Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate

The green side of social innovation: Using sustainable development goals to classify environmental impacts of rural grassroots initiatives

Social innovations are grassroots processes aiming to achieve impacts beyond an individual level and towards a broader societal good. The environmental dimension of impacts refers to any direct change to the environment resulting from social innovation activities, products, or services, which are not addressed by pre-existing systems. In this paper, we determine the role of social innovation in addressing environmental impacts by analyzing a database of social innovation examples in European and circum-Mediterranean rural areas, compiled within the H2020 Project SIMRA. We conceptualize the overall aim of environmentally-focused social innovation initiatives as furthering the sustainable development of their territories. To address the environmental impacts of initiatives in a structured way, we use the Sustainable Development Goals (SDG) classification, to describe social innovation environmental impacts in relation to specific targets. We analyzed 238 initiatives from the SIMRA catalog and associated initiative websites to identify and classify their direct environmental impacts. Our results indicate that 68% of the cases have at least one direct environmental impact that aligns with a SDG target. The most common impacts are related to sustainable natural resource management (SDGs target 12.2), sustainable food production systems (2.4), and equal access to land (2.3). This SDG-based classification proved to be a useful analytical tool for categorizing internationally policy-relevant environmental impacts of social innovations

Exocyclic push-pull conjugated compounds. Part 4. rotational barriers in poorly polarized push-pull ethylenes

The rotational barrier for substituted ethylenes was calculated with MO ab initio methods, both in the Hartree-Fock (HF) single determinant scheme and with multiconfigurational self-consistent field (MCSCF) approaches. The HF model affords reliable results only when applied to molecules substituted with strong electron donor groups, assigning a push-pull character to the molecule. The MCSCF approach was employed for calculating rotational barriers in poorly polarized ethylenes not directly amenable to the HF methods; however, this method was found hard to handle for low symmetry molecules with substituents interacting with the double bond. A method is proposed based on the interpolation of the energy of the perpendicular conformation from a Fourier truncated function constructed with HF molecular energies calculated for frozen conformations twisted up to 60 degrees, The application of HF theory for studying internal rotation in substituted ethylenes with poorly polarized character is discussed and an upper limit of 35-40 kcal/mol can be set up for having reliable barriers from the calculated energy of the rotational transition state at this level of theory. (C) 2001 Elsevier Science B.V. All rights reserved

Exocyclic push-pull conjugated compounds. Part 2. The effect of donor and acceptor substituents on the rotational barrier of push-pull ethylenes

The energy of the rotational barriers and electronic structure of the transition state in substituted ethylenes are discussed in the light of the results obtained from different theoretical MO ab initio approaches. The 6-31G* basis set at Hartree-Fock (HF) level and with second-order Moller-Plesset perturbation theory (MP2) was employed, critical points were localized through Full geometry relaxation and characterized by vibrational analysis. A multiconfigurational approach (MCSCF) with different active spaces was also employed. For alkenes the correct rotational transition state is obtained only from the MCSCF approach, whereas for push-pull olefins the HF approach with correlation corrections at MP2 level provides correct answers for the internal rotation around C(sp(2))-C(sp(2)) bonds. The choice is more critical when only acceptor or donor groups are present, especially when change of hybridization occurs at the atoms at the edges of the C-C bond in the critical points. (C) 2000 Published by Elsevier Science B.V. All rights reserved

Thermoregulatory responses during prolonged swimming with a Wetsuit at 25 °C

The aim of this study was to investigate the effect of prolonged steady state swimming with a wetsuit, on thermoregulatory and behavioural responses, in water of 25°C. Ten male swimmers participated in two 75-min  swim trials, in one wearing a neoprene wetsuit (WS), and the other a conventional swimsuit (SS). The swimming intensity was controlled at 70% of swimmers’ critical velocity (CV). Core (Tcore) and skin (Tsk) temperature, heart rate (HR), and behavioural modulators, were measured before, during and after swimming. A 2-way ANOVA for repeated measures with aFisher’s 2SD test was used for multiple comparisons and a paired t-test with a Tukey post hoc analysis for pre and post measurements at p< 0.05. The results demonstrated an interaction of Tcore (p = 0.039), between time in the water and type of garment worn. Tcore with the WS initially increased until the 45th min and plateaued, while with the SS was continuously decreasing. HR was lower during swimming with the WS and higher with the SS. Thermal sensation and thermal comfort, were more favourable with the WS (p < 0.05). Thus, WS use during prolonged swimming, helped maintain Tcore levels, and improved thermal perceptions, at 25 °C. © 2021 Cardiff Metropolitan University