Analysis of nucleotide binding to Dictyostelium myosin II motor domains containing a single tryptophan near the active site.

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp(501) in the relay loop

The dynamics of the relay loop tryptophan residue in the Dictyostelium myosin motor domain and the origin of spectroscopic signals.

Steady-state and time-resolved fluorescence measurements were performed on a Dictyostelium discoideum myosin II motor domain construct retaining a single tryptophan residue at position 501, located on the relay loop. Other tryptophan residues were mutated to phenylalanine. The Trp-501 residue showed a large enhancement in fluorescence in the presence of ATP and a small quench in the presence of ADP as a result of perturbing both the ground and excited state processes. Fluorescence lifetime and quantum yield measurements indicated that at least three microstates of Trp-501 were present in all nucleotide states examined, and these could not be assigned to a particular gross onformation of the motor domain. Enhancement in emission intensity was associated with a reduction of the contribution from a statically quenched component and an increase in a component with a 5-ns lifetime, with little change in the contribution from a 1-ns lifetime component. Anisotropy measurements indicated that the Trp-501 side chain was relatively immobile in all nucleotide states, and the fluorescence was effectively depolarized by rotation of the whole motor domain with a correlation time on 50-70 ns. Overall these data suggest that the backbone of the relay loop remains structured throughout the myosin ATPase cycle but that the Trp-501 side chain experiences a different weighting in local environments provided by surrounding residues as the adjacent converter domain rolls around the relay loop

Feasibility and reliability of frailty assessment in the critically ill: a systematic review

Background. For healthcare systems, an ageing population poses challenges in the delivery of equitable and effective care. Frailty assessment has the potential to improve care in the intensive care setting, but applying assessment tools in critical illness may be problematic. The aim of this systematic review was to evaluate evidence for the feasibility and reliability of frailty assessment in critical care. Methods. Our primary search was conducted in Medline, Medline In-process, EMBASE, CINAHL, PsycINFO, AMED, Cochrane Database of Systematic Reviews, and Web of Science (January 2001 to October 2017). We included observational studies reporting data on feasibility and reliability of frailty assessment in critical care setting in patients 16 years and older. Feasibility was assessed in terms of timing of evaluation, the background, training and expertise required for assessors, and reliance upon proxy input. Reliability was assessed in terms of inter-rater reliability. Results. Data from 11 study publications are included, representing eight study cohorts and 7761 patients. Proxy involvement in frailty assessment ranged from 58- 100%. Feasibility data were not well-reported overall, but the exclusion rate due to lack of proxy availability ranged from 0 to 45%, the highest rate observed where family involvement was mandatory and the assessment tool relatively complex (Frailty Index, FI). Conventional elements of Frailty Phenotype (FP) assessment required modification prior to use in two studies. Clinical staff tended to use a simple judgement-based tool, the Clinical Frailty Scale (CFS). Inter-rater reliability was reported in one study using the CFS and although a good level of agreement was observed between clinician assessments, this was a small and single centre study. Conclusion. Though of unproven reliability in the critically ill, CFS was the tool used most widely by critical care clinical staff. Conventional FP assessment required modification for general application in critical care, and a FI-based assessment may be difficult to deliver by the critical care team on a routine basis. There is a high reliance on proxies for frailty assessment, and the reliability of frailty assessment tools in critical care needs further evaluation. PROSPERO CRD42016052073

Involvement of Cyclin K Posttranscriptional Regulation in the Formation of Artemia Diapause Cysts

Background: Artemia eggs tend to develop ovoviviparously to yield nauplius larvae in good rearing conditions; while under adverse situations, they tend to develop oviparously and encysted diapause embryos are formed instead. However, the intrinsic mechanisms regulating this process are not well understood. Principal Finding: This study has characterized the function of cyclin K, a regulatory subunit of the positive transcription elongation factor b (P-TEFb) in the two different developmental pathways of Artemia. In the diapause-destined embryo, Western blots showed that the cyclin K protein was down-regulated as the embryo entered dormancy and reverted to relatively high levels of expression once development resumed, consistent with the fluctuations in phosphorylation of position 2 serines (Ser2) in the C-terminal domain (CTD) of the largest subunit (Rpb1) of RNA polymerase II (RNAP II). Interestingly, the cyclin K transcript levels remained constant during this process. In vitro translation data indicated that the template activity of cyclin K mRNA stored in the postdiapause cyst was repressed. In addition, in vivo knockdown of cyclin K in developing embryos by RNA interference eliminated phosphorylation of the CTD Ser2 of RNAP II and induced apoptosis by inhibiting the extracellular signal-regulated kinase (ERK) survival signaling pathway. Conclusions/Significance: Taken together, these findings reveal a role for cyclin K in regulating RNAP II activity during diapause embryo development, which involves the post-transcriptional regulation of cyclin K. In addition, a further role wa

Impact of cognitive stimulation on ripples within human epileptic and non-epileptic hippocampus

Background: Until now there has been no way of distinguishing between physiological and epileptic hippocampal ripples in intracranial recordings. In the present study we addressed this by investigating the effect of cognitive stimulation on interictal high frequency oscillations in the ripple range (80-250 Hz) within epileptic (EH) and non-epileptic hippocampus (NH). Methods: We analyzed depth EEG recordings in 10 patients with intractable epilepsy, in whom hippocampal activity was recorded initially during quiet wakefulness and subsequently during a simple cognitive task. Using automated detection of ripples based on amplitude of the power envelope, we analyzed ripple rate (RR) in the cognitive and resting period, within EH and NH. Results: Compared to quiet wakefulness we observed a significant reduction of RR during cognitive stimulation in EH, while it remained statistically marginal in NH. Further, we investigated the direct impact of cognitive stimuli on ripples (i.e. immediately post-stimulus), which showed a transient statistically significant suppression of ripples in the first second after stimuli onset in NH only. Conclusion: Our results point to a differential reactivity of ripples within EH and NH to cognitive stimulation

Dying well with reduced agency: a scoping review and thematic synthesis of the decision-making process in dementia, traumatic brain injury and frailty

Background In most Anglophone nations, policy and law increasingly foster an autonomy-based model, raising issues for large numbers of people who fail to fit the paradigm, and indicating problems in translating practical and theoretical understandings of ‘good death’ to policy. Three exemplar populations are frail older people, people with dementia and people with severe traumatic brain injury. We hypothesise that these groups face some over-lapping challenges in securing good end-of-life care linked to their limited agency. To better understand these challenges, we conducted a scoping review and thematic synthesis. Methods To capture a range of literature, we followed established scoping review methods. We then used thematic synthesis to describe the broad themes emerging from this literature. Results Initial searches generated 22,375 references, and screening yielded 49, highly heterogeneous, studies that met inclusion criteria, encompassing 12 countries and a variety of settings. The thematic synthesis identified three themes: the first concerned the processes of end-of-life decision-making, highlighting the ambiguity of the dominant shared decision-making process, wherein decisions are determined by families or doctors, sometimes explicitly marginalising the antecedent decisions of patients. Despite this marginalisation, however, the patient does play a role both as a social presence and as an active agent, by whose actions the decisions of those with authority are influenced. The second theme examined the tension between predominant notions of a good death as ‘natural’ and the drive to medicalise death through the lens of the experiences and actions of those faced with the actuality of death. The final theme considered the concept of antecedent end-of-life decision-making (in all its forms), its influence on policy and decision-making, and some caveats that arise from the studies. Conclusions Together these three themes indicate a number of directions for future research, which are likely to be applicable to other conditions that result in reduced agency. Above all, this review emphasises the need for new concepts and fresh approaches to end of life decision-making that address the needs of the growing population of frail older people, people with dementia and those with severe traumatic brain injury

Brain Cortical Mapping by Simultaneous Recording of Functional Near Infrared Spectroscopy and Electroencephalograms from the Whole Brain During Right Median Nerve Stimulation

To investigate relationships between hemodynamic responses and neural activities in the somatosensory cortices, hemodynamic responses by near infrared spectroscopy (NIRS) and electroencephalograms (EEGs) were recorded simultaneously while subjects received electrical stimulation in the right median nerve. The statistical significance of the hemodynamic responses was evaluated by a general linear model (GLM) with the boxcar design matrix convoluted with Gaussian function. The resulting NIRS and EEGs data were stereotaxically superimposed on the reconstructed brain of each subject. The NIRS data indicated that changes in oxy-hemoglobin concentration increased at the contralateral primary somatosensory (SI) area; responses then spread to the more posterior and ipsilateral somatosensory areas. The EEG data indicated that positive somatosensory evoked potentials peaking at 22 ms latency (P22) were recorded from the contralateral SI area. Comparison of these two sets of data indicated that the distance between the dipoles of P22 and NIRS channels with maximum hemodynamic responses was less than 10 mm, and that the two topographical maps of hemodynamic responses and current source density of P22 were significantly correlated. Furthermore, when onset of the boxcar function was delayed 5–15 s (onset delay), hemodynamic responses in the bilateral parietal association cortices posterior to the SI were more strongly correlated to electrical stimulation. This suggests that GLM analysis with onset delay could reveal the temporal ordering of neural activation in the hierarchical somatosensory pathway, consistent with the neurophysiological data. The present results suggest that simultaneous NIRS and EEG recording is useful for correlating hemodynamic responses to neural activity