Nuclear localisation and function of the g protein-coupled receptor kinase 4 subfamily.

G protein-coupled receptor kinases (GRKs) are a family of serine/threonine kinases that phosphorylate and mediate densensitisation of agonist-occupied G protein-coupled receptors (GPCRs). Recent evidence suggests, however, additional cellular functions for these enzymes. The seven mammalian GRKs are divided into the GRK1-, GRK2- and GRK4-like subfamilies. I have discovered that GRK4 subfamily members contain functional nuclear localisation sequences (NLSs). The location of the NLSs of GRK5 and 6 is similar whereas the NLS of GRK4 is more N-terminal. Although all members of the GRK4 subfamily contain NLSs the nuclear localisation of these kinases is differentially regulated by Ca2+/CaM. Furthermore, the NLS of GRKs 5 and 6, but not GRK4, mediate DNA-binding in vitro, suggesting differential nuclear signalling roles for these enzymes. I investigated structural determinants of GRK5 required for DNA-binding and nuclear localisation. The ability of GRK5 to bind DNA requires an intact NLS and an N-terminally located calmodulin (CaM) binding domain. Additionally, CaM- dependent autophosphorylation of GRK5 reduces the affinity of GRK5 for DNA. Overexpression of GRK5 in cultured rat neonatal cardiac myocytes rescues hypertrophy induced by phenylephrine, an oc-adrenergic receptor agonist, or by activation of PLC. Hypertrophy is not rescued by over expression of GRK5 with a mutant NLS (GRK5ANLS), mutant N-terminal CaM binding site (GRK5NTPB) or a catalytically in active version of the kinase (GRK5K215R). That GRK5 and GRK5ANLS show similar kinase activity in vitro suggests that both the kinase activity and the DNA-binding ability of GRK5 contributes to its role in rescuing hypertrophy. Since GRK5 rescues hypertrophy induced downstream of adrenergic receptors, by PLC activation, GRK5 DNA-binding ability and kinase activity in the nucleus would appear to be required for this effect. Clearly, the GRK4 subfamily of GRKs is more diverse than previously appreciated and play a role in cellular signalling in the nucleus distinct from GPCR desensitisation

Variable Effects of Snow Conditions Across Boreal Mesocarnivore Species

Mesocarnivores are increasingly recognized as key drivers of community dynamics, but the effects of bottom-up and abiotic factors on mesocarnivore populations remain poorly understood. We evaluated the effects of snow conditions, prey abundance, and habitat type on the distribution of five sympatric mesocarnivore species in interior Alaska using repeated snow track surveys and occupancy modelling. Snow depth and snow compaction were the best predictors of mesocarnivore occupancy, with differential effects across species. Coyotes (Canis latrans (Say, 1823)) and red foxes (Vulpes vulpes L., 1758) occurred in areas of shallow, compact snow, Canada lynx (Lynx canadensis (Kerr, 1792)) occurred in areas of shallow, fluffy snow, and wolverines (Gulo gulo L., 1758) and marten (Martes americana (Turton, 1806)) occurred in areas of deep, fluffy snow. These findings indicate that altered snow conditions due to climate change may have strong direct effects on the distribution of northern mesocarnivores, with divergent effects across species

Identification and quantification of <i>Acanthamoeba</i> spp. within seawater at four coastal lagoons on the east coast of Australia

Acanthamoeba is an opportunistic free-living heterotrophic protist that is the most predominant amoeba in diverse ecological habitats. Acanthamoeba causes amoebic keratitis (AK), a painful and potentially blinding corneal infection. Major risk factors for AK have been linked to non-optimal contact lens hygiene practices and Acanthamoeba contamination of domestic and recreational water. This study investigated the incidence and seasonal variation of Acanthamoeba spp. within coastal lagoons located on the eastern coast of Australia and then examined the association between Acanthamoeba and water abiotic factors and bacterial species within the water.Water samples were collected from four intermittently closed and open lagoons (ICOLLs) (Wamberal, Terrigal, Avoca and Cockrone) every month between August 2019 to July 2020 except March and April. qPCR was used to target the Acanthamoeba 18S rRNA gene, validated by Sanger sequencing. Water abiotic factors were measured in situ using a multiprobe metre and 16S rRNA sequencing (V3-V4) was performed to characterise bacterial community composition. Network analysis was used to gauge putative associations between Acanthamoeba incidence and bacterial amplicon sequence variants (ASVs).Among 206 water samples analysed, 79 (38.3%) were Acanthamoeba positive and Acanthamoeba level was significantly higher in summer compared with winter, spring, or autumn (p = 0.008). More than 50% (23/45) water samples of Terrigal were positive for Acanthamoeba which is a highly urbanised area with extensive recreational activities while about 32% (16/49) samples were positive from Cockrone that is the least impacted lagoon by urban development. All sequenced strains belonged to the pathogenic genotype T4 clade except two which were of genotype clades T2 and T5. Water turbidity, temperature, intl1 gene concentration, and dissolved O2 were significantly associated with Acanthamoeba incidence (p &lt; 0.05). The ASVs level of cyanobacteria, Pseudomonas spp., Candidatus spp., and marine bacteria of the Actinobacteria phylum and Acanthamoeba 18S rRNA genes were positively correlated (Pearson's r ≥ 0.14). The presence of Acanthamoeba spp. in all lagoons, except Wamberal, was associated with significant differences in the composition of bacterial communities (beta diversity).The results of this study suggest that coastal lagoons, particularly those in urbanised regions with extensive water recreational activities, may pose an elevated risk to human health due to the relatively high incidence of pathogenic Acanthamoeba in the summer. These findings underscore the importance of educating the public about the rare yet devastating impact of AK on vision and quality of life, highlighting the need for collaborative efforts between public health officials and educators to promote awareness and preventive measures, especially focusing lagoons residents and travellers

Life course associations of height, weight, fatness, grip strength, and all-cause mortality for high socioeconomic status Guatemalans

Objectives: The objective of this study was to investigate the association between physical growth in pre-adult life with five outcomes at age 64-76: weight, body mass index, estimated body fat percentage, hand grip strength and mortality. Methods: Super-Imposition by Translation and Rotation (SITAR) growth curves of 40,484 Guatemalan individuals aged 3-19 years were modelled for the parameters of size, timing and intensity (peak growth velocity, e.g. cm/year) of height, weight, body mass index, and grip strength. Associations between the SITAR parameters and old age outcomes were tested using linear and binary logistic regression for a follow-up sample of high socioeconomic status (SES) Guatemalans, of whom 50 were aged 64-76 years old at re-measurement and 45 died prior to the year 2017. Results: SITAR models explained 69-98% of the variance in each outcome, with height the most precise. Individuals in the follow-up sample who had a higher BMI before age 20 years had higher estimated body fat (B=1.4 CI -0.02-2.8) and BMI (B=1.2, CI 0.2-2.2) at the ages 64-76 years. Those who grew slower in height but faster in weight and BMI before age 20 years, had higher BMI and body fat later in life. Conclusions: These findings highlight the importance of a life course perspective on health and mortality risk. Childhood exposures leading to variation in pre-adult growth may be key to better understanding health and mortality risks in old age

Template-stripped gold surfaces with 0.4 nm rms roughness suitable for force measurements. Application to the Casimir force in the 20-100 nm range

Using a template-stripping method, macroscopic gold surfaces with root-mean-square (rms) roughness less than 0.4 nm have been prepared, making them useful for studies of surface interactions in the nanometer range. The utility of such substrates is demonstrated by measurements of the Casimir force at surface separations between 20 and 100 nm, resulting in good agreement with theory. The significance and quantification of this agreement is addressed, as well as some methodological aspects regarding the measurement of the Casimir force with high accuracy.Comment: 7 figure

Action research in physical education: focusing beyond myself through cooperative learning

This paper reports on the pedagogical changes that I experienced as a teacher engaged in an action research project in which I designed and implemented an indirect, developmentally appropriate and child‐centred approach to my teaching. There have been repeated calls to expunge – or at least rationalise – the use of traditional, teacher‐led practice in physical education. Yet despite the advocacy of many leading academics there is little evidence that such a change of approach is occurring. In my role as teacher‐as‐researcher I sought to implement a new pedagogical approach, in the form of cooperative learning, and bring about a positive change in the form of enhanced pupil learning. Data collection included a reflective journal, post‐teaching reflective analysis, pupil questionnaires, student interviews, document analysis, and non‐participant observations. The research team analysed the data using inductive analysis and constant comparison. Six themes emerged from the data: teaching and learning, reflections on cooperation, performance, time, teacher change, and social interaction. The paper argues that cooperative learning allowed me to place social and academic learning goals on an even footing, which in turn placed a focus on pupils’ understanding and improvement of skills in athletics alongside their interpersonal development

Scale-free memory model for multiagent reinforcement learning. Mean field approximation and rock-paper-scissors dynamics

A continuous time model for multiagent systems governed by reinforcement learning with scale-free memory is developed. The agents are assumed to act independently of one another in optimizing their choice of possible actions via trial-and-error search. To gain awareness about the action value the agents accumulate in their memory the rewards obtained from taking a specific action at each moment of time. The contribution of the rewards in the past to the agent current perception of action value is described by an integral operator with a power-law kernel. Finally a fractional differential equation governing the system dynamics is obtained. The agents are considered to interact with one another implicitly via the reward of one agent depending on the choice of the other agents. The pairwise interaction model is adopted to describe this effect. As a specific example of systems with non-transitive interactions, a two agent and three agent systems of the rock-paper-scissors type are analyzed in detail, including the stability analysis and numerical simulation. Scale-free memory is demonstrated to cause complex dynamics of the systems at hand. In particular, it is shown that there can be simultaneously two modes of the system instability undergoing subcritical and supercritical bifurcation, with the latter one exhibiting anomalous oscillations with the amplitude and period growing with time. Besides, the instability onset via this supercritical mode may be regarded as "altruism self-organization". For the three agent system the instability dynamics is found to be rather irregular and can be composed of alternate fragments of oscillations different in their properties.Comment: 17 pages, 7 figur

Anaerobic granular sludge as a biocatalyst for 1,3-propanediol production from glycerol in continuous bioreactors

1,3-propanediol (1,3-PDO) was produced from glycerol in three parallel Expanded Granular Sludge Blanket (EGSB) reactors inoculated with granular sludge (control reactor-R1), heat-treated granular sludge (R2) and disrupted granular sludge (R3) at Hydraulic Retention Times (HRT) between 3 and 24 h. Maximum 1,3-PDO yield (0.52 mol mol-1) and productivity (57 g L-1 d-1) were achieved in R1 at HRTs of 12 h and 3 h, respectively. DGGE profiling of PCR-amplified 16S rRNA gene fragments showed that variations in the HRT had a critical impact in the dominant community of microorganisms. However, no appreciable differences in the bacterial population were observed between R2 and R3 at low HRTs. Production of H2 was observed at the beginning of the operation, but no methane production was observed. This study proves the feasibility of 1,3-PDO production in EGSB reactors and represents a novel strategy to valorise glycerol generated in the biodiesel industry.The financial support given to Roberto Gallardo from Fundacao da Ciencia e da Tecnologia (ref SFRH/BD/42900/2008) is gratefully acknowledged. The authors thank the MIT- Portugal Program for the support given to R. Gallardo and C. Faria