Hedgehog pathway negatively regulated depleted uranium-induced nephrotoxicity.

Depleted uranium (DU) retains the radiological toxicities, which accumulates preferentially in the kidneys. Hedgehog (Hh) pathway plays a critical role in tissue injury. However, the role of Hh in DU-induced nephrotoxicity was still unclear. This study was carried out to investigate the effect of Gli2, which was an important transcription effector of Hh signaling, on DU induced nephrotoxicity. To clarify it, CK19 positive tubular epithelial cells specific Gli2 conditional knockout (KO) mice model was exposed to DU, and then histopathological damage and Hh signaling pathway activation was analyzed. Moreover, HEK-293 T cells were exposed to DU with Gant61 or Gli2 overexpression, and cytotoxicity of DU as analyzed. Results showed that DU caused nephrotoxicity accompanied by activation of Hh signaling pathway. Meanwhile, genetic KO of Gli2 reduced DU-induced nephrotoxicity by normalizing biochemical indicators and reducing Hh pathway activation. Pharmacologic inhibition of Gli1/2 by Gant61 reduced DU induced cytotoxicity by inhibiting apoptosis, ROS formation and Hh pathway activation. However, overexpression of Gli2 aggravated DU-induced cytotoxicity by increasing the levels of apoptosis and ROS formation. Taken together, these results revealed that Hh signaling negatively regulated DU-inducted nephrotoxicity, and that inhibition of Gli2 might serve as a promising nephroprotective target for DU-induced kidney injury.

Metagenomic sequencing expedites diagnosis of disseminated BCG in an infant with BRAFV600E mutation.

INTRODUCTION: Disseminated bacillus Calmette-Guérin (BCG) disease is a rare but serious BCG complication in children. Early diagnosis and timely interventions are essential to improve prognosis. However, its manifestations can closely mimic those of Langerhans cell histiocytosis (LCH), which usually leads to a high rate of misdiagnoses. Herein we report the first case of successful application of biopsy tissue metagenomic next-generation sequencing (mNGS) in the differential diagnosis of disseminated BCG disease and LCH. CASE STUDY: A 5-month-old female infant was transferred to our center for the treatment of paroxysmal cough, intermittent hematochezia and trunk rash. Examination on admission showed moderate anemia, erythropenia, thrombocytopenia and hepatosplenomegaly. The immunohistochemistry of her intestinal biopsy samples showed CD1a (+) and Langerin (+). Genetic testing of both peripheral blood and bone marrow samples suggested BRAFV600E mutation. Hence, she was initially diagnosed with LCH. However, no improvement was observed after a course of systemic chemotherapy. The left axillary lymph node and colonic mucosal biopsy specimens were sent for mNGS which resulted in sequence reads of Mycobacterium bovis-BCG. Triple antimycobacterial therapy was started according to the diagnosis. RESULTS: The diagnosis of this case was corrected as disseminated BCG disease by mNGS. Currently, she is doing well clinically and continues to follow-up at our outpatient clinic. CONCLUSIONS: This case suggests that mNGS is a valuable tool in the differential diagnosis of disseminated BCG disease and LCH, which can improve the early diagnosis rate of disseminated BCG disease.

Nicotinamide mononucleotide impacts HIV-1 infection by modulating immune activation in T lymphocytes and humanized mice.

BACKGROUND: HIV-1-associated immune activation drives CD4+ T cell depletion and the development of acquired immunodeficiency syndrome. We aimed to determine the role of nicotinamide mononucleotide (NMN), the direct precursor of nicotinamide adenine dinucleotide (NAD) co-enzyme, in CD4+ T cell modulation during HIV-1 infection. METHODS: We examined HIV-1 integrated DNA or transcribed RNA, intracellular p24 protein, and T cell activation markers in CD4+ T cells including in vitro HIV-1-infected cells, reactivated patient-derived cells, and in HIV-1-infected humanized mice, under NMN treatment. RNA-seq and CyTOF analyses were used for investigating the effect of NMN on CD4+ T cells. FINDINGS: We found that NMN increased the intracellular NAD amount, resulting in suppressed HIV-1 p24 production and proliferation in infected CD4+ T cells, especially in activated CD25+CD4+ T cells. NMN also inhibited CD25 expression on reactivated resting CD4+ T cells derived from cART-treated people living with HIV-1 (PLWH). In HIV-1-infected humanized mice, the frequency of CD4+ T cells was reconstituted significantly by combined cART and NMN treatment as compared with cART or NMN alone, which correlated with suppressed hyperactivation of CD4+ T cells. INTERPRETATION: Our results highlight the suppressive role of NMN in CD4+ T cell activation during HIV-1 infection. It warrants future clinical investigation of NMN as a potential treatment in combination with cART in PLWH. FUNDING: This work was supported by the Hong Kong Research Grants Council Theme-Based Research Scheme (T11-706/18-N), University Research Committee of The University of Hong Kong, the Collaborative Research with GeneHarbor (Hong Kong) Biotechnologies Limited and National Key R&D Program of China (Grant2021YFC2301900).

ISL1 regulates lung branching morphogenesis via Shh signaling pathway.

Lung branching morphogenesis relies on a complex coordination of multiple signaling pathways and transcription factors. Here, we found that ablation of the LIM homeodomain transcription factor Islet1 (Isl1) in lung epithelium resulted in defective branching morphogenesis and incomplete formation of five lobes. A reduction in mesenchymal cell proliferation was observed in Isl1ShhCre lungs. There was no difference in apoptosis between the wild-type (ShhCre) and Isl1ShhCre embryos. RNA-Seq and in situ hybridization analysis showed that Shh, Ptch1, Sox9, Irx1, Irx2, Tbx2, and Tbx3 were downregulated in the lungs of Isl1ShhCre embryos. ChIP assay implied the Shh gene served as a direct target of ISL1, since the transcription factor ISL1 could bind to the Shh epithelial enhancer sequence (MACS1). Also, activation of the Hedgehog pathway via ectopic gene expression rescued the defects caused by Isl1 ablation, confirming the genetic integration of Hedgehog signaling. In conclusion, our works suggest that epithelial Isl1 regulates lung branching morphogenesis through administrating the Shh signaling mediated epithelial-mesenchymal communications.

Role of NF-κB signaling pathway in hexavalent chromium-induced hepatotoxicity.

Hexavalent chromium Cr (VI) is a primary human carcinogen with damaging toxic effects on multiple organs. Cr (VI) exposure can induce hepatotoxicity through oxidative stress, but its exact mechanism of action was still unclear. In our study, a model of acute Cr (VI) induced liver injury was established by exposing mice to different concentrations (0, 40, 80, and 160 mg/kg) of Cr (VI); RNA-seq was used to characterize changes in liver tissue transcriptome of C57BL/6 mice after exposing to 160 mg/kg Bw of Cr (VI). Changes in liver tissue structures, proteins, and genes were observed by hematoxylin and eosin (H&E), western blot, immunohistochemistry and RT-PCR. After Cr (VI) exposure, abnormal liver tissue structure, hepatocyte injury, and hepatic inflammatory response were observed in mice in a dose-dependent manner. RNA-seq transcriptome results indicated that oxidative stress, apoptosis, and inflammatory response pathways were increased after Cr (VI) exposure; KEGG pathway analysis found that activation of NF-κB signaling pathway was significantly upregulated. Consistent with the RNA-seq results, immunohistochemistry showed that Cr (VI) exposure resulted in infiltrating of Kupffer cells and neutrophils, increasing expression of inflammatory factors (TNF-α, IL-6, IL-1ß), and activating of NF-κB signaling pathways (p-IKKα/ß and p-p65). However, ROS inhibitor, N-acetyl-L-cysteine (NAC), could reduce infiltration of Kupffer cells and neutrophils and expression of inflammatory factors. Besides, NAC could inhibit NF-κB signaling pathway activation, and alleviate Cr (VI)-induced liver tissue damage. Our findings strongly suggested that inhibition of ROS by NAC might help in the development of new strategies for Cr (VI)-associated liver fibrosis. Our findings revealed for the first time that Cr (VI) induced liver tissue damage through the inflammatory response mediated by the NF-κB signaling pathway, and inhibition of ROS by NAC might help in the development of new strategies for Cr (VI)-associated hepatotoxicity.

ISL1/SHH/CXCL12 signaling regulates myogenic cell migration during mouse tongue development.

Migration of myoblasts derived from the occipital somites is essential for tongue morphogenesis. However, the molecular mechanisms of myoblast migration remain elusive. In this study, we report that deletion of Isl1 in the mouse mandibular epithelium leads to aglossia due to myoblast migration defects. Isl1 regulates the expression pattern of chemokine ligand 12 (Cxcl12) in the first branchial arch through the Shh/Wnt5a cascade. Cxcl12+ mesenchymal cells in Isl1ShhCre embryos were unable to migrate to the distal region, but instead clustered in a relatively small proximal domain of the mandible. CXCL12 serves as a bidirectional cue for myoblasts expressing its receptor CXCR4 in a concentration-dependent manner, attracting Cxcr4+ myoblast invasion at low concentrations but repelling at high concentrations. The accumulation of Cxcl12+ mesenchymal cells resulted in high local concentrations of CXCL12, which prevented Cxcr4+ myoblast invasion. Furthermore, transgenic activation of Ihh alleviated defects in tongue development and rescued myoblast migration, confirming the functional involvement of Hedgehog signaling in tongue development. In summary, this study provides the first line of genetic evidence that the ISL1/SHH/CXCL12 axis regulates myoblast migration during tongue development.

Inhibitory Effects of Compounds from Plumula nelumbinis on Biofilm and Quorum Sensing Against P. aeruginosa.

Quorum sensing (QS), which controls the survival and virulence of Pseudomonas aeruginosa, including the formation of biofilm, is considered to be a new target to overcome pathogens. The aim of this study was to identify new QS inhibitors against P. aeruginosa and provide potential treatments for clinical infections. In this study, 25 compounds were isolated from Plumula nelumbini. Among these compounds, C25 showed the most significant biofilm inhibition activity, reaching 44.63% at 100 µM without inhibiting bacterial growth. Furthermore, C25 showed significant inhibition activity of rhamnolipid, pyocyanin, and elastase. Further mechanistic studies have confirmed that C25 could downregulate key genes in the QS system, including lasI, lasR, lasA, lasB, and pqsR, and Molecular docking studies have shown that C25 can bind to the active sites of the LasR and PqsR receptors. The present study suggests that C25 is a promising QS inhibitor for treating P. aeruginosa infections.

Protective effect of Amomum Roxb. essential oils in lipopolysaccharide-induced acute lung injury mice and its metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Several Amomum species are commonly used in food as flavoring agents and traditional Chinese medicine to treat inflammation-related diseases. AIM OF THE STUDY: This study aims to investigate the protective effects of Chinese herbal medicines, including six Amomum Roxb. essential oils (AEOs), against acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice. MATERIALS AND METHODS: The compositions of AEOs were analyzed using gas chromatography - mass spectrometry. RAW264.7 cells were treated with AEOS (0-100 µg/mL) and stimulated with LPS. C57 mice received AEOs (100 mg/kg) via atomization system for seven consecutive days, and then, intratracheal instillation of LPS was applied to establish an in vivo model of acute lung injury. RESULTS: We identified three AEOs demonstrating anti-inflammatory effects and amelioration of LPS-induced lung tissue pathological damage. Furthermore, we found that these AEOs reduced lung wet/dry weight ratios and protein concentrations in the bronchoalveolar lavage fluid of mice with LPS-induced ALI. Additionally, AEOs reduced the levels of malondialdehyde, TNF-α, IL-6, and IL-1ß but increased the levels of superoxide dismutase and catalase in lung tissue, alveolar lavage fluid, and serum samples. We also found that these three AEOs affected proteins related to the TLR4/Myd88/NF-κB pathway. CONCLUSIONS: In summary, our findings revealed that AEOs ameliorate inflammatory and oxidative stress in mice with ALI through the TLR4/Myd88/NF-κB pathway.

The Crohn Disease-associated ATG16L1T300A polymorphism regulates inflammatory responses by modulating TLR- and NLR-mediated signaling.

The mechanisms by which the ATG16L1T300A polymorphism affects cell function and causes an increased risk for the development of Crohn disease remain incompletely understood. Here we report that healthy individuals and mice bearing this polymorphism, even as heterozygotes, manifest enhanced TLR, and NLR cytokine and chemokine responses due to increased activation of NFKB. We elucidated the mechanism of the NFKB abnormality and found that in the ATG16L1T300A cell, there is enhanced polyubiquitination of TRAF6 or RIPK2 resulting from the accumulation of SQSTM1/p62. Indeed, knockout of Sqstm1 in autophagy-deficient cells almost completely normalized TRAF6 or RIPK2 polyubiquitination and NFKB activation in these cells. Thus, by identifying that autophagy is a pathway-intrinsic homeostatic mechanism that restricts excessive TLR- or NLR-mediated inflammatory signaling, our findings shed new light on how the ATG16L1T300A polymorphism sets the stage for the occurrence of Crohn disease.Abbreviations: 3-MA: 3-methyladenine; ATG16L1: autophagy related 16 like 1; ATG7: autophagy related 7; BMDM: bone marrow-derived macrophage; CD: Crohn disease; CXCL: C-X-C motif chemokine ligand; IBD: inflammatory bowel disease; iBMDM: immortalized mouse BMDM; IL1B/IL-1ß: interleukin 1 beta; IL6: interleukin 6; KI: knockin; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPS: lipopolysaccharide; MDP: muramyl dipeptide; MEF: mouse embryonic fibroblast; NFKB/NF-κB: nuclear factor kappa B; NFKBIA/IKBA: NFKB inhibitor alpha; NLR: NOD-like receptor; NOD: nucleotide-binding oligomerization domain containing; RIPK2: receptor interacting serine/threonine kinase 2; SNP: single nucleotide polymorphism; SQSTM1/p62: sequestosome 1; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; Ub: ubiquitin; WT: wild type.

Hexavalent chromium induces hepatocyte apoptosis via regulation of apoptosis signal-regulating kinase 1/c-Jun amino-terminal kinase signaling.

With the spread of hexavalent chromium (Cr(VI)) contamination, Cr(VI)-induced hepatotoxicity has attracted increasing attention in recent years. To date, however, the exact mechanism of Cr(VI) toxicity remains unclear. In this study, we investigated the role of apoptosis signal-regulating kinase 1 (ASK1)/c-Jun amino-terminal kinase (JNK) in Cr(VI)-induced hepatic toxicity and the possible related mechanisms. AML-12 hepatocyte cell-lines were treated with 0, 1, 4, and 16 µmol/Lof Cr(VI) with or without GS-444271 (an ASK1 inhibitor). Adult male mice were administered with 0, 2, 8, and 32 mg/kg body mass (BM)/day of Cr(VI) for 5 days. The level of hepatocyte apoptosis/proliferation, generation of reactive oxygen species (ROS), and expression levels of mRNAs and proteins related to ASK1/JNK and nuclear factor-E2-related factor 2 (Nrf2) signaling were assessed. Results showed that high Cr(VI) exposure induced hepatocyte apoptosis and liver injury by generation of ROS and down-regulation of Nrf2 signaling. In addition, ASK1/JNK signaling activity was upregulated in the Cr(VI)-treated group. Furthermore, GS-444217 treatment significantly rescued Cr(VI)-induced hepatocyte apoptosis and liver dysfunction in vitro and in vivo by down-regulation of ASK1/JNK signaling. Thus, ASK1/JNK signaling appears to play an important role in Cr(VI)-induced hepatocyte apoptosis and liver injury. This study should help improve our understanding of the mechanism of Cr(VI)-induced liver injury and provide support for future investigations on liver disease therapy.

An efficient method of inducing differentiation of mouse embryonic stem cells into primitive endodermal cells.

Primitive Endoderm (PrE) is an extraembryonic structure derived from inner cell mass (ICM) in the blastocysts. Its interaction with the epiblast is critical to sustain embryonic growth and embryonic pattern. In this study, we reported a simple and efficient method to induce the differentiation of mouse Embryonic Stem Cells (mESCs) into PrE cells. In the process of ESC monolayer adherent culture, 1 µM atRA and 10 µM CHIR inducers were used to activate RA and Wnt signaling pathways respectively. After 9 days of differentiation, the proportion of PrE cells was up to 85%. Further studies indicated that Wnt signaling pathway acted as a switch that RA induces mESCs differentiation between SMC and PrE cell. In the presence of only RA signaling, mESCs adopted the fate of smooth muscle cells (SMCs); Simultaneous activation of the Wnt signaling pathway changed the differentiation fate of mESCs into PrE cells. This efficient induction method can provide new cellular resources and models for relevant studies of PrE.

Two new pyrone derivatives from the mangrove-derived endophytic fungus Aspergillus sydowii #2B.

Two new pyrone derivatives, 2-(12S-hydroxypropyl)-3-hydroxy-methyl-6-hydroxy-7-methoxychromone (1), and (±)-pyrenocine S (5), together with five known compounds xanthoradone A (2), (+)-3,3',7,7',8,8'-hexahydroxy-5,5'-dimethyl-bianthra-quinone (3), butyrolactone-I (4), pyrenocine A (6), and (±)-pyrenocine E (7) were obtained from the mangrove endophytic fungus Aspergillus sydowii #2B. Their structures were elucidated on the basis of spectroscopic methods, single-crystal X-ray diffraction analysis and comparison data with of literatures. Compounds 2, 3, 4, 5, 6 and 7 showed cytotoxicities against prostate cancer VCaP cells with IC50 values of 4.19 ± 1.02, 33.36 ± 1.42, 1.92 ± 0.82, 20.06 ± 2.01, 7.92 ± 0.86, and 10.13 ± 0.88 µM respectively, while compound 1 showed no cytotoxic activity. Compounds 1, 3, 6, and 7 exhibited weak inhibition effects against the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced RAW 246.7 cells with IC50 values of 40.15, 28.69, 25.25 and 43.08 µM respectively.

A reaction-based colorimetric and ratiometric chemosensor for imaging identification of HClO in live cells, mung bean sprouts, and paper strips.

Hypochlorous acid (HClO) as well as its ionic form (ClO-), representative of reactive oxygen species (ROS), are essential players in all sorts of biological processes. The abnormal level of each can lead to the onset of various diseases. Besides, Sodium hypochlorite, a commonly-used bleaching agent in our daily lives, could also result in breathing and skin problems when overexposed. Therefore, developing a molecular chemosensor for sensing HClO is of biological and environmental importance. Though many such chemosensors have been reported, new HClO chemosensors with different sensing performances may still come in handy in certain situations. In this work, we have developed a new coumarin-based chemosensor, CM-hbt, for realizing both ratiometric and colorimetric imaging detection of HClO in live cells. Notably, we further explored its application in sensing HClO in plant mung beans as well as fabricated an easy-to-use paper strip apparatus for facilitating its quick detection, which is seldomly seen in other HClO chemosensors. All the analysis results confirmed the high sensitivity and selectivity of this novel chemosensor. DFT calculations were used to decipher the underlying sensing mechanism of CM-hbt. Overall, this work presents a novel chemosensor, CM-hbt, as a colorimetric and ratiometric chemosensor for realizing imaging detection of HClO in a variety of different model systems, which highlights its broad spectrum of application potentials.

Seco-neferine A-F, three new pairs of benzyltetrahydroisoquinoline alkaloid epimers from Plumula Nelumbinis and their activity.

Three new pairs of benzyltetrahydroisoquinoline (BIQ) alkaloid epimers, Seco-neferine A-F (1-6), were isolated from an EtOH extract of Plumula Nelumbinis. The structures of these compounds were identified by a combination of NMR, HR-ESI-MS, circular dichroism, UV spectroscopic analyses and specific rotations. The structure of compounds 1-6 possesses high similarity with neferine, because these three pairs of epimers have the same skeleton as neferine. Compounds 1,2 and 5,6 are open-loop compounds of position 1' and 1 of neferine respectively. The H connects with position 2' N of compounds 1,2 is replaced by methyl, forming the structure of compounds 3,4. Moreover, six compounds were tested for cytotoxicity against MDA-MB-231 breast cancer cell. Compound 6 displayed moderate inhibitory effects on breast cancer with IC50 of 38.96 µM, while compounds 2,3,4 show certain inhibitory effects.

ROCK inhibitor attenuates carbon blacks-induced pulmonary fibrosis in mice via Rho/ROCK/NF-kappa B pathway.

Exposure to carbon blacks (CBs) has been associated with the progression of pulmonary fibrosis, whereas the mechanism is still not clear. We therefore aimed to investigate the effect of RhoA/ROCK pathway on pulmonary fibrosis caused by CBs exposure. Western blot analysis indicated that CBs could promote the activation of RhoA/ROCK pathway and phosphorylation of p65 and IκBα in mice lung. However, ROCK inhibitor Y-27632 could attenuate phosphorylation levels of p65 and IκBα and restore histopathological changes of the lung tissue. Then, we evaluated the effect of RhoA/ROCK pathway on pulmonary fibrosis by detecting the expression levels of α-SMA, vimentin, and Collagen type-I (Col-I), which could be partly inhibited by Y-27632. It was assumed that inhibition of ROCK could be a promising therapeutic candidate for CBs-induced pulmonary fibrosis, which possibly through the blockage of RhoA/ROCK/NF-κB pathway.

Gli2-regulated activation of hepatic stellate cells and liver fibrosis by TGF-ß signaling.

The Hedgehog (Hh) signaling pathway is correlated with hepatic stellate cells (HSCs) activation and liver fibrosis. Gli2 is a key transcription effector of Hh signaling. However, the role of Gli2 in HSC-mediated liver fibrosis progression is largely unknown. In the present study, we investigated the effect of Gli2 on liver fibrogenesis and its possible mechanism using conditional knockout (cKO) Gli2 mice and HSC models. Wild-type (WT) and GFAP-CreERT;Gli2flox/flox male mice were exposed to CCl4 for 1 mo to induce liver fibrosis. Primary HSCs were isolated from mice and the transition of HSCs into a myofibroblastic phenotype was evaluated. Livers from mice underwent histological, immunohistochemical, and immunofluorescence analyses. The expression levels of proteins and genes were evaluated by Western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RNA-seq was used to screen differentially expressed genes. Results showed that CCl4 treatment induced liver fibrosis, promoted HSCs activation and proliferation, and upregulated Hh signaling activity. The cKO of Gli2 in GFAP-CreERT;Gli2flox/flox mice decreased liver fibrosis as well as HSC activation and proliferation. In vitro studies showed that KO of Gli2 in HSCs blocked cell proliferation and activation by decrease of cyclin D1/D2 expression. The RNA-seq results revealed that the expression levels TGF-ß1 ligands were downregulated in Gli2 KO HSCs. Furthermore, overexpression of Gli2 rescued proliferation and activation of HSCs by upregulation of TGF-ß signaling activity. Our data demonstrated that Gli2 regulated HSC activation and liver fibrosis by TGF-ß signaling, thus providing support for future Gli2-based investigations of liver fibrosis therapy.NEW & NOTEWORTHYGli2 is a key transcription effector of Hh signaling. We found that Hh/Gli2 signaling activity was upregulated in CCl4-induced liver fibrosis. Conditional deletion of the Gli2 gene in HSCs ameliorated CCl4-induced liver fibrosis and HSCs activation. Moreover, Gli2 promoted activation of HSCs through upregulation of cyclin expression and TGF-ß signaling activity. Thus, our data provide strong support for future investigations on Gli2 inhibition to slow liver fibrosis progression in humans.

RIPK3 Activates MLKL-mediated Necroptosis and Inflammasome Signaling during Streptococcus Infection.

Community-acquired pneumonia is the most common type of pneumonia and remains a leading cause of morbidity and mortality worldwide. Although many different pathogens can contribute to pneumonia, Streptococcus pneumoniae is one of the common bacterial pathogens that underlie community-acquired pneumonia. RIPK3 (receptor-interacting protein kinase 3) is widely recognized as a key modulator of inflammation and cell death. To elucidate a potential role of RIPK3 in pneumonia, we examined plasma from healthy control subjects and patients positive for streptococcal pneumonia. In human studies, RIPK3 protein concentrations were significantly elevated and were identified as a potential plasma marker of pneumococcal pneumonia. To expand these findings, we used an in vivo murine model of pneumococcal pneumonia to demonstrate that RIPK3 deficiency leads to reduced bacterial clearance, severe pathological damage, and high mortality. Our results illustrated that RIPK3 forms a complex with RIPK1, MLKL (mixed-lineage kinase domain-like protein), and MCU (mitochondrial calcium uniporter) to induce mitochondrial calcium uptake and mitochondrial reactive oxygen species(mROS) production during S. pneumoniae infection. In macrophages, RIPK3 initiated necroptosis via the mROS-mediated mitochondrial permeability transition pore opening and NLRP3 inflammasome activation via the mROS-AKT pathway to protect against S. pneumoniae. In conclusion, our study demonstrated a mechanism by which RIPK3-initiated necroptosis is essential for host defense against S. pneumoniae.

Shh-Yap signaling controls hepatic ductular reactions in CCl4 -induced liver injury.

Carbon tetrachloride (CCl4 ) exposure can induce hepatic ductular reactions. To date, however, the related mechanism remains largely unknown. Sonic hedgehog (Shh) and Yes-associated protein (Yap) signaling are correlated with liver injury and regeneration. Herein, we investigated the role of Shh and Yap signaling in the fate of ductular reaction cells in CCl4 -treated livers and the possible mechanisms. Wild-type and Shh-EGFP-Cre male mice were exposed to CCl4 (2 mL/kg), and then treated with or without the Shh signaling inhibitor Gant61. The level of liver injury, proliferation of ductular reaction cells, and expression levels of mRNA and protein related to the Shh and Yap signaling components were assessed. Results showed that CCl4 treatment induced liver injury and promoted activation and proliferation of ductular reaction cells. In addition, CCl4 induced the expression of Shh ligands in hepatocytes, accompanied by activation of Shh and Yap1 signaling in the liver. Furthermore, administration of Gant61 ameliorated liver regeneration, inhibited hepatic ductular reactions, and decreased Shh and Yap1 signaling activity. Thus, Shh-Yap1 signaling appears to play an integral role in the proliferation of ductular reaction cells in CCl4 -induced liver injury. This study should improve our understanding of the mechanism of CCl4 -induced liver injury and ductular reactions and provide support for future investigations on liver disease therapy.

Effect of an Antagonistic Peptide of CCR5 on the Expression of Autophagy-related Genes and ß-Arrestin 2 in Lung Tissues of Asthmatic Mice.

PURPOSE: The mechanisms of CC chemokine receptor 5 (CCR5) in the process of autophagy remain unknown. In this study, we examined the role of HY peptide, which is an antagonistic peptide specifically binding the second extracellular loop of CCR5, in the expression of autophagy genes and ß-arrestin 2 in lung tissues of asthmatic mice. METHODS: Experimental asthmatic mice were treated with HY peptide and dexamethasone sodium phosphate (Dex). Airway inflammation, autophagy-related genes, autophagic vacuoles (AVs) and ß-arrestin 2 were examined in lung tissues, and the correlation between ß-arrestin 2 and LC3 expression was assessed. RESULTS: HY peptide and Dex treatments alleviate airway inflammation. The expression of autophagy-related genes, such as BECN1, ATG5 and LC3, was decreased in the lung tissues of the asthmatic mice. However, HY peptide and Dex treatments increased the expression of these genes as well as the formation of AVs. Additionally, the expression of the ß-arrestin 2 protein was significantly increased in the HY peptide-treated group, and positive cells expressing ß-arrestin 2 were mainly located in the membrane and cytoplasm of bronchial epithelial cells. The ß-arrestin 2 expression was positively correlated with the expression of LC3 in the model and HY peptide-treated groups. CONCLUSIONS: HY peptide inhibits airway inflammation, autophagic dysfunction exists in asthmatic mice, and targeting HY peptide increases the expression of autophagy-related genes. Thus, ß-arrestin 2 may participate in the mechanisms underlying these processes.

Establishment of drug screening in human embryonic stem cells based on a high-content screening system.

High-content screening (HCS) systems can be used for high-throughput screening of drugs in human embryonic stem cells (hESCs). However, hESCs require immunofluorescence staining with stemness markers (e.g., Oct-4) prior to HCS, which can be time consuming and labor intensive. In this study, we employed transgenic hESCs with enhanced green fluorescent protein driven by stemness gene Oct-4 promoter (Oct-4-EGFP-H9), in which the colony area and relative green fluorescence area inferred a state of hESC proliferation and stemness, respectively. The Oct-4-EGFP-H9 transgenic hESCs were cultured in mTeSR medium with different concentrations of 5-Fluorouracil (5-FU), vitamin C (VC), or retinoic acid (RA) for 5-7 days, followed by repeated imaging using the HCS system. Finally, the hESC colony area and green fluorescence area were calculated. Results showed that 5-FU treatment markedly reduced colony area in a dose-dependent manner, whereas VC and RA treatments did not. MTT assay and flow cytometry indicated that 5-FU inhibited the proliferation of hESCs significantly, verifying reliability of the data from the HCS system based on colony area analysis. The green fluorescence to total colony area ratio decreased with RA treatment, suggesting that RA significantly promoted differentiation, whereas 5-FU and VC had almost no effect, as verified by quantitative real-time polymerase chain reaction and western blot analysis. In conclusion, our study established a rapid and efficient drug screening system without the requirement of staining based on HCS.