Relationship of the lung microbiome with PD-L1 expression and immunotherapy response in lung cancer

IL-35 subunit EBI3 is up-regulated in pulmonary fibrosis tissues. In this study, we investigated the pathological role of EBI3 in pulmonary fibrosis and dissected the underlying molecular mechanism. Bleomycin-induced pulmonary fibrosis mouse model was established, and samples were performed gene expression analyses through RNAseq, qRT-PCR and Western blot. Wild type and EBI3 knockout mice were exposed to bleomycin to investigate the pathological role of IL-35, via lung function and gene expression analyses. Primary lung epithelial cells were used to dissect the regulatory mechanism of EBI3 on STAT1/STAT4 and STAT3. IL-35 was elevated in both human and mouse with pulmonary fibrosis. EBI3 knockdown aggravated the symptoms of pulmonary fibrosis in mice. EBI3 deficiency enhanced the expressions of fibrotic and extracellular matrix-associated genes. Mechanistically, IL-35 activated STAT1 and STAT4, which in turn suppressed DNA enrichment of STAT3 and inhibited the fibrosis process. IL-35 might be one of the potential therapeutic targets for bleomycin-induced pulmonary fibrosis.     

Monitoring the activation of neuronal nitric oxide synthase in brain tissue and cells with a potentiometric immunosensor

An all solid state potentiometric immunosensor (ASPI) has been developed to study the activation process of neuronal nitric oxide synthase (nNOS), the enzyme involved in the synthesis of nitric oxide generated under physiological conditions. At first, an all solid state H⁺-selective ISE was fabricated with the carboxylated poly(vinyl chloride) (PVC-COOH) film containing H⁺ ionophore, antibody was then immobilized on the polymer layer. The immunocomplex formation was detected by monitoring pH change due to interaction between urease labeled secondary antibody and antigen. Experimental parameters such as the amount of phosphorylated nNOS immobilized on the electrode surface and pH responses due to the antibody–antigen reaction were studied in detail. The calibration plot of the potentiometric potential vs. phosphorylated nNOS concentration exhibited a linear relationship in the range of 3.4–340.0 μg/ml. The calibration sensitivity of the phosphorylated nNOS immunosensor was −0.073 ± 0.002 mV/μg ml⁻¹. The detection limit of nNOS was determined to be 0.2 μg/ml based on five-time measurements (95% confidence level, k = 3, n = 5). The reliability of the immunosensor was examined with rat brain tissues as well as neuronal cells, and the results shown were good, implying a promising approach for a novel electrochemical immunosensor platform with potential applications to clinical diagnosis.     

Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood–brain barrier penetration

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood–brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.     

Structure-activity relationship of 5'-substituted fluoro-neplanocin a analogues as potent inhibitors of S-adenosylhomocysteine hydrolase

Four 5'-substituted fluoro-neplanocin A analogues la-d were designed and synthesized, and the inhibitory activity against SAH was in the following order: NH2 > SH > F, N3, indicating a hydrogen bonding donor is essential for inhibitory activity.     

Internalized group V secretory phospholipase A2 acts on the perinuclear membranes.

Mammalian secretory phospholipases A(2) (sPLA(2)) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA(2) mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA(2) acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA(2)s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA(2) hydrolysis products or cytokines, are necessary for the internalization of sPLA(2) into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA(2) internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA(2) induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA(2) acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis.     

Unique Behavioral Characteristics and microRNA Signatures in a Drug Resistant Epilepsy Model

Background

Pharmacoresistance is a major issue in the treatment of epilepsy. However, the mechanism underlying pharmacoresistance to antiepileptic drugs (AEDs) is still unclear, and few animal models have been established for studying drug resistant epilepsy (DRE). In our study, spontaneous recurrent seizures (SRSs) were investigated by video-EEG monitoring during the entire procedure.

Methods/Principal Findings

In the mouse pilocarpine-induced epilepsy model, we administered levetiracetam (LEV) and valproate (VPA) in sequence. AED-responsive and AED-resistant mice were naturally selected after 7-day treatment of LEV and VPA. Behavioral tests (open field, object exploration, elevated plus maze, and light-dark transition test) and a microRNA microarray test were performed. Among the 37 epileptic mice with SRS, 23 showed significantly fewer SRSs during administration of LEV (n = 16, LEV sensitive (LS) group) or VPA (n = 7, LEV resistant/VPA sensitive (LRVS) group), while 7 epileptic mice did not show any amelioration with either of the AEDs (n = 7, multidrug resistant (MDR) group). On the behavioral assessment, MDR mice displayed distinctive behaviors in the object exploration and elevated plus maze tests, which were not observed in the LS group. Expression of miRNA was altered in LS and MDR groups, and we identified 4 miRNAs (miR-206, miR-374, miR-468, and miR-142-5p), which were differently modulated in the MDR group versus both control and LS groups.

Conclusion

This is the first study to identify a pharmacoresistant subgroup, resistant to 2 AEDs, in the pilocarpine-induced epilepsy model. We hypothesize that modulation of the identified miRNAs may play a key role in developing pharmacoresistance and behavioral alterations in the MDR group.     

Complete mitochondrial genome of a forensically important muscid, Hydrotaea chalcogaster (Diptera: Muscidae), with notes on its phylogenetic position

Muscidae are a dipteran family which is important for forensic investigations. However, it has received limited attention in forensic entomological experiments as a reason of identification issues. It is hard to identify specimens by morphological methods, especially in developmental stages. Therefore, complete mitochondrial genome sequences can be important tool in forensic entomology for identifying species. In this study we sequenced and analyzed the first complete mitochondrial genome from a forensically important Muscidae species Hydrotaea (=Ophyra) chalcogaster by next-generation sequencing. The mitochondrial genome of the sequenced species is circular molecules of 17,076?bp which have the typical mitochondrial genome complement of 13 protein-coding genes, 22 tRNAs, two ribosomal RNA genes and a control region. Rearrangements of gene positions are identical with the ancestral insect genome. Furthermore, phylogenetic relationships of the family Muscidae were evaluated in regard to mitochondrial protein coding genes. The inferred trees indicate that the Muscidae is a paraphyletic family. These data provide additional information for molecular identification of muscid species.     

Post‐activation treatment with demecolcine improves development of somatic cell nuclear transfer embryos in pigs by modifying the remodeling of donor nuclei

The objective of this study was to examine the effect of cytochalasin B (CB) and/or demecolcine (Dc) on the remodeling of donor nuclei, nuclear ploidy, and development of somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) pig embryos. SCNT and PA oocytes were either untreated (control), or treated with CB, Dc, or both CB and Dc after electric activation, and then cultured or transferred to surrogates. In SCNT, blastocyst formation was higher after treatment with CB and/or Dc (26–28%) than in the controls (16%). The number of oocytes that formed a single pronucleus (PN) was higher after treatment with Dc (86%) and CB + Dc (86%) than under control conditions (44%) or after treatment with CB (63%). In PA, blastocyst formation was higher after CB treatment (47%) than under control conditions (28%), while the formation of a single PN was higher after treatment with Dc (88%) and CB + Dc (84%) compared to controls (34%). The rate of formation of diploid embryos was higher after treatment with Dc and CB + Dc than under control conditions. Dc treatment resulted in a farrowing rate of 50% with 1.1% production efficiency, while controls showed a farrowing rate of 37.5% and a production efficiency of 0.7%. The results of our study demonstrate that post‐activation treatment with Dc improves preimplantation development and supports normal in vivo development of SCNT pig embryos, probably because Dc induces formation of a single PN and this leads to normal nuclear ploidy. Mol. Reprod. Dev. 76: 611–619, 2009. © 2008 Wiley‐Liss, Inc.     

Anti-apoptotic effect of magnolol in myocardial ischemia and reperfusion injury requires extracellular signal-regulated kinase1/2 pathways in rat in vivo

Magnolol, an active component extracted from Magnolia officinalis, has been reported to have protective effect on ischemia and reperfusion (I/R)-induced injury in experimental animals. The aim of the present investigation was to further evaluate the mechanism(s) by which magnolol reduces I/R-induced myocardial injury in rats in vivo. Under anesthesia, left anterior descending (LAD) coronary artery was occluded for 30 min followed by reperfusion for 24 h (for infarct size and cardiac function analysis). In some experiments, reperfusion was limited to 1 h or 6 h for analysis of biochemical and molecular events. Magnolol and DMSO solution (vehicle) were injected intra-peritoneally 1 h prior to I/R insult. The infarct size was measured by TTC technique and heart function was monitored by Millar Catheter. Apoptosis related events such as p-ERK, p-Bad, Bcl-xl and cytochrome c expression were evaluated by Western blot analysis and myocardial caspase-3 activity was also measured. Magnolol (10 mg/kg) reduced infarct size by 50% (P < 0.01 versus vehicle), and also improved I/R-induced myocardial dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in magnolol-treated rats. Magnolol increased the expression of phosphor ERK and Bad which resulted in inhibition of myocardial apoptosis as evidenced by TUNEL analysis and DNA laddering experiments. Application of PD 98059, a selective MEK1/2 inhibitor, strongly antagonized the effect of magnolol. Taken together, we concluded that magnolol inhibits apoptosis through enhancing the activation of ERK1/2 and modulation of the Bcl-xl proteins which brings about reduction of infarct size and improvement of cardiac function in I/R-induced injury.