Vincenzo Izzo

cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen

A clone (P2) coding for an allergen of Parietaria judaica (Pj) pollen has been isolated and sequenced from a cDNA library in lambda ZAP using a pool of 23 sera from Pj‐allergic patients. The clone contained an insert of 622 nucleotides with an open reading frame of 133 amino acids (aa) and a putative signal peptide of 31 aa giving a deduced mature processed protein of 102 aa with a molecular mass of 11 344 Da. The expressed recombinant protein, named rPar j 2.0101, was a major allergen since it reacted with IgE of 82% (23/28) of the sera of Pj‐allergic subjects analyzed. It was shown to be a new allergen since (i) the amino acid sequence homology with the already reported recombinant allergen Par j 1.0101 was 45% and (ii) there was no cross‐inhibition between rPar j 2.0101 and rPar j 1.0101. In addition, rPar j 2.0101 inhibited 35% of the specific IgE for 10–14 kDa native allergens and preincubation of sera from Pj‐allergic patients with both rPar j 2.0101 and rPar j 1.0101 fully abolished the IgE recognition of the 10–14 kDa native allergen region, suggesting that these two allergens contributed to the region.

cDNA cloning, expression and primary structure of Par j I, a major allergen of Parietaria judaica pollen

A 659 bp cDNA clone1 coding for an allergen of Pj pollen has been isolated from a lambda gt11 library, and its DNA sequence determined. The cDNA insert showed an open reading frame of 429 bp coding for an allergenic protein of 14,866 Da and a deduced amino acid sequence containing 143 residues. The expressed recombinant protein represented the major allergen Par j I since it reacted with 95% of the sera from Pj‐allergic patients (n = 22) and with two Par j I‐specific monoclonal antibodies. No similarity with other known DNA and protein sequences has been detected.

The Allergens of Parietaria

Parietaria is a genus of dicotyledonous weeds of the Urticaceae family including several species and its pollen grain is one of the most important allergenic sources in the Mediterranean area. Species belonging to this genus induce IgE responses in approximately 10 million people. Identification of allergens by means of independent strategies suggest that the allergens of the two more common species, Parietaria judaica and Parietaria officinalis, show molecular weights ranging between 10 and 14 kD and that the allergens of the two extracts are highly cross-reactive. Biochemical analysis and molecular cloning allowed the isolation and immunological characterization of the two major allergens of the P. judaica pollen, Par j 1 and Par j 2. Sequence comparison suggests that the P j major allergens of P. judaica belong to the nonspecific lipid transfer protein family, and three-dimensional modeling by homology has revealed that both proteins present a very conserved structural motif composed of four α-helices. Immunological analysis has shown that Par j 1 and Par j 2 are able to bind most of the P. judaica-specific IgE and some of their IgE determinants have been mapped. Recombinant Par j 1 and Par j 2 allergens have been shown to possess immunological properties equivalent to their natural counterpart and their availability represents a fundamental tool for the diagnosis and therapy of Parietaria pollen allergy.

Isolation and characterization of two cDNA clones coding for isoforms of the Parietaria judaica major allergen Par j 1.0101

Two cDNA clones named P9* and P1* of 794 and 631 bp, respectively, were isolated from a lambda ZAP cDNA expression library using Parietaria judaica (Pj) pollen-specific IgE antibodies from a pool of sera (n = 23) of patients allergic to Pj. Sequence analysis showed open reading frames of 176 and 138 amino acids. Both clones contain a putative signal peptide giving two mature processed proteins named Par j 1.0102 of 14,726 D and Par j 1.0201 of 10,677 D. These proteins represent isoallergenic forms of the major Pj allergen Par j 1.0101 (clone P5) previously reported. The Par j 1.0102 shared 98% amino acid sequence homology with the P5, while the Par j 1.0201 shared 89% homology. Since P1, P5 and P9 clones were expressed in Escherichia coli, and since the three allergenic proteins shared a very high degree of sequence identity and comparable binding to the Pj-specific IgE, we decided to analyze in more detail the immunological properties of only one allergen, the recombinant Par j 1.0101. The allergenic activity determined by the histamine release assay ranged between 9 and 56%, depending on the allergic patient analyzed, while it blocked approximately 40% of all the Pj-specific IgE antibodies, as detected after ELISA and cross-absorption analysis.

FPGA implementation of a high-resolution time-to-digital converter

In the past years, precise measurements of time intervals have been realized using methods such as time- stretching, Vernier and delay line. In this paper, we present two high-resolution time-interval measuring system implemented in a SRAM-based FPGA device. The two methods ought to be used for time interpolation within the system clock cycle. In the first method, dedicated carry lines are used to perform fine time measurement, while in the second one a differential tapped delay line is used. In this paper we compare the two architectures and show their performance in terms of stability and resolution.

Characterizing jitter performance of multi gigabit FPGA-embedded serial transceivers

High-speed serial links are a key component of data acquisition systems for High Energy Physics. They carry physics events data and often also clock, trigger and fast control signals. For the latter applications, the jitter on the clock recovered from the serial stream is a critical parameter since it directly affects the timing performance of data acquisition and trigger systems. Latest Field Programmable Gate Arrays (FPGAs) include multigigabit serial transceivers, which are configurable with various options and support many data encodings. However, an in-depth jitter characterization of those devices is not available yet. In this paper we present measurements of the jitter on the clock recovered by a GTP transceiver (embedded in a Xilinx Virtex 5 FPGA) as a function of the data pattern, coding and logic activity on the transmitter and receiver FPGAs.

High-Speed, Fixed-Latency Serial Links With FPGAs for Synchronous Transfers

Fixed-latency serial links find application in trigger and data acquisition systems of high energy physics (HEP) experiments requiring a predictable data transfer timing. In some architectures, there is the need to clock the data in and out from the link synchronously with a system clock (i.e., synchronous transfers) instead of using the clock recovered from the serial stream. In this work, we present a synchronous link architecture based on high-speed transceivers embedded in latest generation field programmable gate arrays (FPGAs). These transceivers are typically designed for applications that tolerate latency variations. However, we have developed two configurations and a clocking scheme to implement fixed-latency operation. The latency is constant during the transfer, after a loss of lock or a power cycle. Once locked, the link can be considered as a synchronous pipeline. The configurations do not depend on a particular serial encoding, the encoder/decoder being external to the transceiver. We discuss the latency performance for each configuration and show an implementation of the architecture we propose. We also present experimental results showing the stability of the latency of the link.

The Read-out Driver for the RPC of the ATLAS Muon Spectrometer

The spectrometer of the ATLAS experiment has been designed to identify muon tracks with transverse momenta in between 6 and 20 Gev/c. Its barrel section is made of monitored drift tubes and resistive plate chambers (RPCs) arranged in 32 sectors, to form two wheels surrounding the interaction point. The RPC subsystem provides the level-1 trigger in the barrel and it is read out by a specific DAQ system. On-detector electronics pack the RPC data in frames with an event number assigned by the trigger logic and transmit them to the counting room optically. Data from each sector are then routed together to a read-out driver (ROD) board. This is a custom processor that parses the frames, checks their coherence and builds a data structure for the RPCs of one of the 32 sectors of the spectrometer. Each ROD sends the event fragments to a read-out buffer for further event building and analysis. The ROD is a VME64x board, designed around two Xilinx Virtex-II FPGAs and an ARM7 microcontroller. In this paper we describe the system architecture, the event binding algorithms and the monitoring features embedded into the design. The board is in production and will be deployed in the first ATLAS runs.

Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

An inexpensive Plexiglas apparatus which allows a simple and rapid preparation of horizontal polyacrylamide gels of different dimensions for different purposes, is described. Preparation of such gels is as easy and rapid as agarose gel preparation, and polymerized polyacrylamide gels are used to fractionate proteins or small DNA fragments using a common horizontal electrophoretic tank. This apparatus was used to electrophoretically fractionate proteins or DNA for immuno-blot analyses, particularirly in the study of the allergenic response to Parietaria judaica pollen in senescence, for Southern-blot hybridizations and in the study of DNA polymorphisms.

Solvent isotope effect in the monomer–dimer equilibrium of methylene blue

So-called stacking forces, of particular interest in the structural stability of nucleic acids, have been studied in a simple model system of planar, non-hydrogen-bonding dye molecules (Methylene Blue). The dimerization process has been studied by spectral analyses at different concentrations and temperatures in D2O as well as in H2O. Computer-aided data-processing techniques have been used to obtain detailed information on the dimerization process.Results show that the high positive (hydrophobic) contribution to the ‘unitary’ entropy term due to solute–solvent interaction changes appreciably upon H–D substitution. This illustrates the role of hydrophobic interactions in dimerization (stacking) processes.