Structural Cues for Understanding eEF1A2 Moonlighting.

Spontaneous mutations in the EEF1A2 gene cause epilepsy and severe neurological disabilities in children. The crystal structure of eEF1A2 protein purified from rabbit skeletal muscle reveals a post-translationally modified dimer that provides information about the sites of interaction with numerous binding partners, including itself, and maps these mutations onto the dimer and tetramer interfaces. The spatial locations of the side chain carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is uniquely attached via an amide bond, define the anchoring points of eEF1A2 to cellular membranes and interorganellar membrane contact sites. Additional bioinformatic and molecular modeling results provide novel structural insight into the demonstrated binding of eEF1A2 to SH3 domains, the common MAPK docking groove, filamentous actin, and phosphatidylinositol-4 kinase IIIß. In this new light, the role of eEF1A2 as an ancient, multifaceted, and articulated G protein at the crossroads of autophagy, oncogenesis and viral replication appears very distant from the "canonical" one of delivering aminoacyl-tRNAs to the ribosome that has dominated the scene and much of the thinking for many decades.

Pemphigus foliaceus worsened by drugs in a patient with psoriasis, responding to adalimumab.

Pemphigus foliaceus is a blistering autoimmune disease related to the production of autoantibodies against desmoglein 1. We present a patient with psoriasis and pemphigus foliaceus aggravated by enalapril and amlodipine intake, with successful response of both conditions to adalimumab therapy.

El pénfigo foliáceo es una enfermedad autoinmune ampollosa debida a la producción de autoanticuerpos frente a la desmogleína 1. Presentamos el caso de un paciente con psoriasis y pénfigo foliáceo agravado por enalapril y amlodipino, con buena respuesta de ambas patologías a la terapia con adalimumab.Pemphigus foliaceus is a blistering autoimmune disease related to the production of autoantibodies against desmoglein 1. We present a patient with psoriasis and pemphigus foliaceus aggravated by enalapril and amlodipine intake, with successful response of both conditions to adalimumab therapy.

El pénfigo foliáceo es una enfermedad autoinmune ampollosa debida a la producción de autoanticuerpos frente a la desmogleína 1. Presentamos el caso de un paciente con psoriasis y pénfigo foliáceo agravado por enalapril y amlodipino, con buena respuesta de ambas patologías a la terapia con adalimumab.

Muckle-Wells Syndrome: A Case Report with an NLRP3 T348M Mutation.

Autoinflammatory syndromes are a recently described group of conditions caused by mutations in multiple genes that code for proteins of the innate immune system. Cryopyrin-associated periodic syndromes are autoinflammatory diseases comprising three clinically overlapping disorders: familial cold urticaria syndrome, Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease. MWS is characterized by a moderate phenotype with fever, rash, arthralgia, conjunctivitis, sensorineural deafness, and potentially life-threatening amyloidosis. We report a 5-year-old girl with MWS that manifested as a recurrent skin rash without fever episodes or intracranial hypertension with papilledema. Genetic analysis revealed a T348M mutation of the NLRPR 3 gene in the patient and her mother. She was successfully treated with the interleukin-1ß antagonist receptor anakinra.

Systemic contact dermatitis due to amethocaine following digital rectal examination.

Systemic contact dermatitis is a dermatitis that may occur in previously sensitized individuals when they are re-exposed to the allergen. Although many drugs have been implicated as a cause of systemic contact dermatitis, local anesthetics derived from caines have been rarely reported. We present a case of systemic contact dermatitis after a digital rectal examination with a urological lubricant containing amethocaine.

[Effects of chronic smoking on the superior mesenteric artery (SMA)]. / Efecto del tabaquismo crónico sobre la arteria mesentérica superior (AMS).

OBJECTIVE: Evaluate the effect of chronic smoking on peak systolic velocity (PSV) of the superior mesenteric artery, evaluated with pulsed Doppler, determining whether the longer the snuff consumption, the greater the increase in PSV. MATERIAL AND METHODS: This is an analytical cross-sectional study where patients who came to perform abdominal Doppler and had a history of chronic smoking were included. Additional measurements were performed on PSV superior mesenteric artery in 60 patients. Subjects were selected by the non-probability method, taking the total number of patients who presented. Once selected, patients performed pulsed Doppler, and a study and interpretation of these was carried out. RESULTS AND DISCUSSION: We studied 60 patients with a history of chronic smoking between the ages of 25 to 50 years with a median of 38.6±7.1%; chronicity of smoking was measured in years, with a mean of 17.1±8.4 (range 3-37) and PSV was divided into four ranges in cm/sec. CONCLUSIONS: Chronic smoking significantly increases the PSV of the superior mesenteric artery after 21 years of the onset of smoking.

Prevalence of viral hepatitis (B and C) serological markers in healthy working population.

INTRODUCTION AND OBJECTIVES: prevalence of viral hepatitis (B and C) changes geographically. Our aim was to determinate the prevalence of hepatitis B (HBV) and hepatitis C virus (HCV) serological markers in healthy working population and to describe the epidemiological characteristics associated to its presence. METHODS: blood samples and epidemiological data of 5,017 healthy workers from Murcia and Madrid were recorded prospectively. RESULTS: a total of 5,017 healthy volunteers participated. Mean age 39 ± 11 years, men predominance (73 %). Prevalence of serological markers of HCV and HBV was 0.6 % and 0.7 %. Age of patients with HCV antibody was significantly higher (43 + or - 9 years vs. 39 + or - 11 years; p = 0.03). We observed significant differences in liver test values (alanine aminotransferase [ALT] 64 ± 56 IU/L vs. 28 ± 20 IU/L; p < 0.001; aspartate aminotransferase [AST] (51 + or - 45 IU/L vs. 23 + or - 12 IU/L; p < 0.001) and in gamma-glutamyltransferase(GGT) value (104 + or - 122 IU/L vs. 37 + or - 46 IU/L; p < 0.001. The presence of HCV antibody was related significantly to previous transfusion (13 % vs. 5 %; p = 0.03), tattoos (29 % vs. 13 %; p < 0.01), intravenous drug addiction (13 % vs. 0.2 %; p < 0.001) and coexistence with people with positive HCV antibody (16 % vs. 4 %; p < 0.001). In HBV no differences in basal characteristics were observed with exception in AST values (29 + or - 15 IU/L vs. 23 + or - 12 IU/L; p < 0.01). Hepatitis B surface antigen (HBsAg) was related significantly to previous transfusion (15 % vs. 5 %; p < 0.01), tattoos (26 % vs. 14 %; p = 0.04) and coexistence with people with positive HBsAg (17 % vs. 4 %; p < 0.001). CONCLUSIONS: Prevalence of serological markers in healthy working population is low. Risk factors for infection were previous transfusion and tattoos. Intravenous drug addiction was only a risk factor in HCV.

Cell surface shaving of Candida albicans biofilms, hyphae, and yeast form cells.

We used a brief trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS to identify the surface proteins on live Candida albicans organisms growing in biofilms and planktonic yeast cells and hyphae. One hundred thirty-one proteins were present in at least two of the three replicates of one condition and distributed in various combinations of the three growth conditions. Both previously reported and new surface proteins were identified and these were distributed between covalently attached proteins and noncovalently attached proteins of the cell wall.

Lytic activity of LysH5 endolysin secreted by Lactococcus lactis using the secretion signal sequence of bacteriocin Lcn972.

Bacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded by Staphylococcus aureus phage vB_SauS-phi-IPLA88, was expressed and secreted in Lactococcus lactis using the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts.

The tape measure protein of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has an active muramidase domain.

Tailed double-stranded DNA (dsDNA) bacteriophages frequently harbor structural proteins displaying peptidoglycan hydrolytic activities. The tape measure protein from Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has a lysozyme-like and a peptidase_M23 domain. This report shows that the lysozyme-like domain (TG1) has muramidase activity and exhibits in vitro lytic activity against live S. aureus cells, an activity that could eventually find use in the treatment of infections.

Long-term effects of highly active antiretroviral therapy in pretreated, vertically HIV type 1-infected children: 6 years of follow-up.

BACKGROUND: Several studies of children with human immunodeficiency virus (HIV) type 1 infection have demonstrated sustained increases in CD4+ cell count, even when virological failure has occurred after receipt of highly active antiretroviral therapy (HAART), but these studies were of limited duration. Moreover, the CD4+ cell count threshold at which antiretroviral treatment should be initiated is still unsettled. The aim of this study was to define the long-term impact of HAART on CD4+ cell percentage and viral load according to CD4+ cell percentages before HAART was initiated. METHODS: We conducted a retrospective study of 113 pretreated HIV-1-infected children stratified by pre-HAART CD4+ cell percentage (<5%, 5%-15%, 15%-25%, and >25%). The inclusion criteria were as follows: initiating HAART with a protease inhibitor, having 6 years of follow-up after starting HAART, having a CD4+ cell count or viral load recorded before initiation of HAART, and having received mono- or dual-nucleoside therapy before starting HAART. RESULTS: During the first 2 years of HAART, HIV-1-infected children experienced a significant increase in CD4+ cell percentage and a decrease in viral load (P<.05). During their last 4 years of receiving HAART, we found a significant decrease in viral load but not an increase in CD4+ cell percentage, because the CD4+ cell percentage reached a plateau after the second year of HAART. Moreover, children with CD4+ cell percentages of <5% at baseline did not achieve CD4+ cell percentages of >25% after 6 years of HAART. Children with CD4+ cell percentages of 5%-25% at baseline had a strong negative association with achieving CD4+ cell percentages of >30% for at least 6 and 12 months but not with achieving CD4+ cell percentages of >30% for at least 24 months. CONCLUSIONS: Long-term HAART allowed for restoration of CD4+ cell counts and control of viral loads in HIV-1-infected children. However, initiating HAART after severe immunosuppression has occurred is detrimental for the restoration of the CD4+ cell count.

Impact of long-term viral suppression in CD4+ recovery of HIV-children on Highly Active Antiretroviral Therapy.

BACKGROUND: The effects of HAART may differ between children and adults because children have a developing immune system, and the long-term immunological outcome in HIV-infected children on HAART is not well-known. A major aim of our study was to determine CD4+ evolution associated with long-term VL control during 4 years of observation on HAART. METHODS: We carried out a retrospective study on a cohort of 160 vertically HIV-infected children. It was carried out from 1996 to 2004 in six large Spanish pediatric referral hospitals. We compared 33 children who had long-term VL suppression (VL < or = 400 copies/ml) in the first 12 months of follow-up and maintained that level throughout follow-up (Responders-group), and 127 children with persistently detectable VL in spite of ART switches (Non-Responders-group). RESULTS: We observed a quick initial and significant increase in CD4+ counts from the baseline to 12 months on HAART in both groups (p < 0.01). The Non-Responders group sustained CD4+ increases and most of these children maintained high CD4+ level counts (> or = 25%). The Non-Responders group reached a plateau between 26% and 27% CD4+ at the first 12 months of follow-up that remained stable during the following 3 years. However, the Responders group reached a plateau between 30% and 32% CD4+ at 24, 36 and 48 months of follow-up. We found that the Responders group had higher CD4+ count values and higher percentages of children with CD4+ > or = 25% than the Non-Responders group (p < 0.05) after month 12. CONCLUSION: Long-term VL suppression in turn induces large beneficial effects in immunological responses. However, it is not indispensable to recover CD4+ levels.

Construction and characterization of a minimized version of the HIV-1 pNL4-3 plasmid and its application for pseudotyping HIV-1 vectors.

The pUC-based pNL4-3 plasmid is the most widely used vector for in vitro manipulations of the HIV-1 proviral sequences. We have developed a minimal plasmid (pCHUS) based on pNL4-3, which may be useful to facilitate the design of HIV-based constructions. The strategy that has allowed us to construct pCHUS includes the following steps: (1) pNL4-3 digestion by using restriction sites contained within the long terminal repeats (LTRs), (2) recircularization of the fragment containing the pUC18 sequence, (3) amplification of the LTR region restored in the previous step, (4) double digestion of the products obtained in steps 2 and 3, (5) ligation of the fragment containing ColE1+Amp(R) with the LTR fragment, (6) linearization of the intermediate plasmid obtained, and (7) insertion of the fragment containing the proviral genome into the linearized vector. The pCHUS plasmid includes essential information for its replication and antibiotic selection in bacteria, but it lacks all the unnecessary sequences. Our results suggest that pCHUS may be more advantageous than pNL4-3 for in vitro manipulation of the HIV-1 proviral genome. In addition, we describe a potential application of this new vector for pseudotyping HIV-1 particles, using a single plasmid transfection, as a more helpful alternative to the traditionally used cotransfection method.

Once-daily antiretroviral therapy in a cohort of HIV-infected children and adolescents.

We evaluated the evolution over time of once-daily antiretroviral therapy in HIV-infected children and its relationship with adherence. An increase on the prevalence of once-daily antiretroviral therapy was observed over time (from 0.9% in 2002 to 44.2% in 2011). There was no difference in adherence regarding once-daily or BID regimens in 2011. Adherence was related to age and pill burden.

2-DE based proteomic analysis of Saccharomyces cerevisiae wild and K+ transport-affected mutant (trk1,2) strains at the growth exponential and stationary phases.

By using a 2-DE based workflow, the proteome of wild and potassium transport mutant trk1,2 under optimal growth potassium concentration (50mM) has been analyzed. At the exponential and stationary phases, both strains showed similar growth, morphology potassium content, and Vmax of rubidium transport, the only difference found being the Km values for this potassium analogue transport, higher for the mutant (20mM) than for the wild (3-6mM) cells. Proteins were buffer-extracted, precipitated, solubilized, quantified, and subjected to 2-DE analysis in the 5-8 pH range. More differences in protein content (37-64mgg(-1) cell dry weight) and number of resolved spots (178-307) were found between growth phases than between strains. In all, 164 spots showed no differences between samples and a total of 105 were considered to be differential after ANOVA test. 171 proteins, corresponding to 71 unique gene products have been identified, this set being dominated by cytosolic species and glycolitic enzymes. The ranking of the more abundant spots revealed no differences between samples and indicated fermentative metabolism, and active cell wall biosynthesis, redox homeostasis, biosynthesis of amino acids, coenzymes, nucleotides, and RNA, and protein turnover, apart from cell division and growth. PCA analysis allowed the separation of growth phases (PC1 and 2) and strains at the stationary phase (PC3 and 4), but not at the exponential one. These results are also supported by clustering analysis. As a general tendency, a number of spots newly appeared at the stationary phase in wild type, and to a lesser extent, in the mutant. These up-accumulated spots corresponded to glycolitic enzymes, indicating a more active glucose catabolism, accompanied by an accumulation of methylglyoxal detoxification, and redox-homeostasis enzymes. Also, more extensive proteolysis was observed at the stationary phase with this resulting in an accumulation of low Mr protein species.

Variación de las subpoblaciones de células T segun la progresión clínica e inmunológica en niños infectados verticalmente por HIV-1 / T-cell subsets variation during clinical and immunological progression in vertically HIV-infected children

El objetivo de este trabajo es estudiar las subpoblaciones de células T vírgenes, memoria y activadas en sangre periférica de 65 niños infectados verticalmente por el HIV, considerando su categoría clínica, inmunológica y los valores de carga viral (CV). Todos los niños HIV fueron tratados con terapia antirretroviral (AR) (26 niños con terapia AR combinada y 39 en terapia AR altamente agresiva). Las subpoblaciones de linfocitos T se analizaron por citometría de flujo con marcaje triple y se expresaron en porcentaje. Las células T CD4+ vírgenes (CD45RA+CD62L+) estuvieron disminuidas en niños con bajo porcentaje CD4+, pero no en niños en estadios avanzados de la enfermedad o con CV elevadas. Por el contrario, las células T CD8+ vírgenes estuvieron disminuidas en niños con bajo porcentaje CD4+, en estadio avanzado de la enfermedad y con CV elevadas. Las células T CD4+ y CD8+ de memoria (CD45RO+) y activadas (CD38+, HLA-DR+ y CD38+HLA-DR+) estuvieron elevadas en niños con bajo porcentaje CD4+, en estadio avanzado de la enfermedad y con CV elevadas. Sin embargo, las células CD4+CD38+ estuvieron más elevadas en los niños HIV con CD4+>25 por ciento que en el grupo control (p<0,001) y más disminuidas en los niños con bajo porcentaje CD4+. El porcentaje de células T CD4+ y CD8+ vírgenes y memoria depende del porcentaje CD4+ más que de la categoría clínica o el valor de CV. Nuestros datos indican una asociación entre bajo porcentaje CD4+ y CV elevadas con la expresión elevada de marcadores de activación celular, pero no así en estadios clínicos avanzados, posiblemente debido al tratamiento antirretroviral.