Exponential Megapriming PCR (EMP) Cloning-Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.National Institutes of Health (U.S.) (Grant GM077537

Revised Selection Criteria for Candidate Restriction Enzymes in Genome Walking

A new method to improve the efficiency of flanking sequence identification by genome walking was developed based on an expanded, sequential list of criteria for selecting candidate enzymes, plus several other optimization steps. These criteria include: step (1) initially choosing the most appropriate restriction enzyme according to the average fragment size produced by each enzyme determined using in silico digestion of genomic DNA, step (2) evaluating the in silico frequency of fragment size distribution between individual chromosomes, step (3) selecting those enzymes that generate fragments with the majority between 100 bp and 3,000 bp, step (4) weighing the advantages and disadvantages of blunt-end sites vs. cohesive-end sites, step (5) elimination of methylation sensitive enzymes with methylation-insensitive isoschizomers, and step (6) elimination of enzymes with recognition sites within the binary vector sequence (T-DNA and plasmid backbone). Step (7) includes the selection of a second restriction enzyme with highest number of recognition sites within regions not covered by the first restriction enzyme. Step (8) considers primer and adapter sequence optimization, selecting the best adapter-primer pairs according to their hairpin/dimers and secondary structure. In step (9), the efficiency of genomic library development was improved by column-filtration of digested DNA to remove restriction enzyme and phosphatase enzyme, and most important, to remove small genomic fragments (<100 bp) lacking the T-DNA insertion, hence improving the chance of ligation between adapters and fragments harbouring a T-DNA. Two enzymes, NsiI and NdeI, fit these criteria for the Arabidopsis thaliana genome. Their efficiency was assessed using 54 T3 lines from an Arabidopsis SK enhancer population. Over 70% success rate was achieved in amplifying the flanking sequences of these lines. This strategy was also tested with Brachypodium distachyon to demonstrate its applicability to other larger genomes

Potentialities of CdZnTe Quasi-Hemispherical Detectors for Hard X-ray Spectroscopy of Kaonic Atoms at the DAΦNE Collider

Kaonic atom X-ray spectroscopy is a consolidated technique for investigations on the physics of strong kaon-nucleus/nucleon interaction. Several experiments have been conducted regarding the measurement of soft X-ray emission (&lt;20 keV) from light kaonic atoms (hydrogen, deuterium, and helium). Currently, there have been new research activities within the framework of the SIDDHARTA-2 experiment and EXCALIBUR proposal focusing on performing precise and accurate measurements of hard X-rays (&gt;20 keV) from intermediate kaonic atoms (carbon, aluminum, and sulfur). In this context, we investigated cadmium-zinc-telluride (CdZnTe or CZT) detectors, which have recently demonstrated high-resolution capabilities for hard X-ray and gamma-ray detection. A demonstrator prototype based on a new cadmium-zinc-telluride quasi-hemispherical detector and custom digital pulse processing electronics was developed. The detector covered a detection area of 1 cm2 with a single readout channel and interesting room-temperature performance with energy resolution of 4.4% (2.6 keV), 3% (3.7 keV), and 1.4% (9.3 keV) FWHM at 59.5, 122.1, and 662 keV, respectively. The results from X-ray measurements at the DAΦNE collider at the INFN National Laboratories of Frascati (Italy) are also presented with particular attention to the effects and rejection of electromagnetic and hadronic background

New opportunities for kaonic atoms measurements from CdZnTe detectors

We present the tests performed by the SIDDHARTA-2 collaboration at the DA Φ NE collider with a quasi-hemispherical CdZnTe detector. The very good room-temperature energy resolution and efficiency in a wide energy range show that this detector technology is ideal for studying radiative transitions in intermediate and heavy mass kaonic atoms. The CdZnTe detector was installed for the first time in an accelerator environment to perform tests on the background rejection capabilities, which were achieved by exploiting the SIDDHARTA-2 Luminosity Monitor. A spectrum with an 241Am source has been acquired, with beams circulating in the main rings, and peak resolutions of 6% at 60 keV and of 2.2% at 511 keV have been achieved. The background suppression factor, which turned out to be of the order of ≃ 10 5 - 6 , opens the possibility to plan for future kaonic atom measurements with CdZnTe detectors

Relationship between spatial ability, visuospatial working memory and self-assessed spatial orientation ability: a study in older adults

This paper describes some novel spatial tasks and questionnaires designed to assess spatial and orientation abilities. The new tasks and questionnaires were administered to a sample of 90 older adults (41 males, age range 57–90), along with some other tests of spatial ability (Minnesota Paper Form Board, Mental Rotations Test, and Embedded Figures Test) and tests of visuospatial working memory (Corsi’s Block Test and Visual Pattern Test). The internal reliability of the new tasks and questionnaires was analyzed, as well as their relationship with the spatial and working memory tests. The results showed that the new spatial tasks are reliable, correlate with working memory and spatial ability tests and, compared with the latters, show stronger correlations with the self-report questionnaires referring to orientation abilities. A model was also tested (with reference to Allen et al. in Intelligence 22:327–355, 1996) in which the new tasks were assumed to relate to spatial ability and predict orientation abilities as assessed by the self-report measures

The hypergenome in inheritance and development

Both theory and experimentation suggest that during development, the DNA of multicellular organisms, recognized as graced with a lifelong intrinsic stability, is instead target of several modifications (point mutations, larger structural variations, epigenetic marks) and partner of complex interactions with non-DNA moieties (RNAs and proteins). Some of these modifications probably affect a fraction of the genome larger than standard point mutations and are more likely to respond to environmental cues. Thus, the traditional concepts of gene and genome need revision: the structure serving as depository of the overall bioinformation of the cell is more dynamic and less homogeneous than allowed for by the Central Dogma, since in addition to DNA, it includes also RNA and proteins. Each of the individual contributors as well as their stoichiometry undergo modifications. Compared to the traditional unidimensional and static genome, the resulting dynamic aggregate could be more competent to cope with different regulatory requirements: its structural variations may respond to unscheduled macro- and micro-environmental stresses as well as to scheduled genetic programs. A detailed assessment of these variations in time and space should provide a basis for a deeper comprehension of the phenotypic changes punctuating the organism's physiopathological development, aging and transgenerational transmission. The variations of such information storage-delivery system may interest also the germ cells: the inheritance of parental traits and hence their evolutionary transmission would be affected. For the structure featuring all these properties, we propose the term 'hypergenome' to underscore the dynamic composition of a complex nucleoprotein responsive to both unpredictable environmental stimuli and internal built-in program

Time and event based prospective remembering in migraine patients.

Realizing future actions requires that a person remembers what has to be executed and when it has to be carried out, both in the case of time and event based actions. Ten patients suffering from migraine were included in this pilot study. While performing traditional cognitive assessment patients were asked to execute time and event based PM tasks respectively at 1\u2019 and 5\u2019 time interval. An extended neuropsychological battery was administered to all subjects in two sessions in order to assess the role of attentional, executive and memory abilities of the participants. From this pilot study a specific pattern seems to arise, where migraine patients show marked difficulties on time based PM tasks. Prospective remembering difficulties appearing in this group of patients seem to rely on self-initiating processes and executive control of attentional resources rather than on memory deficits