mTORC2 acts as a gatekeeper for mTORC1 deficiency-mediated impairments in ILC3 development.

Group 3 innate lymphoid cells (ILC3s) are mediators of intestinal immunity and barrier function. Recent studies have investigated the role of the mammalian target of rapamycin complex (mTOR) in ILC3s, whereas the mTORC1-related mechanisms and crosstalk between mTORC1 and mTORC2 involved in regulating ILC3 homeostasis remain unknown. In this study, we found that mTORC1 but not mTORC2 was critical in ILC3 development, IL-22 production, and ILC3-mediated intestinal homeostasis. Single-cell RNA sequencing revealed that mTORC1 deficiency led to disruption of ILC3 heterogeneity, showing an increase in differentiation into ILC1-like phenotypes. Mechanistically, mTORC1 deficiency decreased the expression of NFIL3, which is a critical transcription factor responsible for ILC3 development. The activities of both mTORC1 and mTORC2 were increased in wild-type ILC3s after activation by IL-23, whereas inhibition of mTORC1 by Raptor deletion or rapamycin treatment resulted in increased mTORC2 activity. Previous studies have demonstrated that S6K, the main downstream target of mTORC1, can directly phosphorylate Rictor to dampen mTORC2 activity. Our data found that inhibition of mTORC1 activity by rapamycin reduced Rictor phosphorylation in ILC3s. Reversing the increased mTORC2 activity via heterozygous or homozygous knockout of Rictor in Raptor-deleted ILC3s resulted in severe ILC3 loss and complete susceptibility to intestinal infection in mice with mTORC1 deficiency (100% mortality). Thus, mTORC1 acts as a rheostat of ILC3 heterogeneity, and mTORC2 protects ILC3s from severe loss of cells and immune activity against intestinal infection when mTORC1 activity is diminished.

Reducing VEGFB expression regulates the balance of glucose and lipid metabolism in mice via VEGFR1.

In recent years, studies have demonstrated that vascular endothelial growth factor B (VEGFB) can affect the metabolism of fatty acids and glucose, and it is expected to become a target for the diagnosis and treatment of metabolic diseases such as obesity and diabetes. At present, the specific mechanism that VEGFB regulates lipid and glucose metabolism balance is not completely understood. The present study used systemic VEGFB gene­knockout mice to investigate the effects of downregulation of the VEGFB gene on lipid metabolism and insulin secretion, and to explore the mechanism of the VEGFB pathway involved in the regulation of glucose and lipid metabolism. The morphological changes in the liver and pancreas of mice after VEGFB gene deletion were observed under a light microscope and a scanning electron microscope, and the effects of VEGFB gene deletion on lipid metabolism and blood glucose balance were detected by a serological technique. The detection indexes included total cholesterol (TC), triglyceride (TG), low­density lipoprotein cholesterol (LDL­C) and high­density lipoprotein cholesterol. Simultaneously, fasting blood glucose, glycosylated hemoglobin A1c (HbA1c), fasting insulin and glucagon were measured. Insulin sensitivity was assessed by using the insulin tolerance tests and glucose tolerance tests, and function of ß­cell islets was evaluated by using the insulin resistance index (HOMA­IR) and pancreatic ß­cell secretion index (HOMA­ß). Τhe protein expression changes of vascular endothelial growth factor receptor 1 (VEGFR1) and vascular endothelial growth factor receptor 2 (VEGFR2) in mouse islets were detected by western blotting and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) after the VEGFB gene was knocked down to analyze the mechanism of VEGFB that may be involved in glucose and lipid metabolism. It was observed that after VEGFB was knocked down, mouse hepatocytes exhibited steatosis and increased secretory vesicles in islet cells. The lipid metabolism indexes such as TG, TC and LDL increased significantly; however, the levels of FBS, postprandial blood glucose and HbA1c decreased, whereas the glucose tolerance increased. Serum insulin secretion increased and HOMA­IR decreased since VEGFB was knocked down. Western blotting and RT­qPCR results revealed that the expression levels of VEGFR1 and neuropilin­1 decreased after the VEGFB gene was knocked down, while the expression levels of VEGFA and VEGFR2 increased. The absence of VEGFB may be involved in the regulation of glucose and lipid metabolism in mice by activating the VEGFA/VEGFR2 signaling pathway. VEGFB is expected to become a new target for the treatment of metabolic diseases such as obesity and diabetes. At present, the mechanism of VEGFB involved in regulating lipid metabolism and glucose metabolism is not completely clear. It was identified that downregulating VEGFB improved lipid metabolism and insulin resistance. The role of VEGFB/VEGFR1 pathway and other family members in regulating glucose and lipid metabolism was detected, which provided a theoretical and experimental basis for VEGFB to affect the regulation of glucose and lipid metabolism balance.

Insulin-Like Peptide and FoxO Mediate the Trehalose Catabolism Enhancement during the Diapause Termination Period in the Chinese Oak Silkworm (Antheraea pernyi).

In insects, trehalose accumulation is associated with the insulin/insulin-like growth factor signalling (IIS) pathway. However, whether insulin-like peptide is involved in the regulation of the trehalose metabolism during diapause termination remains largely unknown. This study assessed whether insulin-like peptide (ApILP) enhances the trehalose catabolism in the pupae of Antheraeapernyi during their diapause termination process. Injection of 10 µg of bovine insulin triggered diapause termination and synchronous adult eclosion in diapausing pupae. Moreover, treatment with bovine insulin increased the expression of trehalase 1A (ApTre-1A) and trehalase 2 (ApTre-2), as well as the activity of soluble and membrane-bound trehalase, resulting in a decline in trehalose levels in the haemolymph. Silencing ApILP via RNA interference significantly suppressed the expression of ApTre-1A and ApTre-2, thus leading to an increase in the trehalose concentration during diapause termination. However, neither injection with bovine insulin nor ApILP knockdown directly affected trehalase 1B (ApTre-1B) expression. Moreover, overexpression of the transcription factor forkhead box O (ApFoxO) induced an increase in trehalose levels during diapause termination; however, depletion of ApFoxO accelerated the breakdown of trehalose in diapausing pupae by increasing the expression of ApTre-1A and ApTre-2. The results of this study help to understand the contributions of ApILP and ApFoxO to the trehalose metabolism during diapause termination.

Functional and Antioxidant Properties of Plastic Bottle Caps Incorporated with BHA or BHT.

In this study, we prepared new antioxidant active plastic bottle caps by incorporating butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) and 2% (w/w) white masterbatch in high-density polyethylene (HDPE). Fourier-transform infrared (FT-IR) spectrometry revealed that the antioxidants and HDPE were uniformly mixed with noncovalent bonding. In addition, the differential scanning calorimetry (DSC) test revealed that the change in melting point and initial extrapolation temperature of the antioxidant active caps was not significant. Sensory evaluation and removal torque tests validated the suitability of the antioxidant active plastic bottle caps in industrial application. The antioxidant activity increased with a greater concentration of BHA and BHT incorporated in both antioxidant active caps (p < 0.05) and with more impact on the BHA cap compared to BHT cap in terms of antioxidant activity. Migration experiments for 10 days at 40 °C and 2 h at 70 °C showed that active antioxidants in the plastic bottle cap were more easily released into fatty foods and milk products that are highly sensitive to oxidation, and the migration of BHA and BHT did not exceed the maximum amount specified in (EC) No 1333/2008 (<200 mg/kg). As such, the antioxidant active plastic bottle caps inhibited oxidation, thereby ensuring higher food quality.

Vascular endothelial growth factor B inhibits insulin secretion in MIN6 cells and reduces Ca2+ and cyclic adenosine monophosphate levels through PI3K/AKT pathway.

BACKGROUND: Type 2 diabetes (T2D) is characterized by insufficient insulin secretion caused by defective pancreatic ß-cell function or insulin resistance, resulting in an increase in blood glucose. However, the mechanism involved in this lack of insulin secretion is unclear. The level of vascular endothelial growth factor B (VEGF-B) is significantly increased in T2D patients. The inactivation of VEGF-B could restore insulin sensitivity in db/db mice by reducing fatty acid accumulation. It is speculated that VEGF-B is related to pancreatic ß-cell dysfunction and is an important factor affecting ß-cell secretion of insulin. As an in vitro model of normal pancreatic ß-cells, the MIN6 cell line can be used to analyze the mechanism of insulin secretion and related biological effects. AIM: To study the role of VEGF-B in the insulin secretion signaling pathway in MIN6 cells and explore the effect of VEGF-B on blood glucose regulation. METHODS: The MIN6 mouse pancreatic islet ß-cell line was used as the model system. By administering exogenous VEGF-B protein or knocking down VEGF-B expression in MIN6 cells, we examined the effects of VEGF-B on insulin secretion, Ca2+ and cyclic adenosine monophosphate (cAMP) levels, and the insulin secretion signaling pathway. RESULTS: Exogenous VEGF-B inhibited the secretion of insulin and simultaneously reduced the levels of Ca2+ and cAMP in MIN6 cells. Exogenous VEGF-B also reduced the expression of phospholipase C gamma 1 (PLCγ1), phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase (AKT), and other proteins in the insulin secretion pathway. Upon knockdown of VEGF-B, MIN6 cells exhibited increased insulin secretion and Ca2+ and cAMP levels and upregulated expression of PLCγ1, PI3K, AKT, and other proteins. CONCLUSION: VEGF-B can regulate insulin secretion by modulating the levels of Ca2+ and cAMP. VEGF-B involvement in insulin secretion is related to the expression of PLCγ1, PI3K, AKT, and other signaling proteins. These results provide theoretical support and an experimental basis for the study of VEGF-B in the pathogenesis of T2D.

Polycyclic polyprenylated acylphloroglucinol derivatives with neuroprotective effects from Hypericum monogynum.

A new polycyclic polyprenylated acylphloroglucinol (PPAP), hypermonin C (1), along with nine known PPAPs (2-10) were obtained from the leaves and twigs of Hypericum monogynum. The structures of the isolates were determined on the basis of extensive spectroscopic analysis. The neuroprotective effects of the isolates against several chemical-induced injuries in SH-SY5Y and PC12 cells were assessed, and most of the compounds exhibited significant protective effects at 10 µg/ml. Especially, three compounds (1, 3, and 7) showed excellent neuroprotective activity with a cell viability of 92.4% ∼ 95.8% in KCl-induced SH-SY5Y cell injury. Their preliminary structure-activity relationship was also discussed and the configuration of substituent in furohyperforin may be critical for the neuroprotective activity of PPAP derivatives.

The Modulation of Trehalose Metabolism by 20-Hydroxyecdysone in Antheraea pernyi (Lepidoptera: Saturniidae) During its Diapause Termination and Post-Termination Period.

Trehalose plays a crucial role in the diapause process of many insects, serving as an energy source and a stress protectant. Trehalose accumulation has been reported in diapause pupae of Antheraea pernyi; however, trehalose metabolic regulatory mechanisms associated with diapause termination remain unclear. Here, we showed that the enhanced trehalose catabolism was associated with an increase in endogenous 20-hydroxyecdysone (20E) in hemolymph of A. pernyi pupae during their diapause termination and posttermination period. Injection of 20E increased the mRNA level of trehalase 1A (ApTre-1A) and trehalase 2 (ApTre-2) of A. pernyi diapause pupae in a dose-dependent manner but did not affect the mRNA level of trehalase 1B (ApTre-1B). Meanwhile, exogenous 20E increased the enzyme activities of soluble and membrane-bound trehalase, leading to a decline in hemolymph trehalose. Conversely, the expression of ApTre-1A and ApTre-2 were down-regulated after the ecdysone receptor gene (ApEcRB1) was silenced by RNA interference or by injection of an ecdysone receptor antagonist cucurbitacin B (CucB), which inhibits the 20E pathway. Moreover, CucB treatment delayed adult emergence, which suggests that ApEcRB1 might be involved in regulating pupal-adult development of A. pernyi by mediating ApTre-1A and ApTre-2 expressions. This study provides an overview of the changes in the expression and activity of different trehalase enzymes in A. pernyi in response to 20E, confirming the important role of 20E in controlling trehalose catabolism during A. pernyi diapause termination and posttermination period.

Dual Transcriptomic Analysis Reveals a Delayed Antiviral Response of Haliotis diversicolor supertexta against Haliotid Herpesvirus-1.

Haliotid herpesvirus-1 (HaHV-1) is the first identified gastropod herpesvirus, causing a highly lethal neurologic disease of abalone species. The genome of HaHV-1 has been sequenced, but the functions of the putative genes and their roles during infection are still poorly understood. In the present study, transcriptomic profiles of Haliotis diversicolor supertexta at 0, 24 and 60 h post injection (hpi) with HaHV-1 were characterized through high-throughput RNA sequencing. A total of 448 M raw reads were obtained and assembled into 2.08 × 105 unigenes with a mean length of 1486 bp and an N50 of 2455 bp. Although we detected increased HaHV-1 DNA loads and active viral expression at 24 hpi, this evidence was not linked to significant changes of host transcriptomic profiles between 0 and 24 hpi, whereas a rich immune-related gene set was over-expressed at 60 hpi. These results indicate that, at least at the beginning of HaHV-1 infection, the virus can replicate with no activation of the host immune response. We propose that HaHV-1 may evolve more effective strategies to modulate the host immune response and hide during replication, so that it could evade the immune surveillance at the early stage of infection.

Xiao-Xu-Ming Decoction Reduced Mitophagy Activation and Improved Mitochondrial Function in Cerebral Ischemia and Reperfusion Injury.

We investigated whether Xiao-Xu-Ming decoction reduced mitophagy activation and kept mitochondrial function in cerebral ischemia-reperfusion injury. Rats were randomly divided into 5 groups: sham, ischemia and reperfusion (IR), IR plus XXMD (60 g/kg/day) (XXMD60), IR plus cyclosporin A (10 mg/kg/day) (CsA), and IR plus vehicle (Vehicle). Focal cerebral ischemia and reperfusion models were induced by middle cerebral artery occlusion (MCAO). Cerebral infarct areas were measured by triphenyl tetrazolium chloride staining. Cerebral ischemic injury was evaluated by hematoxylin and eosin staining (HE) and Nissl staining. Ultrastructural features of mitochondria and mitophagy in the penumbra of the ischemic cortex were observed by transmission electron microscopy. Mitophagy was detected by immunofluorescence labeled with LC3B and VDAC1. Autophagy lysosome formation was observed by immunofluorescence labeled with LC3B and Lamp1. The expression of LC3B, Beclin1, and Lamp1 was analyzed by Western blot. The rats subjected to MCAO showed worsened neurological score and cell ischemic damage. These were all significantly reversed by XXMD or CsA. Moreover, XXMD/CsA notably downregulated mitophagy and reduced the increase in LC3, Beclin1, and Lamp1 expression induced by cerebral ischemia and reperfusion. The findings demonstrated that XXMD exerted neuroprotective effect via downregulating LC3, Beclin1, Lamp1, and mitochondrial p62 expression level, thus leading to the inhibition of mitophagy.

Development of antibacterial ε-polylysine/chitosan hybrid films and the effect on citrus.

In this study, the ε-polylysine (PL)/chitosan (CS) composite films with the PL concentration of 0%, 1%, 3%, 5% and 7% were prepared via casting method. The Fourier transform infrared (FTIR), scanning electron microscope (SEM), mechanical and antibacterial properties of PL/CS composite films, as well as the effect of PL/CS coatings on citrus were observed. FTIR spectra suggested that there was no strong hydrogen bond between CS and PL. SEM images indicated that the film surface was smooth and the addition of PL to chitosan lead to more holes. The tensile strength was deceasing and the elongation at break of the films was elevating with the increase of PL content. The antibacterial activity against Staphylococcus aureus and Escherichia coli of PL/CS composite films was enhanced with increasing of PL content. Furthermore, it was found that the PL/CS coatings inhibited efficiently the decline of total soluble solids (TSS), ascorbic acid content and reduced the disease incidence of citrus, compared with the control. Also the disease incidence of citrus was decreasing with the increase of PL content. However, the weight loss did not show significant differences between the coated citrus and control during the whole storage period.

Mitophagy is activated in brain damage induced by cerebral ischemia and reperfusion via the PINK1/Parkin/p62 signalling pathway.

This study examined the course of mitophagy following cerebral ischemia with reperfusion and the role of the PTEN-induced kinase 1 (PINK1)/Parkin/p62 signalling pathway. The middle cerebral artery of male Sprague-Dawley rats was occluded for 90 min and was followed by different time-points of reperfusion. Cerebral infarct areas were detected by 2,3,5-triphenyl tetrazolium chloride staining, while brain damage was observed by haematoxylin and eosin staining. Levels of LC3, Beclin1 and LAMP-1 were estimated by western blots. LC3B location was observed in various cells in the neurovascular unit. In addition, PINK1 accumulation in damaged mitochondria and Parkin/p62 mitochondrial translocation were investigated by double immunofluorescence staining. Finally, the levels of PINK1, Parkin and p62 expression in mitochondrial fractions were estimated by western blots. Cerebral ischemia with different time-points of reperfusion resulted in infarct in the territory of the middle cerebral artery accompanied by overall brain damage. In addition, we found up-regulation of LC3B, Beclin1, and LAMP-1, as well as mitophagy activation after reperfusion, with peak expression of these proteins at 24 h after reperfusion. Electron microscopy and immunofluorescence indicated that LC3B was primarily located in neurons, although lower levels of expression were found in astrocytes and even less in vascular endothelial cells. Moreover, significant increases in PINK1 accumulation in the outer membrane of mitochondria and increased Parkin/p62 mitochondrial translocation were shown at 24 h after reperfusion. These findings suggest that the PINK1/Parkin/p62 signalling pathway was involved in the pathophysiological processes following ischemia and reperfusion.

Retinoic acid amide inhibits JAK/STAT pathway in lung cancer which leads to apoptosis.

Small cell lung cancer (SCLC) accounts for 12 to 16% of lung neoplasms and has a high rate of metastasis. The present study demonstrates the antiproliferative effect of retinoic acid amide in vitro and in vivo against human lung cancer cells. The results from MTT assay showed a significant growth inhibition of six tested lung cancer cell lines and inhibition of clonogenic growth at 30 µM. Retinoic acid amide also leads to G2/M-phase cell cycle arrest and apoptosis of lung cancer cells. It caused inhibition of JAK2, STAT3, and STAT5, increased the level of p21WAF1, and decreased cyclin A, cyclin B1, and Bcl-XL expression. Retinoic acid amide exhibited a synergistic effect on antiproliferative effects of methotrexate in lung cancer cells. In lung tumor xenografts, the tumor volume was decreased by 82.4% compared to controls. The retinoic acid amide-treated tumors showed inhibition of JAK2/STAT3 activation and Bcl-XL expression. There was also increase in expression of caspase-3 and caspase-9 in tumors on treatment with retinoic acid amide. Thus, retinoic acid amide exhibits promising antiproliferative effects against human lung cancer cells in vitro and in vivo and enhances the antiproliferative effect of methotrexate.

Involvement of connexin43 in the acute hyperosmotic stimulus­induced synthesis and release of vasopressin in the supraoptic nucleus of rats.

There is evidence that astroglial connexin43 (Cx43) in the supraoptic nucleus (SON) is required for the hyperosmolarity­induced increase in Fos protein expression in magnocellular neurosecretory cells (MNCs). In the present study, the role of astroglial Cx43 in the synthesis and release of vasopressin (VP) by MNCs in the SON subjected to hyperosmotic stimulus was examined. The results revealed that the VP levels in the SON and plasma were increased following acute hyperosmotic stimulus. Treatment of MNCs with Cx43­specific antisense oligodeoxynucleotides (ASODN), which temporarily reduced Cx43 protein production, limited the VP synthesis and release induced by a hyperosmotic stimulus. Similarly, the addition of gap junction and Cx43 hemichannel blockers also attenuated the VP synthesis and release induced by an acute hyperosmotic stimulus. A high extracellular [Ca2+]([Ca2+]o) has been demonstrated to reduce the gap junction activity or opening probability of Cx54 hemichannels. Notably, it was identified that high [Ca2+]o attenuated the VP synthesis and release induced by acute hyperosmotic stimulus, while low [Ca2+]o had a weak or no effect. These results suggested that Cx43 participates in the VP synthesis and release induced by hyperosmotic stimulation in the SON.

Involvement of connexin43 in the infrasonic noise-induced glutamate release by cultured astrocytes.

Infrasonic noise/infrasound is a type of environmental noise that threatens public health as a nonspecific biological stressor. Glutamate-related excitotoxicity is thought to be responsible for infrasound-induced impairment of learning and memory. In addition to neurons, astrocytes are also capable of releasing glutamate. In the present study, to identify the effect of infrasound on astroglial glutamate release, cultured astrocytes were exposed to infrasound at 16 Hz, 130 dB for different times. We found that infrasound exposure caused a significant increase in glutamate levels in the extracellular fluid. Moreover, blocking the connexin43 (Cx43) hemichannel or gap junction, decreasing the probability of Cx43 being open or inhibiting of Cx43 expression blocked this increase. The results suggest that glutamate release by Cx43 hemichannels/gap junctions is involved in the response of cultured astrocytes to infrasound.

[Cumulation and effect of chemokines and CD40L during blood storage--review].

Chemokine is a class of soluble active peptides that attract white blood cells to the inflammatory site. CD40 ligand (CD40L) involves in synthesis of proinflammatory mediators. Accumulation of chemokine and CD40L can induce non-hemolytic reaction after transfusion, transfusion-related acute lung injury (TRALI) and autoimmune disease during blood component storage. Pre-storage leucocyte deletion can prevent the release of leucocyte-derived chemokines, but not prevent the accumulation of platelet-derived chemokines. γ-irradiation or ultraviolet ß irradiation is effective in preventing the increase of chemokines in the storage of platelet, thus prevents non-hemolytic febrile reaction after platelet transfusion. In this review, the recent advance in research of accumulation of chemokines and CD40L during blood component storage, and its effect on blood transfusion, as well as preventive measures are summarized.

[Transfusion-associated microchimerism -- review].

Blood transfusion is a newly recognized cause of microchimerism, it seems to be common in severe traumatic injuries. In this review, the frequency, cause and prevention of transfusion-associated microchimerism (TA-MC), its current progress of knowledge and unanswered questions were summarized. In addition, the pregnancy-associated microchimerism and transplantation-associated microchimerism were discussed in this review.

Down-regulation of apoptosis-inducing factor protein by RNA interference inhibits UVA-induced cell death.

Apoptosis-inducing factor (AIF) is a caspase-independent apoptosis effector. UVA-induced Raji cell death was not completely inhibited by pan-caspase inhibitor zVAD.fmk. Moreover, AIF translocated from its normal location, the mitochondrial intermembrane space, into the nucleus, and induced peripheral chromatin condensation during the early stage of UVA-inducing cell death. Enforced expression of AIF can induce Raji cell death in a caspase-independent manner. Down-regulation of AIF protein level by RNA interference (RNAi) can reduce UVA-induced Raji cell death, but the combination of down-regulation of AIF and zVAD.fmk almost completely inhibited UVA-induced Raji cell death. All these suggest that caspase and AIF are two independent pathways and that UVA-induced Raji cell death is dependent on caspase and AIF.