Tracking Low-Copy Transcription Factors in Living Bacteria:The Case of the lac Repressor

Transcription factors control the expression of genes by binding to specific sites in DNA and repressing or activating transcription in response to stimuli. The lac repressor (LacI) is a well characterized transcription factor that regulates the ability of bacterial cells to uptake and metabolize lactose. Here, we study the intracellular mobility and spatial distribution of LacI in live bacteria using photoactivated localization microscopy combined with single-particle tracking. Since we track single LacI molecules in live cells by stochastically photoactivating and observing fluorescent proteins individually, there are no limitations on the copy number of the protein under study; as a result, we were able to study the behavior of LacI in bacterial strains containing the natural copy numbers (∼40 monomers), as well as in strains with much higher copy numbers due to LacI overexpression. Our results allowed us to determine the relative abundance of specific, near-specific, and non-specific DNA binding modes of LacI in vivo, showing that all these modes are operational inside living cells. Further, we examined the spatial distribution of LacI in live cells, confirming its specific binding to lac operator regions on the chromosome; we also showed that mobile LacI molecules explore the bacterial nucleoid in a way similar to exploration by other DNA-binding proteins. Our work also provides an example of applying tracking photoactivated localization microscopy to studies of low-copy-number proteins in living bacteria

Single-molecule fluorescence analysis of transcription factors in living bacteria

Super-resolution microscopy has become an important tool to image cells at ever increasing resolution. Specifically, localization microscopy, in which single emitters are localized with high accuracy, has allowed us to follow and track single-molecules in live cells. The technique has revealed aspects in the proteinsâ real-time function that were impossible to study previously. Despite their extensive use in recent years, there are still possibilities to improve and validate the technique and the resulting data. This thesis presents the first tracking photo-activation localization microscopy (tracking PALM) on three transcription factors (TFs): the lac repressor (LacI), araC protein and the cAMP receptor protein (CRP). With this work I expand the range of applicability of localization methods and increase in detail an important part of cellular function. TFs control the expression of genes so that cells can adapt to external conditions. In Escherichia coli (E. coli), previous studies have shown that more than 60&percnt; of TFs have less than 100 monomers per genome copy. For example LacI, involved in the lactose utilization control, exists in E. coli in around 40 monomers in contrast to other proteins that exist in the tens of thousands. To study such low-copy numbers we found it necessary increase the throughput of tracking PALM and this thesis summarises our efforts to increase throughput. After constructing a setup capable of tracking PALM with a 3X increased throughput, I studied the diffusion and localization of LacI and LacI relative to its operators. By studying LacI, I found that there were a number of challenges related to low copy numbers, specifically background noise that leads to localisations that can affect the results. I characterized the background tracks and tested the ability of clustering to find a number of tandem operators. This knowledge allowed us to study other TFs: araC, involved in the control of arabinose utilization which also exists in low-copy numbers but functions in multiple genes; and the CRP, which both regulates multiple sites and exists in large copy numbers. In addition I worked towards the understanding of TF binding by simulating the combination of non-specific binding and free diffusion that emulates the search process of TFs. I obtained insight into the analysis of the data generated from tracking PALM. Finally I focused on creating a strategy to use our existing data to extract blinking rates from the fluorescent protein. Our insights into TFs diffusion and binding have shown that tracking PALM can yield important insights at this regime of protein concentrations. Our work can be applied to other TFs and proteins that function in low copy numbers.</p

Single-molecule fluorescence analysis of transcription factors in living bacteria

Super-resolution microscopy has become an important tool to image cells at ever increasing resolution. Specifically, localization microscopy, in which single emitters are localized with high accuracy, has allowed us to follow and track single-molecules in live cells. The technique has revealed aspects in the proteins’ real-time function that were impossible to study previously. Despite their extensive use in recent years, there are still possibilities to improve and validate the technique and the resulting data. This thesis presents the first tracking photo-activation localization microscopy (tracking PALM) on three transcription factors (TFs): the lac repressor (LacI), araC protein and the cAMP receptor protein (CRP). With this work I expand the range of applicability of localization methods and increase in detail an important part of cellular function. TFs control the expression of genes so that cells can adapt to external conditions. In Escherichia coli (E. coli), previous studies have shown that more than 60% of TFs have less than 100 monomers per genome copy. For example LacI, involved in the lactose utilization control, exists in E. coli in around 40 monomers in contrast to other proteins that exist in the tens of thousands. To study such low-copy numbers we found it necessary increase the throughput of tracking PALM and this thesis summarises our efforts to increase throughput. After constructing a setup capable of tracking PALM with a 3X increased throughput, I studied the diffusion and localization of LacI and LacI relative to its operators. By studying LacI, I found that there were a number of challenges related to low copy numbers, specifically background noise that leads to localisations that can affect the results. I characterized the background tracks and tested the ability of clustering to find a number of tandem operators. This knowledge allowed us to study other TFs: araC, involved in the control of arabinose utilization which also exists in low-copy numbers but functions in multiple genes; and the CRP, which both regulates multiple sites and exists in large copy numbers. In addition I worked towards the understanding of TF binding by simulating the combination of non-specific binding and free diffusion that emulates the search process of TFs. I obtained insight into the analysis of the data generated from tracking PALM. Finally I focused on creating a strategy to use our existing data to extract blinking rates from the fluorescent protein. Our insights into TFs diffusion and binding have shown that tracking PALM can yield important insights at this regime of protein concentrations. Our work can be applied to other TFs and proteins that function in low copy numbers.</p

The bone diagnostic instrument II: Indentation distance increase

The bone diagnostic instrument (BDI) is being developed with the long-term goal of providing a way for researchers and clinicians to measure bone material properties of human bone in vivo. Such measurements could contribute to the overall assessment of bone fragility in the future. Here, we describe an improved BDI, the Osteoprobe II™. In the Osteoprobe II™, the probe assembly, which is designed to penetrate soft tissue, consists of a reference probe (a 22 gauge hypodermic needle) and a test probe (a small diameter, sharpened rod) which slides through the inside of the reference probe. The probe assembly is inserted through the skin to rest on the bone. The distance that the test probe is indented into the bone can be measured relative to the position of the reference probe. At this stage of development, the indentation distance increase (IDI) with repeated cycling to a fixed force appears to best distinguish bone that is more easily fractured from bone that is less easily fractured. Specifically, in three model systems, in which previous mechanical testing and∕or tests reported here found degraded mechanical properties such as toughness and postyield strain, the BDI found increased IDI. However, it must be emphasized that, at this time, neither the IDI nor any other mechanical measurement by any technique has been shown clinically to correlate with fracture risk. Further, we do not yet understand the mechanism responsible for determining IDI beyond noting that it is a measure of the continuing damage that results from repeated loading. As such, it is more a measure of plasticity than elasticity in the bone

Terutroban versus aspirin in patients with cerebral ischaemic events (PERFORM): a randomised, double-blind, parallel-group trial

Background: Patients with ischaemic stroke or transient ischaemic attack (TIA) are at high risk of recurrent stroke or other cardiovascular events. We compared the selective thromboxane-prostaglandin receptor antagonist terutroban with aspirin in the prevention of cerebral and cardiovascular ischaemic events in patients with a recent non-cardioembolic cerebral ischaemic event. &lt;p/&gt;Methods: This randomised, double-blind, parallel-group trial was undertaken in 802 centres in 46 countries. Patients who had an ischaemic stroke in the previous 3 months or a TIA in the previous 8 days were randomly allocated with a central interactive response system to 30 mg per day terutroban or 100 mg per day aspirin. Patients and investigators were masked to treatment allocation. The primary efficacy endpoint was a composite of fatal or non-fatal ischaemic stroke, fatal or non-fatal myocardial infarction, or other vascular death (excluding haemorrhagic death). We planned a sequential statistical analysis of non-inferiority (margin 1·05) followed by analysis of superiority. Analysis was by intention to treat. The study was stopped prematurely for futility on the basis of the recommendation of the Data Monitoring Committee. This study is registered, number ISRCTN66157730. &lt;p/&gt;Findings: 9562 patients were assigned to terutroban (9556 analysed) and 9558 to aspirin (9544 analysed); mean follow-up was 28·3 months (SD 7·7). The primary endpoint occurred in 1091 (11%) patients receiving terutroban and 1062 (11%) receiving aspirin (hazard ratio [HR] 1·02, 95% CI 0·94–1·12). There was no evidence of a difference between terutroban and aspirin for the secondary or tertiary endpoints. We recorded some increase in minor bleedings with terutroban compared with aspirin (1147 [12%] vs 1045 [11%]; HR 1·11, 95% CI 1·02–1·21), but no significant differences in other safety endpoints. &lt;p/&gt;Interpretation: The trial did not meet the predefined criteria for non-inferiority, but showed similar rates of the primary endpoint with terutroban and aspirin, without safety advantages for terutroban. In a worldwide perspective, aspirin remains the gold standard antiplatelet drug for secondary stroke prevention in view of its efficacy, tolerance, and cost

Rationale and design of a randomized, double-blind, parallel-group study of terutroban 30 mg/day versus aspirin 100 mg/day in stroke patients: the prevention of cerebrovascular and cardiovascular events of ischemic origin with terutroban in patients with a history of ischemic stroke or transient ischemic attack (PERFORM) study.

BACKGROUND: Ischemic stroke is the leading cause of mortality worldwide and a major contributor to neurological disability and dementia. Terutroban is a specific TP receptor antagonist with antithrombotic, antivasoconstrictive, and antiatherosclerotic properties, which may be of interest for the secondary prevention of ischemic stroke. This article describes the rationale and design of the Prevention of cerebrovascular and cardiovascular Events of ischemic origin with teRutroban in patients with a history oF ischemic strOke or tRansient ischeMic Attack (PERFORM) Study, which aims to demonstrate the superiority of the efficacy of terutroban versus aspirin in secondary prevention of cerebrovascular and cardiovascular events. METHODS AND RESULTS: The PERFORM Study is a multicenter, randomized, double-blind, parallel-group study being carried out in 802 centers in 46 countries. The study population includes patients aged > or =55 years, having suffered an ischemic stroke (< or =3 months) or a transient ischemic attack (< or =8 days). Participants are randomly allocated to terutroban (30 mg/day) or aspirin (100 mg/day). The primary efficacy endpoint is a composite of ischemic stroke (fatal or nonfatal), myocardial infarction (fatal or nonfatal), or other vascular death (excluding hemorrhagic death of any origin). Safety is being evaluated by assessing hemorrhagic events. Follow-up is expected to last for 2-4 years. Assuming a relative risk reduction of 13%, the expected number of primary events is 2,340. To obtain statistical power of 90%, this requires inclusion of at least 18,000 patients in this event-driven trial. The first patient was randomized in February 2006. CONCLUSIONS: The PERFORM Study will explore the benefits and safety of terutroban in secondary cardiovascular prevention after a cerebral ischemic event.Journal ArticleMulticenter StudyRandomized Controlled TrialResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe