Toward a high resolution 2-DE profile of the normal human liver proteome using ultra-zoom gels

The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project (HLPP), the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis (2-DE). We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5–5.5, 5–6, 5.5–6.7, 6–9 and 6–11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3–10 and 4–7 pH gradients respectively, we obtained 5481 protein spots from the multiple (overlapping) narrow pH range ultra-zoom gels in the range of pH 4.5–9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.     

Plasma biomarker screening for liver fibrosis with the N-terminal isotope tagging strategy

A non-invasive diagnostic approach is crucial for the evaluation of severity of liver disease, treatment decisions, and assessing drug efficacy. This study evaluated plasma proteomic profiling via an N-terminal isotope tagging strategy coupled with liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry measurement to detect liver fibrosis staging. Pooled plasma from different liver fibrosis stages, which were assessed in advance by the current gold-standard of liver biopsy, was quantitatively analyzed. A total of 72 plasma proteins were found to be dysregulated during the fibrogenesis process, and this finding constituted a valuable candidate plasma biomarker bank for follow-up analysis. Validation results of fibronectin by Western blotting reconfirmed the mass-based data. Ingenuity Pathways Analysis showed four types of metabolic networks for the functional effect of liver fibrosis disease in chronic hepatitis B patients. Consequently, quantitative proteomics via the N-terminal acetyl isotope labeling technique provides an effective and useful tool for screening plasma candidate biomarkers for liver fibrosis. We quantitatively monitored the fibrogenesis process in CHB patients. We discovered many new valuable candidate biomarkers for the diagnosis of liver fibrosis and also partly identified the mechanism involved in liver fibrosis disease. These results provide a clearer understanding of liver fibrosis pathophysiology and will also hopefully lead to improvement of clinical diagnosis and treatment.     

MiR-223 suppresses cell proliferation by targeting IGF-1R

To study the roles of microRNA-223 (miR-223) in regulation of cell growth, we established a miR-223 over-expression model in HeLa cells infected with miR-223 by Lentivirus pLL3.7 system. We observed in this model that miR-223 significantly suppressed the proliferation, growth rate, colony formation of HeLa cells in vitro, and in vivo tumorigenicity or tumor formation in nude mice. To investigate the mechanisms involved, we scanned and examined the potential and putative target molecules of miR-223 by informatics, quantitative PCR and Western blot, and found that insulin-like growth factor-1 receptor (IGF-1R) was the functional target of miR-223 inhibition of cell proliferation. Targeting IGF-1R by miR-223 was not only seen in HeLa cells, but also in leukemia and hepatoma cells. The downstream pathway, Akt/mTOR/p70S6K, to which the signal was mediated by IGF-1R, was inhibited as well. The relative luciferase activity of the reporter containing wild-type 3'UTR(3'untranslated region) of IGF-1R was significantly suppressed, but the mutant not. Silence of IGF-1R expression by vector-based short hairpin RNA resulted in the similar inhibition with miR-223. Contrarily, rescued IGF-1R expression in the cells that over-expressed miR-223, reversed the inhibition caused by miR-223 via introducing IGF-1R cDNA that didn't contain the 3'UTR. Meanwhile, we also noted that miR-223 targeted Rasa1, but the downstream molecules mediated by Rasa1 was neither targeted nor regulated. Therefore we believed that IGF-1R was the functional target for miR-223 suppression of cell proliferation and its downstream PI3K/Akt/mTOR/p70S6K pathway suppressed by miR-223 was by targeting IGF-1R.     

Dynamics of dynamics within a single data acquisition session: variation in neocortical alpha oscillations in human MEG

Background

Behavioral paradigms applied during human recordings in electro- and magneto- encephalography (EEG and MEG) typically require 1–2 hours of data collection. Over this time scale, the natural fluctuations in brain state or rapid learning effects could impact measured signals, but are seldom analyzed.

Methods and Findings

We investigated within-session dynamics of neocortical alpha (7–14 Hz) rhythms and their allocation with cued-attention using MEG recorded from primary somatosensory neocortex (SI) in humans. We found that there were significant and systematic changes across a single ∼1 hour recording session in several dimensions, including increased alpha power, increased differentiation in attention-induced alpha allocation, increased distinction in immediate time-locked post-cue evoked responses in SI to different visual cues, and enhanced power in the immediate cue-locked alpha band frequency response. Further, comparison of two commonly used baseline methods showed that conclusions on the evolution of alpha dynamics across a session were dependent on the normalization method used.

Conclusions

These findings are important not only as they relate to studies of oscillations in SI, they also provide a robust example of the type of dynamic changes in brain measures within a single session that are overlooked in most human brain imaging/recording studies.     

Adjuvant chemotherapy, with or without taxanes, in early or operable breast cancer: a meta-analysis of 19 randomized trials with 30698 patients

Background

Taxanes have been extensively used as adjuvant chemotherapy for the treatment of early or operable breast cancer, particularly in high risk, node-negative breast cancer. Previous studies, however, have reported inconsistent findings regarding their clinical efficacy and safety. We investigated disease-free survival (DFS), overall survival (OS), and drug-related toxicities of taxanes by a systematic review and meta-analysis.

Methodology and Principal Findings

We systematically searched PubMed, EMBASE, the Cochrane Center Register of Controlled Trials, proceedings of major meetings, and reference lists of articles for studies conducted between January 1980 and April 2011. Randomized controlled trials (RCTs) comparing chemotherapy with and without taxanes in the treatment of patients with early-stage or operable breast cancer were eligible for inclusion in our analysis. The primary endpoint was DFS. Nineteen RCTs including 30698 patients were identified, including 8426 recurrence events and 3803 deaths. Taxanes administration yielded a 17% reduction of hazard ratio (HR) for DFS (HR = 0.83, 95% CI 0.79–0.88, p<0.001) and a 17% reduction of HR for OS (HR = 0.83, 95% CI 0.77–0.90, p<0.001). For high risk, node-negative breast cancer, the pooled HR also favoured the taxane-based treatment arm over the taxane-free treatment arm (HR = 0.82, 95% CI 0.77–0.87, p = 0.022). A significantly increased rate of neutropenia, febrile neutropenia, fatigue, diarrhea, stomatitis, and oedema was observed in the taxane-based treatment arm.

Conclusions/Significance

Adjuvant chemotherapy with taxanes could reduce the risk of cancer recurrence and death in patients with early or operable breast cancer, although the drug-related toxicities should be balanced. Furthermore, we also demonstrated that patients with high risk, node-negative breast cancer also benefited from taxanes therapy, a result that was not observed in previous studies.     

Functional evolution of leptin of Ochotona curzoniae in adaptive thermogenesis driven by cold environmental stress

Background

Environmental stress can accelerate the directional selection and evolutionary rate of specific stress-response proteins to bring about new or altered functions, enhancing an organism''s fitness to challenging environments. Plateau pika (Ochotona curzoniae), an endemic and keystone species on Qinghai-Tibetan Plateau, is a high hypoxia and low temperature tolerant mammal with high resting metabolic rate and non-shivering thermogenesis to cope in this harsh plateau environment. Leptin is a key hormone related to how these animals regulate energy homeostasis. Previous molecular evolutionary analysis helped to generate the hypothesis that adaptive evolution of plateau pika leptin may be driven by cold stress.

Methodology/Principal Findings

To test the hypothesis, recombinant pika leptin was first purified. The thermogenic characteristics of C57BL/6J mice injected with pika leptin under warm (23±1°C) and cold (5±1°C) acclimation is investigated. Expression levels of genes regulating adaptive thermogenesis in brown adipose tissue and the hypothalamus are compared between pika leptin and human leptin treatment, suggesting that pika leptin has adaptively and functionally evolved. Our results show that pika leptin regulates energy homeostasis via reduced food intake and increased energy expenditure under both warm and cold conditions. Compared with human leptin, pika leptin demonstrates a superior induced capacity for adaptive thermogenesis, which is reflected in a more enhanced β-oxidation, mitochondrial biogenesis and heat production. Moreover, leptin treatment combined with cold stimulation has a significant synergistic effect on adaptive thermogenesis, more so than is observed with a single cold exposure or single leptin treatment.

Conclusions/Significance

These findings support the hypothesis that cold stress has driven the functional evolution of plateau pika leptin as an ecological adaptation to the Qinghai-Tibetan Plateau.     

Plant mortality varies with arbuscular mycorrhizal fungal species identities in a self-thinning population

Because arbuscular mycorrhizal fungal (AMF) species differ in stimulating the growth of particular host plant species, AMF species may vary in their effects on plant intra-specific competition and the self-thinning process. We tested this hypothesis using a microcosm experiment with Medicago sativa L. as a model plant population and four AMF species. Our results showed that the AMF species Glomus diaphanum stimulated host plant growth more than the other three AMF species did when the plants were grown individually. Glomus diaphanum also induced the highest rate of mortality in the self-thinning plant populations. We also found a positive correlation between mortality and growth response to colonization. Our results demonstrate that AMF species can affect plant mortality and the self-thinning process by affecting plant growth differently.     

Readmission rates of patients discharged against medical advice: a matched cohort study

Objective

We compared the readmission rates and the pattern of readmission among patients discharged against medical advice (AMA) to control patients discharged with approval over a one-year follow-up period.

Methods

A retrospective matched-cohort study of 656 patients(328 were discharged AMA) who were followed for one year after their initial hospitalization at an urban university-affiliated teaching hospital in Vancouver, Canada that serves a population with high prevalence of addiction and psychiatric disorders. Multivariate conditional logistic regression was used to examine the independent association of discharge AMA on 14-day related diagnosis hospital readmission. We fit a multivariate conditional negative binomial regression model to examine the readmission frequency ratio between the AMA and non-AMA group.

Principal Findings

AMA patients were more likely to be homeless (32.3% vs. 11%) and have co-morbid conditions such as psychiatric illnesses, injection drug use, HIV, hepatitis C and previous gastrointestinal bleeding. Patients discharged AMA were more likely to be readmitted: 25.6% vs. 3.4%, p<0.001 by day 14. The AMA group were more likely to be readmitted within 14 days with a related diagnosis than the non-AMA group (Adjusted Odds Ratio 12.0; 95% Confidence Interval [CI]: 3.7–38.9). Patients who left AMA were more likely to be readmitted multiple times at one year compared to the non-AMA group (adjusted frequency ratio 1.6; 95% CI: 1.3–2.0). There was also higher all-cause in-hospital mortality during the 12-month follow-up in the AMA group compared to non-AMA group (6.7% vs. 2.4%, p = 0.01).

Conclusions

Patients discharged AMA were more likely to be homeless and have multiple co-morbid conditions. At one year follow-up, the AMA group had higher readmission rates, were predisposed to multiple readmissions and had a higher in-hospital mortality. Interventions to reduce discharges AMA in high-risk groups need to be developed and tested.     

Metabolic patterns and biotransformation activities of resveratrol in human glioblastoma cells: relevance with therapeutic efficacies

Background

Trans-resveratrol rather than its biotransformed monosulfate metabolite exerts anti-medulloblastoma effects by suppressing STAT3 activation. Nevertheless, its effects on human glioblastoma cells are variable due to certain unknown reason(s).

Methodology/Principal Findings

Citing resveratrol-sensitive UW228-3 medulloblastoma cell line and primarily cultured rat brain cells/PBCs as controls, the effect of resveratrol on LN-18 human glioblastoma cells and its relevance with metabolic pattern(s), brain-associated sulfotransferase/SULT expression and the statuses of STAT3 signaling and protein inhibitor of activated STAT3 (PIAS3) were elucidated by multiple experimental approaches. Meanwhile, the expression patterns of three SULTs (SULT1A1, 1C2 and 4A1) in human glioblastoma tumors were profiled immunohistochemically. The results revealed that 100 µM resveratrol-treated LN-18 generated the same metabolites as UW228-3 cells, while additional metabolite in molecular weight of 403.0992 in negative ion mode was found in PBCs. Neither growth arrest nor apoptosis was found in resveratrol-treated LN-18 and PBC cells. Upon resveratrol treatment, the levels of SULT1A1, 1C2 and 4A1 expression in LN-18 cells were more up-regulated than that expressed in UW228-3 cells and close to the levels in PBCs. Immunohistochemical staining showed that 42.0%, 27.1% and 19.6% of 149 glioblastoma cases produced similar SULT1A1, 1C2 and 4A1 levels as that of tumor-surrounding tissues. Unlike the situation in UW228-3 cells, STAT3 signaling remained activated and its protein inhibitor PIAS3 was restricted in the cytosol of resveratrol-treated LN-18 cells. No nuclear translocation of STAT3 and PIAS3 was observed in resveratrol-treated PBCs. Treatment with STAT3 chemical inhibitor, AG490, committed majority of LN-18 and UW228-3 cells but not PBCs to apoptosis within 48 hours.

Conclusions/Significance

LN-18 glioblastoma cells are insensitive to resveratrol due to the more inducible brain-associated SULT expression, insufficiency of resveratrol to suppress activated STAT3 signaling and the lack of PIAS3 nuclear translocation. The findings from PBCs suggest that an effective anticancer dose of resveratrol exerts little side effect on normal brain cells.