[A first pilot study on the neonatal screening of primary immunodeficiencies in Spain: TRECS and KRECS identify severe T- and B-cell lymphopenia]. / Primer estudio piloto en España sobre el cribado neonatal de las inmunodeficiencias primarias: TRECS y KRECS identifican linfopenias T y B graves.

INTRODUCTION: Early diagnosis of primary immunodeficiency such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) improves outcome of affected infants/children. The measurement of T-cell receptor excision circles (TRECS) and kappa-deleting recombination excision circles (KRECS) can identify neonates with severe T or B-cell lymphopenia. OBJECTIVES: To determine TRECS and KRECS levels from prospectively collected dried blood spot samples (DBS) and to correctly identify severe T and B-cell lymphopenia. MATERIAL AND METHODS: Determination of TRECS and KRECS by multiplex PCR from neonates born in two tertiary hospitals in Seville between February 2014 and May 2014. PCR cut-off levels: TRECS<15 copies/µl, KRECS<10 copies/µl, ACTB (ß-actin)>1000 copies/µl. Internal (XLA, ataxia telangiectasia) and external (SCID) controls were included. RESULTS: A total of 1068 out of 1088 neonates (mean GA 39 weeks (38-40) and BW 3238g (2930-3520) were enrolled in the study. Mean (median, min/max) copies/µl, were as follows: TRECS 145 (132, 8/503), KRECS 82 (71, 7/381), and ACTB 2838 (2763, 284/7710). Twenty samples (1.87%) were insufficient. Resampling was needed in one neonate (0.09%), subsequently giving a normal result. When using lower cut-offs (TRECS<8 and KRECS<4 copies/µl), all the samples tested were normal and the internal and external controls were correctly identified. CONCLUSION: This is the first prospective pilot study in Spain using TRECS/KRECS/ACTB-assay, describing the experience and applicability of this method to identify severe lymphopenias. The ideal cut-off remains to be established in our population. Quality of sampling, storage and preparation need to be further improved.

Differential expression pattern of microRNAs in CD4+ and CD19+ cells from asymptomatic patients with systemic lupus erythematosus.

OBJECTIVE: The aim of this study was to investigate the pattern of microRNA (miRNA) expression in CD19+ and CD4+ cells from asymptomatic patients with systemic lupus erythematosus (SLE). METHODS: A screening of the expression of 377 miRNAs was performed in human CD4+ and CD19+ cells isolated from the peripheral blood by using a TaqMan Human MicroRNA Array. Validation of differential expression pattern of those was performed using TaqMan assays in these cell populations obtained from a larger cohort of patients and controls. RESULTS: According to the screening assays, three miRNAs were differentially expressed (p value <0.1) in cell populations from both patients and controls: hsa-miR-143, hsa-miR-224 and hsa-miR-576-5p for CD4+ cells, and hsa-miR-10a, hsa-miR-31 and hsa-miR-345 for CD19+ cells. After validation, significant differences (p value <0.05) were confirmed only for hsa-miR-143 and hsa-miR-224 in CD4+ cells and for hsa-miR-10a and hsa-miR-345 in CD19+ cells. In all cases, the miRNAs were over expressed in SLE patients compared with healthy donors. CONCLUSIONS: Our results support a different pattern of miRNA expression in SLE patients.

Antimicrobial effectiveness of intracanal medicaments on Enterococcus faecalis: chlorhexidine versus octenidine.

AIM: To determine the viability of Enterococcus faecalis in infected human root dentine in vitro after exposure to root canal medicaments based on chlorhexidine and octenidine. METHODOLOGY: Human root segments (n = 40) were infected with E. faecalis for 8 weeks. Root dentine samples (rd) collected at week 4 served as individual baseline values. At week 8, the root segments were randomly divided into four test groups (n = 10 each) for the placement of one of the following medicaments in the root canals: calcium hydroxide paste (CH), chlorhexidine gel (CHX-gel) (5.0%), chlorhexidine/gutta-percha points (CHX-GP) (active points(®) ; Roeko, Langenau, Germany) and octenidine gel (OCT-gel) (5.0%) followed by incubation for 4 weeks. The effect on E. faecalis viability was assessed by two fluorescent dyes (syto 9/propidium iodide) to determine the 'proportion of viable bacteria' (PVB%) and number of 'colony-forming units' (CFU). Mean values and 95% confidence intervals (CI) were calculated for PVB% and log CFU, and the difference between groups was established. RESULTS: Viable and dead bacterial cells were detected in all 'rd' samples at weeks 4 and 8. The treatment with CHX-gel, CHX-GP and OCT-gel resulted in significantly lower PVB% values with 15.4%, 3.5% and 0%, respectively. No growth (CFU) was recorded for these samples at week 12. When medicated by CH, the PVB% was increased without a corresponding change in CFUs. CONCLUSIONS: In contrast to calcium hydroxide, both CHX - and octenidine-based intracanal medicaments were effective in decreasing the viability of E. faecalis. OCT showed the most favourable results and may have potential as an endodontic medicament.

LexA-binding sequences in Gram-positive and cyanobacteria are closely related.

The lexA gene of the cyanobacterium Anabaena sp. strain PCC7120 has been cloned by PCR amplification with primers designed after TBLASTN analysis of its genome sequence using the Escherichia coli LexA sequence as a probe. After over-expression in E. coli and subsequent purification, footprinting experiments demonstrated that the Anabaena LexA protein binds to the sequence TAGTACTAATGTTCTA, which is found upstream of its own coding gene. Directed mutagenesis and sequence comparison of promoters of other Anabaena genes, as well as those of several cyanobacteria, allowed us to define the motif RGTACNNNDGTWCB as the LexA box in this bacterial phylum. Substitution of a single nucleotide in this motif present in the Anabena lexA promoter is sufficient to enable it to bind the Bacillus subtilis LexA protein. These data indicate that Cyanobacteria and Gram-positive bacteria are phylogenetically closely related.

The presence of glutamate dehydrogenase is a selective advantage for the Cyanobacterium synechocystis sp. strain PCC 6803 under nonexponential growth conditions.

The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 has two putative pathways for ammonium assimilation: the glutamine synthetase-glutamate synthase cycle, which is the main one and is finely regulated by the nitrogen source; and a high NADP-dependent glutamate dehydrogenase activity (NADP-GDH) whose contribution to glutamate synthesis is uncertain. To investigate the role of the latter, we used two engineered mutants, one lacking and another overproducing NADP-GDH. No major disturbances in the regulation of nitrogen-assimilating enzymes or in amino acids pools were detected in the null mutant, but phycobiline content, a sensitive indicator of the nutritional state of cyanobacterial cells, was significantly reduced, indicating that NADP-GDH plays an auxiliary role in ammonium assimilation. This effect was already prominent in the initial phase of growth, although differences in growth rate between the wild type and the mutants were observed at this stage only at low light intensities. However, the null mutant was unable to sustain growth at the late stage of the culture at the point when the wild type showed the maximum NADP-GDH activity, and died faster in ammonium-containing medium. Overexpression of NADP-GDH improved culture proliferation under moderate ammonium concentrations. Competition experiments between the wild type and the null mutant confirmed that the presence of NADP-GDH confers a selective advantage to Synechocystis sp. strain PCC 6803 in late stages of growth.