Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition.

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.

Semantic Priming from Uncued Distractors in Alzheimer's Disease.

RESEARCH QUESTION: Are semantic impairments in Alzheimer's disease (AD) partially due to deficits in spatial attention? METHODS AND RESULTS: In a target detection task, both older adults (OAs) and AD individuals were facilitated by valid spatial cues, but only OAs were impaired by invalid cues compared to neutral. In a reading task, spatial cues validly or invalidly cued the location of pictures, which were related or unrelated to subsequent, centrally presented, words. OAs showed semantic priming only after valid cues, whereas AD individuals showed priming after valid and invalid cues. DISCUSSION: Failure to inhibit uncued locations results in processing of potentially distracting semantic information in AD.

A systematic review of economic evaluations of preoperative smoking cessation for preventing surgical complications.

BACKGROUND: Whilst there is a substantial body of evidence on the costs and benefits of smoking cessation generally, the benefits of routinely providing smoking cessation for surgical populations are less well known. This review summarises the evidence on the cost-effectiveness of preoperative smoking cessation to prevent surgical complications. MATERIALS AND METHODS: A search of the Cochrane, Econlit, EMBASE, Health Technology Assessment, Medline Complete and Scopus databases was conducted from inception until June 23, 2021. Peer-reviewed, English-language articles describing economic evaluations of preoperative smoking cessation interventions to prevent surgical complications were included. Search results were independently screened for potentially eligible studies. Study characteristics, economic evaluation methods and cost-effectiveness results were extracted by one reviewer and details checked by a second. Two authors independently assessed reporting and methodological quality using the Consolidated Health Economic Evaluation Reporting Standards statement (CHEERS) and the Quality of Health Economic Studies Instrument checklist (QHES) respectively. RESULTS: After removing duplicates, twenty full text articles were screened from 1423 database records, resulting in six included economic evaluations. Studies from the United States (n = 4), France (n = 1) and Spain (n = 1) were reported between 2009 and 2020. Four evaluations were conducted from a payer perspective. Two-thirds of evaluations were well-conducted (mean score 83) and well-reported (on average, 86% items reported). All studies concluded preoperative smoking cessation is cost-effective for preventing surgical complications; results ranged from cost saving to €53,131 per quality adjusted life year gained. CONCLUSIONS: Preoperative smoking cessation is cost-effective for preventing surgical complications from a payer or provider perspective when compared to standard care. There is no evidence from outside the United States and Europe to inform healthcare providers, funders and policy-makers in other jurisdictions and more information is needed to clarify the optimal point of implementation to maximise cost-effectiveness of preoperative smoking cessation intervention. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO 2021 CRD42021257740. RESEARCH REGISTRY REGISTRATION NUMBER: reviewregistry1369.

Protocol for a systematic review of economic evaluations of preoperative smoking cessation interventions for preventing surgical complications.

INTRODUCTION: The short-term economic benefit of embedding best practice tobacco dependence treatment (TDT) into healthcare services prior to surgery across different populations and jurisdictions is largely unknown. The aim of this systematic review is to summarise the cost-effectiveness of preoperative smoking cessation interventions for preventing surgical complications compared with usual care. The results will provide hospital managers, clinicians, healthcare professionals and policymakers with a critical summary of the economic evidence on providing TDT routinely before surgery, aiding the development and dissemination of unified, best practice guidelines, that is, implementation of article 14 of the WHO Framework Convention on Tobacco Control. METHODS AND ANALYSIS: A comprehensive search of peer-reviewed literature will be conducted from database inception until 23 June 2021 (Cochrane, Econlit, Embase, Health Technology Assessment, Medline Complete, Scopus). Published, English-language articles describing economic evaluations of preoperative smoking cessation interventions for preventing surgical complications will be included. One researcher will complete the searches and two researchers will independently screen results for eligible studies. Any disagreement will be resolved by the third researcher. A narrative summary of included studies will be provided. Study characteristics, economic evaluation methods and cost-effectiveness results will be extracted by one reviewer and descriptive analyses will be undertaken. A second reviewer will review data extracted for accuracy from 10% of the included studies. Reporting and methodological quality of the included studies will be evaluated independently by two reviewers using the Consolidated Health Economic Evaluation Reporting Standards statement and the Quality of Health Economic Studies Instrument checklist, respectively. ETHICS AND DISSEMINATION: This research does not require ethics approval because the study is a planned systematic review of published literature. Findings will be presented at health economic, public health and tobacco control conferences, published in a peer-reviewed journal and disseminated via social media. TRIAL REGISTRATION NUMBER: CRD42021257740.

Age-related delay in reduced accessibility of refreshed items.

Previously, we demonstrated that in young adults, briefly thinking of (i.e., refreshing) a just-seen word impairs immediate (100-ms delay) perceptual processing of the word, relative to words seen but not refreshed. We suggested that such reflective-induced inhibition biases attention toward new information. Here, we investigated whether reduced accessibility of refreshed targets dissipates with a longer delay and whether older adults would show a smaller and/or delayed effect compared with young adults. Young adult and older adult participants saw 2 words, followed by a cue to refresh one of these words. After either a 100-ms or 500-ms delay, participants read a word that was the refreshed word (refreshed probe), the nonrefreshed word (nonrefreshed probe), or a new word (novel probe). Young adults were slower to read refreshed probes than nonrefreshed probes at the 100-ms, but not the 500-ms, delay. Conversely, older adults were slower to read refreshed probes than nonrefreshed probes at the 500-ms, but not the 100-ms, delay. The delayed slowing of responses to refreshed probes was primarily observed in older-old adults (75+ years). A delay in suppressing the target of refreshing may disrupt the fluidity with which attention can be shifted to a new target. Importantly, a long-term memory benefit of refreshing was observed for both ages and delays. These results suggest that a full characterization of age-related memory deficits should consider the time course of effects and how specific component cognitive processes affect both working and long-term memory. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes.

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

High-content, high-throughput screening for the identification of cytotoxic compounds based on cell morphology and cell proliferation markers.

Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays.

A high-throughput assay to identify modifiers of premature chromosome condensation.

Premature chromosome condensation (PCC) is a consequence of early mitotic entry, where mitosis begins before completion of DNA replication. Previously we have identified mutations in MCPH1, a DNA damage response and potential tumor suppressor gene, as a cause of primary microcephaly and PCC. Here we describe a high-throughput assay to identify modifiers of PCC. Reverse transfection of control siRNA followed by a forward transfection of MCPH1 small interfering RNA (siRNA) was performed to induce PCC. Condensin II subunits CAPG2 and CAPH2 were validated as PCC modifiers and therefore positive controls. Cell nuclei were detected by DAPI staining using an Operetta imaging system. PCC and nuclei number were determined using Columbus analysis software. Two batches of nine plates were used to determine assay efficacy. Each plate contained four negative (nontargeting) and eight positive control siRNAs. Mean % PCC was 12.35% (n = 72) for negative controls and 4.25% (n = 144) for positive controls. Overall false-positive and false-negative rates were 0% (n = 72) and 2.1% (n = 144), respectively. This assay is currently being used to screen a human druggable genome siRNA library to identify novel therapeutic targets for cancer treatment. The assay can also be used to identify novel compounds and genes that induce PCC.

Foraging for thought: an inhibition-of-return-like effect resulting from directing attention within working memory.

Perceptual processing of a target stimulus may be inhibited if its location has just been cued, a phenomenon of spatial attention known as inhibition of return (IOR). In the research reported here, we demonstrated a striking effect, wherein items that have just been the focus of reflective attention (internal attention to an active representation) also are inhibited. Participants saw two items, followed by a cue to think back to (i.e., refresh, or direct reflective attention toward) one item, and then had to identify either the refreshed item, the unrefreshed item, or a novel item. Responses were significantly slower for refreshed items than for unrefreshed items, although refreshed items were better remembered on a later memory test. Control experiments in which we replaced the refresh event with a second presentation of one of the words did not show similar effects. These results suggest that reflective attention can produce an inhibition effect for attended items that may be analogous to IOR effects in perceptual attention.

Lost thoughts: implicit semantic interference impairs reflective access to currently active information.

Why do we lose, or have trouble accessing, an idea that was in the focus of attention only a moment ago, especially in the absence of any apparent distraction? We tested the hypothesis that accessing a single item that is already active is affected by implicit interference (interference of which we have little or no awareness). We presented masked words that were semantically related or unrelated to a single visible target word that participants were cued to think of (refresh) a half second after its offset. Masked related but not unrelated words increased time to refresh the target but did not influence time required to read a target that was physically present. These findings provide novel evidence that an item in the focus of attention is subject to semantic interference. We suggest that such implicit semantic interference may contribute to the common "lost thought" experience and to cognitive deficits in populations in which refreshing is impaired.

A survey of environmental pollutants and cellular-stress markers of Porites astreoides at six sites in St. John, U.S. Virgin Islands.

Coral communities along the coast of St. John, U.S. Virgin Islands have exhibited site-specific behavior in declines. In order to determine if these specific coral communities are stressed and whether a pollutant or environmental factor present at this site is a probable stressor, we surveyed six near-shore coral communities in St. John, USVI for environmental pollutants and to determine the cellular physiological condition of the coral, Porites astreoides. The six sites within St. John are Cruz Bay, Caneel Bay, Hawksnest Bay, Trunk Bay, Tektite Reef in Beehive Bay, and Red Point. Red Point was considered the reference site because of its abundance and diversity of species, and it was the furthest removed from down-stream and down-current anthropogenic activities. All sites showed distinct cellular-stress marker patterns, indicating that the physiological condition of each population was different. Populations at Cruz, Hawksnest, Trunk, and Tektite were stressed, as indicated by high levels of DNA lesions and expression of stress proteins. Hawksnest and Tektite were contaminated with polyaromatic hydrocarbons (PAHs), while Cruz was contaminated with semi-volatile organochlorines and nitrogen-based biocides. At least for Hawksnest and Tektite, stress-marker patterns were consistent with an exposure to PAHs. Fecal coliform levels were high in Cruz and Trunk, indicating fecal contamination, as well as consideration for management action. Results from this study serve as a justification for a more thorough and methodical investigation into the stressors responsible for declines of coral populations within St. John. Furthermore, this study supports the argument for the importance of local factors contributing to regional coral reef declines; that not all forces impacting coral are global.

Human ASPM participates in spindle organisation, spindle orientation and cytokinesis.

BACKGROUND: Mutations in the Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. ASPM encodes a mitotic spindle pole associated protein. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC). RESULTS: We show that ASPM is a microtubule minus end-associated protein that is recruited in a microtubule-dependent manner to the pericentriolar matrix (PCM) at the spindle poles during mitosis. ASPM siRNA reduces ASPM protein at the spindle poles in cultured U2OS cells and severely perturbs a number of aspects of mitosis, including the orientation of the mitotic spindle, the main determinant of developmental asymmetrical cell division. The majority of ASPM depleted mitotic cells fail to complete cytokinesis. In MCPH patient fibroblasts we show that a pathogenic ASPM splice site mutation results in the expression of a novel variant protein lacking a tripeptide motif, a minimal alteration that correlates with a dramatic decrease in ASPM spindle pole localisation. Moreover, expression of dominant-negative ASPM C-terminal fragments cause severe spindle assembly defects and cytokinesis failure in cultured cells. CONCLUSIONS: These observations indicate that ASPM participates in spindle organisation, spindle positioning and cytokinesis in all dividing cells and that the extreme C-terminus of the protein is required for ASPM localisation and function. Our data supports the hypothesis that the MCPH phenotype caused by ASPM mutation is a consequence of mitotic aberrations during neurogenesis. We propose the effects of ASPM mutation are tolerated in somatic cells but have profound consequences for the symmetrical division of NPCs, due to the unusual morphology of these cells. This antagonises the early expansion of the progenitor pool that underpins cortical neurogenesis, causing the MCPH phenotype.

Age differences in brain activity during perceptual versus reflective attention.

This functional magnetic resonance imaging study presented participants with a face and scene simultaneously on each trial, and assessed the impact of perceptual versus reflective selective attention on activity in parahippocampal place area. Young and older adults showed equivalent activation in parahippocampal place area when cued to attend to the scene when the stimuli were perceptually present and when cued to refresh (briefly think about) the scene after the stimuli were no longer present. The groups also showed equivalent deactivation when cued to attend to the face when the stimuli were perceptually present. However, older adults showed less deactivation than young adults when cued to refresh the face, providing evidence for greater age-related disruption of reflective than perceptual selective attention.

The consequence of refreshing for access to nonselected items in young and older adults.

We examined the effect of competition on briefly thinking of just-seen items. In Experiment 1, participants saw a set of either three related or three unrelated words and then read one of the words again (repeat) or thought briefly of one of the words (refresh). Participants read the set a second time, after which they either refreshed a second word from the set or read a new word. In comparison with reading a new word, response times were slower for refreshing the second item when participants had just refreshed than when they had just repeated the first item. This increase was larger for related words than for unrelated words and for older adults than for younger adults. In Experiment 2, a negative impact of refreshing was observed when participants repeated a different word from the set. The pattern of findings suggests that the negative impact of refreshing comes from increased competition from the refreshed item, rather than from inhibition of the nonrefreshed items.

A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size.

Autosomal recessive primary microcephaly is a potential model in which to research genes involved in human brain growth. We show that two forms of the disorder result from homozygous mutations in the genes CDK5RAP2 and CENPJ. We found neuroepithelial expression of the genes during prenatal neurogenesis and protein localization to the spindle poles of mitotic cells, suggesting that a centrosomal mechanism controls neuron number in the developing mammalian brain.

Preserved spatial memory over brief intervals in older adults.

Two studies compared young and older adults' memory for location information after brief intervals. Experiment 1 found that accuracy of intentional spatial memory for individual locations was similar in young and older participants for set sizes of 3 and 6. Both groups also encoded individual locations in relation to the larger configuration of locations. Experiment 2 showed that like young adults, older adults' latency to respond to a test probe in a letter working memory task was negatively influenced by spatial information that was irrelevant to the task. This interference effect indicated preserved incidental memory for spatial information in older adults. Together, these data suggest that initial encoding of spatial information for relatively small numbers of items is largely preserved in healthy older adults and that representations of spatial information persist over short intervals.