Staphylococcal self-loading helicases couple the staircase mechanism with inter domain high flexibility.

Replication is a crucial cellular process. Replicative helicases unwind DNA providing the template strand to the polymerase and promoting replication fork progression. Helicases are multi-domain proteins which use an ATPase domain to couple ATP hydrolysis with translocation, however the role that the other domains might have during translocation remains elusive. Here, we studied the unexplored self-loading helicases called Reps, present in Staphylococcus aureus pathogenicity islands (SaPIs). Our cryoEM structures of the PriRep5 from SaPI5 (3.3 Å), the Rep1 from SaPI1 (3.9 Å) and Rep1-DNA complex (3.1Å) showed that in both Reps, the C-terminal domain (CTD) undergoes two distinct movements respect the ATPase domain. We experimentally demonstrate both in vitro and in vivo that SaPI-encoded Reps need key amino acids involved in the staircase mechanism of translocation. Additionally, we demonstrate that the CTD's presence is necessary for the maintenance of full ATPase and helicase activities. We speculate that this high interdomain flexibility couples Rep's activities as initiators and as helicases.

A novel DNA primase-helicase pair encoded by SCCmec elements.

Mobile genetic elements (MGEs) are a rich source of new enzymes, and conversely, understanding the activities of MGE-encoded proteins can elucidate MGE function. Here, we biochemically characterize three proteins encoded by a conserved operon carried by the Staphylococcal Cassette Chromosome (SCCmec), an MGE that confers methicillin resistance to Staphylococcus aureus, creating MRSA strains. The first of these proteins, CCPol, is an active A-family DNA polymerase. The middle protein, MP, binds tightly to CCPol and confers upon it the ability to synthesize DNA primers de novo. The CCPol-MP complex is therefore a unique primase-polymerase enzyme unrelated to either known primase family. The third protein, Cch2, is a 3'-to-5' helicase. Cch2 additionally binds specifically to a dsDNA sequence downstream of its gene that is also a preferred initiation site for priming by CCPol-MP. Taken together, our results suggest that this is a functional replication module for SCCmec.

Early Spot Urinary Sodium and Diuretic Efficiency in Acute Heart Failure and Concomitant Renal Dysfunction.

OBJECTIVE: In acute heart failure (AHF), early assessment of spot urinary sodium (UNa) has emerged as a useful biomarker for risk stratification and monitoring. The objective of this study was to investigate (a) whether early spot UNa predicts 24-h diuretic efficiency and (b) the clinical factors associated with early spot UNa in patients with AHF and concomitant renal dysfunction (RD). METHODS: This is a post hoc analysis of the IMPROVE-HF trial, in which 160 patients with AHF and RD (estimated glomerular filtrate rate [eGFR] <60 mL/min/1.73 m2) were included. Diuretic efficiency was calculated as the net fluid output produced per 40 mg of furosemide equivalents in 24 h. The association between early spot UNa and diuretic efficiency and clinical variables associated with UNa were evaluated using multivariate linear regression analysis. The contribution of the exposures in the predictability of the models was assessed with the coefficient of determination (R2). RESULTS: The mean age of the study population was 78 ± 8 years. The median (interquartile range) diuretic efficiency, early spot UNa, aminoterminal pro-brain natriuretic peptide, and eGFR were 747 (490-1,167) mL, 90 mmol/L (65-111), 7,765 pg/mL (3,526-15,369), and 33.7 ± 11.3 mL/min/1.73 m2, respectively. In a multivariate setting, lower UNa was significantly and nonlinearly associated with lower diuretic efficiency (p = 0.001), explaining the 44.4% of the model predictability. Natremia and surrogates of congestion emerged as the main factors related to UNa. CONCLUSIONS: In patients with AHF and RD at presentation, early spot UNa was inversely related to 24-h diuretic efficiency.

Target highlights in CASP13: Experimental target structures through the eyes of their authors.

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.

Use of acetazolamide in the treatment of patients with refractory congestive heart failure.

AIMS: Optimal diuretic treatment of patients with refractory congestive heart failure (CHF) remains to be elucidated. In this work, we aimed to evaluate the serial changes of functional class and surrogates of fluid overload (weight and antigen carbohydrate 125) after addition of oral acetazolamide in patients with refractory CHF. Likewise, serial changes in renal function, serum electrolytes and pH were evaluated. METHOD: This is an observational retrospective study in which 25 ambulatory patients with refractory CHF that received acetazolamide in addition to standard intensive diuretic strategy were evaluated. Longitudinal assessment of New York Heart Association (NYHA) functional class and biomarkers was analyzed using joint modelling of longitudinal and survival data. RESULTS: All patients showed NYHA class III/IV at baseline. After prescription of acetazolamide, a total of 125 outpatient visits were recorded [median visits per patient: 6 (IQR = 3-7)] during a median follow-up of 152 days (IQR = 80-353). A significant decrease in NYHA class, weight, and antigen carbohydrate 125 was observed. On the other hand, estimated glomerular filtration rate increased over time. No significant changes in systolic blood pressure, serum sodium, potassium, amino-terminal pro-brain natriuretic peptide, and pH occurred. CONCLUSION: In a cohort of patients with refractory CHF treated with an intensive diuretic treatment, the addition of acetazolamide was associated with improvement in functional class and surrogates of fluid overload.

Crystal Structure of an Unusual Single-Stranded DNA-Binding Protein Encoded by Staphylococcal Cassette Chromosome Elements.

Methicillin-resistant Staphylococcus aureus is a global public health threat. Methicillin resistance is carried on mobile genetic elements belonging to the staphylococcal cassette chromosome (SCC) family. The molecular mechanisms that SCC elements exploit for stable maintenance and for horizontal transfer are poorly understood. Previously, we identified several conserved SCC genes with putative functions in DNA replication, including lp1413, which we found encodes a single-stranded DNA (ssDNA)-binding protein. We report here the 2.18 Å crystal structure of LP1413, which shows that it adopts a winged helix-turn-helix fold rather than the OB-fold normally seen in replication-related ssDNA-binding proteins. However, conserved residues form a hydrophobic pocket not normally found in winged helix-turn-helix domains. LP1413 also has a conserved but disordered C-terminal tail. As deletion of the tail does not significantly affect cooperative binding to ssDNA, we propose that it mediates interactions with other proteins. LP1413 could play several different roles in vivo.

Pirating conserved phage mechanisms promotes promiscuous staphylococcal pathogenicity island transfer.

Targeting conserved and essential processes is a successful strategy to combat enemies. Remarkably, the clinically important Staphylococcus aureus pathogenicity islands (SaPIs) use this tactic to spread in nature. SaPIs reside passively in the host chromosome, under the control of the SaPI-encoded master repressor, Stl. It has been assumed that SaPI de-repression is effected by specific phage proteins that bind to Stl, initiating the SaPI cycle. Different SaPIs encode different Stl repressors, so each targets a specific phage protein for its de-repression. Broadening this narrow vision, we report here that SaPIs ensure their promiscuous transfer by targeting conserved phage mechanisms. This is accomplished because the SaPI Stl repressors have acquired different domains to interact with unrelated proteins, encoded by different phages, but in all cases performing the same conserved function. This elegant strategy allows intra- and inter-generic SaPI transfer, highlighting these elements as one of nature's most fascinating subcellular parasites.

Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus.

DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages.

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative.

Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3'-to-5' helicase and that the adjacent ORF encodes a single-stranded DNA-binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-family replicative helicases, belongs to the pre-sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.

Virus Satellites Drive Viral Evolution and Ecology.

Virus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hosts.

Phage dUTPases control transfer of virulence genes by a proto-oncogenic G protein-like mechanism.

dUTPases (Duts) have emerged as promising regulatory molecules controlling relevant cellular processes. However, the mechanism underlying this regulatory function remains enigmatic. Using staphylococcal pathogenicity island (SaPI) repression as a model, we report here that phage Duts induce the transfer of SaPI-encoded virulence factors by switching between active (dUTP-bound) and inactive (apo state) conformations, a conversion catalyzed by their intrinsic dUTPase activity. Crystallographic and mutagenic analyses demonstrate that binding to dUTP reorders the C-terminal motif V of the phage-encoded Duts, rendering these proteins into the active conformation required for SaPI derepression. By contrast, the conversion to the apo state conformation by hydrolysis of the bound dUTP generates a protein that is unable to induce the SaPI cycle. Because none of the requirements involving Duts in SaPI transfer are exclusive to the phage-encoded proteins, we propose that Duts are widespread cellular regulators acting in a manner analogous to the eukaryotic G proteins.

Control of Staphylococcus aureus pathogenicity island excision.

Staphylococcus aureus pathogenicity islands (SaPIs) are a group of related 15-17 kb mobile genetic elements that commonly carry genes for superantigen toxins and other virulence factors. The key feature of their mobility is the induction of SaPI excision and replication by certain phages and their efficient encapsidation into specific small-headed phage-like infectious particles. Previous work demonstrated that chromosomal integration depends on the SaPI-encoded recombinase, Int. However, although involved in the process, Int alone was not sufficient to mediate efficient SaPI excision from chromosomal sites, and we expected that SaPI excision would involve an Xis function, which could be encoded by a helper phage or by the SaPI, itself. Here we report that the latter is the case. In vivo recombination assays with plasmids in Escherichia coli demonstrate that SaPI-coded Xis is absolutely required for recombination between the SaPI att(L) and att(R) sites, and that both sites, as well as their flanking SaPI sequences, are required for SaPI excision. Mutational analysis reveals that Xis is essential for efficient horizontal SaPI transfer to a recipient strain. Finally, we show that the master regulator of the SaPI life cycle, Stl, blocks expression of int and xis by binding to inverted repeats present in the promoter region, thus controlling SaPI excision.

Comparación del somatotipo, evaluación nutricional e ingesta alimentaria entre estudiantes universitarios deportistas y sedentarios / Comparison of the somatotype, nutritional assessment and food intake among university sport and sedentary students

Fundamento y objetivo: El estudio del somatotipo y de la ingesta alimentaria entre la población universitaria es importante para implantar políticas internas de mejora de la salud y de la prevención de enfermedades. En este estudio se ha realizado la comparación del somatotipo, evaluación energética-nutricional e ingesta alimentaria entre estudiantes universitarios deportistas frente a los sedentarios. Sujetos y método: La muestra está formada por 1.299 jóvenes universitarios (420 varones y 879 mujeres) de la Universitat de València, durante 2008-2009 y 2009-2010, evaluándose en ellos diversos parámetros antropométricos, el somatotipo y la ingesta nutricional y alimentaria mediante un registro del consumo alimentario de 7 días. Resultados: El somatotipo fue Endo-Mesomorfo, Meso-Endomorfo, Endomorfo Balanceado y Meso-Endomorfo para varones deportistas y sedentarios, y mujeres deportistas y sedentarias, respectivamente. Los cuatro grupos presentan un porcentaje alto para proteínas y grasas y bajo para hidratos de carbono porcentualmente con respecto a las calorías totales establecidos por los objetivos nutricionales para la población española. Se observa un déficit de vitamina E para los varones deportistas, de vitaminas A, D y E, folato y biotina para los varones sedentarios y de vitaminas D y E, folato y biotina para las mujeres deportistas y sedentarias. En los cuatro grupos hay una elevada ingesta de sodio y un déficit de potasio, calcio, yodo y magnesio. Para todos ellos, el grupo de cereales es el grupo de alimentos que más contribuye energéticamente al día. Conclusiones: Los desequilibrios tanto en macronutrientes como en micronutrientes reflejan la importancia de desarrollar adecuadas políticas de educación alimentaria universitaria que pueden mejorar esta situación (AU)

Background and objective: The study of the somatotype and food intake among university students is important to carry out internal policies about the improvement of health and prevention of diseases. The aim of this study was to compare the somatotype, nutritional assessment and food intake of university sport and sedentary students. Subjects and method: The sample were 1,299 university students (420 males and 879 females) from University of Valencia, during 2008-2009 and 2009-2010, who were evaluated for several anthropometric parameters, the somatotype and the nutritional and food intakes with a 7 day dietary diary. Results: The somatotype was Endo-Mesomorph, Meso-Endomorph, Balanced Endomorph and Meso-Endomorph for sport and sedentary males, and sports and sedentary females, respectively. All groups had a high and low percentage of total calories in comparison with proteins plus lipids, and carbohydrates, respectively, as established in the nutritional objectives for the Spanish population. Low vitamin intakes, including E (in the sport males), A, D and E, folate and biotine (in the sedentary males) and A, D, E, folate and biotine (in the sport and sedentary females) were observed. All groups had a high intake of sodium and a deficit of potassium, calcium, iodine and magnesium. On the other hand, cereal groups were the most important in the energy dairy intake. Conclusions: Disequilibrium in macronutrients and micronutrients reflects the importance of developing food policies among University people to improve this situation (AU)

[Comparison of the somatotype, nutritional assessment and food intake among university sport and sedentary students]. / Comparación del somatotipo, evaluación nutricional e ingesta alimentaria entre estudiantes universitarios deportistas y sedentarios.

BACKGROUND AND OBJECTIVE: The study of the somatotype and food intake among university students is important to carry out internal policies about the improvement of health and prevention of diseases. The aim of this study was to compare the somatotype, nutritional assessment and food intake of university sport and sedentary students. SUBJECTS AND METHOD: The sample were 1,299 university students (420 males and 879 females) from University of Valencia, during 2008-2009 and 2009-2010, who were evaluated for several anthropometric parameters, the somatotype and the nutritional and food intakes with a 7 day dietary diary. RESULTS: The somatotype was Endo-Mesomorph, Meso-Endomorph, Balanced Endomorph and Meso-Endomorph for sport and sedentary males, and sports and sedentary females, respectively. All groups had a high and low percentage of total calories in comparison with proteins plus lipids, and carbohydrates, respectively, as established in the nutritional objectives for the Spanish population. Low vitamin intakes, including E (in the sport males), A, D and E, folate and biotine (in the sedentary males) and A, D, E, folate and biotine (in the sport and sedentary females) were observed. All groups had a high intake of sodium and a deficit of potassium, calcium, iodine and magnesium. On the other hand, cereal groups were the most important in the energy dairy intake. CONCLUSIONS: Disequilibrium in macronutrients and micronutrients reflects the importance of developing food policies among University people to improve this situation.

Spanish experience in autologous chondrocyte implantation.

INTRODUCTION: The Spanish Ministry of Health commissioned the Galician Agency for Health Technology Assessment to monitor and follow-up Autologous Chondrocyte Implantation (ACI) used to treat chondral lesions of the knee in Spain. The objective of this monitoring was to assess efficacy and safety of the technique. DESIGN: One-hundred and eleven consecutive patients with knee chondral lesions were included in a multi-center study between January 2001 and January 2005. ACI was used in these patients as a second-line treatment option (or a first-line treatment option if the cause was Osteocondritis dissecans). The Cincinnati score and the Short Form 36 (SF-36) questionnaire were used to assess the patients' self-reported satisfaction with the outcomes of ACI. A descriptive analysis was performed and non-parametric tests were used to establish correlations and compare results among subgroups. A multivariate analysis was also performed to measure the effect of different variables on changes in the condition of the knee. RESULTS: Eighty men (72%) and 31 women (21%) with an age range from 16 to 49 years, underwent ACI surgery. Among these subjects, the most common previous first-line treatment was debridement (64 individuals, 74.4%). The mean size of the lesion treated with ACI was 3.82 cm(2), and the most frequent location of the lesion was the inner femoral condyle (53.6%). The patient satisfaction was high or very high in 36 subjects (66.7%). Overall knee joint assessment improved from 4.32 points to 6.78. All SF-36 questionnaire categories improved, notably those related to physical condition. CONCLUSIONS: The results of this study indicate that ACI is safe; however, further studies are mandated to assess the efficacy of ACI compared to alternative treatment options.

Inestabilidad postraumática tras luxación del pisiforme en un jugador de balonmano / Postraumatic inestability after pisiform dislocation in a handball player

Las lesiones traumáticas del pisiforme se producen por caídas con la mano en dorsiflexión. Para producir una luxación se debe asociar una contracción activa del tendón cubital anterior. La luxación aislada del hueso pisiforme es una entidad poco frecuente, aunque en la literatura se pueden encontrar cada vez un mayor número de referencias. La movilidad del pisiforme es bastante variable de un individuo a otro. En algunos casos no presenta prácticamente movilidad. Sin embargo, en la mayoría de las ocasiones podemos demostrar su movilidad mediante dos proyecciones: una proyección en semisupinación de 10°-30° que muestra el paralelismo entre el pisiforme y el piramidal y la segunda que muestra el canal carpiano en un corte transverso. En flexión podemos apreciar un aumento del espacio piso-triquetral y en extensión una disminución del espacio piso-triquetral. Presentamos el caso de un varón de 18 años de edad, jugador de balonmano que, tras caída con la mano en extensión, presentó dolor, deformidad e impotencia funcional a nivel de la muñeca derecha, siendo diagnosticado de contusión de muñeca y tratado mediante un vendaje compresivo. Ante mínimos traumatismos presentaba dolor y deformidad en cara volar, borde cubital muñeca derecha. Se diagnosticó de luxación recidivante del pisiforme optándose por el tratamiento quirúrgico que consistió en la exéresis del mismo. Se accede a nivel de la eminencia hipoténar, longitudinalmente sobre el tendón del músculo cubital anterior y de la cara palmar del pisiforme. Se debe ser respetuoso sobre las partes blandas preservando el aparato ligamentoso. Tras la cirugía se inmovilizó el antebrazo mediante un vendaje compresivo que se mantuvo durante dos semanas. Tras este periodo se retiraron los puntos y se procedió a la rehabilitación de la muñeca. A los pocos meses de la cirugía los problemas habían cedido y el paciente se reincorporó a su práctica deportiva sin molestias (AU)