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Background: It is well-established that serum testosterone in men decreases with age, yet the underlying mechanism of this change remains elusive.Methods: The expression patterns of Fancd2 opposite-strand (Fancd2os) in BALB/c male mice and testicular tissue derived cell lines (GC-1, GC-2, TM3, and TM4) were assessed using real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence. The Fancd2os-overexpressing or knockdown TM3 cells were constructed by infecting them with lentivirus particles and were used to evaluated the function of Fancd2os. The testosterone production was measured using enzyme linked immunosorbent assay (ELISA) and the steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) were analysed using RT-PCR. The apoptosis of TM3 cells induced by ultraviolet light or testicular tissues was detected using flow cytometry, Western blot or dUTP-biotin nick end labeling (TUNEL) assays. Pearson correlation analysis was used to assess the correlation between the Fancd2os expression and TUNEL-positive staining in mouse testicular Leydig cells.Results: The Fancd2os protein was predominantly expressed in mouse testicular Leydig cells and its expression increased with age. Fancd2os overexpression inhibited testosterone levels in TM3 Leydig cells, whereas knockdown of Fancd2os elevated testosterone production. Fancd2os overexpression downregulated the levels of StAR, P450scc and 3β-HSD, while Fancd2os knockdown reversed this effect. Fancd2os overexpression promoted ultraviolet light-induced apoptosis of TM3 cells. In contrast, Fancd2os knockdown restrained apoptosis in TM3 cells. In vivo assays revealed that higher Fancd2os levels and mouse age were associated with increased apoptosis in Leydig cells and decreased serum testosterone levels. Pearson correlation analysis exhibited a strong positive correlation between the expression of Fancd2os and TUNEL-positive staining in mouse testicular Leydig cells.Conclusion: Our findings suggest that Fancd2os regulates testosterone synthesis via both steroidogenic enzymes and the apoptotic pathway.

Polyketide synthase-terpenoid synthase (PKS-TPS) hybrid pathways for biosynthesis of unique sesquiterpenyl epoxy-cyclohexenoids (SECs) have been found to be widely distributed in plant pathogenic fungi. However, the natural and ecological functions of these pathways and their metabolites still remain cryptic. In this study, the whole PKS-TPS hybrid pathway in the predominant nematode-trapping fungus Arthrobotrys oligospora was first proposed according to all the intermediates and their derivatives from all the A. oligospora mutants with a deficiency in each gene involved in SEC biosynthesis. Most mutants displayed significantly increased trap formation which was correlated with alteration of the ammonia level. Further analysis revealed that the main metabolites involved in ammonia metabolism were largely increased in most mutants. However, significantly retarded colonization in soil were observed in most mutants compared to the wild-type strain due to significantly decreased antibacterial activities. Our results suggested that A. oligospora used the PKS-TPS hybrid pathway for fungal soil colonization via decreasing fungal nematode-capturing ability. This also provided solid evidence that boosting fungal colonization in soil was the secondary metabolite whose biosynthesis depended on a PKS-TPS hybrid pathway.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease of unknown etiology that affects the colon and rectum. It has evolved into a global burden due to the high incidence in developed countries and the highly-increased incidence in developing countries. Non-starch polysaccharides (NSPs) from natural resources, as a type of functional carbohydrates, have a significant therapeutic effect on UC because of their good anti-inflammatory and immunomodulatory activities. Based on the etiology and pathogenesis of UC, this review summarizes the intervention effects and mechanisms of NSPs in the prevention and treatment of UC. The results showed that NSPs can improve UC by protecting the intestinal mucosal barrier, regulating the immune response of the intestinal mucosa, and remodeling the intestinal flora and metabolites. These contents provide theoretical basis for the application of polysaccharides in the prevention and treatment of UC.

Cardiac fibrosis is one of the common pathological processes in many cardiovascular diseases characterized by excessive extracellular matrix deposition. SerpinE2 is a kind of protein that inhibits peptidase in extracellular matrix and up-regulated tremendously in mouse model of cardiac fibrosis induced by pressure-overloaded via transverse aortic constriction (TAC) surgery. However, its effect on cardiac fibroblasts (CFs), collagen secretion and the underlying mechanism remains unclear. In this study, DyLight® 488 green fluorescent dye or His-tagged proteins were used to label the exogenous serpinE2 protein. It was showed that extracellular serpinE2 translocated into CFs by low-density lipoprotein receptor-related protein 1 (LRP1) and urokinase plasminogen activator receptor (uPAR) of cell membrane through endocytosis. Knockdown of LRP1 or uPAR reduced the level of serpinE2 in CFs and down-regulated the collagen expression. Inhibition of the endocytosis of serpinE2 could inhibit ERK1/2 and β-catenin signaling pathways and subsequently attenuated collagen secretion. Knockdown of serpinE2 attenuates cardiac fibrosis in TAC mouse. We conclude that serpinE2 could be translocated into cardiac fibroblasts due to endocytosis through directly interact with the membrane protein LRP1 and uPAR, and this process activated the ERK1/2, β-catenin signaling pathways, consequently promoting collagen production.

Background: Mitochondrial transcription factor A (TFAM) plays multiple pathophysiologic roles in mitochondrial DNA (mtDNA) maintenance. However, the role of TFAM in sepsis-induced acute kidney injury (AKI) remains largely unknown. Methods: Lipopolysaccharide (LPS) treatment of HK-2 cells mimics the in vitro model of AKI inflammation. pcDNA3.1 plasmid was used to construct pcDNA3.1-TFAM. sh-TFAM-543, sh-TFAM-717, sh-TFAM-765, sh-TFAM-904 and pcDNA3.1-TFAM were transfected into HK-2 cells using Lipofectamine 2000. MtDNA transcriptional levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was performed to assess the cell viability. Changes in reactive oxygen species (ROS) and mitochondrial membrane potential in HK-2 cells were detected using the corresponding kits. Immunofluorescence experiment was used to investigate the displacement of TFAM. mRNA and protein expression levels of TFAM and its related genes were measured by qRT-PCR and western blot respectively. Mice in sepsis were administered cecal ligation and puncture surgery. Results: LPS treatment was a non-lethal influencing factor, leading to the upregulation of ROS levels and downregulation of mtDNA copy number and NADH dehydrogenase subunit-1 (ND1) expression, and caused damage to the mitochondria. As the LPS treatment time increased, TFAM was displaced from the periphery of the nucleus to cytoplasm. TFAM reduced ROS and P38MAPK levels by inhibiting toll-like receptor 4 (TLR4) expression, ultimately inhibiting inflammation and repairing mtDNA. Conclusions: Our results indicate that TFAM repairs mtDNA by blocking the TLR4/ROS/P38MAPK signaling pathway in inflammatory cells, thereby repairing septic tubular epithelial cells, and TFAM may serve as a new target for sepsis therapy.

Sepsis is a life-threatening condition that can lead to several organ failures including kidney. In this study, we investigated the roles of GAS5 and miR-579-3p in regulating cell pyroptosis in the sepsis-induced renal injury model. Lipopolysaccharide (LPS) treatment or cecal ligation and puncture (CLP) surgery was used to create the in vitro and in vivo sepsis-induced renal injury model. The interactions between GAS5 and miR-579-3p, miR-579-3p and SIRT1 were determined by bioinformatic prediction, luciferase reporter assay and RIP assay. In vitro cell pyroptosis was examined by flow cytometry marked with active caspase-1 and PI. The protein levels of IL-1β and IL-18 induced by cell pyroptosis were quantified using ELISA assay. In vivo renal injuries were evaluated with HE and TUNEL stainings, bacterial load in serum and creatinine and blood urea nitrogen content analyses. Expression levels of GAS5, miR-579-3p, pyroptosis and SIRT1/PGC-1a/Nrf2 pathway-related molecules were evaluated by qRT-PCR or western blot. GAS5 and SIRT1 were downregulated while miR-579-3p was upregulated in in vitro and in vivo sepsis-induced renal injury models. GAS5 negatively and directly regulated miR-579-3p to reduce cell pyroptosis via activation of SIRT1/PGC-1a/Nrf2 pathway. In addition, miR-579-3p suppressed PGC-1a/Nrf2 pathway to induce cell pyroptosis by directly targeting SIRT1. What's more, overexpression of GAS5, or knockdown of miR-579-3p, enhanced SIRT1 expression that led to the improved survival rate, reduced the weight loss, and relieved renal injuries in septic mice. Overexpression of GAS5 demonstrated protective effects against sepsis-induced renal injury via downregulating miR-579-3p and activating SIRT1/PGC-1α/Nrf2 pathway to inhibit cell pyroptosis.

A new polysaccharide (cDFP-W1) with high immunostimulatory activities was isolated from the stems of Dendrobium fimbriatum Hook. The analysis of the physicochemical properties showed that cDFP-W1 consisted of mannose and glucose in a molar ratio of 1 : 3.84, and its relative molecular weight was 4.0 × 104 Da. Structural analysis implied that the linear backbone of cDFP-W1 was composed of α-1,4-D-Glcp, β-1,4-D-Manp, 3-O-acetyl-α-1,4-D-Glcp and α-1,4,6-D-Glcp, and its branches were the terminal β-D-Manp that was attached to the C-6 position of α-1,4,6-D-Glcp. An in vivo immunostimulatory assay exhibited that cDFP-W1 at 200 mg kg−1 could significantly increase the proportions of CD4+ T-cell subpopulations, B cells, natural killer cells and dendritic cells, decrease the proportion of CD8+ T-cell subpopulations, and upregulate the percentage of activated macrophages (p < 0.01) in the spleen of mice. An in vitro immunostimulatory assay revealed that cDFP-W1 could effectively promote the proliferation of spleen lymphocytes, enhance the proliferation and phagocytosis of macrophage RAW264.7 cells, and stimulate the mRNA expression and extracellular release of NO, TNF-α and IL-1β of RAW264.7 cells. The western blot experiment suggested that the immunostimulatory activities of cDFP-W1 were closely related to the activation of MAPKs, NF-κB and PI3K/Akt signaling pathways.

Sepsis-induced inflammatory damage is a crucial cause of acute kidney injury (AKI), and AKI is an ecumenical fearful complication in approximately half of patients with sepsis. CCAAT/enhancer-binding protein β (C/EBPβ) plays roles in regulating acute phase responses and inflammation. However, the role and mechanism of C/EBPβ in AKI are unclear. LPS combined with ATP-treated renal epithelial cells HK2 and cecal ligation-peferation (CLP)-mice were used as models of AKI in vitro and in vivo. Cell damage, the secretion of interleukin-1 beta (IL-1β), IL-18 and cysteinyl aspartate specific proteinase 1 (caspase-1) activity were tested by LDH, ELISA assay and flow cytometry analysis, respectively. The expression levels of TFAM, C/EBPβ, and pyroptosis-related molecules were tested by qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assessed the interaction between C/EBPβ with TFAM. Hematoxylin-Eosin (H&E) staining detected pathological changes of kidney tissues, and immunohistochemistry measured TFAM and C/EBPβ in mice kidney tissues. C/EBPβ or TFAM were up-regulated in LPS combined with ATP -induced HK2 cells. Knockdown of C/EBPβ could suppress cell injury and the secretion of IL-1β and IL-18 induced by LPS combined with ATP. Furthermore, C/EBPβ up-regulated the expression levels of TFAM via directly binding to TFAM promoter. Overexpression of TFAM reversed the effects of C/EBPβ deficiency on pyroptosis. Knockdown of C/EBPβ could inhibit NLRP3 inflammasome-mediated caspase-1 signaling pathway by inactivating TFAM/RAGE pathway. It was further confirmed in the AKI mice that C/EBPβ and TFAM promoted AKI by activating NLRP3-mediated pyroptosis. The interaction of between C/EBPβ and TFAM facilitated pyroptosis by activating NLRP3/caspase-1 signal axis, thereby promoting the occurrence of AKI.

Mice are widely used as experimental models for gut microbiome (GM) studies, yet the majority of mouse GM members remain uncharacterized. Here, we report the construction of a mouse gut microbial biobank (mGMB) that contains 126 species, represented by 244 strains that have been deposited in the China General Microorganism Culture Collection. We sequence and phenotypically characterize 77 potential new species and propose their nomenclatures. The mGMB includes 22 and 17 species that are significantly enriched in ob/ob and wild-type C57BL/6J mouse cecal samples, respectively. The genomes of the 126 species in the mGMB cover 52% of the metagenomic nonredundant gene catalog (sequence identity ≥ 60%) and represent 93–95% of the KEGG-Orthology-annotated functions of the sampled mouse GMs. The microbial and genome data assembled in the mGMB enlarges the taxonomic characterization of mouse GMs and represents a useful resource for studies of host-microbe interactions and of GM functions associated with host health and diseases., Here, the authors established and characterized the mouse gut microbial biobank (mGMB), which includes 244 strains and 126 species that enlarges previous mouse intestinal bacterial collections and represents a resource for studies using mouse models to investigate microbiome-associated health and disease.

Background and Aim Emerging evidence indicates that psychological stress is involved in the pathogenesis of irritable bowel syndrome, which is characterized by visceral hypersensitivity and may be accompanied by gut dysbiosis. However, how such stress contributes to the development of visceral hypersensitivity is incompletely understood. Here, we aimed to investigate the influence that stress‐induced microbial changes exert on visceral sensitivity, as well as the possible underlying mechanisms associated with this effect. Methods Male Sprague–Dawley rats underwent chronic water avoidance stress (WAS) to induce visceral hypersensitivity. Visceral sensitivity, colonic tight junction protein expression, and short‐chain fatty acids of cecal contents were measured. Fecal samples were collected to characterize microbiota profiles. In a separate study, oral gavage of Roseburia in WAS rats was conducted to verify its potential role in the effectiveness on visceral hypersensitivity. Results Repeated WAS caused visceral hypersensitivity, altered fecal microbiota composition and function, and decreased occludin expression in the colon. Stressed rats exhibited reduced representation of pathways involved in the metabolism of butyrate and reduced abundance of several operational taxonomic units associated with butyrate‐producing bacteria, such as Lachnospiraceae. Consistently, supplementation with Roseburia hominis, a species belonging to Lachnospiraceae, significantly increased cecal butyrate content. Moreover, Roseburia supplementation alleviated visceral hypersensitivity and prevented the decreased expression of occludin. Conclusions Reduction in the abundance of butyrate‐producing Lachnospiraceae, which is beneficial for the intestinal barrier, was involved in the formation of visceral hypersensitivity. R. hominis is a potential probiotic for treating stress‐induced visceral hypersensitivity.

Purpose We sought to determine the diagnostic accuracy of in vivo confocal microscopy (IVCM) for ocular demodicosis. Design Reliability and validity analysis. Methods This was a single-center study of consecutive patients presenting to Zhongnan Hospital of Wuhan University, Hubei, China, between February 2017 and February 2018 with blepharitis. After examination, the blepharitis was scanned by IVCM. The regrading of the shuffled image set was performed by grader 1 (experienced) and grader 2 (inexperienced). The regrading of the shuffled image set was performed by 2 graders 3 weeks later. Eyelash samples were collected for light microscopy. The main outcome measures were sensitivity, specificity, and positive and negative predictive values of IVCM compared with those of positive light microscopy under 2 definitions. Sensitivities and specificities for multiple graders were pooled and 95% confidence intervals (CIs) were calculated. Results Sensitivity of IVCM grader 1 and grader 2 based on the definition of “light microscopy–positive demodex” was 100% (95% CI 94.84–100%) and 98.8% (95% CI 93.02–99.94%), respectively. Sensitivity of IVCM grader 1, grader 2, and light microscopy to the definition of “definite diagnosis demodex” definition was 100% (95% CI 97.02–100%), 93.63% (95% CI 88.28–96.73%), and 56.69% (95% CI 48.55–64.49%). Conclusions IVCM is better than traditional methods in detecting the number and sensitivity of demodex, and it is both highly sensitive and specific when performed by an experienced operator.

Summary We demonstrated the metabolic benefits of Parabacteroides distasonis (PD) on decreasing weight gain, hyperglycemia, and hepatic steatosis in ob/ob and high-fat diet (HFD)-fed mice. Treatment with live P. distasonis (LPD) dramatically altered the bile acid profile with elevated lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) and increased the level of succinate in the gut. In vitro cultivation of PD demonstrated its capacity to transform bile acids and production of succinate. Succinate supplementation in the diet decreased hyperglycemia in ob/ob mice via the activation of intestinal gluconeogenesis (IGN). Gavage with a mixture of LCA and UDCA reduced hyperlipidemia by activating the FXR pathway and repairing gut barrier integrity. Co-treatment with succinate and LCA/UDCA mirrored the benefits of LPD. The binding target of succinate was identified as fructose-1,6-bisphosphatase, the rate-limiting enzyme in IGN. The succinate and secondary bile acids produced by P. distasonis played key roles in the modulation of host metabolism.

It is a great challenge to develop drugs for treatment of metabolic syndrome. With ganomycin I as a leading compound, 14 meroterpene derivatives were synthesized and screened for their α-glucosidase and HMG-CoA reductase inhibitory activities. As a result, a α-glucosidase and HMG-CoA reductase dual inhibitor (( R, E)-5-(4-( tert-butyl)phenyl)-3-(4,8-dimethylnona-3,7-dien-1-yl)furan-2(5 H)-one, 7d) with improved chemical stability and long-term safety was obtained. Compound 7d showed multiple and strong in vivo efficacies in reducing weight gain, lowering HbAlc level, and improving insulin resistance and lipid dysfunction in both ob/ob and diet-induced obesity (DIO) mice models. Compound 7d was also found to reduce hepatic steatosis in ob/ob model. 16S rRNA gene sequencing, SCFA, and intestinal mucosal barrier function analysis indicated that gut microbiota plays a central and causative role in mediating the multiple efficacies of 7d. Our results demonstrate that 7d is a promising drug candidate for metabolic syndrome.

The CD4+CD25+ regulatory T cells (Tregs), an innate immunomodulator, suppress cerebral inflammation and maintain immune homeostasis in multiple central nervous system injury, but its role in intracerebral hemorrhage (ICH) has not been fully characterized. This study investigated the effect of Tregs on brain injury using the mouse ICH model, which is established by autologous blood infusion. The results showed that tail intravenous injection of Tregs significantly reduced brain water content and Evans blue dye extravasation of perihematoma at day (1, 3 and 7), and improved short- and long-term neurological deficits following ICH in mouse model. Tregs treatment reduced the content of pro-inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and malondialdehyde, while increasing the superoxide dismutase (SOD) enzymatic activity at day (1, 3 and 7) following ICH. Furthermore, Tregs treatment obviously reduced the number of NF-κB+, IL-6+, TUNEL+ and active caspase-3+ cells at day 3 after ICH. These results indicate that adoptive transfer of Tregs may provide neuroprotection following ICH in mouse models.

Several studies showed that the up-regulation of glial glutamate transporter-1 (GLT-1) participates in the acquisition of brain ischemic tolerance induced by cerebral ischemic preconditioning or ceftriaxone pretreatment in rats. To explore whether GLT-1 plays a role in the acquisition of brain ischemic tolerance induced by intermittent hypobaric hypoxia (IH) preconditioning (mimicking 5,000 m high-altitude, 6 h per day, once daily for 28 days), immunohistochemistry and western blot were used to observe the changes in the expression of GLT-1 protein in hippocampal CA1 subfield during the induction of brain ischemic tolerance by IH preconditioning, and the effect of dihydrokainate (DHK), an inhibitor of GLT-1, on the acquisition of brain ischemic tolerance in rats. The basal expression of GLT-1 protein in hippocampal CA1 subfield was significantly up-regulated by IH preconditioning, and at the same time astrocytes were activated by IH preconditioning, which appeared normal soma and aplenty slender processes. The GLT-1 expression was decreased at 7 days after 8-min global brain ischemia. When the rats were pretreated with the IH preconditioning before the global brain ischemia, the down-regulation of GLT-1 protein was prevented clearly. Neuropathological evaluation by thionin staining showed that 200 nmol DHK blocked the protective role of IH preconditioning against delayed neuronal death induced normally by 8-min global brain ischemia. Taken together, the up-regulation of GLT-1 protein participates in the acquisition of brain ischemic tolerance induced by IH preconditioning in rats.

Gold nanoparticles (AuNPs) confined in liposomes of diameters around 200 nm produce strong scattering signal owing to surface plasmon resonance, and therefore bright-field optical tracking of the AuNP-encapsulating liposomes can be conducted in living cells. Using an optical profiling technique called noninterferometric wide-field optical profilometry and a bright-field tracking algorithm, the polynomial-fit Gaussian weight method, we analyze three-dimensional (3D) motion of such liposomes in living fibroblasts. The positioning accuracy in three dimensions is nearly 20 nm. We tag the liposome membranes with fibroblast growth factor-1 and reveal the intracellular transportation processes toward or away from the nucleus. On the basis of a temporal analysis of the intracellular 3D trajectories of AuNP-encapsulating liposomes, we identify directed and diffusive motions in the transportation processes.

To monitor the dynamic changes of extracellular quantum dots (QDs) in vivo in the livers of anesthetized rats, we developed an automatic online analytical system comprising push-pull perfusion (PPP) sampling, the established in-tube solid phase extraction (SPE) procedure, and inductively coupled plasma mass spectrometry (ICPMS). The method takes advantage of the retention of QDs onto the interior surface of a polytetrafluoroethylene (PTFE) tube as a means of extracting the QDs from complicated push-pull perfusates. For the injected QDs present in the liver extracellular fluid (ECF) at low picomolar levels, a temporal resolution of 10 min was required to collect sufficient amounts of QDs to meet the sensitivity requirements of the ICPMS system. To the best of our knowledge, this study is the first to exploit the PPP technique for the collection of QDs from living animals and PTFE tubing as a SPE adsorbent for the online extraction of QDs and the removal of biological matrix prior to ICPMS analysis of cadmium-containing inorganic nanocrystal. We confirmed the analytical reliability of this method from measurements of the spike recoveries of saline samples; in addition, we demonstrated the systems' applicability through in vivo monitoring of the time-dependent concentration profile of liver extracellular QDs in living rats after intravenous administration.

Objective— Loss-of-function mutations in genes coding for transforming growth factor-β/bone morphogenetic protein receptors and changes in nitric oxide • (NO • ) bioavailability are associated with hereditary hemorrhagic telangiectasia and some forms of pulmonary arterial hypertension. How these abnormalities lead to seemingly disparate pulmonary pathologies remains unknown. Endoglin (Eng), a transforming growth factor-β coreceptor, is mutated in hereditary hemorrhagic telangiectasia and involved in regulating endothelial NO • synthase (eNOS)-derived NO • production and oxidative stress. Because some patients with pulmonary arterial hypertension harbor ENG mutations leading to haplo insufficiency, we investigated the pulmonary vasculature of Eng +/− mice and the potential contribution of abnormal eNOS activation to pulmonary arterial hypertension. Methods and Results— Hemodynamic, histological, and biochemical assessments and x-ray micro-CT imaging of adult Eng +/− mice indicated signs of pulmonary arterial hypertension including increased right ventricular systolic pressure, degeneration of the distal pulmonary vasculature, and muscularization of small arteries. These findings were absent in 3-week-old Eng +/− mice and were attributable to constitutively uncoupled eNOS activity in the pulmonary circulation, as evidenced by reduced eNOS/heat shock protein 90 association and increased eNOS-derived superoxide ( • O 2 − ) production in a BH 4 -independent manner. These changes render eNOS unresponsive to regulation by transforming growth factor-β/bone morphogenetic protein and underlie the signs of pulmonary arterial hypertension that were prevented by Tempol. Conclusion— Adult Eng +/− mice acquire signs of pulmonary arterial hypertension that are attributable to uncoupled eNOS activity and increased • O 2 − production, which can be prevented by antioxidant treatment. Eng links transforming growth factor/bone morphogenetic protein receptors to the eNOS activation complex, and its reduction in the pulmonary vasculature leads to increased oxidative stress and pulmonary arterial hypertension.

A simple and rapid MEKC method was developed for the simultaneous determination of myo-inositol, scyllo-inositol, and glucose. Prior to electrophoretic separation, the nonfluorescent inositols and glucose were derivatized by N-methylisatoic anhydride at 25 degrees C for 10 min so that they could be detected by a fluorescence detector during separation. The good separation with high efficiency by MEKC was achieved in 13 min with a glycine buffer containing SDS and PEG 4000. Several parameters affecting the separation were studied, including the pH of BGE, the concentrations of glycine, SDS, and PEG 4000, and the applied voltage. Using glycerol as an internal standard, the linear ranges of the method for myo-inositol, scyllo-inositol, and glucose were 0.03-10, 0.01-5, and 0.05-20 mM; the concentration LODs of myo-inositol, scyllo-inositol, and glucose were 0.020, 0.0078, and 0.026 mM, respectively. The method was applied to analyze extracellular myo-inositol and glucose in the microdialysates from rat brain cortex of ischemia animal model and intracellular myo-inositol and scyllo-inositol in the rat brain extract.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that affects humans and various vertebrate livestock and causes serious economic losses. To develop an effective vaccine against T. gondii infection, we constructed a DNA vaccine encoding the T. gondii rhoptry protein 17 (TgROP17) and evaluated its immune protective efficacy against acute T. gondii infection in mice. The DNA vaccine (p3×Flag-CMV-14-ROP17) was intramuscularly injected to BALB/c mice and the immune responses of the vaccinated mice were determined. Compared to control mice treated with empty vector or PBS, mice immunized with the ROP17 vaccine showed a relatively high level of specific anti-T. gondii antibodies, and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a specific lymphocyte proliferative response, a Th1-type cellular immune response with production of IFN-γ and interleukin-2, and increased number of CD8(+) T cells. Immunization with the ROP17 DNA significantly prolonged the survival time (15.6 ± 5.4 days, P < 0.05) of mice after challenge infection with the virulent T. gondii RH strain (Type I), compared with the control groups which died within 8 days. Therefore, our data suggest that DNA vaccination with TgROP17 triggers significant humoral and cellular responses and induces effective protection in mice against acute T. gondii infection, indicating that TgROP17 is a promising vaccine candidate against acute toxoplasmosis.

Nanoparticles were designed to encapsulate drugs to alter their pharmacological behaviors, therefore, it is very essential to monitor the pharmacokinetic profile of drug encapsulated in nanoparticles in order to clarify and predict their efficacy and side effects. In this paper, we reported a simple, rapid μ-elution 96-well solid phase extraction (μSPE) method combining with ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for determination of nanoformulated drug in rat plasma. This method presented satisfactory results in terms of sensitivity, precision, accuracy, and recovery, for the first time, of quantitatively analyzing clarithromycin (CLA) in rat plasma after intravenous administration CLA-loaded ultrafine PLGA nanoparticles for pharmacokinetic study. This method has been proved to be fast, reliable and reproducible to accurately analyze drug encapsulated in polymeric nanoparticles sample for a pharmacokinetic study.

Iron oxide nanoparticles can serve as a heating source upon alternative magnetic field (AMF) exposure. Iron oxide nanoparticles can be mixed with thermosensitive nanovehicles for hyperthermia-induced drug release, yet such a design and mechanism may not be suitable for controllable drug release applications in which the tissues are susceptible to environmental temperature change such as brain tissue. In the present study, iron oxide nanoparticles were entrapped inside of thermosensitive liposomes for AMF-induced drug release while the environmental temperature was maintained at a constant level. Carboxyfluorescein was co-entrapped with the iron oxide nanoparticles in the liposomes as a model compound for monitoring drug release and environmental temperature was maintained with a water circulator jacket. These experiments have been successfully performed in solution, in phantom and in anesthetized animals. Furthermore, the thermosensitive liposomes were administered into rat forearm skeletal muscle, and the release of carboxylfluorescein triggered by the external alternative magnetic field was monitored by an implanted microdialysis perfusion probe with an on-line laser-induced fluorescence detector. In the future such a device could be applied to simultaneous magnetic resonance imaging and non-invasive drug release in temperature-sensitive applications.