A novel method for DNA delivery into bacteria using cationic copolymers.

Amphiphilic copolymers have a wide variety of medical and biotechnological applications, including DNA transfection in eukaryotic cells. Still, no polymer-primed transfection of prokaryotic cells has been described. The reversible addition-fragmentation chain transfer (RAFT) polymer synthesis technique and the reversible deactivation radical polymerization variants allow the design of polymers with well-controlled molar mass, morphology, and hydrophilicity/hydrophobicity ratios. RAFT was used to synthesize two amphiphilic copolymers containing different ratios of the amphiphilic poly[2-(dimethyl-amino) ethyl methacrylate] and the hydrophobic poly [methyl methacrylate]. These copolymers bound to pUC-19 DNA and successfully transfected non-competent Escherichia coli DH5α, with transformation efficiency in the range of 103 colony-forming units per µg of plasmid DNA. These results demonstrate prokaryote transformation using polymers with controlled amphiphilic/hydrophobic ratios.

A novel method for DNA delivery into bacteria using cationic copolymers

Amphiphilic copolymers have a wide variety of medical and biotechnological applications, including DNA transfection in eukaryotic cells. Still, no polymer-primed transfection of prokaryotic cells has been described. The reversible addition-fragmentation chain transfer (RAFT) polymer synthesis technique and the reversible deactivation radical polymerization variants allow the design of polymers with well-controlled molar mass, morphology, and hydrophilicity/hydrophobicity ratios. RAFT was used to synthesize two amphiphilic copolymers containing different ratios of the amphiphilic poly[2-(dimethyl-amino) ethyl methacrylate] and the hydrophobic poly [methyl methacrylate]. These copolymers bound to pUC-19 DNA and successfully transfected non-competent Escherichia coli DH5α, with transformation efficiency in the range of 103 colony-forming units per µg of plasmid DNA. These results demonstrate prokaryote transformation using polymers with controlled amphiphilic/hydrophobic ratios.

Estudio de riesgo de recurrencia de trastorno del espectro autista (TEA) en hermanos de niños con diagnóstico TEA / Study on recurrence risk of autism spectrum disorders (ASD) in siblings of children diagnosed with ASD

El trastorno del espectro autista (TEA) es un trastorno del desarrollo, común de la niñez, con una fuerte predisposición genética y alta heredabilidad. El riesgo de recurrencia en hermanos oscila entre 10-20% y en caso de familias con dos o más niños afectados el riesgo de recurrencia aumenta hasta un 35%. Dentro de las pruebas complementarias para el diagnóstico, el gold standard es la escala ADOS, existe además una prueba de pesquisa, el M-CHAT. Objetivo: evaluar riesgo de recurrencia de TEA en hermanos menores de niños con diagnóstico de TEA. Materiales y Métodos: se realizó un estudio de tipo transversal, observacional y descriptivo. Fueron estudiados niños entre 18-36 meses, hermanos de pacientes con diagnóstico de TEA. La evaluación del desarrollo se realizó utilizando: Escalas CAT/CLAMS, M-CHAT y ADOS 2. Resultados: se estudiaron 39 hermanos. 25 fueron varones y 14 fueron mujeres. Se identificaron 5 niños con diagnóstico de TEA, por lo que el riesgo de recurrencia en la población estudiada fue de 13%, con una relación varón/mujer de 4/1. Del resto de la población estudiada, 13% reunieron criterios para fenotipo ampliado del autismo (Broader Autism Phenotype ­BAP­ en su sigla en inglés), 31% presentaron retraso del lenguaje(RL) y 7%retraso global del desarrollo (RGD). Solo el 36% presentó desarrollo típico. Conclusión: Los hermanos de niños afectados representan un grupo de riesgo para problemas del desarrollo, que debe ser tenido en cuenta por los profesionales de la salud que siguen longitudinalmente a niños con diagnóstico confirmado de TEA (AU)

Autism spectrum disorder (ASD) is a developmental disorder that is common in childhood with a strong genetic predisposition and high heritability. The risk of recurrence in siblings is found to be between 10-20% and in families with two or more affected children recurrence risk is as high as 35%. Among the complementary diagnostic tests, the gold standard is the ADOS scale, and additionally the M-CHAT screening test. Objective: To evaluate the recurrence risk of ASD in younger siblings of children diagnosed with ASD. Material and Methods: A cross-sectional, observational, descriptive study was conducted. Children between 18- 36 months of age, siblings of children diagnosed with ASD were studied. Development was assessed using the CAT/CLAMS, MCHAT, and ADOS 2 scales. Results: 39 siblings were studied; 25 were male and 14 female. Five children with ASD were identified, accounting for a recurrence risk of 13% in the study population and a male/female ratio of 4/1. Of the remaining children, 13% met the criteria for the broader autism phenotype (BAP), 31% had language delay (LD), and 7% global developmental delay (GDD). Only 36% had normal development. Conclusion: Siblings of affected children are at risk for developmental disorders that should be taken into account by health professionals that ongitudinally follow children with a confirmed diagnosis of ASD (AU)

Current smoking is associated with lower seminal vesicles and ejaculate volume, despite higher testosterone levels, in male subjects of infertile couples.

STUDY QUESTION: What is the impact of smoking behaviour on seminal, hormonal and male genital tract ultrasound parameters in subjects seeking medical care for couple infertility? STUDY ANSWER: In males of infertile couples, current smokers (CS), when compared with non-smokers, show lower ejaculate and ultrasound-derived seminal vesicles (SV) volume, despite higher testosterone levels. WHAT IS KNOWN ALREADY: Data on the effects of smoking on male fertility are conflicting. A correlation between smoking and reduced semen parameters has been reported, however, with a high heterogeneity among studies. An association between smoking behaviour and higher testosterone levels in men has been described in several, but not all, the previous studies. No study has systematically evaluated the impact of smoking on the male genital tract ultrasound characteristics. STUDY DESIGN, SIZE AND DURATION: Retrospective cross-sectional analysis of a consecutive series of 426 subjects seeking medical care for couple infertility from January 2010 to July 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: From the entire cohort, 394 men (age 36.0 ± 8.0 years) free of genetic abnormalities were selected. All subjects underwent a complete andrological and physical examination, biochemical and hormonal assessment, scrotal and transrectal colour-Doppler ultrasound and semen analysis (including seminal interleukin-8 levels, sIL-8) within the same day. MAIN RESULTS AND THE ROLE OF CHANCE: Among the patients evaluated, 229 were never smokers (NS), 56 past smokers (PS) and 109 CS. When CS were compared with the rest of the sample (non-smokers, NS + PS), in a multivariate model (analysis of covariance, ANCOVA) adjusted for age, lifestyle (including alcohol, cannabis and physical activity), BMI and sex hormone-binding globulin, significantly higher androgen (total testosterone, P = 0.001; calculated free testosterone, P < 0.005) and lower FSH (P < 0.05) levels were observed in CS. However, when total testosterone was also included in the multivariate model as a further covariate, the difference in FSH levels was not confirmed. In a similar model, a lower ejaculate volume (P < 0.01) and a higher prevalence of normal sperm morphology (P < 0.02) were also detected in CS in comparison with the rest of the sample. However, when total testosterone was also included in the multivariate model as a further covariate, only the difference in ejaculate volume between CS and non-smokers was confirmed (-0.61 ± 0.23 ml, P < 0.01). Finally, CS showed lower total SV volume, before and after ejaculation, even after adjusting for confounders (P = 0.02 and <0.01, respectively). Similar results were observed when the reported number of cigarettes smoked or the number of pack-years was considered separately. LIMITATIONS, REASONS FOR CAUTION: The present results are derived from patients consulting an Andrology Clinic for couple infertility, who could have different characteristics from the general male population or males consulting general practitioners for reasons other than couple infertility. In addition, we did not have a true control group composed of age-matched, apparently healthy, fertile men, and therefore true normative data of sonographic parameters cannot be inferred. Due to the cross-sectional nature of our study, neither a causality hypothesis nor mechanistic models can be drawn. Finally, this is a retrospective study, and further prospective studies are required. WIDER IMPLICATIONS OF THE FINDINGS: We report an apparent paradox in CS: lower SV volume despite higher testosterone levels. Our data suggest that smoking may negatively affect SV volume in an independent manner, as the difference between CS and non-smokers retained significance after adjusting for confounders including testosterone. This is the first study reporting such ultrasound evidence. How this new smoking-related alteration, along with low semen volume, impacts male fertility needs to be addressed by further studies. STUDY FUNDING/COMPETING INTERESTS: No funding was received for the study. None of the authors have any conflict of interest to declare.

The 17ß-hydroxysteroid dehydrogenase type 3 deficiency: a case report of an 18-year patient and review of the literature.

UNLABELLED: Deficiency of 17ß-hydroxysteroid dehydrogenase type 3 (17ßHSD3), an enzyme converting androstenedione (A) to testosterone (T), is a rare cause of autosomal recessive 46,XY disorder of sexual development (DSD). A 18-years phenotypically female patient from southern Italy presented with primary amenorrhea. She had deep voice, macrocephaly, enlarged and bulbous nasal tip, macrostomia, facial acne, breast asymmetry, hypoplasia of the first finger of right hand, proximal implant of the fifth metatarsus bilaterally as well as an increased muscle mass and hirsutism, with hair distribution on face, neck, chest, abdomen, pubic region and on upper and lower limbs. Genital exam showed thickened labra majora with absence of labra minora and a blind-ending pseudo-vagina with clitoris enlargement. Karyotype analysis showed a male genotype (46,XY). Hormonal evaluation showed decreased T (188 ng/dL-6.5 nmol/L) and increased A (10 ng/mL-34,96 nmol/L), considering male reference ranges, resulting in a decreased T/A ratio (0,186). MRI identified testicles in inguinal regions. Human Chorionic Gonadotropin test showed T/A ratio permanently under 0,8. These evidences were suggestive of a 46,XY DSD due to 17ßHSD3 deficiency. An homozygous mutation (IVS3 -1 G>C or c.326-1G>C) of the 17ßHSD3 gene was discovered. Psychologist identified a well determined female gender identity. It was decided to proceed with gonadectomy and vaginal enlargement by use of dilatators. CONCLUSION: The case described represents a new case of DSD due to 17ßHSD3 deficiency. This patient, raised as a girl, is diagnosed in a very late stage. The identified mutation, previously reported only in Dutch and Brazilian population, is one of 27 presently known mutations of 17ßHSD3 gene and is never reported in Italian population.

Selective COX-1 inhibition: A therapeutic target to be reconsidered.

Since cyclooxygenase (COX) isozymes discovery, many papers and reviews have been published to describe the structural bases of COX inhibition, and to debate on the therapeutic and adverse effects of worldwide clinically used nonsteroidal anti-inflammatory drugs (NSAIDs), included COX-2 selective inhibitors (well known as Coxibs). COX-2 inhibition has been widely investigated, whereas the role of COX-1 in human pathophysiology is mostly not yet well ascertained. As time goes on, the cliché that the constitutively expressed isoform COX-1 is only involved in normal physiological functions, such as platelet aggregation, gastric mucosa protection and renal electrolyte homeostasis is going to be shattered. Low-dose aspirin, behaving as a preferential inhibitor of platelet COX-1, allowed to enlighten the role exerted by this isoenzyme in many mammalian cell types. This review would elucidate the most recent findings on selective COX-1 inhibition and their relevance to human pathology such as cancer, neuro-inflammation, cardioprotection, fever and pain. It would also focus on the design and development of new highly selective COX-1 inhibitors, useful tools in pharmacological studies aimed at gaining a deeper insight of the role of COX-1 in human health and disease. Among the traditional NSAIDs, other then aspirin and indomethacin, only few examples of selective COX-1 inhibitors (SC-560, FR122047, mofezolac, P6 and TFAP) have been so far identified. This review has also the scope to stimulate the development of novel drugs, which activity is COX-1 mediated.

General anaesthesia in a patient affected by Brugada syndrome.

This case report describes an asymptomatic patient with positive familiar anamnesis of Brugada syndrome (BrS) who elected to undergo surgery. The anaesthesiological technique using propofol, fentanyl, atracurium, air/oxygen did not induce any electrocardiographic alteration during the operation; the intraoperation use of a biphasic defibrillator was critical here. The cerebral state index and adhesive plaques connected with a biphasic defibrillator having PM capabilities allowed us to monitor the operation and continually assess the patient's cardiac stability. Afterwards, the patient was transferred to the intensive care unit and was monitored for 24 hours. This anesthesiological technique was performed in place of ARL, which the patient refused.

EphA/ephrin-A interactions during optic nerve regeneration: restoration of topography and regulation of ephrin-A2 expression.

During visual system development, interactions between Eph tyrosine kinase receptors and their ligands, the ephrins, guide retinal ganglion cell (RGC) axons to their topographic targets in the optic tectum. Here we show that Eph/ephrin interactions are also involved in restoring topography during RGC axon regeneration in goldfish. Following optic nerve crush, EphA/ephrin-A interactions were blocked by intracranial injections of recombinant Eph receptor (EphA3-AP) or phospho-inositol phospholipase-C. Topographic errors with multiple inputs to some tectal loci were detected electrophysiologically and increased projections to caudal tectum demonstrated by RT-97 immunohistochemistry. In EphA3-AP-injected fish, ephrin-A2-expressing cells in the retino-recipient tectal layers were reduced in number compared to controls and their distribution was no longer graded. The findings, supported by in vitro studies, implicate EphA/ephrin-A interactions in restoring precise topography and in regulating ephrin-A2 expression during regeneration.

Mechanisms of transcriptional activation of the col6a1 gene during Schwann cell differentiation.

A transgenic mouse line expressing the lacZ reporter under the control of a regulatory region of the col6a1 gene has been used to investigate differentiation of Schwann cells. The data suggest that: (1) activation of col6a1 gene transcription in the peripheral nervous system is part of the differentiation program of Schwann cells from neural crest cells stimulated by neuregulins; (2) once the Schwann cell precursors have acquired the competence of transcribing the col6a1 gene, transcriptional regulation becomes independent from neuregulins and is modulated by different mechanisms, including cell cycle; (3) activation of transgene expression after birth in sciatic nerves corresponds to the time of withdrawal of immature Schwann cells from the cell cycle and the beginning of their differentiation into myelinating Schwann cells.

Critical exponents of the Gross-Neveu model from the effective average action.

The phase transition of the Gross-Neveu model with N fermions is investigated by means of a nonperturbative evolution equation for the scale dependence of the effective average action. The critical exponents and scaling amplitudes are calculated for various values of N in d = 3. It is also explicitly verified that the Neveu-Yukawa model belongs to the same universality class as the Gross-Neveu model.

Analysis of transcription of the Col6a1 gene in a specific set of tissues suggests a new variant of enhancer region.

The region extending from -5.4 to -3.9 kilobase pairs from the transcription start site of the Col6a1 gene has been previously shown to contain sequences activating tissue-specific transcription in articular cartilage, intervertebral disks, subepidermal, and vibrissae mesenchyme and peripheral nervous system (Braghetta, P., Fabbro, C., Piccolo, S., Marvulli, D., Bonaldo, P., Volpin, D., and Bressan, G. M. (1996) J. Cell Biol. 135, 1163-1177). Analysis of expression of deletions of this region in transgenic mice has identified the 383-base pair fragment E-L as the most active sequence of the region. Linker-scanning mutagenesis analysis of segment E-J, which spans the 5' 245 base pairs of E-L and is sufficient for high frequency expression in articular cartilage, showed that all the mutations reduced transcription considerably, suggesting that the integrity of the entire cluster of elements is necessary for enhancer activity. Electrophoretic mobility shift assays with nuclear extracts derived from various sources showed that fragment E-J binds numerous transcription factors (at least 22). These factors are present in most cells, expressing and nonexpressing alpha1(VI) collagen mRNA, but in different relative proportions, and none of them appears to be cell type-specific. Several lines of evidence indicate that sequence elements of the enhancer may have different functional roles in various cells. The data configure the -5.4/-3.9 region of the Col6a1 gene as a new type of tissue-specific enhancer, characterized by a variety of tissues supporting its activation and by the dependence of its function only on ubiquitous transcription factors. This type of enhancer is postulated to be particularly important for genes such as those of the extracellular matrix, which are often expressed with broad tissue specificity.

Tissue-specific expression of promoter regions of the alpha1(VI) collagen gene in cell cultures and transgenic mice.

Cis-acting regions regulating transcription of the alpha1(VI) collagen chain have been investigated in vitro by transfection of promoter-CAT (where CAT is chloramphenicol acetyltransferase) constructs in different types of cultured cells and in vivo in transgenic mice carrying the same CAT constructs or minigenes derived from the fusion of genomic and cDNA sequences in which small deletions of the collagenous domain had been engineered. 215 bp of 5'-flanking sequence showed promoter activity in vitro, yet were not expressed in any tissue of six transgenic lines, indicating that this fragment contains the basal promoter, but not activator sequences. Constructs with 0.6 and 1.4 kb of the 5'-flanking region produced significantly higher CAT activity in transfected cells and were expressed in tissues of about 30% of transgenic lines. Although CAT activity was totally unrelated to the pattern of expression of the alpha1(VI) mRNA, these results suggest the presence of an activator(s) between -0.2 and -0.6 kb from the transcription start site. When the promoter size was increased to 5.4 or 6.5 kb, CAT activity was stimulated severalfold relative to the construct p1.4CAT and p4.0CAT in NIH3T3 fibroblasts and chick embryo chondroblasts. This stimulation was, however, not observed in C2C12 myoblasts. Transgenic mice generated with 6.5CAT construct or minigenes, containing 6.2 kb of promoter, exhibited very high levels of expression, which was similar to the relative amount alpha1(VI) mRNA in the majority of tissues, with the exception of lung, adrenal gland and uterus. CAT activity in tissues was 100-1000-fold higher than that measured in transgenic mice with shorter promoter (0.6 or 1.4 kb). Since expression of minigenes was determined by RNase protection assay, the levels of mRNA per transgene copy were compared to those of the chromosomal gene and found to be always less than one quarter. These data suggest that the region -4.0/-5.4 contains an important activator(s) sequence which induces transcription in several, but not all, type VI collagen-producing tissues. Finally, analysis with the longest promoter fragment (7.5 kb) revealed a complex effect of the region -6.5/-7.5 on alpha1(VI) chain transcription. The sequence was inhibitory in NIH3T3 cells, indifferent in myoblasts and activating in chondroblasts in vitro, whereas transgenic animals generated with 7.5CAT construct produced a pattern of expression comparable to that of 6.5CAT and minigenes. During postnatal development transcription from both the endogenous gene and the transgenes decreased. However, the ratio of transgene/chromosomal gene expression was not constant, but varied in a way dependent on the tissue. This observation suggests that the fragment studied contains key sequences for the age-dependent regulation of the alpha1(VI) gene. No phenotypic alterations were induced by the presence of mutations in the minigenes.

Transcriptional activation of the alpha 1(VI) collagen gene during myoblast differentiation is mediated by multiple GA boxes.

During differentiation of ClC12 myoblasts in vitro, expression of alpha 1(VI) collagen mRNA was transiently stimulated severalfold. Promoter assays on cells transfected with chloramphenicol acetyltransferase (CAT) chimeric constructs have identified a region of the alpha 1(VI) a collagen promoter that increases CAT activity about 8-fold during differentiation. The region, which overlaps with transcription initiation sites, was shown to contain three protected segments (A, B, and C) in DNase I footprinting assays. The contact points between nuclear factors and the protected segments were determined by methylation interference assay and included the sequence GGGAGGG (GA box) in all segments. Experiments in which CAT constructs were cotransfected with double-stranded oligonucleotides containing the GA box suggested that this motif was necessary for induction. Transfections with deletion constructs of the natural promoter and with minipromoters made of three copies of A, B, or C showed that the elements have inducing activity and that elements C and, to a lower extent, B are stimulatory for basal transcription, whereas the contribution of A in this process is limited. Electrophoretic mobility shift assays with nuclear extracts from C2C12 cells indicated that the three GA box-containing elements bound several transcription factors, including Sp1. Comparison of the properties of the bands shifted under different experimental conditions (presence of 10 mM EDTA, heating of the nuclear extracts, addition of different concentrations of competitor oligonucleotides) established that A, B, and C probes form nine, eight and five main retarded complexes, respectively, and indicated that nuclear factors binding to C and B are subsets of proteins binding to A. UV cross-linking assays identified several peptides (seven with probe A, six with B, And five with C) in the range of 150-32 kDa. Comparison of the gel retardation pattern obtained with nuclear extracts from proliferating and differentiating cells revealed a particular increased intensity of two retarded bands. The data establish that multiple GA boxes mediate induction of the alpha 1(VI) collagen promoter during myoblast differentiation and suggest the attractive hypothesis that the effect may be related to variations of expression of transcription factors binding to these motifs.

[Surgical treatment of incessant atrial tachycardia: description of a case]. / Trattamento chirurgico della tachicardia atriale incessante: descrizione di un caso.

A 50 year old woman with no evidence of structural heart disease was referred for ectopic incessant repetitive atrial tachycardia uncontrolled by medical therapy. Intracavitary and transesophageal simultaneous recordings revealed the earliest atrial electrical activity to be located in the left atrium. Intraoperative electrophysiologic mapping demonstrated that the site of earliest atrial activation was in a small diverticulum of the left atrial appendage. Excision of the appendage and isolation of left atrium was carried out with restoration of sinus rhythm. The patient was arrhythmia-free till 24 months later. Surgical treatment appears to be an effective therapeutic option for drug-resistant ectopic atrial tachycardia.

[Double ventricular response to a single atrial stimulation caused by simultaneous anterograde conduction through two atrioventricular nodal pathways]. / Duplice risposta ventricolare ad un singolo stimolo atriale dovuta a conduzione anterograda simultanea in due vie del nodo A-V.

In a patient with atrioventricular nodal reentrant tachycardia, during programmed stimulation, an atrial extrastimulus induced a double ventricular response due to a single atrial depolarization, with simultaneous and delayed anterograde conduction through fast and slow pathways, and induced the tachycardia. Pacing-induced type I block involving both pathways put these pathways out of phase, so that the distal conduction system and the ventricle responded to both the fast and slow pathways anterograde impulses.

Computerized screening for appropriate dosing of renally eliminated medications.

A method of rapid screening for appropriateness of certain drug doses is described. The program extracts patient information from a hospital mainframe computer system, performs an estimation of creatinine clearance (CrCl), and prints a report of patients, drugs, doses, and CrCl for patients within a specified CrCl range.

Reactance theory and alcohol consumption laws: further confirmation among collegiate alcohol consumers.

By the late 1980s, the United States legal drinking age had increased to 21 years. Based on psychological reactance theory, one would predict that these law changes would cause underage collegiate consumers to drink more alcohol because of the belief that their behavioral freedom was being reduced. It was hypothesized that underage collegiate alcohol consumers (UC) would drink more than their legal-age peers (LC) if psychological reactance was a contributing factor to consumption, whereas no differences would be present between the UC and LC groups' usage of illicit drugs, as these had not been affected by recent law changes. To test this hypothesis, a sample of 2,142 college students from 10 midwestern postsecondary educational facilities responded to the Alcohol and Other Drug Use Needs Assessment Survey in the spring of 1990. Mann-Whitney U analyses revealed significant differences between groups on alcohol use measures, but no differences were present on illicit substance use measures. These results are interpreted as supporting reactance theory.

Paraneoplastic pemphigus appearing as bullous pemphigoid-like eruption after palliative radiation therapy.

Paraneoplastic pemphigus is a recently described disease in which patients have polymorphous skin lesions suggestive of both erythema multiforme major and pemphigus vulgaris in association with internal neoplasms, especially non-Hodgkin's lymphoma. These patients have characteristic autoantibodies that bind specific epidermal proteins. A Nikolsky-negative bullous pemphigoid-like eruption developed within the radiation therapy field in a 72-year-old man receiving palliative treatment for recurrent large cell lymphoma. The eruption rapidly progressed to a Nikolsky-positive bullous process more typical of pemphigus vulgaris with extensive involvement of respiratory epithelia. Despite aggressive treatment with high-dose corticosteroids and antibiotics, the patient rapidly succumbed. Results of immunofluorescence studies and autopsy findings confirmed the diagnosis of paraneoplastic pemphigus.