Leukaemic cells from chronic lymphocytic leukaemia patients undergo apoptosis following microtubule depolymerization and Lyn inhibition by nocodazole.

Functional abnormalities of chronic lymphocytic leukaemia (CLL) cells may be related to the microtubular network of cell cytoskeleton; specifically tubulin involvement in cells after B-cell receptor engagement. As microtubule inhibitors could represent a therapeutic strategy for CLL, this study investigated the capability of nocodazole, a synthetic depolymerizing agent, to kill CLL leukaemic cells. We demonstrated that nocodazole was highly specific for the in vitro induction of apoptosis in leukaemic cells from 90 CLL patients, without affecting the viability of T-cells and/or mesenchymal stromal cells (MSCs) recovered from the same patients. Nocodazole was observed to overcome the pro-survival signals provided by MSCs. Competing with ATP for the nucleotide-binding site, nocodazole has been observed to turn off the high basal tyrosine phosphorylation of leukaemic cells mediated by the Src-kinase Lyn. Considering that most anti-microtubule drugs have limited clinical use because of their strong toxic effects, the high selectivity of nocodazole for leukaemic cells in CLL and its capability to bypass microenvironmental pro-survival stimuli, suggests the use of this inhibitor for designing new therapeutic strategies in CLL treatment.

A cluster of factor XI-deficient patients due to a new mutation (Ile 436 Lys) in northeastern Italy.

A new mutation (Ile 436 Lys) was found in a cluster of patients in northeastern Italy. The mutation was present in five patients at the homozygote level and in one patient as a compound heterozygote with an already known mutation namely Glu 117 stop. All these patients showed a mild bleeding tendency mainly associated with deliveries or surgery. The first two patients were two sisters, and their parents were consanguineous. The third patient was the only homozygote in the family, and parents apparently were not consanguineous. The fourth and fifth patients were a brother and a sister, and in this case too, parents were not consanguineous. The sixth patient, a compound heterozygote, negated also the existence of consanguinity between his parents. There were also seven heterozygotes among the family members of the patients homozygous for this new mutation (Ile 436 Lys). Finally, there were two heterozygotes for the Glu 117 stop mutation in the family of the sixth patient. The heterozygotes, regardless of the mutation, were asymptomatic. The Ile436Lys mutation is characterized by low factor XI activity and antigen, namely is a cross-reaction material negative form. Molecular modeling indicates that the Ile436Lys mutation causes a large conformational change within the 432-442 loop. No relation could be traced among the different families; however, all their ancestors were autochthonous of the same two small towns. Furthermore, no Jewish ancestry could be found. The close geographical area in which all these patients were found and the absence of the same mutation in the general population of the area strongly suggests a founder effect and that the mutation is responsible for the defect. The compound heterozygosis with the Glu 117 stop mutation, common among Jews, was not surprising because of the past strict ties of the Republic of Venice with the Middle East.

New Trends in Inspecting GPCR-ligand Recognition Process: the Contribution of the Molecular Modeling Section (MMS) at the University of Padova.

In this review, we present a survey of the recent advances carried out by our research groups in the field of ligand-GPCRs recognition process simulations recently implemented at the Molecular Modeling Section (MMS) of the University of Padova. We briefly describe a platform of tools we have tuned to aid the identification of novel GPCRs binders and the better understanding of their binding mechanisms, based on two extensively used computational techniques such as molecular docking and MD simulations. The developed methodologies encompass: (i) the selection of suitable protocols for docking studies, (ii) the exploration of the dynamical evolution of ligand-protein interaction networks, (iii) the detailed investigation of the role of water molecules upon ligand binding, and (iv) a glance at the way the ligand might go through prior reaching the binding site.

ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors.

ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

Carboxylation-dependent conformational changes of human osteocalcin.

Osteocalcin (OCN) is a small noncollagenous protein mainly produced by osteoblasts and is highly represented in bones of most vertebrates. Human OCN contains up to three gamma-carboxyglutamic acid (Gla-OCN) residues at positions 17, 21 and 24 which are thought to increase calcium binding strength, improving mechanical properties of the bone matrix. Recent studies revealed that OCN exerts also important endocrine functions, affecting energy metabolism and male fertility. The latter effect seems to be mediated by the uncarboxylated form of OCN (Glu-OCN). We employed human and mouse OCN as models of fully carboxylated and uncarboxylated OCN forms to investigate, by the use of circular dichroism and molecular dynamics simulations, the respective conformational properties and Ca2+ affinity. Ca2+ binding was found to trigger a similar conformational transition in both Glu-OCN and Gla-OCN, from a disordered structure to a more compact/stable form. Notably, gamma-carboxylation increases the affinity of OCN for Ca2+ by > 30 fold suggesting that, in physiological conditions, Gla-OCN is essentially Ca2+-bound, whereas Glu-OCN circulates mainly in the Ca2+-free form.

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.

Validation of a novel molecular dynamics simulation approach for lipophilic drug incorporation into polymer micelles.

Polymer micelles can be used to facilitate the aqueous solubilization of lipophilic, poorly water-soluble compounds and drugs. Even if the evaluation of the efficiency of drug incorporation into such micelles can be tested experimentally, a theoretical approach based on molecular simulation can constitute a useful tool that reduces time and cost. Here we present a promising method, based on molecular dynamics simulation, for the calculation of the Flory-Huggins interaction parameters as a measure of the potential for drug incorporation into polymer micelles. The data from modeling are validated on four drug compounds with different physical-chemical properties by means of a comparison with the data obtained from experiments.

Ecografía axilar con punción citológica positiva en pacientes con cáncer de mama: Análisis de su utilidad para indicar linfadenectomía axilar en la era del acozog Z0011: Experiencia en la Unidad de Mastología del Hospital Español de Montevideo, Uruguay / Axillary ultrasound with puncture positive cytology in patients with breast cancer: Analysis of its usefulness to indicate axillary lymphadenectomy in the era of acozog Z0011: Experience in the Mastology Unit of the Spanish Hospital of Montevideo, Uruguay

Objetivo Evaluar la validez de la ecografía axilar (ea) con punción citológica para indicar linfadenectomía axilar en pacientes con cáncer de mama, dentro del marco de los criterios propuestos por el estudio acosog Z0011. Material y método Estudio prospectivo, descriptivo. Se presentan los resultados del análisis de 123 pacientes con diagnóstico de cáncer de mama pertenecientes a la Unidad de Mastología del Hospital Español de Montevideo (Uruguay). En todas las pacientes se evaluó la axila con ecografía y, cuando existían ganglios sospechosos, se procedió a realizar punción con aguja fina. Cuando esta modalidad resultó positiva, en todas las pacientes se realizó linfadenectomía axilar de inicio. Cuantificamos la carga tumoral axilar de estas pacientes y analizamos la indicación de linfadenectomía axilar dentro del marco de propuestas del acozog Z0011. Resultados Los resultados obtenidos para predecir alta carga tumoral fueron los siguientes: porcentaje de verdaderos positivos (vp) 88,88%; porcentaje de falsos negativos (fn) 32%; sensibilidad (s) 80%; especificidad 80,95%; valor predictivo positivo (vpp) 88,89%; valor predictivo negativo (vpn) 68%; cociente de probabilidad positivo 4,20; cociente de probabilidad negativo 0,25; pacientes correctamente diagnosticados 80,33%. Discusión Desde hace varios años, la ecografía axilar con punción citológica ha demostrado elevada especificidad para predecir metástasis ganglionares. Publicaciones recientes afirman que la positividad de esta modalidad de estudio evidencia alta carga tumoral axilar y que, por lo tanto, se debe excluir a la paciente de las conductas conservadoras sobre la axila propuestas por el estudio acosog Z0011. Conclusiones Nuestros resultados concuerdan con la literatura internacional: la ecografía axilar con punción citológica es una metodología de elevada especificidad para predecir alta carga tumoral axilar. Esta metodología permite individualizar a aquellas pacientes que deben ser tratadas de inicio con linfadenectomía axilar, inclusive bajo los nuevos postulados del acosog Z0011

Objectives Evaluate the validity of ultrasound-guided fine needle aspiration (ugfna) of lymph nodes to determine axillary lymphadenectomy in patients with breast cancer, following the criteria proposed by acosog Z0011. Materials and method A prospective descriptive design was utilized. We present the analysis and results of 123 patients who were treated in the Unidad de Mastología at Hospital Español in Montevideo (Uruguay). All patients underwent axillary ultrasonography in order to evaluate lymphnodes. When suspicious lymph nodes were found, ugfna was performed. Patients who's ugfna yielded positive results were subjected to immediate axillary lymphadenectomy. We then quantified tumoral load and analyzed it according to the guidelines proposed by acosog Z0011. Results The following results were obtained for predicting tumoral load: True Positive Value of 88.88%; False Negative Value of 32%; Sensitivity of 80%; Specificity of 80.95%; Positive Predictive Value of 88.89%; Negative Predictive Value of 68%; Positive Likelihood Ratio of 4.20; Negative Likelihood Ratio of 0.25; accurately diagnosed patients 80.33%. Discussion ugfna has shown to have elevated specificity when predicting lymph node metastases. Recent investigations have shown that positive results of ugfna of lymph nodes determines elevated tumoral load; therefore, conservative treatment proposed by acosog Z0011 should not be followed. Conclusions Our results are comparable to research published internationally. ugfna is a method which has high specificity for the prediction of elevated axillary lymph node tumoral load. ugfna allows the tailoring of treatment for patients which must be treated initially with axillary lymphadenectomy, even under the new evidence put forth by acosog Z0011.

ClickMD: an intuitive web-oriented molecular dynamics platform.

BACKGROUND: Molecular dynamics (MD) simulations are broadly used to reproduce protein motions at an atomic level of detail. Running MD simulations is generally considered an expert-driven task. DISCUSSION: ClickMD provides an integrated web-oriented platform for processing and analyzing the time-dependent behavior of a biomolecular system in an MD workflow. ClickMD is freely available online and can be easily integrated into the drug-design process, in particular in combination with molecular docking simulations. CONCLUSION: ClickMD has the potential to reduce the shuffling among various software applications and to facilitate the seamless processing of the MD trajectories.

The role of the N-terminal domain in the regulation of the "constitutively active" conformation of protein kinase CK2α: insight from a molecular dynamics investigation.

Protein kinase CK2 is an extremely well-conserved pleiotropic protein kinase with a growing list of substrates, the majority of which are proteins implicated in signal transduction, gene expression, and transcription-related functions. Protein kinase CK2 is a ubiquitous heterotetrameric serine/threonine protein kinase made up of two α or α' catalytic subunits and two ß regulatory subunits. Moreover, protein kinase CK2 is defined as a "constitutively active" protein kinase in contrast to most other protein kinases characterized by the presence of distinct conformations associated with the active and inactive states. As previously demonstrated by in vitro mutation studies, CK2 activity is substantially regulated by the interaction between the N-terminal tail and the kinase domain. In fact, progressive deletions of the N-terminal tail show a decrease in the activity of the kinase. Even if the detrimental effects of Δ2-12 deletion can be partially reversed by the addition of a CK2ß subunit, deletions Δ2-12 and Δ2-30 progressively decrease the basal activity of CK2. In particular, as experimentally demonstrated, the Δ2-12 N-terminal deletion affects both the K(M) value for ATP and for the substrate peptide, and the k(cat) value of CK2α. In this work, molecular dynamics (MD) simulations were carried out on wild-type (wt), Δ2-12 and Δ2-30 deletion mutants of CK2α in order to explore the role of the N-terminal tail on the conformational behavior of CK2. Furthermore, classical MD simulations were carried out to assess the anticipated impact of conformational changes in a novel set of CK2α mutant forms, such as the triple mutant Y206F-R10A-Y261F and the single mutant Y125F.

A real-time and self-calibrating algorithm based on triaxial accelerometer signals for the detection of human posture and activity.

Assessment of human activity and posture with triaxial accelerometers provides insightful information about the functional ability: classification of human activities in rehabilitation and elderly surveillance contexts has been already proposed in the literature. In the meanwhile, recent technological advances allow developing miniaturized wearable devices, integrated within garments, which may extend this assessment to novel tasks, such as real-time remote surveillance of workers and emergency operators intervening in harsh environments. We present an algorithm for human posture and activity-level detection, based on the real-time processing of the signals produced by one wearable triaxial accelerometer. The algorithm is independent of the sensor orientation with respect to the body. Furthermore, it associates to its outputs a "reliability" value, representing the classification quality, in order to launch reliable alarms only when effective dangerous conditions are detected. The system was tested on a customized device to estimate the computational resources needed for real-time functioning. Results exhibit an overall 96.2% accuracy when classifying both static and dynamic activities.