Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration

We present a method for using long-term organotypic slice co-cultures of the entorhino-hippocampal formation to analyze the axon-regenerative properties of a determined compound. The culture method is based on the membrane interphase method, which is easy to perform and is generally reproducible. The degree of axonal regeneration after treatment in lesioned cultures can be seen directly using green fluorescent protein (GFP) transgenic mice or by axon tracing and histological methods. Possible changes in cell morphology after pharmacological treatment can be determined easily by focal in vitro electroporation. The well-preserved cytoarchitectonics in the co-culture facilitate the analysis of identified cells or regenerating axons. The protocol takes up to a month.

The transcriptional co-activator PCAF regulates cdk2 activity.

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G2/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G2/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity.

Reversible ferromagnetic switching in ZnO:(Co,Mn) powders

We report here on the magnetic properties of ZnO:Mn- and ZnO:Co-doped nanoparticles. We have found that the ferromagnetism of ZnO:Mn can be switched on and off by consecutive low-temperature annealings in O2 and N2, respectively, while the opposite phenomenology was observed for ZnO:Co. These results suggest that different defects (presumably n-type for ZnO:Co and p-type for ZnO:Mn) are required to induce a ferromagnetic coupling in each case. We will argue that ferromagnetism is likely to be restricted to a very thin, nanometric layer at the grain surface. These findings reveal and give insight into the dramatic relevance of surface effects to the occurrence of ferromagnetism in ZnO-doped oxides.

The bZIP transcription factor Rca1p is a central regulator of a novel CO₂ sensing pathway in yeast.

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO(2) concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO(2) and identify the bZIP transcription factor Rca1p as the first CO(2) regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO(2) build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO(2) sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO(2) availability in environments as diverse as the phagosome, yeast communities or liquid culture.

Size effects in the magneto-optical response of Co nanoparticles

A study of the magneto-optical (MO) spectral response of Co nanoparticles embedded in MgO as a function of their size and concentration in the spectral range from 1.4 to 4.3 eV is presented. The nanoparticle layers were obtained by sputtering at different deposition temperatures. Transmission electron microscopy measurements show that the nanoparticles have a complex structure which consists of a crystalline core having a hexagonal close-packed structure and an amorphous crust. Using an effective-medium approximation we have obtained the MO constants of the Co nanoparticles. These MO constants are different from those of continuous Co layers and depend on the size of the crystalline core. We associate these changes with the size effect of the intraband contribution to the MO constants, related to a reduction of the relaxation time of the electrons into the nanoparticles.

p21 as a transcriptional co-repressor of S-phase and mitotic control genes.

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes.

Concurrent or Sequential Adjuvant Hormonoradiotherapy after Conservative Surgery for Early-Stage Breast Cancer: Clinical Results of the CO-HO-RT Phase II Randomized Trial.

Purpose: Letrozole (LET) has recently been shown to be superior to tamoxifen for postmenopausal patients (pts). In addition, LET radiosensitizes breast cancer cells in vitro. We conducted a phase II randomized study to evaluate concurrent and sequential radiotherapy (RT)-LET in the adjuvant setting. We present here clinical results with a minimum follow-up of 24 months. Patients and Methods: Postmenopausal pts with early-stage breast cancer were randomized after conservative surgery to either: A) concurrent RT-LET (LET started 3 weeks before the first day of RT) or B) sequential RT-LET (LET started 3 weeks after the end of RT). Whole breast RT was delivered to a total dose of 50 Gy. A 10-16 Gy boost was allowed according to age and pathological prognostic factors. Pts were stratified by center, adjuvant chemotherapy, boost, and radiation-induced CD8 apoptosis (RILA). RILA was performed before RT as previously published (Ozsahin et al. Clin Cancer Res, 2005). An independent monitoring committee reviewed individual safety data. Skin toxicities were evaluated by two different clinicians at each medical visit (CTCAE v3.0). Lung CT-scan and functional pulmonary tests were performed regularly. DNA samples were screened for SNPs in candidate genes as recently published (Azria et al., Clin Cancer Res, 2008). Results: A total of 150 pts were randomized between 01/05 and 02/07. Median follow-up is 26 months (range, 3-40 months). No statistical differences were identified between the two arms in terms of mean age; initial TNM; median surgical bed volume; post surgical breast volume. Chemotherapy and RT boost were delivered in 19% and 38% of pts, respectively. Nodes received 50 Gy in 23% of patients without differences between both arms. During RT and within the first 6 weeks after RT, 10 patients (6.7%) presented grade 3 acute skin dermatitis during RT but no differences were observed between both arms (4 and 6 patients in arm A and B, respectively). At 26 month of follow-up, grade 2 and more radiation-induced subcutaneous fibrosis (RISCF) was present in 4 patients (3%) without any difference between arm A (n = 2) and B (n = 2), p=0.93. In both arms, all patients that presented a RICSF had a RILA lower than 16%. Sensitivity and specificity were 100% and 39%, respectively.No acute lung toxicities were observed and quality of life was good to excellent for all patients.SNPs analyses are still on-going (Pr Rosenstein, NY). Conclusion: Acute and early late grade 2 dermatitis were similar in both arms. The only factor that influenced RISCF was a low radiation-induced lymphocyte apoptosis yield. We confirmed prospectively the capacity of RILA for identifying hypersensitive patients to radiation. Indeed, patients with RILA superior to 16% did not present late effects to radiation and confirmed the first prospective trial we published in 2005 (Ozsahin et al., Clin Cancer Res).

Numerical signs fos a transition in the 2d Random Field Ising Model at T=0

Intensive numerical studies of exact ground states of the two-dimensional ferromagnetic random field Ising model at T=0, with a Gaussian distribution of fields, are presented. Standard finite size scaling analysis of the data suggests the existence of a transition at ¿c=0.64±0.08. Results are compared with existing theories and with the study of metastable avalanches in the same model.

Substance P and calbindin D-28k-immunoreactivity in primary sensory neurons of chick embryos: differential neuronal birthdates and transient co-localization.

During the ontogenesis of dorsal root ganglia (DRG), the immunoreactivity to substance P (SP) and calbindin D-28k (CaBP) appears in chickens at embryonic day 5 (E5) and E10 respectively. To establish the birthdates of primary sensory neurons expressing SP or CaBP, chick embryos were given repetitive intra-amniotic injections of [3H]-thymidine. The neuroblasts giving rise to SP-expressing neurons were labeled up to E6 while those generating CaBP-immunoreactive neurons stopped to incorporate [3H]-thymidine before E5.5. This finding indicates that neurons exhibiting distinct phenotypes may originate from neuroblasts which arrest to proliferate at close but distinct stages of development. To determine whether SP and CaBP are co-expressed or not in DRG neurons, chick embryos at E12, E18, and chickens two weeks after hatching were perfused and fixed to detect simultaneously SP- and CaBP-immunoreactivity in DRG sections. The results showed that SP and CaBP were transiently co-expressed by a subset of neurons at E12. Later, however, the SP-immunoreactivity was gradually lost by these ganglion cells, so that the SP- and CaBP-immunoreaction defined two distinct neuronal subpopulations after hatching. In conclusion, most CaBP-immunoreactive DRG cells derive from a subset of neurons in which SP and CaBP are transiently co-localized.

Heat capacity measurements on the equiatomic compounds in Ni-X (X = Al, In, Si, Ge and Bi) and M-Sb (with M = Ni, Co and Fe) systems

Molar heat capacities of the binary compounds NiAl, NiIn, NiSi, NiGe, NiBi, NiSb, CoSb and FeSb were determined every 10 K by differential scanning calorimetry in the temperature range 310-1080 K. The experimental results have been fitted versus temperature according to C-p = a + b . T + c . T-2 + d . T-2. Results are given, discussed and compared to estimations found in the literature. Two compounds, NiBi and FeSb, are subject to transformations between 460 and 500 K. (C) 1999 Elsevier Science Ltd. All rights reserved.

Coordenades clàssiques de Shadowlands de Richard Attenborough (sobre la vida i obra de C. S. Lewis)

L'objectiu d'aquest article és mostrar els parámetres clàssics de Shadowlands de R. Attenborough, amb guió de W. Nicholson, sobre la vida i obra de C. S. Lewis. Basant-se en una anàlisi acurada dels textos de Lewis, l'autor proposa d'interpretar l'oposició Lewis / Gresham com la traducció en la vida real de la oposició entre els temperaments platònic o idealista i aristotèlic o materialista que ja mencionava Coleridge. En qualsevol cas, són moltes les referències clàssiques que cal tenir en compte si volem comprendre fins a quin punt el cristianisme de Lewis és també un cristianisme clàssic, és a dir, grecollatí.