Sleep Disturbance Affects Immune Factors in Clinical Liver Cancer Patients.

BACKGROUND: Sleep-wake disturbance is prevalent in patients with liver cancer, but there is no direct evidence of its association and related biological mechanisms. Our study was to assess quality of sleep and to describe prevalence of sleep disturbances in patients with different etiologies of liver cancer, especially to explore whether sleep quality influences immune factors. METHODS: A total of 210 patients with liver cancer from August 2015 to December 2015 were randomly divided into two groups including HBV cirrhosis and non-HBV cirrhosis. The Pittsburgh Sleep Quality Index (PSQI) was used to evaluate their sleep quality, and then 202 patients enrolled in this study were divided into two groups according to their PSQI scores: PSQI ≤ 5 and PSQI > 5. The association between sleep disturbances and immune factors was analyzed by logistic regression models. RESULTS: A total of 56.9% of liver cancer patients experienced poor sleep quality (PSQI > 5). The prevalence of sleep disturbances was significantly higher in patients with liver cancer of non-hepatitis B virus (HBV) cirrhosis than with that evolving from HBV cirrhosis (66.7% vs. 50%, p = 0.018). In non-HBV cirrhosis liver cancer patients, the PSQI > 5 group had a higher percentage of CD3+ T cells (71.06 ± 11.07 vs. 63.96 ± 14.18, p = 0.014) and lower natural killer (NK) cells (14.67 ± 9.65 vs. 20.5 ± 10.77, p = 0.014) compared with patients with PSQI ≤ 5. Logistic regression further confirmed that liver cancer patients without HBV cirrhosis are more prone to experience poor sleep with increased CD3+ T cells (OR = 1.07, 95% CI = 1.01-1.13, p = 0.030) and decreased NK cells (OR = 0.92, 95% CI = 0.85-0.98, p = 0.014). Our results indicate that increased CD3+ T cells and decreased NK cells are both associated with sleep disturbances in patients with liver cancer of non-HBV cirrhosis. CONCLUSIONS: Most liver cancer patients suffer from sleep disturbances, especially evolving from non-HBV cirrhosis. A rise in CD3+ T cells and a reduction in NK cells are associated with sleep disturbances in patients with liver cancer of non-HBV cirrhosis.

Neuropeptide Bursicon Influences Reproductive Physiology in Tribolium Castaneum.

Bursicon is a neuropeptide belonging to the cystine knot family and is composed of burs and partner of burs (pburs) subunits. It can form heterodimers or homodimers to execute different biological functions. Bursicon heterodimers regulate cuticle sclerotization and wing maturation, whereas bursicon homodimers mediate innate immunity and midgut stem cell proliferation. A recent study has shown that bursicon potentially induces the expression of vitellogenin (Vg) in the black tiger shrimp Penaeus monodon; however, the underlying mechanism remains unknown. In this study, we investigated the role of bursicon in the reproductive physiology of the red flour beetle, Tribolium castaneum. The knockdown of burs, pburs, or its receptor T. castaneum rickets (Tcrk) in 2-day pupae significantly downregulated the expression levels of Vg1, Vg2, and Vg receptor (VgR) genes in females 3- and 5-day post-adult emergence, leading to abnormal oocytes with limited Vg content. The silencing of burs repressed the number of eggs laid and completely inhibited egg hatch, whereas the silencing of pburs dramatically decreased the number of eggs laid, hatch rate, and offspring larval size, and this RNA interference (RNAi) effects persisted to the next generation. Furthermore, the knockdown of burs or pburs downregulated the expression of the insulin/insulin-like signaling/target of rapamycin (TOR) signaling genes encoding insulin receptor (InR), protein kinase B (Akt), TOR, and ribosomal protein S6 kinase (S6K). Most importantly, the injection of recombinant pburs (r-pburs) protein was able to upregulate the expression of Vg, VgR, InR, Akt, TOR, S6K, JH synthesis (JHAMT), Methoprene-tolerant (Met), and Taiman (Tai) in normal females and rescue the expression of Vg and VgR in pburs RNAi females but failed to rescue Vg and VgR in Tcrk knockdown females. We infer that bursicon homodimers influence Vg expression via the receptor Tcrk, possibly by mediating the expression of the juvenile hormone (JH) and IIS/TOR pathway genes, thereby regulating reproduction in T. castaneum.

Sleep Deprivation Disturbs Immune Surveillance and Promotes the Progression of Hepatocellular Carcinoma.

Sleep disturbance is common in patients with cancer and is associated with poor prognosis. However, the effects of sleep deprivation (SD) on immune surveillance during the development of hepatocellular carcinoma (HC) and the underlying mechanisms are not known. This was investigated in the present study using mouse models of SD and tumorigenesis. We determined that acute and chronic sleep deprivation (CSD) altered the relative proportions of various immune cell types in blood and peripheral organs. CSD increased tumor volume and weight, an effect that was enhanced with increasing CSD time. Expression of the cell proliferation marker Ki-67 was elevated in tumor tissues, and tumor cell infiltration into adjacent muscles was enhanced by CSD. Multicolor flow cytometry analysis revealed that CSD significantly reduced the numbers of antitumor CD3+ T cells and natural killer (NK) cells and increased that of immunosuppressive CD11b+ cells infiltrating into the tumor microenvironment from the spleen via the peripheral blood. These results indicate that CSD impairs immune surveillance mechanisms and promotes immunosuppression in the tumor microenvironment to accelerate tumor growth, underscoring the importance of alleviating sleep disturbance in HC patients in order to prevent HC progression.

Role of juvenile hormone receptor Methoprene-tolerant 1 in silkworm larval brain development and domestication.

The insect brain is the central part of the neurosecretory system, which controls morphology, physiology, and behavior during the insect's lifecycle. Lepidoptera are holometabolous insects, and their brains develop during the larval period and metamorphosis into the adult form. As the only fully domesticated insect, the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process. Hormonal regulation in insects is a key factor in multiple processes. However, how juvenile hormone (JH) signals regulate brain development in Lepidoptera species, especially in the larval stage, remains elusive. We recently identified the JH receptor Methoprene tolerant 1 ( Met1) as a putative domestication gene. How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin's theory on domestication. Here, CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain, unlike precocious pupation of the cuticle. At the whole transcriptome level, the ecdysteroid (20-hydroxyecdysone, 20E) signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain. Pathways related to cell proliferation and specialization processes, such as extracellular matrix (ECM)-receptor interaction and tyrosine metabolism pathways, were suppressed in the brain. Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B. mori, which decreased transcriptional binding activity. The B. mori MET1 protein showed a changed structure and dynamic features, as well as a weakened co-expression gene network, compared with B. mandarina. Based on comparative transcriptomic analyses, we proposed a pathway downstream of JH signaling (i.e., tyrosine metabolism pathway) that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.

20E-mediated regulation of BmKr-h1 by BmKRP promotes oocyte maturation.

BACKGROUND: Krüppel homolog 1 (Kr-h1) is a critical transcription factor for juvenile hormone (JH) signaling, known to play a key role in regulating metamorphosis and adult reproduction in insects. Kr-h1 can also be induced by molting hormone 20-hydroxyecdysone (20E), however, the underlying mechanism of 20E-induced Kr-h1 expression remains unclear. In the present study, we investigated the molecular mechanism of Kr-h1 induction by 20E in the reproductive system of a model lepidopteran insect, Bombyx mori. RESULTS: Developmental and tissue-specific expression analysis revealed that BmKr-h1 was highly expressed in ovaries during the late pupal and adult stages and the expression was induced by 20E. RNA interference (RNAi)-mediated depletion of BmKr-h1 in female pupae severely repressed the transcription of vitellogenin receptor (VgR), resulting in the reduction in vitellogenin (Vg) deposition in oocytes. BmKr-h1 specifically bound the Kr-h1 binding site (KBS) between - 2818 and - 2805 nt upstream of BmVgR and enhanced the transcription of BmVgR. A 20E cis-regulatory element (CRE) was identified in the promoter of BmKr-h1 and functionally verified using luciferase reporter assay, EMSA and DNA-ChIP. Using pull-down assays, we identified a novel transcription factor B. mori Kr-h1 regulatory protein (BmKRP) that specifically bound the BmKr-h1 CRE and activated its transcription. CRISPR/Cas9-mediated knockout of BmKRP in female pupae suppressed the transcription of BmKr-h1 and BmVgR, resulting in arrested oogenesis. CONCLUSION: We identified BmKRP as a new transcription factor mediating 20E regulation of B. mori oogenesis. Our data suggests that induction of BmKRP by 20E regulates BmKr-h1 expression, which in turn induces BmVgR expression to facilitate Vg uptake and oogenesis.

RIC7 plays a negative role in ABA-induced stomatal closure by inhibiting H2O2 production.

When plants encounter environmental stresses, phytohormone abscisic acid (ABA) accumulates quickly and efficiently reduces water loss by inducing stomatal closure. Reactive oxygen species (ROS) is an important regulator in ABA-induced stomatal closure, and ROS generation is modulated by multiple components in guard-cell ABA signaling. ROP interactive CRIB-containing protein 7 (RIC7) has been found to negatively regulate ABA-induced stomatal closure. However, the molecular details of the RIC7 function in this process are unclear. Here, by using two RIC7 overexpressing mutants, we confirmed the negative role of RIC7 in ABA-induced stomatal closure and found that guard cells of RIC7 overexpressing mutants generated less H2O2 than the wild type with ABA treatment, which were consistent with the reduced expression levels of ROS generation related NADPH oxidase genes AtRBOHD and AtRBOHF, and cytosolic polyamine oxidase genes PAO1 and PAO5 in the RIC7 overexpressing mutants. Furthermore, external applied H2O2 failed to rescue the defects of stomatal closure in RIC7 overexpressing mutants. These results suggest that RIC7 affects H2O2 generation in guard cells, and the function of H2O2 is dependent on RIC7 in ABA-induced stomatal closure, indicative of interdependency between RIC7 and H2O2 in ABA guard-cell signaling.

Transcriptomic analysis reveals the regulation network of BmKrüppel homolog 1 in the oocyte development of Bombyx mori.

Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor, is involved in the metamorphosis and adult reproduction of insects. However, the role of Kr-h1 in reproduction of holometabolic insects remains to be elucidated. The regulation network of Kr-h1-associated genes in the reproduction in Bombyx mori was investigated in this study. The higher expression level of BmKr-h1 in the ovaries was detected during the late pupal stage and adults. RNA interference (RNAi)-mediated depletion of BmKr-h1 in the female at day 6 of pupae resulted in abnormal oocytes at 48 h post-double-stranded RNA treatment, which showed less yolk protein deposition and partially transparent chorion. RNA-seq and subsequent differentially expressed transcripts analysis showed that knockdown of BmKr-h1 caused a decrease in the expression of 2882 genes and an increase in the expression of 2565 genes in the oocytes at day 8 of pupae. Totally, 27 genes coding for transcription factors were down-regulated, while six genes coding for other transcription factors were up-regulated. BmKr-h1 bound to the Kr-h1 binding site of the transcription factors AP-1 (activating protein-1) and FOXG1 to increase their messenger RNA transcripts in the BmN cells, respectively. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses of that positively co-expressed with AP-1 and FOXG1 transcripts showed mainly enrichment in the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes. These data suggested that BmKr-h1 might directly regulate the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes or probably through AP-1 and /or FOXG1 to regulate oocyte development.

Bombyx mori transcription factors FoxA and SAGE divergently regulate the expression of wing cuticle protein gene 4 during metamorphosis.

Stage-specific gene expression governs metamorphosis of the silkworm, Bombyx mori. B. mori wing cuticle protein gene 4 (BmWCP4) is an essential gene for wing disc development expressed specifically during pupation. BmWCP4 transcription is suppressed at the larval stage by unknown mechanisms, which we sought to elucidate here. Bioinformatics analysis predicted seven potential Forkhead box (Fox) cis-regulatory elements (CREs) in the BmWCP4 promoter region, and we found that Fox CRE6 contributes to suppression of BmWCP4 expression. Electrophoretic mobility shift (EMSA) and DNA pull-down assays revealed that BmFoxA suppressed activity at the BmWCP4 promoter by specifically binding to the Fox CRE6. The expression level of BmFoxA in the wing discs was higher during the larval stage than at the pupal stage. In contrast, expression of another transcription factor, BmSAGE, increased over the course of development. Of note, the hormone 20-hydroxyecdysone (20E), which governs molting in insects, suppressed BmFoxA expression in the wing discs and up-regulated that of BmSage EMSA and cell co-transfection assays indicated that BmSAGE interacted with BmFoxA and suppressed its binding to the Fox CRE6, thereby releasing BmFoxA-mediated suppression of BmWCP4 In summary, higher BmFoxA expression during the larval stage suppresses BmWCP4 expression by binding to the Fox CRE6 on the BmWCP4 promoter. During metamorphosis, BmSAGE forms a complex with BmFoxA to relieve this repression, initiating BmWCP4 expression. Taken together, this study reveals a switchlike role for BmFoxA in regulating BmWCP4 expression and provides new insights into the regulatory regulation of wing disc development in insects.

Identification of the binding domains and key amino acids for the interaction of the transcription factors BmPOUM2 and BmAbd-A in Bombyx mori.

The transcription factor BmPOUM2 interacted with another transcription factor BmAbd-A to regulate the expression of the wing cuticle protein gene BmWCP4 in Bombyx mori. In this study, the binding domains and amino acids for the interaction between BmPOUM2 and BmAbd-A were reported. Two isoforms of BmPOUM2 were identified. The short isoform (BmPOUM2-S) lacks a 114-amino acid sequence containing a POU-homeodomain and a nuclear localization signal peptide (NLS), as compared to the full-length isoform (BmPOUM2). Both BmPOUM2 and BmPOUM2-S proteins bound to the BmAbd-A through the POU-specific domain. When the six amino acids (Lys166, Gly173, Gln176, Ser192, Glu200 and Asn208) that are highly conserved in POU family genes were mutated, BmPOUM2 did not bind to BmAbd-A. BmAbd-A interacted with BmPOUM2 by the homeobox domain or the LCR2 (low complexity region) domain. When seven amino acids (Phe156/248, His158/250, Ala175/263, Cys180/265, Glu190/268, Trp196/274 and Val214/289) that are shared in the homeobox and LCR2 domains were mutated, BmAbd-A did not bind to BmPOUM2. Overexpression of either BmPOUM2 or BmAbd-A or both increased the activity of BmWCP4 promoter in CHO cells. ChIP assay and EMSA showed that BmAbd-A protein bound to the Hox cis-regulatory element in the BmWCP4 promoter, while the BmPOUM2 bound to the nearby POU CRE. A model for the interaction and action of BmPOUM2 and BmAbd-A in regulation of the BmWCP4 expression is proposed.