Investigating the cell division protein FtsZ and its regulation in Bacillus subtilis

University of Technology, Sydney. Faculty of Science.As with all organisms, bacterial cells divide with amazing precision. The first stage of this process is marked by the polymerisation of the essential tubulin-like FtsZ protein at midcell into a ring, the Z ring. Understanding its formation and regulation provides an insight into how the crucial event of cell division is controlled. However, despite intense investigation, these molecular mechanisms are not fully understood. One factor believed to play a role in midcell Z ring placement is the coordination between DNA replication and cell division. Previously it has been shown that when initiation of DNA replication is allowed, but DNA synthesis is inhibited by two different methods (thymine starvation or addition of HPUra), Z rings are able to form at midcell in one case, and not in the other. Both conditions block DNA synthesis at the same stage, the beginning of DNA chain elongation. In an attempt to understand these incongruous results, the possibility of the drug HPUra playing a nonreplicative role, leading to di placement of the Z ring, was examined. It was found that Z ring positioning in an HPUra-resistant strain was not significantly different to that of wild type. Z rings formed at midcell in both conditions. Thu in the wild type strain, the effect of HPUra on Z ring positioning is dependant on its ability to inhibit replication. Hence the block to the elongation stage of DNA replication mediated by the addition of HPUra is capable of misplacing the Z ring, strong evidence for a link between these essential processe of DNA replication and cell division. Ten years ago it was proposed that the Z ring forms by bidirectional growth from a midcell nucleation site. Work presented in this thesis now suggests this may not be the case. Using a modified immunofluorescence protocol it was discovered that, in addition to forming a Z ring, FtsZ forms a helical structure along the length of the cell in vegetatively growing wild type Bacillus subtilis cells. Time-lapse experiments in live cells using an inducible FtsZYFP fusion, showed that the helical FtsZ structure is highly dynamic and undergoes cell cycle-regulated changes in localization. The monitoring of a complete cell cycle revealed the early appearance of a pole-to-pole FtsZ helix, a subsequent short helix spatially restricted to midcell, and finally this redistributed to produce a sharp midcell Z ring. These observations led to the proposal of a novel assembly tnechanism for Z ring formation involving a cell-cycle mediated multi-step remodelling of FtsZ polymers. This was the first report of an FtsZ helix in B. subtilis during vegetative growth. The new model for Z ring formation predicts that in order for the cell to assemble a Z ring, FtsZ must go through long helix-to-short helix-to-Z ring polymerisation changes. How is the Min system, a known negative regulator of FtsZ responsible for inhibiting aberrant Z ring assembly at the cell poles, involved in regulating these FtsZ polymerisation transitions? To address this, FtsZ polymer remodelling was examined whilst modifying the effect of the Min system. Time-lapse studies of a strain carrying a deletion of the minCD genes showed FtsZ polymerising at the poles in the same fashion as wild type; that is going through a short helical intetmediate prior to Z ring formation. This indicated that the helical form of FtsZ is in fact a true intennediate, required for Z rings to form even at non-midcell locations. A minCD over-expression strain showed a marked decrease in Z ring formation and time-lapse imaging wa conducted to asses at which transitional stage FtsZ assembly was affected. Intere tingly, it wa found that both the long and hort helical polymerisation of FtsZ can actually form as wild type in the over-expression experiments. The excess of MinCO in the cell appeared to be able to severely impair division by hampering and prolonging the transition of the short midcell helix to a ring. It is proposed that this i mediated by the inhibition of lateral interactions of FtsZ protofilaments. Indeed a model is put forth etnphasizing the importance of lateral interactions in the helix -to-ring remodelling, and thus in stable Z ring formation. To examine the in vivo FtsZ helix with higher resolution, the advanced microscopic techniques of 4Pi and STED imaging were employed. Using alternative methods has the advantages of confirming the helical structure and extracts further information, for example is the helix continuous? STED microscopy breaks the diffraction barrier and lateral resolution is increased to ~ 100 ntn, ~2. 5 times that of normal confocal microscopy. Using STED, FtsZ localization showed a distinct periodicity, consistent with a helical conformation. Additionally FtsZ staining was revealed to be extremely punctate and discontinuous, suggesting that the helical structure of FtsZ may depend on a cellular track. Visualising cells and their sub-cellular structure in ever increasing detail ensures novel insights into the regulation of Z ring assembly in bacteria

Temperature-dependent changes in neuronal dynamics in a patient with an SCN1A mutation and hyperthermia induced seizures

Dravet syndrome is the prototype of SCN1A-mutation associated epilepsies. It is characterised by prolonged seizures, typically provoked by fever. We describe the evaluation of an SCN1A mutation in a child with early-onset temperature-sensitive seizures. The patient carries a heterozygous missense variant (c3818C > T; pAla1273Val) in the NaV1.1 brain sodium channel. We compared the functional effects of the variant vs. wild type NaV1.1 using patch clamp recordings from channels expressed in Chinese Hamster Ovary Cells at different temperatures (32, 37, and 40 °C). The variant channels produced a temperature-dependent destabilization of activation and fast inactivation. Implementing these empirical abnormalities in a computational model predicts a higher threshold for depolarization block in the variant, particularly at 40 °C, suggesting a failure to autoregulate at high-input states. These results reveal direct effects of abnormalities in NaV1.1 biophysical properties on neuronal dynamics. They illustrate the value of combining cellular measurements with computational models to integrate different observational scales (gene/channel to patient)

A Candidate Sub-Parsec Supermassive Binary Black Hole System

We identify SDSS J153636.22+044127.0, a QSO discovered in the Sloan Digital Sky Survey, as a promising candidate for a binary black hole system. This QSO has two broad-line emission systems separated by 3500 km/sec. The redder system at z=0.3889 also has a typical set of narrow forbidden lines. The bluer system (z=0.3727) shows only broad Balmer lines and UV Fe II emission, making it highly unusual in its lack of narrow lines. A third system, which includes only unresolved absorption lines, is seen at a redshift, z=0.3878, intermediate between the two emission-line systems. While the observational signatures of binary nuclear black holes remain unclear, J1536+0441 is unique among all QSOs known in having two broad-line regions, indicative of two separate black holes presently accreting gas. The interpretation of this as a bound binary system of two black holes having masses of 10^8.9 and 10^7.3 solar masses, yields a separation of ~ 0.1 parsec and an orbital period of ~100 years. The separation implies that the two black holes are orbiting within a single narrow-line region, consistent with the characteristics of the spectrum. This object was identified as an extreme outlier of a Karhunen-Loeve Transform of 17,500 z < 0.7 QSO spectra from the SDSS. The probability of the spectrum resulting from a chance superposition of two QSOs with similar redshifts is estimated at 2X10^-7, leading to the expectation of 0.003 such objects in the sample studied; however, even in this case, the spectrum of the lower redshift QSO remains highly unusual.Comment: 8 pages, 2 figures, Nature in pres

Associations between health-related quality of life, physical function and fear of falling in older fallers receiving home care

Falls and injuries in older adults have significant consequences and costs, both personal and to society. Although having a high incidence of falls, high prevalence of fear of falling and a lower quality of life, older adults receiving home care are underrepresented in research on older fallers. The objective of this study is to determine the associations between health-related quality of life (HRQOL), fear of falling and physical function in older fallers receiving home care

The Statistical Mechanics of Horizons and Black Hole Thermodynamics

Although we know that black holes are characterized by a temperature and an entropy, we do not yet have a satisfactory microscopic ``statistical mechanical'' explanation for black hole thermodynamics. I describe a new approach that attributes the thermodynamic properties to ``would-be gauge'' degrees of freedom that become dynamical on the horizon. For the (2+1)-dimensional black hole, this approach gives the correct entropy. (Talk given at the Pacific Conference on Gravitation and Cosmology, Seoul, February 1996.)Comment: 11 pages, LaTe

A falls prevention programme to improve quality of life, physical function and falls efficacy in older people receiving home help services: study protocol for a randomised controlled trial

BACKGROUND: Falls and fall-related injuries in older adults are associated with great burdens, both for the individuals, the health care system and the society. Previous research has shown evidence for the efficiency of exercise as falls prevention. An understudied group are older adults receiving home help services, and the effect of a falls prevention programme on health-related quality of life is unclear. The primary aim of this randomised controlled trial is to examine the effect of a falls prevention programme on quality of life, physical function and falls efficacy in older adults receiving home help services. A secondary aim is to explore the mediating factors between falls prevention and health-related quality of life. METHODS: The study is a single-blinded randomised controlled trial. Participants are older adults, aged 67 or older, receiving home help services, who are able to walk with or without walking aids, who have experienced at least one fall during the last 12 months and who have a Mini Mental State Examination of 23 or above. The intervention group receives a programme, based on the Otago Exercise Programme, lasting 12 weeks including home visits and motivational telephone calls. The control group receives usual care. The primary outcome is health-related quality of life (SF-36). Secondary outcomes are leg strength, balance, walking speed, walking habits, activities of daily living, nutritional status and falls efficacy. All measurements are performed at baseline, following intervention at 3 months and at 6 months' follow-up. Sample size, based on the primary outcome, is set to 150 participants randomised into the two arms, including an estimated 15-20% drop out. Participants are recruited from six municipalities in Norway. DISCUSSION: This trial will generate new knowledge on the effects of an exercise falls prevention programme among older fallers receiving home help services. This knowledge will be useful for clinicians, for health managers in the primary health care service and for policy makers

Six-Month Mortality among HIV-Infected Adults Presenting for Antiretroviral Therapy with Unexplained Weight Loss, Chronic Fever or Chronic Diarrhea in Malawi.

In sub-Saharan Africa, early mortality is high following initiation of antiretroviral therapy (ART). We investigated 6-month outcomes and factors associated with mortality in HIV-infected adults being assessed for ART initiation and presenting with weight loss, chronic fever or diarrhea, and with negative TB sputum microscopy

Searches for Gravitational Waves from Binary Neutron Stars: A Review

A new generation of observatories is looking for gravitational waves. These waves, emitted by highly relativistic systems, will open a new window for ob- servation of the cosmos when they are detected. Among the most promising sources of gravitational waves for these observatories are compact binaries in the final min- utes before coalescence. In this article, we review in brief interferometric searches for gravitational waves emitted by neutron star binaries, including the theory, instru- mentation and methods. No detections have been made to date. However, the best direct observational limits on coalescence rates have been set, and instrumentation and analysis methods continue to be refined toward the ultimate goal of defining the new field of gravitational wave astronomy.Comment: 30 pages, 5 Figures, to appear in "Short-Period Binary Stars: Observations, Analyses, and Results", Ed.s Eugene F. Milone, Denis A. Leahy, David W. Hobil

DNA adducts in fish following an oil spill exposure

On 12 December 1999, one third of the load of the Erika tanker, amounting to about 10,000 t crude oil flowed into sea waters close to the French Atlantic Coast. This oil contained polycyclic aromatic compounds (PAC) that are known to be genotoxic. Genotoxic effects induce DNA adducts formation, which can thus be used as pollution biomarkers. Here, we assessed the genotoxic impact of the “Erika” oil spill by DNA adducts detection in the liver of immature fishes (Solea solea) from four locations of the French Brittany coasts. Two months after the spill, a high amount of DNA adducts was found in samples from all locations, amounting to 92–290 DNA adduct per 109 nucleotides. Then total DNA adduct levels decreased to reach about 50 adducts per 109 nucleotides nine months after the spill. In vitro experiments using human cell cultures and fish liver microsomes evidence the genotoxicity of the Erika fuel. They also prove the formation of reactive species able to create DNA adducts. Furthermore, in vitro and in vivo DNA adducts fingerprints are similar, thus confirming that DNA adducts are a result of the oil spill

Spatially Explicit Data: Stewardship and Ethical Challenges in Science

Scholarly communication is at an unprecedented turning point created in part by the increasing saliency of data stewardship and data sharing. Formal data management plans represent a new emphasis in research, enabling access to data at higher volumes and more quickly, and the potential for replication and augmentation of existing research. Data sharing has recently transformed the practice, scope, content, and applicability of research in several disciplines, in particular in relation to spatially specific data. This lends exciting potentiality, but the most effective ways in which to implement such changes, particularly for disciplines involving human subjects and other sensitive information, demand consideration. Data management plans, stewardship, and sharing, impart distinctive technical, sociological, and ethical challenges that remain to be adequately identified and remedied. Here, we consider these and propose potential solutions for their amelioration