Hepatic hydrothorax does not increase the risk of death after transjugular intrahepatic portosystemic shunt in cirrhosis patients.

OBJECTIVES: Hepatic hydrothorax (HH) is a predictor of poor survival in cirrhosis patients. However, whether HH increases the mortality risk of cirrhosis patients treated with transjugular intrahepatic portosystemic shunt (TIPS) is unknown. Our objective was to evaluate the influence of HH on the survival of cirrhosis patients after TIPS. METHODS: Cirrhosis patients with portal hypertension complications were selected from a prospective database of consecutive patients treated with TIPS in Xijing Hospital from January 2015 to June 2021. Cirrhosis patients with HH were treated as the experimental group. A control group of cirrhosis patients without HH was created using propensity score matching. Survival after TIPS and the related risk factors were analysed. RESULTS: There were 1292 cirrhosis patients with portal hypertension complications treated with TIPS, among whom 255 patients had HH. Compared with patients without HH, patients with HH had worse liver function (MELD, 12 vs. 10, p < 0.001), but no difference in survival after TIPS was observed. After propensity score matching, 243 patients with HH and 243 patients without HH were enrolled. There was no difference in cumulative survival between patients with and without HH. Cox regression analysis showed that HH was not associated with survival after TIPS, and main portal vein thrombosis (> 50%) was a prognostic factor of long-term survival after TIPS in cirrhosis patients (hazard ratio, 1.386; 95% CI, 1.030-1.865, p = 0.031). CONCLUSION: Hepatic hydrothorax does not increase the risk of death after TIPS in cirrhosis patients. KEY POINTS: • Hepatic hydrothorax is a decompensated event of cirrhosis and increases the risk of death. • Hepatic hydrothorax is associated with worse liver function. • Hepatic hydrothorax does not increase the mortality of cirrhosis treated with TIPS.

Effects of hyperbaric oxygen pretreatment on brain antioxidant capacity in rats with decompression sickness.

OBJECTIVE: Decompression sickness (DCS) causes serious brain hypoxic-ischemic injury. This experiment was designed to observe whether hyperbaric oxygen (HBO2) pretreatment played a neuroprotective effect in decompression sickness rat models and to explore the mechanism of protective effects. METHODS: Sprague-Dawley (SD) male rats were pretreated with HBO2 and then underwent decompression to establish the DCS rat model. Antioxidant capacities were evaluated by detecting peroxides (GPx), superoxide dismutase (SOD), catalase (CAT) activity and malondialdehyde (MDA) content in brains. The levels of metal elements manganese (Mn), zinc (Zn), iron (Fe) and magnesium (Mg) in brain tissues were assessed by flame atomic absorption spectrometry. Necrosis and apoptosis of neurons were assessed by H-E staining and immunohistochemical staining. RESULTS: HBO2 pretreatment reduced the degree of necrosis and apoptosis in brain tissues of decompression sickness rat models. In addition, HBO2 pretreatment increased GPx, SOD and CAT activities and reduced MDA accumulation. It also increased the content of Mn, Zn, Fe and Mg in brain tissue, which are all related to free radical metabolism. CONCLUSION: These results suggested that HBO2 pretreatment has protective effects on brain injury of rats with decompression sickness. The mechanism of the protective effects may be related to reducing oxidative damage by affecting metal elements in vivo.

Bifidobacterium infantis Relieves Allergic Asthma in Mice by Regulating Th1/Th2.

BACKGROUND Bifidobacteria are among the probiotics used in treating intestinal diseases and are rarely used for allergic asthma treatment. The present study investigated the mechanism of B. infantis in treating allergic asthma in mice. MATERIAL AND METHODS A total of 40 male Balb/c mice were randomized into control, ovalbumin (OVA), montelukast (Mon), and B. infantis (B10) groups, and allergic asthma was induced in the OVA, Mon, and B10 groups. Airway reactivity was measured on day 29 by methacholine at various doses. The numbers of total cells and inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted by blood cell counter and Diff-Quik staining. Hematoxylin-eosin (HE) staining was performed to observe inflammatory cell infiltration in lung tissues. Total IgE and OVA-specific IgE in serum were measured by ELISA. Mucin 5AC expression was detected by Western blot to evaluate airway obstruction. The levels of Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-13) cytokines in BALF and tissues were detected by ELISA and qRT-PCR, respectively. RESULTS The mice in the OVA group had airway hyperreactivity, while the symptoms in the B10 group and Mon group were effectively relieved. B10 reduced the number of inflammatory cells in BALF as well as inflammatory cell infiltration in tissues. Moreover, the levels of total serum IgE, OVA-specific IgE, and Mucin 5AC were increased in the OVA group, but were reduced in the Mon group and B10 group. B. infantis increased the levels of Th1 cytokines and decreased those of Th2 cytokines. CONCLUSIONS B. infantis can reduce the infiltration of inflammatory cells induced by OVA-specific antibodies in mice. B. infantis has therapeutic effects on allergic asthma by promoting Th1 and inhibiting Th2 immune responses.

Probing electron transfer between hemin and riboflavin using a combination of analytical approaches and theoretical calculations.

Extracellular electron transfer (EET) occurs from outer-membrane proteins to electron acceptors. Heme(ii) is the active center of outer-membrane proteins and delivers electrons to acceptors or mediators such as riboflavin, a redox active chromophore present in organisms. However, the EET mechanism via mediators, especially the electron transfer process from outer-membrane proteins to mediators, has not been well documented yet. In this work, the mechanism behind the electron transfer from heme(ii) to riboflavin is investigated by using in situ ultraviolet visible and fluorescence spectroelectrochemical analysis, which provides the information regarding the structural change and electrochemical characteristics of species in the electron transfer process. It is found that hemin(iii), the oxidized form of heme(ii), is electrolyzed to an intermediate "hemx(ii)" without structural changes, and is then transformed to heme(ii) by conjugating with riboflavin and its radicals. Heme(ii) is able to activate riboflavin reduction via a two-electron two-proton pathway in aqueous solution. The mechanisms proposed on the basis of experimental results are further confirmed by density functional theory calculations. The results about the electron transfer from hemx(ii) (or heme(ii)) to riboflavin are useful not only for understanding the EET mechanisms, but also for maximizing the role of riboflavin in biogeochemical cycling and environmental bioremediation.

Effects of hyperbaric oxygen on hippocampal neuronal apoptosis in rats with acute carbon monoxide poisoning.

INTRODUCTION: Acute carbon monoxide (CO) poisoning causes serious health problems such as neuropsychological sequelae. This study aimed to investigate neuronal apoptosis and the effects of hyperbaric oxygen (HBO2) on different regions of the rat hippocampus after CO poisoning. METHODS: 90 mature male Sprague Dawley rats were randomly divided into three groups: the normal control group (NC group), the acute carbon monoxide-poisoned group (CO group) and the hyperbaric oxygen treatment group (HBO2 group). CO exposure included 0, 1, 3, 7, 14 and 21 treatment days, one exposure on the first day, and sacrifice on each of the following days. HBO2 exposure included treatment for 0, 1, 3, 7, 14 and 21 days, daily treatment after CO exposure, and sacrifice after the last HBO2 treatment on each of those days. Hematoxylin-eosin staining, immunohistochemical staining, immunofluorescence staining, and western blot analysis were performed to detect apoptosis in brain tissue samples. RESULTS: MMP-9 and caspase-3 were prominently increased by CO exposure and inhibited by HBO2 in the CA3 region in the hippocampus at one, three and seven days (immunohistochemical staining [IHC]: P ⟨ 0.05). Neu N and the ratio of Bcl-2/ BAX were prominently decreased by CO exposure and rescued by HBO2 in the CA3 region after seven days of treatment (IHC: P ⟨ 0.05). CONCLUSION: These findings indicated that neuronal apoptosis in the rat hippocampus could be induced by acute CO exposure, especially in the CA3 region. HBO2 could effectively inhibit neuronal apoptosis, especially in the CA3 region after seven days of treatment. The application of HBO2 to inhibit MMP-9 and apoptosis may contribute to brain recovery after acute CO poisoning.

POU2F2-oriented network promotes human gastric cancer metastasis.

BACKGROUND AND AIMS: Aberrant upregulation of POU2F2 expression has been discovered in metastatic gastric cancer (GC). However, the mechanisms underlying the aberrant upregulation and the potential functions of POU2F2 remain uncertain. DESIGN: The role and mechanism of POU2F2 in GC metastasis were investigated in gastric epithelial cells, GC cell lines and an experimental metastasis animal model by gain of function and loss of function. Upstream and downstream targets of POU2F2 were selected by bioinformatics and identified by luciferase reporter assay, electrophoretic mobility shift assay and chromatin immunoprecipitation PCR. The influence of miR-218 on its putative target genes (POU2F2, ROBO1 and IKK-ß) and GC metastasis was further explored via in vitro and in vivo approaches. RESULTS: Increased POU2F2 expression was detected in metastatic GC cell lines and patient samples. POU2F2 was induced by the activation of nuclear factor (NF)-κB and, in turn, regulated ROBO1 transcription, thus functionally contributing to GC metastasis. Finally, miR-218 was found to suppress GC metastasis by simultaneously mediating multiple molecules in the POU2F2-oriented network. CONCLUSIONS: This study demonstrated that NF-κB and the SLIT2/ROBO1 interaction network with POU2F2 as the central part may exert critical effects on tumour metastasis. Blocking the activation of the POU2F2-oriented metastasis network using miR-218 precursors exemplified a promising approach that sheds light on new strategies for GC treatment.

Identification of early gene expression changes in primary cultured neurons treated with topoisomerase I poisons.

Topoisomerase 1 (TOP1) poisons like camptothecin (CPT) are currently used in cancer chemotherapy but these compounds can have damaging, off-target effects on neurons leading to cognitive, sensory and motor deficits. To understand the molecular basis for the enhanced sensitivity of neurons to CPT, we examined the effects of compounds that inhibit TOP1-CPT, actinomycin D (ActD) and ß-lapachone (ß-Lap)-on primary cultured rat motor (MN) and cortical (CN) neurons as well as fibroblasts. Neuronal cells expressed higher levels of Top1 mRNA than fibroblasts but transcript levels are reduced in all cell types after treatment with CPT. Microarray analysis was performed to identify differentially regulated transcripts in MNs in response to a brief exposure to CPT. Pathway analysis of the differentially expressed transcripts revealed activation of ERK and JNK signaling cascades in CPT-treated MNs. Immediate-early genes like Fos, Egr-1 and Gadd45b were upregulated in CPT-treated MNs. Fos mRNA levels were elevated in all cell types treated with CPT; Egr-1, Gadd45b and Dyrk3 transcript levels, however, increased in CPT-treated MNs and CNs but decreased in CPT-treated fibroblasts. These transcripts may represent new targets for the development of therapeutic agents that mitigate the off-target effects of chemotherapy on the nervous system.

RNA sequencing analysis reveals new findings of hyperbaric oxygen treatment on rats with acute carbon monoxide poisoning.

OBJECTIVE: To elucidate the altered gene network in the brains of carbon monoxide (CO) poisoned rats after treatment with hyperbaric oxygen (HBO2). METHODS: RNA sequencing (RNA-seq) analysis was performed to examine differentially expressed genes (DEGs) in brain tissue samples from nine male rats: a normal control group; a CO poisoning group; and an HBO2 treatment group (three rats/group). Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR were used for validation of the DEGs in another 18 male rats (six rats/group). RESULTS: RNA-seq revealed that two genes were upregulated (4.18 and 8.76 log to the base 2 fold change) (p⟨0.05) in the CO-poisoned rats relative to the control rats; two genes were upregulated (3.88 and 7.69 log to the base 2 fold change); and 23 genes were downregulated (3.49-15.12 log to the base 2 fold change) (p⟨0.05) in the brains of the HBO2-treated rats relative to the CO-poisoned rats. Target prediction of DEGs by gene network analysis and analysis of pathways affected suggested that regulation of gene expressions of dopamine metabolism and nitric oxide (NO) synthesis were significantly affected by CO poisoning and HBO2 treatment. Results of RT-PCR and real-time quantitative PCR indicated that four genes (Pomc, GH-1, Pr1 and Fshß) associated with hormone secretion in the hypothalamic-pituitary system have potential as markers for prognosis of CO. CONCLUSION: This study is the first RNA-seq analysis profile of HBO2 treatment on rats with acute CO poisoning. It concludes that changes of hormone secretion in the hypothalamic-pituitary system, dopamine metabolism and NO synthesis involved in brain damage and behavior abnormalities after CO poisoning and HBO2 therapy may regulate these changes.

Effects of hydrogen-rich saline on rats with acute carbon monoxide poisoning.

BACKGROUND: Studies have shown that inhalation of hydrogen gas, which acts as an antioxidant, can protect the brain against free radicals in rats with ischemia-reperfusion. The neuronal damage caused by acute carbon monoxide (CO) poisoning is partly free radical mediated. We hypothesize that hydrogen may prevent neurological damage from CO poisoning. OBJECTIVES: This study is designed to test whether hydrogen (H(2))-rich saline will have a protective effect on rats with acute CO poisoning. METHODS: Male Sprague-Dawley rats were subjected to CO poisoning. H(2)-rich saline was administered by peritoneal injection (6 mL/kg/24 h). We used the Morris water maze and the open field test to determine cognitive function. After cognitive function studies, rats were decapitated and the levels of trace elements copper (Cu), zinc (Zn), and iron (Fe) in serum and brain were assessed by flame atomic absorption spectrometry. Necrosis, apoptosis, and autophagy of neurons were assessed by H-E staining and immunohistochemical staining in another group of rats. RESULTS: H(2)-rich saline treatment improved the cognitive deficits and reduced the degree of necrosis, apoptosis, and cell autophagy in rats. Additionally, H(2)-rich saline decreased the content of Fe in serum and brain in these rats, and increased the content of serum Cu related to free radical metabolism. CONCLUSIONS: H(2)-rich saline may effectively protect the brain from injury after acute CO poisoning. The mechanism of this protection may be related to lessening oxidative damage by affecting trace elements in vivo.

Transjugular intrahepatic collateral-systemic shunt is effective for cavernous transformation of the portal vein with variceal bleeding.

BACKGROUND: The transjugular intrahepatic portal collateral-systemic shunt (transcollateral TIPS) is used to treat portal hypertension-related complications in patients with cavernous transformation of the portal vein (CTPV) and whose main portal vein cannot be recanalized. It is still not clear whether transcollateral TIPS can be as effective as portal vein recanalization-transjugular intrahepatic portosystemic shunt (PVR-TIPS). This study aimed to evaluate the efficacy and safety of transcollateral TIPS in the treatment of refractory variceal bleeding with CTPV. METHODS: Patients with refractory variceal bleeding caused by CTPV were selected from the database of consecutive patients treated with TIPS in Xijing Hospital from January 2015 to March 2022. They were divided into the transcollateral TIPS group and the PVR-TIPS group. The rebleeding rate, overall survival, shunt dysfunction, overt hepatic encephalopathy (OHE) and operation-related complications were analyzed. RESULTS: A total of 192 patients were enrolled, including 21 patients with transcollateral TIPS and 171 patients with PVR-TIPS. Compared with the patients with PVR-TIPS, the patients with transcollateral TIPS had more noncirrhosis (52.4 vs. 19.9%, p = 0.002), underwent fewer splenectomies (14.3 vs. 40.9%, p = 0.018), and had more extensive thromboses (38.1 vs. 15.2%, p = 0.026). There were no differences in rebleeding, survival, shunt dysfunction, or operation-related complication rates between the transcollateral TIPS and PVR-TIPS groups. However, the OHE rate was significantly lower in the transcollateral TIPS group (9.5 vs. 35.1%, p = 0.018). CONCLUSION: Transcollateral TIPS is an effective treatment for CTPV with refractory variceal bleeding.

Identification of the phosphorylation sites in the survival motor neuron protein by protein kinase A.

The survival motor neuron (SMN) protein plays an essential role in the assembly of uridine-rich small nuclear ribonuclear protein complexes. Phosphorylation of SMN can regulate its function, stability, and sub-cellular localization. This study shows that protein kinase A (PKA) phosphorylates SMN both in vitro and in vivo. Bioinformatic analysis predicts 12 potential PKA phosphorylation sites in human SMN. Mass spectrometric analysis of a tryptic digest of SMN after PKA phosphorylation identified five distinct phosphorylation sites in SMN (serines 4, 5, 8, 187 and threonine 85). Mutagenesis of this subset of PKA-phosphorylated sites in SMN affects association of SMN with Gemin2 and Gemin8. This result indicates that phosphorylation of SMN by PKA may play a role in regulation of the in vivo function of SMN.

MicroRNA 322-5p reduced neuronal inflammation via the TLR4/TRAF6/NF-κB axis in a rat epilepsy model.

This study aimed to determine whether microRNA-322-5p regulates seizure and seizure damage by targeting the TLR4/TRAF6/NF-κB-associated inflammatory signaling pathway. In a pilocarpine-induced epileptic rat model, the expressions of miR-322-5p, TLR4, NF-κB, TRAF6, IRF5, IL-1ß, and GABA were assessed by a quantitative polymerase chain reaction and western blotting. Tunel detects hippocampal neuron apoptosis. The results showed that the expression of miR-322-5p significantly decreased in status epilepticus (SE) rats. The reduction of miR-322-5p was accompanied by increased levels of pro-inflammatory cytokines, an increased NF-κB expression, and reduced γ-aminobutyric acid (GABA) levels. Exogenous miR-322-5p reduced the expression of inflammatory molecules and increased the GABA levels in SE rats, and also reduced hippocampal neuronal cell apoptosis caused by epilepsy. In conclusion, the miR-322-5p significantly inhibited the TLR4/TRAF6/NF-κB-associated inflammation and reduced neuronal apoptosis, suggesting that its induction may be of potential interest for novel antiseizure medications.

MiR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.

Background: Gastric cancer (GC) is one of the most malignant and lethal cancers worldwide. Multiple microRNAs (miRNAs) have been identified as key regulators in the progression of GC. However, the underlying pathogenesis that miRNAs govern GC malignancy remains uncertain. Here, we identified a novel miR-585-5p as a key regulator in GC development. Methods: The expression of miR-585-5p in the context of GC tissue was detected by in situ hybridization for GC tissue microarray and assessed by H-scoring. The gain- and loss-of-function analyses comprised of Cell Counting Kit-8 assay and Transwell invasion and migration assay. The expression of downstream microphthalmia-associated transcription factor (MITF), cyclic AMP-responsive element-binding protein 1 (CREB1) and mitogen-activated protein kinase 1 (MAPK1) were examined by Immunohistochemistry, quantitative real-time PCR and western blot. The direct regulation between miR-585-5p and MITF/CREB1/MAPK1 were predicted by bioinformatic analysis and screened by luciferase reporter assay. The direct transcriptional activation of CREB1 on MITF was verified by luciferase reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs). The interaction between MAPK1 and MITF was confirmed by co-immunoprecipitation (Co-IP) and immunofluorescent double-labelled staining. Results: MiR-585-5p is progressively downregulated in GC tissues and low miR-585-5p levels were strongly associated with poor clinical outcomes. Further gain- and loss-of-function analyses showed that miR-585-5p possesses strong anti-proliferative and anti-metastatic capacities in GC. Follow-up studies indicated that miR-585-5p targets the downstream molecules CREB1 and MAPK1 to regulate the transcriptional and post-translational regulation of MITF, respectively, thus controlling its expression and cancer-promoting activity. MiR-585-5p directly and negatively regulates MITF together with CREB1 and MAPK1. According to bioinformatic analysis, promotor reporter gene assays, ChIP and EMSAs, CREB1 binds to the promotor region to enhance transcriptional expression of MITF. Co-IP and immunofluorescent double-labelled staining confirmed interaction between MAPK1 and MITF. Protein immunoprecipitation revealed that MAPK1 enhances MITF activity via phosphorylation (Ser73). MiR-585-5p can not only inhibit MITF expression directly, but also hinder MITF expression and pro-cancerous activity in a CREB1-/MAPK1-dependent manner indirectly. Conclusions: In conclusion, this study uncovered miR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.

Proteomic assessment of a cell model of spinal muscular atrophy.

BACKGROUND: Deletion or mutation(s) of the survival motor neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), a neuromuscular disease characterized by spinal motor neuron death and muscle paralysis. Complete loss of the SMN protein is embryonically lethal, yet reduced levels of this protein result in selective death of motor neurons. Why motor neurons are specifically targeted by SMN deficiency remains to be determined. In this study, embryonic stem (ES) cells derived from a severe SMA mouse model were differentiated into motor neurons in vitro by addition of retinoic acid and sonic hedgehog agonist. Proteomic and western blot analyses were used to probe protein expression alterations in this cell-culture model of SMA that could be relevant to the disease. RESULTS: When ES cells were primed with Noggin/fibroblast growth factors (bFGF and FGF-8) in a more robust neural differentiation medium for 2 days before differentiation induction, the efficiency of in vitro motor neuron differentiation was improved from ~25% to ~50%. The differentiated ES cells expressed a pan-neuronal marker (neurofilament) and motor neuron markers (Hb9, Islet-1, and ChAT). Even though SMN-deficient ES cells had marked reduced levels of SMN (~20% of that in control ES cells), the morphology and differentiation efficiency for these cells are comparable to those for control samples. However, proteomics in conjunction with western blot analyses revealed 6 down-regulated and 14 up-regulated proteins with most of them involved in energy metabolism, cell stress-response, protein degradation, and cytoskeleton stability. Some of these activated cellular pathways showed specificity for either undifferentiated or differentiated cells. Increased p21 protein expression indicated that SMA ES cells were responding to cellular stress. Up-regulation of p21 was confirmed in spinal cord tissues from the same SMA mouse model from which the ES cells were derived. CONCLUSION: SMN-deficient ES cells provide a cell-culture model for SMA. SMN deficiency activates cellular stress pathways, causing a dysregulation of energy metabolism, protein degradation, and cytoskeleton stability.

Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan.

Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5-500 kDa were investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffer systems was determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample as well as calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa at sample loads of 0.5 µg (for polyacrylamide) to 2.5 µg (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1-IGD-G2 domains) to a 150-kDa HA standard.

Effects of immune reaction in rats after acute carbon monoxide poisoning.

This study is designed to observe the immune reaction in rats after acute carbon monoxide (CO) poisoning. We observed brain injury, cognitive impairment, a variety of microglias and expression of immune factors, including major histocompatibility complex II (MHCII), CD4, vascular cell adhesion molecule-1 (VCAM-1) and interferon-gamma (IFN-gamma) in the brain tissues of CO-poisoned rats. Then relationships between cognitive impairment and immune factors were explored. We found that there were extensive neuronal degeneration and necrosis in the brains of CO-poisoned rats, and the escape latency of the CO Group in a Morris water maze became significantly longer than that of the Control Group (11.63 +/- 3.54s vs. 7.06 +/- 3.13s, p < 0.05) after six days of CO poisoning. Microglias, as immune effector cells, underwent activation and proliferation which reached 35.0 +/- 5.7 cells per five high-power fields (HPF) in the seventh day after CO poisoning, but 20.3 +/- 2.9 cells/5HPF in the Control Group (p < 0.05). Expression levels of immune factors increased in the brains of CO-poisoned rats. VCAM-1-positive cells peaked in quantity the first day, IFN-gamma-positive cells and MHCII-positive cells the third day and CD4-positive cells the seventh day. The results indicate that immune reaction plays an important role on CO-mediated neuropathology.

The lncRNA PVT1/miR-590-5p/FSTL1 axis modulates the proliferation and migration of airway smooth muscle cells in asthma.

OBJECTIVE: Asthma is a prevalent chronic inflammatory airway disease that is characterised by airway remodelling and airway hyperresponsiveness. Abnormal proliferation and migration of airway smooth muscle cells (ASMCs) contribute to airway remodelling in asthma. However, the molecular mechanism underlying an increased ASMC mass in asthma remains elusive. Herein, we aimed at investigating the regulation of lncRNA PVT1 on ASMCs and focussing on the mechanism in the proliferation and migration. METHODS: Expression levels of lncRNA PVT1 and miR-590-5p in the serum collected from 24 children with asthma and 10 control children were determined by qRT-PCR. ASMCs proliferation and migration prior to and post platelet-derived growth factor subunit B (PDGF-BB) stimulation were examined by CCK-8 test and transwell assay. Dual-luciferase reporter assay was performed to determine miR-590-5p interaction with lncRNA PVT1 and follistatin-like 1 (FSTL1). Expression of lncRNA PVT1, miR-590-5p, FSTL1, C-Myc, cyclin D1, and cyclin-dependent kinase 1 (CDK1) was tested by quantitative real-time PCR (qRT-PCR) and immunoblotting analysis. RESULTS: The expression level of lncRNA PVT1 was higher but the expression level of miR-590-5p was lower in the serum of children with asthma than in control children. The expression level of lncRNA PVT1 was negatively correlated with the expression level of miR-590-5p in asthma. LncRNA PVT1 was upregulated upon PDGF-BB stimulation. LncRNA PVT1 knockdown by its specific shRNA repressed PDGF-BB-induced promotion of proliferation and migration in ASMCs and triggered an elevated miR-590-5p along with declined C-Myc, cyclin D1, and CDK1. The effects of lncRNA PVT1 knockdown on PDGF-BB-induced ASMCs were lost upon miR-590-5p inhibition. MiR-590-5p targeted FSTL1 gene and declined its expression, thus suppressing ASMC proliferation and migration following PDGF-BB stimulation and downregulating C-Myc, cyclin D1, and CDK1 expressions. The effects of miR-590-5p on PDGF-BB-induced ASMCs were lost upon FSTL1 overexpression. CONCLUSION: These results support the notion that the lncRNA PVT1/miR-590-5p/FSTL1 axis modulates ASMCs proliferation and migration following PDGF-BB stimulation, providing a potential therapeutic target to attenuate airway remodelling in asthma.

Crystal structure of the N-terminal domain of ryanodine receptor from the honeybee, Apis mellifera.

Ryanodine receptors (RyRs) are the molecular target of diamides, a new chemical class of insecticides. Diamide insecticides are used to control lepidopteran pests and were considered relatively safe for mammals and non-targeted beneficial insects, including honey bees. However, recent studies showed that exposure to diamides could cause long-lasting locomotor deficits of bees. Here we report the crystal structure of RyR N-terminal domain A (NTD-A) from the honeybee, Apis mellifera, at 2.5 Å resolution. It shows a similar overall fold as the RyR NTD-A from mammals and the diamondback moth (DBM), Plutella xylostella, and still several loops located at the inter-domain interfaces show insect-specific or bee-specific structural features. A potential insecticide-binding pocket formed by loop9 and loop13 is conserved in lepidopteran but different in both mammals and bees, making it a good candidate targeting site for the development of pest-selective insecticides. Furthermore, a conserved intra-domain disulfide bond was observed in both DBM and bee RyR NTD-A crystal structures, which explains their higher thermal stability compared to mammalian RyR NTD-A. This work provides a basis for the development of novel insecticides with better selectivity between pests and bees by targeting a distinct site on pest RyRs, which would be a promising strategy to overcome the current toxicity problem.

Increased susceptibility of spinal muscular atrophy fibroblasts to camptothecin is p53-independent.

BACKGROUND: Deletion or mutation(s) of the survival motor neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA). The SMN protein is known to play a role in RNA metabolism, neurite outgrowth, and cell survival. Yet, it remains unclear how SMN deficiency causes selective motor neuron death and muscle atrophy seen in SMA. Previously, we have shown that skin fibroblasts from SMA patients are more sensitive to the DNA topoisomerase I inhibitor camptothecin, supporting a role for SMN in cell survival. Here, we examine the potential mechanism of camptothecin sensitivity in SMA fibroblasts. RESULTS: Camptothecin treatment reduced the DNA relaxation activity of DNA topoisomerase I in human fibroblasts. In contrast, kinase activity of DNA topoisomerase I was not affected by camptothecin, because levels of phosphorylated SR proteins were not decreased. Upon camptothecin treatment, levels of p53 were markedly increased. To determine if p53 plays a role in the increased sensitivity of SMA fibroblasts to camptothecin, we analyzed the sensitivity of SMA fibroblasts to another DNA topoisomerase I inhibitor, beta-lapachone. This compound is known to induce death via a p53-independent pathway in several cancer cell lines. We found that beta-lapachone did not induce p53 activation in human fibroblasts. In addition, SMA and control fibroblasts showed essentially identical sensitivity to this compound. By immunofluorescence staining, SMN and p53 co-localized in gems within the nucleus, and this co-localization was overall reduced in SMA fibroblasts. However, depletion of p53 by siRNA did not lessen the camptothecin sensitivity in SMA fibroblasts. CONCLUSION: Even though p53 and SMN are associated, the increased sensitivity of SMA fibroblasts to camptothecin does not occur through a p53-dependent mechanism.

3-(1H-Imidazo[4,5-f][1,10]phenanthrolin-2-yl)benzonitrile methanol solvate.

In the title compound, C(20)H(11)N(5)·CH(3)OH, the benzene ring is twisted by a small dihedral angle of 1.89 (11)° with respect to the imidazo[4,5-f][1,10]phenanthroline ring system. N-H⋯O and O-H⋯N hydrogen bonding is present in the crystal structure.