Fluorescent indicators for live-cell and in vitro detection of inorganic cadmium dynamics.

Cadmium contamination is a severe threat to the environment and food safety. Thus, there is an urgent need to develop highly sensitive and selective cadmium detection tools. The engineered fluorescent indicator is a powerful tool for the rapid detection of inorganic cadmium in the environment. In this study, the development of yellow fluorescent indicators of cadmium chloride by inserting a fluorescent protein at different positions of the high cadmium-specific repressor and optimizing the flexible linker between the connection points is reported. These indicators provide a fast, sensitive, specific, high dynamic range, and real-time readout of cadmium ion dynamics in solution. The excitation and emission wavelength of this indicator used in this work are 420/485 and 528 nm, respectively. Fluorescent indicators N0C0/N1C1 showed a linear response to cadmium concentration within the range from 10/30 to 50/100 nM and with a detection limit of 10/33 nM under optimal condition. Escherichia coli cells containing the indicator were used to further study the response of cadmium ion concentration in living cells. E. coli N1C1 could respond to different concentrations of cadmium ions. This study provides a rapid and straightforward method for cadmium ion detection in vitro and the potential for biological imaging.

Construction of luminescent Escherichia coli via expressing lux operons and their application on toxicity test.

Recombinant luminescent Escherichia coli strains could be used to detect the toxicity of pure or mixed contaminants as a light-off sensor. In this work, the lux operon of Photobacterium phosphoreum T3 was identified for the first time. Recombinant luminescent E. coli strains were constructed via expressing the lux operons of P. phosphoreum T3 and Vibrio qinghaiensis Q67 in E. coli MG1655, and the optimal protectant containing 10% (w/v) trehalose and 4% sucrose was used to prepare the freeze-dried recombinant luminescent E. coli cells. Then, these freeze-dried E. coli cells were subjected to acute toxicity detection. The results showed that luminescent E. coli strains displayed sensitive toxic responses to BPA, nFe2O3, Cd, Pb, As, and Hg, for example, the EC50 values of BPA and nFe2O3 to luminescent E. coli strains ranged from 1.54 to 50.19 mg/l and 17.50 to 21.52 mg/l, respectively. Indeed, luminescent E. coli strains exhibited more sensitive responses to Cd, Pb, and Hg than the natural strain Q67. The results suggested that recombinant luminescent E. coli strains could be used for the detection of acute toxicity. Furthermore, the combined toxicities of BPA and nFe2O3, Hg, and Pb were measured, and the joint effects of these mixtures were evaluated with luminescent E. coli. The results indicated that the joint effects of BPA and nFe2O3 suggested to be synergistic or additive to luminescent E. coli, while the joint effects of heavy metals and nFe2O3 exhibited additivities. The cellular endocytosis for Fe2O3 nanoparticles was not observed, which could explain the additive instead of synergistic effects between heavy metals and nFe2O3. KEY POINTS: • Sequence of the lux operon from P. phosphoreum T3 was reported for the first time. • Recombinant luminescent E. coli was more sensitive to Cd, Pb, and Hg than Q67. • Joint effects of BPA and nFe2O3 were synergistic or additive to luminescent E. coli.

Dendrobium nobile-derived polysaccharides stimulate the glycolytic pathway by activating SIRT2 to regulate insulin resistance in polycystic ovary syndrome granulosa cells.

Insulin resistance (IR) is one of the major complications of polycystic ovary syndrome (PCOS). This study aimed to investigate the effects and the molecular regulatory mechanism by which Dendrobium nobile-derived polysaccharides (DNP) improve IR in rats with letrozole and high-fat-diet induced PCOS. In vivo, DNP (200 mg/kg/d) administration not only reduced body weight, blood glucose, and insulin levels in PCOS rats, but also improve the disrupted estrous cycle. In addition, DNP treatment reduced atretic and cystic follicles and enhanced granulosa cell layer thickness, thereby restoring follicle development. In vitro, DNP treatment (100 µM) increased lactate levels and decreased pyruvate levels in insulin-treated (8 µg/mL) KGN cells. Additionally, DNP also decreased the expression of IGF1 and increased that of IGF1R, SIRT2, LDHA, PKM2 and HK2 both in vivo and in vitro. Also, SIRT2 expression was specifically inhibited by AGK2, while DNP significantly improved IR and glycolysis by reversing the effect of AGK2 treatment on lactate and pyruvate production, upregulating the expression levels of IGF1R, LDHA, HK2, and PKM2 and downregulating the expression level of IGF1. The results indicate that DNP can effectively improve IR and restore glycolytic pathway by activating SIRT2, which may provide a potential therapeutic approach for PCOS patients.

Napabucasin deactivates STAT3 and promotes mitoxantrone-mediated cGAS-STING activation for hepatocellular carcinoma chemo-immunotherapy.

The immune resistance of tumor microenvironment (TME) causes immune checkpoint blockade therapy inefficient to hepatocellular carcinoma (HCC). Emerging strategies of using chemotherapy regimens to reverse the immune resistance provide the promise for promoting the efficiency of immune checkpoint inhibitors. The induction of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) in tumor cells evokes the adaptive immunity and remodels the immunosuppressive TME. In this study, we report that mitoxantrone (MIT, a chemotherapeutic drug) activates the cGAS-STING signaling pathway of HCC cells. We provide an approach to augment the efficacy of MIT using a signal transducer and activator of transcription 3 (STAT3) inhibitor called napabucasin (NAP). We prepare an aminoethyl anisamide (AEAA)-targeted polyethylene glycol (PEG)-modified poly (lactic-co-glycolic acid) (PLGA)-based nanocarrier for co-delivery of MIT and NAP. The resultant co-nanoformulation can elicit the cGAS-STING-based immune responses to reshape the immunoresistant TME in the mice orthotopically grafted with HCC. Consequently, the resultant co-nanoformulation can promote anti-PD-1 antibody for suppressing HCC development, generating long-term survival, and inhibiting tumor recurrence. This study reveals the potential of MIT to activate the cGAS-STING signaling pathway, and confirms the feasibility of nano co-delivery for MIT and NAP on achieving HCC chemo-immunotherapy.

A tetramethylpyrazine-loaded hyaluronic acid-based hydrogel modulates macrophage polarization for promoting wound recovery in diabetic mice.

The failure of wound healing often causes lower limb disability and amputation of diabetic patients. Current strategies for diabetic wound management often fail to achieve the expected outcomes, and emerging alternatives are urgently needed. Recent advances in the identification of active compounds from traditional herbal medicines provide promising therapeutics for tissue repair and regeneration. In this study, the pro-healing effects of tetramethylpyrazine (TMP, a natural alkaloid found in Ligusticum chuanxiong Hort) for diabetic wounds were for the first time demonstrated. The cutaneous healing was mainly achieved by TMP-mediated macrophage polarization from pro-inflammatory to pro-healing phenotype. In addition, the topical administration of TMP was facilitated by the hyaluronic acid (HA) hydrogel for promoting the full-thickness wounds in the experimental diabetic mice. Consequently, TMP-loaded HA hydrogel (TMP-HA) profoundly accelerated the wound closure in comparison with TMP-loaded INTRASITE Gel (it is a commercial hydrogel), which was evident with the inflammation mitigation, the angiogenesis enhancement, and the collagen deposition. Our work reveals the macrophage-modulatory function of TMP for diabetic wound healing and demonstrates great potential of TMP-HA for clinical application.

Nanodelivery of scutellarin induces immunogenic cell death for treating hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) causes the immunosuppressive tumor microenvironment (TME) resistant to current immunotherapy. The immunogenic apoptosis (currently termed immunogenic cell death, ICD) of cancer cells may induce the adaptive immunity against tumors, thereby providing great potential for treating HCC. In this study, we have confirmed the potential of scutellarin (SCU, a flavonoid found in Erigeron breviscapus) for triggering ICD in HCC cells. To facilitate in vivo application of SCU for HCC immunotherapy, an aminoethyl anisamide-targeted polyethylene glycol-modified poly(lactide-co-glycolide) (PLGA-PEG-AEAA) was produced to facilitate SCU delivery in this study. The resultant nanoformulation (PLGA-PEG-AEAA.SCU) remarkably promoted blood circulation and tumor delivery in the orthotopic HCC mouse model. Consequently, PLGA-PEG-AEAA.SCU reversed the immune suppressive TME and achieved the immunotherapeutic efficacy, resulting in significantly longer survival of mice, without inducing toxicity. These findings uncover the ICD potential of SCU and provide a promising strategy for HCC immunotherapy.

d-lactate modulates M2 tumor-associated macrophages and remodels immunosuppressive tumor microenvironment for hepatocellular carcinoma.

The polarization of tumor-associated macrophages (TAMs) from M2 to M1 phenotype demonstrates great potential for remodeling the immunosuppressive tumor microenvironment (TME) of hepatocellular carcinoma (HCC). d-lactate (DL; a gut microbiome metabolite) acts as an endogenous immunomodulatory agent that enhances Kupffer cells for clearance of pathogens. In this study, the potential of DL for transformation of M2 TAMs to M1 was confirmed, and the mechanisms underlying such polarization were mainly due to the modulation of phosphatidylinositol 3-kinase/protein kinase B pathway. A poly(lactide-co-glycolide) nanoparticle (NP) was used to load DL, and the DL-loaded NP was modified with HCC membrane and M2 macrophage-binding peptide (M2pep), forming a nanoformulation (DL@NP-M-M2pep). DL@NP-M-M2pep transformed M2 TAMs to M1 and remodeled the immunosuppressive TME in HCC mice, promoting the efficacy of anti-CD47 antibody for long-term animal survival. These findings reveal a potential TAM modulatory function of DL and provide a combinatorial strategy for HCC immunotherapy.

Acute and Chronic Toxicity of Binary Mixtures of Bisphenol A and Heavy Metals.

Bisphenol A (BPA) and heavy metals are widespread contaminants in the environment. However, the combined toxicities of these contaminants are still unknown. In this study, the bioluminescent bacteria Vibrio qinghaiensis Q67 was used to detect the single and combined toxicities of BPA and heavy metals, then the joint effects of these contaminants were evaluated. The results show that chronic toxicities of chromium (Cr), cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg), nickel (Ni), and BPA were time−dependent; in fact, the acute toxicities of these contaminants were stronger than the chronic toxicities. Furthermore, the combined toxicities of BPA and heavy metals displayed BPA + Hg > BPA + Cr > BPA + As > BPA + Ni > BPA + Pb > BPA + Cd in the acute test and BPA + Hg > BPA + Cd > BPA + As > BPA + Cd in the chronic test, which suggested that the combined toxicity of BPA and Hg was stronger than that of other mixtures in acute as well as chronic tests. Additionally, both CA and IA models underestimated the toxicities of mixtures at low concentrations but overestimated them at high concentrations, which indicates that CA and IA models were not suitable to predict the toxicities of mixtures of BPA and heavy metals. Moreover, the joint effects of BPA and heavy metals mainly showed antagonism and additive in the context of acute exposure but synergism and additive in the context of chronic exposure. Indeed, the difference in the joint effects on acute and chronic exposure can be explained by the possibility that mixtures inhibited cell growth and luminescence in chronic cultivation. The chronic toxicity of the mixture should be considered if the mixture results in the inhibition of the growth of cells.

Preparation of freeze-dried bioluminescent bacteria and their application in the detection of acute toxicity of bisphenol A and heavy metals.

Current chemical analysis approaches for contaminants have failed to reveal their biotoxicity. Moreover, conventional bioassays are time consuming and exhibit poor repeatability. In this study, we performed the acute toxicity detection of various contaminants (chromium (Cr), cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg), tin (Sn), nickel (Ni), and bisphenol A (BPA)) with four bioluminescent bacteria (Vibrio qinghaiensis Q67, V. fischeri, Photobacterium phosphoreum T3, and P. phosphoreum 502) using a rapid, flexible, and low-cost bioassay. We found that the temperature affected the bacterial luminescence, and freeze-dried cells exhibited sensitive toxic responses to contaminants. Indeed, the optimized protectants containing 12% (w/v) trehalose, 4% sucrose, and 2% sorbitol displayed better luminescence and toxic sensitivity. Furthermore, freeze-dried powders of these strains were prepared and subjected to acute toxicity detection. The results showed that all contaminants exhibited acute toxicity toward Q67, but the other strains did not show obvious response to nickel and tin. The relative half-maximal effective concentration (EC50) values of BPA, Cr, Cd, Pb, As, Hg, Ni, and Sn to Q67 were 0.674, 1.313, 11.137, 5.921, 4.674, 0.911, 5.941, and 54.077 mg/L, respectively. In addition, the EC50 values of contaminants toward different strains were suggested to be statistically significant. Freeze-dried Q67 exhibited toxic responses to more contaminants than the other bioluminescent strains; therefore, Q67 was selected to be more suitable than the other strains for single and mixture toxicity detection tests. Compared with other strains, Q67 was more appropriate for the rapid screening of the mixture toxicity of contaminants in samples as a nonspecific screening sensor before the use of standard analysis approaches.

Local Administration of Ginkgolide B Using a Hyaluronan-Based Hydrogel Improves Wound Healing in Diabetic Mice.

The delayed and incomplete healing of diabetic wounds remains a major concern of global healthcare. The complex biological processes within the diabetic wound, such as chronic inflammation, impaired blood vessel growth and immature collagen remodeling, dramatically cause the failure of current treatments. Thus, emerging therapeutic strategies are highly desirable. Ginkgolide B (GB, a natural product extracted from the leaves of Ginkgo biloba L.) has been applied in the treatment of cerebrovascular and cardiovascular disorders, which is mainly due to the anti-oxidative, anti-inflammatory and proliferative effects. In this study, the role of GB in facilitating the anti-inflammatory and pro-healing effects on diabetic wounds was for the first time confirmed using in vitro, ex vivo and in vivo experimental methods. As a consequence, GB was able to significantly achieve the activities of anti-inflammation, re-epithelialization, and pro-angiogenesis. Previously, a hydrogel has been developed using the high molecular weight hyaluronan (hyaluronic acid, HA) in our laboratory. In this study, this hydrogel was utilized in vivo for local administration of GB to the full-thickness wounds of diabetic mice. The resultant hydrogel formulation (HA-GB) resulted in the reduction of inflammation, the enhancement of re-epithelialization and angiogenesis, and the modulation of collagens from type III to type I, significantly promoting the healing outcome as compared with a commercially available wound dressing product (INTRASITE Gel). This study confirms a great therapeutic promise of HA-GB for the chronic wounds of diabetic patients.

Nano co-delivery of Plumbagin and Dihydrotanshinone I reverses immunosuppressive TME of liver cancer.

Hepatocellular carcinoma (HCC) is resistant to current immunotherapy. This poor outcome mainly results from the immunosuppressive characteristics of tumor microenvironment (TME). Accumulating evidence indicates that some chemotherapy agents trigger immunogenic cell death (ICD), providing a promising strategy to remodel the immunosuppressive TME. The role of Plumbagin (PLB, a naphthoquinone compound from Plumbago zeylanica L.) as the ICD inducer for HCC cells was confirmed in this study. Dihydrotanshinone I (DIH, a phenanthraquinone compound of Salvia miltiorrhiza) functioned as the ICD enhancer by generating the reactive oxygen species (ROS). A poly(D,L-lactic-co-glycolic acid) (PLGA)-based nanoparticle (NP) was used to co-encapsulate PLB, DIH and NH4HCO3 (a pH sensitive adjuvant). This NP was further coated with the mannose-inserted erythrocyte membrane to produce a nanoformulation. This nanoformulation significantly increased the half-life and tumor targeting of two drugs in orthotopic HCC mice, generating chemo-immunotherapeutic effects for reversal of immunosuppressive TME. Consequently, the biomimetic nanoformulation loaded with low doses of PLB and DIH achieved significantly longer survival of HCC mice, without causing toxic signs. Our study demonstrates a promising strategy for remodeling the immunosuppressive TME of liver cancer.

The role of adiponectin in type 2 diabetes-related periodontitis / 口腔疾病防治

@#Adiponectin, an adipocytokine secreted by adipocytes, has emerged as a potential treatment agent for type 2 diabetes. Adiponectin plays a variety of physiological roles in regulating glucolipid metabolism, oxidative stress, inflammatory responses and bone metabolism by binding to its receptors expressed on a variety of cells and tissues. Numerous studies have confirmed the strong association of adiponectin with type 2 diabetes-related periodontitis. Adiponectin can improve systemic insulin resistance by increasing insulin sensitivity and promoting insulin secretion. It improves the periodontal inflammatory response by inhibiting the expression of proinflammatory cytokines induced by Porphyromonas gingivalis lipopolysaccharide and promoting M2-type polarization of macrophages. In addition, adiponectin inhibits osteoclast differentiation and maturation through various pathways, such as Wnt/β-catenin and NF-κ, and promotes osteoblast differentiation to regulate bone metabolism, thus improving periodontal bone resorption and destruction. Therefore, adiponectin is expected to become a therapeutic target for type 2 diabetes-related periodontitis. Due to the physiological characteristics of adiponectin, its clinical application has been somewhat limited. This article reviews the latest research progress on adiponectin in type 2 diabetes-related periodontitis, aiming to elucidate the possible effects of adiponectin on type 2 diabetes-related periodontitis in terms of glycemic control, anti-inflammation and bone metabolism and to provide some opinions on the treatment of this disease and the development of relevant drugs.