A point mutation within the ATP-binding site inactivates both catalytic functions of the ATP-dependent protease La (Lon) from Escherichia coli

AbstractA point mutant in the ATP-binding motif (GPPGVGK362T) of the ATP-dependent protease La from Escherichia coli was investigated in which the lysine at position 362 was replaced by an alanine. The catalytic efficiency of the K362A mutant is at least two orders of magnitude lower than that of wild-type protease La due to a decreased Vmax and an increased KM for ATP. Simultaneously, the peptidase activity of La K362A is almost completely eliminated. Since selective inactivation of the peptidase activity of La does not affect its intrinsic ATPase activity, coupling of proteolysis with ATP hydrolysis is only uni-directional in this energy-dependent protease

Swinging of red blood cells under shear flow

We reveal that under moderate shear stress (of the order of 0.1 Pa) red blood cells present an oscillation of their inclination (swinging) superimposed to the long-observed steady tanktreading (TT) motion. A model based on a fluid ellipsoid surrounded by a visco-elastic membrane initially unstrained (shape memory) predicts all observed features of the motion: an increase of both swinging amplitude and period (1/2 the TT period) upon decreasing the shear stress, a shear stress-triggered transition towards a narrow shear stress-range intermittent regime of successive swinging and tumbling, and a pure tumbling motion at lower shear stress-values.Comment: 4 pages 5 figures submitted to Physical Review Letter

A Machine Learning-Based Raman Spectroscopic Assay for the Identification of Burkholderia mallei and Related Species

Burkholderia (B.) mallei, the causative agent of glanders, and B. pseudomallei, the causative agent of melioidosis in humans and animals, are genetically closely related. The high infectious potential of both organisms, their serological cross-reactivity, and similar clinical symptoms in human and animals make the differentiation from each other and other Burkholderia species challenging. The increased resistance against many antibiotics implies the need for fast and robust identification methods. The use of Raman microspectroscopy in microbial diagnostic has the potential for rapid and reliable identification. Single bacterial cells are directly probed and a broad range of phenotypic information is recorded, which is subsequently analyzed by machine learning methods. Burkholderia were handled under biosafety level 1 (BSL 1) conditions after heat inactivation. The clusters of the spectral phenotypes and the diagnostic relevance of the Burkholderia spp. were considered for an advanced hierarchical machine learning approach. The strain panel for training involved 12 B. mallei, 13 B. pseudomallei and 11 other Burkholderia spp. type strains. The combination of top- and sub-level classifier identified the mallei-complex with high sensitivities (>95%). The reliable identification of unknown B. mallei and B. pseudomallei strains highlighted the robustness of the machine learning-based Raman spectroscopic assay

Low awareness of transient ischemic attacks and risk factors of stroke in a Swiss urban community

Background and purpose: Time delays from stroke onset to arrival at the hospital are the main obstacles for widespread use of thrombolysis. In order to decrease the delays, educational campaigns try to inform the general public how to act optimally in case of stroke. To determine the content of such a campaign, we assessed the stroke knowledge in our population. Methods: The stroke knowledge was studied by means of a closed-ended questionnaire. 422 randomly chosen inhabitants of Bern, Switzerland, were interviewed. Results: The knowledge of stroke warning signs (WS) was classified as good in 64.7%. A good knowledge of stroke risk factors (RF) was noted in 6.4%. 4.2% knew both the WS and the RF of stroke indicating a very good global knowledge of stroke. Only 8.3% recognized TIA as symptoms of stroke resolving within 24hours, and only 2.8% identified TIA as a disease requiring immediate medical help. In multivariate analysis being a woman, advancing age, and having an afflicted relative were associated with a good knowledge of WS (p=0.048, p<0.001 and p=0.043). Good knowledge of RF was related to university education (p<0.001). The good knowledge of TIA did not depend on age, sex, level of education or having an afflicted relative. Conclusions: The study brings to light relevant deficits of stroke knowledge in our population. A small number of participants could recognize TIA as stroke related symptoms resolving completely within 24hours. Only a third of the surveyed persons would seek immediate medical help in case of TIA. The information obtained will be used in the development of future educational campaign

Metabolic Futile Cycles and Their Functions: A Systems Analysis of Energy and Control

It has long been hypothesized that futile cycles in cellular metabolism are involved in the regulation of biochemical pathways. Following the work of Newsholme and Crabtree, we develop a quantitative theory for this idea based on open-system thermodynamics and metabolic control analysis. It is shown that the {\it stoichiometric sensitivity} of an intermediary metabolite concentration with respect to changes in steady-state flux is governed by the effective equilibrium constant of the intermediate formation, and the equilibrium can be regulated by a futile cycle. The direction of the shift in the effective equilibrium constant depends on the direction of operation of the futile cycle. High stoichiometric sensitivity corresponds to ultrasensitivity of an intermediate concentration to net flow through a pathway; low stoichiometric sensitivity corresponds to super-robustness of concentration with respect to changes in flux. Both cases potentially play important roles in metabolic regulation. Futile cycles actively shift the effective equilibrium by expending energy; the magnitude of changes in effective equilibria and sensitivities is a function of the amount of energy used by a futile cycle. This proposed mechanism for control by futile cycles works remarkably similarly to kinetic proofreading in biosynthesis. The sensitivity of the system is also intimately related to the rate of concentration fluctuations of intermediate metabolites. The possibly different roles of the two major mechanisms for cellular biochemical regulation, namely reversible chemical modifications via futile cycles and shifting equilibrium by macromolecular binding, are discussed.Comment: 11 pages, 5 figure