Studies of Kinetochore Mechanobiology in Drosophila

Kinetochores are large multiprotein structures through which chromosomes engage with the microtubules of the mitotic spindle. All kinetochore pairs must ultimately adopt a bioriented configuration, with their associated sister chromatids linked to opposite spindle poles and poised to segregate equally between two daughter cells. Erroneous, non-bioriented attachments that are left uncorrected lead to chromosome mis-segregation, producing aneuploid daughter cells with unequal numbers of chromosomes. Before anaphase onset, bioriented attachments are selectively stabilized whereas non-bioriented attachments remain unstable and are eliminated. This error correction process relies heavily on the extent of outer kinetochore phosphorylation by an Aurora B kinase activity centered at the inner centromere, between the two members of a kinetochore pair. Phosphorylation of Aurora B substrates at the outer kinetochore tends to destabilize kinetochore-microtubule attachments by lowering the affinity with which outer kinetochore proteins bind to spindle microtubules. Reduced phosphorylation, and attachment stabilization, are associated with the kinetochore’s capacity to stretch when placed under tension by the pulling force exerted by microtubule plus end dynamics. Such intrakinetochore stretch in response to mechanical tension generally occurs only at bioriented attachments. This Dissertation examines kinetochore-microtubule attachment stabilization and error correction in Drosophila melanogaster. Experimental deletion of an extendible, structurally disordered segment of the Drosophila kinetochore protein CENP-C is found to be associated with attachment instability and an increased incidence of chromosome misalignment. That association can be explained in terms of reduced intrakinetochore stretch and an inability of the metaphase spindle to pull the outer kinetochore away from inner centromere-based Aurora B. On that view, selective stabilization of bioriented attachments in wild type cells depends on changes in the distance between the inner centromere and the kinetochore-microtubule binding interface. Separately, it is shown that erroneous, syntelic kinetochore-microtubule attachments can be stabilized, and the phosphorylation-based error correction system overridden, by overexpressing the Drosophila chromokinesin motor NOD. NOD overexpression artificially increases the magnitude of the polar ejection force that pushes chromosome arms away from the spindle poles. In turn, the elevated polar ejection force increases the tension experienced by syntelically attached kinetochore pairs, stabilizing what would ordinarily be a highly unstable form of attachment

Chromosome Biorientation Produces Hundreds of Piconewtons at a Metazoan Kinetochore

High-fidelity transmission of the genome through cell division requires that all sister kinetochores bind to dynamic microtubules (MTs) from opposite spindle poles. The application of opposing forces to this bioriented configuration produces tension that stabilizes kinetochore–microtubule (kt–MT) attachments. Defining the magnitude of force that is applied to kinetochores is central to understanding the mechano-molecular underpinnings of chromosome segregation; however, existing kinetochore force measurements span orders of magnitude. Here we measure kinetochore forces by engineering two calibrated force sensors into the Drosophila kinetochore protein centromere protein (CENP)-C. Measurements of both reporters indicate that they are, on average, under ∼1–2 piconewtons (pNs) of force at metaphase. Based on estimates of the number of CENP-C molecules and MTs per Drosophila kinetochore and envisioning kinetochore linkages arranged such that they distribute forces across them, we propose that kinetochore fibres (k-fibres) exert hundreds of pNs of poleward-directed force to bioriented kinetochores

Sensorineural hearing loss after neonatal meningitis: a single-centre retrospective study

Babies in intensive care are at higher risk for meningitis and sensorineural hearing loss (SNHL). We reviewed the rate of SNHL among definite cases of bacterial/fungal meningitis in our neonatal intensive care unit over a 16-year period (2006–2021). We identified 16 confirmed meningitis cases among 16 070 admissions: 8 of 10 surviving infants with available diagnostic audiology had normal/satisfactory hearing while 2 of 10 had SNHL. Both infants with permanent hearing loss had been born extremely preterm and received potentially ototoxic antimicrobials. Larger studies are needed to clarify whether SNHL occurs mainly due to meningitis itself or to its antimicrobial drug treatment

CD4+ T cell surface alpha enolase is lower in older adults

To identify novel cell ageing markers in order to gain insight into ageing mechanisms, we adopted membrane enrichment and comparison of the CD4+ T cell membrane proteome (purified by cell surface labelling using Sulfo-NHS-SS-Biotin reagent) between healthy young (n=9, 20-25y) and older (n=10; 50-70y) male adults. Following two-dimensional gel electrophoresis (2DE) to separate pooled membrane proteins in triplicates, the identity of protein spots with age-dependent differences (p1.4 fold difference) was determined using liquid chromatography-mass spectrometry (LC-MS/MS). Seventeen protein spot density differences (ten increased and seven decreased in the older adult group) were observed between young and older adults. From spot intensity analysis, CD4+ T cell surface α-enolase was decreased in expression by 1.5 fold in the older age group; this was verified by flow cytometry (n=22) and qPCR with significantly lower expression of cellular α-enolase mRNA and protein compared to young adult CD4+ T cells (p<0.05). In an independent age-matched case-control study, lower CD4+ T cell surface α-enolase expression was observed in age-matched patients with cardiovascular disease (p<0.05). An immune-modulatory role has been proposed for surface α-enolase and our findings of decreased expression suggest that deficits in surface α-enolase merit investigation in the context of immune dysfunction during ageing and vascular disease

Understanding practice: the factors that influence management of mild traumatic brain injury in the emergency department-a qualitative study using the Theoretical Domains Framework

Background: Mild traumatic brain injury is a frequent cause of presentation to emergency departments. Despite the availability of clinical practice guidelines in this area, there is variation in practice. One of the aims of the Neurotrauma Evidence Translation program is to develop and evaluate a targeted, theory- and evidence-informed intervention to improve the management of mild traumatic brain injury in Australian emergency departments. This study is the first step in the intervention development process and uses the Theoretical Domains Framework to explore the factors perceived to influence the uptake of four key evidence-based recommended practices for managing mild traumatic brain injury. Methods: Semi-structured interviews were conducted with emergency staff in the Australian state of Victoria. The interview guide was developed using the Theoretical Domains Framework to explore current practice and to identify the factors perceived to influence practice. Two researchers coded the interview transcripts using thematic content analysis. Results: A total of 42 participants (9 Directors, 20 doctors and 13 nurses) were interviewed over a seven-month period. The results suggested that (i) the prospective assessment of post-traumatic amnesia was influenced by: knowledge; beliefs about consequences; environmental context and resources; skills; social/professional role and identity; and beliefs about capabilities; (ii) the use of guideline-developed criteria or decision rules to inform the appropriate use of a CT scan was influenced by: knowledge; beliefs about consequences; environmental context and resources; memory, attention and decision processes; beliefs about capabilities; social influences; skills and behavioral regulation; (iii) providing verbal and written patient information on discharge was influenced by: beliefs about consequences; environmental context and resources; memory, attention and decision processes; social/professional role and identity; and knowledge; (iv) the practice of providing brief, routine follow-up on discharge was influenced by: environmental context and resources; social/professional role and identity; knowledge; beliefs about consequences; and motivation and goals. Conclusions: Using the Theoretical Domains Framework, factors thought to influence the management of mild traumatic brain injury in the emergency department were identified. These factors present theoretically based targets for a future intervention

Attentional bias retraining in cigarette smokers attempting smoking cessation (ARTS): study protocol for a double bline randomised controlled trial

YesSmokers attend preferentially to cigarettes and other smoking-related cues in the environment, in what is known as an attentional bias. There is evidence that attentional bias may contribute to craving and failure to stop smoking. Attentional retraining procedures have been used in laboratory studies to train smokers to reduce attentional bias, although these procedures have not been applied in smoking cessation programmes. This trial will examine the efficacy of multiple sessions of attentional retraining on attentional bias, craving, and abstinence in smokers attempting cessation. This is a double-blind randomised controlled trial. Adult smokers attending a 7-session weekly stop smoking clinic will be randomised to either a modified visual probe task with attentional retraining or placebo training. Training will start 1 week prior to quit day and be given weekly for 5 sessions. Both groups will receive 21 mg transdermal nicotine patches for 8–12 weeks and withdrawal-orientated behavioural support for 7 sessions. Primary outcome measures are the change in attentional bias reaction time and urge to smoke on the Mood and Physical Symptoms Scale at 4 weeks post-quit. Secondary outcome measures include differences in withdrawal, time to first lapse and prolonged abstinence at 4 weeks post-quit, which will be biochemically validated at each clinic visit. Follow-up will take place at 8 weeks, 3 months and 6 months post-quit. This is the first randomised controlled trial of attentional retraining in smokers attempting cessation. This trial could provide proof of principle for a treatment aimed at a fundamental cause of addiction.National Institute for Health Research (NIHR) Doctoral Research Fellowship (DRF) awarded to RB (DRF-2009-02-15

Virological failure and development of new resistance mutations according to CD4 count at combination antiretroviral therapy initiation

Objectives: No randomized controlled trials have yet reported an individual patient benefit of initiating combination antiretroviral therapy (cART) at CD4 counts > 350 cells/μL. It is hypothesized that earlier initiation of cART in asymptomatic and otherwise healthy individuals may lead to poorer adherence and subsequently higher rates of resistance development. Methods: In a large cohort of HIV-positive individuals, we investigated the emergence of new resistance mutations upon virological treatment failure according to the CD4 count at the initiation of cART. Results: Of 7918 included individuals, 6514 (82.3%), 996 (12.6%) and 408 (5.2%) started cART with a CD4 count ≤ 350, 351-499 and ≥ 500 cells/μL, respectively. Virological rebound occurred while on cART in 488 (7.5%), 46 (4.6%) and 30 (7.4%) with a baseline CD4 count ≤ 350, 351-499 and ≥ 500 cells/μL, respectively. Only four (13.0%) individuals with a baseline CD4 count > 350 cells/μL in receipt of a resistance test at viral load rebound were found to have developed new resistance mutations. This compared to 107 (41.2%) of those with virological failure who had initiated cART with a CD4 count < 350 cells/μL. Conclusions: We found no evidence of increased rates of resistance development when cART was initiated at CD4 counts above 350 cells/μL. HIV Medicin