Mössbauer, X-ray and Magnetic Studies of Black Sand from the Italian Mediterranean Sea

The study of natural magnetic sands is instrumental to investigate the geological aspects of their formation and of the origin of their territory. In particular, Mössbauer spectroscopy provides unique information on their iron content and on the oxidation state of iron in their mineral composition. The Italian coast on the Mediterranean Sea near Rome is known for the presence of highly magnetic black sands of volcanic origin. A study of the room temperature Mössbauer spec- trum, powder X-ray diffraction, energy dispersive X-ray spectroscopy, and magnetic measurements of a sample of black magnetic sand collected on the seashore of the town of Ladispoli is performed. This study reveals magnetite as main constituent with iron in both tetrahedral and octahedral sites. Minor constituents are the iron minerals hematite and ilmenite, the iron containing minerals diopsite, gossular, and allanite, as well as ubiquitous sanidine, quartz, and calcite.

Climate and chemistry effects of a regional scale nuclear conflict

Previous studies have highlighted the severity of detrimental effects for life on earth after an assumed regionally limited nuclear war. These effects are caused by climatic, chemical and radiative changes persisting for up to one decade. However, so far only a very limited number of climate model simulations have been performed, giving rise to the question how realistic previous computations have been. This study uses the coupled chemistry climate model (CCM) SOCOL, which belongs to a different family of CCMs than previously used, to investigate the consequences of such a hypothetical nuclear conflict. In accordance with previous studies, the present work assumes a scenario of a nuclear conflict between India and Pakistan, each applying 50 warheads with an individual blasting power of 15 kt ("Hiroshima size") against the major population centers, resulting in the emission of tiny soot particles, which are generated in the firestorms expected in the aftermath of the detonations. Substantial uncertainties related to the calculation of likely soot emissions, particularly concerning assumptions of target fuel loading and targeting of weapons, have been addressed by simulating several scenarios, with soot emissions ranging from 1 to 12 Tg. Their high absorptivity with respect to solar radiation leads to a rapid self-lofting of the soot particles into the strato- and mesosphere within a few days after emission, where they remain for several years. Consequently, the model suggests earth's surface temperatures to drop by several degrees Celsius due to the shielding of solar irradiance by the soot, indicating a major global cooling. In addition, there is a substantial reduction of precipitation lasting 5 to 10 yr after the conflict, depending on the magnitude of the initial soot release. Extreme cold spells associated with an increase in sea ice formation are found during Northern Hemisphere winter, which expose the continental land masses of North America and Eurasia to a cooling of several degrees. In the stratosphere, the strong heating leads to an acceleration of catalytic ozone loss and, consequently, to enhancements of UV radiation at the ground. In contrast to surface temperature and precipitation changes, which show a linear dependence to the soot burden, there is a saturation effect with respect to stratospheric ozone chemistry. Soot emissions of 5 Tg lead to an ozone column reduction of almost 50% in northern high latitudes, while emitting 12 Tg only increases ozone loss by a further 10%. In summary, this study, though using a different chemistry climate model, corroborates the previous investigations with respect to the atmospheric impacts. In addition to these persistent effects, the present study draws attention to episodically cold phases, which would likely add to the severity of human harm worldwide. The best insurance against such a catastrophic development would be the delegitimization of nuclear weapons.

Regulation of visfatin by microbial and biomechanical signals in PDL cells

OBJECTIVES This in vitro study was established to examine whether visfatin thought to be a link between periodontitis and obesity is produced by periodontal ligament (PDL) cells and, if so, whether its synthesis is modulated by microbial and/or biomechanical signals. MATERIALS AND METHODS PDL cells seeded on BioFlex® plates were exposed to the oral pathogen Fusobacterium nucleatum ATCC 25586 and/or subjected to biomechanical strain for up to 3 days. Gene expression of visfatin and toll-like receptors (TLR) 2 and 4 was analyzed by RT-PCR, visfatin protein synthesis by ELISA and immunocytochemistry, and NFκB nuclear translocation by immunofluorescence. RESULTS F. nucleatum upregulated the visfatin expression in a dose- and time-dependent fashion. Preincubation with neutralizing antibodies against TLR2 and TLR4 caused a significant inhibition of the F. nucleatum-upregulated visfatin expression at 1 day. F. nucleatum stimulated the NFκB nuclear translocation. Biomechanical loading reduced the stimulatory effects of F. nucleatum on visfatin expression at 1 and 3 days and also abrogated the F. nucleatum-induced NFκB nuclear translocation at 60 min. Biomechanical loading inhibited significantly the expression of TLR2 and TLR4 at 3 days. The regulatory effects of F. nucleatum and/or biomechanical loading on visfatin expression were also observed at protein level. CONCLUSIONS PDL cells produce visfatin, and this production is enhanced by F. nucleatum. Biomechanical loading seems to be protective against the effects of F. nucleatum on visfatin expression. CLINICAL RELEVANCE Visfatin produced by periodontal tissues could play a major role in the pathogenesis of periodontitis and the interactions with obesity and other systemic diseases.

Molecular phylogenetic analyses identify Alpine differentiation and dysploid chromosome number changes as major forces for the evolution of the European endemic Phyteuma (Campanulaceae)

Phyteuma is a chromosomally and ecologically diverse vascular plant genus and constitutes an excellent system for studying both the role of chromosomal change for species diversification and the evolution of high-mountain biota. This kind of research is, however, hampered by the lack of a sound phylogenetic framework exacerbated by the notoriously low predictive power of traditional taxonomy with respect to phylogenetic relationships in Campanulaceae. Based on a comprehensive taxon sampling and analyses of nuclear and plastid sequence and AFLP fingerprint data, Phyteuma is confirmed as a monophyletic group sister to the monotypic Physoplexis, which is in line with their peculiar flower morphologies. Within Phyteuma two clades, largely corresponding to previously recognized sections, are consistently found. The traditional circumscription of taxonomic series is largely rejected. Whereas distinctness of the currently recognized species is mostly corroborated, some interspecific relationships remain ambiguous due to incongruences between nuclear and plastid data. Major forces for diversification and evolution of Phyteuma are descending dysploidy (i.e., a decrease in chromosome base number) as well as allopatric and ecological differentiation within the Alps, the genus' center of species diversity. (C) 2013 Elsevier Inc. All rights reserved.

Mesenchymal stromal cells improve survival during sepsis in the absence of heme oxygenase-1: the importance of neutrophils

The use of mesenchymal stromal cells (MSCs) for treatment of bacterial infections, including systemic processes like sepsis, is an evolving field of investigation. This study was designed to investigate the potential use of MSCs, harvested from compact bone, and their interactions with the innate immune system, during polymicrobial sepsis induced by cecal ligation and puncture (CLP). We also wanted to elucidate the role of endogenous heme oxygenase (HO)-1 in MSCs during a systemic bacterial infection. MSCs harvested from the bones of HO-1 deficient (-/-) and wild-type (+/+) mice improved the survival of HO-1(-/-) and HO-1(+/+) recipient mice when administered after the onset of polymicrobial sepsis induced by CLP, compared with the administration of fibroblast control cells. The MSCs, originating from compact bone in mice, enhanced the ability of neutrophils to phagocytize bacteria in vitro and in vivo and to promote bacterial clearance in the peritoneum and blood after CLP. Moreover, after depleting neutrophils in recipient mice, the beneficial effects of MSCs were entirely lost, demonstrating the importance of neutrophils for this MSC response. MSCs also decreased multiple organ injury in susceptible HO-1(-/-) mice, when administered after the onset of sepsis. Taken together, these data demonstrate that the beneficial effects of treatment with MSCs after the onset of polymicrobial sepsis is not dependent on endogenous HO-1 expression, and that neutrophils are crucial for this therapeutic response.

CCND1/CyclinD1 status in metastasizing bladder cancer: a prognosticator and predictor of chemotherapeutic response

The CCND1 gene encodes the protein CyclinD1, which is an important promoter of the cell cycle and a prognostic and predictive factor in different cancers. CCND1 is amplified to a substantial proportion in various tumors, and this may contribute to CyclinD1 overexpression. In bladder cancer, information about the clinical relevance of CCND1/CyclinD1 alterations is limited. In the present study, amplification status of CCND1 and expression of CyclinD1 were evaluated by fluorescence in situ hybridization and immunohistochemistry on tissue microarrays from 152 lymph node-positive urothelial bladder cancers (one sample each from the center and invasion front of the primary tumors, two samples per corresponding lymph node metastasis) treated by cystectomy and lymphadenectomy. CCND1 amplification status and the percentage of immunostained cancer cells were correlated with histopathological tumor characteristics, cancer-specific survival and response to adjuvant chemotherapy. CCND1 amplification in primary tumors was homogeneous in 15% and heterogeneous in 6% (metastases: 22 and 2%). Median nuclear CyclinD1 expression in amplified samples was similar in all tumor compartments (60-70% immunostained tumor nuclei) and significantly higher than in non-amplified samples (5-20% immunostained tumor nuclei; P<0.05). CCND1 status and CyclinD1 expression were not associated with primary tumor stage or lymph node tumor burden. CCND1 amplification in primary tumors (P=0.001) and metastases (P=0.02) and high nuclear CyclinD1 in metastases (P=0.01) predicted early cancer-related death independently. Subgroup analyses showed that chemotherapy was particularly beneficial in patients with high nuclear CyclinD1 expression in the metastases, whereas expression in primary tumors and CCND1 status did not predict chemotherapeutic response. In conclusion, CCND1 amplification status and CyclinD1 expression are independent risk factors in metastasizing bladder cancer. High nuclear CyclinD1 expression in lymph node metastases predicts favorable response to chemotherapy. This information may help to personalize prognostication and administration of adjuvant chemotherapy.

Phylogeography of silver pheasant (Lophura nycthemera L.) across China: aggregate effects of refugia, introgression, and riverine barriers

The role of Pleistocene glacial cycles in forming the contemporary genetic structure of organisms has been well studied in China with a particular focus on the Tibetan Plateau. However, China has a complex topography and diversity of local climates, and how glacial cycles may have shaped the subtropical and tropical biota of the region remains mostly unaddressed. To investigate the factors that affected the phylogeography and population history of a widely distributed and nondeciduous forest species, we analysed morphological characters, mitochondrial DNA sequences and nuclear microsatellite loci in the Silver Pheasant (Lophura nycthemera). In a pattern generally consistent with phenotypic clusters, but not nominal subspecies, deeply divergent mitochondrial lineages restricted to different geographic regions were detected. Coalescent simulations indicated that the time of main divergence events corresponded to major glacial periods in the Pleistocene and gene flow was only partially lowered by drainage barriers between some populations. Intraspecific cytonuclear discordance was revealed in mitochondrial lineages from Hainan Island and the Sichuan Basin with evidence of nuclear gene flow from neighbouring populations into the latter. Unexpectedly, hybridization was revealed in Yingjiang between the Silver Pheasant and Kalij Pheasant (Lophura leucomelanos) with wide genetic introgression at both the mtDNA and nuclear levels. Our results highlight a novel phylogeographic pattern in a subtropical area generated from the combined effects of climate oscillation, partial drainage barriers and interspecific hybridization. Cytonuclear discordance combined with morphological differentiation implies that complex historical factors shaped the divergence process in this biodiversity hot spot area of southern China.

GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1.

Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C (nuclear factor 1-C) to the first 227 bp of 5'flanking DNA (-227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express -227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at -64/-58 and an SF-1 site at -58/-50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated -227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in -227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at -196/-188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected -227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells.

The sequence of addition of IL-3 and IgE-dependent/IgE-independent stimuli regulates RANKL expression in human basophils

Background: Receptor Activator of Nuclear Factor kappaB Ligand (RANKL), a member of the TNF superfamily, contributes to the imbalance of bone resorption and immunoregulation in rheumatoid arthritis. In mice, collagen induced arthritis was exacerbated by IL-3 and anti-IgER antibodies, two mediators activating basophils that are known as effector cells of allergy. Interestingly, our unpublished microarray data revealed that IL-3 induces RANKL mRNA in human basophils. Here we further investigate under which conditions human basophils express surface and/or soluble RANKL. Methods: One part of purified human basophils was co-stimulated with IL-3 and either IgE-dependent or IgE-independent stimuli. The other part of purified basophils was first primed with IL-3 and subsequently triggered with IgE-dependent or IgE-independent stimuli. Expression of surface and soluble RANKL were detected by flow cytometry, ELISA and real-time PCR. Results: By flow cytometry we show that IL-3 induces de novo expression of surface RANKL on human basophils in a time and dose dependent manner. Co-stimulation of basophils with IL-3 and an IgE-dependent stimulus reduces IL-3-induced expression of surface RANKL in a dose dependent manner while IgE-independent stimuli have no effect. In contrast, both IgE-dependent and IgE-independent stimuli enhance expression of surface and soluble RANKL in basophils that were first primed with IL-3 and then triggered. Real-time PCR analysis shows that surface hRANKL1 and soluble hRANKL3 are induced by IL-3 and reduced by co-stimulation with IL-3 and an IgE-dependent stimulus and thus confirms our flow cytometry data. Conclusion: RANKL expression in human basophils is not only dependent on IL-3 and IgE-dependent/IgE-independent stimuli but also on the sequence of their addition to cell culture. Based on our data, we suggest that basophils might have previously unidentified functions in bone resorption or immunoregulation via RANKL.

Analysis of the 207Bi, 194Hg/Au and 173Lu distribution in the irradiated MEGAPIE target

Samples obtained from different locations within the prototype liquid metal spallation target MEGAPIE irradiated in 2006 at PSI were analysed using γ-spectrometry. A variety of radionuclides formed by reaction of the target material, lead–bismuth eutectic (LBE), with the proton beam and secondary particles were identified. While nuclides representing the target material itself (207Bi) and nuclides of noble metals were found in LBE samples throughout the target, nuclides of electropositive metals were found to be quantitatively deposited on free surfaces and material interfaces within the target system. This behaviour is analysed in more detail based on results obtained for three nuclides representing groups of elements with distinct chemical behaviour, namely 207Bi, 194Hg/Au and 173Lu. Quantitative analysis results are given and compared with predictions obtained using nuclear physics calculations for those nuclides showing rather homogeneous distribution within the target. Possible reasons for the separation of radionuclides from the liquid metal and their deposition on surfaces are given, and consequences arising for nuclear facilities utilizing liquid metals are discussed.

Comparison of 68Ga-DOTANOC and 68Ga-DOTATATE PET/CT within patients with gastroenteropancreatic neuroendocrine tumors

Somatostatin receptor PET tracers such as [68Ga-DOTA,1-Nal3]-octreotide (68Ga-DOTANOC) and [68Ga-DOTA,Tyr3]-octreotate (68Ga-DOTATATE) have shown promising results in patients with neuroendocrine tumors, with a higher lesion detection rate than is achieved with 18F-fluorodihydroxyphenyl-l-alanine PET, somatostatin receptor SPECT, CT, or MR imaging. 68Ga-DOTANOC has high affinity for somatostatin receptor subtypes 2, 3, and 5 (sst2,3,5). It has a wider receptor binding profile than 68Ga-DOTATATE, which is sst2-selective. The wider receptor binding profile might be advantageous for imaging because neuroendocrine tumors express different subtypes of somatostatin receptors. The goal of this study was to prospectively compare 68Ga-DOTANOC and 68Ga-DOTATATE PET/CT in the same patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs) and to evaluate the clinical impact of 68Ga-DOTANOC PET/CT. Methods: Eighteen patients with biopsy-proven GEP-NETs were evaluated with 68Ga-DOTANOC and 68Ga-DOTATATE using a randomized crossover design. Labeling of DOTANOC and DOTATATE with 68Ga was standardized using a fully automated synthesis device. PET/CT findings were compared with 3-phase CT scans and in some patients with MR imaging, 18F-FDG PET/CT, and histology. Uptake in organs and tumor lesions was quantified and compared by calculation of maximum standardized uptake values (SUVmax) using volume computer-assisted reading. Results: Histology revealed low-grade GEP-NETs (G1) in 4 patients, intermediate grade (G2) in 7, and high grade (G3) in 7. 68Ga-DOTANOC and 68Ga-DOTATATE were false-negative in only 1 of 18 patients. In total, 248 lesions were confirmed by cross-sectional and PET imaging. The lesion-based sensitivity of 68Ga-DOTANOC PET was 93.5%, compared with 85.5% for 68Ga-DOTATATE PET (P = 0.005). The better performance of 68Ga-DOTANOC PET is attributed mainly to the significantly higher detection rate of liver metastases rather than tumor differentiation grade. Multivariate analysis revealed significantly higher SUVmax in G1 tumors than in G3 tumors (P = 0.009). This finding was less pronounced with 68Ga-DOTANOC (P > 0.001). Altogether, 68Ga-DOTANOC changed treatment in 3 of 18 patients (17%). Conclusion: The sst2,3,5-specific radiotracer 68Ga-DOTANOC detected significantly more lesions than the sst2-specific radiotracer 68Ga-DOTATATE in our patients with GEP-NETs. The clinical relevance of this finding has to be proven in larger studies.

Targeted radiotherapy of prostate cancer with a gastrin-releasing peptide receptor antagonist is effective as monotherapy and in combination with rapamycin

UNLABELLED The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. METHODS To determine the effect of treatment with rapamycin and radiotherapy with a novel (177)Lu-labeled GRPr antagonist ((177)Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and (177)Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on (177)Lu-RM2 tumor uptake, in vivo small-animal PET studies with (68)Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of (177)Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with (177)Lu-RM2 alone or after pretreatment with rapamycin. RESULTS Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with (177)Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of (177)Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. CONCLUSION Radiotherapy using a (177)Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

Promises of cyclotron-produced 44Sc as a diagnostic match for trivalent β--emitters: in vitro and in vivo study of a 44Sc-DOTA-folate conjugate

In recent years, implementation of 68Ga-radiometalated peptides for PET imaging of cancer has attracted the attention of clinicians. Herein, we propose the use of 44Sc (half-life = 3.97 h, average β+ energy [Eβ+av] = 632 keV) as a valuable alternative to 68Ga (half-life = 68 min, Eβ+av = 830 keV) for imaging and dosimetry before 177Lu-based radionuclide therapy. The aim of the study was the preclinical evaluation of a folate conjugate labeled with cyclotron-produced 44Sc and its in vitro and in vivo comparison with the 177Lu-labeled pendant. Methods: 44Sc was produced via the 44Ca(p,n)44Sc nuclear reaction at a cyclotron (17.6 ± 1.8 MeV, 50 μA, 30 min) using an enriched 44Ca target (10 mg 44CaCO3, 97.00%). Separation from the target material was performed by a semiautomated process using extraction chromatography and cation exchange chromatography. Radiolabeling of a DOTA-folate conjugate (cm09) was performed at 95°C within 10 min. The stability of 44Sc-cm09 was tested in human plasma. 44Sc-cm09 was investigated in vitro using folate receptor–positive KB tumor cells and in vivo by PET/CT imaging of tumor-bearing mice Results: Under the given irradiation conditions, 44Sc was obtained in a maximum yield of 350 MBq at high radionuclide purity (>99%). Semiautomated isolation of 44Sc from 44Ca targets allowed formulation of up to 300 MBq of 44Sc in a volume of 200–400 μL of ammonium acetate/HCl solution (1 M, pH 3.5–4.0) within 10 min. Radiolabeling of cm09 was achieved with a radiochemical yield of greater than 96% at a specific activity of 5.2 MBq/nmol. In vitro, 44Sc-cm09 was stable in human plasma over the whole time of investigation and showed folate receptor–specific binding to KB tumor cells. PET/CT images of mice injected with 44Sc-cm09 allowed excellent visualization of tumor xenografts. Comparison of cm09 labeled with 44Sc and 177Lu revealed almost identical pharmacokinetics. Conclusion: This study presents a high-yield production and efficient separation method of 44Sc at a quality suitable for radiolabeling of DOTA-functionalized biomolecules. An in vivo proof-of-concept study using a DOTA-folate conjugate demonstrated the excellent features of 44Sc for PET imaging. Thus, 44Sc is a valid alternative to 68Ga for imaging and dosimetry before 177Lu-radionuclide tumor therapy.

Morphoproteomic evidence of constitutively activated and overexpressed mTOR pathway in cervical squamous carcinoma and high grade squamous intraepithelial lesions.

Human papilloma virus (HPV) infection of the uterine cervix is linked to the pathogenesis of cervical cancer. Preclinical in vitro and in vivo studies using HPV-containing human cervical carcinoma cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, erlotinib, can induce growth delay of xenografts. Activation of Akt and mTOR are also observed in cervical squamous cell carcinoma and, the expression of phosphorylated mTOR was reported to serve as a marker to predict response to chemotherapy and survival of cervical cancer patients. Therefore, we investigated: a) the expression level of EGFR in cervical squamous cell carcinoma (SCC) and high-grade squamous intraepithelial lesions (HSIL) versus non-neoplastic cervical squamous epithelium; b) the state of activation of the mTOR pathway in these same tissues; and c) any impact of these signal transduction molecules on cell cycle. Formalin-fixed paraffin-embedded tissue microarray blocks containing 20 samples each of normal cervix, HSIL and invasive SCC, derived from a total of 60 cases of cervical biopsies and cervical conizations were examined. Immunohistochemistry was utilized to detect the following antigens: EGFR; mTOR pathway markers, phosphorylated (p)-mTOR (Ser2448) and p-p70S6K (Thr389); and cell cycle associated proteins, Ki-67 and S phase kinase-associated protein (Skp)2. Protein compartmentalization and expression were quantified in regard to proportion (0-100%) and intensity (0-3+). Mitotic index (MI) was also assessed. An expression index (EI) for pmTOR, p-p70S6K and EGFR, respectively was calculated by taking the product of intensity score and proportion of positively staining cells. We found that plasmalemmal EGFR expression was limited to the basal/parabasal cells (2-3+, EI = 67) in normal cervical epithelium (NL), but was diffusely positive in all HSIL (EI = 237) and SCC (EI 226). The pattern of cytoplasmic p-mTOR and nuclear p-p70S6K expression was similar to that of EGFR; all showed a significantly increased EI in HSIL/SCC versus NL (p<0.02). Nuclear translocation of p-mTOR was observed in all SCC lesions (EI = 202) and was significantly increased versus both HSIL (EI = 89) and NL (EI = 54) with p<0.015 and p<0.0001, respectively. Concomitant increases in MI and proportion of nuclear Ki-67 and Skp2 expression were noted in HSIL and SCC. In conclusion, morphoproteomic analysis reveals constitutive activation and overexpression of the mTOR pathway in HSIL and SCC as evidenced by: increased nuclear translocation of pmTOR and p-p70S6K, phosphorylated at putative sites of activation, Ser2448 and Thr389, respectively; correlative overexpression of the upstream signal transducer, EGFR, and increases in cell cycle correlates, Skp2 and mitotic indices. These results suggest that the mTOR pathway plays a key role in cervical carcinogenesis and targeted therapies may be developed for SCC as well as its precursor lesion, HSIL.

The role of peroxisome proliferator-activated receptors in the development and physiology of gametes and preimplantation embryos.

In several species, a family of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs) composed of three isotypes, is expressed in somatic cells and germ cells of the ovary as well as the testis. Invalidation of these receptors in mice or stimulation of these receptors in vivo or in vitro showed that each receptor has physiological roles in the gamete maturation or the embryo development. In addition, synthetic PPAR gamma ligands are recently used to induce ovulation in women with polycystic ovary disease. These results reveal the positive actions of PPAR in reproduction. On the other hand, xenobiotics molecules (in herbicides, plasticizers, or components of personal care products), capable of activating PPAR, may disrupt normal PPAR functions in the ovary or the testis and have consequences on the quality of the gametes and the embryos. Despite the recent data obtained on the biological actions of PPARs in reproduction, relatively little is known about PPARs in gametes and embryos. This review summarizes the current knowledge on the expression and the function of PPARs as well as their partners, retinoid X receptors (RXRs), in germ cells and preimplantation embryos. The effects of natural and synthetic PPAR ligands will also be discussed from the perspectives of reproductive toxicology and assisted reproductive technology.