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Background During the COVID‐19 pandemic in‐person visits for patients with cardiac implantable electronic devices should be replaced by remote monitoring (RM), in order to prevent viral transmission. A direct home‐delivery service of the RM communicator has been implemented at 49 Italian arrhythmia centers. Methods According to individual patient preference or the organizational decision of the center, patients were assigned to the home‐delivery group or the standard in‐clinic delivery group. In the former case, patients received telephone training on the activation process and use of the communicator. In June 2020, the centers were asked to reply to an ad hoc questionnaire to describe and evaluate their experience in the previous 3 months. Results RM was activated in 1324 patients: 821 (62%) received the communicator at home and the communicator was activated remotely. Activation required one additional call in 49% of cases, and the median time needed to complete the activation process was 15 min [25th‐75th percentile: 10–20]. 753 (92%) patients were able to complete the correct activation of the system. At the time when the questionnaire was completed, 743 (90%) communicators were regularly transmitting data. The service was generally deemed useful (96% of respondents) in facilitating the activation of RM during the COVID‐19 pandemic and possibly beyond. Conclusions Home delivery of the communicator proved to be a successful approach to system activation, and received positive feedback from clinicians. The increased use of a RM protocol will reduce risks for both providers and patients, while maintaining high‐quality care.

El ámbito educativo, indudablemente, puede beneficiarse de los aportes de las neurociencias, enfoque desde el cual, en este estudio, se plantearon los objetivos de explorar la relación existente entre la capacidad intelectual (CI), las funciones ejecutivas (FE) y el rendimiento académico (RA) en adolescentes; analizar si la CI y las FE constituyen variables predictoras del RA, e indagar el funcionamiento ejecutivo de la muestra según la CI fuera baja o alta. Para ello, se realizó un estudio no experimental, transversal, descriptivo-correlacional, en el que participaron 36 sujetos de 13-14 años. Los instrumentos de evaluación utilizados fueron WISC-IV, BANFE-2 y el registro de calificaciones provisto por la institución educativa. Los datos obtenidos fueron sometidos a análisis descriptivos, de correlación y de regresión. Los resultados arrojaron correlación positiva significativa entre las variables CI y FE, y entre la prueba WISC-IV y RA, determinándose el CI como variable predictiva del RA, mientras que en relación con las FE evaluadas con BANFE-2 solo se encontró asociación estadísticamente significativa con las subpruebas correspondientes al área prefrontal anterior. Además se observaron diferencias significativas entre los grupos de alta y baja CI en las FE de planificación, memoria de trabajo (MT) y fluidez verbal, en favor del grupo de alto desempeño intelectual. La conclusión se centra en la importancia de involucrar los hallazgos sobre los diferentes procesos cognitivos en el diseño de las estrategias pedagógicas para favorecer al máximo el RA del estudiante. Finalmente, las limitaciones versaron en relación con el tamaño muestral, y se proponen futuras líneas de estudio que posibiliten generalizar los resultados.

Durante el nivel inicial, los infantes suelen expresar respuestas emocionales y conductuales de manera impulsiva, dado que carecen de la aptitud de abstenerse de efectuar conductas que no son apropiadas a la ocasión. En este punto, las Habilidades Sociales (HHSS) y las Funciones Ejecutivas (FE) serán facilitadoras de la interacción asertiva con otros, favoreciendo en control comportamental y evitando los Problemas de Conducta (PC). El objetivo de este estudio fue analizar la correlación entre las FE, la percepción parental en relación a las emociones, y la percepción de docentes respecto de las HHSS y la conducta en niños del nivel inicial de educación; a tal fin se realizó un estudio cuantitativo, transversal, descriptivo-correlacional, en base a un diseño no experimental. La muestra estuvo integrada por 36 niños/as de nivel inicial, a quienes se les administró una batería neuropsicológica, además se incluyeron dos cuestionarios, uno para padres y otro para docentes. Se encontró correlación entre HHSS y PC; entre Planeación y Emociones; y entre Atención y Concentración y Memoria de Trabajo. Se concluye que los niños de nivel inicial poseen una conducta socialmente habilidosa, y manejan sus emociones sin dificultad indistintamente del sexo, según la perspectiva parental y docente. Fil: Mucchiut, Alvaro. Centro de Estudios Cognitivos; Argentina Fil: Dri, Constanza Ayelen. Centro de Estudios Cognitivos; Argentina Fil: Vaccaro, Paola. Centro de Estudios Cognitivos; Argentina Fil: Pietto, Marcos Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. CEMIC-CONICET. Centro de Educaciones Médicas e Investigaciones Clínicas "Norberto Quirno". CEMIC-CONICET; Argentina

Aims During the COVID-19 pandemic in-person visits for patients with cardiac implantable electronic devices should be replaced by remote monitoring (RM), in order to prevent viral transmission. A direct home-delivery service of the RM communicator has been implemented at 49 Italian arrhythmia centres. Methods and results According to individual patient preference or the organizational decision of the centre, patients were assigned to the home-delivery group or the standard in-clinic delivery group. In the former case, patients received telephone training on the activation process and use of the communicator. In June 2020, the centres were asked to reply to an ad hoc questionnaire to describe and evaluate their experience in the previous 3 months. RM was activated in 1324 patients: 821 (62%) received the communicator at home and the communicator was activated remotely. Activation required one additional call in 49% of cases, and the median time needed to complete the activation process was 15 min (25th–75th percentile: 10–20). 753 (92%) patients were able to complete the correct activation of the system. At the time when the questionnaire was completed, 743 (90%) communicators were regularly transmitting data. The service was generally deemed useful (96% of respondents) in facilitating the activation of RM during the COVID-19 pandemic and possibly beyond. Conclusions Home delivery of the communicator proved to be a successful approach to system activation, and received positive feedback from clinicians. The increased use of a RM protocol will reduce risks for both providers and patients, while maintaining high-quality care.

Resumen Considerando los procesos de memoria como uno de los factores facilitadores en los procesos de enseñanza-aprendizaje se propuso este estudio de diseño preexperimental, desarrollado en dos etapas, con el objetivo central de explorar qué conocimientos tiene el cuerpo docente respecto a los procesos de memoria y sus etapas, esto con el fin de establecer las bases para estudios posteriores de mayor profundidad. Para sondear los conocimientos del docente al respecto se confeccionó una encuesta en dos partes, la cual fue completada por un total de 196 docentes de diferentes niveles educativos asistentes a las jornadas de neuroeducación desarrolladas en la ciudad de Resistencia en el año 2017. El análisis de los datos se elaboró mediante el empleo de planillas de cálculo Excel. Los resultados revelaron que antes del inicio de la capacitación la mayoría de docentes reportaron conocer poco (65%) - o nada (15%)- en relación con el concepto de memoria; a su vez que el 72% presenta mínimos conocimientos sobre la relación existente entre memoria y aprendizaje y un 13% la desconoce. Finalizada la capacitación, se observó un incremento significativo en el conocimiento de cada participante sobre el concepto de memoria (aumentó en un 76%), los tipos de memoria (52%) y su relación con el aprendizaje (55%). Se concluye que conocer en general cómo aprende el cerebro del estudiantado, y en particular los procesos de la memoria, resulta esencial en la formación docente, pues permitirá planificar y ejecutar su práctica pedagógica con mayor efectividad. Abstract Memory facilitates teaching and learning. This pre-experimental design study explores how knowledgeable teachers are about memory and consists of two stages that serve as baselines for future studies. A group of 196 teachers that taught all types of grade levels were asked to answer a two-part survey. The teachers were attendees at a Neuroeducation training session that was held in the city of Resistencia, Argentina in 2017. The data was analyzed on Excel spreadsheets. According to the results obtained, 65% of the teachers stated having knowledge about the concept of memory and 15% reported having none. On the other hand, 72% said that they had minimal knowledge about memory as it relates to learning whereas 13% stated having none. The workshop proved to significantly increase the level of knowledge among the attendees regarding concept of memory (76%), different types of memory (52%) and how it relates to learning (55%). It can be concluded that knowledge about how the student brain learns and, specifically, how the memory process is interrelated to learning, is essential for teacher training since it allows for better planning and more effective teaching.

Cuando se incluyen las Funciones Ejecutivas (FE) en educación se suelen extrapolar programas clínicos al aula, obviando aspectos propios del ambiente educativo como las prácticas pedagógicas (PP). El presente trabajo, es un estudio pre-experimental con diseño pre-post prueba. En este sentido, se procuró describir el posible impacto de PP orientadas a favorecer las FE en adolescentes, desde un enfoque de intervención ecológica. Para tal fin, participaron 32 estudiantes de nivel secundario, que fueron evaluados con BANFE antes y después de la intervención. Dicha prueba, incluyó diferentes PP implementadas durante 7 meses en 7 espacios curriculares diferentes. Por consiguiente, los resultados mostraron cambios en el desempeño de la fase post-PP en comparación a la pre-PP, evidenciándose menor tiempo de reacción, disminución de errores e incremento de aciertos en algunas pruebas de inhibición, planificación, memoria de trabajo, flexibilidad cognitiva y metafunciones. En general, los cambios entre las fases del estudio indicaron un mejor desempeño en las pruebas. Estos hallazgos van en sentido de lo esperado, considerando que la efectividad obtenida a partir de la intervención con las PP tuvo como consecuencia un mejor rendimiento de las FE de los adolescentes. Se concluye que la implementación de intervenciones ecológicas que estimulan las FE mediante PP, podrían favorecer el funcionamiento ejecutivo del estudiante. Además, se insta a continuar profundizando acerca de la influencia de las PP en el desarrollo de las FE, para un mejor entendimiento de ésta dinámica.

Aims There are conflicting data on the benefit of cardiac resynchronization therapy (CRT) in heart failure (HF) patients with permanent atrial fibrillation (AF). We aimed to compare patient outcomes according to the presence or absence of permanent AF at device implantation. Methods and results We retrospectively analysed remote monitoring data from 1141 CRT defibrillators. Propensity score with inverse‐probability weighting method was used to balance AF and sinus rhythm (SR) groups. Analysis endpoints included total mortality, appropriate defibrillation shocks, and CRT percentage. There were 229 patients (20.1%) in the AF group and 912 patients (79.9%) in the SR group. Compared with SR patients, AF patients were older (median age, 77 vs. 72 years, P

Objective: An intra-aortic balloon pump (IABP) is used routinely in high-risk patients undergoing cardiac surgery to prevent or treat low-cardiac-output syndrome and to reduce perioperative mortality. The insertion and management of IABP carry the risk of major vascular complications. The authors reviewed their database to ascertain the incidence of IABP-related severe complications. Design: A retrospective study. Setting: A teaching hospital. Participants: Ten thousand three hundred sixty-five patients scheduled for elective or emergency cardiac surgery over a 12-year period at a single center. Interventions: Four hundred twenty-three patients received an IABP perioperatively. Careful preoperative screening for peripheral arterial disease, strict postoperative control, and the sheathless insertion technique to spare the arterial flow to the lower limb were performed routinely. Measurements and Main Results: The use of a perioperative IABP was 0.7% at the beginning of the observation period in 1999 and 7.3% in 2010, showing a fluctuating trend. Two patients (0.47%) died of direct complications, arterial wall damage and bleeding. Immediate surgical exploration and control of bleeding were followed by multiple-organ failure and death. Vascular complications, leading to lower-limb ischemia, occurred in 4 of 423 patients (0.94%). All of them underwent urgent vascular surgery and survived. Local sepsis occurred in 2 other patients (0.47%). Conclusions: These data indicate that an IABP is a valuable option in high-risk patients undergoing cardiac surgery even if not devoid of intrinsic risks for vascular complications (0.94%), septic complications (0.47%) and mortality (0.47%). (C) 2012 Elsevier Inc. All rights reserved.

On November 25, 2014, an Italian physician infected by Ebola virus in Sierra Leone was admitted to the "Lazzaro Spallanzani" National Institute for Infectious Diseases in Rome, Italy. He was the first Italian case and was successfully cured in 38 days. The staff responsible for communication had a critical role ensuring that this challenging mission went smoothly. The Institutional Press Office working together with the press offices of the Ministry of Health was able to provide the high level of expertise necessary within both medical and communication contexts. Communication strategy, tools and procedures adopted before and after the arrival of the patient are summarized.

Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals.

The mapping of monoclonal antibody epitopes is now predominantly carried out using molecular diversity techniques, phage display in particular. However, until very recently, phage display methods have been inappropriate for the analysis of epitopes that require a free carboxy terminus. Here we describe the use of two different techniques to analyze the known C terminal epitope specificity of MN423, a monoclonal antibody specifically staining truncated tau in Alzheimer's brain. Using a lambda phage based C-terminal random peptide library, and an intracellular expression library based on truncated tau, we show that this antibody has an absolute requirement for a glycine at position −3 with respect to the C terminus. Both methods give similar results, and identify other important residues in the binding site. However, affinity analysis of synthetic peptides revealed that the affinity of the antibody for identified tripeptides was far lower than the pentapeptide sequence in the native target, and that this in turn was considerably below the affinity for the native target itself. This suggests that molecular diversity methods may define minimum, but not necessarily complete epitopes. The methods described here have a general application to the analysis of antibody epitopes suspected to be found at the C terminus.

Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.

Generation of human recombinant antibody libraries displayed on the surface of the filamentous phage and selection of specific antibodies against desirable targets allows production of fully human antibodies usable for repeated administration in humans. Various lymphoid tissues from immunized donors, such as lymph nodes or peripheral blood lymphocytes from individuals with tumor or lymphocytes infiltrating tumor masses may serve as a source of specific anti-tumor antibody repertoire for generation of tumor-focused phage display libraries. In the case of lack of tumor-associated antigens in the purified form, high affinity anti-tumor antibodies can be isolated through library panning on whole cells expressing these antigens. However, affinity selection against cell surface specific antigens within highly heterogeneous population of molecules is not a very efficient process that often results in the selection of unspecific antibodies or antibodies against intracellular antigens that are generally useless for targeted immunotherapy. In this work, we developed a new cell-based antibody selection protocol that, by eliminating the contamination of dead cells from the cell suspension, dramatically improves the selection frequency of anti-tumor antibodies recognizing cell surface antigens.

BackgroundCEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration.MethodsThe human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, byin vitromutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized.ResultsThe scFv MA39 antibody was affinity-maturated byin vitromutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells.ConclusionThe binding affinity of the scFv E8 is in a range for efficient,in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance.

Objective : In this article we provide evidence of a significant spontaneous humoral response in cancer patients. Methods : A panel of tumor-associated antigens, previously identified through serological screening of phage-displayed cDNA libraries from solid human tumors, breast carcinoma cell lines and human testis by employing breast cancer patient sera, was used in this study to survey sera from 182 patients with known disease histories and clinical stages. Results : This analysis reveals a statistically significant association between tumor disease and presence in peripheral blood of IgG antibodies against four autoantigens. One of these antigens (D7-1) is particularly interesting in that the antibody response against it grows with cancer progression from stages I through IV, with an incidence of 13.2, 13.5, 18.2 and 27%, respectively. The significance of this stage-dependent increase in the incidence is confirmed by the Mantel–Haenszel Chi-squared test ( P =0.001). Conclusions : Our data confirm association between breast cancer diagnosis of patients and presence in their peripheral blood of antibodies against several autoantigens identified by phage display.

In the present work we demonstrate the efficient display of functional scFv antibodies on the bacteriophage lambda capsid. A single-chain (scFv) anti-CEA antibody gene was cloned in two different vectors to obtain fusion of the scFv antibody to the N- or C-terminus of the bacteriophage lambda capsid protein D (gpD). Lambda bacteriophage assembly occurs in the reducing environment of the cytoplasm; despite this the lambda-displayed anti-CEA antibody fragments retain the capacity to recognize the antigen, indicating correct single-chain antibody folding. Efficient production of functional scFv exposed on lambda capsid with viable antigen binding specificity allowed us to study and compare the capacity of display, the stability of recombinant antibody expression and the assembly efficiency of bacteriophage particles decorated with recombinant antibody fused to the amino- or carboxy-terminus of lambda D protein.

Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals.

PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Proteins containing multiple PDZ domains often bind to different trans-membrane and intracellular proteins, playing a central role as organizers of multimeric complexes. To characterize the rules underlying the binding specificity of different PDZ domains, we have assembled a novel repertoire of random peptides that are displayed at high density at the carboxyl terminus of the capsid D protein of bacteriophage lambda. We have exploited this combinatorial library to determine the peptide binding preference of the seven PDZ domains of human INADL, a multi-PDZ protein that is homologous to the INAD protein of Drosophila melanogaster. This approach has permitted the determination of the consensus ligand for each PDZ domain and the assignment to class I, class II, and to a new specificity class, class IV, characterized by the presence of an acidic residue at the carboxyl-terminal position. Homology modeling and site-directed mutagenesis experiments confirmed the involvement of specific residues at contact positions in determining the domain binding preference. However, these experiments failed to reveal simple rules that would permit the association of the chemical characteristics of any given residue in the peptide binding pocket to the preference for specific amino acid sequences in the ligand peptide. Rather, they suggested that to infer the binding preference of any PDZ domain, it is necessary to simultaneously take into account all contact positions by using computational procedures. For this purpose we extended the SPOT algorithm, originally developed for SH3 domains, to evaluate the probability that any peptide would bind to any given PDZ domain.

1. Synapsin I, a major synaptic vesicle (SV)-associated phosphoprotein, is involved in the regulation of neurotransmitter release and synapse formation. By binding to both phospholipid and protein components of SV with high affinity and in a phosphorylation-dependent fashion, synapsin I is believed to cluster SV and to attach them to the actin-based cytoskeleton of the nerve terminal. 2. In the present study we have investigated the kinetic aspects of synapsin I-SV interactions and the mechanisms of their modulation by ionic strength and site-specific phosphorylation, using fluorescence resonance energy transfer between suitable fluorophores linked to synapsin I and to the membrane bilayer. 3. The binding of synapsin I to the phospholipid and protein components of SV has fast kinetics: mean time constants ranged between 1 and 4 s for association and 9 and 11's for ionic strength-induced dissociation at 20 degrees C. The interaction with the phospholipid component consists predominantly of a hydrophobic binding with the core of the membrane which may account for the membrane stabilizing effect of synapsin I. 4. Phosphorylation of synapsin I by either SV-associated or purified exogenous Ca2+/calmodulin-dependent protein kinase II (CaMPKII) inhibited the association rate and the binding to SV at steady state by acting on the ionic strength-sensitive component of the binding. When dephosphorylated synapsin I was previously bound to SV, exposure of SV to Ca2+/calmodulin in the presence of ATP triggered a prompt dissociation of synapsin I with a time constant similar to that of ionic strength-induced dissociation. 5. In conclusion, the reversible interactions between synapsin I and SV are highly regulated by site-specific phosphorylation and have kinetics of the same order of magnitude as the kinetics of SV recycling determined in mammalian neurons under comparable temperature conditions. These findings are consistent with the hypothesis that synapsin I associates with, and dissociates from, SV during the exo-endocytotic cycle. The on-vesicle phosphorylation of synapsin I by the SV-associated CaMPKII, and the subsequent dissociation of the protein from the vesicle membrane, though not involved in mediating exocytosis of primed vesicles evoked by a single stimulus, may represent a prompt and efficient mechanism for the modulation of neurotransmitter release and presynaptic plasticity.

The synapsins are a family of major neuron-specific synaptic vesicle-associated phosphoproteins which play important roles in synaptic function. In an effort to identify molecular tools which can be used to perturb the activity of the synapsins in in vitro as well as in vivo experiments, we have localized the epitopes of a panel of monoclonal antibodies (mAbs) raised against synapsins I and II and have characterized their ability to interfere with the interactions of the synapsins with protein kinases, actin and Src homology-3 (SH3) domains. The epitopes of the six mAbs were found to be concentrated in the N-terminal region within domains A and B for the synapsin II-reactive mAbs 19.4, 19.11, 19.51 and 19.21, and in two C-terminal clusters in the proline-rich domains D for synapsin I (mAbs 10.22, 19.51, 19.11 and 19.8) and G for synapsin II (mAb 19.8). The synapsin II-specific mAbs 19.4 and 19.21, whose overlapping epitopes are adjacent to phosphorylation site 1, specifically inhibited synapsin II phosphorylation by endogenous or exogenous cAMP-dependent protein kinase. While all the anti-synapsin I mAbs were unable to affect the interactions of synapsin I both with Ca2+/calmodulin-dependent protein kinase II and with actin monomers and filaments, mAbs 19.8 and 19.51 were found to inhibit the binding of Grb2 SH3 domains to the proline-rich C-terminal region of synapsin I.

Classi¢cation of protein interaction domains on the basis of the chemical characteristics of binding pocket residues is a di¤cult task, because multiple contact positions are usually involved in the recognition speci¢city mechanism. On the other hand, target peptides may be classi¢ed according to the (few) speci¢c residues that constitute the binding motif, through analysis of molecular repertoires (libraries of synthetic peptides; phage display; two hybrid, etc.) that allow identifying collections of diierent ligands. We have recently pointed out that, in order to characterize PDZ domains and to infer their binding speci¢city, it is necessary to exploit computational procedures, which simultaneously take into account all the contact positions of the domain binding pocket and the corresponding residues of the ligand [1]. PDZ domains are protein interaction modules that recognize and bind the C-terminal four residues of their target. The solution of X-ray crystallographic structure of PDZ domains complexed with their peptide ligands reveals at least 23 contact positions, whose interacting atoms are at a distance shorter than the sum of the van der Waals radii (r) +3 Ai . Some of these positions contain residues that are highly conserved in the PDZ domain family: for example the `GLGF' loop and a positively charged residue that accommodate the terminal carboxylate group. The majority of the PDZ domains (identi¢ed in the proteome up to now) recognize ligands of class I (Table 1). These PDZ domains are provided with a hydrophilic pocket, where residues of the LB strand and of the KB helix (in particular a histidine, highly conserved at position KB1) are involved in contacting the peptide ligand. Most of the remaining PDZ domains recognize a varied class of ligand peptides, characterized by aromatic or hydrophobic residues at position P32. Even residues mimicking part of a hydrophobic moiety at position P32 (such as the arginine at P32 of the peptide ligand in the crystallized structure of hCASK) can be accommodated in the large hydrophobic pocket that characterizes PDZ domains binding to class II peptides [2]. Main determinants of the binding, as derived from the contacts in the crystal structure, are residues at LB5, KB1 and KB3 positions. Bezprozvanny and Maximov have recently proposed a classi¢cation of the PDZ domains listed in the SMART Website, based upon the type of residues present in only two contact positions ^ LB5 and KB1 ^ of the binding pocket [3]. By grouping the couples of residues on the basis of their polarity and/or bulkiness, they de¢ned 25 groups and correlated them to experimentally determined ligands. Unfortunately, ligand sequences are available only for nine out of 25 groups and, while the ¢rst group (G,H) is enforced by the presence of 68 PDZ domains (those binding to class I motifs), the others are less clearly determined. Two of them (G,n) and (a,p) do not correspond to known PDZ domains; other 14 (G,h), (G,a), (n,H), (n,n), (n,p), (Sp,n), (Sp,h), (Sp,a), (Lh,H), (Lh,n), (Lh,p), (a,H), (a,n), (a,h) are not correlated to any ligand sequence; four other groups (p,H), (Sp,p), (Lh,h), (Lh,a) can be uni¢ed into canonical class II binding domains, because they all recognize ligands conforming to the consensus ([x/8]Xx*) (Table 1). One group (n,a) corresponds to the previously de¢ned group of PDZ domains that bind to class III ligands (consensus [D/E]XV*). Another group (n,h) includes PDZ domains characterized by dual speci¢city. On this purpose we have to emphasize that, in some cases, diierent PDZ domain lists, available on the Web, may induce confusion in group assignment. In particular, in the course of the characterization of the PDZ domains present in the full-length clone hINADL (accession number AJ224747) we used the `Pfam v5.5 version sequence alignment' [1]. Only seven PDZ domains were found by Pfam search tool, because the score of the region among residues 555 and 638 resulted less signi¢cant than the required threshold. By screening a combinatorial phage library, we classi¢ed the ¢rst four domains of hINADL as class II binding PDZ, and the last three domains as class I binding PDZ. In contrast, Bezprozvanny and Maximov used a SMART classi¢cation that considers eight PDZ domains in hINADL. As a consequence of this diierent numbering, the binding preferences of hINADL-5 (hINADL-4 in our list) de¢ned by Bezprozvanny and Maximov, using diierent techniques, results in agreement with the class II binding consensus, identi¢ed by phage library screening [1]. Therefore, the binding to neurexin Ia, whose terminus is `EYYV*' (class II), is not surprising, while it is interesting to note that the same domain binds also to peptide `DHWC' at the end of NC4 (N-type Ca2‡ channel). Anyway, as previously mentioned, the large hydrophobic pocket of a class II binding domain can accommodate also residues that in part mimic a hydrophobic moiety [2]. Dual speci¢city is not a rare event in PDZ binding mode: we have recently described a new class of binding motifs, characterized by the presence of a negative residue at the C-terminus, which were selected by hINADL-3. This PDZ domain can bind with the same a¤nity also to class II peptide ligands [1]. In conclusion, we believe that a classi¢cation of PDZ domains, exclusively based on the chemical properties of only two positions, is not su¤cient to describe the complexity of the domain family and to predict the speci¢city of binding. Several experimental data con¢rm this statement: we have shown that the substitution of the histidine at the crucial position KB1 of hINADL-7 (a class I binding PDZ) is not su¤cient to change its binding speci¢city and to confer a clear ligand preference [1]. Furthermore, the ¢rst PDZ of MUPP1 is a member of the most abundant group (G,H); therefore it should bind to class I motifs. In contrast, our computer-aided analysis [1] and two hybrid (in vitro) and co-immunoprecipitation (in vivo) assays [4] indicate a preference of MUPP1-1 PDZ domain for class II ligands, such as NG2 proteoglycan. Another example is CIPP-3 PDZ domain that is classi¢ed as

The sexual and asexual phases of reproductive cycles of two sponges, Tethya citrina and T. aurantium, living sympatrically in a Mediterranean coastal lagoon (Stagnone di Marsala, NW Sicily) were studied from samples collected over an 18-mo period. Both species are oviparous and gonochoric. They have a summer, partially overlapping, period of oocyte production, although T. citrina appear to mature earlier. No males were found, possibly due to the very short period of spermatogenesis. Both species produce asexual buds during the autumn/winter months. However, they seem to follow different reproductive strategies, with T. citrina showing a significantly lower production of buds than T. aurantium; by contrast, egg production is significantly lower in the latter species. The difference in the reproductive resource allocation is consistent with data reported in the literature on the anatomy features, genetic population structure and ecological distribution.

Filamentous phage has been extensively used to implement various aspects of phage display technology. The success of these organisms as vectors to present foreign peptides and to link them to their coding sequences is a consequence of their structural and biological characteristics. Some of these properties, however, represent a limitation when one attempts to display proteins that cannot be efficiently exported through the bacterial membrane or do not fold properly in the periplasm. Thus, the desirability of developing alternative display systems was recognised recently and led to the development of a different class of display vectors that assemble their capsid in the cytoplasm and are released via cell lysis. This review describes and compares the properties of these alternative display systems.

Background Consistent progress in the development of bacteriophage lambda display platform as an alternative to filamentous phage display system was achieved in the recent years. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA. Results In the present work we describe an original method for dual display of large proteins on the surface of lambda particles. An anti-CEA single-chain antibody fragment and green fluorescent protein or alkaline phosphatase were simultaneously displayed by engineering both gpD and gpV lambda proteins. Conclusions Here we show that such modified phage particles can be used for the detection of target molecules in vitro and in vivo. Dual expression of functional moieties on the surface of the lambda phage might open the way to generation of a new class of diagnostic and therapeutic targeted nanoparticles.