Principal component analysis to identify the major contributors to task-activated neurovascular responses.

Background: Consensus on the optimal metrics for neurovascular coupling (NVC) is lacking. The aim of this study was to use principal component analysis (PCA) to determine the most significant contributors to NVC responses in healthy adults (HC), Alzheimer's disease (AD), and mild cognitive impairment (MCI). New method: PCA was applied to three datasets: 1) 69 HC, 2) 30 older HC, 34 AD, and 22 MCI, 3) 1&2 combined. Data were extracted on peak percentage change in cerebral blood flow velocity (CBFv), variance ratio (VR), cross-correlation function peak (CCF), and blood pressure, for five cognitive tasks. An equamax rotation was applied and factors were significant where the eignevalue was ≥1. Rotated factor loadings ≥0.4 determined significant NVC variables. Results: PCA identified 12 significant factors accounting for 78% of variance (all datasets). Contributing variables loaded differently on the factors across the datasets. In datasets 1&2, peak percentage change in CBFv contributed to factors explaining the most variance (45-58%), whereas cognitive test scores, fluency and memory domains contributed the least (15-37%). In the combined dataset, CBFv, CCF and fluency domain contributed the majority (33-43%), whereas VR and attention the least (6-24%). Conclusions: Peak percentage change in CBFv and the visuospatial task consistently accounted for a large proportion of the variance, suggesting these are robust NVC markers for future studies.

DDX5 potentiates HIV-1 transcription as a co-factor of Tat.

BACKGROUND: HIV-1 does not encode a helicase and hijacks those of the cell for efficient replication. We and others previously showed that the DEAD box helicase, DDX5, is an essential HIV dependency factor. DDX5 was recently shown to be associated with the 7SK snRNP. Cellular positive transcription elongation factor b (P-TEFb) is bound in an inactive form with HEXIM1/2 on 7SK snRNP. The Tat/P-TEFb complex is essential for efficient processivity of Pol II in HIV-1 transcription elongation and Tat competes with HEXIM1/2 for P-TEFb. We investigated the precise role of DDX5 in HIV replication using siRNA mediated knockdown and rescue with DDX5 mutants which prevent protein-protein interactions and RNA and ATP binding. RESULTS: We demonstrate a critical role for DDX5 in the Tat/HEXIM1 interaction. DDX5 acts to potentiate Tat activity and can bind both Tat and HEXIM1 suggesting it may facilitate the dissociation of HEXIM1/2 from the 7SK-snRNP complex, enhancing Tat/P-TEFb availability. We show knockdown of DDX5 in a T cell line significantly reduces HIV-1 infectivity and viral protein production. This activity is unique to DDX5 and cannot be substituted by its close paralog DDX17. Overexpression of DDX5 stimulates the Tat/LTR promoter but suppresses other cellular and viral promoters. Individual mutations of conserved ATP binding, RNA binding, helicase related or protein binding motifs within DDX5 show that the N terminal RNA binding motifs, the Walker B and the glycine doublet motifs are essential for this function. The Walker A and RNA binding motifs situated on the transactivation domain are however dispensable. CONCLUSION: DDX5 is an essential cellular factor for efficient HIV transcription elongation. It interacts with Tat and may potentiate the availability of P-TEFb through sequestering HEXIM1.

The Effects of Healthy Ageing on Cerebral Blood Flow Responses to Cognitive Testing.

BACKGROUND: Transcranial Doppler Ultrasonography (TCD) can be utilised to measure the tight coupling of cerebral blood flow velocity (CBFv) in response to cognitive demand by task activation, termed neurovascular coupling. AIMS: To investigate the differences in neurovascular coupling between healthy older (>50 years) and younger (18-49 years) adults in response to cognitive testing. METHODS: Fifty-four older (n=25) and younger (n=29) adults underwent continuous bilateral TCD, beat-to-beat blood pressure (MAP; Finapres), heart rate (HR; electrocardiogram), and end-tidal CO2 (ETCO2; capnography) monitoring. After a 5-min baseline period, memory (M1-4: recalling three learned words, learning a name and address, recalling US presidents and UK prime ministers, and recalling the previously learned name and address) and visuospatial (V1-4: drawing a cube and infinity diagram, drawing a clock face, counting dots, and recognising obscured letters) tasks from the Addenbrooke's Cognitive Examination (ACE-III) were performed. Data are mean (standard deviation). RESULTS: In the memory paradigms, the peak percentage change in CBFv differed significantly between younger and older groups only in the dominant hemisphere during the M1 task, (2.17 (9.16)% vs. 8.38 (9.27)%, respectively, p=0.017). In the visuospatial paradigm, there were also significant differences in peak percentage change in CBFv between younger and older groups in the V1 (5.87 (8.32)% vs. 11.89 (6.60)%, p=0.005) and V2 tasks (6.30 (8.72)% vs. 11.30 (7.77)%, p=0.032). CONCLUSION: Healthy older adults demonstrate augmented cerebrovascular physiology in response to cognitive challenge compared to younger adults. The impact of abnormal ageing on cerebrovascular physiology, for example, related to cognitively impaired states, requires further investigation.

DDX17 Specifically, and Independently of DDX5, Controls Use of the HIV A4/5 Splice Acceptor Cluster and Is Essential for Efficient Replication of HIV.

HIV splicing involves five splice donor and eight splice acceptor sequences which, together with cryptic splice sites, generate over 100 mRNA species. Ninety percent of both partially spliced and fully spliced transcripts utilize the intrinsically weak A4/A5 3' splice site cluster. We show that DDX17, but not its close paralog DDX5, specifically controls the usage of this splice acceptor group. In its absence, production of the viral envelope protein and other regulatory and accessory proteins is grossly reduced, while Vif, which uses the A1 splice acceptor, is unaffected. This is associated with a profound decrease in viral export from the cell. Loss of Vpu expression causing upregulation of cellular Tetherin compounds the phenotype. DDX17 utilizes distinct RNA binding motifs for its role in efficient HIV replication, and we identify RNA binding motifs essential for its role, while the Walker A, Walker B (DEAD), Q motif and the glycine doublet motif are all dispensable. We show that DDX17 interacts with SRSF1/SF2 and the heterodimeric auxiliary factor U2AF65/35, which are essential splicing factors in the generation of Rev and Env/Vpu transcripts.

Neurovascular coupling response to cognitive examination in healthy controls: a multivariate analysis.

Cognitive testing with transcranial Doppler ultrasonography (TCD) has been used to assess neurovascular coupling (NVC), but few studies address its multiple contributions. Subcomponent analysis considers the relative myogenic (resistance area product, RAP) and metabolic (critical closing pressure (CrCP)) contributors. The aim of this study was to investigate the changes in subcomponents that occur with cognitive stimulation with the Addenbrooke's Cognitive Examination (ACE-III) in healthy controls. Healthy volunteers underwent continuous recording of bilateral TCD, heart rate (HR, three-lead ECG), end-tidal CO2 (ETCO2 , capnography), and mean arterial pressure (MAP, Finometer). The study comprised a 5-min baseline recording, followed by all 20 paradigms from the ACE-III. The cerebral blood flow velocity (CBFv) response was decomposed into the relative contributions (subcomponents); VBP (MAP), VCrCP (CrCP), and VRAP (RAP). Data are presented as peak population normalized mean changes from baseline, and median area under the curve (AUC). Forty bilateral datasets were obtained (27 female, 37 right hand dominant). VBP increased at task initiation in all paradigms but differed between tasks (range (SD): 4.06 (8.92)-16.04 (12.23) %, P < 0.05). HR, but not ETCO2 , also differed significantly (P < 0.05). Changes in VRAP reflected changes in MAP, but in some paradigms atypical responses were seen. VCrCP AUC varied significantly within paradigm sections (range [SD]: 18.4 [24.17] to 244.21 [243.21] %*s, P < 0.05). All paradigms demonstrated changes in subcomponents with cognitive stimulation, and can be ranked based on their relative presumed metabolic demand. The integrity of NVC requires further investigation in patient populations.

The assessment of neurovascular coupling with the Addenbrooke's Cognitive Examination: a functional transcranial Doppler ultrasonographic study.

Cerebrovascular dysfunction occurs early in dementia and can be identified by transcranial Doppler ultrasonography (TCD). Few studies have examined cerebral blood flow velocity (CBFv) responses to a detailed cognitive battery. This study aimed to characterize all CBFv responses, and the effect of hemispheric dominance, to the Addenbrooke's Cognitive Examination (ACE-III) in healthy volunteers. Forty volunteers underwent continuous bilateral TCD, beat-to-beat blood pressure (MAP; Finapres), heart rate (HR; electrocardiogram), and end-tidal CO2 (ETCO2; capnography) monitoring. After a 5-min baseline period, all tasks from the ACE-III were performed in 3 sections (A: attention, fluency, memory; B: language; C: visuospatial, memory). Data are population mean normalized percentage (PM%) change from a 20-s baseline period before task initiation. Forty bilateral data sets were obtained (27 women, 37 right-hand dominant). All paradigms produced a sharp increase in CBFv in both dominant (PM% range: 3.29 to 9.70%) and nondominant (PM% range: 4.34 to 11.63%) hemispheres at task initiation, with associated increases in MAP (PM% range: 3.06 to 16.04%). ETCO2 did not differ significantly at task initiation (PM% range: -1.1 to 2.4%, P > 0.05). HR differed significantly across A and C tasks at initiation (PM% range: -1.1 to 2.4%, P < 0.05), but not B tasks. In conclusion, all tasks resulted in increases in CBFv, differing significantly between paradigms. These results require further investigation in a cognitively impaired population. NEW & NOTEWORTHY This study is the first to provide a normative data set of cerebral blood flow velocity (CBFv) responses to a complete cognitive assessment (Addenbrooke's Cognitive Examination, ACE-III) in a large sample ( n = 40) of healthy volunteers. All tasks produced peak and sustained increases in CBFv to different extents. The ACE-III is a feasible tool to assess neurovascular coupling with transcranial Doppler ultrasonography. These data can be used to inform the most appropriate cognitive task to elicit CBFv responses for future studies.

Identification of RNA helicases in human immunodeficiency virus 1 (HIV-1) replication - a targeted small interfering RNA library screen using pseudotyped and WT HIV-1.

Central to the development of new treatments for human immunodeficiency virus 1 (HIV-1) is a more thorough understanding of the viral life cycle and the cellular cofactors upon which this depends. Targeting cellular proteins and their interaction with HIV-1 has the potential to reduce the problem of emerging viral resistance to drugs as mutational escape is more difficult. We performed a short interfering RNA (siRNA) library screen targeting 59 cellular RNA helicases, assessing the effect on both viral capsid protein production and infectious virion formation. Five RNA helicases were identified which, when knocked down, reproducibly decreased infectious particle production: DDX5, DDX10, DDX17, DDX28 and DDX52. Two of these proteins (DDX5 and DDX17) have known roles in HIV-1 replication. A further helicase (DDX10) was a positive hit from a previous genome-wide siRNA screen; however, DDX28 and DDX52 have not previously been implicated as essential cofactors for HIV-1.

The Cochran-Weiss-Shanteau performance index as an indicator of upper limb risk assessment expertise.

Ergonomists and many other professionals apply ergonomics principles to musculoskeletal health problems. This study examines whether there are differences when it comes to judgement expertise concerning upper limb disorders (ULDs) between ergonomists and those with less ergonomics training. The Cochran-Weiss-Shanteau (CWS) performance index combines judgement consistency with discrimination into one CWS index. Fifty-eight professionals working in the musculoskeletal health area, from four different professions, judged the likelihood of staff complaining of ULDs in a number of written work scenarios containing ULD risk factors. A student group (n = 148) taking an introductory ergonomics module was used as a reference. The ergonomists scored higher on the CWS index than all of the other groups, performing significantly better than all but the occupational health advisors. Performance improved with increased training level but not with experience. This study suggests that ergonomists are quantifiably different from other ergonomics advisors in their judgement performance in this context. Given the global cost of musculoskeletal disorders, assessing the expertise of those giving ergonomics advice for the management of musculoskeletal health is of great significance. This study presents a method for assessing judgement performance in ULD risk assessment, an important part of musculoskeletal health management.