Kinetic modeling of molecular motors: pause model and parameter determination from single-molecule experiments

Single-molecule manipulation experiments of molecular motors provide essential information about the rate and conformational changes of the steps of the reaction located along the manipulation coordinate. This information is not always sufficient to define a particular kinetic cycle. Recent single-molecule experiments with optical tweezers showed that the DNA unwinding activity of a Phi29 DNA polymerase mutant presents a complex pause behavior, which includes short and long pauses. Here we show that different kinetic models, considering different connections between the active and the pause states, can explain the experimental pause behavior. Both the two independent pause model and the two connected pause model are able to describe the pause behavior of a mutated Phi29 DNA polymerase observed in an optical tweezers single-molecule experiment. For the two independent pause model all parameters are fixed by the observed data, while for the more general two connected pause model there is a range of values of the parameters compatible with the observed data (which can be expressed in terms of two of the rates and their force dependencies). This general model includes models with indirect entry and exit to the long-pause state, and also models with cycling in both directions. Additionally, assuming that detailed balance is verified, which forbids cycling, this reduces the ranges of the values of the parameters (which can then be expressed in terms of one rate and its force dependency). The resulting model interpolates between the independent pause model and the indirect entry and exit to the long-pause state model

Assessment of the sensitivity and specificity of serological (IFAT) and molecular (direct PCR) techniques for diagnosis of leishmaniasis in lagomorphs using a Bayesian approach

Leishmaniasis, caused by Leishmania infantum, is a vector-borne zoonotic disease that is endemic to the Mediterranean basin. The potential of rabbits and hares to serve as competent reservoirs for the disease has recently been demonstrated, although assessment of the importance of their role on disease dynamics is hampered by the absence of quantitative knowledge on the accuracy of diagnostic techniques in these species. A Bayesian latent-class model was used here to estimate the sensitivity and specificity of the Immuno-fluorescence antibody test (IFAT) in serum and a Leishmania-nested PCR (Ln-PCR) in skin for samples collected from 217 rabbits and 70 hares from two different populations in the region of Madrid, Spain. A two-population model, assuming conditional independence between test results and incorporating prior information on the performance of the tests in other animal species obtained from the literature, was used. Two alternative cut-off values were assumed for the interpretation of the IFAT results: 1/50 for conservative and 1/25 for sensitive interpretation. Results suggest that sensitivity and specificity of the IFAT were around 70–80%, whereas the Ln-PCR was highly specific (96%) but had a limited sensitivity (28.9% applying the conservative interpretation and 21.3% with the sensitive one). Prevalence was higher in the rabbit population (50.5% and 72.6%, for the conservative and sensitive interpretation, respectively) than in hares (6.7% and 13.2%). Our results demonstrate that the IFAT may be a useful screening tool for diagnosis of leishmaniasis in rabbits and hares. These results will help to design and implement surveillance programmes in wild species, with the ultimate objective of early detecting and preventing incursions of the disease into domestic and human populations.