Classification of subcellular location by comparative proteomic analysis of native and density-shifted lysosomes

One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore the necessity of balance sheet analysis for localization studies.     

血清S-100B蛋白和NSE含量变化评估脑损伤程度的意义

目的探讨颅脑损伤后血清S-100B蛋白、神经元特异性烯醇化酶(NSE)水平变化及其在损伤程度的临床意义。方法采用酶联免疫测定法检测90例颅脑损伤患者入院时及入院后6、12、24h和3、7天血清S-100B蛋白、NSE水平,并分析其与颅脑损伤严重程度的关系。同时选择30例健康体检者作为对照组进行比较分析。结果颅脑损伤后血清S-100B蛋白、NSE水平较对照组明显升高(P0.05或P0.01);重型组患者的血清S-100B蛋白和NSE水平明显高于轻、中型组患者(P0.05或P0.01);动态检测结果显示,血清S-100B蛋白在伤后12h达峰值(P0.05),NSE含量在伤后24h达峰值(P0.05),然后缓慢下降。GCS评分与S-100B、NSE浓度呈负相关(分别为r=-0.814,P0.01;r=-0.726,P0.05)。结论颅脑损伤后血清S-100B蛋白、NSE水平升高,其与脑损伤程度有较好的相关性,2种标记物水平对颅脑损伤程度有较高的评估价值。     

A Strategy for Sensitive,Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.  Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.     

AMPK regulates homeostasis of invasion and viability in trophoblasts by redirecting glucose metabolism: Implications for pre‐eclampsia

Pre‐eclampsia (PE) is deemed an ischemia‐induced metabolic disorder of the placenta due to defective invasion of trophoblasts during placentation; thus, the driving role of metabolism in PE pathogenesis is largely ignored. Since trophoblasts undergo substantial glycolysis, this study aimed to investigate its function and regulatory mechanism by AMPK in PE development. Metabolomics analysis of PE placentas was performed by gas chromatography–mass spectrometry (GC–MS). Trophoblast‐specific AMPKα1‐deficient mouse placentas were generated to assess morphology. A mouse PE model was established by Reduced Uterine Perfusion Pressure, and placental AMPK was modulated by nanoparticle‐delivered A769662. Trophoblast glucose uptake was measured by 2‐NBDG and 2‐deoxy‐d‐[³H] glucose uptake assays. Cellular metabolism was investigated by the Seahorse assay and GC–MS.PE complicated trophoblasts are associated with AMPK hyperactivation due not to energy deficiency. Thereafter, AMPK activation during placentation exacerbated PE manifestations but alleviated cell death in the placenta. AMPK activation in trophoblasts contributed to GLUT3 translocation and subsequent glucose metabolism, which were redirected into gluconeogenesis, resulting in deposition of glycogen and accumulation of phosphoenolpyruvate; the latter enhanced viability but compromised trophoblast invasion. However, ablation of AMPK in the mouse placenta resulted in decreased glycogen deposition and structural malformation. These data reveal a novel homeostasis between invasiveness and viability in trophoblasts, which is mechanistically relevant for switching between the ‘go’ and ‘grow’ cellular programs.

Pre‐eclampsia (PE) is associated with trophoblast AMPK hyperactivation, presumably due to LKB1 phosphorylation, and glucose uptake is consequently increased via trafficking of GLUT3 from the cytosol to the plasma membrane. Such translocation enhances glycolytic flux and redirects glucose metabolic intermediates into gluconeogenesis, resulting in PEP accumulation, which not only benefits cell survival but also suppresses invasion by repressing MMPs, and thus in turn modulates switching between the ‘go’ and ‘grow’ cellular programs.     

Liquid-Solid Phase-Inversion PLGA Implant for the Treatment of Residual Tumor Tissue after HIFU Ablation

BackgroundHIFU has been shown to be a more suitable alternative for the treatment of primary solid tumors and metastatic diseases than other focal heat ablation techniques due to its noninvasive and extracorporeal nature. However, similar to other focal heat ablation techniques, HIFU is still in need of refinements due to tumor recurrence.MethodsIn this work, we investigated the effectiveness of an adjunct treatment regimen using doxorubicin (DOX)-loaded, injectable, in situ-forming, and phase-inverting PLGA as the second line of defense after HIFU ablation to destroy detrimental residual tumors and to prevent tumor recurrence. All of the statistical analyses were performed using the Statistical Package for the Social Sciences 18.0(SPSS, Inc., Chicago, IL, USA), and p< 0.05 was considered statistically significant. All of the results are presented as the means ± STDEV (standard deviation). For multiple comparisons, ANOVA (differences in tumor volumes, growth rates, apoptosis, proliferation indexes, and Bcl-2 and Bax protein levels) was used when the data were normally distributed with homogenous variance, and rank sum tests were used otherwise. Once significant differences were detected, Student-t tests were used for comparisons between two groups.ResultsOur results revealed that DOX diffused beyond the ablated tissue regions and entered tumor cells that were not affected by the HIFU ablation. Our results also show that HIFU in concert with DOX-loaded PLGA led to a significantly higher rate of tumor cell apoptosis and a lower rate of tumor cell proliferation in the areas beyond the HIFU-ablated tissues and consequently caused significant tumor volume shrinkage (tumor volumes:0.26±0.1,1.09±0.76, and 1.42±0.9cm³ for treatment, sham, and no treatment control, respectively).ConclusionsFrom these results, we concluded that the intralesional injection of DOX-loaded PLGA after HIFU ablation is significantly more effective than HIFU alone for the treatment of solid tumors.     

New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators

It had been known for decades that primordial follicles in mammalian ovaries are assembled with definite numbers and represent the ovarian reserve throughout the reproductive life. Intra-oocyte PI3K/mTOR pathways have been indicated to play a central role on the activation of primordial follicles. Genetic modified mouse models with chronic activation of PI3K/mTOR signals in primordial oocytes showed premature activation of all primordial follicles and eventually their exhaustion. On the other hand, this may suggest that, unlike chronic activation of PI3K/mTOR, its acute activation in infertility would activate primordial follicles, permitting fertility during the treatment. Previously, PI3K stimulators were reported as a temporary measure to accelerate primordial follicle activation and follicular development in both mouse and human, and were applied in the treatment of infertility in premature ovarian failure (POF) patients. To address whether mTOR stimulators could play similar role in the process, we transiently treated neonatal and aged mouse ovaries with mTOR stimulators-phosphatidic acid (PA) and propranolol. Our results demonstrated the stimulators increased activation of primordial follicles and the production of progeny. Human ovarian cortex cubes were also treated with mTOR or/and PI3K stimulators in vitro. When they were used separately, both of them showed similar promotive effects on primordial follicles. Surprisingly, after joint-treatment with the 2 kinds of stimulators together, synergistic effects on follicular development were observed. Based on increased efficiency of follicular activation in humans, here we propose in vitro transient treatment with mTOR and PI3K stimulators as an optimized protocol for the application in different clinical conditions with limited follicle reserve.     

Cerebrospinal fluid Th1/Th2 cytokine profiles in children with enterovirus 71‐associated meningoencephalitis

Enterovirus 71 (EV71) infection can cause severe neurological complications including meningoencephalitis (ME) in some patients with hand, foot and mouth disease (HFMD). However, to date no studies have reported changes in cytokine concentrations and their correlations with clinical variables in patients with ME following EV71 infection. In this study, responses of Th1/Th2 cytokine, including IL‐2, IL‐4, IL‐6, IL‐10, TNF‐α and IFN‐γ, in cerebrospinal fluid (CSF) from patients with EV71‐related HFMD with ME and patients with febrile convulsions (FC) were analyzed using cytometric bead array technology. It was found that CSF IL‐6 and IFN‐γ concentrations were significantly higher in patients with EV71‐related ME than in those with FC. Additionally, both CSF IL‐6 and IFN‐γ concentrations were correlated with CSF cytology, fever duration and duration of hospital stay. More interestingly, a positive correlation between CSF IL‐6 and IFN‐γ concentrations was observed. Finally, receiver operating characteristic analysis revealed that when a cutoff value of 9.40 pg/mL was set for IL‐6, the sensitivity and specificity were 84.5% and 85.5%, respectively, for discriminating EV71‐related ME from FC. In conclusion, IL‐6 and IFN‐γ may be associated with EV71‐induced neuropathology.     

Overexpression of an Arabidopsis magnesium transport gene, AtMGT1, in Nicotiana benthamiana confers Al tolerance

Aluminium (Al) toxicity is the most important limiting factor for crop production in acid soil environments worldwide. In some plant species, application of magnesium (Mg(2+)) can alleviate Al toxicity. However, it remains unknown whether overexpression of magnesium transport proteins can improve Al tolerance. Here, the role of AtMGT1, a member of the Arabidopsis magnesium transport family involved in Mg(2+) transport, played in Al tolerance in higher plants was investigated. Expression of 35S::AtMGT1 led to various phenotypic alterations in Nicotiana benthamiana plants. Transgenic plants harbouring 35S::AtMGT1 exhibited tolerance to Mg(2+) deficiency. Element assay showed that the contents of Mg, Mn, and Fe in 35S::AtMGT1 plants increased compared with wild-type plants. Root growth experiment revealed that 100 microM AlCl(3) caused a reduction in root elongation by 47% in transgenic lines, whereas root growth in wild-type plants was inhibited completely. Upon Al treatment, representative transgenic lines also showed a much lower callose deposition, an indicator of increased Al tolerance, than wild-type plants. Taken together, the results have demonstrated that overexpression of ATMGT1 encoding a magnesium transport protein can improve tolerance to Al in higher plants.     

Metabolism in 1,3‐propanediol fed‐batch fermentation by a D‐lactate deficient mutant of Klebsiella pneumoniae

Klebsiella pneumoniae HR526, a new isolated 1,3‐propanediol (1,3‐PD) producer, exhibited great productivity. However, the accumulation of lactate in the late‐exponential phase remained an obstacle of 1,3‐PD industrial scale production. Hereby, mutants lacking D ‐lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D ‐lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed‐batch fermentation. In experiments using pure glycerol as feedstock, the 1,3‐PD concentrations, conversion, and productivity increased from 95.39 g L^?1, 0.48 and 1.98 g L^?1 h^?1 to 102. 06 g L^?1, 0.52 mol mol^?1 and 2.13 g L^?1 h^?1, respectively. The diol (1,3‐PD and 2,3‐butanediol) conversion increased from 0.55 mol mol^?1 to a maximum of 0.65 mol mol^?1. Lactate would not accumulate until 1,3‐PD exceeded 84 g L^?1, and the final lactate concentration decreased dramatically from more than 40 g L^?1 to <3 g L^?1. Enzymic measurements showed LDH activity decreased by 89–98% during fed‐batch fermentation, and other related enzyme activities were not affected. NADH/NAD⁺ enhanced more than 50% in the late‐exponential phase as the D ‐lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3‐PD industrial production. Biotechnol. Bioeng. 2009; 104: 965–972. © 2009 Wiley Periodicals, Inc.