Buoyant plumes from solute gradients generated by non-motile Escherichia coli.

The effect of hydrodynamic mixing in bacterial populations due to bacterial chemotaxis is a well-described phenomenon known as bioconvection. Here we report the observation of buoyant plumes that result in hydrodynamic mixing, but in contrast to bioconvection the plumes form in the absence of bacterial motility. We propose that the buoyant flow originates from solute gradients created by bacterial metabolism, similar to solute-induced buoyant flow around growing protein crystals. In our experiments, metabolically-active non-motile Escherichia coli were layered along the bottom of flat-bottomed containers. The E. coli consumed glucose in the medium creating a lighter fluid beneath a heavier fluid. The situation is an example of Rayleigh-Taylor instability, in which a lighter fluid pushes on a heavier one. We developed a numerical model to study the effect of E. coli nutrient consumption and by-product excretion on extracellular solute gradients. The model solutions showed reduced-density fluid along the bottom of the fluid domain leading to buoyant plumes, which were qualitatively similar to the experimental plumes. We also used scaling analyses to study the dependence of plume formation on container size and cell size, and to investigate the effect of reduced gravity, such as the microgravity conditions encountered during spaceflight.

MTMDAT: Automated analysis and visualization of mass spectrometry data for tertiary and quaternary structure probing of proteins.

UNLABELLED: In structural biology and -genomics, nuclear magnetic resonance (NMR) spectroscopy and crystallography are the methods of choice, but sample requirements can be hard to fulfil. Valuable structural information can also be obtained by using a combination of limited proteolysis and mass spectrometry, providing not only knowledge of how to improve sample conditions for crystallization trials or NMR spectrosopy by gaining insight into subdomain identities but also probing tertiary and quaternary structure, folding and stability, ligand binding, protein interactions and the location of post-translational modifications. For high-throughput studies and larger proteins, however, this experimentally fast and easy approach produces considerable amounts of data, which until now has made the evaluation exceedingly laborious if at all manually possible. MTMDAT, equipped with a browser-like graphical user interface, accelerates this evaluation manifold by automated peak picking, assignment, data processing and visualization. AVAILABILITY: MTMDAT can be downloaded from the following page: http://www.cms.liu.se/chemistry/molbiotech/maria_sunnerhagens_group/mtmdat by clicking on the corresponding links (windows- or unix-based) together with the manual and example files. The program is free for academic/non-commercial purposes only.

The fellowship of the RING: the RING-B-box linker region interacts with the RING in TRIM21/Ro52, contains a native autoantigenic epitope in Sjögren syndrome, and is an integral and conserved region in TRIM proteins.

Ro52 is a major autoantigen that is targeted in the autoimmune disease Sjögren syndrome and belongs to the tripartite motif (TRIM) protein family. Disease-related antigenic epitopes are mainly found in the coiled-coil domain of Ro52, but one such epitope is located in the Zn(2+)-binding region, which comprises an N-terminal RING followed by a B-box, separated by a approximately 40-residue linker peptide. In the present study, we extend the structural, biophysical, and immunological knowledge of this RING-B-box linker (RBL) by employing an array of methods. Our bioinformatic investigations show that the RBL sequence motif is unique to TRIM proteins and can be classified into three distinct subtypes. The RBL regions of all three subtypes are as conserved as their known flanking domains, and all are predicted to comprise an amphipathic helix. This helix formation is confirmed by circular dichroism spectroscopy and is dependent on the presence of the RING. Immunological studies show that the RBL is part of a conformation-dependent epitope, and its antigenicity is likewise dependent on a structured RING domain. Recombinant Ro52 RING-RBL exists as a monomer in vitro, and binding of two Zn(2+) increases its stability. Regions stabilized by Zn(2+) binding are identified by limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry. Furthermore, the residues of the RING and linker that interact with each other are identified by analysis of protection patterns, which, together with bioinformatic and biophysical data, enabled us to propose a structural model of the RING-RBL based on modeling and docking experiments. Sequence similarities and evolutionary sequence patterns suggest that the results obtained from Ro52 are extendable to the entire TRIM protein family.

Transendoscopic ultrasound in ventricular lesions.

BACKGROUND: After transendoscopic sonocatheters had been tested in the laboratory for imaging characteristics and practicability, clinical application was studied with special reference to imaging and navigation capabilities, practicability, safety, and preliminary indications. METHODS: Intraoperative ENS images prepared during surgery on 75 selected patients between 1996 and 2005 were examined. There were 35 female and 40 male patients, and their mean age was 42 years (range, 2-69 years). Within this series, there were 28 cases of ventricular lesions (ventricular hematomas, tumors, and colloid cyst included, 35 cases) with different diagnosis. In most cases, Aloka sono equipment (Aloka Deutschland, Düsseldorf, Germany) was used because equipment supplied by this company had yielded superior imaging results in the laboratory. This work with patients differed from the laboratory work in that 2 sizes (diameters) of catheters were used: 6-F catheters for block-shaft endoscopes and 8-F for hollow-shaft endoscopes. RESULTS: Imaging: In clinical use, the sonocatheter has superior imaging and navigation abilities to those seen in anatomical laboratory work. Real-time and online characteristics represent changes such as shifting, pulsation, CSF flow, blood flow, and changes in size and form of structures. When confronted with clinical problems, this technique still has some limitations such as short penetration depth of 3-cm radius and lack of scanning anterior to the endoscope. Navigation: The scan is radial 360 degrees and in an orthogonal plane to the axis of the endoscope. At the tip of the endoscope it delivers an image that looks geometrically like a "brain radar." Because of its real-time characteristic, ENS has a navigation capacity that markedly differs from usual neuronavigation, but is intuitively usable. Endoneurosonography was applied in 8 hydrocephali, 3 colloid cysts, 5 intraventricular hematomas, 1 septostomy, 11 ETVs, 2 cystostomies, 4 multiple cysts, and 1 tumor biopsy cases. Three illustrative cases are presented. CONCLUSION: Endoneurosonography is a tool for intraoperative real-time and online high-resolution imaging, and neuronavigation of endoscopes with a working channel at least 2 mm in diameter; it also has application in a wide variety of ventricular lesions. Endoneurosonography is limited by short penetration depth and not scanning ahead to the endoscope anteriorly.

Endoneurosonography: technique and equipment, anatomy and imaging, and clinical application.

OBJECTIVE: To evaluate the usefulness of transendoscopic ultrasound in neurosurgery, we studied two new sonoprobes measuring 6 and 8 French in diameter in 20 fresh specimens. The application and indication are discussed in the first clinical series of 75 patients. METHODS: Sonocatheters (ALOKA, Meerbusch, Germany) 1.9 mm (6 French) and 2.4 mm (8 French) in diameter were introduced into the working channel of an endoscope. The preparations were done in nonfixed skulls in a surgical simulation-setting laboratory. Based on these experiences with imaging possibilities, intraoperative transendoscopic ultrasound was applied in 75 patients and a variety of lesions. It was used for imaging (41 patients), targeting (18 patients), and neuronavigation (16 patients) in neuroendoscopy. RESULTS: The sonoprobe adds a transverse scan at the tip of the probe to the anterior endoscopic view. This axial scan to the longitudinal axis of the endoscope is geometrically comparable with radar scanning. Three probes working with 10, 15, and 20 MHz were used, resulting in a short penetration with a radius of 3 cm. The orthogonal scanning plane had limitations, which were documented. We observed precise imaging of well known anatomic structures and, moreover, achieved an additional dimension in endoscopy. The axial scan presents the anatomic landmarks like a map at the tip of the endoscope where the endoscope is represented as a spot. The real-time imaging and representation of the tip of the endoscope showed a capacity for navigation. This preclinical study rectified clinical application. The real-time imaging of this technique showed the ability of the navigation of endoscopes to detect more overall movements, such as blood flow or change of ventricle size during endoscopy. The primary benefit in this first clinical series was witnessed in difficult endoscopy cases and complex lesions, but benefit was also observed in cases in which vision through the endoscope alone was obscured. The main limitation was the result of little penetration depth and lack of anterior scanning. CONCLUSION: Application of transendoscopic ultrasound is appropriate in neurosurgery. Training is necessary to understand the imaging and the geometry of scans because this technique does not scan along the axis of the endoscope. Further development to overcome the current limits of this technique and more clinical experience are needed.

Considerations for non-invasive in-flight monitoring of astronaut immune status with potential use of MEMS and NEMS devices.

The dynamics of how astronauts' immune systems respond to space flight have been studied extensively, but the complex process has not to date been thoroughly characterized, nor have the underlying principles of what causes the immune system to change in microgravity been fully determined. Statistically significant results regarding overall immunological effects in space have not yet been established due to the relatively limited amount of experimental data available, and are further complicated by the findings not showing systematically reproducible trends. Collecting in vivo data during flight without affecting the system being measured would increase understanding of the immune response process. The aims of this paper are to briefly review the current knowledge regarding how the immune system is altered in space flight; to present a group of candidate biomarkers that could be useful for in-flight monitoring and give an overview of the current methods used to measure these markers; and finally, to further establish the need and usefulness of incorporating real-time analytical techniques for in-flight assessment of astronaut health, emphasizing the potential application of MEMS/NEMS devices.

Microbial antibiotic production aboard the International Space Station.

Previous studies examining metabolic characteristics of bacterial cultures have mostly suggested that reduced gravity is advantageous for microbial growth. As a consequence, the question of whether space flight would similarly enhance secondary metabolite production was raised. Results from three prior space shuttle experiments indicated that antibiotic production was stimulated in space for two different microbial systems, albeit under suboptimal growth conditions. The goal of this latest experiment was to determine whether the enhanced productivity would also occur with better growth conditions and over longer durations of weightlessness. Microbial antibiotic production was examined onboard the International Space Station during the 72-day 8A increment. Findings of increased productivity of actinomycin D by Streptomyces plicatus in space corroborated with previous findings for the early sample points (days 8 and 12); however, the flight production levels were lower than the matched ground control samples for the remainder of the mission. The overall goal of this research program is to elucidate the specific mechanisms responsible for the initial stimulation of productivity in space and translate this knowledge into methods for improving efficiency of commercial production facilities on Earth.

Endo-neuro-sonography: first clinical series (52 cases).

OBJECTIVE: A sono catheter for transendoscopic imaging was applied in neurosurgery for the first time in 52 patients with a broad variety of lesions. METHODS: A transendoscopic sono catheter (Aloka Deutschland GmbH, Düsseldorf, Germany) with a diameter of 1.9 mm (6F) was used and introduced into the working canal of an endoscope. The image produced by the probe is a 360 degrees scan ("brain radar") displayed on a monitor, on which some parameters can be varied to get the best view of the different anatomical structures. RESULTS: In 39 patients intraoperative imaging was the main reason for investigation and in 13 patients neuronavigation was the focus of interest. In 18 cases of tumor resection control targeting a visualized remnant was necessary. There are limitations and artifacts, which should reveal themselves in laboratory and clinical experience. CONCLUSION: In this small series, endo-neuro-sonography proved to make neuroendoscopy safer and easier by online and real-time imaging with high resolution.

The effect of space flight on the production of actinomycin D by Streptomyces plicatus.

The effect of space flight on production of the antibiotic actinomycin D by Streptomyces plicatus WC56452 was examined onboard the US Space Shuttle mission STS-80. Paired space flight and ground control samples were similarly prepared using identical hardware, media, and inoculum. The cultures were grown in defined and complex media under dark, anaerobic, thermally controlled (20 degrees C) conditions with samples fixed after 7 and 12 days in orbit, and viable residuals maintained through landing at 17 days, 15 h. Postflight analyses indicated that space flight had reduced the colony-forming unit (CFU) per milliliter count of S. plicatus and increased the specific productivity (pg CFU(-1)) of actinomycin D. The antibiotic compound itself was not affected, but its production time course was altered in space. Viable flight samples also maintained their sporulation ability when plated on agar medium postflight, while the residual ground controls did not sporulate.

Postmortem inspection for neurosurgery: a training model for endoscopic dissection technique.

In 63 specimens, 74 aneurysms, and five other lesions, postmortem microsurgical and endoscopic inspection (PMI) was done. This work not only allowed for safe pathoanatomic findings, but moreover showed characteristics of a training method developed according to a model with clear standards. PMI gives training in: 1. Understanding of pathoanatomic topography and syntopy. 2. Analysis of imaging findings. 3. Analysis of approaches (approach planning). 4. Paraendoscopic methods (video surgery). 5. Clipping training. 6. Analyzing the ergonomy of the setting and instrumentation. In the series presented, aneurysms were the focus of attention. Postmortem inspection trains nearly all manipulative and cognitive abilities necessary for operative management of this difficult lesion. The acceptance and applicability of this method for resident training must be evaluated in the future.

Clinostats and bioreactors.

The environment created on Earth within a clinostat or Rotating Wall Vessel (RWV) bioreactor is often referred to as "simulated microgravity". Both devices utilize constant reorientation to effectively nullify cumulative sedimentation of particles. Neither, however, can fully reproduce the concurrent lack of structural deformation, displacement of intercellular components and/or reduced mass transfer in the extracellular fluid that occur in actual weightlessness. Parameters including density, viscosity, and even container geometry must each be considered to determine the overall gravity-dependent effects produced by either a clinostat or the RWV bioreactor; in addition, the intended application of these two devices differs considerably. A state of particle "motionlessness" relative to the surrounding bulk fluid, which is nearly analogous to the extracellular environment encountered under weightless conditions, can theoretically be achieved through clinorotation. The RWV bioreactor, on the other hand, while similarly maintaining cells in suspension as they continually "fall" through the medium under 1 g conditions, can also purposefully induce a perfusion of nutrients to and waste from the culture. A clinostat, therefore, is typically used in an attempt to reproduce the quiescent, unstirred fluid conditions achievable on orbit; while the RWV bioreactor ideally creates a low shear, but necessarily mixed, fluid environment that is optimized for suspension culture and tissue growth. Other techniques for exploring altered inertial environments, such as freefall, neutral buoyancy and electromagnetic levitation, can also provide unique insight into how gravity affects biological systems. Ultimately, all underlying biophysical principles thought to give rise to gravity-dependent physiological responses must be identified and thoroughly examined in order to accurately interpret data from flight experiments or ground-based microgravity analogs.

Bacterial growth in space flight: logistic growth curve parameters for Escherichia coli and Bacillus subtilis.

Previous investigations have reported that bacterial suspension cultures grow to higher stationary concentrations in space flight than on Earth; however, none of these investigations included extensive ground controls under varied inertial conditions. This study includes extensive controls and cell-growth data taken at several times during lag phase, log phase, and stationary phase of Escherichia coli and Bacillus subtilis. The Marquardt-Levenberg, least-squares fitting algorithm was used to calculate kinetic growth parameters from the logistic bacterial growth equations for space-flight and control growth curves. Space-flight cultures grew to higher stationary-phase concentrations and had shorter lag-phase durations. Also, evidence was found for increased exponential growth rate in space.

Microgravity and its implication for fermentation biotechnology.

Fermentation processes are highly dependent upon physical and chemical environmental parameters, many of which are influenced by gravity. Extending biotechnology into the realm of space flight provides researchers with an opportunity to investigate the role that gravity plays in natural growth processes. Physical factors governing cell sedimentation, nutrient mixing and byproduct dispersion are altered in the absence of the constant sedimenting force of gravity. In addition, space flight has also been shown to give rise to a wide variety of indirect consequences associated with the physiology of the organisms themselves.

The effects of space flight on the production of monorden by Humicola fuscoatra WC5157 in solid-state fermentation.

The effect of space flight on the production of the antibiotic monorden on two types of agar media, T8 and PG, by Humicola fuscoatra WC5157 was examined on board the US Space Shuttle mission STS-77 in May 1996. Paired space-flight and ground control samples were prepared using identical hardware, protocol, media, and inoculum. Inoculation occurred simultaneously for both groups 2.5 after launch. The flight and ground samples were allowed to grow for the entire 10-day mission in a dark, thermally controlled (22 degrees C) environment. Post-flight HPLC analysis of the flight and ground sample extracts indicated that the production of monorden by H. fuscoatra WC5157 in the flight samples was higher than in the ground samples in both agar media. In the T8 medium, the production of monorden in the flight and ground samples was 11.6 +/- 3.5 micrograms and 8.9 +/- 1.1 micrograms respectively (30% increase). In the PG medium, the production of monorden in the flight and ground samples was 23.8 +/- 3.3 micrograms and 8.2 +/- 2.2 micrograms respectively (190% increase). The production of monorden in the flight and ground control samples was confirmed by HPLC-MS analysis.

Cellular responses to gravity: extracellular, intracellular and in-between.

Our understanding of gravitational effects (inertial effects in the vicinity of 1 x g) on cells has matured to a stage at which it is possible to define, on the basis of experimental evidence, extracellular effects on small cells and intracellular effects on eukaryotic gravisensing cells. Yet undetermined is the nature of response, if any, of those classes of cells that are not governed solely by extracellular physical events (as are prokaryotes) and are devoid of obvious mechanical devices for sensing inertial forces (such as those possessed by certain plant cells and sensory cells of animals). This "in-between" class of cells needs to be understood on the basis of the combination of intracellular and extracellular gravity-dependent processes that govern experimentally-measurable variables that are relevant to the cell's responses to modified inertial forces. The forces that certain cell types generate or respond to are therefore compared to those imposed by approximately 1 x g in the context of cytoskeletal action and symmetry-breaking pathways.

Functional weightlessness during clinorotation of cell suspensions.

A clinostat is a device often used in gravitational biology studies. Selecting an appropriate speed of rotation, however, is a frequently debated topic, particularly for suspended cells. In an attempt to define the necessary criteria for determining an acceptable revolution speed, the primary forces governing particle behavior during clinorotation--gravity, diffusion and centrifugation--were mathematically assessed. In support of the theoretical exercise, bacterial growth experiments indicated that results obtained using a clinostat followed trends resembling previous space flight results. It is suspected that this is due, in part at least, to similarly altered external transport processes in each environment.

Autonomous Biological System (ABS) experiments.

Three space flight experiments have been conducted to test and demonstrate the use of a passively controlled, materially closed, bioregenerative life support system in space. The Autonomous Biological System (ABS) provides an experimental environment for long term growth and breeding of aquatic plants and animals. The ABS is completely materially closed, isolated from human life support systems and cabin atmosphere contaminants, and requires little need for astronaut intervention. Testing of the ABS marked several firsts: the first aquatic angiosperms to be grown in space; the first higher organisms (aquatic invertebrate animals) to complete their life cycles in space; the first completely bioregenerative life support system in space; and, among the first gravitational ecology experiments. As an introduction this paper describes the ABS, its flight performance, advantages and disadvantages.

Theories and models on the biology of cells in space.

A wide variety of observations on cells in space, admittedly made under constraining and unnatural conditions in many cases, have led to experimental results that were surprising or unexpected. Reproducibility, freedom from artifacts, and plausibility must be considered in all cases, even when results are not surprising. The papers in the symposium on "Theories and Models on the Biology of Cells in Space" are dedicated to the subject of the plausibility of cellular responses to gravity--inertial accelerations between 0 and 9.8 m/s2 and higher. The mechanical phenomena inside the cell, the gravitactic locomotion of single eukaryotic and prokaryotic cells, and the effects of inertial unloading on cellular physiology are addressed in theoretical and experimental studies.

Four educational programs in Space Life Sciences.

Four different educational programs impacting Space Life Sciences are described: the NASA/USRA Advanced Design Program, the NASA Specialized Center of Research and Training (NSCORT) Program, the Centers for the Commercial Development of Space (CCDS) Program, and the NASA Graduate Research Fellow Program. Each program makes somewhat different demands on the students engaged in them. Each program, at the University of Colorado, involves Space Life Sciences training. While the Graduate Student Research Fellow and NSCORT Programs are discipline oriented, the Advanced Design and CCDS Programs are focused on design, technologies and applications. Clearly, the "training paradigms" differ for these educational endeavors. But, these paradigms can be made to mutually facilitate enthusiasm and motivation. Discipline-oriented academic programs, ideally, must be flexible enough to accommodate the emergent cross-disciplinary needs of Space Life Sciences students. Models for such flexibility and resultant student performance levels are discussed based upon actual academic and professional records.