Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death.

VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.

APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.

Mental retardation in Down syndrome (DS) appears to be related to severe neurogenesis impairment during critical phases of brain development. Recent lines of evidence in the cerebellum of a mouse model for DS (the Ts65Dn mouse) have shown a defective responsiveness to Sonic Hedgehog (Shh), a potent mitogen that controls cell division during brain development, suggesting involvement of the Shh pathway in the neurogenesis defects of DS. Based on these premises, we sought to identify the molecular mechanisms underlying derangement of the Shh pathway in neural precursor cells (NPCs) from Ts65Dn mice. By using an in vitro model of NPCs obtained from the subventricular zone and hippocampus, we found that trisomic NPCs had an increased expression of the Shh receptor Patched1 (Ptch1), a membrane protein that suppresses the action of a second receptor, Smoothened (Smo), thereby maintaining the pathway in a repressed state. Partial silencing of Ptch1 expression in trisomic NPCs restored cell proliferation, indicating that proliferation impairment was due to Ptch1 overexpression. The overexpression of Ptch1 in trisomic NPCs resulted from increased levels of AICD [a transcription-promoting fragment of amyloid precursor protein (APP)] and increased AICD binding to the Ptch1 promoter. Our data provide novel evidence that Ptch1 overexpression underlies derangement of the Shh pathway in trisomic NPCs with consequent proliferation impairment. The demonstration that Ptch1 overexpression in trisomic NPCs is due to an APP fragment provides a link between this trisomic gene and the defective neuronal production that characterizes the DS brain.

Differing risk of cancer death among patients with lymph node metastasis after radical prostatectomy and pelvic lymph node dissection: identification of risk categories according to number of positive nodes and Gleason score.

OBJECTIVES: To evaluate the outcomes in patients with node-positive prostate cancer (PCa) after radical prostatectomy (RP) and pelvic lymph node dissection (PLND) according to the number of positive lymph nodes (LNs). To identify different risk groups among patients with node-positive PCa. PATIENTS AND METHODS: We evaluated 98 consecutive patients with pN1M0 PCa who underwent RP between November 1995 and May 2011. Kaplan-Meier and Cox proportional univariable and multivariable regression models were used to analyse the survival rates. Patients were divided into two groups according to number of positive LNs using the most informative positive LN theshold for predicting survival, then into three different risk groups according to number of positive LNs and pathological Gleason score (GS). RESULTS: Mean (range) follow-up was 68.4 (10-192) months. Patients with 1-3 positive LNs (n = 75; 76.5%) had significantly better cancer-specific survival (CSS) and overall survival (OS) compared with those with >3 positive nodes (n = 23; 23.4%; P < 0.01). Patients with 1-3 positive LNs and pathological GS ≤7 (Group 1) had significantly better CSS than those with >3 positive LNs or GS 8-10 (Group 2 [P = 0.015]). Group 2 patients, moreover, had significantly better CSS (P = 0.019) and OS (P = 0.021) than those with >3 positive LNs and GS 8-10 (Group 3). CONCLUSIONS: Patients with 1-3 positive LNs have higher CSS and OS rates than those with >3 metastatic LNs. Taking into account the pathological GS, as well as the number of positive nodes, three risk group categories with considerable differences in terms of survival can be found. Patients with LN-positive PCa should be stratified into different groups according to these two measures, to obtain a better prediction of oncological outcomes.

Novel Thienoduocarmycin-Trastuzumab ADC Demonstrates Strong Antitumor Efficacy with Favorable Safety Profile in Preclinical Studies.

New antibodies-drug conjugate (ADC) payloads overcoming chemoresistance and killing also poorly proliferating tumors at well-tolerated doses are much desired. Duocarmycins are a well-known class of highly potent cytotoxic agents, with DNA minor groove-binding and alkylation properties, active also in chemoresistant tumors. Although different duocarmycin derivatives have been used during the years as payloads for ADC production, unfavorable physicochemical properties impaired the production of ADCs with optimal features. Optimization of the toxin to balance reactivity and stability features and best linker selection allowed us to develop the novel duocarmycin-like payload-linker NMS-P945 suitable for conjugation to mAbs with reproducible drug-antibody ratio (DAR) >3.5. When conjugated to trastuzumab, it generated an ADC with good internalization properties, ability to induce bystander effect and immunogenic cell death. Moreover, it showed strong target-driven activity in cells and cytotoxic activity superior to trastuzumab deruxtecan tested, in parallel, in cell lines with HER2 expression. High in vivo efficacy with cured mice at well-tolerated doses in HER2-driven models was also observed. A developed pharmacokinetic/pharmacodynamic (PK/PD) model based on efficacy in mice and cynomolgus monkey PK data, predicted tumor regression in patients upon administration of 2 doses of trastuzumab-NMS-P945-ADC at 0.5 mg/kg. Thus, considering the superior physicochemical features for ADC production and preclinical results obtained with the model trastuzumab ADC, including bystander effect, immunogenic cell death and activity in chemoresistant tumors, NMS-P945 represents a highly effective, innovative payload for the creation of novel, next-generation ADCs.

An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma.

Enthusiasm for the use of antibody-drug conjugates (ADCs) in cancer therapy has risen over the past few years. The success of this therapeutic approach relies on the identification of cell surface antigens that are widely and selectively expressed on tumor cells. Studies have shown that native ALK protein is expressed on the surface of most neuroblastoma cells, providing an opportunity for development of immune-targeting strategies. Clinically relevant antibodies for this target have not yet been developed. Here, we describe the development of an ALK-ADC, CDX-0125-TEI, which selectively targets both wild-type and mutated ALK-expressing neuroblastomas. CDX-0125-TEI exhibited efficient antigen binding and internalization, and cytotoxicity at picomolar concentrations in cells with different expression of ALK on the cell surface. In vivo studies showed that CDX-0125-TEI is effective against ALK wild-type and mutant patient-derived xenograft models. These data demonstrate that ALK is a bona fide immunotherapeutic target and provide a rationale for clinical development of an ALK-ADC approach for neuroblastomas and other ALK-expressing childhood cancers such as rhabdomyosarcomas.

Adverse Features and Competing Risk Mortality in Patients With High-Risk Prostate Cancer.

PURPOSE: To assess survival and competing causes of mortality in prostate cancer (PCa) patients referred to radical prostatectomy through a combination of unfavorable characteristics. PATIENTS AND METHODS: We evaluated 615 PCa patients referred to radical prostatectomy and pelvic lymph node dissection at single tertiary-care center with at least one adverse feature (AF): preoperative prostate-specific antigen ≥ 20 ng/mL, pathologic Gleason score 8 to 10, and no organ-confined disease at final pathology (seminal vesicle involvement, positive surgical margins, and/or lymph node invasion). Kaplan-Meier analyses were used to assess cancer-specific mortality (CSM)-free survival rates by stratifying patients into 3 risk categories according to the number of AFs (namely, 1, 2, and 3 AFs). Multivariable competing risk Cox regression analyses were used to assess CSM and other cause of mortality. RESULTS: Significant differences were found in terms of preoperative and pathologic tumor characteristics, adjuvant therapies, and biochemical recurrence (BCR). Men with 1 AF had higher CSM-free survival estimates compared to those with 2 and 3 AFs (92.8% vs. 84.2% vs. 27.7% at 10 years' follow-up, P < .001). Moreover, the presence of 3 AFs (hazard ratio [HR], 2.96), postoperative adjuvant treatment status (HR, 2.44), and time to BCR (HR, 0.96) were all independent predictors of CSM (P ≤ .04). Age at surgery and time to BCR were the only independent predictors of other causes of mortality (P ≤ .0009). CONCLUSION: The risk group stratification according to the number of AFs could help physicians to accurately predict oncologic outcomes and to select PCa patients for the most appropriate postoperative strategies.

The Prognostic Impact of Tumor Size on Cancer-Specific and Overall Survival Among Patients With Pathologic T3a Renal Cell Carcinoma.

BACKGROUND: We aimed to determine the prognostic role of tumor size in patients with stage pT3a renal cell carcinoma (RCC). PATIENTS AND METHODS: We analyzed our database of patients who underwent radical nephrectomy for RCC between July 2000 and December 2013. Clinical and pathologic data were obtained for each patient. Patients with stage pT3a disease were divided into 2 subgroups according to the most informative threshold for pathologic tumor dimension that was able to predict survival outcomes (group 1, ≤ 8 cm; group 2, > 8 cm). RESULTS: Globally, 185 consecutive patients were evaluated. The median (interquartile range [IQR]) follow-up was 32 months (18-62 months). The median (IQR) pathologic tumor size was 7.5 cm (5.7-10 cm). Seventy (34.3%) patients died of RCC during the follow-up period. Patients in group 2 experienced worse cancer-specific survival (CSS) rates compared with those in group 1, (5- and 10- year CSS, 52% and 40% vs. 67% and 63%, respectively; P = .001). Overall survival (OS) rates were significantly lower for patients included in group 2 compared with patients in group 1 (5- and 10- year OS rates, 46% and 38% vs. 60% and 57%, respectively; P = .01). Subgroup stratification (hazard ratio [HR], 3.65; P < .001), presence of positive surgical margins (HR, 3.86; P = .22), high Fuhrman grade (HR, 4.33; P < .001), and the presence of sarcomatoid cells (HR, 2.61; P = .02) were found to be independent predictors of CSS. CONCLUSION: Worse oncologic outcomes are observed in patients with stage pT3a RCC tumors > 8 cm. The current TNM classification still does not precisely correlate with CSS. Tumor size should be taken into account in a future revision of the TNM staging system.

Nodal occult metastases in intermediate- and high-risk prostate cancer patients detected using serial section, immunohistochemistry, and real-time reverse transcriptase polymerase chain reaction: prospective evaluation with matched-pair analysis.

BACKGROUND: The purpose of the study was to prospectively evaluate the incidence of nodal OCM assessed using SS, IHC, and RT-PCR in prostate cancer patients compared with the standard pathological evaluation (SPE), and to evaluate short-term oncological outcomes of patients with OCM. PATIENTS AND METHODS: Fifty-four consecutive patients with intermediate- or high-risk prostate cancer treated with radical prostatectomy and extended pelvic LN dissection comprised the study population (StP). The central sections with the largest diameter of each LN of the StP and a matched-pair population (MpP) with identical characteristics as the StP were used to assess the improved detection rate of OCM. Pathological characteristics and biochemical recurrence-free survival were assessed according to the presence of macroscopic or OCM. RESULTS: A total of 1064 LNs were processed in the 54 patients of the StP, with 11 (20.4%) patients with evident metastases at SPE and 7 with OCM (13.0% additional patients); the percentage of positive patients improved from 16.6% (18 of 108) of the MpP to 33.3% (18 of 54) of the StP (16% additional patients). The mean diameter of the 10 additional LNs with OCM found at SS only and of the 6 additional LNs found at IHC only was significantly lower than the mean diameter of the 28 metastases found at routine pathologic examination (RPE) (P < .0001). Patients with OCM showed risk of biochemical recurrence similar to patients with no LN metastases (P = .008). CONCLUSION: SS, IHC, and RT-PCR can detect a not negligible percentage of OCM missed at RPE. Patients with OCM showed short-term oncological outcomes more similar to those with macroscopic metastases. Longer follow-up studies considering cancer-specific survival are needed.

Most immunoglobulin heavy chain switch mu rearrangements in B-cell chronic lymphocytic leukemia are internal deletions.

We investigated 38 cases of B-cell chronic lymphocytic leukemia (B-CLL) for the presence of non-productive rearrangements in the S(mu) regions and defined for the first time the molecular nature of these rearrangements. Southern blot analysis revealed S(mu) region rearrangements in 13 cases (34%) and polymerase chain reactions (PCRs) indicated that these rearrangements consisted of internal deletions of the S(mu) region. Long-distance PCRs localized the S(mu) deletions in the V(H)DJ(H) rearranged allele in most cases. We investigated if S(mu) deletions were related to V(H) somatic mutations that, together with isotype switch recombination, are indicative of the B-cell maturation stage. No significant correlation between the presence of S(mu) deletions and V(H) somatic mutations was found, indicating that the two processes are independent in B-CLL. Moreover no significant correlation between S(mu) deletions and prognosis was observed. Having shown that S(mu) internal deletions are not chromosome translocations rules out their involvement in the onset of malignancy, while their localization in the V(H)DJ(H) rearranged alleles suggests a possible role in the stabilization of the isotype of the expressed immunoglobulin.

Search for the insertion element IS256 within the ica locus of Staphylococcus epidermidis clinical isolates collected from biomaterial-associated infections.

Staphylococcus epidermidis biofilm-forming strains produce a polysaccharide intercellular adhesin (PIA), which mediates bacterial cell aggregation and favours the colonisation on prosthetic implants. PIA synthesis is regulated by the icaADBC locus. In vitro, by repeated subcultures of a biofilm-producing strain, the loss of the ability to produce biofilm appears associated with the insertion of the IS256 element into the ica locus. This study was aimed (i) to investigate if the five genes of ica locus are always all present in different strains of S. epidermidis, and (ii) to search if IS256 insertion naturally occurs in ica locus without making recourse to the experimental procedure of repeated subcultures of strains. 120 S. epidermidis clinical isolates from peri-prosthesis infections were investigated both by an original multiplex PCR analysis of the ica genes and by PCR amplification of the IS256 element. Also two reference strains (the biofilm-negative S. epidermidis ATCC 12228 and the biofilm-forming ATCC 35984 [RP62A]) and two biofilm-negative RP62A-derived acriflavin mutants (D9 and HAM892) were analysed. D9 e HAM892 were for the first time shown to contain in ica locus, at the base 3319, a 1300-bp insertion with a DNA sequence corresponding to IS256. Among the 120 clinical isolates, 51 (43%) turned out completely ica-positive, 69 completely ica-negative (57%). The genes of the ica locus appear, in all cases of the present collection, strictly linked each other, so they are either all present or all absent. In this collection, IS256 was present in eight out of the 69 ica-negative strains and in 34 out of the 51 ica-positive strains. IS256, also when present in bacterial genomic DNA, was never found inside the ica locus, thus suggesting that insertion/excision of this element is not a natural occurring mechanism for off/on switching of biofilm production.

Evaluation of bacterial adhesion of Streptococcus mutans on dental restorative materials.

Bacterial adhesion to the surface of composite resins and other dental restorative materials is an important parameter in the aetiology of secondary caries formation. The aim of the present study was to investigate the adhesion of a Streptococcus mutans strain (ATCC 25175) on the surface of different restorative materials. The test materials examined included three flowable composites (Filtek Flow, Tetric Flow, and Arabesk Flow), three microhybrid composites (Clearfil APX, Solitaire 2, and Z250), two glass-ionomers (Fuji IX, Fuji IX fast), a compomer (F2000), an ormocer (Admira), and a control reference material (tissue culture grade, surface treated polystyrene). The adhesion tests were carried out in 24-well plates. Quantitative turbidimetric measurements were finally performed in order to indirectly evaluate the amount of bacteria retained on the material surface after in vitro exposure to the bacteria suspension. Under these conditions, with the exception of the Admira ormocer and the Fuji IX fast glass ionomer, which were found to be more adhesive, all the other material surfaces showed a similar susceptibility to bacterial adhesion, exhibiting values not significantly different than the reference polystyrene control. Furthermore, the release of fluoride from some of the test surfaces did not appear capable to reduce early bacterial adhesion.

[Predictors of positive surgical margins after nephron-sparing surgery for renal cell carcinoma: retrospective analysis on 298 consecutive patients]. / Fattori predittivi di margini chirurgici positivi dopo chirurgia conservativa per neoplasia renale: analisi retrospettiva monocentrica su 298 pazienti.

OBJECTIVES: Aim of our study was to evaluate the predictive factors of positive surgical margins (PSM) in a cohort of patients who underwent partial nephrectomy (PN) for renal cell carcinoma. MATERIAL AND METHODS: We retrospectively evaluated our Institutional database of patients treated with open or laparoscopic PN between 200 and 2013. Categorical variables were compared using Pearson's chi-square test and linear-by-linear association. Multivariable Cox analysis was used in order to evaluate independent predictors of PSM. RESULTS: Surgical margins were found to be negative in 274 out of 298 patients (91.9%), and the remaining 24 (8.1%) patients had PSM at the final pathological exam. The median clinical size was significantly lower in patients with PSM than those with negative margins (2.6 vs. 3 cm, p=0.03). At univariable analysis, a shorter operative time (p=0.04), a malignant histotype (p=0.04) and higher Fuhrman grade (p=0.02) were observed in patients with positive surgical margins compared to those without PSM. At multivariable analysis, median tumor dimension (p=0.02), the malignant histotype (p=0.01) and the high Fuhrman grade (3-4) (p=0.01) were found to be independent predictive factors of PSM. CONCLUSIONS: The most important goal of any PN is to reach negative surgical margins. In our study, clinical tumor dimensions, malignant tumor histotype and the high Fuhrman grade demonstrated to be independent predictive factors of PSM after nephron sparing surgery for renal cell carcinoma. Other prospective, multi-institutional studies are needed in order to confirm these results.

Revised Gleason grading system is a better predictor of indolent prostate cancer at the time of diagnosis: retrospective clinical-pathological study on matched biopsy and radical prostatectomy specimens.

INTRODUCTION/BACKGROUND: The increase of prostate cancer diagnosis after the introduction of prostate-specific antigen (PSA) screening resulted in overtreatment of patients with low risk tumors. The histological Gleason score (GS) revised in 2005 by the International Society of Urological Pathology (ISUP) is currently the most reliable tool to separate aggressive from indolent prostate cancer. MATERIALS AND METHODS: Using the new 2005 GS criteria we retrospectively evaluated biopsy and surgical samples of 1344 patients who underwent radical prostatectomy in our institution. According to the new GS criteria we then selected 134 patients who would have been suitable for active surveillance at the time of biopsy (at least 2 positive cores, PSA < 10 ng/mL, GS ≤ 6). We finally assessed the accuracy of the revised GS in biopsy to predict indolent cancer in the prostatectomy specimens. RESULTS: The mean GS increased from 6 to 7 after histological revision in biopsy and prostatectomy specimens. Histological revision determined a significant decrease of patients with GS ≤ 6 and an increase of those with GS ≥ 7 (all P < .001). The average of pathologically indolent disease (organ-confined, GS ≤ 6 at surgery, tumor of any volume) significantly decreased after histological revision (P < .001). CONCLUSION: The revised ISUP 2005 criteria for Gleason grading provided better stratification of GS ≤ 6 prostate cancer and improved the accuracy for the histological diagnosis of indolent prostate cancer in biopsy and radical prostatectomy specimens.

Discovery of 2-(cyclohexylmethylamino)pyrimidines as a new class of reversible valosine containing protein inhibitors.

Valosine-containing protein (VCP), also known as p97 or cdc48 in yeast, is a highly abundant protein belonging to the AAA ATPase family involved in a number of essential cellular functions, including ubiquitin-proteasome mediated protein degradation, Golgi reassembly, transcription activation, and cell cycle control. Altered expression of VCP has been detected in many cancer types sometimes associated with poor prognosis. Furthermore, VCP mutations are causative of some neurodegenerative disorders. In this paper we report the discovery, synthesis, and structure-activity relationships of substituted 2-aminopyrimidines, representing a new class of reversible VCP inhibitors. This class of compounds, identified in a HTS campaign against recombinant VCP, has been progressively expanded and manipulated to increase biochemical potency and gain cellular activity.

Alkylsulfanyl-1,2,4-triazoles, a new class of allosteric valosine containing protein inhibitors. Synthesis and structure-activity relationships.

Valosine containing protein (VCP), also known as p97, is a member of AAA ATPase family that is involved in several biological processes and plays a central role in the ubiquitin-mediated degradation of misfolded proteins. VCP is an ubiquitously expressed, highly abundant protein and has been found overexpressed in many tumor types, sometimes associated with poor prognosis. In this respect, VCP has recently received a great deal of attention as a potential new target for cancer therapy. In this paper, the discovery and structure-activity relationships of alkylsulfanyl-1,2,4-triazoles, a new class of potent, allosteric VCP inhibitors, are described. Medicinal chemistry manipulation of compound 1, identified via HTS, led to the discovery of potent and selective inhibitors with submicromolar activity in cells and clear mechanism of action at consistent doses. This represents a first step toward a new class of potential anticancer agents.

NMS-P937, an orally available, specific small-molecule polo-like kinase 1 inhibitor with antitumor activity in solid and hematologic malignancies.

Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase considered to be the master player of cell-cycle regulation during mitosis. It is indeed involved in centrosome maturation, bipolar spindle formation, chromosome separation, and cytokinesis. PLK1 is overexpressed in a variety of human tumors and its overexpression often correlates with poor prognosis. Although five different PLKs are described in humans, depletion or inhibition of kinase activity of PLK1 is sufficient to induce cell-cycle arrest and apoptosis in cancer cell lines and in xenograft tumor models. NMS-P937 is a novel, orally available PLK1-specific inhibitor. The compound shows high potency in proliferation assays having low nanomolar activity on a large number of cell lines, both from solid and hematologic tumors. NMS-P937 potently causes a mitotic cell-cycle arrest followed by apoptosis in cancer cell lines and inhibits xenograft tumor growth with clear PLK1-related mechanism of action at well-tolerated doses in mice after oral administration. In addition, NMS-P937 shows potential for combination in clinical settings with approved cytotoxic drugs, causing tumor regression in HT29 human colon adenocarcinoma xenografts upon combination with irinotecan and prolonged survival of animals in a disseminated model of acute myelogenous leukemia in combination with cytarabine. NMS-P937, with its favorable pharmacologic parameters, good oral bioavailability in rodent and nonrodent species, and proven antitumor activity in different preclinical models using a variety of dosing regimens, potentially provides a high degree of flexibility in dosing schedules and warrants investigation in clinical settings.

Impact of environmental enrichment on neurogenesis in the dentate gyrus during the early postnatal period.

Accumulating evidence shows that environmental enrichment increases neurogenesis in the adult hippocampal dentate gyrus. The goal of the current study was to examine the effect of environmental enrichment on hippocampal neurogenesis during early life stages. We used as an animal model the guinea pig, a precocious rodent that is early independent from maternal care. Animals were assigned to either a standard (control) or an enriched environment a few days after birth (P5-P6). Between P14 and P17 animals received one daily bromodeoxyuridine (BrdU) injection, to label dividing cells, and were sacrificed either on P18, to evaluate cell proliferation or on P45, to evaluate cell survival and differentiation. In 18-day old enriched animals, there was a larger number of BrdU-positive cells compared to that found in controls. At P45, enriched animals had more surviving cells and more cells with a neuronal phenotype than controls. Unbiased stereology revealed that enriched animals had more granule cells (+37% at P18 and +31% at P45). Results show that environmental enrichment in the early postnatal period notably increases cell proliferation and survival, with a large increase in the number of neurons forming the granule cell layer. The impact of environmental enrichment in the early postnatal period emphasizes the relevance of extrinsic factors in the modulation of neurogenesis during critical time windows of hippocampal development.

Phosphorylation of TCTP as a marker for polo-like kinase-1 activity in vivo.

Polo-like kinase 1 (PLK1) is the master regulator of mitosis and a target for anticancer therapy. To develop a marker of PLK1 activity in cells and tumour tissues, this study focused on translational controlled tumour protein (TCTP) and identified serine 46 as a site phosphorylated by PLK1 in vitro. Using an antibody raised against phospho-TCTP-Ser46, it was demonstrated that phosphorylation at this site correlates with PLK1 level and kinase activity in cells. Moreover, PLK1 depletion by siRNA or inactivation by specific inhibitors caused a correspondent decrease in phospho-TCTP-Ser46 signal validating this site as a direct marker of PLK1. Using this marker, the study characterized PLK1 inhibitors in cells by setting up a high-content assay and finally immunohistochemical assay suitable for following inhibitor activity in preclinical tumour models and possibly in clinical studies was developed.

Neonatal isolation impairs neurogenesis in the dentate gyrus of the guinea pig.

In the current study we examined the effects of early isolation rearing on cell proliferation, survival and differentiation in the dentate gyrus of the guinea pig. Animals were assigned to either a standard (control) or an isolated environment a few days after birth (P5-P6), taking advantage of the precocious independence from maternal care of the guinea pig. On P14-P17 animals received one daily bromodeoxyuridine injection, to label dividing cells, and were sacrificed either on P18, to evaluate cell proliferation or on P45, to evaluate cell survival and differentiation. In P18 isolated animals we found a reduced cell proliferation (-35%) compared to controls and a lower expression of brain-derived neurotrophic factor (BDNF). Though in absolute terms P45 isolated animals had less surviving cells, they showed no differences in survival rate and phenotype percent distribution compared to controls. Looking at the location of the new neurons, we found that while in control animals 76% of them had migrated to the granule cell layer, in isolated animals only 55% of the new neurons had reached this layer. Examination of radial glia cells of P18 and P45 animals by vimentin immunohistochemistry showed that in isolated animals radial glia cells were reduced in density and had less and shorter processes. Granule cell count revealed that P45 isolated animals had less (-42%) granule cells than controls. Results show that isolation rearing reduces hippocampal cell proliferation, likely by reducing BDNF expression and hampers migration of the new neurons to the granule cell layer, likely by altering density/morphology of radial glia cells. The large reduction in granule cell number following isolation rearing emphasizes the role of environmental cues as relevant modulators of neurogenesis.

Evidence of a linkage between matrilin-1 gene (MATN1) and idiopathic scoliosis.

BACKGROUND: In a previous study, a number of genes, associated with spine musculoskeletal deformity phenotypes in mouse and in synteny between mouse and man, were identified as candidate genes for IS. Among these genes, MATN1, which carries a polymorphic microsatellite marker within its sequence, was selected for a linkage analysis. MATN1 is localised at 1p35 and is mainly expressed in cartilage. The objective of this study was to assess a linkage disequilibrium between the matrilin-1 (MATN1) gene and the idiopathic scoliosis (IS). METHODS: The genetic study was conducted on a population of 81 trios, each consistent of a daughter/son affected by idiopathic scoliosis (IS) and both parents. In all trios components, the region of MATN1 gene containing the microsatellite marker was amplified by a polymerase chain reaction. The amplicons were analysed by a DNA sequencer-genotyper. The statistical linkage analysis was performed using the extended transmission/disequilibrium test. RESULTS: Three microsatellite polymorphisms, respectively consisting of 103 bp, 101 bp and 99 bp, were identified. ETDT evidenced a significant preferential transmission for the 103 bp allele (Chi-square = 5.058, df = 1, P = 0.024) CONCLUSION: The results suggest that the familial idiopathic scoliosis is associated to the MATN1 gene.