Acute toxicity of binary mixtures for quorum sensing inhibitors and sulfonamides against Aliivibrio fischeri: QSAR investigations and joint toxic actions.

Quorum sensing inhibitors (QSIs), as a kind of ideal antibiotic substitutes, have been recommended to be used in combination with traditional antibiotics in medical and aquaculture fields. Due to the co-existence of QSIs and antibiotics in environmental media, it is necessary to evaluate their joint risk. However, there is little information about the acute toxicity of mixtures for QSIs and antibiotics. In this study, 10 QSIs and 3 sulfonamides (SAs, as the representatives for traditional antibiotics) were selected as the test chemicals, and their acute toxic effects were determined using the bioluminescence of Aliivibrio fischeri (A. fischeri) as the endpoint. The results indicated that SAs and QSIs all induced S-shaped dose-responses in A. fischeri bioluminescence. Furthermore, SAs possessed greater acute toxicity than QSIs, and luciferase (Luc) might be the target protein of test chemicals. Based on the median effective concentration (EC50) for each test chemical, QSI-SA mixtures were designed according to equitoxic (EC50(QSI):EC50(SA) = 1:1) and non-equitoxic ratios (EC50(QSI):EC50(SA) = 1:10, 1:5, 1:0.2, and 1:0.1). It could be observed that with the increase of QSI proportion, the acute toxicity of QSI-SA mixtures enhanced while the corresponding TU values decreased. Furthermore, QSIs contributed more to the acute toxicity of test binary mixtures. The joint toxic actions of QSIs and SAs were synergism for 23 mixtures, antagonism for 12 mixtures, and addition for 1 mixture. Quantitative structure-activity relationship (QSAR) models for the acute toxicity QSIs, SAs, and their binary mixtures were then constructed based on the lowest CDOCKER interaction energy (Ebind-Luc) between Luc and each chemical and the component proportion in the mixture. These models exhibited good robustness and predictive ability in evaluating the toxicity data and joint toxic actions of QSIs and SAs. This study provides reference data and applicable QSAR models for the environmental risk assessment of QSIs, and gives a new perspective for exploring the joint effects of QSI-antibiotic mixtures.

Transcriptome Analysis Reveals Dynamic Microglial-Induced A1 Astrocyte Reactivity via C3/C3aR/NF-κB Signaling After Ischemic Stroke.

Microglia and astrocytes are key players in neuroinflammation and ischemic stroke. A1 astrocytes are a subtype of astrocytes that are extremely neurotoxic and quickly kill neurons. Although the detrimental A1 astrocytes are present in many neurodegenerative diseases and are considered to accelerate neurodegeneration, their role in the pathophysiology of ischemic stroke is poorly understood. Here, we combined RNA-seq, molecular and immunological techniques, and behavioral tests to investigate the role of A1 astrocytes in the pathophysiology of ischemic stroke. We found that astrocyte phenotypes change from a beneficial A2 type in the acute phase to a detrimental A1 type in the chronic phase following ischemic stroke. The activated microglial IL1α, TNF, and C1q prompt commitment of A1 astrocytes. Inhibition of A1 astrocytes induction attenuates reactive gliosis and ameliorates morphological and functional defects following ischemic stroke. The crosstalk between astrocytic C3 and microglial C3aR contributes to the formation of A1 astrocytes and morphological and functional defects. In addition, NF-κB is activated following ischemic stroke and governs the formation of A1 astrocytes via direct targeting of inflammatory cytokines and chemokines. Taken together, we discovered that A2 astrocytes and A1 astrocytes are enriched in the acute and chronic phases of ischemic stroke respectively, and that the C3/C3aR/NF-κB signaling leads to A1 astrocytes induction. Therefore, the C3/C3aR/NF-κB signaling is a novel therapeutic target for ischemic stroke treatment.

An iron-dependent form of non-canonical ferroptosis induced by labile iron.

Ferroptosis is a recently identified iron-dependent form of nonapoptotic cell death characterized by reactive oxygen species (ROS) generation and lipid peroxidation. Here, we report a novel iron-dependent form of ferroptosis induced by labile iron and investigate the mechanism underlying this process. We find that labile iron-induced ferroptosis is distinct from canonical ferroptosis and is linked to the mitochondrial pathway. Specifically, the mitochondrial calcium uniporter mediates the ferroptosis induced by labile iron. Interestingly, cells undergoing labile iron-induced ferroptosis exhibit cytoplasmic features of oncosis and nuclear features of apoptosis. Furthermore, labile iron-induced ferroptosis involves a unique set of genes. Finally, labile iron-induced ferroptosis was observed in liver subjected to acute iron overload in vivo. Our study reveals a novel form of ferroptosis that may be implicated in diseases caused by acute injury.

Th17/Regulatory T-Cell Imbalance and Acute Kidney Injury in Patients with Sepsis.

To analyze the predictive value of the Th17/Treg ratio for renal injury in sepsis patients, a prospective observational study was conducted. Adult patients with sepsis were enrolled and divided into a sepsis-induced acute kidney injury (SAKI) group and a sepsis-without-AKI group. Logistic regression was used to analyze the independent predictors of SAKI, and the ROC curve was plotted to evaluate the predictive value of the Th17/Treg ratio for renal injury in patients with sepsis. A total of 124 patients were enrolled in this study, including 60 cases (48.39%) of SAKI. Patients who developed sepsis-induced acute kidney injury had a higher Th17/Treg ratio level compared to patients without it (0.11 [0.07, 0.28] versus 0.06 [0.05, 0.16], p < 0.05, respectively. The area under the receiver operating characteristic curve of the Th17/Treg ratio to predict sepsis-induced acute kidney injury was 0.669 (95% CI 0.574−0.763, p < 0.05). The Th17/Treg ratio was associated with SAKI (OR 1.15, 95%CI [1.06−1.24], p < 0.05, non-adjusted and R 1.12, 95%CI [1.00−1.25], p < 0.05, adjusted). The use of the Th17/Treg ratio improved the prediction performance of the prediction model of NAGL. The median Th17/Treg ratio significantly increased with the stratified KDIGO stage (p < 0.05). Th17/Treg imbalance was associated with occurrence of acute kidney injury and AKI severity in patients with sepsis. The Th17/Treg ratio could be a potential predictive marker of sepsis-induced acute kidney injury.

Adipose-Derived Stem Cells From Patients With Ulcerative Colitis Exhibit Impaired Immunosuppressive Function.

Adipose-derived stem cells (ADSCs) are able to modulate the immune response and are used for treating ulcerative colitis (UC). However, it is possible that ADSCs from patients with inflammatory or autoimmune disorders may show defective immunosuppression. We investigated the use of ADSCs from UC patients for autologous cell treatment, specifically, ADSCs from healthy donors (H-ADSCs) and UC patients (P-ADSCs) in terms of various functions, including differentiation, proliferation, secretion, and immunosuppression. The efficacy of P-ADSCs for treating UC was examined in mouse models of acute or chronic colitis. Both H-ADSCs and P-ADSCs were similar in cell morphology, size, adipogenic differentiation capabilities, and cell surface markers. We found that P-ADSCs had lower proliferative capacity, cloning ability, and osteogenic and chondrogenic differentiation potential than H-ADSCs. P-ADSCs exhibited a diminished capacity to inhibit peripheral blood mononuclear cell proliferation, suppress CD25 and CD69 marker expression, decrease the production of inflammation-associated cytokines interferon-γ and tumor necrosis factor-α, and reduce their cytotoxic effect on A549 cells. When primed with inflammatory cytokines, P-ADSCs secreted lower levels of prostaglandin E2, indoleamine 2, 3-dioxygenase, and tumor necrosis factor-α-induced protein 6, which mediated their reduced immunopotency. Moreover, P-ADSCs exhibited weaker therapeutic effects than H-ADSCs, determined by disease activity, histology, myeloperoxidase activity, and body weight. These findings indicate that the immunosuppressive properties of ASCs are affected by donor metabolic characteristics. This study shows, for the first time, the presence of defective ADSC immunosuppression in UC, indicating that autologous transplantation of ADSCs may be inappropriate for patients with UC.

Effects of Dietary Supplementation of gEGF on the Growth Performance and Immunity of Broilers.

EGF has been shown to stimulate the growth of animals. In this study, the content of EGF in chicken embryos (gallus EGF, gEGF) aged from 1 to 20 days of incubation were determined by ELISA kit, and the 5-day-old chicken embryos with the highest content of 5593 pg/g were selected to make gEGF crude extracts. A total of 1500 1-day-old Xianju chickens were randomly divided into five groups with six replicates of 50 chickens each. The control group was fed a basal diet, and other treatment diets were supplemented with 4, 8, 16 and 32 ng/kg gEGF crude extract, respectively. The experiment lasted for 30 days. Chicks were harvested at the end of the experiment, and liver, spleen, thymus, bursa and serum samples were collected. Results showed that average daily gain (ADG) and average daily feed intake (ADFI) of 16 ng/kg group were higher than those in the control group (p < 0.05). The serum uric acid (UA) of the 16 ng/kg group was reduced (p < 0.01), and the serum alkaline phosphatase (AKP) of the 16 ng/kg group increased (p < 0.01). The gEGF extract also increased chick's antioxidant capacity, decreased malondialdehyde (MDA) and increased catalase (CAT) in the liver and serum of 16 ng/kg groups in compared to the control group (p < 0.01). Furthermore, immunity was improved by the addition of gEGF to broiler diets. The serum immunoglobin A (IgA) content of 8 and 16 ng/kg groups and the serum immunoglobin M (IgM) content of 4 and 8 ng/kg groups were increased (p < 0.05) compared to the control group. The bursa index of each experimental group was higher than the control group (p < 0.01). These findings demonstrate that the crude extract of gEGF prepared in this experiment could improve the growth performance, antioxidant capacity and immunity of broilers.

14-kDa phosphohistidine phosphatase is a potential therapeutic target for liver fibrosis.

Liver fibrosis, a major cause of morbidity and mortality worldwide, leads to liver damage, seriously threatening human health. In our previous study, we demonstrated that 14 kDa phosphohistidine phosphatase (PHP14) was upregulated in fibrotic liver tissue and involved in the migration and lamellipodia formation of hepatic stellate cells (HSCs). In this study, we evaluated PHP14 as a therapeutic target for liver fibrosis and investigated the mechanism by which it mediates liver fibrosis. AAV-shPhpt1 administration significantly attenuates CCl4-induced liver fibrosis in mice. In particular, fibrosis-associated inflammatory infiltration was significantly suppressed after PHP14 knockdown. Mechanistically, PHP14 regulated macrophage recruitment, infiltration, and migration by affecting podosome formation of macrophages. Inhibition of PHP14 decreased the expression of the fibrogenic signature at the early stage of liver fibrogenesis and the activation of HSCs in vivo. Thus, PHP14 can be considered a potential therapeutic target for liver fibrosis.NEW & NOTEWORTHY PHP14 inhibition via adeno-associated virus (AAV)-mediated gene silencing could potently attenuate carbon tetrachloride (CCl4)-induced liver fibrosis. PHP14 could regulate the migration of macrophages to the site of injury in vivo. PHP14 knockdown in vivo influenced the environment of fibrogenesis and relevant signaling pathways, subsequently affecting myofibroblast activation.

Benzene Derivatives from Ink Lead to False Positive Results in Neonatal Hyperphenylalaninemia Screening with Ninhydrin Fluorometric Method.

Ninhydrin-based fluorometric quantification of phenylalanine is one of the most widely used methods for hyperphenylalaninemia (HPA) screening in neonates due to its high sensitivity, high accuracy, and low cost. Here we report an increase of false positive cases in neonatal HPA screening with this method, caused by contamination of blood specimen collection devices during the printing process. Through multiple steps of verification, the contaminants were identified from ink circles printed on the collection devices to indicate the positions and sizes of blood drops. Blood specimens from HPA-negative persons collected on these contaminated collection devices showed positive results in the fluorometric tests, but negative results in tandem mass spectroscopy (MS/MS) experiments. Contaminants on the collection devices could be extracted by 80% ethanol and showed an absorption peak around 245 nm, suggesting that these contaminants may contain benzene derivatives with similar structure to phenylalanine. High-performance liquid chromatography (HPLC) analysis of the ethanol extracts from contaminated collection devices identified two prominent peaks specifically from the devices. Methyl-2-benzoylbenzoate (MBB, CAS#606-28-0) was found as one of the major chemicals from contaminated collection devices. This report aims to remind colleagues in the field of this potential contamination and call for tighter regulation and quality control of specimen collection devices.

KLF5 regulates epithelial-mesenchymal transition of liver cancer cells in the context of p53 loss through miR-192 targeting of ZEB2.

Krüppel-like factor 5 (KLF5) can both promote and suppress cell migration, but the underlying mechanisms have not been elucidated. In this study, we show that the function of KLF5 in epithelial-mesenchymal transition (EMT) and migration of liver cancer cells depends on the status of the cellular tumor antigen p53 (p53). Furthermore, zinc finger E-box-binding homeobox 2 (ZEB2) is the main regulator of KLF5 in EMT in liver cancer cells in the context of p53 loss. Most importantly, the regulation of ZEB2 by p53 and KLF5 is indirect and that miR-192 mediates this regulation. Finally, we find that in invasive liver cancer, KLF5 is absent in the context of p53 loss or mutation.

Correlation of Blood FoxP3+ Regulatory T Cells and Disease Activity of Atopic Dermatitis.

OBJECTIVES: To investigate CD4+CD25+FoxP3+ T regulatory cells (Tregs) in the peripheral blood of patients with atopic dermatitis (AD) and its correlation with disease severity. METHODS: Blood samples from 79 AD patients before and after four-week conventional treatment were collected. Cell counts of CD4+CD25+FoxP3+Tregs, CD4+CD25+FoxP3-T effector cells (Teffs), and CD4+IL-10+Tregs were analyzed by flow cytometry. Serum levels of IL-4, IL-10, IL-12, IL-13, IFN-γ, and TGF-ß were measured by ELISA. RESULTS: The pretreatment cell count of CD4+CD25+FoxP3+Tregs positively correlated with disease severity in all patients (P < 0.0001). However, when that correlation was rechecked based on the treatment response, a much stronger correlation of that was found in those patients with remission after treatment, while no correlation of that was found in patients without remission. Both the cell count and proportions of peripheral CD4+CD25+FoxP3+Tregs and CD4+CD25+FoxP3-Teffs reduced significantly after treatment in patients with remission, but remained unchanged in patients without remission. The cell count and proportion of CD4+IL-10+Tregs did not change after treatment in both groups. In patients with remission, serum levels of IL-4 and IL-13 significantly reduced (all P < 0.05); IL-12 and IFN-γ levels increased significantly (all P < 0.05); IL-10 and TGF-ß levels remained unchanged after treatment. None of those cytokine levels changed in patients without remission. CONCLUSIONS: CD4+CD25+FoxP3+Tregs is associated with AD development and severity in some patients but not in others. AD maybe divided into CD4+CD25+FoxP3+Treg-associated subtype, which CD4+CD25+FoxP3+Treg is parallel to the activity of AD, and nonassociated subtype, which CD4+CD25+FoxP3+Treg is not related. This subgroup difference may contribute partly to the nonidentical markers that have been found in AD and should be studied further.

ARHGEF4-mediates the actin cytoskeleton reorganization of hepatic stellate cells in 3-dimensional collagen matrices.

The actin cytoskeleton of hepatic stellate cells (HSCs) is reorganized when they are cultured in 3D collagen matrices. Here, we investigated the molecular mechanism of actin cytoskeleton reorganization in HSCs cultured in 3D floating collagen matrices (FCM) compared to those on 2D polystyrene surfaces (PS). First, we found that the generation of dendritic cellular processes was controlled by Rac1. Next, we examined the differential gene expression of HSCs cultured on 2D PS and in 3D FCM by RNA-Seq and focused on the changes of actin cytoskeleton reorganization-related molecular components and guanine nucleotide exchange factors (GEFs). The results showed that the expression of genes associated with actin cytoskeleton reorganization-related cellular components, filopodia and lamellipodia, were significantly decreased, but podosome-related genes was significantly increased in 3D FCM. Furthermore, we found that a Rac1-specific GEF, ARHGEF4, played roles in morphological changes, migration and podosome-related gene expression in HSCs cultured in 3D FCM. Abbreviations: 2D PS: 2-dimensional polystyrene surface; 3D FCM: 3-dimensional floating collagen matrices; ARHGEF4: Rho guanine nucleotide exchange factor 4; ARHGEF6: Rho guanine nucleotide exchange factor 6; GEF: guanine nucleotide exchange factor; HSC: hepatic stellate cell.

PHP14 regulates hepatic stellate cells migration in liver fibrosis via mediating TGF-ß1 signaling to PI3Kγ/AKT/Rac1 pathway.

Hepatic fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Migration of the activated HSCs to the site of injury is one of the key characteristics during the wound healing process. We have previously demonstrated that 14 kDa phosphohistidine phosphatase (PHP14) is involved in migration and lamellipodia formation of HSCs. However, the role of PHP14 in liver fibrosis remains unknown. In this study, we first assessed PHP14 expression and distribution in liver fibrotic tissues using western blot, immunohistochemistry, and double immunofluorescence staining. Next, we investigated the role of PHP14 in liver fibrosis and, more specifically, the migration of HSCs by Transwell assay and 3D collagen matrices assay. Finally, we explored the possible molecular mechanisms of the effects of PHP14 on these processes. Our results show that the PHP14 expression is up-regulated in fibrotic liver and mainly in HSCs. Importantly, TGF-ß1 can induce PHP14 expression in HSCs accompanied with the activation of HSCs. Consistent with the previous study, PHP14 promotes HSCs migration, especially, promotes 3D floating collagen matrices contraction but inhibits stressed-released matrices contraction. Mechanistically, the PI3Kγ/AKT/Rac1 pathway is involved in migration regulated by PHP14. Moreover, PHP14 specifically mediates the TGF-ß1 signaling to PI3Kγ/AKT pathway and regulates HSC migration, and thus participates in liver fibrosis. Our study identified the role of PHP14 in liver fibrosis, particularly HSC migration, and suggested a novel mediator of transducting TGF-ß1 signaling to PI3Kγ/AKT/Rac1 pathway. KEY MESSAGES: PHP14 is up-regulated in fibrotic liver and activated hepatic stellate cells. The expression of PHP14 is induced by TGF-ß1. The migration of hepatic stellate cells is regulated by PHP14. PHP14 is a mediator of TGF-ß1 signaling to PI3Kγ/AKT/Rac1 pathway in hepatic stellate cells.