Eukaryotic initiation factor eIF6 modulates the expression of Kermit 2/XGIPC in IGF- regulated eye development.

The eukaryotic initiation translation factor eIF6 is a highly conserved, essential protein implicated in translation. eIF6 is regulated in vivo by extracellular signals, such as IGF signaling (for a review see Miluzio et al., 2009). In Xenopus, eif6 over-expression causes a delay in eye development (De Marco et al., 2011). In this study we showed that eif6 co-immunoprecipitates with the insulin-like growth factor receptor (igfr) and may function downstream of igf in eye formation. The relationship between eif6 and gipc2, a protein partner of a variety of molecules including membrane proteins, was investigated. gipc2 is required for maintaining igf-induced akt activation on eye development (Wu et al., 2006). Significantly eif6 and gipc2 have opposite effects in eye development. While eif6 is required for eye formation below threshold levels, gipc2 knockdown impairs eye development (De Marco et al., 2011; Wu et al., 2006). In this study, it was shown that in eif6 over-expressors, the delay in eye morphogenesis is reversed by gipc2 injection, while the injection of eif6 down-regulates gipc2 expression. Real-time-PCR indicates that eif6 regulates gipc2 expression in a dose-dependent manner. In contrast, gipc2 knockdown has no significant effect on eif6 mRNA levels. These results suggest that eif6 regulation of gipc2 enables correct morphogenesis of Xenopus eye and stimulate questions on the molecular network implicated in this process.

Super-Resonant Intracavity Coherent Absorption.

The capability of optical resonators to extend the effective radiation-matter interaction length originates from a multipass effect, hence is intrinsically limited by the resonator's quality factor. Here, we show that this constraint can be overcome by combining the concepts of resonant interaction and coherent perfect absorption (CPA). We demonstrate and investigate super-resonant coherent absorption in a coupled Fabry-Perot (FP)/ring cavity structure. At the FP resonant wavelengths, the described phenomenon gives rise to split modes with a nearly-transparent peak and a peak whose transmission is exceptionally sensitive to the intracavity loss. For small losses, the effective interaction pathlength of these modes is proportional respectively to the ratio and the product of the individual finesse coefficients of the two resonators. The results presented extend the conventional definition of resonant absorption and point to a way of circumventing the technological limitations of ultrahigh-quality resonators in spectroscopy and optical sensing schemes.

Mode-splitting cloning in birefringent fiber Bragg grating ring resonators.

In this Letter, we report the theoretical model and the experimental evidence of a mode-splitting cloning effect due to the resonant coupling between modes having different polarizations in weakly birefringent fiber Bragg grating (FBG) ring resonators. This modal coupling depends on the fiber birefringence and the FBG reflectivity. In the ideal case of the absence of birefringence, a single split-mode resonant structure can be observed in the resonator transmission spectrum due to the degeneracy removal of the two counter-propagating modes. In the presence of FBG birefringence, a secondary split doublet resulting in a clone of the initial one is generated. The described effect can be exploited for spectroscopic-sensing applications based on more complex split-mode dynamics.

Fiber Bragg grating laser sensor with direct radio-frequency readout.

A fiber Bragg grating (FBG)-coupled ring laser sensor is demonstrated. In the proposed configuration the interrogating source, the sensing head and the readout instrument are integrated in a single fiber-optic device. An FBG inserted within a bidirectional fiber ring couples the two counterpropagating modes of the cavity, generating a splitting of the resonant wavelengths proportional to the FBG reflectivity. When the cavity gain is brought beyond threshold, the two peaks of the split resonances simultaneously lase, leading to a beat note in the emission spectrum whose frequency tracks any small shift of the FBG reflectivity spectrum. Such a beat note can be simply monitored by a frequency counter, without the need for an optical spectrometer, allowing to significantly reduce size and costs of the sensor setup. The sensing performance compares well to the state-of-the-art thermo-mechanical fiber sensors.

Fiber Bragg grating ring resonators under rotation for angular velocity sensing.

In this paper we investigate the possibility of using hybrid resonators based on fiber Bragg grating ring resonators (FBGRRs) and π-shifted FBGRRs (i.e., defective FBGRRs) as rotation sensitive elements for gyroscope applications. In particular, we model the conventional fiber Bragg grating (FBG) with the coupled mode theory by taking into account how the Sagnac effect, induced by the rotation, modifies the eigenvalues, the photonic band gap, and the spectral response of the FBG. Then, on the basis of the FBG model under rotation conditions, the spectral responses of the FBGRR and π-FBGRR have been evaluated, confirming that the Sagnac effect manifests itself with a spectral shift of the eigensolutions. This physical investigation can be exploited for opening new ways in the optical gyroscope platforms.

Enhanced spectral response of π-phase shifted fiber Bragg gratings in closed-loop configuration.

The transmission spectrum of a ring resonator enclosing a π-phase shifted fiber Bragg grating (π-FBG) shows a spectral feature at the Bragg wavelength that is much sharper than resonance of the π-FBG alone, and that can be detected with a simple integrated cavity output technique. Hence, the resolution of any sensor based on the fitting of the π-FBG spectral profile can be largely improved by the proposed configuration at no additional fabrication costs and without altering the sensor robustness. A theoretical model shows that the resolution enhancement attainable in the proposed closed-loop geometry depends on the quality factor of the ring resonator. With a commercial grating in a medium-finesse ring, a spectral feature 12 times sharper than the π-FBG resonance is experimentally demonstrated. A larger enhancement is expected in a low-loss, polarization maintaining setup.

Split-mode fiber Bragg grating sensor for high-resolution static strain measurements.

We demonstrate a strain sensor with very high sensitivity in the static and low frequency regime based on a fiber ring cavity that includes a π phase-shifted fiber Bragg grating. The grating acts as a partial reflector that couples the two counter-propagating cavity modes, generating a splitting of the resonant frequencies. The presence of a sharp transition within the π phase-shifted fiber Bragg grating's spectral transmittance makes this frequency splitting extremely sensitive to length, temperature, and the refractive index of the fiber in the region where the grating is written. The splitting variations caused by small mechanical deformations of the grating are tracked in real time by interrogating a cavity resonance with a locked-carrier scanning-sideband technique. The measurable strain range and bandwidth are characterized, and a resolution of 320 pϵ/Hz(1/2) at 0 Hz is experimentally demonstrated, the highest achieved to date with a fiber Bragg grating sensor.

Localized strain sensing with fiber Bragg-grating ring cavities.

We report the theoretical description and the experimental demonstration of an optical resonator formed by inserting a Fiber Bragg Grating (FBG) in a closed fiber loop. The spectral characteristics of such a resonator strongly depend on the reflectivity of the FBG. In the wavelength region where the FBG reflectivity R is negligible, the system behaves like a conventional ring resonator. On the other hand, when R is not vanishing, a split-mode structure can be observed, associated to the degeneracy removal of two counterpropagating resonant modes. The magnitude of the mode splitting can be used to sense small variations of the FBG physical parameters, such as length, temperature or group index. An example of strain sensing with this setup is reported, showing that the mode splitting is sensitive to a mechanical strain applied to the FBG, while it is almost insensitive to a strain applied to any other point of the resonator. This peculiar feature allows to perform cavity-enhanced, local strain measurements with a reduced sensitivity to environmental perturbations, which represents an important improvement in the framework of the fiber-optic sensors.

Human dental pulp cell apoptosis: immunohistochemical study after applying orthodontic traction.

The aim of this study was to compare human dental pulp stress and programmed cell death after 3 and 6 months of orthodontic treatments by assessing the degree of apoptosis and related proteins. Human dental pulps were collected from twenty young patients orthodontically treated by Straight Wire technique. Samples were fixed, paraffin-embedded and processed for histology and immunohistochemistry using anti-heat shock protein 60 kDa (Hsp60), -caspase 3, -caspase 9, and -PCNA antibodies, as well as TUNEL reactions. Moreover, we performed immunoprecipitation for Hsp60 and caspase 3, and for Hsp60 and caspase 9, from paraffin extracted tissues. Increased levels of both caspases and Hsp60 occurred in 6-months treated samples; at the same time, we found increased levels of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells. Immunoprecipitation showed that Hsp60 forms a complex with both Pro-caspase 3 and Caspase 3, and this may accelerate Pro-caspase 3 activation, especially in the 6-months treated group. On the contrary, no complex between Hsp60 and Pro-caspase 9 was detected. The orthodontic tractions may be a cause of stress, apoptosis and proliferation in pulp tissue. These results suggest the need of further studies about the effects of long term orthodontic treatments on the dental pulp.

Expression of XNOA 36 in the mitochondrial cloud of Xenopus laevis oocytes.

In Xenopus laevis oocytes a mitochondrial cloud (MC) is found between the nucleus and the plasma membrane at stages I-II of oogenesis. The MC contains RNAs that are transported to the future vegetal pole at stage II of oogenesis. In particular, germinal plasm mRNAs are found in the Message Transport Organiser (METRO) region, the MC region opposite to the nucleus. At stages II-III, a second pathway transports Vg1 and VegT mRNAs to the area where the MC content merges with the vegetal cortex. Microtubules become polarized at the sites of migration of Vg1 and VegT mRNAs through an unknown signalling mechanism. In early meiotic stages, the centrioles are almost completely lost with their remnants being dispersed into the cytoplasm and the MC, which may contain a MTOC to be used in the later localization pathway of the mRNAs. In mammals, XNOA 36 encodes a member of a highly conserved protein family and localises to the nucleolus or in the centromeres. In the Xenopus late stage I oocyte, XNOA 36 mRNA is transiently segregated in one half of the oocyte, anchored by a cytoskeletal network that contains spectrin. Here we found that XNOA 36 transcript also localises to the nucleoli and in the METRO region. XNOA 36 protein immunolocalization, using an antibody employed for the library immunoscreening that depicted XNOA 36 expression colonies, labels the migrating MC, the cytoplasm of stage I oocytes and in particular the vegetal cortex facing the MC. The possible role of XNOA 36 in mRNA anchoring to the vegetal cortex or in participating in early microtubule reorganization is discussed.

Eukaryotic initiation factor 6 (eif6) overexpression affects eye development in Xenopus laevis.

The translation initiation factor eif6 has been implicated as a regulator of ribosome assembly, selective mRNA translation and apoptosis. Many of these activities depend upon the phosphorylation of eif6 serine 235 by PKC. Previous data showed that eif6 binds to the 60S ribosomal subunit when unphosphorylated, inhibiting assembly with the 40S subunit. Phosphorylation of Ser235 releases eif6 from the 60S subunit and allows assembly. eif6 acts as an anti-apoptotic factor via regulation of the bcl2/bax balance and acts selectively upstream of bcl2. This activity also depends upon phosphorylation of eif6 Ser235. One of the consequences of eif6 overexpression in Xenopus embryos is aberrant eye development. Here we evaluate the eye phenotype and show that it is transient. We show that the whole eye, particularly the retina layers, of the embryos injected with eif6-encoding mRNA recover by stage 42. Embryos over-expressing eif6 have normal expression of anterior- and brain-specific markers, indicating that outside the eye field, other neural regions appear unaffected by the eif6 injection. No eye defect was detected when morpholinos were used to reduce eif6 protein synthesis. We tested how two known pathways of eif6 function with respect to alteration of eye development. We found that injection of bcl2 did not produce the eye phenotype and eif6-bax co-injection did not rescue the eye defect, suggesting that the eye phenotype is not bearing on the anti-apoptotic role played by eif6 is not linked to its role as an anti-apoptotic factor. We also determined that PKC-dependant phosphorylation of Ser235 in eif6 is not required to produce defective eye development. These results indicate that the aberrant eye phenotype, produced by eif6 overexpression, is not directly linked to the PKC-regulated effects of eif6 on translation and ribosomal subunit interaction or on eif6 anti-apoptotic properties.

Lipovitellin constitutes the protein backbone of glycoproteins involved in sperm-egg interaction in the amphibian Discoglossus pictus.

Our knowledge of the molecules that interact with sperm at the egg membrane is restricted to a short list. In the eggs of Discoglossus pictus, fusion with sperm is limited to a differentiated structure, the dimple, offering several advantages for detecting molecules involved in fertilization. Previous studies have identified fucosylated glycoproteins of 200, 260, and 270 kDa located at the surface of the dimple that are able to bind sperm in vitro. Here, we show that dimple glycoproteins and a protein represented by a 120-kDa band released following gel-into-gel SDS-PAGE of both glycoproteins share the same N-terminal amino acid sequence, which itself is similar to the N-termini of Xenopus liver-synthesized vitellogenin (VTG) and the lipovitellin 1. MALDI/MS mass spectrometry indicated that the 120-kDa band is part of both gps 200 and 270/260. A 117-kDa major protein of the egg lysate exhibits the same MALDI/MS spectrum, and LC-MSMS indicates that this is a lipovitellin 1 (DpLIV) that coincides with the 120-kDa band and is responsible for the formation of the 200-270-kDa dimers. Therefore, lipovitellin 1 constitutes the protein backbone of the dimple glycoconjugates. In vitro assays using polystyrene beads coated with DpLIV or with its dimers indicate that significant sperm binding occurs only with DpLIV dimers. In amphibians, VTG is taken up by the oocyte, where it releases lipovitellins destined to form yolk. In Discoglossus, our data suggest that yolk proteins are also synthesized by the oocyte. The dimple forms in the ovulated oocyte following the exocytosis of vesicles that likely expose DpLIVs at their membrane. Indeed, in whole mounts of immunostained eggs, anti-vitellogenin antibodies label only the surface of the dimple.

In X. laevis embryos high levels of the anti-apoptotic factor p27BBP/eIF6 are stage-dependently found in BrdU and TUNEL-reactive territories.

p27BBP/eIF6 (ß4 binding protein/eukaryotic initiation factor 6) is a highly conserved protein necessary for cell life. In adult eIF6 mice, a 50% decrease in the protein levels in all tissues is accompanied by a reduction in cell proliferation only in the liver, fat cells and cultured fibroblasts. During X. laevis embryogenesis expression of p27BBP/eIF6 is abundant in high proliferative territories. However, in Xenopus cell proliferation appears unaffected following p27BBP/eIF6 over-expression or down-regulation. Indeed, p27BBP/eIF6 is an anti-apoptotic factor acting upstream of Bcl2 that reduces endogenous apoptosis. We studied p27BBP/eIF6 protein localization in wild type embryos and compared it to proliferation and apoptosis. At the beginning of embryogenesis, high levels of p27BBP/eIF6, proliferation and apoptosis overlap. In later development stages high proliferation levels are present in the same regions where higher p27BBP/eIF6 expression is observed, while apoptosis does not appear specifically concentrated in the same sites. The higher presence of p27BBP/eIF6 would appear related to an increased need of apoptosis control in the regions where cell death is essential for normal development.

Cetuximab plus chronomodulated irinotecan, 5-fluorouracil, leucovorin and oxaliplatin as neoadjuvant chemotherapy in colorectal liver metastases: POCHER trial.

BACKGROUND: We assessed the effectiveness of cetuximab plus chronomodulated irinotecan, 5-fluorouracil (5-FU), leucovorin (FA) and oxaliplatin (L-OHP) (chrono-IFLO) administered as neoadjuvant chemotherapy to increase the resectability of colorectal liver metastases. METHODS: This was a phase II prospective trial with rate of liver metastases resection as primary end point. Forty-three patients with unresectable metastases were enroled: 9 with metastases >5 cm; 29 with multinodular (>4) disease; 1 with hilar location; 4 with extrahepatic lung disease. Treatment consisted of cetuximab at day 1 plus chronomodulated irinotecan 5-FU, FA and L-OHP for 2-6 days every 2 weeks. After the first 17 patients, doses were reduced for irinotecan to 110 mg m⁻², 5-FU to 550 mg m⁻² per day and L-OHP to 15 mg m⁻² per day. RESULTS: Macroscopically complete resections were performed in 26 out of 43 patients (60%) after a median of 6 (range 3-15) cycles. Partial response was noticed in 34 patients (79%). Median overall survival was 37 months (95% CI: 21-53 months), with a 2-year survival of 68% in the entire population, 80.6% in resected patients and 47.1% in unresected patients (P=0.01). Grade 3/4 diarrhoea occurred in 93% and 36% of patients before and after dose reduction. CONCLUSION: Cetuximab plus chrono-IFLO achieved 60% complete resectability of colorectal liver metastases.

A transient asymmetric distribution of XNOA 36 mRNA and the associated spectrin network bisects Xenopus laevis stage I oocytes along the future A/V axis.

In Xenopus oogenesis, the mechanisms governing the localisation of molecules crucial for primary axis determination have been uncovered in recent years. In stage I oocytes, the mitochondrial cloud (MC) entraps RNAs implicated in germ line specification and other RNAs, such as Xwnt-11 and Xlsirts, that are later delivered to the vegetal pole. Microfilaments and microtubules gradually develop in the cytoplasm, sustaining organelles as well as the MC. At stage III, other mRNAs migrate to the vegetal hemisphere through a microtubule-dependent mechanism. We report here the isolation of a cDNA encoding XNOA 36, a highly conserved protein, whose function is to date not fully understood. The XNOA 36 transcript is abundantly accumulated in stage I oocytes where it decorates a filamentous network. At the end of stage I the transcript gradually segregates in a sector of the oocyte surrounding the MC and opposite the ovarian hylum. Here, XNOA 36 mRNA distributes in a gradient-like pattern extending from a peripheral network towards the interior of the oocyte. This distribution is similar to that of alpha-spectrin mRNA. Both mRNAs are segregated in one half of the 250 microm oocytes, with the MC located between the XNOA 36/alpha-spectrin mRNA-labelled and unlabelled regions. XNOA 36 mRNA localisation was uncoupled from that of alpha-spectrin mRNA by cytochalasin B or ice-nocodazole treatments, suggesting that the two transcripts rely on different mechanisms for their localisation. However, immunolocalisation experiments coupled with in situ hybridisation revealed that the XNOA 36 transcript co-localises with the protein spectrin. This observation, together with the finding that XNOA 36 mRNA co-precipitates with spectrin, indicates that these two molecules interact physically. In conclusion, our data suggest that XNOA 36 mRNA is localized and/or anchored in the oocyte through a cytoskeletal network containing spectrin. The putative implications of this finding are discussed.

Fast light generation through velocity manipulation in two vertically-stacked ring resonators.

Speed manipulation of optical pulses is a very attractive research challenge enabling next-generation high-capacity all-optical communication networks. Pulses can be effectively slowed by using different integrated optical structures such as coupled-resonator waveguiding structures or photonic crystal cavities. Fast light generation by means of integrated photonic devices is currently a quite unexplored research field in spite of its crucial importance for all-optical pulse processing. In this paper, we report on the first theoretical demonstration of fast light generation in an ultra-compact double vertical stacked ring resonator coupled to a bus waveguide. Periodic coupling between the two rings leads to splitting and recombining of symmetric and anti-symmetric resonant modes. Re-established degenerate modes can form when a symmetric and an anti-symmetric mode having different resonance order exhibit the same resonance wavelength. Under degenerate mode conditions, wide wavelength ranges where the group velocity is negative or larger than the speed of light in vacuum are generated. The paper proves how this physical effect can be exploited to design fast light resonant devices. Moreover, conditions are also derived to obtain slow light operation regime.

p27(BBP)/eIF6 acts as an anti-apoptotic factor upstream of Bcl-2 during Xenopus laevis development.

p27(BBP)/eIF6 (beta4-binding protein/eukaryotic initiation factor 6) regulates the joining of 40S and 60S ribosomal subunits, on receptor for activated C kinase 1 binding and protein kinase C phosphorylation in serine 235. In Xenopus, p27(BBP)/eIF6 is abundantly expressed in the majority of the embryonic anlagen. Although p27(BBP)/eIF6 abundance may be required for a general regulation of protein synthesis, our data suggest that p27(BBP)/eIF6 may target the translation of specific mRNAs. We injected Xp27(BBP)/eIF6 mRNA in one blastomere of two-cell-stage embryos and obtained a bent phenotype, the curvature being lateral with respect to the embryo antero-posterior axis. The injected side had fewer apoptotic cells than the uninjected side, whereas cell proliferation appeared unaffected. Accordingly, in Xp27(BBP)/eIF6 morphants, endogenous apoptosis increased. Injection of Xp27(BBP)/eIF6 point mutants indicated that the anti-apoptotic action of Xp27(BBP)/eIF6 requires the conserved S235. The bent phenotype was also obtained with B-cell lymphoma gene-2 (Bcl-2) overexpression and was rescued by Bcl-2-associated X protein (Bax)/Xp27(BBP)/eIF6 co-injection. In addition, embryos overexpressing Xp27(BBP)/eIF6 had a higher amount of Bcl-2 and an unchanged amount of Bax with respect to controls. In Xp27(BBP)/eIF6 morphants, Bcl-2 levels were unaffected and Bax levels were higher than in the controls. Thus, we propose that Xp27(BBP)/eIF6 is part of a mechanism acting on the specific translation of messengers regulating cell survival. In particular, we suggest that Xp27(BBP)/eIF6 may regulate the translation of factors upstream of Bcl-2/Bax.

A comparative analysis of the products of GROEL-1 gene from Chlamydia trachomatis serovar D and the HSP60 var1 transcript from Homo sapiens suggests a possible autoimmune response.

Chlamydia trachomatis serovar D produces large quantities of HSP60-1 during infections, which accumulate inside the host cell inducing autoimmunity. We compare the aminoacid sequences of the human HSP60 with the bacterial counterpart to better elucidate how CTHSP60 may simulate HSP60 from human origin during infection and may induce an autoimmune response. As a result of the comparison we suggest several possible epitopes of the CTHSP60, which may induce autoimmunity.

Upon oxidative stress, the antiapoptotic Hsp60/procaspase-3 complex persists in mucoepidermoid carcinoma cells.

Hsp60, a mitochondrial chaperonin highly conserved during evolution, has been found elevated in the cytosol of cancer cells, both in vivo and in vitro, but its role in determining apoptosis during oxidative stress (OS) has not yet been fully elucidated. The aim of the present work was to study the effects of OS on Hsp60 levels and its interactions with procaspase- 3 (p-C3) and p53 in tumor cells. NCI-H292 (mucoepidermoid carcinoma) cells were exposed to various concentrations of hydrogen peroxide (H2O2) for 24 hours. Cell viability was determined by Trypan blue and MTT assays. DNA damage was assessed by the Comet assay, and apoptosis was measured by the AnnexinV cytofluorimetric test. Exposure to increasing concentrations of H2O2 resulted in a reduction of cell viability, DNA damage, and early apoptotic phenomena. Hsp60, p-C3, p53, and p21 were assessed by Western blotting and immunocytochemistry before and after OS. Hsp60 and p-C3 were present before and after OS induction. Immunoprecipitation experiments showed an Hsp60/p-C3 complex before OS that persisted after it, while an Hsp60/p53 complex was not detected in either condition. The presence of wild type (wt) p53 was confirmed by RT-PCR, and p21 detection suggested p53 activation after OS. We postulate that, although OS may induce early apoptosis in NCI-H292 cells, Hsp60 exerts an anti-apoptotic effect in these cells and, by extension, it may do so in other cancer cells.

P53 and bcl-2 in colorectal cancer arising in patients under 40 years of age: distribution and prognostic relevance.

Young people (40 years of age) with colorectal cancer (CRC) represent a distinct subgroup with more aggressive disease behaviour compared to older patients. We evaluate whether p53 and bcl-2 could be useful in identifying young patients at higher risk of tumour progression. We reviewed 1340 CRC patients with 58 patients 40 years (4.2%). They had more frequent moderately or poorly differentiated mucinous adenocarcinomas (26% versus 12.3%, p=0.03); higher advanced stage at diagnosis; shorter 5-year overall survival (49.8% versus 71%; p=0.02); more frequent p53 positive (89.8% versus 72.6%, p<0.05) and bcl-2 negative (88.0% versus 66.2%, p<0.05) tumours; no difference in DNA content or proliferation indexes. Moreover, p53+ and bcl-2- resulted in being independent predictors of survival with shorter survival for the p53+/bcl-2- patients. Combining p53 and bcl-2, we could identify young CRC patients at higher risk of progression, who probably require development of a more sophisticated therapeutic approach based on identification of predictive factors.