Semi-targeted plasma proteomics discovery workflow utilizing two-stage protein depletion and off-line LC-MALDI MS/MS

A quantitative proteomics workflow was implemented that provides extended plasma protein coverage by extensive protein depletion in combination with the sensitivity and breadth of analysis of two-dimensional LC-MS/MS shotgun analysis. Abundant proteins were depleted by a two-stage process using IgY and Supermix depletion columns in series. Samples are then extensively fractionated by two-dimensional chromatography with fractions directly deposited onto MALDI plates. Decoupling sample fractionation from mass spectrometry facilitates a targeted MS/MS precursor selection strategy that maximizes measurement of a consistent set of peptides across experiments. Multiplexed stable isotope labeling provides quantification relative to a common reference sample and ensures an identical set of peptides measured in the set of samples (set of eight) combined in a single experiment. The more extensive protein depletion provided by the addition of the Supermix column did not compromise overall reproducibility of the measurements or the ability to reliably detect changes in protein levels between samples. The implementation of this workflow is presented for a case study aimed at generating molecular signatures for prediction of first heart attack.     

ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF-1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2alpha or by a dominant-negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.     

Role of p-glycoprotein expression in predicting response to neoadjuvant chemotherapy in breast cancer-a prospective clinical study

Background

Lung cancer still remains one of the most commonly occurring solid tumors and even in stage Ia, surgery fails in 30% of patients who develop distant metastases. It is hypothesized that these must have developed from occult circulating tumor cells present at the time of surgery, or before. The aim of the present study was to detect such cells in the peripheral blood and to monitor these cells following surgery.

Methods

30 patients treated for lung cancer with surgery were monitored for circulating epithelial cells (CEC) by taking peripheral blood samples before, 2 weeks and 5 months after surgery and/or radiotherapy (RT) chemotherapy (CT) or combined RT/CT using magnetic bead enrichment and laser scanning cytometry (MAINTRAC^®) for quantification of these cells.

Results

In 86% of the patients CEC were detected before surgery and in 100% at 2 weeks and 5 months after surgery. In the control group, which consisted of 100 normal donors without cancer, 97 % were negative for CEC. A significantly higher number of CEC was found preoperatively in patients with squamous cell carcinoma than in those with adenocarcinoma. In correlation to the extent of parenchymal manipulation 2 weeks after surgery, an increase in numbers of CEC was observed with limited resections (18/21) whereas pneumonectomy led to a decrease (5/8) of CEC, 2 weeks after surgery. The third analysis done 5 months after surgery identified 3 groups of patients. In the group of 5 patients who received neo- or adjuvant chemo/radiotherapy there was evidence that monitoring of CEC can evaluate the effects of therapy. Another group of 7 patients who underwent surgery only showed a decrease of CEC and no signs of relapse. A third group of 11 patients who had surgery only, showed an increase of CEC (4 with an initial decrease after surgery and 7 with continuous increase). In the group with a continuous increase during the following 24 months, 2 early relapses in patients with stage Ia adenocarcinoma were observed. The increase of CEC preceded clinical detection by six months.

Conclusion

We consider, therefore, that patients with adenocarcinoma and a continuous increase of CEC after complete resection for lung cancer are at an increased risk of early relapse.     

Chemopreventive effect of vanadium in a rodent model of chemical hepatocarcinogenesis: reflections in oxidative DNA damage, energy-dispersive X-ray fluorescence profile and metallothionein expression

In the present study, we investigated the antitumour efficacy of vanadium in a defined rodent model of experimental hepatocarcinogenesis. Hepatic preneoplasia was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal injection of diethylnitrosamine (DEN) (200 mg/kg body weight) followed by promotion with phenobarbital (PB). The levels of modified DNA bases 8-hydroxy-2′-deoxyguanosine (8-OHdG), a potential marker involved in the initiation of carcinogenesis, were measured by high-performance liquid chromatography, whereas tissue trace element status and expression of metallothionein (MT), a Cu–Zn metalloprotein associated with neoplastic cell growth and subsequent development of premalignant phenotype of the cell, were studied by energy-dispersive X-ray fluorescence spectrometry and enzyme-coupled immunohistochemistry, respectively. There was a significant and steady elevation of modified bases (8-OHdG) along with substantial increase in MT immunoexpression and disturbance in trace element homeostasis following DEN exposure. Supplementation of vanadium at a dose of 0.5 ppm for four consecutive weeks strictly abated the formation of 8-OHdG (P < 0.0001; 81.28%) in preneoplastic rat liver. In a long-term DEN plus PB regimen, vanadium was able to limit in situ MT expression with a concomitant decrease in MT immunoreactivity (P < 0.05). Furthermore, vanadium treatment throughout the study restored hepatic levels of essential trace elements and decreased nodular incidence (58.34%) and nodule multiplicity (P < 0.001; 66.89%) in rats treated with DEN plus PB. Taken together, the study provides evidence in support of the chemopreventive potential of vanadium in limiting neoplastic transformation during the preneoplastic stages of hepatocarcinogenesis in rats.     

Fungal endophyte assemblages from ethnopharmaceutically important medicinal trees

Endophytic fungi represent an interesting group of microorganisms associated with the healthy tissues of terrestrial plants. They represent a large reservoir of genetic diversity. Fungal endophytes were isolated from the inner bark segments of ethnopharmaceutically important medicinal tree species, namely Terminalia arjuna, Crataeva magna, Azadirachta indica, Holarrhena antidysenterica, Terminalia chebula, and Butea monosperma (11 individual trees), growing in different regions of southern India. Forty-eight fungal species were recovered from 2200 bark segments. Mitosporic fungi represented a major group (61%), with ascomycetes (21%) and sterile mycelia (18%) the next major groups. Species of Fusarium, Pestalotiopsis, Myrothecium, Trichoderma, Verticillium, and Chaetomium were frequently isolated. Exclusive fungal taxa were recovered from five of the six plant species considered for the study of endophytic fungi. Rarefaction indices for species richness indicated the highest expected number of species for bark segments were isolated from T. arjuna and A. indica (20 species each) and from C. magna (18 species).     

p57kip2 regulates glial fate decision in adult neural stem cells

Our recent studies revealed p57kip2 as an intrinsic regulator of late gliogenesis and demonstrated that in oligodendroglial precursor cells p57kip2 inhibition leads to accelerated maturation. Adult neural stem cells have been described as a source of glial progenitors; however, the underlying mechanisms of cell fate specification are still poorly understood. Here, we have investigated whether p57kip2 can influence early events of glial determination and differentiation. We found that Sox2/GFAP double-positive cells express p57kip2 in stem cell niches of the adult brain. Short-hairpin RNA-mediated suppression of p57kip2 in cultured adult neural stem cells was found to strongly reduce astroglial characteristics, while oligodendroglial precursor features were increased. Importantly, this anti-astrogenic effect of p57kip2 suppression dominated the bone morphogenetic protein-mediated promotion of astroglial differentiation. Moreover, we observed that in p57kip2 knockdown cells, the BMP antagonist chordin was induced. Finally, when p57kip2-suppressed stem cells were transplanted into the adult spinal cord, fewer GFAP-positive cells were generated and oligodendroglial markers were induced when compared with control cells, demonstrating an effect of in vivo relevance.     

Semi-synthesis and bio-evaluation of polybrominated diphenyl ethers from the sponge Dysidea herbacea

The sponge Dysidea herbacea was collected from the Mandapam Coast, Tamilnadu, India. Isolated gram quantities of hydroxylated polybrominated diphenyl ether (HO-PBDE) and semi-synthesized a series of new PBDEs derivatives and tested them for antibacterial and cytotoxic activities.