Force determination in lateral magnetic tweezers combined with TIRF microscopy

Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA:protein interactions. In particular, combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of the flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)-TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation.We thank the financial support from the Spanish MINECO (FIS2014-58328-P) and from (ERC) under the European Union’s Horizon2020 Research and Innovation programme (grant agreement No 681299). J. M. M. acknowledges a Predoctoral PhD fellowship from the Basque Country Government Department of Education, Language Policy and Culture (ref. PRE_2013_11_1174). ). G. L. M. F was supported by the Biotechnology and Biological Sciences Research Council (1363883). M. S. D was supported by the Wellcome Trust (100401 and 077368).Peer reviewe

Purified Smc5/6 complex exhibits DNA substrate recognition and compaction

Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.The work in the Aragon laboratory was supported by a Wellcome Trust Senior Investigator award to L.A. (100955, “Functional dissection of mitotic chromatin”) and the London Institute of Medical Research (LMS), which receives its core funding from the UK Medical Research Council (MC-A652-5PY00). F.M.-H. acknowledges support from the European Research Council (ERC) under the European Union Horizon 2020 Research and Innovation Program (grant agreement 681299). Work in the Moreno-Herrero laboratory was also supported by Spanish Ministry of Economy and Competitiveness grant BFU2017-83794-P (AEI/FEDER, UE to F.M.-H.) and Comunidad de Madrid grants Tec4Bio – S2018/NMT-4443 and NanoBioCancer – Y2018/BIO-4747 (to F.M.-H.). Work in the J.T.-R. lab was supported by grants BFU2015-71308-P and PGC2018-097796-B-I00 from the Ministerio de Ciencia, Innovación y Universidades and grant 2017-SGR-569 from AGAUR-Generalitat de Catalunya. The IRBLLEIDA Institute is part of the CERCA Programme-Generalitat de Catalunya

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Vigilancia epidemiológica de parasitosis zoonóticas en un área centinela desde la perspectiva de Una Salud

El abordaje de las enfermedades zoonóticas debe realizarse desde un enfoque multisectorial. Desde el concepto de Una Salud, las acciones realizadas por un solo sector no resultan eficaces para controlar este tipo de infecciones. En ese sentido, los distintos agentes de enfermedades transmisibles zoonóticas, afectan tanto a animales como a humanos y habitualmente el ecosistema actúa como reservorio o transmisor directo o indirecto, en la interfaz hombre-animal-ambiente. Los caninos pueden diseminar con sus heces enteroparásitos transmisibles a humanos y como animales centinela, pueden utilizarse para realizar vigilancia de la circulación de patógenos. Algunas helmintiasis, varias protozoosis y el alga parásita Blastocystis sp., son comunes en caninos y humanos. Nematodes del género Toxocara spp., enteroparásito de animales, ocasionan en personas toxocarosis, enfermedad de elevada seroprevalencia en la ciudad de La Plata y otras regiones. Sus formas neurológica y ocular tienen generalmente serias consecuencias. Su presencia en humanos se ve influenciada favorablemente por el lugar de residencia y su tejido suburbano. Giardia lamblia, enteroparásito zoonótico, ocasiona síndrome de malabsorción y modificación de moléculas de fármacos, también se correlaciona giardiasis con enfermedad de Whipple y otros disturbios gastroentéricos. El enfoque "Una salud" tiene en su estructura elementos esenciales, ellos son voluntad política (compromiso con las normas internacionales y los Objetivos de Desarrollo Sostenible), planes de financiación sostenibles; comunicación (entre sectores y disciplinas a nivel internacional, regional, nacional y subnacional). Los aprendizajes socialmente productivos y significativos “resignifica el papel social, cultural y económico social del conocimiento” y son aquellos “modifican a los sujetos enseñándoles a transformar su naturaleza y su cultura, enriqueciendo el capital cultural de la sociedad y de las comunidades” (Orozco B, 2009: 88). Los objetivos de este trabajo fueron: Realizar vigilancia y alertas tempranas en cuanto a parasitosis humanas animales y zoonóticas. Ejecutar acciones tendientes a su control. Implementar aprendizajes socialmente significativos.Facultad de Ciencias Veterinaria

Comparison of four polymerase chain reaction assays for the detection of Brucella spp. in clinical samples from dogs

Aim: This study aimed to compare the sensitivity (S), specificity (Sp), and positive likelihood ratios (LR+) of four polymerase chain reaction (PCR) assays for the detection of Brucella spp. in dog's clinical samples. Materials and Methods: A total of 595 samples of whole blood, urine, and genital fluids were evaluated between October 2014 and November 2016. To compare PCR assays, the gold standard was defined using a combination of different serological and microbiological test. Bacterial isolation from urine and blood cultures was carried out. Serological methods such as rapid slide agglutination test, indirect enzyme-linked immunosorbent assay, agar gel immunodiffusion test, and buffered plate antigen test were performed. Four genes were evaluated: (i) The gene coding for the BCSP31 protein, (ii) the ribosomal gene coding for the 16S-23S intergenic spacer region, (iii) the gene coding for porins omp2a/omp2b, and (iv) the gene coding for the insertion sequence IS711. Results: The results obtained were as follows: (1) For the primers that amplify the gene coding for the BCSP31 protein: S: 45.64% (confidence interval [CI] 39.81-51.46), Sp: 95.62% (CI 93.13-98.12), and LR+: 10.43 (CI 6.04-18); (2) for the primers that amplify the ribosomal gene of the 16S-23S rDNA intergenic spacer region: S: 69.80% (CI 64.42-75.18), Sp: 95.62 % (CI 93.13-98.12), and LR+: 11.52 (CI 7.31-18.13); (3) for the primers that amplify the omp2a and omp2b genes: S: 39.26% (CI 33.55-44.97), Sp: 97.31% (CI 95.30-99.32), and LR+ 14.58 (CI 7.25-29.29); and (4) for the primers that amplify the insertion sequence IS711: S: 22.82% (CI 17.89 - 27.75), Sp: 99.66% (CI 98.84-100), and LR+ 67.77 (CI 9.47-484.89). Conclusion: We concluded that the gene coding for the 16S-23S rDNA intergenic spacer region was the one that best detected Brucella spp. in canine clinical samples