Circadian rhythms are core regulatory mechanisms that evolved to align biological functions with the Earth's rotation. These rhythms are conserved across organisms from unicellular life to multicellular species and play essential roles in metabolism, immune responses, and sleep-wake cycle. Circadian disruptions are strongly associated with various diseases. Over the past decades, genetic studies in Drosophila and mice have identified key conserved clock genes and uncovered transcription-translation feedback loops governing circadian regulation. Additionally, rhythmic neurons in the brain integrate complex neural circuits to precisely regulate physiological and behavioral rhythms. This review highlights recent advances in understanding the neuronal circuit mechanisms of rhythmic neurons in the Drosophila brain and discusses future directions for translating circadian rhythm research into chronomedicine and precision therapies.
Animals ; Circadian Rhythm/genetics* ; Neurons/physiology* ; Drosophila/physiology* ; Brain/physiology* ; Nerve Net/physiology*

OBJECTIVES: To investigate how larval feeding regimens influence development and deltamethrin resistance of Aedes albopictus to provide evidence for standardizing larval feeding protocols in studies of insecticide resistance. METHODS: Aedes albopictus larvae of a laboratory resistant strain were divided into 3 groups (n=500) and reared with high, medium, and low food availability (100, 50, or 25 mg daily for the 1st and 2nd instars, and 500 mg 250, or 125 mg daily for 3rd and 4th instars). The developmental time, pupation rate, adult emergence rate, adult body weight, and wing length were recorded in each group, and deltamethrin resistance of the mosquitoes was assessed using larval bioassays and contact tube tests for adults. RESULTS: Significant developmental differences were observed across the 3 feeding groups. Larval development time decreased as the food availability increased, and both high- and low-food groups showed reduced pupation rates (χ²=16.282, 7.440) and emergence rates (χ²=4.093, 6.977) compared to the medium-food group. Adult body weight and wing length were positively correlated with the amount of larval food intake (P<0.05). In high, medium and low food intake groups, larval LC₅₀ values for deltamethrin were 0.110, 0.072 and 0.064 mg/L, adult KDT50 values were 97.404, 68.964 and 65.005 min, and adult mosquitoe mortality rates at 24 h after deltamethrin exposure were 12%, 16% and 19%, respectively. CONCLUSIONS The feeding amount during larval stage significantly impacts the development and deltamethrin resistance of Aedes albopictus, suggesting the importance of standardization of larval nutrition for ensuring comparability of resistance test data across laboratories.
Animals ; Aedes/physiology* ; Pyrethrins/pharmacology* ; Nitriles/pharmacology* ; Larva/physiology* ; Insecticide Resistance ; Insecticides/pharmacology* ; Feeding Behavior

Sleep, an essential and evolutionarily conserved behavior, is regulated by numerous neurotransmitter systems. In mammals, glutamate serves as the wake-promoting signaling agent, whereas in Drosophila, it functions as the sleep-promoting signal. However, the precise molecular and cellular mechanisms through which glutamate promotes sleep remain elusive. Our study reveals that disruption of glutamate signaling significantly diminishes nocturnal sleep, and a neural cell-specific knockdown of the glutamate-gated chloride channel (GluClα) markedly reduces nocturnal sleep. We identified two pairs of neurons in the ventral nerve cord (VNC) that receive glutamate signaling input, and the GluClα derived from these neurons is crucial for sleep promotion. Furthermore, we demonstrated that GluClα mediates the glutamate-gated inhibitory input to these VNC neurons, thereby promoting sleep. Our findings elucidate that GluClα enhances nocturnal sleep by mediating the glutamate-gated inhibitory input to two pairs of VNC neurons, providing insights into the mechanism of sleep promotion in Drosophila.
Animals ; Sleep/physiology* ; Neurons/metabolism* ; Chloride Channels/genetics* ; Drosophila Proteins/genetics* ; Drosophila ; Glutamic Acid/metabolism* ; Animals, Genetically Modified

Human keratinocyte growth factor-1 (hKGF-1), a member of the fibroblast growth factor (FGF) family, plays crucial roles in organ development, cell proliferation, wound healing, and tissue repair, representing one of the most effective and specific growth factors for skin repair. However, obtaining recombinant hKGF-1 remains challenging due to its universally low expression efficiency in vitro. This study employs the Bombyx mori baculovirus expression system to establish a technological platform that utilizes the economically important insect Bombyx mori as a bioreactor for high-efficiency and low-cost expression and production of recombinant human keratinocyte growth factor 1 (hKGF-1) protein, ultimately achieving high-level expression of hKGF-1 in Bombyx mori ovary cell line (BmN). In this study, we optimized the hKGF-1 sequence based on the codon preference of baculovirus. By fusing hKGF-1 with polyhedrin (highly expressed in this system) and adding extra promoters and enhancers, we significantly improved the expreesion level of hKGF-1 in Bombyx mori cells. The results demonstrated that the aforementioned strategies significantly enhanced the expression level of hKGF-1 in Bombyx mori cells. SDS-PAGE and Western blotting results revealed that the highest hKGF-1 expression (accounting for 8.7% of total cellular protein) was achieved when the Polh promoter was combined in tandem with the P6.9 promoter and hKGF-1 was fused with a 15-residue polyhedrin fragment for co-expression. The optimal harvest time was determined to be 120 h post transfection. This study achieved the efficient expression of hKGF-1 in Bombyx mori cells, establishing an ideal technological platform for the industrial utilization of recombinant hKGF-1. The developed methodology not only provides valuable technical references for the production of other growth factors and complex proteins, but also demonstrates significant implications for employing silkworms as bioreactors for recombinant human protein expression.
Bombyx/metabolism* ; Animals ; Baculoviridae/metabolism* ; Humans ; Fibroblast Growth Factor 7/biosynthesis* ; Recombinant Proteins/genetics* ; Cell Line ; Genetic Vectors/genetics*

Bombyx mori nucleopolyhedrovirus (BmNPV) is extremely harmful to the silk industry. The polyhedrin, which encodes the polyhedrin (Polh), can be expressed at ultra-high levels and form occlusion bodies in the nucleus, embedding the progeny virus within it. However, the detailed mechanism by which polyhedrin is transported into the host cell nucleus remains unknown. Clarifying the nuclear import mechanisms of viral proteins can help us develop better prevention and treatment measures against baculoviruses. This study employed molecular cloning, co-immunoprecipitation, and immunofluorescence to analyze in detail the expression pattern of the highly expressed polyhedrin in the very late stage of the virus, and further revealed that the host protein importin α participates in the nuclear import of polyhedrin through protein interactions. This study provides a reference for further elucidating the nuclear import mechanisms of the baculovirus proteins including polyhedrin that can enter the nucleus.
Nucleopolyhedroviruses/metabolism* ; Active Transport, Cell Nucleus ; Animals ; Bombyx/virology* ; alpha Karyopherins/metabolism* ; Cell Nucleus/metabolism* ; Viral Structural Proteins/metabolism* ; Occlusion Body Matrix Proteins

Introduction: Folkloric claims have surrounded essential oils, including their enhancement of learning and memory through inhalational exposure. Few studies in humans have shown a benefit in cognition, albeit incremental. However, this benefit may not be entirely attributable to the essential oil aroma but may be confounded by psychological associations. We investigated rosemary, peppermint, lemon, and coffee aromas in a learning and memory model of Drosophila melanogaster to eliminate this confounder. Methods: We screened for concentrations of the four treatments that are non-stimulatory for altered locomotory behavior in the flies. At these concentrations, we determined if they were chemoneutral (i.e., neither chemoattractant nor chemorepellent) to the flies. Learning and memory of the flies exposed to these aromas were determined using an Aversive Phototaxis Suppression (APS) assay. Results: The aromas of rosemary, peppermint, and lemon that did not elicit altered mobility in the flies were from dilute essential oil solutions that ranged from 0.2 to 0.5% v/v; whereas for the aroma in coffee, it was at a higher concentration of 7.5% m/v. At these concentrations, the aromas used were found to be chemoneutral towards the flies. We observed no improvement in both learning and memory in the four aromas tested. While a significant reduction (p < 0.05) in learning was observed when flies were treated with the aromas of rosemary, peppermint, and coffee, a significant reduction (p < 0.05) in memory was only observed in the peppermint aroma treatment. Conclusion This study demonstrated that in the absence of psychological association, the four aromas do not enhance learning and memory
Drosophila melanogaster ; Learning ; Memory ; Rosmarinus ; Mentha piperita ; Citrus ; Coffea

Background: Consumption and abuse of alcohol remains a significant cause of concern worldwide. Furthermore, there is evidence of the association between chronic alcohol use and reduced life expectancy. Objectives: To study the effects of Garcinia binucao extract (GBE) supplementation on lifespan of Drosophila melanogaster, in the presence or absence of chronic alcohol exposure. Methods: D. melanogaster was mass cultured and given GBE supplementation in high (1 mg/mL) and low (200 µg/mL) sublethal doses. D. melanogaster flies were divided into groups - with and without chronic alcohol exposure, and their respective lifespans were monitored. Results: In D. melanogaster without alcohol exposure, mean lifespan was highest in the control flies (38.15 days), followed by high-dose GBE (34.42 days), low-dose GBE (33.24 days), and DMSO (22.29 days). In D. melanogaster chronically exposed to alcohol, the longest mean lifespan was observed in flies treated with high-dose GBE (33.80 days), followed by low-dose GBE (33.63 days), the DMSO group (30.30), and the control group (29.65 days), but the differences were not statistically significant. Comparing groups with and without chronic alcohol exposure, the mean lifespan of the control group chronically exposed to alcohol significantly decreased by 9.51 days (p < 0.05). In GBE treatment groups, mean lifespan significantly decreased by 0.82 days in high-dose set-up (p < 0.05), and significantly increased by 0.39 days in the low-dose set-up (p < 0.05) upon chronic alcohol exposure. Conclusion Garcinia binucao extract supplementation ameliorated the observed reduction in lifespan of Drosophila melanogaster chronically exposed to alcohol.
Drosophila melanogaster ; Longevity

Human epidermal growth factor (hEGF) can be applied in the treatment of surgical trauma (burns, scalds), tissue repair, skin moisturizing, beauty, skincare, etc. However, the low expression and high cost limit the application of hEGF. In order to improve the expression level of hEGF and reduce the production cost, considering the high expression of polyhedrin, this study fused a partial sequence of polyhedrin with hEGF and expressed the fused sequence by using a silkworm baculovirus expression vector system. In view of the small molecular weight of hEGF, we connected hEGF genes in series and optimized the codons to construct multiple fusion expression vectors by fusing different partial sequences of polyhedrin at the N-terminus. The results showed that through the above strategy, the protein expression level of hEGF was significantly increased. The expression vector containing three concatenated hEGF genes with optimized codons and fused with the sequence encoding 25 or 35 residues at the N-terminus of polyhedrin showed the highest expression level.
Humans ; Epidermal Growth Factor/biosynthesis* ; Genetic Vectors/genetics* ; Recombinant Fusion Proteins/biosynthesis* ; Animals ; Bombyx/metabolism* ; Occlusion Body Matrix Proteins/genetics* ; Nucleopolyhedroviruses/genetics* ; Amino Acid Sequence

This study aims to investigate the effect of Bombyx Batryticatus extract(BBE) on behaviors of rats with global cerebral ischemia reperfusion(I/R) and the underlying mechanism. The automatic coagulometer was used to detect the four indices of human plasma coagulation after BBE intervention for quality control of the extract. Sixty 4-week-old male SD rats were randomized into sham operation group(equivalent volume of normal saline, ip), model group(equivalent volume of normal saline, ip), positive drug group(900 IU·kg~(-1) heparin, ip), and low-, medium-, and high-dose BBE groups(0.45, 0.9, and 1.8 mg·g~(-1)·d~(-1) BBE, ip). Except the sham operation group, rats were subjected to bilateral common carotid artery occlusion followed by reperfusion(BCCAO/R) to induce I/R. The administration lasted 7 days for all the groups. The behaviors of rats were examined by beam balance test(BBT). Morphological changes of brain tissue were observed based on hematoxylin-eosin(HE) staining. Immunofluorescence method was used to detect common leukocyte antigen(CD45), leukocyte differentiation antigen(CD11b), and arginase-1(Arg-1) in cerebral cortex(CC). The protein expression of interleukin-1β(IL-1β), interleukin-4(IL-4), interleukin-6(IL-6), and interleukin-10(IL-10) was detected by enzyme-linked immunosorbent assay(ELISA). The non-targeted metabonomics was employed to detect the levels of metabolites in plasma and CC of rats after BBE intervention. The results of quality control showed that the BBE prolonged the activated partial thromboplastin time(APTT), prothrombin time(PT), and thrombin time(TT) of human plasma, which was similar to the anticoagulation effect of BBE obtained previously. The results of behavioral test showed that the BBT score of the model group increased compared with that of the sham operation group. Compared with the model group, BBE reduced the BBT score. As for the histomorphological examination, compared with the sham operation group, the model group showed morphological changes of a lot of nerve cells in CC. The nerve cells with abnormal morphology in CC decreased after the intervention of BBE compared with those in the model group. Compared with the sham operation group, the model group had high average fluorescence intensity of CD45 and CD11b in the CC. The average fluorescence intensity of CD11b decreased and the average fluorescence intensity of Arg-1 increased in CC in the low-dose BBE group compared with those in the model group. The average fluorescence intensity of CD45 and CD11b decreased and the average fluorescence intensity of Arg-1 increased in medium-and high-dose BBE groups compared with those in the model group. The expression of IL-1β and IL-6 was higher and the expression of IL-4 and IL-10 was lower in the model group than in the sham operation group. The expression of IL-1β and IL-6 was lower and the expression of IL-4 and IL-10 was higher in the low-dose, medium-dose, and high-dose BBE groups than in the model group. The results of non-targeted metabonomics showed that 809 metabolites of BBE were identified, and 57 new metabolites in rat plasma and 45 new metabolites in rat CC were found. BBE with anticoagulant effect can improve the behaviors of I/R rats, and the mechanism is that it promotes the polarization of microglia to M2 type, enhances its anti-inflammatory and phagocytic functions, and thus alleviates the damage of nerve cells in CC.
Humans ; Rats ; Male ; Animals ; Interleukin-10 ; Rats, Sprague-Dawley ; Interleukin-4/metabolism* ; Bombyx ; Interleukin-6/metabolism* ; Microglia/metabolism* ; Saline Solution/metabolism* ; Reperfusion Injury/metabolism* ; Brain Ischemia/metabolism* ; Cerebral Infarction ; Reperfusion ; Neurons

OBJECTIVE: To analyze the density, distribution and insecticide resistance of Aedes albopictus in Ningbo City in 2021, so as to provide insights into formulation of dengue fever control strategies. METHODS: Four administrative villages were randomly selected from each county (district) in Ningbo City from April to November, 2021, to investigate the indoor population density of Aedes larvae, and the Breteau index (BI) was calculated. The population density of adult mosquitoes was investigated in residential areas, parks/bamboo forests, waste tire stacking sites/waste stations/construction sites in each county (district). On June 2021, larvae of the natural strain A. albopictus were collected from epidemic sites of dengue fever in Ningbo City in 2018, and raised in laboratory. Then, larvae and female mosquitoes without blood feeding were selected for insecticide resistance bioassays, while insecticide-sensitive strains of A. albopictus served as controls. The resistance of A. albopictus larvae to deltamethrin, beta-cypermethrin, propoxur, temephos and dichlorvos using the impregnation method, and the medium lethal concentration (LC₅₀) and resistance ratio (RR) were calculated. The resistance of adult A. albopictus to beta-cypermethrin, permethrin, deltamethrin, propoxur and malathion was determined using the tube bioassay, and the mosquito mortality was calculated. RESULTS: A total of 10 072 small water containers from 9 935 households were investigated in Ningbo City in 2021, and there were 1 276 containers with Aedes larvae detected, with an average BI of 12.89. Totally 1 422 mosquito nets were allocated and 954 female A. albopictus were captured, with an average net trapping index of 1.34 mosquitoes/(net·hour). Both larval and adult A. albopictus mosquitoes were found from April to November, and the density of larval A. albopictus peaked in September (BI = 21.21), while the density of adult A. albopictus peaked in August, with a net trapping index of 2.38 mosquitoes/(net·hour). The LC₅₀ values of delta-methrin, beta-cypermethrin, propoxur, temephos and dichlorvos were 0.017 4, 0.000 9, 0.364 1, 0.038 1 mg/L and 0.001 6 mg/L against larvae of natural strains of A. albopicchus, with RRs of 49.66, 25.53, 9.65, 2.24 and 6.06, and the mortality rates of adult mosquitoes were 66.00% (66/100), 69.39% (68/98), 25.00% (25/100), 98.97% (96/97) and 100.00% (98/98) 24 hours post-treatment with 0.08% beta-cypermethrin, 0.03% deltamethrin, 0.4% permethrin, 0.05% propoxur, and 0.5% malathion for 24 h, respectively. CONCLUSIONS A. albopictus is widely distributed in Ningbo City, with a high population density and presents high-level resistance to common pyrethroid insecticides. The population density and insecticide resistance of A. albopictus requires to be reinforced.
Animals ; Female ; Malathion ; Temefos ; Aedes ; Propoxur ; Permethrin ; Dichlorvos ; Mosquito Vectors ; Larva ; Dengue/prevention & control*