Double-Strand Break Repair by Non-homologous End-Joining

Non-homologous end-joining (NHEJ) is an important mechanism of double-strand break repair (DSBR) which is largely conserved from yeast to humans. Mammalian NHEJ is dependent on the Ku, DNA-PKcs, XRCC4 and DNA ligase IV proteins. NHEJ-defective rodent cells are highly sensitive to ionising radiation, implicating NHEJ as a critical mechanism for the repair of radiation-induced double-strand breaks (DSBs). DNA termini at sites of radiation-induced DSBs exhibit modifications, including the presence of 5'-hydroxyl and 3'-phosphate or phosphoglycolate groups. During DSBR, these termini must undergo enzymatic processing to return the normal 5'-phosphate and 3'-hydroxyl groups required for ligation. Despite the activities of the core NHEJ proteins being well documented, little is known about the auxiliary processing factors required for repair of ionising radiation-induced breaks. Using an in vitro DNA end-joining assay catalysed by human cell-free extracts I have investigated the wider protein requirements of NHEJ and the ability of extracts to join certain modified DNA termini. In contrast to the essential role for the Mre11 and Xrs2 proteins in the joining of complementary termini in Saccharomyces cerevisiae, the human homologues, MRE11 and NBS1 were not required for the joining of complementary protruding 5' termini. Joining of modified and non-complementary termini implicated requirements for exonuclease and polymerase activities during extract-catalysed end-joining. Moreover, end-joining reactions with DNA molecules containing 5'-hydroxyl termini demonstrated a role for polynucleotide kinase (PNK) in the repair of radiation-induced breaks. The defective end-joining observed with PNK-depleted extracts was complemented by addition of physiological amounts of purified human PNK, but not T4 PNK. In addition, phosphorylation of 5'-hydroxy termini was not observed when DNA end-joining was blocked, either by use of XRCC4 antibodies or DNA-PKcs-defective extracts, implicating co-ordination between the phosphorylation and end-joining reactions. Finally, gel filtration and coimmunoprecipitation experiments indicated that PNK might be associated with the NHEJ proteins in vivo

Validation of the Ambassador Questionnaire for Undergraduate Students Conducting Engineering Outreach

Although K-12 engineering outreach commonly involves college students, the young professionals who act as ambassadors for their field are less likely to be studied than the students they serve. Yet, outreach activities may offer opportunities for undergraduate students to develop aspects of their professional selves. As there is currently no comprehensive measure that allows researchers, program evaluators, and outreach advisors to examine ambassadors\u27 professional development and growth, this study sought to develop and validate an Ambassador Questionnaire (AQ). The multi-step process included the selection and adaptation of items from extant measures of engineering students\u27 motivation, beliefs, professional skills, and perceptions of ambassador training. After an expert panel evaluated the initial group of items, the 57-item AQ was completed by a diverse group of 350 undergraduate engineering students engaged in ambassadorship. Exploratory and confirmatory factor analyses were used to examine construct validity, and internal consistency reliability analyses followed. The findings indicated a five-factor model that accounted for 53% of the variance and demonstrated strong internal consistency reliability. Potential uses for the measure are discussed

Little Association between Intracranial Arterial Stenosis and Lacunar Stroke

Atheromatous middle cerebral artery (MCA) stenosis could cause lacunar stroke by occluding lenticulostriate artery origins, but atheroma is common, and previous studies lacked suitable controls. We aimed to determine if intracranial atheroma was more common in lacunar than in cortical ischaemic stroke. We recruited patients with lacunar stroke and controls with mild cortical stroke, confirmed the stroke subtype with magnetic resonance imaging and used transcranial Doppler ultrasound imaging to record flow velocity and focal stenoses in the basal intracranial arteries 1 month after stroke. We compared ipsi- and contralateral MCA mean flow velocities between stroke subtypes and tested for associations using linear mixed models. Amongst 67 lacunar and 67 mild cortical strokes, mean age 64 and 67 years, respectively, we found no difference in MCA mean flow velocity between cortical and lacunar patients. Increasing age and white matter lesion scores were independently associated with lower MCA flow velocities (0.2 cms−1 fall in velocity per year increase in age, p = 0.045; 3.75 cms−1 fall in flow velocity per point increase in white matter lesion score, p = 0.004). We found no intracranial arterial stenoses. MCA atheromatous stenosis is unlikely to be a common cause of lacunar stroke in white populations. Falling velocities with increasing white matter lesion scores may reflect progressive brain tissue loss leaving less tissue to supply

A review of feto-placental vasculature flow modelling

The placenta provides the vital nutrients and removal of waste products required for fetal growth and development. Understanding and quantifying the differences in structure and function between a normally functioning placenta compared to an abnormal placenta is vital to provide insights into the aetiology and treatment options for fetal growth restriction and other placental disorders. Computational modelling of blood flow in the placenta allows a new understanding of the placental circulation to be obtained. This structured review discusses multiple recent methods for placental vascular model development including analysis of the appearance of the placental vasculature and how placental haemodynamics may be simulated at multiple length scales

Parameter identifiability of fundamental pharmacodynamic models

Issues of parameter identifiability of routinely used pharmacodynamics models are considered in this paper. The structural identifiability of 16 commonly applied pharmacodynamic model structures was analyzed analytically, using the input-output approach. Both fixed-effects versions (non-population, no between-subject variability) and mixed-effects versions (population, including between-subject variability) of each model structure were analyzed. All models were found to be structurally globally identifiable under conditions of fixing either one of two particular parameters. Furthermore, an example was constructed to illustrate the importance of sufficient data quality and show that structural identifiability is a prerequisite, but not a guarantee, for successful parameter estimation and practical parameter identifiability. This analysis was performed by generating artificial data of varying quality to a structurally identifiable model with known true parameter values, followed by re-estimation of the parameter values. In addition, to show the benefit of including structural identifiability as part of model development, a case study was performed applying an unidentifiable model to real experimental data. This case study shows how performing such an analysis prior to parameter estimation can improve the parameter estimation process and model performance. Finally, an unidentifiable model was fitted to simulated data using multiple initial parameter values, resulting in highly different estimated uncertainties. This example shows that although the standard errors of the parameter estimates often indicate a structural identifiability issue, reasonably “good” standard errors may sometimes mask unidentifiability issues

Cerebral perivascular spaces visible on magnetic resonance imaging: Development of a qualitative rating scale and its observer reliability

BACKGROUND: Perivascular spaces (PVS) are an important component of cerebral small vessel disease (SVD), several inflammatory disorders, hypertension and blood-brain barrier breakdown, but are difficult to quantify. A recent international collaboration of SVD experts has highlighted the need for a robust, easy-to-use PVS rating scale for the effective investigation of the diagnostic and prognostic significance of PVS. The purpose of the current study was to develop and extend existing PVS scales to provide a more comprehensive scale for the measurement of PVS in the basal ganglia, centrum semiovale and midbrain, and to test its intra- and inter-rater agreement, assessing reasons for discrepancy. METHODS: We reviewed previously published PVS scales, including site of PVS assessed, rating method, and size and morphological criteria. Retaining key features, we devised a more comprehensive scale in order to improve the reliability of PVS rating. Two neuroradiologists tested the new scale in MRI brain scans of 60 patients from two studies (stroke, ageing population), chosen to represent a full range of PVS, and demonstrating concomitant features of SVD such as lacunes and white matter hyperintensities. We rated basal ganglia, centrum semiovale, and midbrain PVS. Basal ganglia and centrum semiovale PVS were rated 0 (none), 1 (1–10), 2 (11–20), 3 (21–40) and 4 (>40), and midbrain PVS were rated 0 (none visible) or 1 (visible). We calculated kappa statistics for rating, assessed consistency in use of PVS categories (Bhapkar test) and reviewed sources of discrepancy. RESULTS: Intra- and inter-rater kappa statistics were highest for basal ganglia PVS (range 0.76–0.87 and 0.8–0.9, respectively) than for centrum semiovale PVS (range 0.68–0.75 and 0.61–0.8, respectively) or midbrain PVS (inter-rater range 0.51–0.52). Inter-rater consistency was better for basal ganglia compared to centrum semiovale PVS (Bhapkar statistic 2.49–3.72, compared to 6.79–21.08, respectively). Most inter-rater disagreements were due to very faint PVS, coexisting extensive white matter hyperintensities (WMH) or the presence of lacunes. CONCLUSIONS: We developed a more inclusive and robust visual PVS rating scale allowing rating of all grades of PVS severity on structural brain imaging. The revised PVS rating scale has good observer reliability for basal ganglia and centrum semiovale PVS, best for basal ganglia PVS, and moderate reliability for midbrain PVS. Agreement is influenced by PVS severity and the presence of background features of SVD. The current scale can be used in further studies to assess the clinical implications of PVS

Neuropsychiatric symptoms as a sign of small vessel disease progression in cognitive impairment

BACKGROUND: Neuropsychiatric symptoms associate cross-sectionally with cerebral small vessel disease but it is not clear whether these symptoms could act as early clinical markers of small vessel disease progression. We investigated whether longitudinal change in Neuropsychiatric Inventory (NPI) scores associated with white matter hyperintensity (WMH) progression in a memory clinic population. MATERIAL AND METHODS: We included participants from the prospective Sunnybrook Dementia Study with Alzheimer's disease and vascular subtypes of mild cognitive impairment and dementia with two MRI and ≥ 1 NPI. We conducted linear mixed-effects analyses, adjusting for age, atrophy, vascular risk factors, cognition, function, and interscan interval. RESULTS: At baseline (n=124), greater atrophy, age, vascular risk factors and total NPI score were associated with higher baseline WMH volume, while longitudinally, all but vascular risk factors were associated. Change in total NPI score was associated with change in WMH volume, χ2 = 7.18, p = 0.007, whereby a one-point change in NPI score from baseline to follow-up was associated with a 0.0017 change in normalized WMH volume [expressed as cube root of (WMH volume cm³ as % intracranial volume)], after adjusting for age, atrophy, vascular risk factors and interscan interval. CONCLUSIONS: In memory clinic patients, WMH progression over 1–2 years associated with worsening neuropsychiatric symptoms, while WMH volume remained unchanged in those with stable NPI scores in this population with low background WMH burden

Association between Striatal Brain Iron Deposition, Microbleeds and Cognition 1 Year After a Minor Ischaemic Stroke

Brain iron deposits (IDs) are inversely associated with cognitive function in community-dwelling older people, but their association with cognition after ischemic stroke, and whether that differs from microbleeds, is unknown. We quantified basal ganglia IDs (BGID) and microbleeds (BMBs) semi-automatically on brain magnetic resonance images from patients with minor stroke (NIHSS < 7), at presentation and 12 months after stroke. We administered the National Adult Reading Test (NART, estimates premorbid or peak adult cognition) and the Revised Addenbrooke’s Cognitive Examination (ACE-R; current cognition) at 1 and 12 months after stroke. We adjusted analyses for baseline cognition, age, gender, white matter hyperintensity (WMH) volume and vascular risk factors. In 200 patients, mean age 65 years, striatal IDs and BMBs volumes did not change over the 12 months. Baseline BGID volumes correlated positively with NART scores at both times (ρ = 0.19, p < 0.01). Baseline and follow-up BGID volumes correlated positively with age (ρ = 0.248, p < 0.001 and ρ = 0.271, p < 0.001 respectively), but only baseline (and not follow-up) BMB volume correlated with age (ρ = 0.129, p < 0.05). Both smoking and baseline WMH burden predicted verbal fluency and visuospatial abilities scores (B = −1.13, p < 0.02 and B = −0.22, p = 0.001 respectively) at 12 months after stroke. BGIDs and BMBs are associated differently with cognition post-stroke; studies of imaging and post-stroke cognition should adjust for premorbid cognition. The positive correlation of BGID with NART may reflect the lower premorbid cognition in patients with stroke at younger vs older ages