MSCs alleviate LPS-induced acute lung injury by inhibiting the proinflammatory function of macrophages in mouse lung organoid-macrophage model.

Acute lung injury (ALI) is an inflammatory disease associated with alveolar injury, subsequent macrophage activation, inflammatory cell infiltration, and cytokine production. Mesenchymal stem cells (MSCs) are beneficial for application in the treatment of inflammatory diseases due to their immunomodulatory effects. However, the mechanisms of regulatory effects by MSCs on macrophages in ALI need more in-depth study. Lung tissues were collected from mice for mouse lung organoid construction. Alveolar macrophages (AMs) derived from bronchoalveolar lavage and interstitial macrophages (IMs) derived from lung tissue were co-cultured, with novel matrigel-spreading lung organoids to construct an in vitro model of lung organoids-immune cells. Mouse compact bone-derived MSCs were co-cultured with organoids-macrophages to confirm their therapeutic effect on acute lung injury. Changes in transcriptome expression profile were analyzed by RNA sequencing. Well-established lung organoids expressed various lung cell type-specific markers. Lung organoids grown on spreading matrigel had the property of functional cells growing outside the lumen. Lipopolysaccharide (LPS)-induced injury promoted macrophage chemotaxis toward lung organoids and enhanced the expression of inflammation-associated genes in inflammation-injured lung organoids-macrophages compared with controls. Treatment with MSCs inhibited the injury progress and reduced the levels of inflammatory components. Furthermore, through the nuclear factor-κB pathway, MSC treatment inhibited inflammatory and phenotypic transformation of AMs and modulated the antigen-presenting function of IMs, thereby affecting the inflammatory phenotype of lung organoids. Lung organoids grown by spreading matrigel facilitate the reception of external stimuli and the construction of in vitro models containing immune cells, which is a potential novel model for disease research. MSCs exert protective effects against lung injury by regulating different functions of AMs and IMs in the lung, indicating a potential mechanism for therapeutic intervention.

Mitochondrial abnormality in ovarian granulosa cells of patients with polycystic ovary syndrome.

The quality of oocytes in patients with polycystic ovary syndrome (PCOS) decreases, which is closely related to the function of oocytes' mitochondria. If mitochondrial dysfunction is involved in PCOS, it is likely to affect the function of cumulus cells. However, the mechanism of mitochondrial dysfunction remains unclear. In the present study, granulosa cells were collected from women attending the Hebei Reproductive Health Hospital and were divided into the normal ovarian reserve group (CON group) and the PCOS group. The mitochondrial ultrastructure was observed by transmission electron microscope, and the mitochondrial function was determined by detecting the ATP content, reactive oxygen species levels, the number of mitochondria and the mitochondrial membrane potential. Additionally, western blotting was used to compare the expression levels of mitochondrial kinetic protein, the related channel protein, between the two groups. In the present study, it was found that patients with PCOS had abnormal granulosa cell morphology, increased mitochondrial abnormalities, decreased mitochondrial function and disturbed mitochondrial dynamics. In addition, the silent information regulator 1/phosphorylated­AMP­activated protein kinase/peroxisome proliferator­activated receptor­Î³ coactivator 1α pathway expression was decreased, and it was hypothesized that the decreased mitochondrial mass in the PCOS group was associated with it.

BRSK1 confers cisplatin resistance in cervical cancer cells via regulation of mitochondrial respiration.

PURPOSE: Although cisplatin-containing chemotherapy has been utilized as a front-line treatment for cervical cancer, intrinsic and acquired resistance of cisplatin remains a major hurdle for the durable and curative therapeutic response. We thus aim to identify novel regulator of cisplatin resistance in cervical cancer cells. METHODS: Real-time PCR and western blotting analysis were employed to determine the expression of BRSK1 in normal and cisplatin-resistant cells. Sulforhodamine B assay was conducted to assess the sensitivity of cervical cancer cells to cisplatin. Seahorse Cell Mito Stress Test assay was utilized to evaluate the mitochondrial respiration in cervical cancer cells. RESULTS: BRSK1 expression was upregulated in cisplatin-treated cervical cancer patient tumors and cell lines compared with untreated tumors and cell lines. Depletion of BRSK1 significantly enhanced the sensitivity of both normal and cisplatin-resistant cervical cancer cells to cisplatin treatment. Moreover, BRSK1-mediated regulation of cisplatin sensitivity is conducted by a subpopulation of BRSK1 residing in the mitochondria of cervical cancer cells and is dependent on its kinase enzymatic activity. Mechanistically, BRSK1 confers cisplatin resistance via the regulation of mitochondrial respiration. Importantly, treatment with mitochondrial inhibitor in cervical cancer cells phenocopied the BRSK1 depletion-mediated mitochondria dysfunction and cisplatin sensitization. Of note, we observed that high BRSK1 expression is correlated with poor prognosis in cisplatin-treated cervical cancer patients. CONCLUSION: Our study defines BRSK1 as a novel regulator of cisplatin sensitivity, identifying that targeting BRSK1-regulated mitochondrial respiration could be a useful approach for enhancing the efficacy of cisplatin-based chemotherapy in cervical cancer patients.

Engineered ATG8-binding motif-based selective autophagy to degrade proteins and organelles in planta.

Protein-targeting technologies represent essential approaches in biological research. Protein knockdown tools developed recently in mammalian cells by exploiting natural degradation mechanisms allow for precise determination of protein function and discovery of degrader-type drugs. However, no method to directly target endogenous proteins for degradation is currently available in plants. Here, we describe a novel method for targeted protein clearance by engineering an autophagy receptor with a binder to provide target specificity and an ATG8-binding motif (AIM) to link the targets to nascent autophagosomes, thus harnessing the autophagy machinery for degradation. We demonstrate its specificity and broad potentials by degrading various fluorescence-tagged proteins, including cytosolic mCherry, the nucleus-localized bZIP transcription factor TGA5, and the plasma membrane-anchored brassinosteroid receptor BRI1, as well as fluorescence-coated peroxisomes, using a tobacco-based transient expression system. Stable expression of AIM-based autophagy receptors in Arabidopsis further confirms the feasibility of this approach in selective autophagy of endogenous proteins. With its wide substrate scope and its specificity, our concept of engineered AIM-based selective autophagy could provide a convenient and robust research tool for manipulating endogenous proteins in plants and may open an avenue toward degradation of cytoplasmic components other than proteins in plant research.

Colocalization Assay with Fluorescent-tagged ATG8 Using a Nicotiana benthamiana -based Transient System.

Autophagy is an evolutionarily conserved intracellular degradation process. During autophagy, a set of autophagy-related (ATG) proteins orchestrate the formation of double-bound membrane vesicles called autophagosomes to engulf cytoplasmic material and deliver it to the vacuole for breakdown. Among ATG proteins, the ATG8 is the only one decorating mature autophagosomes and therefore is regarded as a bona fide autophagic marker; colocalization assays with ATG8 are wildly used as a reliable method to identify the components of autophagy machinery or autophagic substrates. Here, we describe a colocalization assay with fluorescent-tagged ATG8 using a tobacco ( Nicotiana benthamiana )-based transient expression system.

Psychrobacter halodurans sp. nov. and Psychrobacter coccoides sp. nov., two new slightly halophilic bacteria isolated from marine sediment.

In this study, two bacterial strains designated F2608T and F1192T, isolated from marine sediment sampled in Weihai, PR China, were characterized using a polyphasic approach. Strains were aerobic, Gram-stain-negative and motile. According to the results of phylogenetic analyses based on their 16S rRNA genes, these two strains should be classified under the genus Psychrobacter and they both show <98.5% sequence similarity to their closest relative, Psychrobacter celer JCM 12601T. Moreover, strain F2608T showed 97.5% sequence similarity to strain F1192T. Strain F2608T grew at 4-37 °C (optimum, 30-33 °C) and at pH 6.0-9.0 (optimum, pH 6.5-7.0) in the presence of 0-12% (w/v) NaCl (optimum, 4.0-5.0%). Strain F1192T grew at 4-37 °C (optimum, 30 °C) and at pH 5.5-9.0 (optimum, pH 7.0-7.5) in the presence of 0.5-12% (w/v) NaCl (optimum, 3.0-4.0%). The genomic DNA G+C contents of strain F2608T and strain F1192T were 47.4 and 44.9 %, respectively. Genomic characteristics including average nucleotide identity and digital DNA-DNA hybridization values clearly separated strain F2608T from strain F1192T. The sole isoprenoid quinone in these two strains was ubiquinone 8 and the major cellular fatty acids (>10.0%) were C18:1 ω9c and C17:1 ω8c. The major polar lipids of these two strains were phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Based on the results of polyphasic analysis, the two strains represent two novel species of the genus Psychrobacter, for which the names Psychrobacter halodurans sp. nov. and Psychrobacter coccoides sp. nov. are proposed. The type strains are F2608T (=MCCC 1K05774T=KCTC 82766T) and F1192T (=MCCC 1K05775T=KCTC 82765T), respectively.

Gelidibacter maritimus sp. nov., isolated from marine sediment.

A Gram-stain-negative, yellow, strictly aerobic, non-flagellated, gliding, rod-shaped bacterial strain, was isolated from costal sediment, designated as F6074T. The strain F6074T grows optimally at 30 °C, pH 7.5, and 3.0% (w/v) NaCl. Cells of strain F6074T are 0.2-0.5 µm wide and 1.0-2.0 µm long. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain F6074T belonged to the genus Gelidibacter, with the highest sequence similarity to Gelidibacter japonicus JCM 31967T (98.0%), followed by G. flavus JCM 31135T (97.7%), and similarity between strain F6074T and the type species G. algens DSM 12408T was 96.0%. Genome sequencing results revealed a genome size of 47,07,621 bp. The DNA G + C content was 37.8 mol%. The ANI and dDDH values between strain F6074T and G. japonicus JCM 31967T were 83.9 and 27.8%, the values between strain F6074T and G. algens DSM 12408T were 77.5% and 31.5%, and the values between strain F6074T and G. flavus JCM 31135T were 84.3 and 27.9%, respectively. The predominant quinone was MK-6 and the major fatty acids were iso-C15:0, iso-C15:1G, iso-C17:0 3-OH, anteiso-C15:0 and summed feature 3. The polar lipids were consisted of phosphatidylethanolamine (PE), two unidentified aminolipids (AL) and three unidentified lipids (L1, L2, L3). Based on the phenotypic, phylogenetic and chemotaxonomic data, strain F6074T was considered to represent a novel species of the genus Gelidibacter, for which the name Gelidibacter maritimus sp. nov., is proposed. The type strain is F6074T (MCCC 1H00427T = KCTC 72942T).

Inhibition of IL-32 Expression Ameliorates Cerebral Ischemia-Reperfusion Injury via the NOD/MAPK/NF-κB Signaling Pathway.

Cerebral ischemia represents a major cause of disability, yet its precise mechanism remains unknown. In addition, ischemia-reperfusion injury which occurs during the blood recovery process increases the risk of mortality, and is not adequately addressed with current treatment. To improve therapeutic options, it is important to explore the vital substances that play a pivotal role in ischemia-reperfusion injury. This study is the first to investigate the role of IL-32, a vital pro-inflammatory factor, in models of cerebral ischemia-reperfusion injury. The results showed that IL-32 was highly expressed in both in vivo and in vitro models. The proteins of the NOD/MAPK/NF-κB pathway were also up-regulated, indicating a potential signaling pathway mechanism. Inhibition of IL-32 and blocking of the NOD/MAPK/NF-κB pathway increased cell survival, decreased the level of inflammatory factors and inflammasomes, and attenuated nitrosative stress. Taken together, the results show that inhibition of IL-32 expression ameliorates cerebral ischemia-reperfusion injury via the NOD/MAPK/NF-κB signaling pathway. The findings in this study reveal that IL-32 is a vital target of ischemia-reperfusion injury, providing a new avenue for treatment development.

A novel RAB39B gene mutation in X-linked juvenile parkinsonism with basal ganglia calcification.

OBJECTIVES: Mutations in RAB39B have been reported as a potential cause of X-linked Parkinson's disease (PD), a rare form of familial PD. We conducted a genetic analysis on RAB39B to evaluate whether RAB39B mutations are related to PD in the Chinese population. METHODS: In this study, 2 patients from an X-linked juvenile parkinsonism pedigree were clinically characterized and underwent whole-exome sequencing. A comprehensive screening for RAB39B mutations in 505 sporadic patients with PD and 510 healthy controls in a Chinese population was also performed. RESULTS: A novel mutation, c. 536dupA (p.E179fsX48), in RAB39B was identified in the juvenile parkinsonism pedigree. Brain MRI and CT scans in the 2 patients revealed calcification within the bilateral globus pallidus. No other potentially disease-causing RAB39B mutations were found in sporadic PD patients and controls. CONCLUSIONS: X-linked juvenile parkinsonism could be caused by a RAB39B mutation, and basal ganglia calcification may be a novel clinical feature of RAB39B-related parkinsonism. © 2016 International Parkinson and Movement Disorder Society.

Ethylene/propylene copolymerization catalyzed by vanadium complexes containing N-heterocyclic carbenes.

Various vanadium complexes containing N-heterocyclic carbenes, VOCl3[1,3-R2(NCH=)2C:] (V1, R = 2,6-Me2C6H3; V2, R = 2,6-Et2C6H3; V3, R = 2,6-(i)Pr2C6H3; V4, R = 2,4,6-Me3C6H2), have been synthesized and employed as catalyst precursors for ethylene/propylene copolymerization after activation by Et3Al2Cl3. Complex V4 showed higher catalytic activity of ca. 38 kg copolymer per (mol of V) per h and an ethylene/propylene copolymer with random monomer distribution could be prepared. Complex V3 consumed more cocatalyst than its analogues to reach higher catalytic activity. The obtained copolymers exhibit relatively narrow polydispersity and contain more randomly distributed monomer units than that the copolymers prepared by using the traditional vanadium catalytic system.

Baicalein suppresses 17-ß-estradiol-induced migration, adhesion and invasion of breast cancer cells via the G protein-coupled receptor 30 signaling pathway.

Flavonoids are structurally similar to steroid hormones, particularly estrogens, and therefore have been studied for their potential effects on hormone-dependent cancers. Baicalein is the primary flavonoid derived from the root of Scutellaria baicalensis Georgi. In the present study, we investigated the effects of baicalein on 17ß-estradiol (E2)-induced migration, adhesion and invasion of MCF-7 and SK-BR-3 breast cancer cells. The results demonstrated that baicalein suppressed E2-stimulated wound-healing migration and cell­Matrigel adhesion, and ameliorated E2-promoted invasion across a Matrigel-coated Transwell membrane. Furthermore, baicalein interfered with E2-induced novel G protein-coupled estrogen receptor (GPR30)-related signaling, including a decrease in tyrosine phosphorylation of epidermal growth factor receptor (EGFR) as well as phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine kinase Akt, without affecting GPR30 expression. The results also showed that baicalein suppressed the expression of GPR30 target genes, cysteine-rich 61 (CYR61) and connective tissue growth factor (CTGF) induced by E2. Furthermore, baicalein prevented GPR30-related signaling activation and upregulation of CYR61 and CTGF mRNA levels induced by G1, a specific GPR 30 agonist. The results suggest that baicalein inhibits E2-induced migration, adhesion and invasion through interfering with GPR30 signaling pathway activation, which indicates that it may act as a therapeutic candidate for the treatment of GPR30-positive breast cancer metastasis.

Cytotoxicity of ultrafine monodispersed nanoceria on human gastric cancer cells.

The safety and toxicity of CeO2 nanoparticles (nanoceria) are of growing concern due to their potential applications in biological and medical fields based on the radical scavenging and UV-filtering properties. In this paper, the ultrafine monodisperse (2-5 nm) water-insoluble (CeO2-P) and water-soluble nanoceria modified with various functional groups of dextran (CeO2-dextran), polyacrylic acid (CeO2-PAA) and ethylenediamine (CeO2-EDA) on surface were synthesized via alkaline-based precipitation and inverse microemulsion methods. The cell uptaking, oxidative stress and cytotoxicity of these nanoceria on human gastric cancer cell line (BGC-803) were systematically investigated. It is found that the cell uptaking of nanoceria is largely relied on the function groups on its surfaces and followed the order: CeO2-P > CeO2-EDA > CeO2-dextran > CeO2-PAA. Moreover, the oxidative stress of BGC-803 cells is obviously affected by the antioxidant capacity of nanoceria determined by Ce3+/Ce4+ ratio, which eventually causes the cell viability variable once the nanoceria entered into BGC-803 cells. In addition, the cell viability is also closely correlated with the concentration and surface characteristics of nanoceria. The cytotoxicity of nanoceria on BGC-803 cells is largely dependent on its surface functional groups. Our work may provide guidance on the cytotoxicity of ultrafine monodisperse nanoceria for their uses in biological and medical fields.

Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell-matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis.

Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer.

Mutations of the EPHA2 receptor tyrosine kinase gene cause autosomal dominant congenital cataract.

Congenital cataracts (CCs) are clinically and genetically heterogeneous. Mutations in the same gene may lead to CCs differing in inheritance, morphology and severity. Loci for autosomal dominant posterior polar CC and total CC have both been mapped to the chromosomal 1p36 region harboring the EPHA2 receptor tyrosine kinase gene. Here, we report mutations of EPHA2 in three CC families from different ancestral groups. In a Chinese family with posterior polar CC, we identified a missense mutation, c.2819C>T (p.T940I), replacing a critical amino acid that functions at the receptor oligomerization interface. In a British family with posterior polar CC and an Australian family with total CC, we found a frameshift mutation (c.2915_2916delTG) and a splicing mutation (c.2826-9G>A), respectively. These two mutations are predicted to produce novel C-terminal polypeptides with 39 identical amino acids. Yeast two-hybrid analysis showed stronger interaction between the total CC-associated mutant EPHA2 and low molecular weight protein-tyrosine phosphatase, a negative regulator of EPHA2 signaling. Our results implicate the Eph-ephrin signaling system in development of human cataract and provide a novel insight into the molecular mechanism underlying the pathogenesis of human CCs.