A precise DNA bend angle is essential for the function of the phage φ29 transcriptional regulator

Bacteriophage φ29 protein p4 is essential for the regulation of the switch from early to late phage transcription. The protein binds to two regions of the phage genome located between the regulated promoters. Each region contains two inverted repeats separated by 1 bp. We used circular permutation assays to study the topology of the DNA upon binding of the protein and found that p4 induced the same extent of bending independent of the topology of the binding region. In addition, the results revealed that the p4-induced bending is not dependent on the affinity to the binding site but is intrinsic to p4 binding. Independent binding sites were identified through the characterization of the minimal sequence required for p4 binding. The protein has different affinity for each of its binding sites, with those overlapping the A2c and A2b promoter cores (sites 1 and 3), having the highest affinity. The functionality of the p4 binding sites and the contribution of p4-mediated promoter restructuring in transcription regulation is discussed

Prognostic value of ACE I/D, AT1R A1166C, PAI-I 4G/5G and GPIIIa a1/a2 polymorphisms in myocardial infarction

Background: Coronary artery disease (CAD) has turned into a prevalent cause of morbi-mortality contributing some polymorphisms in the recurrence of major adverse cardiac events (MACE).Methods: Three hundred and fifty six patients with first myocardial infarction (MI) were followed up during a 60-month period to find out if ACE I/D, AT1R A1166C, PAI-I 4G/5G and GPIIIa a1/a2 polymorphisms, in combination with other classical cardiovascular risk factors, can contribute to the relapse of MACE.Results: Two hundred and eighty five (80.1%) men and 71 (19.9%) women were followed up after first MI. The primary clinical endpoint, a composite of MACE, was reached in 106 (29.8%) patients. In the Cox univariate survival analysis those risk factors influencing a poorer prognosis were age (p = 0.004), a positive family history of CAD (p = 0.007), diabetes (p = 0.004), smoking (p = 0.024), fibrinolytic therapy (p = 0.012) and having 2 or 3 vessels CAD (p = 0.046). Cox proportional hazards regression model showed that patients with the DD genotype had a 1.5 increased risk of having an unfavorable outcome when compared with No-DD genotype patients (RR 1.561, 95% CI 1.048–2.326, p = 0.028) and that patients with the ACE DD genotype plus the AT1R No-AA genotype, the GPIIIa No-a1a1 genotype or a combination of both, had a twice higher risk than any other genotype of MACE in the follow-up (RR 1.978, 95% CI 1.286–3.043, p = 0.002).Conclusions: Patients with the ACE DD genotype plus 1 or 2 unfavorable genotypes, the AT1R No-AA, the GPIIIa No-a1a1 or a combination of both, have twice higher the risk of MACE during their clinical follow-up.

DNA sequence-specific recognition by a transcriptional regulator requires indirect readout of A-tracts

The bacteriophage Ø29 transcriptional regulator p4 binds to promoters of different intrinsic activities. The p4–DNA complex contains two identical protomers that make similar interactions with the target sequence 5′-AACTTTTT-15 bp-AAAATGTT-3′. To define how the various elements in the target sequence contribute to p4's affinity, we studied p4 binding to a series of mutated binding sites. The binding specificity depends critically on base pairs of the target sequence through both direct as well as indirect readout. There is only one specific contact between a base and an amino acid residue; other contacts take place with the phosphate backbone. Alteration of direct amino acid–base contacts, or mutation of non-contacted A·T base pairs at A-tracts abolished binding. We generated three 5 ns molecular dynamics (MD) simulations to investigate the basis for the p4–DNA complex specificity. Recognition is controlled by the protein and depends on DNA dynamic properties. MD results on protein–DNA contacts and the divergence of p4 affinity to modified binding sites reveal an inherent asymmetry, which is required for p4-specific binding and may be crucial for transcription regulation

Simplified Model to Survey Tuberculosis Transmission in Countries Without Systematic Molecular Epidemiology Programs

Systematic molecular/genomic epidemiology studies for tuberculosis surveillance cannot be implemented in many countries. We selected Panama as a model for an alternative strategy. Mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) analysis revealed a high proportion (50%) of Mycobacterium tuberculosis isolates included in 6 clusters (A–F) in 2 provinces (Panama and Colon). Cluster A corresponded to the Beijing sublineage. Whole-genome sequencing (WGS) differentiated clusters due to active recent transmission, with low single-nucleotide polymorphism–based diversity (cluster C), from clusters involving long-term prevalent strains with higher diversity (clusters A, B). Prospective application in Panama of 3 tailored strain–specific PCRs targeting marker single-nucleotide polymorphisms identified from WGS data revealed that 31.4% of incident cases involved strains A–C and that the Beijing strain was highly represented and restricted mainly to Colon. Rational integration of MIRU-VNTR, WGS, and tailored strain–specific PCRs could be a new model for tuberculosis surveillance in countries without molecular/genomic epidemiology programsSystematic molecular/genomic epidemiology studies for tuberculosis surveillance cannot be implemented in many countries. We selected Panama as a model for an alternative strategy. Mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) analysis revealed a high proportion (50%) of Mycobacterium tuberculosis isolates included in 6 clusters (A–F) in 2 provinces (Panama and Colon). Cluster A corresponded to the Beijing sublineage. Whole-genome sequencing (WGS) differentiated clusters due to active recent transmission, with low single-nucleotide polymorphism–based diversity (cluster C), from clusters involving long-term prevalent strains with higher diversity (clusters A, B). Prospective application in Panama of 3 tailored strain–specific PCRs targeting marker single-nucleotide polymorphisms identified from WGS data revealed that 31.4% of incident cases involved strains A–C and that the Beijing strain was highly represented and restricted mainly to Colon. Rational integration of MIRU-VNTR, WGS, and tailored strain–specific PCRs could be a new model for tuberculosis surveillance in countries without molecular/genomic epidemiology program

Mycobacterium tuberculosis acquires limited genetic diversity in prolonged infections, reactivations and transmissions involving multiple hosts

Publisher Copyright: © 2018 Herranz, Pole, Ozere, Chiner-Oms, Martínez-Lirola, Pérez-García, Gijón, Serrano, Romero, Cuevas, Comas, Bouza, Pérez-Lago and García-de-Viedma.Background: Mycobacterium tuberculosis (MTB) has limited ability to acquire variability. Analysis of its microevolution might help us to evaluate the pathways followed to acquire greater infective success. Whole-genome sequencing (WGS) in the analysis of the transmission of MTB has elucidated the magnitude of variability in MTB. Analysis of transmission currently depends on the identification of clusters, according to the threshold of variability (<5 SNPs) between isolates. Objective: We evaluated whether the acquisition of variability in MTB, was more frequent in situations which could favor it, namely intrapatient, prolonged infections or reactivations and interpatient transmissions involving multiple sequential hosts. Methods: We used WGS to analyze the accumulation of variability in sequential isolates from prolonged infections or translations from latency to reactivation. We then measured microevolution in transmission clusters with prolonged transmission time, high number of involved cases, simultaneous involvement of latency and active transmission. Results: Intrapatient and interpatient acquisition of variability was limited, within the ranges expected according to the thresholds of variability proposed, even though bursts of variability were observed. Conclusions: The thresholds of variability proposed for MTB seem to be valid in most circumstances, including those theoretically favoring acquisition of variability. Our data point to multifactorial modulation of microevolution, although further studies are necessary to elucidate the factors underlying this modulation.publishersversionPeer reviewe

Novel dimeric structure of phage ϕ29-encoded protein p56: insights into uracil-DNA glycosylase inhibition

Protein p56 encoded by the Bacillus subtilis phage ϕ29 inhibits the host uracil-DNA glycosylase (UDG) activity. To get insights into the structural basis for this inhibition, the NMR solution structure of p56 has been determined. The inhibitor defines a novel dimeric fold, stabilized by a combination of polar and extensive hydrophobic interactions. Each polypeptide chain contains three stretches of anti-parallel β-sheets and a helical region linked by three short loops. In addition, microcalorimetry titration experiments showed that it forms a tight 2:1 complex with UDG, strongly suggesting that the dimer represents the functional form of the inhibitor. This was further confirmed by the functional analysis of p56 mutants unable to assemble into dimers. We have also shown that the highly anionic region of the inhibitor plays a significant role in the inhibition of UDG. Thus, based on these findings and taking into account previous results that revealed similarities between the association mode of p56 and the phage PBS-1/PBS-2-encoded inhibitor Ugi with UDG, we propose that protein p56 might inhibit the enzyme by mimicking its DNA substrate

A novel strategy based on genomics and specific PCR reveals how a multidrug resistant Mycobacterium tuberculosis strain became prevalent in Equatorial Guinea 15 years after its emergence.

OBJECTIVE: Molecular epidemiology techniques in tuberculosis (TB) can identify high-risk strains that are actively transmitted. We aimed to implement a novel strategy to optimize the identification and control of multidrug-resistant (MDR) TB in a specific population. METHODS: We developed a strain-specific PCR tailored from whole genome sequencing (WGS) data to track a specific MDR prevalent strain in Equatorial Guinea (EG-MDR). RESULTS: The PCR was applied prospectively on remnants of GeneXpert reaction mixtures owing to the lack of culture facilities in Equatorial Guinea. In 147 (93%) of 158 cases, we were able to differentiate between infection by the EG-MDR strain or by any other strain and found that 44% of all rifampicin-resistant TB cases were infected by EG-MDR. We also analysed 93 isolates obtained from Equatorial Guinea 15 years ago, before MDR-TB had become the problem it is today. We found that two of the scarce historical MDR cases were infected by EG-MDR. WGS revealed low variability-six single nucleotide polymorphisms acquired by this strain over 15 years-likely because of the lack in the country of a specific program to treat MDR-TB. CONCLUSIONS: Our novel strategy, which integrated WGS analysis and strain-specific PCRs, represents a low-cost, rapid and transferable strategy that allowed a prospective efficient survey and fast historical analysis of MDR-TB in a population

Detección de Mycobacterium tuberculosis, linaje Beijing, en Ecuador

Introduction: Mycobacterium tuberculosis Beijing lineage isolates are considered to be especially virulent, transmissible and prone to acquire resistances. Beijing strains have been reported worldwide, but studies in Latin America are still scarce. The only multinational study performed in the region indicated a heterogeneous distribution for this lineage, which was absent in Chile, Colombia and Ecuador, although further studies found the lineage in Chile and Colombia.Objective: To search for the presence of the Beijing lineage in Ecuador, the only country in the region where it remains unreported.Materials and methods: We obtained a convenience sample (2006-2012) from two hospitals covering different populations. The isolates were genotyped using 24-MIRU-VNTR. Lineages were assigned by comparing their patterns to those in the MIRU-VNTRplus platform. Isolates belonging to the Beijing lineage were confirmed by allele-specific PCR.Results: We identified the first Beijing isolate in Ecuador in an unexpected epidemiological scenario: A patient was infected in the Andean region, in a population with low mobility and far from the borders of the neighboring countries where Beijing strains had been previously reported.Conclusion: This is the first report of the presence of the Beijing lineage in Ecuador in an unusual epidemiological context that deserves special attention.Introducción. Los aislamientos de Mycobacterium tuberculosis pertenecientes al linaje Beijing se consideran especialmente virulentos y transmisibles, y con mayor tendencia a la adquisición de resistencia. El linaje Beijing se ha reportado en todo el mundo; sin embargo, en Latinoamérica los estudios al respecto son más escasos. En el único estudio multinacional llevado a cabo en la región, se detectó una distribución heterogénea del linaje, y no se le encontró en Chile, Colombia y Ecuador,aunque en estudios nacionales posteriores se identificaron aislamientos en Chile y Colombia.Objetivo. Rastrear la presencia del linaje Beijing de M. tuberculosis en Ecuador, único país en la región en el que aún no se reporta.Materiales y métodos. Se analizó una muestra de conveniencia (2006-2012) en dos hospitales que atendían poblaciones diferentes. La genotipificación de los aislamientos de M. tuberculosis se hizo mediante la plataforma 24-MIRU-VNTR. La asignación de linajes se hizo mediante la comparación de los patrones genotípicos con los incluidos en la plataforma MIRU-VNTRplus, y aquellos pertenecientes al linaje Beijing fueron confirmados mediante reacción en cadena de la polimerasa específica de alelo.Resultados. Se detectó el primer aislamiento Beijing en Ecuador, en una circunstancia epidemiológica inesperada: un paciente de la región andina, proveniente de una comunidad con escasa movilidad y alejada de las fronteras con los países limítrofes, Perú y Colombia, en los que ya se han identificado aislamientos de M. tuberculosis pertenecientes al linaje Beijing.Conclusiones. En este trabajo se reporta por primera vez la presencia del linaje Beijing de M. tuberculosis en Ecuador en un contexto epidemiológico inusual que merece especial atención

Integración de dispositivos de salud personal en la plataforma de telecuidado para diabetes PERSONA

La plataforma de telecuidado PERSONA se ha desarrollado en el marco del CIBER-BBN y tiene por objetivo soportar el autocuidado diario de pacientes con diabetes tipo 1. La plataforma proporciona acceso a herramientas de soporte a la decisión, de procesado automático de la información, de monitorización de las variables que afectan a la enfermedad y facilita la comunicación entre los agentes involucrados en el cuidado del paciente. La integración de dispositivos médicos interoperables es un requisito principal de la plataforma PERSONA. En este trabajo presentamos las soluciones adoptadas en cuanto a la integración de dispositivos médicos y analizamos las características de los protocolos de comunicación inalámbrica de los dispositivos considerados y los recursos necesarios para la comunicación con dispositivos móviles de telefonía

Simplified Model to Survey Tuberculosis Transmission in Countries Without Systematic Molecular Epidemiology Programs

Systematic molecular/genomic epidemiology studies for tuberculosis surveillance cannot be implemented in many countries. We selected Panama as a model for an alternative strategy. Mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) analysis revealed a high proportion (50%) of Mycobacterium tuberculosis isolates included in 6 clusters (A–F) in 2 provinces (Panama and Colon). Cluster A corresponded to the Beijing sublineage. Whole-genome sequencing (WGS) differentiated clusters due to active recent transmission, with low single-nucleotide polymorphism–based diversity (cluster C), from clusters involving long-term prevalent strains with higher diversity (clusters A, B). Prospective application in Panama of 3 tailored strain–specific PCRs targeting marker single-nucleotide polymorphisms identified from WGS data revealed that 31.4% of incident cases involved strains A–C and that the Beijing strain was highly represented and restricted mainly to Colon. Rational integration of MIRU-VNTR, WGS, and tailored strain–specific PCRs could be a new model for tuberculosis surveillance in countries without molecular/genomic epidemiology programs.Systematic molecular/genomic epidemiology studies for tuberculosis surveillance cannot be implemented in many countries. We selected Panama as a model for an alternative strategy. Mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) analysis revealed a high proportion (50%) of Mycobacterium tuberculosis isolates included in 6 clusters (A–F) in 2 provinces (Panama and Colon). Cluster A corresponded to the Beijing sublineage. Whole-genome sequencing (WGS) differentiated clusters due to active recent transmission, with low single-nucleotide polymorphism–based diversity (cluster C), from clusters involving long-term prevalent strains with higher diversity (clusters A, B). Prospective application in Panama of 3 tailored strain–specific PCRs targeting marker single-nucleotide polymorphisms identified from WGS data revealed that 31.4% of incident cases involved strains A–C and that the Beijing strain was highly represented and restricted mainly to Colon. Rational integration of MIRU-VNTR, WGS, and tailored strain–specific PCRs could be a new model for tuberculosis surveillance in countries without molecular/genomic epidemiology programs