The circadian clock affects starvation resistance through the pentose phosphate pathway in silkworm, Bombyx mori.

Disruption of the circadian clock can affect starvation resistance, but the molecular mechanism is still unclear. Here, we found that starvation resistance was significantly reduced in the core gene BmPer deficient mutant silkworms (Per-/-), but the mutant's starvation resistance increased with larval age. Under natural physiological conditions, the weight of mutant 5th instar larvae was significantly increased compared to wild type, and the accumulation ability of triglycerides and glycogen in the fat bodies was upregulated. However, under starvation conditions, the weight consumption of mutant larvae was increased and cholesterol utilization was intensified. Transcriptome analysis showed that beta-oxidation was significantly upregulated under starvation conditions, fatty acid synthesis was inhibited, and the expression levels of genes related to mitochondrial function were significantly changed. Further investigations revealed that the redox balance, which is closely related to mitochondrial metabolism, was altered in the fat bodies, the antioxidant level was increased, and the pentose phosphate pathway, the source of reducing power in cells, was activated. Our findings suggest that one of the reasons for the increased energy burden observed in mutants is the need to maintain a more robust redox balance in metabolic tissues. This necessitates the diversion of more glucose into the pentose phosphate pathway to ensure an adequate supply of reducing power.

Nonsteaming method improves the nutritional value and utilization efficiency of silkworm artificial diets.

Artificial diets for silkworms overcome the seasonal limitations of traditional rearing methods with fresh mulberry leaves. However, the current wet artificial diets, steamed at high temperatures, are not favored by silkworms, and they are cumbersome and challenging to preserve. These conditions adversely affected the development of artificial diet-based sericulture production. In this study, we disinfected dry powder diets with radiation and added distilled water without steaming before use. Then, the nutritional value of finished diets and their impact on silkworm development was assessed. Compared with steamed diets, nonsteamed diets were more attractive to silkworms. Chemical assays showed significantly more essential nutrients for silkworms, including l-ascorbic acid, vitamin B1, vitamin B2, and urease in nonsteamed diets than in steamed diets. Feeding fifth-instar silkworm larvae with nonsteamed diets significantly improved the ammonia utilization efficiency of the diet and increased the cocoon shell rate and diet/silk protein conversion efficiency by 5.9% and 13.3%, respectively. When fed with nonsteamed diets, the abundance of aerobic microorganisms in silkworm intestines increased and the abundance of pathogenic bacteria decreased. Furthermore, the vitality of the silkworm, measured by the dead worm cocoon rate, significantly improved by 16.90%. In summary, preparing sterile wet diets without high-temperature steaming effectively improved the nutritional value of the diet and enhanced silkworm growth.

Mechanism of hyperproteinemia-induced damage to female reproduction in a genetic silkworm model.

Hyperproteinemia is a metabolic disorder characterized by abnormally elevated plasma protein concentrations (PPC) in humans and animals. Here, a genetic silkworm model with high PPC was employed to investigate the effect of elevated PPC on female reproduction. Transcriptomic analysis revealed that high PPC induces downregulation of the ovarian development-related genes and disrupts ovarian sugar metabolism. Biochemical and endocrinal analyses revealed that high PPC increases trehalose and glucose levels in hemolymph and glycogen content in the fat body through activation of the gluconeogenic pathway and inhibition of the Insulin/Insulin-like growth factor signaling pathway-the serine/threonine kinase (IIS-AKT) pathway, thus disrupting characteristic metabolic homeostasis of sugar in the ovary. These resulted in ovarian developmental delay as well as reduced number and poor quality of eggs. Insulin supplementation effectively increased egg numbers by lowering blood sugar. These collective results provide new insights into the mechanisms by which high PPC negatively affects female reproduction and support the potential therapeutic effects of insulin.

The clock gene Cryptochrome 1 is involved in the photoresponse of embryonic hatching behavior in Bombyx mori.

The hatching of insect eggs is a classic circadian behavior rhythm controlled by the biological clock. Its function is considered to impose a daily rhythm on the embryo, allowing it to hatch within a permissible time window. However, the molecular pathways through which the clock affects embryonic hatching behavior remain unclear. Here, we utilized a clock gene Cryptochrome1 (Cry1) knockout mutant to dissect the pathways by which the circadian clock affects embryonic hatching rhythm in the silkworm. In the Cry1 mutant, the embryo hatching rhythm was disrupted. Under the constant light or constant dark incubation conditions, mutant embryos lost their hatching rhythm, while wild-type embryos hatch exhibiting free-running rhythm. In the light-dark cycle (LD), the hatching rhythm of CRY1-deficient silkworms could not be entrained by the LD photoperiod during the incubation period. The messenger RNA levels and enzymatic activities of Cht and Hel in the mutant embryos were significantly reduced at circadian time 24 (CT24). Transcriptome analysis revealed significant differences in gene expression at CT24 between the Cry1 knockout mutant and the wild-type, with 2616 differentially expressed genes identified. The enriched Gene Ontology pathway includes enzyme activity, energy availability, and protein translation. Short neuropeptide F signaling was reduced in the CT24 embryonic brain of the mutant, the expression of the neuropeptide PTTH was also reduced and the rhythm was lost, which further affects ecdysteroid signaling. Our results suggested that the silkworm circadian clock affects neuropeptide-hormone signaling as well as physiological functions related to hatching, which may regulate the hatching rhythm.

[Corrigendum] Comparison of the biological characteristics of human mesenchymal stem cells derived from exfoliated deciduous teeth, bone marrow, gingival tissue, and umbilical cord.

Subsequently to the publication of this paper, an interested reader drew to the authors' attention that, in Fig. 3 on p. 4973, the data panels shown for the "Osteogenesis" row of data for the GMSC and BMSC experiments appeared to be overlapping, such the data may have been derived from the same original source. After having examined their original data, the authors have realized that the data panel selected for the GMSC "Osteogenesis" experiment was inadvertently chosen incorrectly. The corrected version of Fig. 3 is shown below. Note that this error did not significantly affect the results or the conclusions reported in this paper, and all the authors agree to this Corrigendum. The authors are grateful to the editor of Molecular Medicine Reports for allowing them the opportunity to publish this corrigendum, and apologize to the readership for any inconvenience caused.[Molecular Medicine Reports 18: 4969­4977, 2018; DOI: 10.3892/mmr.2018.9501].

Temporal transcriptome reveals that circadian clock is involved in the dynamic regulation of immune response to bacterial infection in Bombyx mori.

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.

Mechanism of hyperproteinemia-induced blood cell homeostasis imbalance in an animal model.

Hyperproteinemia is a metabolic disorder associated with increased plasma protein concentration (PPC) and is often clinically complicated by malignant diseases or severe infections. At present, however, research on the molecular mechanism underlying high PPC (HPPC) is scant. Here, an animal model of primary hyperproteinemia was constructed in an invertebrate ( Bombyx mori) to investigate the effects of HPPC on circulating blood cells. Results showed that HPPC affected blood cell homeostasis, leading to increased reactive oxygen species levels, and induced programmed cell death dependent on the endoplasmic reticulum-calcium ion signaling pathway. HPPC induced the proliferation of blood cells, mainly granulocytes, by activating the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. Supplementation with the endocrine hormone active substance 20E significantly reduced the impact of HPPC on blood cell homeostasis. Thus, we identified a novel signaling pathway by which HPPC affects blood cell homeostasis, which differs from hyperglycemia, hyperlipidemia, and hypercholesterolemia. In addition, we showed that down-regulation of gene expression of the hematopoietic factor Gcm could be used as a potential early detection indicator for hyperproteinemia.

Circadian Clock Gene Period Contributes to Diapause via GABAeric-Diapause Hormone Pathway in Bombyx mori.

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.

Personality types of patients with glaucoma: A systematic review of observational studies.

OBJECTIVES: To synthesize recent empirical research on the association between personality and glaucoma among this sub-population. METHODS: PubMed/MEDLINE, Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, Scopus and ScienceDirect databases were searched to identify eligible studies published between January 1950 and March 2019 in any language. The quality of included observational studies was assessed using an 11-item checklist which was recommended by Agency for Healthcare Research and Quality (AHRQ). After using the checklist, 12 papers are included into the systematic review. RESULTS: There are some differences on the studies about the negative personality of glaucoma patients. In spite of these differences, most included studies significantly showed that glaucoma patients tend to or do have some specific personality. CONCLUSION: The extant research could demonstrate that glaucoma patients tend to have some negative personality in some extent. Future studies are needed to provide more convincing support to personality of glaucoma patients.

Bombyx mori can synthesize ascorbic acid through the l-gulose pathway to varying degrees depending on developmental stage.

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.

Influence of Hyperproteinemia on Insect Innate Immune Function of the Circulatory System in Bombyx mori.

Metabolic disorders of the circulatory system of animals (e.g., hyperglycemia and hyperlipidemia) can significantly affect immune function; however, since there is currently no reliable animal model for hyperproteinemia, its effects on immunity remain unclear. In this study, we established an animal model for hyperproteinemia in an invertebrate silkworm model, with a controllable plasma protein concentration (PPC) and no primary disease effects. We evaluated the influence of hyperproteinemia on innate immunity. The results showed that high PPC enhanced hemolymph phagocytosis via inducing a rapid increase in granulocytes. Moreover, while oenocytoids increased, the plasmacytes quickly dwindled. High PPC inhibited hemolymph melanization due to decreased phenoloxidase (PO) activity in the hemolymph via inhibiting the expression of the prophenoloxidase-encoding genes, PPO1 and PPO2. High PPC upregulated the gene expression of antimicrobial peptides via differential activation of the Toll and Imd signaling pathways associated with NF-κB signaling, followed by an induction of inconsistent antibacterial activity towards Gram-positive and Gram-negative bacteria in an animal model of high PPC. Therefore, high PPC has multiple significant effects on the innate immune function of the silkworm circulatory system.

Oct4 Regulates the Transition of Cancer Stem-Like Cells to Tumor Endothelial-Like Cells in Human Liver Cancer.

Octamer-binding transcription factor 4 (Oct4) has been recently implicated as a proangiogenic regulator in several induced pluripotent stem cells (iPSCs), however, its role in cancer stem-like cells (CSCs) remain unclear. We report here that Oct4 participates in tumor vasculogenesis in liver CSCs (LCSCs). We identify that LCSCs possess the potential of endothelial trans-differentiation under endothelial induction, present endothelial specific markers and their functions in vitro, and participate in neovasculogenesis in vivo. The knockdown of the Oct4A by short hairpin RNA (shRNA) in LCSCs represses endothelial trans-differentiation potential, but induces endothelial lineage-restricted differentiation, the latter is positively regulated by Oct4B1. Furthermore, Oct4 regulates vasculogenesis in LCSCs may be via the AKT-NF-κB-p65 signaling pathway. This work reveals Oct4, which is a crucial regulator, plays a critical role in tumor endothelial-like cells transition of LCSCs through Oct4A and Oct4B1 by different ways. The simultaneous inhibition of both the isoforms of Oct4 is hence expected to help regress neovascularization derived from CSCs. Our findings may provide insights to the possible new mechanisms of tumor vasculogenesis for primary liver cancer.

Yu Gan Long Ameliorates Hepatic Fibrosis by Inhibiting PI3K/AKT, Ras/ERK and JAK1/STAT3 Signaling Pathways in CCl4-induced Liver Fibrosis Rats.

Yu Gan Long (YGL) is a Chinese traditional herbal formula which has been reported to attenuate liver fibrosis for many years and we have explored its anti-fibrotic mechanism through blocking transforming growth factor (TGF-ß) in the previous study. But the mechanisms associated with platelet-derived growth factor (PDGF)-BB remain obscure. In this study, we further investigated the mechanism of YGL reducing carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Our results showed that YGL suppressed CCl4-induced upregulation of collagen IV (Col IV), type HI precollagen (PCHI), hyaluronuc acid (HA) and laminin (LN), which are implicated in liver fibrosis. Also, YGL reduced the α-smooth muscle actin (α-SMA) expression, which acts as the indicator of liver fibrosis. Furthermore, YGL decreased the serum levels of hepatic stellate cell (HSC) mitogen PDGF-BB and inflammation cytokines, including TNF-α, IL-1ß, IL-6. Markers involved in liver fibrosis, such as Ras, p-Raf-1, p-ERK1/2, p-JNK, p-P38, p-PI3K, p-AKT, p-JAKl, p-STAT3 were downregulated significantly after treatment with YGL. Our results indicated that YGL ameliorated CCl4-induced liver fibrosis by reducing inflammation cytokines production, and suppressing Ras/ERK, PI3K/AKT, and JAK1/STAT3 signaling pathways, which provided further evidence towards elucidation of the anti-fibrotic mechanism of YGL.

Impact of adding glucose-coated water-soluble silver nanoparticles to the silkworm larval diet on silk protein synthesis and related properties.

Biological modifications of the silk fibroin (silk) material have broad applications in textiles, biomedical materials and other industrial materials. It is economical to incorporate nanoparticles to the biosynthesis of silk fibroin by adding them to silkworm larval diets. This strategy may result in the rapid stable production of modified silk. Glucose-coated silver nanoparticles (AgNPs) were used to improve the AgNPs' biocompatibility, and the AgNPs were efficiently incorporated into silk by feeding. Larvae fed with AgNPs produced silk with significantly improved antibacterial properties and altered silk secondary structures. Both positive and negative effects on the growth and synthesis of silk proteins were observed after different AgNPs doses. Larvae feeding with low concentration of 0.02% and medium 0.20% AgNPs have greater transfer efficiencies of AgNPs to silk compared with feeding high concentration of 2.00% AgNPs. In addition, the elongation and tensile strength of the produced silk fibers were also significantly increased, with greater mammalian cell compatibility. The appropriate AgNPs concentration in the diet of silkworms can promote the synthesis of silk proteins, enhance their mechanical properties, improve their antibacterial property and inhibit the presence of Gram-negative bacteria.

Influence of hyperproteinemia on reproductive development in an invertebrate model.

Hyperproteinemia is a severe metabolic disease characterized by abnormally elevated plasma protein concentrations (PPC). However, there is currently no reliable animal model for PPC, and the pathological mechanism of hyperproteinemia thus remains unclear. In this study, we evaluated the effects of hyperproteinemia on reproductive development in an invertebrate silkworm model with a controllable PPC and no primary disease effects. High PPC inhibited the synthesis of vitellogenin and 30K protein essential for female ovarian development in the fat body of metabolic tissues, and inhibited their transport through the hemolymph to the ovary. High PPC also induced programmed cell death in testis and ovary cells, slowed the development of germ cells, and significantly reduced the reproductive coefficient. Furthermore, the intensities and mechanisms of high-PPC-induced reproductive toxicity differed between sexes in this silkworm model.

Inhibition of Period Gene Expression Causes Repression of Cell Cycle Progression and Cell Growth in the Bombyx mori Cells.

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.

Comparison of the biological characteristics of human mesenchymal stem cells derived from exfoliated deciduous teeth, bone marrow, gingival tissue, and umbilical cord.

Different sources of mesenchymal stem cells (MSCs) may differ in their biological characteristics, which are important for their clinical application. In the present study, MSCs were isolated from human exfoliated deciduous teeth (SHED), bone marrow, gingival tissue and umbilical cord tissue, and their biological characteristics including surface markers, proliferation capacity, tumorigenicity and immunogenicity were analyzed by flow cytometric analysis, ELISA and co­culture with human lymphocytes, respectively. The results indicated that all four types of stem cells obtained from different sources expressed MSC surface markers, and they did not show tumorigenicity either in vivo or in vitro. Stem cells from SHED exhibited the strongest proliferation capacity. Umbilical cord­derived MSCs displayed the strongest immunomodulatory ability, while bone marrow MSCs exhibited the best antigen­presenting potential in response to interferon­Î³ stimulation. These results provide information on MSCs derived from different tissues, which may be helpful in their clinical application.

Induced Hyperproteinemia and Its Effects on the Remodeling of Fat Bodies in Silkworm, Bombyx mori.

Hyperproteinemia, which is characterized by an abnormally elevated plasma protein concentration (PPC), is a high-mortality, metabolic complication associated with severe liver and kidney disease. It is difficult to clinically distinguish the difference between the impacts of primary diseases and hyperproteinemia on tissues and organs, and there are no available animal models of hyperproteinemia. Here, we constructed an animal model of hyperproteinemia with a controllable PPC and no primary disease effects in the silkworm Bombyx mori that has attracted interest owing to its potential use in the pathological analysis of model animals. Silkworm have an open circulatory system in which each organ is directly immersed in hemolymph. The fat body (FB) of a silkworm, as a major organ for nutrient storage and energy metabolism, can effectively reflect hyperproteinemia-induced metabolic abnormalities in damaged visceral tissues. A pathogenesis study showed that hyperproteinemia attenuated cell autophagy and apoptosis by attenuating an endocrine hormone, thereby preventing FB remodeling during metamorphosis. Meanwhile, hyperproteinemia increased oxidative stress in the FB and resulted in a dysfunction of amino acid conversion. Supplementation with exogenous 20-hydroxyecdysone effectively mitigated the hyperproteinemia-mediated inhibition of FB remodeling.

Treatment of gingival defects with gingival mesenchymal stem cells derived from human fetal gingival tissue in a rat model.

BACKGROUND: The study aimed to evaluate the efficacy and safety of gingival mesenchymal stem cells (GMSCs) from human fetal gingival tissue used for treating gingival defects in a rat model. METHODS: GMSCs were isolated from human fetal gingival tissue and identified by flow cytometry for nestin, Oct4, vimentin, NANOG, CD105, and CD90. The immunogenicity of GMSCs was analyzed by mixed lymphocyte reactions; the tumorigenicity of GMSCs was evaluated by xenotransplanting into nude mice. The gingival defect animal model was established by mechanical resection in rats. GMSCs were transplanted into the defective area, and the regeneration of gingival tissue was observed twice weekly. Four weeks after transplantation, the gingival tissue was surgically cut down, and the graft was analyzed by immunohistochemistry staining for human mitochondrial antigens and rat CD3 and CD20. RESULTS: GMSCs from human fetal gingival tissue positively expressed nestin, Oct4, vimentin, NANOG, CD105, and CD90. There was no cell aggregation after mixed lymphocyte reactions, and interleukin-2 did not increase. Inoculation of GMSCs into nude mice for 6 months showed no tumor formation. GMSCs were transplanted into the gingiva defects of rats. One week after transplantation, the defect area was reduced, and after 3 weeks the morphology and color of local gingival tissue was similar to normal gingival tissue, and gingival height was the same as the normal control group. CONCLUSIONS: Using GMSCs from human fetal gingival tissue to treat gingival defects is a safe and effective innovative treatment method.

Differences in gut microbiota between silkworms (Bombyx mori) reared on fresh mulberry (Morus alba var. multicaulis) leaves or an artificial diet.

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.