Clinical and pathological factors influencing survival in a large cohort of triple-negative breast cancer patients.

BACKGROUND: To provide further information on the clinical and pathological prognostic factors in triple-negative breast cancer (TNBC), for which limited and inconsistent data are available. METHODS: Pathological characteristics and clinical records of 841 TNBCs diagnosed between 1994 and 2015 in four major oncologic centers from Sardinia, Italy, were reviewed. Multivariate hazard ratios (HRs) for mortality and recurrence according to various clinicopathological factors were estimated using Cox proportional hazards models. RESULTS: After a mean follow-up of 4.3 years, 275 (33.3%) TNBC patients had a progression of the disease and 170 (20.2%) died. After allowance for study center, age at diagnosis, and various clinicopathological factors, all components of the TNM staging system were identified as significant independent prognostic factors for TNBC mortality. The HRs were 3.13, 9.65, and 29.0, for stage II, III and IV, respectively, vs stage I. Necrosis and Ki-67 > 16% were also associated with increased mortality (HR: 1.61 and 1.99, respectively). Patients with tumor histotypes other than ductal invasive/lobular carcinomas had a more favorable prognosis (HR: 0.40 vs ductal invasive carcinoma). No significant associations with mortality were found for histologic grade, tumor infiltrating lymphocytes, and lymphovascular invasion. Among lymph node positive TNBCs, lymph node ratio appeared to be a stronger predictor of mortality than pathological lymph nodes stage (HR: 0.80 for pN3 vs pN1, and 3.05 for >0.65 vs <0.21 lymph node ratio), respectively. Consistent results were observed for cancer recurrence, except for Ki-67 and necrosis that were not found to be significant predictors for recurrence. CONCLUSIONS: This uniquely large study of TNBC patients provides further evidence that, besides tumor stage at diagnosis, lymph node ratio among lymph node positive tumors is an additional relevant predictor of survival and tumor recurrence, while Ki-67 seems to be predictive of mortality, but not of recurrence.

Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer.

Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.

A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells.

The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T-cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNAagainst FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4(+) T-cell lineages.

Human pallial MGE-type GABAergic interneuron cell therapy for chronic focal epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy. One-third of patients have drug-refractory seizures and are left with suboptimal therapeutic options such as brain tissue-destructive surgery. Here, we report the development and characterization of a cell therapy alternative for drug-resistant MTLE, which is derived from a human embryonic stem cell line and comprises cryopreserved, post-mitotic, medial ganglionic eminence (MGE) pallial-type GABAergic interneurons. Single-dose intrahippocampal delivery of the interneurons in a mouse model of chronic MTLE resulted in consistent mesiotemporal seizure suppression, with most animals becoming seizure-free and surviving longer. The grafted interneurons dispersed locally, functionally integrated, persisted long term, and significantly reduced dentate granule cell dispersion, a pathological hallmark of MTLE. These disease-modifying effects were dose-dependent, with a broad therapeutic range. No adverse effects were observed. These findings support an ongoing phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE.

The T-box brain 1 (Tbr1) transcription factor inhibits astrocyte formation in the olfactory bulb and regulates neural stem cell fate.

The T-box brain 1 (Tbr1) gene encodes a transcription factor necessary for the maintenance and/or differentiation of glutamatergic cells in the olfactory bulb (OB) and cortex, although its precise function in the development of glutamatergic neurons is not known. Furthermore, Tbr1 has not been reported to regulate the formation of glial cells. We show that Tbr1 is expressed during the initial stages in the generation of glutamatergic mitral neurons from dividing progenitors in the E12.5 mouse OB. Retroviral-mediated overexpression of Tbr1 in cultured embryonic and adult OB stem cells (OBSC) produces a marked increase in the number of TuJ1(+) neurons (including VGLUT1(+) glutamatergic and GABA(+) neurons) and O4(+) oligodendrocytes. Moreover, transduction of Tbr1 inhibits the production of GFAP(+) astrocytes from both cultured OBSC and dividing progenitor cells in vivo. These results show that the expression of Tbr1 in neural stem and progenitor cells prevents them from following an astrocyte fate during OB development. Our findings suggest that the transduction of Tbr1 into neural stem cells could be useful to increase the production of neurons and oligodendrocytes in studies of neuroregeneration.

Coexpression of CD163 and CD141 identifies human circulating IL-10-producing dendritic cells (DC-10).

Tolerogenic dendritic cells (DCs) are key players in maintaining immunological homeostasis, dampening immune responses, and promoting tolerance. DC-10, a tolerogenic population of human IL-10-producing DCs characterized by the expression of HLA-G and ILT4, play a pivotal role in promoting tolerance via T regulatory type 1 (Tr1) cells. Thus far, the absence of markers that uniquely identify DC-10 has limited in vivo studies. By in vitro gene expression profiling of differentiated human DCs, we identified CD141 and CD163 as surface markers for DC-10. The coexpression of CD141 and CD163 in combination with CD14 and CD16 enables the ex vivo isolation of DC-10 from the peripheral blood. CD14+CD16+CD141+CD163+ cells isolated from the peripheral blood of healthy subjects (ex vivo DC-10) produced spontaneously and upon activation of IL-10 and limited levels of IL-12. Moreover, in vitro stimulation of allogeneic naive CD4+ T cells with ex vivo DC-10 induced the differentiation of alloantigen-specific CD49b+LAG-3+ Tr1 cells. Finally, ex vivo DC-10 and in vitro generated DC-10 exhibited a similar transcriptional profile, which are characterized by an anti-inflammatory and pro-tolerogenic signature. These results provide new insights into the phenotype and molecular signature of DC-10 and highlight the tolerogenic properties of circulating DC-10. These findings open the opportunity to track DC-10 in vivo and to define their role in physiological and pathological settings.

IGF-I promotes neuronal migration and positioning in the olfactory bulb and the exit of neuroblasts from the subventricular zone.

While insulin-like growth factor-I (IGF-I) supports neuronal and glial differentiation in the CNS, it is largely unknown whether IGF-I also influences neuronal migration and positioning. We show here that the pattern of olfactory bulb (OB) layering is altered in Igf-I (-/-) mice. In these animals, Tbr1(+)-glutamatergic neurons are misplaced in the mitral cell layer (ML) and the external plexiform layer (EPL). In addition, there are fewer interneurons in the glomerular layer and the EPL of the Igf-I (-/-) mice, and fewer newborn neurons are incorporated into the OB from the forebrain subventricular zone (SVZ). Indeed, neuroblasts accumulate in the postnatal/adult SVZ of Igf-I (-/-) mice. Significantly, the positioning of Tbr1(+)-cells in a primitive ML is stimulated by IGF-I in cultured embryonic OB slices, an effect that is partially repressed by the phosphoinositide 3-kinase (PI3K) inhibitor. In OB cell cultures, IGF-I increases the phosphorylation of disabled1 (P-Dab1), an adaptor protein that is a target of Src family kinases (SFK) in the reelin signalling pathway, whereas reduced P-Dab1 levels were found in Igf-I (-/-) mice. Neuroblast migration from the rostral migratory stream (RMS) explants of postnatal Igf-I (-/-) was similar to that from Igf-I (+/+) explants. However, cell migration was significantly enhanced by IGF-I added to the explants, an effect that was repressed by PI3K and SFK inhibitors. These findings suggest that IGF-I promotes neuronal positioning in the OB and support a role for IGF-I in stimulating neuroblast exit from the SVZ into the RMS, thereby promoting the incorporation of newly formed neurons into the OB.

Prospective isolation of functionally distinct radial glial subtypes--lineage and transcriptome analysis.

Since the discovery of radial glia as the source of neurons, their heterogeneity in regard to neurogenesis has been described by clonal and time-lapse analysis in vitro. However, the molecular determinants specifying neurogenic radial glia differently from radial glia that mostly self-renew remain ill-defined. Here, we isolated two radial glial subsets that co-exist at mid-neurogenesis in the developing cerebral cortex and their immediate progeny. While one subset generates neurons directly, the other is largely non-neurogenic but also gives rise to Tbr2-positive basal precursors, thereby contributing indirectly to neurogenesis. Isolation of these distinct radial glia subtypes allowed determining interesting differences in their transcriptome. These transcriptomes were also strikingly different from the transcriptome of radial glia isolated at the end of neurogenesis. This analysis therefore identifies, for the first time, the lineage origin of basal progenitors and the molecular differences of this lineage in comparison to directly neurogenic and gliogenic radial glia.

The proneural gene ASCL1 governs the transcriptional subgroup affiliation in glioblastoma stem cells by directly repressing the mesenchymal gene NDRG1.

Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.

Spatial and temporal expression of POF1B, a gene expressed in epithelia.

Mammalian epithelia possess specialized cellular components that provide an impermeable barrier between two different environments. In particular, in the skin, mitotically dividing cells undergo a programmed set of morphological and biochemical changes leading to the establishment of the epidermal permeability barrier (EPB) to prevent escape of moisture and entrance of toxic molecules. Many different skin proteins are involved in the process but not all have been identified. We report here the results of the expression studies of a novel gene, highly and specifically expressed in the granular layer of the epidermis and in the epithelia of the oro-pharyngeal and gastro-intestinal tracts. Our data show that during mouse development Pof1b expression is activated in the external layers of the epidermis just prior to formation of the EPB.

AntiHunter 2.0: increased speed and sensitivity in searching BLAST output for EST antisense transcripts.

An increasing number of eukaryotic and prokaryotic genes are being found to have natural antisense transcripts (NATs). There is also growing evidence to suggest that antisense transcription could play a key role in many human diseases. Consequently, there have been several recent attempts to set up computational procedures aimed at identifying novel NATs. Our group has developed the AntiHunter program for the identification of expressed sequence tag (EST) antisense transcripts from BLAST output. In order to perform an analysis, the program requires a genomic sequence plus an associated list of transcript names and coordinates of the genomic region. After masking the repeated regions, the program carries out a BLASTN search of this sequence in the selected EST database, reporting via email the EST entries that reveal an antisense transcript according to the user-supplied list. Here, we present the newly developed version 2.0 of the AntiHunter tool. Several improvements have been added to this version of the program in order to increase its ability to detect a larger number of antisense ESTs. As a result, AntiHunter can now detect, on average, >45% more antisense ESTs with little or no increase in the percentage of the false positives. We also raised the maximum query size to 3 Mb (previously 1 Mb). Moreover, we found that a reasonable trade-off between the program search sensitivity and the maximum allowed size of the input-query sequence could be obtained by querying the database with the MEGABLAST program, rather than by using the BLAST one. We now offer this new opportunity to users, i.e. if choosing the MEGABLAST option, users can input a query sequence up to 30 Mb long, thus considerably improving the possibility to analyze longer query regions. The AntiHunter tool is freely available at http://bioinfo.crs4.it/AH2.0.

Generation of Human Induced Pluripotent Stem Cell-Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy.

Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains. Stem Cells Translational Medicine 2017;6:352-368.

Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex.

The developing neocortex contains two types of progenitor cells for glutamatergic, pyramidal-projection neurons. The first type, radial glia, produce neurons and glia, divide at the ventricular surface, and express Pax6, a homeodomain transcription factor. The second type, intermediate progenitor cells, are derived from radial glia, produce only neurons, and divide away from the ventricular surface. Here we show that the transition from radial glia to intermediate progenitor cell is associated with upregulation of Tbr2, a T-domain transcription factor, and downregulation of Pax6. Accordingly, Tbr2 expression in progenitor compartments (the subventricular zone and ventricular zone) rises and falls with cortical plate neurogenesis. The subsequent transition from intermediate progenitor cell to postmitotic neuron is marked by downregulation of Tbr2 and upregulation of Tbr1, another T-domain transcription factor. These findings delineate the transcription factor sequence Pax6 --> Tbr2 --> Tbr1 in the differentiation of radial glia --> intermediate progenitor cell --> postmitotic projection neuron. This transcription factor sequence is modified in preplate neurons, in which Tbr2 is transiently coexpressed with Tbr1, and in the direct differentiation pathway from radial glia --> postmitotic projection neuron, in which Tbr2 is expressed briefly or not at all.

Telencephalic embryonic subtractive sequences: a unique collection of neurodevelopmental genes.

The vertebrate telencephalon is composed of many architectonically and functionally distinct areas and structures, with billions of neurons that are precisely connected. This complexity is fine-tuned during development by numerous genes. To identify genes involved in the regulation of telencephalic development, a specific subset of differentially expressed genes was characterized. Here, we describe a set of cDNAs encoded by genes preferentially expressed during development of the mouse telencephalon that was identified through a functional genomics approach. Of 832 distinct transcripts found, 223 (27%) are known genes. Of the remaining, 228 (27%) correspond to expressed sequence tags of unknown function, 58 (7%) are homologs or orthologs of known genes, and 323 (39%) correspond to novel rare transcripts, including 48 (14%) new putative noncoding RNAs. As an example of this latter group of novel precursor transcripts of micro-RNAs, telencephalic embryonic subtractive sequence (TESS) 24.E3 was functionally characterized, and one of its targets was identified: the zinc finger transcription factor ZFP9. The TESS transcriptome has been annotated, mapped for chromosome loci, and arrayed for its gene expression profiles during neural development and differentiation (in Neuro2a and neural stem cells). Within this collection, 188 genes were also characterized on embryonic and postnatal tissue by in situ hybridization, demonstrating that most are specifically expressed in the embryonic CNS. The full information has been organized into a searchable database linked to other genomic resources, allowing easy access to those who are interested in the dissection of the molecular basis of telencephalic development.

Developmental expression of the T-box transcription factor T-bet/Tbx21 during mouse embryogenesis.

A novel type of DNA-binding domain, the 'T-box' domain, characterizes an increasingly large family of transcription factors (Trends Genet. 15 (1999) 154). We have identified and characterized the expression pattern of a new member of the Tbr1 subfamily of T-box genes; this gene has been recently named T-bet/Tbx21 (Genomics 70 (2000) 41; Cell 17 (2000) 655; Science 292 (2001) 1907; Science 295 (2002) 338). The sequence and expression of Tbr1 and eomesodermin/Tbr2 are closely related to T-bet/Tbx21. The expression of Tbr1 (Neuron 15 (1995) 63) and Tbr2 (Mech Dev 84 (1999) 133) have virtually identical onset, at around E10.5, and expression domains in the mouse telencephalon. While Tbr1 is expressed in postmitotic neurons, Tbr2 (which is also expressed during gastrulation is also expressed in neural progenitors. We have used in situ hybridization to determine the temporal and spatial distribution of T-bet/Tbx21 expression during mouse development. T-bet/Tbx21 expression is exclusively restricted to the olfactory bulb and the thymus. To assess the distribution of T-BET/TBX21 expression in the haematopoietic compartment we used reverse transcriptase-polymerase chain reaction and found its expression in several human blood cell lineages, including progenitors/stem cells, immature B cells and peripheral T cells.

Pcp4l1, a novel gene encoding a Pcp4-like polypeptide, is expressed in specific domains of the developing brain.

We report the cloning of a novel mouse gene (Pcp4l1) that encodes a polypeptide with significant sequence similarity to the Purkinje cell protein 4 gene (Pcp4) and describe its expression pattern during mouse development. Similar to Pcp4, the Pc4l1 gene product is characterized by the presence of an IQ domain and is highly conserved across evolution. RNA in situ hybridization reveals instead that Pcp4l1 has a distinct pattern of expression: it is only expressed in the central nervous system (CNS), and is first detected at E9.5 in the mesencephalic and metencephalic roof plate as well as in the isthmus, in a region that overlaps the expression domains of Pax2, Fgf8 and Wnt1. Thus, the early Pcp4l1 expression pattern coincides with the regional expression of well-characterized patterning molecules in the organizing centers of the developing brain. Starting at midgestation, Pcp4l1 is mainly expressed in the structures of the circumventricular organs, including the subcommissural organ, the rhombencephalic and telencephalic choroid plexi, and the pineal gland. In the adult brain, this transcript is also detected in laminar as well as in several nuclear structures of the CNS.

Role of citron kinase in dendritic morphogenesis of cortical neurons.

Small GTPases of the rho family regulate the extensive rearrangements of the cytoskeleton that characterize neuronal differentiation. Citron kinase is a target molecule for activated rhoA, previously implicated in control of cytokinesis. We have found that, in addition, it could play an important role in modulating the extension of neuronal processes. Using constitutively active and dominant negative mutants, we showed that citron kinase is involved in the morphologic differentiation of N1E-115 neuroblastoma cells induced by serum starvation. More importantly, quantitative analysis of citron kinase knockout cerebral cortex displayed that this molecule may differentially regulate the morphology of the dendritic compartment in corticocollicular versus callosally-projecting pyramidal neurons.

BAT2 and BAT3 polymorphisms as novel genetic risk factors for rejection after HLA-related SCT.

The genetic background of donor and recipient is an important factor determining the outcome of allogeneic hematopoietic SCT (allo-HSCT). We applied whole-genome analysis to investigate genetic variants-other than HLA class I and II-associated with negative outcome after HLA-identical sibling allo-HSCT in a cohort of 110 ß-Thalassemic patients. We identified two single-nucleotide polymorphisms (SNPs) in BAT2 (A/G) and BAT3 (T/C) genes, SNP rs11538264 and SNP rs10484558, both located in the HLA class III region, in strong linkage disequilibrium between each other (R(2)=0.92). When considered as single SNP, none of them reached a significant association with graft rejection (nominal P<0.00001 for BAT2 SNP rs11538264, and P<0.0001 for BAT3 SNP rs10484558), whereas the BAT2/BAT3 A/C haplotype was present at significantly higher frequency in patients who rejected as compared to those with functional graft (30.0% vs 2.6%, nominal P=1.15 × 10(-8); and adjusted P=0.0071). The BAT2/BAT3 polymorphisms and specifically the A/C haplotype may represent a novel immunogenetic factor associated with graft rejection in patients undergoing allo-HSCT.

Prevalence, tumorigenic role, and biochemical implications of rare BRAF alterations.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the thyroid gland, accounting for 74-80% of all thyroid cancers. The 1799T>A transversion is an activating mutation of the BRAF oncogene that is common in and specific to conventional PTC. We studied the prevalence, tumorigenic role, and biochemical implications of rare BRAF variants in a large cohort of patients. METHODS: A total of 2131 fine-needle aspiration biopsy samples were collected and subjected to BRAF mutation analysis. BRAF genetic variants were analyzed by Western blot, immunofluorescence, and in silico analysis. RESULTS: BRAF mutations were found in 50% (347/700) of thyroid cancers (644 PTCs, 22 anaplastic thyroid carcinomas, 34 follicular thyroid carcinomas). They were the classic (c.1799T>A, p.V600E) mutation in 96.8% (336/347) and rare genetic variants in 3.2% (11/347). In all, five infrequent BRAF alterations were detected: (i) c.1795_1797dupACA (p.T599dup); (ii) c.1801A>G (p.K601E); (iii) c.1799_1801delTGA (p.V600_K601>E); (iv) c.1799_1814>A (p.V600_S605>D); and (v) c.1798_1810delinsA (p.V600_W604>R). The last BRAF variant has never been described in the literature. Western blot analysis and immunofluorescence both revealed a variegated reactivity pattern, again emphasizing the peculiar role of every specific BRAF genetic alteration. In silico analysis of the samples studied revealed a stabilization of the "active" geometrical conformation of the B-raf enzyme associated with the activated and productive state of the kinase domain. CONCLUSIONS: Rare BRAF variants were found in 1.6% of all thyroid malignancies, all clustered around the codon V600, in the binding pocket named A-loop, confirming its crucial role in the enzymatic activation of the B-Raf protein. These mutations were associated mainly with the activation of key effectors in the mitogen-activated protein kinase pathway, but a simultaneous stimulation of the PI3k/Akt cascade was demonstrated in some cases. The rare BRAF variants were not generally associated with an aggressive behavior of the PTC. To our knowledge, this is the largest series of thyroid cancers analyzed to identify and functionally characterize rare BRAF variants.

Conformational Changes of Congenital FVII Variants with Defective Binding to Tissue Factor ARG304GLN (FVII Padua), ARG 304TRP (FVII Nagoya) and ARG79GLN (FVII Shinjo or Tondabayashi).

BACKGROUND: The relation between Factor VII (FVII) and tissue thromboplastin is not completely clarified, yet. Three FVII abnormalities, FVII Padua (Arg304Gln), FVII Nagoya (Arg304Trp) and FVII Shinjo or Tondabayshi (Arg79Gln) show different FVII activity according to the tissue Tissue Factor (TF) used in the assay system (rabbit brain, human placenta or human recombinant and ox brain). OBJECTIVES: To investigate the possible existence of common conformational changes with regard to different tissue factors in these three FVII variants. MATERIAL AND METHODS: Crystal structure analysis and "visual inspection" of FVII were deeply performed to select a crystallographic template for the in silico mutagenesis procedure of FVII Arg79Gln, Arg304Gln and Arg304Trp.100ns 300K NVT large-scale molecular dynamics simulation on GPU were applied to the models of FVII. The aims of this run was to describe at molecular level the influence of the mutation on the protein structure and function. RESULTS: The molecular modelling of those three variants has shown common features in spite of the different location of the mutation involved (the first epidermal growth factor for the Arg79Gln and the catalytic region for the Arg304Gln or Arg304Trp). Molecular dynamics studies have shown in fact that the mutant FVII, shows a decreased flexibility or freezing of the protein conformation of FVIIa with regard to TF. This results in the formation of a defective FVIIa-TF complex that justifies the different clotting results observed in these variants according to the TF used. CONCLUSIONS: The conformational studies may supply useful information on the structure- function relation of clotting factors.