked at 6 and 24 h after infection, during which "transcription storms" occurred, and the expression levels of the immune response genes, PGRP and antimicrobial peptides (AMPs), were significantly upregulated in WT. In contrast, cry1 knockout did not effectively activate Toll, Imd, or NF-κB signaling pathways during the immune adjustment period from 12 to 18 h after infection, resulting in failure to initiate the immune responsiveness peak at 24 h after infection. This may be related to inhibited silkworm fat body energy metabolism. These results demonstrated the dynamic regulation of circadian clock on silkworm immune response to bacterial infection and provided important insights into host antimicrobial defense mechanisms.

Acetaminophen (APAP) is an effective and widely used analgesic. However, APAP overdose is the principal cause of acute liver failure (ALF) in many countries. Here, we report the phenomenon of liver melanization which occurred before APAP-induced ALF in mice. A melanic surface induced by APAP which was time- and dose-dependent in the silkworm invertebrate model was observed. In addition, an APAP-induced acute tissue failure model (ATF) was established using a metabolic detoxification tissue fat body which simulated mouse liver. An investigation of the anabolic mechanism of melanin in experimental animals showed that dopaquinone and dopamine which were synthesized from tyrosine via dopa in silkworms were further metabolized to melanin, while in mice, epinephrine was synthesized via the dopamine branch and melanin was only synthesized via the dopaquinone branch. On this basis, it is proposed that melanin-metabolic levels in plasma could be used as an early diagnostic marker of APAP overdose and the black spots on insect epidermis could be used as a fast detection model of toxicity.

Vitamin C (VC) is an essential nutrient for many animals. However, whether insects, including Bombyx mori, can synthesize VC remains unclear. In this article, the optimized HPLC method was used to determine the content of l-ascorbic acid (AsA) in silkworm eggs, larvae and pupae, and the activity of l-gulono-1,4-lactone oxidase (GULO), a key enzyme in VC synthesis. The RNA interference method was used to determine the effect of the BmGulo-like gene on embryonic development and GULO activity in the pupal fat body. The AsA content increased significantly during E144 h-E168 h in the late embryonic stage and P48 h-P144 h in the middle-late pupal stage, in which exogenous VC was not ingested. Furthermore, the body AsA content in larvae fed VC-free feed also increased with larval stage. The GULO enzymatic activity was present in eggs and the fat bodies of larvae and pupae, even when the larvae were reared with fresh mulberry leaves. Moreover, the activity was higher in the later embryonic stages (E144 h-E168 h) and the early pupal stage (before P24 h). The GULO activity in the pupal fat body dramatically decreased when the screened BmGulo-like gene (BGIBMGA005735) was knocked down with small interfering RNA; in addition, the survival rate and hatching rate of eggs significantly decreased 21% and 44%, respectively, and embryonic development was delayed. Thus, Bombyx mori can synthesize AsA through the l-gulose pathway, albeit with low activity, and this synthesis ability varies with developmental stages.

Artificial diets for silkworms have many potential applications and they are important in sericulture. However, the challenges of weak larvae and low silk protein synthesis efficiency in silkworms reared on artificial diets have not been resolved. Here, we used high-throughput sequencing to analyse the differences between the gut microbiota of 5th-instar larvae reared on mulberry leaves and larvae reared on an artificial diet. The results showed that at the phylum level, Cyanobacteria, Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria are the dominant bacteria in the intestines of silkworm larvae of all the strains. But the abundance of dominant bacteria in the gut microbiota differed between the silkworm strains that were reared on mulberry leaves, as well as between the silkworm strains that were reared on the artificial diet, while the gut microbiota diversity was lower in the silkworm strains that were reared on the artificial diet. Prediction of the functions of the gut microbiota in the hosts indicated that there was no significant difference between the silkworm strains that were reared on mulberry leaves, while there were significant differences between silkworm strains reared on the artificial diet. When the silkworm diet changed from mulberry leaves to the artificial diet, changes in gut microbiota in the silkworms affected host nutrient metabolism and immune resistance. These changes may be related to the adaptation of silkworms to their long evolutionary history of eating mulberry leaves.

Diapause is a developmental transition in insects based on seasonal adaptation to adversity, it is regulated by a circadian clock system and the endocrine system. However, the molecular node and its mechanism underlying the effects of these systems are still unclear. Here, a mutant of Bombyx mori with the circadian clock gene Period (Per) knocked out was constructed, which dramatically changed the classic diapause-destined pathway. Per-knockout silkworms powerfully attenuated, but could not completely block, the predetermined effects of temperature and photoperiod on diapause determination, and this effect depended on the diapause hormone (DH) pathway. The impaired transcription-translation feedback loop of the circadian clock system lacking the Per gene caused direct up-regulation of the expression of GRD, a receptor of γ-aminobutyric acid (GABA), by changing expression level of Cycle. The synthesis of GABA in the tissue complex of brain-suboesophageal ganglion then increased and restricted the decomposition, which continuously promoted the GABAergic signal to play a role, and finally inhibiting (delaying) the release of DH to the hemolymph, and reducing the diapause-inducing effect of DH. The results provided an example to explain the regulatory mechanism of the circadian clock on endocrine hormones in the silkworm.

Purpose. With the development of digital X-ray imaging and processing methods, the categorization and analysis of massive digital radiographic images need to be automatically finished. What is crucial in this processing is the automatic retrieval and recognition of radiographic position. To address these concerns, we developed an automatic method to identify a patient’s position and body region using only frequency curve classification and gray matching. Methods. Our new method is combined with frequency analysis and gray image matching. The radiographic position was determined from frequency similarity and amplitude classification. The body region recognition was performed by image matching in the whole-body phantom image with prior knowledge of templates. The whole-body phantom image was stitched by radiological images of different parts. Results. The proposed method can automatically retrieve and recognize the radiographic position and body region using frequency and intensity information. It replaces 2D image retrieval with 1D frequency curve classification, with higher speed and accuracy up to 93.78%. Conclusion. The proposed method is able to outperform the digital X-ray image’s position recognition with a limited time cost and a simple algorithm. The frequency information of radiography can make image classification quicker and more accurate.

Circadian clock system disorders can lead to uncontrolled cell proliferation, but the molecular mechanism remains unknown. We used a Bombyx mori animal model of single Period gene (BmPer) expression to investigate this mechanism. A slow growing developmental cell model (Per-KD) was isolated from a B. mori ovarian cell line (BmN) by continuous knock down of BmPer expression. The effects of BmPer expression knockdown (Per-KD) on cell proliferation and apoptosis were opposite to those of m/hPer1 and m/hPer2 in mammals. The knockdown of BmPer expression led to cell cycle deceleration with shrinking of the BmN cell nucleus, and significant inhibition of nuclear DNA synthesis and cell proliferation. It also promoted autophagy via the lysosomal pathway, and accelerated apoptosis via the caspase pathway.

The physiology and metabolism of poikilothermic insects are under the control of environmental temperature. Temperature is the primary cue for the circadian rhythm. Reports on the timing mechanisms of temperature in lepidopterans are limited. This study used Bombyx mori BmN ovarian cells to investigate the effect of temperature on expression of the main circadian clock genes in a negative feedback loop. A 37°C, 30-min high-temperature stimulation induced transcription of the circadian clock genes Cry1, Cry2, Per, and Tim. The gene expression profiles showed rhythmic oscillations, with shortened oscillatory periods for Cry1 and altered oscillatory phases for Cry1 and Per. Cyclical increases in temperature of 2°C starting at 26°C, 5°C starting at 20 or 25°C, or 10°C starting at 20°C induced cyclical changes in expression and protein from the four circadian clock genes. Cyclical temperature changes with a difference of 10°C had the most influence. In conclusion, cyclical changes in temperature with differences from 2 to 10°C reset and synchronized the circadian clock of silkworm BmN cells. Transcription of the genes for and protein from Cry2 and Per showed a better reset and synchronization with cyclical temperature changes.

A novel one-pot sequential transformation via decarboxylative Mannich reaction (DMR) and oxidative C-H amination of cyclic imines with β-ketoacids is described. This methodology has been utilized to provide access to fused aziridines with excellent diastereoselectivity. Several examples of catalytic enantioselective sequential transformation are presented.

Three novel acidic polysaccharides termed PRM1, PRM3 and PRM5 were purified from Rhynchosia minima root using DEAE-52 cellulose and sephadex G-150 column chromatography. Their structures were characterized by ultraviolet (UV) and Fourier transform infrared (FTIR) spectrometry, gel permeation chromatography (GPC), gas chromatography-mass spectrometry (GC-MS), and differential scanning colorimeter (DSC) analysis. The uronic acid contents of PRM1, PRM3 and PRM5 were 30.7%, 12.7% and 47.7%, respectively. PRM1 (143.2 kDa), PRM3 (105.3 kDa) and PRM5 (162.1 kDa) were heteropolysaccharides because they were composed of arabinose, mannose, glucose and galactose. Their enthalpy values were 201.0, 111.0 and 206.8 J/g, respectively. PRM3 and PRM1 exhibited strong in vitro anticancer activity against lung cancer A549 and liver cancer HepG2 cells in a dose-dependent manner. These findings suggested that PRM1 and PRM3 could be potentially developed as natural anticancer agents.

Artificial diets for silkworms have many potential applications and they are important in sericulture. However, the challenges of weak larvae and low silk protein synthesis efficiency in silkworms reared on artificial diets have not been resolved. Here, we used high-throughput sequencing to analyse the differences between the gut microbiota of 5th-instar larvae reared on mulberry leaves and larvae reared on an artificial diet. The results showed that at the phylum level, Cyanobacteria, Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria are the dominant bacteria in the intestines of silkworm larvae of all the strains. But the abundance of dominant bacteria in the gut microbiota differed between the silkworm strains that were reared on mulberry leaves, as well as between the silkworm strains that were reared on the artificial diet, while the gut microbiota diversity was lower in the silkworm strains that were reared on the artificial diet. Prediction of the functions of the gut microbiota in the hosts indicated that there was no significant difference between the silkworm strains that were reared on mulberry leaves, while there were significant differences between silkworm strains reared on the artificial diet. When the silkworm diet changed from mulberry leaves to the artificial diet, changes in gut microbiota in the silkworms affected host nutrient metabolism and immune resistance. These changes may be related to the adaptation of silkworms to their long evolutionary history of eating mulberry leaves.

Silkworms (Bombyx mori) reared on artificial diets have great potential applications in sericulture. However, the mechanisms underlying the enhancement of metabolic utilization by altering silkworm nutrition are unclear. The aim of this study was to investigate the mechanisms responsible for the poor development and low silk protein synthesis efficiency of silkworms fed artificial diets. After multi-generational selection of the ingestive behavior of silkworms to artificial diets, we obtained two strains, one of which developed well and another in which almost all its larvae starved to death on the artificial diets. Subsequently, we analyzed the metabolomics of larval hemolymph by gas chromatography/liquid chromatography–mass spectrometry, and the results showed that vitamins were in critically short supply, whereas the nitrogen metabolic end product of urea and uric acid were enriched substantially, in the hemolymph of the silkworms reared on the artificial diets. Meanwhile, amino acid metabolic disorders, as well as downregulation of carbohydrate metabolism, energy metabolism, and lipid metabolism, co-occurred. Furthermore, 10 male-dominant metabolites and 27 diet-related metabolites that differed between male and female silkworms were identified. These findings provide important insights into the regulation of silkworm metabolism and silk protein synthesis when silkworms adapt to an artificial diet.

Hatching behavior is a key target in silkworm (Bombyx mori) rearing, especially for the control of Lepidoptera pests. According to previous research, hatching rhythms appear to be controlled by a clock mechanism that restricts or "gates" hatching to a particular time. However, the underlying mechanism remains elusive. Under 12-h light:12-h dark photoperiod (LD) conditions, the transcriptional levels of the chitinase5 (Cht5) and hatching enzyme-like (Hel) genes, as well as the enzymatic activities of their gene products, oscillated in time with ambient light cycles, as did the transcriptional levels of the cryptochrome 1, cryptochrome 2, period (per), and timeless genes, which are key components of the negative feedback loop of the circadian rhythm. These changes were related to the expression profile of the ecdysteroid receptor gene and the hatching behavior of B. mori eggs. However, under continuous light or dark conditions, the hatching behavior, the expression levels of Cht5 and Hel, as well as the enzymatic activities of their gene products, were not synchronized unlike under LD conditions. In addition, immunohistochemistry experiments showed that light promoted the translocation of PER from the cytoplasm to the nucleus. In conclusion, LD cycles regulate the hatching rhythm of B. mori via negative feedback loop of the circadian oscillator.

Abnormalities in the circadian clock system are known to affect the body’s metabolic functions, though the molecular mechanisms responsible remain uncertain. In this study, we achieved continuous knockdown of B. mori Period (BmPer) gene expression in the B. mori ovary cell line (BmN), and generated a Per-KD B. mori model with developmental disorders including small individual cells and slow growth. We conducted cell metabolomics assays by gas chromatography/liquid chromatography-mass spectrometry and showed that knockdown of BmPer gene expression resulted in significant inhibition of glycometabolism. Amino acids that used glucose metabolites as a source were also down-regulated, while lipid metabolism and nucleotide metabolism were significantly up-regulated. Metabolite correlation analysis showed that pyruvate and lactate were closely related to glycometabolism, as well as to metabolites such as aspartate, alanine, and xanthine in other pathways. Further validation experiments showed that the activities of the key enzymes of glucose metabolism, hexokinase, phosphofructokinase, and citrate synthase, were significantly decreased and transcription of their encoding genes, as well as that of pyruvate kinase, were also significantly down-regulated. We concluded that inhibition of the circadian clock gene BmPer repressed glycometabolism, and may be associated with changes in cellular amino acid metabolism, and in cell growth and development.

Insects possess an innate immune system that responds to invading microorganisms. In this study, a subtractive cDNA library was constructed to screen for immune response-related genes in the fat bodies of Antheraea pernyi (Lepidoptera: Saturniidae) pupa challenged with Escherichia coli. Four hundred putative EST clones were identified by suppression subtractive hybridization (SSH), including 50 immune response-related genes, three cytoskeleton genes, eight cell cycle and apoptosis genes, five respiration and energy metabolism genes, five transport genes, 40 metabolism genes, ten stress response genes, four transcription and translation regulation genes and 77 unknown genes. To verify the reliability of the SSH data, the transcription of a set of randomly selected immune response-related genes were confirmed by semi-quantitative reverse transcription-PCR (RT-PCR) and real-time quantitative reverse transcription-PCR (qRT-PCR). These identified immune response-related genes provide insight into understanding the innate immunity in A. pernyi.

Sexual glands are key sites affected by nanotoxicity, but there is no sensitive assay for measuring reproductive toxicity in animals. The aim of this study was to investigate the toxic effects of cadmium telluride quantum dots (CdTe-QDs) on gonads in a model organism, Bombyx mori. After dorsal vein injection of 0.32 nmol of CdTe-QDs per individual, the QDs passed through the outer membranes of gonads via the generation of ROS in the membranes of spermatocysts and ovarioles, as well as internal germ cells, thereby inducing early germ cell death or malformations via complex mechanisms related to apoptosis and autophagy through mitochondrial and lysosomal pathways. Histological observations of the gonads and quantitative analyses of germ cell development showed that the reproductive toxicity was characterized by obvious male sensitivity. Exposure to QDs in the early stage of males had severe adverse effects on the quantity and quality of sperm, which was the main reason for the occurrence of unfertilized eggs. Ala- or Gly-conjugated QDs could reduce the nanotoxicity of CdTe-QDs during germ cell development and fertilization of their offspring. The results demonstrate that males are preferable models for evaluating the reproductive toxicity of QDs in combined in vivo/in vitro investigations.

Perilla frutescens (L.) Britt., a worldwide distributed plant, is an important economic crop and with a long cultivation history in China as well as some other countries in Asia. Except for the edible applications, the plant of P. frutescens is also traditionally used as a medicinal herb in China for thousands years. The leaves, seeds and stems of P. frutescens are recommended by the Chinese Pharmacopeia as three medicinal materials for various therapeutic applications. In the past decades, amount investigations have been done about different aspects for P. frutescens. However, no literature review about these works has been compiled. This review aims to present the findings of research conducted up-to-date (2015) on the traditional use, phytochemicals, pharmacological activities and toxicities of P. frutescens to provide scientific evidence for well-understanding and future research of P. frutescens. It was found that more than 100 compounds have been reported for P. frutescens and most of them are contributed to its medical benefits such as anti-allergic, anti-inflammatory, anti-oxidant, anticancer, anti-microbial, anti-depressive and anti-cough effects. Toxicology studies have been conducted to evaluate the safety of P. frutescens to provide information on their dosages and usages.

A novel one-pot sequential transformation via decarboxylative Mannich reaction (DMR) and oxidative C–H amination of cyclic imines with β-ketoacids is described. This methodology has been utilized to provide access to fused aziridines with excellent diastereoselectivity. Several examples of catalytic enantioselective sequential transformation are presented.

Panax ginseng (Asian ginseng), Panax quinquefolium (American ginseng) and Panax notoginseng (notoginseng) are highly valuable tonic herbs derived from the Panax genus that have similar morphological appearances and odors but different pharmacological activities and clinical indications. Thus, the authentication of these three Panax species is crucial for ensuring the quality, safety and efficacy of medication. In the present study, a static headspace gas chromatography — mass spectrometry (GC-MS) followed by a multivariate statistical analysis was developed to globally characterize the volatile organic compound (VOC) profiles in P. ginseng, P. quinquefolium, and P. notoginseng, and subsequently to discover differentiating chemical markers. Under the optimized conditions, the headspace VOCs of a total of 49 batches of Panax herbs derived from the three Panax species were profiled, and the dataset of sample code, [Formula: see text]-m/z pair and ion intensity was processed by unsupervised principal component analysis (PCA) and by supervised partial least squared discriminant analysis (PLS-DA) to comprehensively compare the chemical differences in Panax across the species. The results demonstrated that Panax herbs derived from three species possess obviously diverse chemical characteristics of VOCs, PCA, and PLS-DA. According to their VOC profiles, 49 tested samples could be clearly differentiated according to species. Chemomarker 1, 2, and 4 might be used as unique chemical markers of P. ginseng, P. notoginseng and P. quinquefolium, respectively. Our findings indicate that static headspace GC-MS-based VOC profiling, combined with multivariate statistical analysis, provide a reliable tool to discriminate between the three Panax species and to identify their differentiation markers, which will be helpful for ensuring their quality, safety and efficacy.

The physiology and metabolism of poikilothermic insects are under the control of environmental temperature. Temperature is the primary cue for the circadian rhythm. Reports on the timing mechanisms of temperature in lepidopterans are limited. This study used Bombyx mori BmN ovarian cells to investigate the effect of temperature on expression of the main circadian clock genes in a negative feedback loop. A 37°C, 30-min high-temperature stimulation induced transcription of the circadian clock genes Cry1, Cry2, Per, and Tim. The gene expression profiles showed rhythmic oscillations, with shortened oscillatory periods for Cry1 and altered oscillatory phases for Cry1 and Per. Cyclical increases in temperature of 2°C starting at 26°C, 5°C starting at 20 or 25°C, or 10°C starting at 20°C induced cyclical changes in expression and protein from the four circadian clock genes. Cyclical temperature changes with a difference of 10°C had the most influence. In conclusion, cyclical changes in temperature with differences from 2 to 10°C reset and synchronized the circadian clock of silkworm BmN cells. Transcription of the genes for and protein from Cry2 and Per showed a better reset and synchronization with cyclical temperature changes.

We recently proposed a technique able to analyze the trend of the seismic activity by combining neural network model and seismic factors. In this paper, a variation of seismicity was introduced to reflect the corresponding vary of the frequency of earthquake. This variation was used as the precursor of future moderate earthquake. The time intervals of earthquake will be obtained through training the neural network. Then we judge the occurrence of strong earthquakes according to the time intervals. Finally, some statistical researches were made by using the method for the earthquake catalog in Sichuan and Taiwan in China. Through this above, we verify the validity of this method and state the general steps of this method.

Multi-origin Chinese herbal medicines, with herbs originating from more than one species of plants, is a common phenomenon but an important issue in Traditional Chinese Medicines (TCMs). In the present study, a gas chromatography-mass spectrometry (GC-MS)—based fatty acid profiling approach to rapidly discriminate multi-origin Chinese medicines in terms of species and medicinal parts was proposed and validated using tuberous roots (Curcumae Radix) and rhizomes (Curcumae Rhizoma and Curcumae Longae Rhizoma) derived from four Curcuma species (e.g., C. wenyujin, C. kwangsiensis, C. phaeocaulis and C. longa) as models. Both type and content of fatty acids varied among different species of either tuberous roots or rhizomes, indicating each species has its own fatty acid pattern. Orthogonal partial least squares discriminant analysis (OPLS-DA) and hierarchical clustering analysis (HCA) based on dataset of global fatty acid profiling showed that either tuberous roots or rhizomes samples could be clearly classified into four clusters according to their species. Furthermore, those tested samples could also be discriminated in terms of their medicinal parts (e.g., tuberous root and rhizome). Our findings suggest that the proposed GC-MS-based fatty acid profiling followed by multivariate statistical analysis provides a reliable platform to discriminate multi-origin Chinese herbal medicines according to species and medicinal parts, which will be helpful for ensuring their quality, safety and efficacy.