[Self-limited focal epilepsies of childhood]. / Epilepsias focales autolimitadas de la infancia.

Self-limited Focal Epilepsies of Childhood (SELFEs) are the most prevalent electroclinical syndromes in pediatric age, whose typical evolution, with age-dependent onset and remission, has allowed the ILAE Nosology and Definitions Working Group (2022) to define them as "Selflimited Focal Epilepsies of Childhood", thus establishing alert and exclusion criteria to standardize their diagnosis. These syndromes include: Self-limited Epilepsy with Centrotemporal Spikes (previously Rolandic Epilepsy), Self-limited Epilepsy with Autonomic Seizures (previously Panayiotopoulos Syndrome), Childhood Occipital Visual Epilepsy, (previously Gastaut Syndrome), and Photosensitive Occipital Lobe Epilepsy. Using the term "benign" to refer to them is no longer recommended, as this would ignore the comorbidities some individuals suffer. Also, the term "idiopathic" is now only used to refer to the syndromes classified as Idiopathic Generalized Epilepsies.

Las Epilepsias Focales Autolimitadas de la Infancia (SELFEs - siglas en inglés) son los síndromes electroclínicos más prevalentes en edad pediátrica, cuya evolución típica, con inicio y remisión dependientes de la edad, ha permitido que el Grupo de Trabajo de Nosología y Definiciones de la ILAE (2022) las defina como "Epilepsias focales autolimitadas de la infancia", estableciendo así, criterios de alerta y exclusión para estandarizar su diagnóstico. Dentro de estos síndromes se incluyen: la Epilepsia Autolimitada con Espigas Centrotemporales (previamente Epilepsia Rolándica), Epilepsia Autolimitada con Crisis Autonómicas. (previamente Síndrome de Panayiotopoulos), Epilepsia Visual Occipital Infantil (previamente Síndrome de Gastaut), y Epilepsia Fotosensible del Lóbulo occipital. Ya no se recomienda utilizar el término "benignas" para referirse a ellas, ya que esto haría caso omiso de las comorbilidades que padecen algunos individuos. Asimismo, el término "idiopático" sólo se utiliza ahora para denominar a los síndromes clasificados como Epilepsias Generalizadas Idiopáticas.

Laboratory metabolic evolution improves acetate tolerance and growth on acetate of ethanologenic Escherichia coli under non-aerated conditions in glucose-mineral medium.

In this work, Escherichia coli MG1655 was engineered to produce ethanol and evolved in a laboratory process to obtain an acetate tolerant strain called MS04 (E. coli MG1655: ΔpflB, ΔadhE, ΔfrdA, ΔxylFGH, ΔldhA, PpflB::pdc ( Zm ) -adhB ( Zm ), evolved). The growth and ethanol production kinetics of strain MS04 were determined in mineral medium, mainly under non-aerated conditions, supplemented with glucose in the presence of different concentrations of sodium acetate at pH 7.0 and at different values of acid pH and a constant concentration of sodium acetate (2 g/l). Results revealed an increase in the specific growth rate, cell mass formation, and ethanol volumetric productivity at moderate concentrations of sodium acetate (2-10 g/l), in addition to a high tolerance to acetate because it was able to grow and produce a high yield of ethanol in the presence of up to 40 g/l of sodium acetate. Genomic analysis of the ΔpflB evolved strain identified that a chromosomal deletion of 27.3 kb generates the improved growth and acetate tolerance in MG1655 ΔpflB derivative strains. This deletion comprises genes related to the respiration of nitrate, repair of alkylated DNA and synthesis of the ompC gene coding for porin C, cytochromes C, thiamine, and colonic acid. Strain MS04 is advantageous for the production of ethanol from hemicellulosic hydrolysates that contain acetate.

Structure, morphology, adhesion and in vitro biological evaluation of antibacterial multi-layer HA-Ag/SiO2/TiN/Ti coatings obtained by RF magnetron sputtering for biomedical applications.

Biocompatible and antibacterial multi-layer coatings of hydroxyapatite (HA)-Ag/SiO2/TiN/Ti were obtained on the Ti-6Al-4V alloy, by means of the magnetron sputtering technique. During characterization of the coatings, the chemical composition was evaluated by energy dispersive X-ray spectroscopy and the phase analysis was carried out by X-ray diffraction. The morphology of the coatings was observed by field emission scanning electron microscopy, while transmission electron microscopy was used to appreciate their structure. The adhesion of the coatings to the substrate was evaluated by micro scratch test. The in vitro biological response was evaluated in terms of cytotoxicity, adhesion and differentiation of mouse mesenchymal stem cells, as well as adhesion and bacterial viability of Staphylococcus aureus strain. Through the compositional study carried out, the deposition of the HA phase was verified, with a Ca/P ratio close to 1.67 and the characteristic diffraction peaks of this compound. The structural study of the coatings evidenced the obtention of multi-layer architectures. The use of an intermediate SiO2/TiN/Ti trilayer was found to improve adhesion between HA-Ag and the substrate by 84%. Finally, the in vitro biological tests carried out indicated a potentially non-toxic character in the coatings. Additionally, an antibacterial effect was registered at low concentrations of Ag (<0.25 mg/L).

Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin.

A gene for somatostatin, a mammalian peptide (14 amino acid residues) hormone, was synthesized by chemical methods. This gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pBR322. Transformation of E. coli with the chimeric plasmid DNA led to the synthesis of a polypeptide including the sequence of amino acids corresponding to somatostatin. In vitro, active somatostatin was specifically cleaved from the large chimeric protein by treatment with cyanogen bromide. This represents the first synthesis of a functional polypeptide product from a gene of chemically synthesized origin.

The role of penicillin amidases in nature and in industry.

Penicillin amidase (PA) is the enzyme used commercially for the production of semisynthetic penicillins. During the past decade, a detailed picture of the structure and regulation of the gene encoding this enzyme has emerged, revealing a variety of interesting features that are unique among microorganisms. Clues to the biological role of this enzyme have been provided, as well as new strategies for the commercial production and utilization of PA.

Epilepsias focales autolimitadas de la infancia / Self-limited Focal Epilepsies of Childhood

Resumen Las Epilepsias Focales Autolimitadas de la Infancia (SELFEs - siglas en inglés) son los síndromes electroclíni cos más prevalentes en edad pediátrica, cuya evolución típica, con inicio y remisión dependientes de la edad, ha permitido que el Grupo de Trabajo de Nosología y Definiciones de la ILAE (2022) las defina como "Epilep sias focales autolimitadas de la infancia", estableciendo así, criterios de alerta y exclusión para estandarizar su diagnóstico. Dentro de estos síndromes se incluyen: la Epilepsia Autolimitada con Espigas Centrotemporales (previamente Epilepsia Rolándica), Epilepsia Autolimi tada con Crisis Autonómicas. (previamente Síndrome de Panayiotopoulos), Epilepsia Visual Occipital Infantil (previamente Síndrome de Gastaut), y Epilepsia Fotosen sible del Lóbulo occipital. Ya no se recomienda utilizar el término "benignas" para referirse a ellas, ya que esto haría caso omiso de las comorbilidades que padecen algunos individuos. Asimismo, el término "idiopático" sólo se utiliza ahora para denominar a los síndromes clasificados como Epilepsias Generalizadas Idiopáticas.

Abstract Self-limited Focal Epilepsies of Childhood (SELFEs) are the most prevalent electroclinical syndromes in pe diatric age, whose typical evolution, with age-dependent onset and remission, has allowed the ILAE Nosology and Definitions Working Group (2022) to define them as "Self-limited Focal Epilepsies of Childhood", thus establishing alert and exclusion criteria to standardize their diagno sis. These syndromes include: Self-limited Epilepsy with Centrotemporal Spikes (previously Rolandic Epilepsy), Self-limited Epilepsy with Autonomic Seizures (previ ously Panayiotopoulos Syndrome), Childhood Occipital Visual Epilepsy, (previously Gastaut Syndrome), and Photosensitive Occipital Lobe Epilepsy. Using the term "benign" to refer to them is no longer recommended, as this would ignore the comorbidities some individuals suffer. Also, the term "idiopathic" is now only used to refer to the syndromes classified as Idiopathic General ized Epilepsies.

Pathway engineering for the production of aromatic compounds in Escherichia coli.

Glucose is the preferred substrate for certain fermentation processes. During its internalization and concomitant formation of glucose-6-phosphate through the glucose phosphotransferase system (PTS), one molecule of phosphoenolpyruvate (PEP) is consumed. Together with erythrose 4-phosphate (E4P), PEP is condensed to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP), the first intermediate of the common segment of the aromatic pathway. From this metabolic route, several commercially important aromatic compounds can be obtained. We have selected Escherichia coli mutants that can transport glucose efficiently by a non-PTS uptake system. In theory, this process should increase the availability of PEP for other biosynthetic reactions. Using these mutants, in a background where the DAHP synthase (the enzyme that catalyzes the condensation of PEP and E4P into DAHP) was amplified, we were able to show that at least some of the PEP saved during glucose transport, can be redirected into the aromatic pathway. This increased carbon commitment to the aromatic pathway was enhanced still further upon amplification of the E. coli tktA gene that encodes for a transketolase involved in the biosynthesis of E4P.

Selection for Hyoscyamine and Cinnamoyl Putrescine Overproduction in Cell and Root Cultures of Hyoscyamus muticus.

Hairy root cultures of Hyoscyamus muticus have been shown to produce stable levels of tropane alkaloids comparable to those found in whole plants. In contrast, cell cultures of this and other solanaceous species produce only trace amounts of alkaloids but can be used for selection of metabolic variants. We have taken advantage of both systems and the ability to convert between them in vitro in an effort to select for increased production of the tropane alkaloid hyoscyamine. Hairy roots were converted into cell suspensions by addition of 1 mg/L 2,4-dichlorophenoxyacetic acid to Murashige-Skoog medium (T. Murashige and F. Skoog [1962] Physiol Plant 15: 473-497) and screened for resistance to the amino acid analog p-fluorophenylalanine (PFP). Cells that could grow in media containing 400 [mu]M PFP were selected and cloned from single cells. The resistant cells accumulated high levels of cinnamoyl putrescines, which share the same biosynthetic precursors as hyoscyamine. Hairy root cultures were regenerated from both PFP-sensitive and PFP-resistant cells by removing 2,4-dichlorophenoxyacetic acid from the medium. Resistance to PFP continued to be expressed in regenerated roots. Higher levels of hyoscyamine were found in hairy roots regenerated from PFP-resistant cells than were found in controls. We suggest that the precursors overproduced by the PFP-resistant cells can be diverted into the hyoscyamine pathway upon the regeneration of root cultures.

[Knowledge level and professional attitudes to the Living Will: Differences between different professionals and provinces in the same autonomous community]. / Grado de conocimiento y actitudes de los profesionales ante el Documento de Voluntades Anticipadas: diferencias entre distintos profesionales y provincias de una misma autonomía.

OBJECTIVES: Primary: To determine the differences, by occupational category and province, in the knowledge and attitudes of health professionals on the Living Wills Document (LWD) in 4 Andalusian provinces: Cordoba, Jaen, Cadiz, and Granada. Secondary: To determine the number of documents prepared in these areas and the number consulted in terminal situations. MATERIAL AND METHODS: Descriptive observational multicenter study, with 17 health areas in 4 Andalusian provinces. TARGET POPULATION: Family doctors, nurses and social workers of the areas studied (n=340). Interventions Validated self-administered questionnaire about advance directives. Descriptive and bivariate (×2) analysis of data was performed. RESULTS: Mean age 46±8.8 years, 53.2% women. Physicians 56.1%, nurses 41.1%, social workers 2.6%. The mean score (0-10) of their knowledge was 5.42±2.41, with 78.4% believing that LWD are regulated in Andalusia (provinces differences, P=.001). More than one-third (36.7%) had read the document (differences by occupation, P=.001). The mean score on the advantage of preparing a LWD for the patient was 8.27±2.16 (significant differences between provinces P=.02). Mean score about the practitioner would respect the wishes of a patient in a LWD was 9.14±1.64 (significant difference between provinces P=.03). The mean score of the question about expressing the desires of the professional on preparing their LWD in the following year was 4.85±3.74 (P=.02). CONCLUSIONS: There are different behaviors between professions on reading the LWD. There are differences between provinces in the following aspects: whether the documents are regulated, whether the professionals prepare the LWD, and whether the professionals respects the provisions of the LWD.

[Characteristics of smoking, nicotine dependence and motivation for change in specialists training in health sciences (residents) in Andalusia (Spain)]. / Características del consumo de tabaco, dependencia y motivación para el cambio de los especialistas internos residentes de Andalucía (España).

OBJECTIVE: As there are few studies on the smoking habits of specialists training in health sciences (residents), it is of interest to determine the prevalence of smoking, nicotine dependence and motivation for change, and their relationship with other variables (personal, work and consumption of other drugs). MATERIAL AND METHODS: A multicentre, cross-sectional study using a questionnaire was conducted in 2012. All the residents who were studying in Teaching Health Centres in Andalusia (Spain) completed a questionnaire, which was sent by e-mail, collecting: age, sex, specialty, country of origin, qualitative-quantitative consumption of tobacco, age of onset/cessation, Fagerström test and stage of change (Proschaka). RESULTS: A total of 2667 residents (63% of total) completed the questionnaire. The mean age was 29.1 years (± 5.2), 69% female, 89% Spanish, and 86% physicians. Of the 17% who smoked (daily pattern-47%, intermittently-41%, related to leisure-3%), starting at 17.4 years (±3.5) and mean of 7.5 cigarettes per day (±7.1), higher medical specialties (P=.067 ANOVA), and in men (P=.074, Student-t). More than three-quarters (82%) had a low nicotine dependence, being higher in hospital medical specialties (P=.078 χ(2)). Of the total, 7% were former smokers, and 48% wanted to quit smoking (contemplation 38%, preparation 10%). In the multivariate analysis there was a link between smoking and alcohol consumption (OR 2.84) and illegal drugs (OR 3.57). There were no differences by age or country. CONCLUSIONS: The consumption of tobacco in residents is less than the general population, with a low dependence and better willingness to change. The period of specialised training is a good time to offer tobacco interventions.

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.

Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication.

The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328. This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981). In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.

Construction and characterization of new cloning vehicles. VII. Construction of plasmid pBR327par, a completely sequenced, stable derivative of pBR327 containing the par locus of pSC101.

In vitro recombinant DNA experiments, using plasmid pBR327 and a DNA fragment derived from plasmid pSC101 containing the par region, resulted in the construction of plasmid pBR327par. This new cloning vehicle has all the cloning properties of the parental plasmid, and is more stable than pBR327. Since the nucleotide sequence of the par region has been determined, this new vector is completely characterized. Some features of the sequence with possible functional significance are discussed.

Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325.

In vitro recombinant DNA experiments involving restriction endonuclease fragments derived from the plasmids pBR322 and pBR325 resulted in the construction of two new cloning vehicles. One of these plasmids, designated pBR327, was obtained after an EcoRII partial digestion of pBR322. The plasmid pBR327 confers resistance to tetracycline and ampicillin, contains 3273 base pairs (bp) and therefore is 1089 bp smaller than pBR322. The other newly constructed vector, which has been designated pBR328, confers resistance to chloramphenicol as well as the two former antibiotics. This plasmid contains unique HindIII, BamHI and SalI sites in the tetracycline resistance gene, unique PvuI and PstI sites in the ampicillin resistance gene and unique EcoRI, PvuII and BalI sites in the chloramphenicol resistance gene. The pBR328 plasmid contains approx. 4900 bp.

Structural organization of the genes that encode two glutamate synthase subunits of Escherichia coli.

Plasmid pRSP20, a recombinant plasmid isolated from the Clarke-Carbon Escherichia coli gene bank, contains the two genes coding for the subunits of glutamate synthase (GOGAT). We have constructed several derivatives of pRSP20, and analyzed the direction of transcription and genetic organization of these genes. Unexpectedly, we have found that although they are tightly linked and are transcribed in the same direction, each of them has its own promoter.

Plasmid pBRINT: a vector for chromosomal insertion of cloned DNA.

Plasmid pBRINT is a pBR322 derivative [Bolivar et al., Gene 2 (1977) 95-113; Balbás et al., Gene 50 (1986) 3-40] that allows the insertion and replacement of DNA sequences into the Escherichia coli chromosome by homologous recombination. This method uses the inability of E. coli strain ATCC47002 (JC7623) to replicate covalently closed circular (ccc) pBR322-derived plasmids, and the convenience of XGal+IPTG screening for recombinants. The vector also contains suitable selection markers (Ap and Cm), as well as a multiple cloning site (MCS) derived from the pUC vectors [Yanisch-Perron et al., Gene 33 (1985) 103-119] to facilitate cloning. A simple PCR scheme was developed to scan for DNA insertions into the bacterial chromosome. Once introduced into the chromosome, the inserted DNA sequences can be transferred to other strains by bacteriophage P1-mediated transduction.

Deletion of a repetitive extragenic palindromic (REP) sequence downstream from the structural gene of Escherichia coli glutamate dehydrogenase affects the stability of its mRNA.

A deletion that removes one repetitive extragenic palindromic sequence downstream of the structural gene of Escherichia coli glutamate dehydrogenase, reduces twofold the half-life of gdhA mRNA.

Identification of a functional promoter for the Escherichia coli gdhA gene and its regulation.

Glutamate dehydrogenase (GDH) catalyzes the synthesis of L-glutamate from 2-oxoglutarate and ammonia. The complete nucleotide sequence of the Escherichia coli gdhA gene, as well as its 5' and 3' flanking regions have been previously reported [Valle et al., Gene 23 (1983) 199-209; 27 (1984) 193-199]. In this paper we present data on the GDH specific activities using both excess and limiting concentrations of ammonia as nitrogen sources. Evidence is presented on the regulation of the mRNA levels for this enzyme by the ammonia concentration in the growth medium. We have identified a single and apparently invariant transcript for several metabolic growth conditions. We also report the identification of a functional promoter and the corresponding transcription start point under several growth conditions. Finally, possible regulatory sequences located at the 5' flanking region of the gdhA gene are discussed.

Repetitive extragenic palindromic (REP) sequences in the Escherichia coli gdhA gene.

Deletions of the 3' flanking DNA region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, have been produced on a plasmid that carries the complete gdhA gene. Those deletions include part of the repetitive extragenic palindromic (REP) sequences proposed by Stern et al. [Cell 37 (1984) 1015-1026], as a novel and major feature of the bacterial genome. The effect of these deletions on the final GDH level in the cell, has been determined. A broader compilation, analysis and alternative functions of the REP sequences, is also presented.

A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome.

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211-213]. A family of related plasmids containing different antibiotic-resistance markers (CmR or GmR or KmR) and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.