S2 subunit plays a critical role in pathogenesis of TW-like avian coronavirus infectious bronchitis virus.

To investigate the critical role of the S gene in determining pathogenesis of TW-like avian infectious bronchitis virus (IBV), we generated two recombinant IBVs (rGDaGD-S1 and rGDaGD-S2) by replacing either the S1 or S2 region of GD strain with the corresponding regions from an attenuated vaccine candidate aGD strain. The virulence and pathogenicity of these recombinant viruses was assessed both in vitro and in vivo. Our results indicated the mutations in the S2 region led to decreased virulence, as evidenced by reduced virus replication in embryonated chicken eggs and chicken embryonic kidney cells as well as observed clinical symptoms, gross lesions, microscopic lesions, tracheal ciliary activity, and viral distribution in SPF chickens challenged with recombinant IBVs. These findings highlight that the S2 subunit is a key determinant of TW-like IBV pathogenicity. Our study established a foundation for future investigations into the molecular mechanisms underlying IBV virulence.

Cu-MOFs/GOx Bifunctional Probe-Based Synergistic Signal Amplification Strategy: Toward Highly Sensitive Closed Bipolar Electrochemiluminescence Immunoassay.

A closed bipolar electrochemiluminescence (BP-ECL) platform for sensitive prostate specific antigen (PSA) detection was proposed based on a novel synergistic signal amplification strategy. Specifically, glucose oxidase-loaded Cu-based metal-organic frameworks (Cu-MOFs/GOx) as bifunctional probes were bridged on the anodic interface with the target PSA as the intermediate unit. In virtue of the large loading capacity of Cu-MOFs, a large amount of a co-reactant, i.e., H2O2 in this L-012-based ECL system and gluconic acid were generated on the anodic pole in the presence of glucose. The generated gluconic acid could effectively degrade the Cu-MOFs to release Cu2+ which greatly accelerates the formation of highly active intermediates from co-reactant H2O2, boosting the ECL intensity. As for the cathodic pole, K3Fe(CN)6 with a lower reduction potential is used to reduce the driving voltage and speed up the reaction rate, further strengthening the ECL intensity. Thanks to the synergistic signal amplification effect at both two electrode poles of the BP-ECL system, highly sensitive detection of PSA was realized with a detection limit of 5.0 × 10-14 g/mL and a wide linear range of 1.0 × 10-13-1.0 × 10-7 g/mL. The strategy provides a novel way for signal amplification in the BP-ECL biosensing field.

Polydopamine functionalized graphene oxide membrane with the sandwich structure for osmotic energy conversion.

Ion-selective membrane is the key component for osmotic energy conversion. Nanofluid channels based on two-dimensional materials have advantages of facile preparation, tunable channel size, and easy upscaling, which is promising for efficient osmotic energy harvesting. However, further improvement of the output power is hindered by the low ion sensitivity for the limited charge density. Herein, we demonstrate the preparation of a cation-selective polydopamine-coated graphene oxide composite membrane with the sandwich structure by a simple interfacial polymerization technique, which greatly improves the surface charge density and further generates a power density of 3.4 W/m2 under river water and seawater. The GO membrane is firstly fabricated to function as the supporting layer and provide the reaction sites. And the ultrathin selective layer of the polydopamine membrane is chemically bonded with the GO layer by the in-situ polymerization on both sides of the GO membrane. The sandwiched nanofluidic membrane with ultrahigh charge density exhibits both high cation selectivity and ionic conductivity, benefiting the performance of osmotic energy conversion. The economic, easy-prepared method of the sandwiched nanofluidic membrane provides a promising strategy for high-performance osmotic energy conversion.

Well-Defined Nanostructures by Block Copolymers and Mass Transport Applications in Energy Conversion.

With the speedy progress in the research of nanomaterials, self-assembly technology has captured the high-profile interest of researchers because of its simplicity and ease of spontaneous formation of a stable ordered aggregation system. The self-assembly of block copolymers can be precisely regulated at the nanoscale to overcome the physical limits of conventional processing techniques. This bottom-up assembly strategy is simple, easy to control, and associated with high density and high order, which is of great significance for mass transportation through membrane materials. In this review, to investigate the regulation of block copolymer self-assembly structures, we systematically explored the factors that affect the self-assembly nanostructure. After discussing the formation of nanostructures of diverse block copolymers, this review highlights block copolymer-based mass transport membranes, which play the role of "energy enhancers" in concentration cells, fuel cells, and rechargeable batteries. We firmly believe that the introduction of block copolymers can facilitate the novel energy conversion to an entirely new plateau, and the research can inform a new generation of block copolymers for more promotion and improvement in new energy applications.

Construction and Ion Transport-Related Applications of the Hydrogel-Based Membrane with 3D Nanochannels.

Hydrogel is a type of crosslinked three-dimensional polymer network structure gel. It can swell and hold a large amount of water but does not dissolve. It is an excellent membrane material for ion transportation. As transport channels, the chemical structure of hydrogel can be regulated by molecular design, and its three-dimensional structure can be controlled according to the degree of crosslinking. In this review, our prime focus has been on ion transport-related applications based on hydrogel materials. We have briefly elaborated the origin and source of hydrogel materials and summarized the crosslinking mechanisms involved in matrix network construction and the different spatial network structures. Hydrogel structure and the remarkable performance features such as microporosity, ion carrying capability, water holding capacity, and responsiveness to stimuli such as pH, light, temperature, electricity, and magnetic field are discussed. Moreover, emphasis has been made on the application of hydrogels in water purification, energy storage, sensing, and salinity gradient energy conversion. Finally, the prospects and challenges related to hydrogel fabrication and applications are summarized.

Rice Stripe Virus Modulates the Feeding Preference of Small Brown Planthopper from the Stems to Leaves of Rice Plants to Promote Virus Infection.

Research on plant-virus-vector interactions has revealed that viruses can enhance their spread to new host plants by attracting nonviruliferous vectors to infected plants or driving viruliferous vectors to noninfected plants. However, whether viruses can also modulate the feeding preference of viruliferous vectors for different plant parts remains largely unknown. Here, by using rice stripe virus (RSV) and its vector, the small brown planthopper (SBPH), as a model, the effect of the virus on the feeding preference of its vector was studied by calculating the number of nonviruliferous and viruliferous SBPHs settling on different parts of rice plants. The results showed that the RSV-free SBPHs significantly preferred feeding on the stems of rice plants, whereas RSV-carrying SBPHs fed more on rice leaves. Moreover, the rice plants inoculated with RSV on the leaves showed more severe symptoms, with enhanced disease incidence and virus accumulation compared with rice plants inoculated at the top and bottom of stems, suggesting that the leaves are more susceptible to RSV than the stems of rice plants. These results demonstrate that RSV modulates the feeding preference of its transmitting vector SBPH from the stems to leaves of rice plants to promote virus infection. Interestingly, we also found that the leaves were more susceptible than the stems to rice black-streaked dwarf virus. This study proves that the feeding preference of insect vectors can be modulated by plant viruses to facilitate virus transmission.

Analyses on the Infection Process of Rice Virus and the Spatiotemporal Expression Pattern of Host Defense Genes Based on a Determined-Part Inoculation Approach.

Rice viral diseases adversely affect crop yield and quality. Most rice viruses are transmitted through insect vectors. However, the traditional whole-plant inoculation method cannot control the initial inoculation site in rice plants because the insect feeding sites in plants are random. To solve this problem, we established a determined-part inoculation approach in this study that restricted the insect feeding sites to specific parts of the rice plant. Rice stripe virus (RSV) was used as the model virus and was inoculated at the bottom of the stem using our method. Quantitative real-time PCR and Western blot analyses detected RSV only present at the bottom of the Nipponbare (NPB) stem at 1 day post-inoculation (dpi), indicating that our method successfully controlled the inoculation site. With time, RSV gradually moved from the bottom of the stem to the leaf in NPB rice plants, indicating that systemic viral spread can also be monitored using this method. In addition, a cultivar resistant to RSV, Zhendao 88 (ZD88), was inoculated using this method. We found that RSV accumulation in ZD88 was significantly lower than in NPB. Additionally, the expression level of the resistant gene STV11 in ZD88 was highly induced at the initial invasion stage of RSV (1 dpi) at the inoculation site, whereas it remained relatively stable at non-inoculated sites. This finding indicated that STV11 directly responded to RSV invasion to inhibit virus accumulation at the invasion site. We also proved that this approach is suitable for other rice viruses, such as Rice black-streaked dwarf virus (RBSDV). Interestingly, we determined that systemic infection with RSV was faster than that with RBSDV in NPB, which was consistent with findings in field trails. In summary, this approach is suitable for characterizing the viral infection process in rice plants, comparing the local viral accumulation and spread among different cultivars, analyzing the spatiotemporal expression pattern of resistance-associated genes, and monitoring the infection rate for different viruses.

Effects of microbial community and disease resistance against Vibrio splendidus of Yesso scallop (Patinopecten yessoensis) fed supplementary diets of tussah immunoreactive substances and antimicrobial peptides.

This study was conducted to investigate the effects of dietary supplementation of tussah immunoreactive substances (TIS) and antimicrobial peptides (AMPs) on microbial community and resistance against Vibrio splendidus of Yesso scallop Patinopecten yessoensis. Scallops were fed with the basal diets supplemented with TIS (T group), AMPs (A group), or both of the two (TA group). After the feeding trial, the microbial community changes were evaluated, and the challenge test with V. splendidus was conducted, as well as the immune parameters and digestive enzyme activities were determined. The results revealed that the TA group was more capable of modulating the bacterial community composition of scallops by increasing the potentially beneficial bacteria and suppressing the pathogenic microorganism during the feeding trial. After injection, the cumulative mortality rate in TA group was notably lower than others. In addition, the TA group showed better digestive and immune parameters involved in digestive capacity, phagocyte function, phosphatase-responsiveness, and oxidation resistance. These results collectively confirmed that dietary TIS and AMPs in diet could effectively modulate the microflora structure and improve disease resistance against V. splendidus of scallop, and the positive effects were more obvious when dietary supplementation of them in combination.

A membraneless self-powered photoelectrochemical biosensor based on Bi2S3/BiPO4 heterojunction photoanode coupling with redox cycling signal amplification strategy.

The photoelectrochemical (PEC) self-powered system has attracted great attention in disease detection. The determination of a simple and efficient approach for disease-related biomarkers is highly interesting and appealing. Herein, an ingenious visible light-induced membraneless self-powered PEC biosensing platform was constructed, integrating a signal amplification strategy for ultrasensitive split-type PEC bioanalysis. The system was comprised of a Bi2S3/BiPO4 heterojunction photoanode and a platinum (Pt) cathode in a one compartment chamber. An alkaline phosphatase (ALP)-loaded sandwich immunoassay was used to generate the signal reporter ascorbic acid (AA) in a 96-well plate, and myoglobin (Myo) was used as a model protein. In the presence of AA, ferrocene (Fc), and Tris (2-carboxyethyl) phosphine (TCEP), the chemical-chemical redox cycling scheme was operated upon the initial oxidation of Fc by the holes in the Bi2S3/BiPO4 photoelectrode, and Fc was regenerated from Fc+ by AA. Subsequently, AA was regenerated by TCEP after its oxidation, and cycling was triggered. As a result, the proposed self-powered PEC sensing exhibited excellent performance with a wide linear range from 5.0 × 10-13 to 1.0 × 10-7 g/mL, and a low detection limit of 2.0 × 10-13 g/mL for Myo. This work provided a new design of a redox cycling strategy in the self-powered PEC biosensor, and showed an effective approach for the clinical diagnosis.

Genome Editing of Rice eIF4G Loci Confers Partial Resistance to Rice Black-Streaked Dwarf Virus.

Rice black-streaked dwarf disease, caused by rice black-streaked dwarf virus (RBSDV), is a serious constraint in Chinese rice production. Breeding disease-resistant varieties through multigene aggregation is considered an effective way to control diseases, but few disease-resistant resources have been characterized thus far. To develop novel resources for resistance to RBSDV through CRISPR/Cas9-mediated genome editing, a guide RNA sequence targeting exon 1 of eIF4G was designed and cloned into a binary vector, pHUE401. This recombinant vector was used to generate mutations in the rice cultivar Nipponbare via Agrobacterium-mediated transformation. This approach produced heritable homozygous mutations in the transgene-free T1 generation. Sequence analysis of the eIF4G target region from T1 transgenic plants identified 3 bp deletion mutants, and analysis of the predicted amino acid sequence identified one amino acid deletion in mutants that possess near full-length eIF4G. Furthermore, our data suggest that eIF4G may plays an important role in rice normal development, as there were no eIF4G knock-out homozygous mutants in T1 generation plants. When homozygous mutant lines were inoculated with RBSDV, they exhibited enhanced tolerance to virus infection, without visibly affecting plant growth and development. However, the eif4g mutant plants showed the same sensitivity to rice stripe virus (RSV) infection as wild-type plants. Notably, the wild-type and mutant N-termini of eIF4G interacted directly with RBSDV P8 in yeast and in planta. Additionally, compared to wild-type plants, the eIF4G transcript level was reduced twofold in the mutant plants. These results indicate that site-specific mutation of rice eIF4G successfully conferred partial resistance specific to RBSDV associated with less transcription of eIF4G in mutants. Therefore, this study demonstrates that the novel eIF4G alleles generated by CRISPR/Cas9 represent valuable disease-resistant resources that can be used to develop RBSDV-resistant varieties.

Photogenerated Hole-Induced Chemical-Chemical Redox Cycling Strategy on a Direct Z-Scheme Bi2S3/Bi2MoO6 Heterostructure Photoelectrode: Toward an Ultrasensitive Photoelectrochemical Immunoassay.

To achieve high sensitivity for biomolecule detection in photoelectrochemical (PEC) bioanalysis, the ideal photoelectrode and ingenious signaling mechanism play crucial roles. Herein, the feasibility of the photogenerated hole-induced chemical-chemical redox cycling amplification strategy on a Z-scheme heterostructure photoelectrode was validated, and the strategy toward enhanced multiple signal amplification for advanced PEC immunoassay application was developed. Specifically, a direct Z-scheme Bi2S3/Bi2MoO6 heterostructure was synthesized via a classic hydrothermal method and served as a photoelectrode for the signal response. Under the illumination, the PEC chemical-chemical redox cycling (PECCC) among 4-aminophenol generated by the enzymatic catalysis from a sandwich immunoassay, ferrocene as a mediator, and tris (2-carboxyethyl) phosphine as a reducing agent was run on the Z-scheme Bi2S3/Bi2MoO6 heterostructure photoelectrode. Exemplified by interleukin-6 (IL-6) as the target, the applicability of the strategy was studied in a PEC immunoassay. Thanks to the multiple signal amplification originating from the high efficiency of the PECCC redox cycling system, the enzymatic amplification, and the fine performance of the Z-scheme Bi2S3/Bi2MoO6 heterostructure photoelectrode, the assay for IL-6 exhibits a very low detection limit of 2.0 × 10-14 g/mL with a linear range from 5.0 × 10-14 to 1.0 × 10-8 g/mL. This work first validates the feasibility of the PECCC redox cycling on the Z-scheme heterostructure photoelectrode and the good performance of the strategy in PEC bioanalysis. We envision that it would provide a new prospective for highly sensitive PEC bioanalysis on the basis of a Z-scheme heterostructure.

Liposome-assisted enzyme catalysis: toward signal amplification for sensitive split-type electrochemiluminescence immunoassay.

Developing an efficient signal amplification strategy is very important to improve the sensitivity of bioanalysis. In this paper, a liposome-assisted enzyme catalysis signal amplification strategy was developed for electrochemiluminescence (ECL) immunoassay of prostate specific antigen (PSA) in a split-type mode. The sandwich immunoreaction occurred in a 96-well plate, and glucose oxidase (GOx) encapsulated and antibody-modified liposomes were used as labels. The ECL detection was carried out using a rGO-Au NP modified glassy carbon electrode (GCE). The large amount of generated H2O2, i.e. the coreactant of the luminol system, and the excellent catalytic behavior of rGO-Au NPs greatly boosted the ECL signal, resulting in the signal amplification. The developed ECL immunosensor for detecting PSA achieved a wider linear range from 1.0 × 10-13 to 1.0 × 10-8 g mL-1 and a detection limit of 1.7 × 10-14 g mL-1. The application of the proposed strategy was demonstrated by analyzing PSA in human serum samples with recoveries from 89.0% to 113.0%, and relative standard deviations (RSDs) were less than 6.6%. This work provides a new horizon to expand the application of liposomes for ECL bioanalysis.

Enzymatic Hydrolysis of Defatted Antheraea pernyi (Lepidoptera: Saturniidae) Pupa Protein by Combined Neutral Protease Yield Peptides With Antioxidant Activity.

In this study, peptides were prepared from defatted Antheraea pernyi (Lepidoptera: Saturniidae) pupa protein via hydrolysis with combined neutral proteases. Single-factor tests and response surface methodology (RSM) were used to determine the optimal hydrolysis condition suitable for industrial application. Optimal hydrolysis of the defatted pupa protein was found to occur at an enzyme concentration of 4.85 g/liter, a substrate concentration of 41 g/liter, a hydrolysis temperature of 55°C, and a hydrolysis time of 10 h and 40 min. Under these conditions, the predicted and actual rates of hydrolysis were 45.82% and 45.75%, respectively. Peptides with a molecular weight of less than 2,000 Da accounted for 90.5% of the total peptides generated. Some of the peptides were antioxidant peptides as revealed by sequencing and functional analysis. The antioxidant activity of the mixed peptides was subsequently confirmed by an antioxidant activity assay. The results showed that peptides with high antioxidant activity could be obtained from the hydrolysis of A. pernyi pupa protein.

An attenuated TW-like infectious bronchitis virus strain has potential to become a candidate vaccine and S gene is responsible for its attenuation.

TW-like infectious bronchitis virus (IBV) with high pathogenicity is becoming the predominant IBV type circulating in China. To develop vaccines against TW-like IBV strains and investigate the critical genes associated with their virulence, GD strain was attenuated by 140 serial passages in specific-pathogen-free embryonated eggs and the safety and efficacy of the attenuated GD strain (aGD) were examined. The genome sequences of GD and aGD were also compared and the effects of mutations in the S gene were observed. The results revealed that aGD strain showed no obvious pathogenicity with superior protective efficacy against TW-like and QX-like virulent IBV strains. The genomes of strains aGD and GD shared high similarity (99.87 %) and most of the mutations occurred in S gene. Recombinant IBV strain rGDaGD-S, in which the S gene was replaced with the corresponding regions from aGD, showed decreased pathogenicity compared with its parental strain. In conclusion, attenuated TW-like IBV strain aGD is a potential vaccine candidate and the S gene is responsible for its attenuation. Our research has laid the foundation for future exploration of the attenuating molecular mechanism of IBV.

Enhanced protein phosphorylation in Apostichopus japonicus intestine triggered by tussah immunoreactive substances might be involved in the regulation of immune-related signaling pathways.

The sea cucumber Apostichopus japonicus is an economically important species owing to its high nutritive and medicinal value. In order to avoid the pollution resulting from the overuse of antibiotics in A. japonicus aquaculture, various immunostimulants have been used as an alternative to improve the efficiency of A. japonicus farming. Our previous proteomic investigation has shown that several proteins participating in the immune-related physiology of A. japonicus were differentially expressed in the intestinal tissue in response to tussah immunoreactive substances (TIS). This study further explored the immunostimulation mechanism of TIS in A. japonicus. Phosphoproteomics technology was used to investigate the effect of TIS on protein phosphorylation in the intestine of A. japonicus following feeding with a TIS-supplemented diet. A total of 213 unique phosphoproteins were detected from 225 unique phosphopeptides. KEGG pathway analysis showed that majority of the phosphoproteins are involved in endocytosis, carbon metabolism and spliceosome functional group. Sixteen of the phosphoproteins exhibited differential phosphorylation in response to TIS and 12 of these were found to associate with biological functions. Of these 12 phosphoproteins, eight exhibited enhanced phosphorylation while four displayed reduced phosphorylation. These 12 proteins were further analyzed and all were found to play a role in regulating some aspects of the immune system and the growth of sea cucumbers, especially in phagocytosis, energy metabolism and disease resistance. The findings of this study could therefore shed new light on the immune pathways of sea cucumber that are affected by TIS. This could help us to better understand the underlying mechanism linked to the immunoenhancement of A. japonicus in response to TIS, one that is associated with the change in protein phosphorylation.

A ternary CdS@Au-g-C3N4 heterojunction-based photoelectrochemical immunosensor for prostate specific antigen detection using graphene oxide-CuS as tags for signal amplification.

Photoactive materials with high photo-electron transfer efficiency and stable signal output hold a key role in constructing the photoelectrochemical (PEC) biosensing systems. In this study, the ternary CdS@Au-g-C3N4 heterojunction was first prepared and characterized, and its application in PEC bioanalysis was explored. The gold nanoparticles sandwiched between CdS and g-C3N4, acting as both plasmonic photosensitizer and electron relay, significantly boosted the light absorption and accelerated the charge transfer from g-C3N4 to CdS, both of which contributed to the enhancement of photoelectric conversion efficiency. Signal quenching with graphene oxide-CuS efficiently weakened the photocurrent from CdS@Au-g-C3N4. The combination of the excellent PEC properties of CdS@Au-g-C3N4 and the remarkable quenching effects of graphene oxide-CuS enabled construction of a sandwich-type PEC immunosensor for prostate specific antigen (PSA) detection. This immunosensor achieved sensitive PSA analysis by multiple signal amplification mechanisms, with a detection limit of 0.6 pg mL-1 and a wide linear range from 1.0 pg mL-1 to 10 ng mL-1. This work not only demonstrates the great potential of noble metal sandwiched ternary heterojunctions in the PEC field, but also lays a foundation for developing the general PEC immunoassays.

Copper ion modified graphitic C3N4 nanosheets enhanced luminol-H2O2 chemiluminescence system: Toward highly selective and sensitive bioassay of H2S in human plasma.

A high-efficient chemiluminescence (CL) platform for highly selective and sensitive H2S detection was constructed on the basis of the quenching effect of S2- on the copper ion modified graphitic carbon nitride nanosheets (Cu2+-g-C3N4 NSs) enhanced luminol-H2O2 system. Cu2+-g-C3N4 NSs with horseradish peroxidase-like catalytic activity were prepared and provide a great improvement for luminol-H2O2 system. The presence of S2- induced the formation of CuS precipitate on g-C3N4 NSs surface. The precipitate can block the catalytic Cu2+ sites on the g-C3N4 NSs surface, resulting in a great CL decrease of CL system. Based on such a mechanism, a simple, highly selective and sensitive CL biosensor for H2S detection was designed. Under the optimized conditions, luminol-H2O2-Cu2+-g-C3N4 NSs system gave a decrease of CL intensity with the Na2S concentration increasing. The CL biosensor is in a linear range of 10.0 pM-50.0 nM and the detection limit for detecting Na2S is as low as 2.0 pM. Moreover, the method here has enjoyed a successful application for determining H2S in human plasma samples and the recovery is between 95.7% and 110.0%.

A novel nitrogen and sulfur co-doped carbon dots-H2O2 chemiluminescence system for carcinoembryonic antigen detection using functional HRP-Au@Ag for signal amplification.

A novel nitrogen and sulfur co-doped carbon dots (NS-CDs)-H2O2 chemiluminescence (CL) system was developed to detect carcinoembryonic antigen (CEA) by taking advantage of dual-signal amplification of functional Au@Ag nanoparticles (NPs) nanoprobes. Horseradish peroxidase (HRP) and the complementary DNA were co-immobilized onto Au@Ag NPs surface to shape the functional nanoprobes (HRP-Au@Ag-cDNA) for signal amplification. In this proposal, HRP-Au@Ag-cDNA was specifically hybridized with CEA aptamer-functionalized magnetic beads to form the double-strand hybridization nanocomposites (HRP-Au@Ag-dsDNA-MB). Upon the addition of CEA, the CEA aptamer preferred to bind with CEA instead of double-strand hybridization interaction, thus HRP-Au@Ag-dsDNA-MB was dehybridized and the HRP-Au@Ag-cDNA nanoprobe was released. The synergistic catalytic effects of HRP and Au @Ag NPs endow the nanoprobe producing a dual CL signal amplification in the NS-CDs-H2O2 CL system. The CL intensity of the developed strategy enhanced with CEA concentration increasing in the range of 0.3-80 ng mL-1. Benefiting from the synergistic effect, a detection limit as low as 94 pg mL-1 was obtained. Moreover, successful application of this CL sensing platform was achieved for the determination of CEA in human serum samples, demonstrating the promising prospect in the early tumor warning and therapeutic monitoring.

Tussah silkmoth pupae improve anti-tumor properties of Cordyceps militaris (L.) Link by increasing the levels of major metabolite cordycepin.

Silkworms have been reported to promote the growth and production of the stromata of C. militaris (L.) Link as a parasite insect medium and may improve its metabolites. The effects of Tussah silkmoth pupae (TG group) and rice (RG group) on the metabolic profile of C. militaris (L.) Link were compared by metabolomics. Meanwhile, the profile of natural C. sinensis (NG group) was also analyzed. The functions of these metabolites from different groups and cordycepin were tested using breast cancer cells and an animal model. 292 metabolites were detected, including 51, 31 and 23 unique metabolites from the TG, RG and NG groups, respectively. The level of 3-deoxyadenosine (cordycepin with anti-tumor activity) was highest in the TG group. Tussah silkmoth pupae induced the biosynthesis of cordycepin and unsaturated fatty acids, which may be beneficial in the prevention of breast cancer. The TG group and cordycepin had significant inhibitory activities on breast cancer cells and in animal models when compared with the two other groups. Tussah silkmoth pupae improved the metabolic profile of C. militaris (L.) Link, which has more pharmaceutical metabolites than C. sinensis.

BMAL1 but not CLOCK is associated with monochromatic green light-induced circadian rhythm of melatonin in chick pinealocytes.

The avian pineal gland, an independent circadian oscillator, receives external photic cues and translates them for the rhythmical synthesis of melatonin. Our previous study found that monochromatic green light could increase the secretion of melatonin and expression of CLOCK and BMAL1 in chick pinealocytes. This study further investigated the role of BMAL1 and CLOCK in monochromatic green light-induced melatonin secretion in chick pinealocytes using siRNAs interference and overexpression techniques. The results showed that si-BMAL1 destroyed the circadian rhythms of AANAT and melatonin, along with the disruption of the expression of all the seven clock genes, except CRY1. Furthermore, overexpression of BMAL1 also disturbed the circadian rhythms of AANAT and melatonin, in addition to causing arrhythmic expression of BMAL1 and CRY1/2, but had no effect on the circadian rhythms of CLOCK, BMAL2 and PER2/3. The knockdown or overexpression of CLOCK had no impact on the circadian rhythms of AANAT, melatonin, BMAL1 and PER2, but it significantly deregulated the circadian rhythms of CLOCK, BMAL2, CRY1/2 and PER3. These results suggested that BMAL1 rather than CLOCK plays a critical role in the regulation of monochromatic green light-induced melatonin rhythm synthesis in chicken pinealocytes. Moreover, both knockdown and overexpression of BMAL1 could change the expression levels of CRY2, it indicated CRY2 may be involved in the BMAL1 pathway by modulating the circadian rhythms of AANAT and melatonin.