Investigation of genomic DNA methylation by ultraviolet resonant Raman spectroscopy

Cytosine plays a preeminent role in DNA methylation, an epigenetic mechanism that regulates gene expression, the misregulation of which can lead to severe diseases. Several methods are nowadays employed for assessing the global DNA methylation levels, but none of them combines simplicity, high sensitivity, and low operating costs to be translated into clinical applications. Ultraviolet (UV) resonant Raman measurements at excitation wavelengths of 272 nm, 260 nm, 250 nm, and 228 nm have been carried out on isolated deoxynucleoside triphosphates (dNTPs), on a dNTP mixture as well as on genomic DNA (gDNA) samples, commercial from salmon sperm and non-commercial from B16 murine melanoma cell line. The 228 nm excitation wavelength was identified as the most suitable energy for enhancing cytosine signals over the other DNA bases. The UV Raman measurements performed at this excitation wavelength on hyper-methylated and hypo-methylated DNA from Jurkat leukemic T-cell line have revealed significant spectral differences with respect to gDNA isolated from salmon sperm and mouse melanoma B16 cells. This demonstrates how the proper choice of the excitation wavelength, combined with optimized extraction protocols, makes UV Raman spectroscopy a suitable technique for highlighting the chemical modifications undergone by cytosine nucleotides in gDNA upon hyper- and hypo-methylation events

Short-wavelength four wave mixing experiments using single and two-color schemes at FERMI

The development of ultra-bright extreme ultraviolet (EUV) and X-ray free electron laser (FEL) sources has enabled the extension of wave-mixing approaches into the short wavelength regime. Such a class of experiments relies upon nonlinear interactions among multiple light pulses offering a unique tool for exploring the dynamics of ultrafast processes and correlations between selected excitations at relevant length and time scales adding elemental and site selectivity as well. Besides the availability of a suitable photon source, the implementation of wave mixing methodology requires efforts in developing the instrumental set-up. We have realized at the FERMI FEL two dedicated set-ups to handle multiple FEL beams with preselected parameters in a non-collinear fashion and control their interaction sequence at the target. These unique apparatuses, combined with the exceptional characteristics of the seeded FERMI FEL, have allowed us to make the first steps into this field and further advances are foreseen in the near future

Smallsat high-energy missions using ablative pulsed plasma thrusters

Faculdade UnB Gama (FGA

Probing the molecular connectivity of water confined in polymer hydrogels

The molecular connectivity and the extent of hydrogen-bond patterns of water molecules confined in the polymer hydrogels, namely cyclodextrin nanosponge hydrogels, are here investigated by using vibrational spectroscopy experiments. The proposed spectroscopic method exploits the combined analysis of the vibrational spectra of polymers hydrated with water and deuterated water that allows us to separate and selectively investigate the temperature-evolution of the HOH bending mode of engaged water molecules and of the vibrational modes assigned to specific chemical groups of the polymer matrix involved in the physical interactions with water. As main results, we find a strong experimental evidence of a liquid-like behaviour of water molecules confined in the nano-cavities of hydrogel and we observe a characteristic destructuring effect on the hydrogen-bonds network of confined water induced by thermal motion. More interestingly, the extent of this temperature-disruptive effect is found to be selectively triggered by the cross-linking degree of the hydrogel matrix. These results give a more clear picture of the molecular mechanism of water confinement in the pores of nanosponge hydrogel and open the possibility to exploit the spectroscopic method here proposed as investigating tools for water-retaining soft materials

Tunability experiments at the FERMI@Elettra free-electron laser

FERMI@Elettra is a free electron-laser (FEL)-based user facility that, after two years of commissioning, started preliminary users' dedicated runs in 2011. At variance with other FEL user facilities, FERMI@Elettra has been designed to deliver improved spectral stability and longitudinal coherence. The adopted scheme, which uses an external laser to initiate the FEL process, has been demonstrated to be capable of generating FEL pulses close to the Fourier transform limit. We report on the first instance of FEL wavelength tuning, both in a narrow and in a large spectral range (fine- and coarse-tuning). We also report on two different experiments that have been performed exploiting such FEL tuning. We used fine-tuning to scan across the 1s–4p resonance in He atoms, at ≈23.74 eV (52.2 nm), detecting both UV–visible fluorescence (4p–2s, 400 nm) and EUV fluorescence (4p–1s, 52.2 nm). We used coarse-tuning to scan the M4,5 absorption edge of Ge (∼29.5 eV) in the wavelength region 30–60 nm, measured in transmission geometry with a thermopile positioned on the rear side of a Ge thin foil

Early somatosensory evoked potentials in supratentorial brain tumors.

Early somatosensory evoked potentials were recorded in 33 patients with supratentorial brain tumors (9 benign tumors, 17 glioblastomas and 7 metastases). All the cases were studied with CT scan and all but 2 were surgically explored and histologically classified. Evoked potentials were statistically analyzed regarding the nature and site of the tumors. The temporal malignant tumors showed the most significant alterations in latencies, particularly for waves P25, N34 and P44

Longitudinal acoustic compliance and tagged particle susceptibility in liquid and supercooled glycerol

Brillouin spectra of glycerol measured in the visible, ultraviolet and X-ray frequency regions allow us to reckon the imaginary part of acoustic compliance,J"(omega), over a broad frequency range from fraction of GHz to tens of THz. We observe that J"(omega) suitably mimic the shape of the tagged particle susceptibility, chi"(INS)(omega), measured by incoherent neutron spectra for both the liquid and supercooled states. The proportionality between these two quantities suggests a strict relationship between acoustic dissipation and generalized density of states. (C) 2010 Elsevier B.V. All rights reserved. [Comez, L.] Univ Perugia, IOM CNR, I-06123 Perugia, Italy; [Comez, L.; Paciaroni, A.; Scarponi, F.; Fioretto, D.] Univ Perugia, Dipartimento Fis, I-06123 Perugia, Italy; [Monaco, G.] European Synchrotron Radiat Facil, F-38043 Grenoble, France; [Masciovecchio, C.; Gessini, A.] Sincrotrone Trieste, I-34012 Basovizza Trieste, Italy; [Ruocco, G.] Univ Roma La Sapienza, Dipartimento Fis, I-00185 Rome, Italy Comez, L (reprint author), Univ Perugia, IOM CNR, I-06123 Perugia, Italy. [email protected] Ruocco, Giancarlo/A-6245-2010; paciaroni, alessandro/J-2447-2012 Ruocco, Giancarlo/0000-0002-2762-9533; 34 1 1 ELSEVIER SCIENCE BV AMSTERDAM PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS 0022-3093 J NON-CRYST SOLIDS J. Non-Cryst. Solids JAN 15 2011 357 2 SI 515 517 10.1016/j.jnoncrysol.2010.05.096 3 Materials Science, Ceramics; Materials Science, Multidisciplinary Materials Science 725JK WOS:000287640800049 J Gregori, B; Papazachariadis, O; Farruggia, A; Accornero, N Gregori, Bruno; Papazachariadis, Odysseas; Farruggia, Alfonsa; Accornero, Neri A differential color flicker test for detecting acquired color vision impairment in multiple sclerosis and diabetic retinopathy JOURNAL OF THE NEUROLOGICAL SCIENCES English Article Critical fusion frequency; Flicker test; Color vision disorders; Optic neuritis; Retinopathy; Diabetes OPTIC NEURITIS; CONTRAST SENSITIVITY; MACULAR DEGENERATION; EVOKED-POTENTIALS; FUSION FREQUENCY; AGE; LUMINANCE; DISEASE; LOSSES; DISCRIMINATION Background: Optic neuritis related to multiple sclerosis and diabetic retinopathy are relatively selective postretinal and retinal vision disorders. Vision impairment in both conditions is reliably measured by testing critical fusion frequency (CFF). Methods: To examine color vision, we measured the CFF in response to red and blue stimuli, and tested CFF values in patients without evident vision impairment. To ensure that differences in CFF values in a given subject depended only on color perception we displayed red and blue flickering stimuli at equal luminance. CFF to red or blue stimuli were compared in patients with medical history of optic neuritis related to multiple sclerosis (post-retinal vision impairment), patients with diabetic retinopathy (retinal vision impairment) and healthy subjects. Results: The test procedure disclosed altered CFF values for red and blue stimuli in both groups of patients studied. The comparison between the two groups disclosed a prevalent CFF impairment for red stimuli in patients with optic neuritis related to multiple sclerosis and for blue stimuli in patients with diabetic retinopathy. Conclusions: The differential color flicker test appears highly accurate in detecting color vision impairment. Comparison of the two color CFFs differentiates retinal from post-retinal visual disorders. (c) 2010 Elsevier B.V. All rights reserved. [Papazachariadis, Odysseas] Univ Roma La Sapienza, Dept Physiol & Pharmacol, I-00185 Rome, Italy; [Gregori, Bruno; Papazachariadis, Odysseas; Farruggia, Alfonsa; Accornero, Neri] Univ Roma La Sapienza, Dept Neurol Sci, I-00185 Rome, Italy; [Gregori, Bruno] UO Neurol, Clin Nuova Itor, I-00158 Rome, Italy Papazachariadis, O (reprint author), Univ Roma La Sapienza, Dept Physiol & Pharmacol, Piazzale Aldo Moro 5, I-00185 Rome, Italy. [email protected] 42 2 2 ELSEVIER SCIENCE BV AMSTERDAM PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS 0022-510X J NEUROL SCI J. Neurol. Sci. JAN 15 2011 300 1-2 130 134 10.1016/j.jns.2010.09.002 5 Clinical Neurology; Neurosciences Neurosciences & Neurology 714ZT WOS:000286850900024 J Bernabei, M; Moreno, AJ; Zaccarelli, E; Sciortino, F; Colmenero, J Bernabei, Marco; Moreno, Angel J.; Zaccarelli, Emanuela; Sciortino, Francesco; Colmenero, Juan From caging to Rouse dynamics in polymer melts with intramolecular barriers: A critical test of the mode coupling theory JOURNAL OF CHEMICAL PHYSICS English Article SPATIALLY HETEROGENEOUS DYNAMICS; GLASS-TRANSITION; SUPERCOOLED LIQUIDS; SIMULATION; RELAXATION; SCATTERING; ADSORPTION; CHAINS By means of computer simulations and solution of the equations of the mode coupling theory ( MCT), we investigate the role of the intramolecular barriers on several dynamic aspects of nonentangled polymers. The investigated dynamic range extends from the caging regime characteristic of glass-formers to the relaxation of the chain Rouse modes. We review our recent work on this question, provide new results, and critically discuss the limitations of the theory. Solutions of the MCT for the structural relaxation reproduce qualitative trends of simulations for weak and moderate barriers. However, a progressive discrepancy is revealed as the limit of stiff chains is approached. This disagreement does not seem related with dynamic heterogeneities, which indeed are not enhanced by increasing barrier strength. It is not connected either with the breakdown of the convolution approximation for three-point static correlations, which retains its validity for stiff chains. These findings suggest the need of an improvement of the MCT equations for polymer melts. Concerning the relaxation of the chain degrees of freedom, MCT provides a microscopic basis for time scales from chain reorientation down to the caging regime. It rationalizes, from first principles, the observed deviations from the Rouse model on increasing the barrier strength. These include anomalous scaling of relaxation times, long-time plateaux, and nonmonotonous wavelength dependence of the mode correlators. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3525147] [Moreno, Angel J.; Colmenero, Juan] Univ Basque Country, CSIC, Ctr Fis Mat, E-20018 San Sebastian, Spain; [Moreno, Angel J.; Colmenero, Juan] Mat Phys Ctr MPC, E-20018 San Sebastian, Spain; [Bernabei, Marco; Colmenero, Juan] Donostia Int Phys Ctr, E-20018 San Sebastian, Spain; [Zaccarelli, Emanuela; Sciortino, Francesco] Univ Roma La Sapienza, Dipartimento Fis, I-00185 Rome, Italy; [Zaccarelli, Emanuela; Sciortino, Francesco] Univ Roma La Sapienza, CNR ISC, I-00185 Rome, Italy; [Colmenero, Juan] Univ Basque Country, Dept Fis Mat, E-20080 San Sebastian, Spain Moreno, AJ (reprint author), Univ Basque Country, CSIC, Ctr Fis Mat, Paseo Manuel Lardizabal 5, E-20018 San Sebastian, Spain. [email protected] Moreno, Angel/C-7313-2011; Sciortino, Francesco/B-4768-2012; CSIC-UPV/EHU, CFM/F-4867-2012 EU [FP7-PEOPLE-2007-1-1-ITN, MAT2007-63681, IT-436-07, ERC-226207-PATCHYCOLLOIDS, ITN-234810-COMPLOIDS] We thank S.-H. Chong, T. Franosch, M. Fuchs, M. Sperl, and J. Baschnagel for useful discussions. We acknowledge financial support from projects FP7-PEOPLE-2007-1-1-ITN (DYNACOP, EU), MAT2007-63681 (Spain), IT-436-07 (GV, Spain), ERC-226207-PATCHYCOLLOIDS (EU), and ITN-234810-COMPLOIDS (EU). 50 2 2 AMER INST PHYSICS MELVILLE CIRCULATION & FULFILLMENT DIV, 2 HUNTINGTON QUADRANGLE, STE 1 N O 1, MELVILLE, NY 11747-4501 USA 0021-9606 J CHEM PHYS J. Chem. Phys. JAN 14 2011 134 2 024523 10.1063/1.3525147 12 Physics, Atomic, Molecular & Chemical Physics 709WK WOS:000286471900064 J Zammataro, M; Chiechio, S; Montana, MC; Traficante, A; Copani, A; Nicoletti, F; Gereau, RW Zammataro, Magda; Chiechio, Santina; Montana, Michael C.; Traficante, Anna; Copani, Agata; Nicoletti, Ferdinando; Gereau, Robert W. mGlu2 metabotropic glutamate receptors restrain inflammatory pain and mediate the analgesic activity of dual mGlu2/mGlu3 receptor agonists MOLECULAR PAIN English Article NAAG PEPTIDASE INHIBITORS; MESSENGER-RNA EXPRESSION; RAT FORMALIN TEST; GROUP-II; N-ACETYLASPARTYLGLUTAMATE; SPINAL-CORD; UP-REGULATION; PERSISTENT; MODELS; PRETREATMENT Group II metabotropic glutamate receptors (mGluRs) couple to the inhibitory G-protein Gi. The group II mGluRs include two subtypes, mGlu2 and mGlu3, and their pharmacological activation produces analgesic effects in inflammatory and neuropathic pain states. However, the specific contribution of each one of the two subtypes has not been clarified due to the lack of selective orthosteric ligands that can discriminate between mGlu2 and mGlu3 subtypes. In this study we used mGlu2 or mGlu3 knock-out mice to dissect the specific role for these two receptors in the endogenous control of inflammatory pain and their specific contribution to the analgesic activity of mixed mGlu2/3 receptor agonists. Our results showed that mGlu2(-/-) mice display a significantly greater pain response compared to their wild type littermates. Interestingly the increased pain sensitivity in mGlu2(-/-) mice occurred only in the second phase of the formalin test. No differences were observed in the first phase. In contrast, mGlu3(-/-) mice did not significantly differ from their wild type littermates in either phase of the formalin test. When systemically injected, a single administration of the mGlu2/3 agonist, LY379268 (3 mg/kg, ip), showed a significant reduction of both phases in wild-type mice and in mGlu3(-/-) but not in mGlu2(-/-) mice. However tolerance to the analgesic effect of LY379268 (3 mg/kg, ip) in mGlu3(-/-) mice developed following 5 consecutive days of injection. Taken together, these results demonstrate that: (i) mGlu2 receptors play a predominant role over mGlu3 receptors in the control of inflammatory pain in mice; (ii) the analgesic activity of mixed mGlu2/3 agonists is entirely mediated by the activation of the mGlu2 subtype and (iii) the development of tolerance to the analgesic effect of mGlu2/3 agonists develops despite the lack of mGlu3 receptors. [Chiechio, Santina; Copani, Agata] Univ Catania, Dept Drug Sci, I-95124 Catania, Italy; [Zammataro, Magda] Univ Catania, Ph D Program Neuropharmacol, I-95124 Catania, Italy; [Montana, Michael C.; Gereau, Robert W.] Washington Univ, Sch Med, Dept Anesthesiol, St Louis, MO 63110 USA; [Montana, Michael C.; Gereau, Robert W.] Washington Univ, Sch Med, Pain Ctr, St Louis, MO 63110 USA; [Traficante, Anna; Nicoletti, Ferdinando] INM Neuromed, Pozzilli, Italy; [Nicoletti, Ferdinando] Univ Roma La Sapienza, Dept Physiol & Pharmacol, Rome, Italy Chiechio, S (reprint author), Univ Catania, Dept Drug Sci, I-95124 Catania, Italy. [email protected] National Institutes of Health [R01NS42595] We thank Professor S. Nakanishi for providing the mGlu2-/- mice. This work was supported by funds from the National Institutes of Health (R01NS42595) to RWG. 37 2 2 BIOMED CENTRAL LTD LONDON 236 GRAYS INN RD, FLOOR 6, LONDON WC1X 8HL, ENGLAND 1744-8069 MOL PAIN Mol. Pain JAN 14 2011 7 6 10.1186/1744-8069-7-6 5 Neurosciences Neurosciences & Neurology 712PJ WOS:000286678400001 J Wang, K; Diskin, SJ; Zhang, HT; Attiyeh, EF; Winter, C; Hou, CP; Schnepp, RW; Diamond, M; Bosse, K; Mayes, PA; Glessner, J; Kim, C; Frackelton, E; Garris, M; Wang, Q; Glaberson, W; Chiavacci, R; Nguyen, L; Jagannathan, J; Saeki, N; Sasaki, H; Grant, SFA; Iolascon, A; Mosse, YP; Cole, KA; Li, HZ; Devoto, M; McGrady, PW; London, WB; Capasso, M; Rahman, N; Hakonarson, H; Maris, JM Wang, Kai; Diskin, Sharon J.; Zhang, Haitao; Attiyeh, Edward F.; Winter, Cynthia; Hou, Cuiping; Schnepp, Robert W.; Diamond, Maura; Bosse, Kristopher; Mayes, Patrick A.; Glessner, Joseph; Kim, Cecilia; Frackelton, Edward; Garris, Maria; Wang, Qun; Glaberson, Wendy; Chiavacci, Rosetta; Nguyen, Le; Jagannathan, Jayanti; Saeki, Norihisa; Sasaki, Hiroki; Grant, Struan F. A.; Iolascon, Achille; Mosse, Yael P.; Cole, Kristina A.; Li, Hongzhe; Devoto, Marcella; McGrady, Patrick W.; London, Wendy B.; Capasso, Mario; Rahman, Nazneen; Hakonarson, Hakon; Maris, John M. Integrative genomics identifies LMO1 as a neuroblastoma oncogene NATURE English Article COPY NUMBER; CHROMOSOMAL TRANSLOCATIONS; ACTIVATING MUTATIONS; ALK KINASE; GENE; TRANSCRIPTION; CANCER; INTERACTS; TARGET; CELLS Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths(1,2). To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 x 10(-16), odds ratio of risk allele = 1.34 (95% confidence interval 1.25-1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression. [Wang, Kai; Zhang, Haitao; Hou, Cuiping; Glessner, Joseph; Kim, Cecilia; Frackelton, Edward; Glaberson, Wendy; Chiavacci, Rosetta; Grant, Struan F. A.; Hakonarson, Hakon] Childrens Hosp Philadelphia, Ctr Appl Genom, Philadelphia, PA 19104 USA; [Diskin, Sharon J.; Attiyeh, Edward F.; Winter, Cynthia; Schnepp, Robert W.; Diamond, Maura; Bosse, Kristopher; Mayes, Patrick A.; Garris, Maria; Wang, Qun; Nguyen, Le; Jagannathan, Jayanti; Mosse, Yael P.; Cole, Kristina A.; Maris, John M.] Childrens Hosp Philadelphia, Div Oncol, Philadelphia, PA 19104 USA; [Diskin, Sharon J.; Attiyeh, Edward F.; Winter, Cynthia; Schnepp, Robert W.; Diamond, Maura; Bosse, Kristopher; Mayes, Patrick A.; Garris, Maria; Wang, Qun; Nguyen, Le; Jagannathan, Jayanti; Mosse, Yael P.; Cole, Kristina A.; Maris, John M.] Childrens Hosp Philadelphia, Ctr Childhood Canc Res, Philadelphia, PA 19104 USA; [Nguyen, Le; Li, Hongzhe; Devoto, Marcella] Univ Penn, Sch Med, Dept Biostat & Epidemiol, Philadelphia, PA 19104 USA; [Saeki, Norihisa; Sasaki, Hiroki] Natl Canc Ctr, Res Inst, Div Genet, Tokyo 1040045, Japan; [Nguyen, Le; Grant, Struan F. A.; Mosse, Yael P.; Cole, Kristina A.; Devoto, Marcella; Hakonarson, Hakon; Maris, John M.] Univ Penn, Sch Med, Dept Pediat, Philadelphia, PA 19104 USA; [Grant, Struan F. A.; Devoto, Marcella; Hakonarson, Hakon] Childrens Hosp Philadelphia, Div Human Genet, Philadelphia, PA 19104 USA; [Iolascon, Achille; Capasso, Mario] CEINGE Biotecnol Avanzate, I-80145 Naples, Italy; [Devoto, Marcella] Univ Roma La Sapienza, Dept Expt Med, I-00185 Rome, Italy; [McGrady, Patrick W.] Univ Florida, Dept Stat, Gainesville, FL 32603 USA; [McGrady, Patrick W.] Childrens Oncol Grp, Gainesville, FL 32603 USA; [London, Wendy B.] Dana Farber Childrens Hosp, Ctr Canc, Boston, MA 02115 USA; [London, Wendy B.] Childrens Oncol Grp, Boston, MA 02115 USA; [Iolascon, Achille; Capasso, Mario] Univ Naples Federico 2, Dept Biochem & Med Biotechnol, I-80131 Naples, Italy; [Rahman, Nazneen] Inst Canc Res, Sect Canc Genet, Sutton SM2 5NG, Surrey, England Hakonarson, H (reprint author), Childrens Hosp Philadelphia, Ctr Appl Genom, Philadelphia, PA 19104 USA. [email protected]; [email protected] National Institutes of Health [R01-CA124709]; Giulio D'Angio Endowed Chair; Alex's Lemonade Stand Foundation; Evan Dunbar Foundation; Rally Foundation; Andrew's Army Foundation; Abramson Family Cancer Research Institute; Howard Hughes Medical Institute; Associazione Oncologia Pediatrica e Neuroblastoma; Cotswold Foundation; Children's Hospital of Philadelphia; [UL1-RR024134-03] We acknowledge the Children's Oncology Group for providing most blood and tumour specimens and clinical and outcome data (U10-CA98543 and U10-CA98413) from neuroblastoma patients. We thank G. P. Tonini for providing neuroblastoma DNA samples in the Italian replication cohort. This work was supported in part by National Institutes of Health grant R01-CA124709 (to J.M.M.), the Giulio D'Angio Endowed Chair (J.M.M.), the Alex's Lemonade Stand Foundation (J.M.M.), the Evan Dunbar Foundation (J.M.M.), the Rally Foundation (J.M.M.), Andrew's Army Foundation (J.M.M.), the Abramson Family Cancer Research Institute (J.M.M.), a Howard Hughes Medical Institute Research Training Fellowship (K. B.), a fellowship from Associazione Oncologia Pediatrica e Neuroblastoma (M. C.), a Research Development Award from the Cotswold Foundation (H. H.), UL1-RR024134-03 (H. H.) and an Institutional Development Award to the Center for Applied Genomics from the Children's Hospital of Philadelphia (H.H.). 22 30 32 NATURE PUBLISHING GROUP LONDON MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND 0028-0836 NATURE Nature JAN 13 2011 469 7329 216 220 10.1038/nature09609 5 Multidisciplinary Sciences Science & Technology - Other Topics 705NO WOS:000286143400039 J Nottola, SA; Cecconi, S; Bianchi, S; Motta, C; Rossi, G; Continenza, MA; Macchiarelli, G Nottola, Stefania A.; Cecconi, Sandra; Bianchi, Serena; Motta, Cecilia; Rossi, Gianna; Continenza, Maria A.; Macchiarelli, Guido Ultrastructure of isolated mouse ovarian follicles cultured in vitro REPRODUCTIVE BIOLOGY AND ENDOCRINOLOGY English Article TRANSMISSION ELECTRON-MICROSCOPY; GRANULOSA-CELL PROLIFERATION; BOVINE PREANTRAL FOLLICLES; PRE-ANTRAL FOLLICLES; LONG-TERM CULTURE; STIMULATING-HORMONE; DEVELOPMENTAL COMPETENCE; FOLLICULAR DEVELOPMENT; PRIMORDIAL FOLLICLES; LUTEINIZING-HORMONE Background: In vitro maturation of ovarian follicles, in combination with cryopreservation, might be a valuable method for preserving and/or restoring fertility in mammals with impaired reproductive function. Several culture systems capable of sustaining mammalian follicle growth in vitro have been developed and many studies exist on factors influencing the development of in vitro grown oocytes. However, a very few reports concern the ultrastructural morphology of in vitro grown follicles. Methods: The present study was designed to evaluate, by transmission and scanning electron microscopy, the ultrastructural features of isolated mouse preantral follicles cultured in vitro for 6 days in a standard medium containing fetal calf serum (FCS). The culture was supplemented or not with FSH. Results: The follicles cultured in FCS alone, without FSH supplementation (FCS follicles), did not form the antral cavity. They displayed low differentiation (juxta-nuclear aggregates of organelles in the ooplasm, a variable amount of microvilli on the oolemma, numerous granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). Eighty (80)% of FSH-treated follicles formed the antral cavity (FSH antral follicles). These follicles showed various ultrastructural markers of maturity (spreading of organelles in ooplasm, abundant microvilli on the oolemma, scarce granulosa cell-oolemma contacts, granulosa cell proliferation). Areas of detachment of the innermost granulosa cell layer from the oocyte were also found, along with a diffuse granulosa cell loosening compatible with the antral formation. Theca cells showed an immature morphology for the stage reached. Twenty (20)% of FSH-treated follicles did not develop the antral cavity (FSH non-antral follicles) and displayed morphological differentiation features intermediate between those shown by FCS and FSH antral follicles (spreading of organelles in the ooplasm, variable amount of microvilli, scattered granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). Conclusions: It is concluded that FSH supports the in vitro growth of follicles, but the presence of a diffuse structural granulosa cell-oocyte uncoupling and the absence of theca development unveil the incomplete efficiency of the system. The present study contributes to explain, from a morphological point of view, the effects of culture conditions on the development of mouse in vitro grown follicles and to highlight the necessity of maintaining efficient intercellular communications to obtain large numbers of fully-grown mature germ cells. [Cecconi, Sandra; Bianchi, Serena; Rossi, Gianna; Continenza, Maria A.; Macchiarelli, Guido] Univ Aquila, Dept Hlth Sci, I-67100 Laquila, Italy; [Nottola, Stefania A.; Motta, Cecilia] Univ Roma La Sapienza, Dept Anat Histol Forens Med & Orthopaed, Rome, Italy; [Macchiarelli, Guido] Univ Aquila, Ctr Microscopy, I-67100 Laquila, Italy Macchiarelli, G (reprint author), Univ Aquila, Dept Hlth Sci, I-67100 Laquila, Italy. [email protected] Italian Ministry of Education, University and Research, "La Sapienza" University, Rome; Italian Ministry of Education, University and Research, University of L'Aquila T