Investigation of photoelectrons from molecules in a strong field

When intense and few-cycle laser pulses interact with an atomic/molecular beam highorder Above Threshold Ionisation (HATI) can take place. The resulting electrons can be re-scattered from the parent atoms/molecules and gain kinetic energy. The HATI electrons contain information about the atomic/molecular structure thus providing a method to probe atomic and molecular dynamics with sub-fs temporal and sub-angström spatial resolution. In this thesis the development of a Velocity Map Imaging apparatus capable of measuring the two-dimensional (2D) projection of the velocity distribution of electrons with energies up to 400 eV is described. This device was implemented with a molecular beam apparatus to study the electron re-scattering process that occurs when atoms/molecules are subjected to strong laser fields. Time-of-fight measurements were carried out to find the molecular beam. To perform the experiments a method to generate intense and few-cycle pulses based on hollow fibre pulse compression has been implemented. Pulses of 14 fs with energies of 500 μJ have been measured in a differentially pumped fibre set-up, with input pulses of 42 fs and 700 μJ using a home-made Frequency-Resolved Optical Gating device. The performance of the VMI apparatus was investigated by first studying the ATI rings formed by low energy electrons. Then, a study of the high energy electrons was carried out in different gases and re-scattered electrons with energies up to 100 eV were measured. The photoelectron spectra recorded with linearly polarised laser exhibit a plateau with a cut-off at 10 UP that is a characteristic of the re-scattering process. The observation of rescattered electrons was confirmed by two techniques: comparison of the data obtained with vertical polarisation (re-scattering) and circular polarisation (no re-scattering) and analysis of the structure in the angular distribution obtained in Xenon

Peripartum cardiomyopathy

This issue of eMedRef provides information to clinicians on the pathophysiology, diagnosis, and therapeutics of peripartum cardiomyopathy

Binary polypeptide system for permanent and oriented protein immobilization.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling. RESULTS: Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. This irreversible protein attachment system (IPAS) uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. CONCLUSIONS: IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.Published versio

Investigating the Heart of a Community: Archaeological Excavations at the African Meeting House, Boston, Massachusetts

In collaboration with the Museum of African American History, an archaeological research team from the University of Massachusetts Boston carried out a data recovery excavation at the African Meeting House on Beacon Hill. The African Meeting House was a powerful social institution for 19thcentury Boston’s free black community. The site played an important role in the abolition movement, the creation of educational opportunity, and other community action for social and political equality. The Meeting House was originally built in 1806, and renovations in preparation for the 2006 bi-centennial celebration prompted an investigation of areas of the property to be impacted by the proposed construction. Archaeological fieldwork, conducted under Massachusetts Historical Commission Permit Number 2750, was spread over seven weeks in May through July 2005. The field team opened and explored about 19 m2 of the site in the backlot south of the Meeting House and alley to the west. These excavations recorded information about a series of significant features and deposits, and collected over 38,000 artifacts and a series of soil samples for a detailed archaeobiological research program. These excavations met the requirements of the data recovery program as outlined in 950 CMR 70.00 and in the Memorandum of Agreement for the project, and the proposed renovation work proceeded with a finding of no adverse effect (36 CFR 800.5(b)). The depositional history and the nature of the archaeological record allow us to separate the overall excavation into three sub-areas: 1) the west alley between the AMH and 2 Smith Court; 2) the historic Meeting House backlot; and 3) the south yard, which originally belonged to the 44 Joy Street property. In terms of significant features and deposits, the west alley was almost entirely a series of builders’ trenches reflecting the historic sequence of construction and remodeling of the Meeting House and adjacent buildings to the west. In the backlot, the units against the south wall of the Meeting House contained similar builders’ trenches. The backlot also contained a series of stone and brick drains and a trash-rich midden layer. The vast majority of artifacts in the Meeting House backlot date from about 1806–1840. The ceramics assemblage is particularly large, and reflects both community meals at the Meeting House and business of Domingo Williams, a caterer who rented a basement apartment. Finally, only one feature was studied in the south yard, a privy (outhouse) that was for the 44 Joy Street property. The bottommost layer of the privy was an artifact rich nightsoil layer, dating to about 1811–1838, and containing the trash of African American tenants living at 44 Joy Street. Together, the archaeological deposits in the backlot provide a variety of insights into living conditions, economic opportunity, foodways, health, and daily life for 19th-century Boston’s free black community. These results thus provide information to help further the research, interpretation, and public education goals of the Museum of African American History

Assembly of Protein Building Blocks Using a Short Synthetic Peptide

Combining proteins or their defined domains offers new enhanced functions. Conventionally, two proteins are either fused into a single polypeptide chain by recombinant means or chemically cross-linked. However, these strategies can have drawbacks such as poor expression (recombinant fusions) or aggregation and inactivation (chemical cross-linking), especially in the case of large multifunctional proteins. We developed a new linking method which allows site-oriented, noncovalent, yet irreversible stapling of modified proteins at neutral pH and ambient temperature. This method is based on two distinct polypeptide linkers which self-assemble in the presence of a specific peptide staple allowing on-demand and irreversible combination of protein domains. Here we show that linkers can either be expressed or be chemically conjugated to proteins of interest, depending on the source of the proteins. We also show that the peptide staple can be shortened to 24 amino acids still permitting an irreversible combination of functional proteins. The versatility of this modular technique is demonstrated by stapling a variety of proteins either in solution or to surfaces

Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

FTY-720 (Fingolimod) was one of the first compounds authorized for the treatment of multiple sclerosis. Among its other activities, this sphingosine analogue enhances exocytosis in neuroendocrine chromaffin cells, altering the quantal release of catecholamines. Surprisingly, the size of chromaffin granules is reduced within few minutes of treatment, a process that is paralleled by the homotypic fusion of granules and their heterotypic fusion with mitochondria, as witnessed by dynamic confocal and TIRF microscopy. Electron microscopy studies support these observations, revealing the fusion of several vesicles with individual mitochondria to form large, round mixed organelles. This cross-fusion is SNARE-dependent, being partially prevented by the expression of an inactive form of SNAP-25. Fused mitochondria exhibit an altered redox potential, which dramatically enhances cell death. Therefore, the cross-fusion of intracellular organelles appears to be a new mechanism to be borne in mind when considering the effect of FTY-720 on the survival of neuroendocrine cells

Re-Assembled Botulinum Neurotoxin Inhibits CNS Functions without Systemic Toxicity

The therapeutic potential of botulinum neurotoxin type A (BoNT/A) has recently been widely recognized. BoNT/A acts to silence synaptic transmission via specific proteolytic cleavage of an essential neuronal protein, SNAP25. The advantages of BoNT/A-mediated synaptic silencing include very long duration, high potency and localized action. However, there is a fear of possible side-effects of BoNT/A due to its diffusible nature which may lead to neuromuscular blockade away from the injection site. We recently developed a “protein-stapling” technology which allows re-assembly of BoNT/A from two separate fragments. This technology allowed, for the first time, safe production of this popular neuronal silencing agent. Here we evaluated the re-assembled toxin in several CNS assays and assessed its systemic effects in an animal model. Our results show that the re-assembled toxin is potent in inhibiting CNS function at 1 nM concentration but surprisingly does not exhibit systemic toxicity after intraperitoneal injection even at 200 ng/kg dose. This shows that the re-assembled toxin represents a uniquely safe tool for neuroscience research and future medical applications