DNA methylases for site-selective inhibition of type IIS restriction enzyme activity

DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques

Subthreshold depression as a risk indicator for major depressive disorder: a systematic review of prospective studies.

Objective: In order to examine whether the incidence of major depressive disorder (MDD) is increased in subjects with subthreshold depression, or sD (clinically relevant depressive symptoms, without meeting criteria for a full-blown MDD), we conducted a review of prospective studies examining the incidence of MDD in subjects with sD. Method: A systematic literature search was conducted. For all studies, the relative risk of developing MDD was calculated, based on person-years. Results: Twenty studies (23 comparisons) were found, based on community samples, general medical patients and high-risk subjects. Most comparisons showed that subjects with sD had a consistently larger chance of developing MDD. The studies differed considerably in the definition of sD, the recency (occurrence of the last sD) and the in-/ exclusion of lifetime MDD. Conclusion: The incidence of MDD in subjects with sD is larger than in subjects without sD. Otherwise, the concept of sD is too broad to be used. In future studies, some consensus should be reached regarding the definition of sD

A model for optimizing hooked end steel fibre reinforcements in cracked cement composites

The addition of metallic fibres in concrete is a widely used technique to increase its resistance to crack opening, energy absorption capacity, and durability. The properties of the resulting composite strongly depend on the geometry of the fibres, which usually dispose of an hooked end, as well as on the mechanical properties of both the fibre and the concrete. The optimization of the fibre reinforcement mechanisms can only be achieved by an approach that takes into account adequately these aspects. Therefore, in this work, a computational model incorporating the key features of a hooked end fibre embedded in a cement matrix is proposed. The fibre is modelled as a Timoshenko beam, whereas a cohesive interface is used to model the interaction with the surrounding concrete. Different failure mechanisms are defined including fibre debonding or fibre tensile rupture and concrete spalling at fibre exit point. The model is calibrated by using the results of an experimental campaign conducted by authors. A multi-step optimization algorithm is used to find the optimal geometry and model’s constitutive parameters that maximize the peak pull-out force and the energy absorption capacity in a fibre pull-out test. The analysis suggests that the use of concrete with high strength has the potential to increase both peak force and energy absorption capacity by designing the proper geometry of the fibreFEDER through the Operational Program for Competitiveness Factors - COMPETE and Internationalization Program (POCI), under the project NG_ TPfib POCI-01-0247-FEDER-03371

COVID-19 restrictions increased perceptions of social isolation for older people discharged home after rehabilitation: A mixed-methods study

ObjectiveTo explore older persons perceptions of the impact of COVID-19 restrictions on participating in community activities after discharge from inpatient rehabilitation. MethodsMixed-methods study design. Participants were older adults who were discharged home following inpatient rehabilitation. Interviews were conducted with 70 participants, with a variety of diagnoses, 8 weeks after discharge from inpatient rehabilitation. Frequency of participation in domestic, leisure/work and outdoor activities was measured using the Frenchay Activities Index (FAI). Qualitative analysis was completed using qualitative content analysis and triangulated with FAI scores. ResultsIn all, 70 older adults (mean age: 73.0 years, SD: 9.9; 59% female) participated in the study. The overarching theme was that participants felt socially isolated following discharge from rehabilitation, with COVID-19 restrictions increasing perceptions of social isolation and complicating their return to participating in community activities. The four categories informing the overarching theme were as follows: physical health was the primary limitation to participation in community activities; COVID-19 restrictions limited participation in social activities and centre-based physical rehabilitation; low uptake of videoconferencing to facilitate socialisation and rehabilitation; and reduced incidental physical activity. Mean FAI score was 21.2 (SD 7.8), indicating that participants were moderately active. Participants most commonly performed domestic activities (mean: 10.0, SD: 4.1), followed by outdoor activities (mean: 6.6, SD: 3.5) and leisure/work activities (mean: 4.5, SD: 2.5). ConclusionsCOVID-19 restrictions exacerbated perceptions of social isolation and the limitations already imposed by poor physical health after discharge from rehabilitation. The findings highlight the need for rehabilitation that addresses the psychological and social dimensions of community reintegration

The need to promote behaviour change at the cultural level: one factor explaining the limited impact of the MEMA kwa Vijana adolescent sexual health intervention in rural Tanzania. A process evaluation

Background - Few of the many behavioral sexual health interventions in Africa have been rigorously evaluated. Where biological outcomes have been measured, improvements have rarely been found. One of the most rigorous trials was of the multi-component MEMA kwa Vijana adolescent sexual health programme, which showed improvements in knowledge and reported attitudes and behaviour, but none in biological outcomes. This paper attempts to explain these outcomes by reviewing the process evaluation findings, particularly in terms of contextual factors. Methods - A large-scale, primarily qualitative process evaluation based mainly on participant observation identified the principal contextual barriers and facilitators of behavioural change. Results - The contextual barriers involved four interrelated socio-structural factors: culture (i.e. shared practices and systems of belief), economic circumstances, social status, and gender. At an individual level they appeared to operate through the constructs of the theories underlying MEMA kwa Vijana - Social Cognitive Theory and the Theory of Reasoned Action – but the intervention was unable to substantially modify these individual-level constructs, apart from knowledge. Conclusion - The process evaluation suggests that one important reason for this failure is that the intervention did not operate sufficiently at a structural level, particularly in regard to culture. Recently most structural interventions have focused on gender or/and economics. Complementing these with a cultural approach could address the belief systems that justify and perpetuate gender and economic inequalities, as well as other barriers to behaviour change

A frequentist framework of inductive reasoning

Reacting against the limitation of statistics to decision procedures, R. A. Fisher proposed for inductive reasoning the use of the fiducial distribution, a parameter-space distribution of epistemological probability transferred directly from limiting relative frequencies rather than computed according to the Bayes update rule. The proposal is developed as follows using the confidence measure of a scalar parameter of interest. (With the restriction to one-dimensional parameter space, a confidence measure is essentially a fiducial probability distribution free of complications involving ancillary statistics.) A betting game establishes a sense in which confidence measures are the only reliable inferential probability distributions. The equality between the probabilities encoded in a confidence measure and the coverage rates of the corresponding confidence intervals ensures that the measure's rule for assigning confidence levels to hypotheses is uniquely minimax in the game. Although a confidence measure can be computed without any prior distribution, previous knowledge can be incorporated into confidence-based reasoning. To adjust a p-value or confidence interval for prior information, the confidence measure from the observed data can be combined with one or more independent confidence measures representing previous agent opinion. (The former confidence measure may correspond to a posterior distribution with frequentist matching of coverage probabilities.) The representation of subjective knowledge in terms of confidence measures rather than prior probability distributions preserves approximate frequentist validity.Comment: major revisio

Temporal Networks

A great variety of systems in nature, society and technology -- from the web of sexual contacts to the Internet, from the nervous system to power grids -- can be modeled as graphs of vertices coupled by edges. The network structure, describing how the graph is wired, helps us understand, predict and optimize the behavior of dynamical systems. In many cases, however, the edges are not continuously active. As an example, in networks of communication via email, text messages, or phone calls, edges represent sequences of instantaneous or practically instantaneous contacts. In some cases, edges are active for non-negligible periods of time: e.g., the proximity patterns of inpatients at hospitals can be represented by a graph where an edge between two individuals is on throughout the time they are at the same ward. Like network topology, the temporal structure of edge activations can affect dynamics of systems interacting through the network, from disease contagion on the network of patients to information diffusion over an e-mail network. In this review, we present the emergent field of temporal networks, and discuss methods for analyzing topological and temporal structure and models for elucidating their relation to the behavior of dynamical systems. In the light of traditional network theory, one can see this framework as moving the information of when things happen from the dynamical system on the network, to the network itself. Since fundamental properties, such as the transitivity of edges, do not necessarily hold in temporal networks, many of these methods need to be quite different from those for static networks

Visually Guided Avoidance in the Chameleon (Chamaeleo chameleon): Response Patterns and Lateralization

The common chameleon, Chamaeleo chameleon, is an arboreal lizard with highly independent, large-amplitude eye movements. In response to a moving threat, a chameleon on a perch responds with distinct avoidance movements that are expressed in its continuous positioning on the side of the perch distal to the threat. We analyzed body-exposure patterns during threat avoidance for evidence of lateralization, that is, asymmetry at the functional/behavioral levels. Chameleons were exposed to a threat approaching horizontally from the left or right, as they held onto a vertical pole that was either wider or narrower than the width of their head, providing, respectively, monocular or binocular viewing of the threat. We found two equal-sized sub-groups, each displaying lateralization of motor responses to a given direction of stimulus approach. Such an anti-symmetrical distribution of lateralization in a population may be indicative of situations in which organisms are regularly exposed to crucial stimuli from all spatial directions. This is because a bimodal distribution of responses to threat in a natural population will reduce the spatial advantage of predators