The Global Influence of Sodium on Cyanobacteria in Resuscitation from Nitrogen Starvation.

Dormancy and resuscitation are key to bacterial survival under fluctuating environmental conditions. In the absence of combined nitrogen sources, the non-diazotrophic model cyanobacterium Synechocystis sp. PCC 6803 enters into a metabolically quiescent state during a process termed chlorosis. This state enables the cells to survive until nitrogen sources reappear, whereupon the cells resuscitate in a process that follows a highly orchestrated program. This coincides with a metabolic switch into a heterotrophic-like mode where glycogen catabolism provides the cells with reductant and carbon skeletons for the anabolic reactions that serve to re-establish a photosynthetically active cell. Here we show that the entire resuscitation process requires the presence of sodium, a ubiquitous cation that has a broad impact on bacterial physiology. The requirement for sodium in resuscitating cells persists even at elevated CO2 levels, a condition that, by contrast, relieves the requirement for sodium ions in vegetative cells. Using a multi-pronged approach, including the first metabolome analysis of Synechocystis cells resuscitating from chlorosis, we reveal the involvement of sodium at multiple levels. Not only does sodium play a role in the bioenergetics of chlorotic cells, as previously shown, but it is also involved in nitrogen compound assimilation, pH regulation, and synthesis of key metabolites.

Diurnal metabolic control in cyanobacteria requires perception of second messenger signaling molecule c-di-AMP by the carbon control protein SbtB.

Because of their photosynthesis-dependent lifestyle, cyanobacteria evolved sophisticated regulatory mechanisms to adapt to oscillating day-night metabolic changes. How they coordinate the metabolic switch between autotrophic and glycogen-catabolic metabolism in light and darkness is poorly understood. Recently, c-di-AMP has been implicated in diurnal regulation, but its mode of action remains elusive. To unravel the signaling functions of c-di-AMP in cyanobacteria, we isolated c-di-AMP receptor proteins. Thereby, the carbon-sensor protein SbtB was identified as a major c-di-AMP receptor, which we confirmed biochemically and by x-ray crystallography. In search for the c-di-AMP signaling function of SbtB, we found that both SbtB and c-di-AMP cyclase­deficient mutants showed reduced diurnal growth and that c-di-AMP­bound SbtB interacts specifically with the glycogen-branching enzyme GlgB. Accordingly, both mutants displayed impaired glycogen synthesis during the day and impaired nighttime survival. Thus, the pivotal role of c-di-AMP in day-night acclimation can be attributed to SbtB-mediated regulation of glycogen metabolism.

The essential role of sodium bioenergetics and ATP homeostasis in the developmental transitions of a cyanobacterium.

The ability to resume growth after a dormant period is an important strategy for the survival and spreading of bacterial populations. Energy homeostasis is critical in the transition into and out of a quiescent state. Synechocystis sp. PCC 6803, a non-diazotrophic cyanobacterium, enters metabolic dormancy as a response to nitrogen starvation. We used Synechocystis as a model to investigate the regulation of ATP homeostasis during dormancy, and we unraveled a critical role for sodium bioenergetics in dormant cells. During nitrogen starvation, cells reduce their ATP levels and engage sodium bioenergetics to maintain the minimum ATP content required for viability. When nitrogen becomes available, energy requirements rise, and cells immediately increase ATP levels, employing sodium bioenergetics and glycogen catabolism. These processes allow them to restore the photosynthetic machinery and resume photoautotrophic growth. Our work reveals a precise regulation of the energy metabolism essential for bacterial survival during periods of nutrient deprivation.

Antimicrobial Effect of Biocompatible Silicon Nanoparticles Activated Using Therapeutic Ultrasound.

In this study, we report a method for the suppression of Escherichia coli (E. coli) vitality by means of therapeutic ultrasound irradiation (USI) using biocompatible silicon nanoparticles as cavitation sensitizers. Silicon nanoparticles without (SiNPs) and with polysaccharide (dextran) coating (DSiNPs) were used. Both types of nanoparticles were nontoxic to Hep 2 cells up to a concentration of 2 mg/mL. The treatment of bacteria with nanoparticles and application of 1 W/cm2 USI resulted in the reduction of their viabilities up to 35 and 72% for SiNPs and DSiNPs, respectively. The higher bacterial viability reduction for DSiNPs as compared with SiNPs can be explained by the fact that the biopolymer shell of the polysaccharide provides a stronger adhesion of nanoparticles to the bacterial surface. Transmission electron microscopy (TEM) studies showed that the bacterial lipid shell was partially perforated after the combined treatment of DSiNPs and USI, which can be explained by the lysis of bacterial membrane due to the cavitation sensitized by the SiNPs. Furthermore, we have shown that 100% inhibition of E. coli bacterial colony growth is possible by coupling the treatments of DSiNPs and USI with an increased intensity of up to 3 W/cm2. The observed results reveal the application of SiNPs as promising antimicrobial agents.

Studies of silicon nanoparticles uptake and biodegradation in cancer cells by Raman spectroscopy.

In-vitro Raman micro-spectroscopy was used for diagnostics of the processes of uptake and biodegradation of porous silicon nanoparticles (SiNPs) in breast cancer cells (MCF-7 cell line). Two types of nanoparticles, with and without photoluminescence in the visible spectral range, were investigated. The spatial distribution of photoluminescent SiNPs within the cells obtained by Raman imaging was verified by high-resolution structured-illumination optical microscopy. Nearly complete biodegradation of SiNPs inside the living cells was observed after 13days of the incubation. The results reveal new prospects of multi-modal visualization of SiNPs inside cancer cells for theranostic applications.

Organogels based on 12-hydroxy stearic acid as a leitmotif: Dependence of gelation properties on chemical modifications.

Various compounds based on the structural leitmotif of 12-hydroxy stearic acid (HSA) were studied with respect to their ability to form organogels. They were modified by ethoxylation in order to avoid the acid group of HSA, which is unwanted for many of the applications of organogels. In this paper, it is shown that the rheological performance of organogels depends strongly on the extent of ethoxylation, exhibiting an optimum at intermediate degrees of ethoxylation. Furthermore, we reveal that the ability for gelation as well as the mechanical properties are substantially reduced by the presence of stearic acid (SA) in the original reaction mixture, which is a typical contamination of HSA. This is quantified by the amount of gelator required for gelation and the elastic moduli observed for the gels. At the same time the mesoscopic structure, as probed by small-angle neutron scattering (SANS), is almost unchanged for different degrees of ethoxylation or the addition of SA--and similarly thick fibres are observed, while the viscoelastic parameters evolve. Accordingly the elastic efficiency of the individual structural units is responsible for the observed changes in the gelation properties. These findings are relevant for the application of such low molecular weight organogelators in practical formulations, as one can optimise the rheological properties of organogelators by appropriately choosing the degree of ethoxylation.

Pelvic fracture in multiple trauma: are we still up-to-date with massive fluid resuscitation?

Until today the mortality of complex pelvic trauma remains unacceptably high. On the one hand this could be attributed to a biological limit of the survivable trauma load, on the other hand side an ongoing inadequate treatment might be conceivable too. For the management of multiple trauma patients with life-threatening pelvic fractures, there is ongoing international debate on the adequate therapeutic strategy, e.g. arterial embolization or pelvic packing, as well as aggressive or restrained volume therapy. Whereas traditional pelvis-specific trauma algorithms still recommend massive fluid resuscitation, there is upcoming evidence that a restrained volume therapy in the preclinical setting may improve trauma outcomes. Less intravenous fluid administration may also reduce haemodilution and concomitant trauma-associated coagulopathy. After linking the data of the TraumaRegister DGU(®) and the German Pelvic Injury Register, for the first time, the initial fluid management for complex pelvic traumas as well as for different Tile/OTA types of pelvic ring fractures could be addressed. Unfortunately, the results could not answer the question of the adequate fluid resuscitation but confirmed the actuality of massive fluid resuscitation in the prehospital and emergency room setting. Low-volume resuscitation seems not yet accepted in practice in managing multiple trauma patients with pelvic fractures at least in Germany. Nevertheless, prevention of exsanguination and of complications like multiple organ dysfunction syndrome still poses a major challenge in the management of complex pelvic ring injuries. Even nowadays, fluid management for trauma, not only for pelvic fractures, remains a controversial area and further research is mandatory.

Compression and contact area of anterior strut grafts in spinal instrumentation: a biomechanical study.

BACKGROUND: Anterior bone grafts are used as struts to reconstruct the anterior column of the spine in kyphosis or following injury. An incomplete fusion can lead to later correction losses and compromise further healing. Despite the different stabilizing techniques that have evolved, from posterior or anterior fixating implants to combined anterior/posterior instrumentation, graft pseudarthrosis rates remain an important concern. Furthermore, the need for additional anterior implant fixation is still controversial. In this bench-top study, we focused on the graft-bone interface under various conditions, using two simulated spinal injury models and common surgical fixation techniques to investigate the effect of implant-mediated compression and contact on the anterior graft. METHODS: Calf spines were stabilised with posterior internal fixators. The wooden blocks as substitutes for strut grafts were impacted using a "pressfit" technique and pressure-sensitive films placed at the interface between the vertebral bone and the graft to record the compression force and the contact area with various stabilization techniques. Compression was achieved either with posterior internal fixator alone or with an additional anterior implant. The importance of concomitant ligament damage was also considered using two simulated injury models: pure compression Magerl/AO fracture type A or rotation/translation fracture type C models. RESULTS: In type A injury models, 1 mm-oversized grafts for impaction grafting provided good compression and fair contact areas that were both markedly increased by the use of additional compressing anterior rods or by shortening the posterior fixator construct. Anterior instrumentation by itself had similar effects. For type C injuries, dramatic differences were observed between the techniques, as there was a net decrease in compression and an inadequate contact on the graft occurred in this model. Under these circumstances, both compression and the contact area on graft could only be maintained at high levels with the use of additional anterior rods. CONCLUSIONS: Under experimental conditions, we observed that ligamentous injury following type C fracture has a negative influence on the compression and contact area of anterior interbody bone grafts when only an internal fixator is used for stabilization. Because of the loss of tension banding effects in type C injuries, an additional anterior compressing implant can be beneficial to restore both compression to and contact on the strut graft.

Trauma registry record linkage: methodological approach to benefit from complementary data using the example of the German Pelvic Injury Register and the TraumaRegister DGU(®).

BACKGROUND: In Germany, hospitals can deliver data from patients with pelvic fractures selectively or twofold to two different trauma registries, i.e. the German Pelvic Injury Register (PIR) and the TraumaRegister DGU(®) (TR). Both registers are anonymous and differ in composition and content. We describe the methodological approach of linking these registries and reidentifying twofold documented patients. The aim of the approach is to create an intersection set that benefit from complementary data of each registry, respectively. Furthermore, the concordance of data entry of some clinical variables entered in both registries was evaluated. METHODS: PIR (4,323 patients) and TR (34,134 patients) data from 2004-2009 were linked together by using a specific match code including code of the trauma department, dates of admission and discharge, patient's age, and sex. Data entry concordance was evaluated using haemoglobin and blood pressure levels at emergency department arrival, Injury Severity Score (ISS), and mortality. RESULTS: Altogether, 420 patients were identified as documented in both data sets. Linkage rates for the intersection set were 15.7% for PIR and 44.4% for TR. Initial fluid management for different Tile/OTA types of pelvic ring fractures and the patient's posttraumatic course, including intensive care unit data, were now available for the PIR population. TR is benefiting from clinical use of the Tile/OTA classification and from correlation with the distinct entity "complex pelvic injury." Data entry verification showed high concordance for the ISS and mortality, whereas initial haemoglobin and blood pressure data showed significant differences, reflecting inconsistency at the data entry level. CONCLUSIONS: Individually, the PIR and the TR reflect a valid source for documenting injured patients, although the data reflect the emphasis of the particular registry. Linking the two registries enabled new insights into care of multiple-trauma patients with pelvic fractures even when linkage rates were poor. Future considerations and development of the registries should be done in close bilateral consultation with the aim of benefiting from complementary data and improving data concordance. It is also conceivable to integrate individual modules, e.g. a pelvic fracture module, into the TR likewise a modular system in the future.

Acute management and outcome of multiple trauma patients with pelvic disruptions.

INTRODUCTION: Data on prehospital and trauma-room fluid management of multiple trauma patients with pelvic disruptions are rarely reported. Present trauma algorithms recommend early hemorrhage control and massive fluid resuscitation. By matching the German Pelvic Injury Register (PIR) with the TraumaRegister DGU (TR) for the first time, we attempt to assess the initial fluid management for different Tile/OTA types of pelvic-ring fractures. Special attention was given to the patient's posttraumatic course, particularly intensive care unit (ICU) data and patient outcome. METHODS: A specific match code was applied to identify certain patients with pelvic disruptions from both PIR and TR anonymous trauma databases, admitted between 2004 and 2009. From the resulting intersection set, a retrospective analysis was done of prehospital and trauma-room data, length of ICU stay, days of ventilation, incidence of multiple organ dysfunction syndrome (MODS), sepsis, and mortality. RESULTS: In total, 402 patients were identified. Mean ISS was 25.9 points, and the mean of patients with ISS ≥ 16 was 85.6%. The fracture distribution was as follows: 19.7% type A, 29.4% type B, 36.6% type C, and 14.3% isolated acetabular and/or sacrum fractures. The type B/C, compared with type A fractures, were related to constantly worse vital signs that necessitated a higher volume of fluid and blood administration in the prehospital and/or the trauma-room setting. This group of B/C fractures were also related to a significantly higher presence of concomitant injuries and related to increased ISS. This was related to increased ventilation and ICU stay, increased rate of MODS, sepsis, and increased rate of mortality, at least for the type C fractures. Approximately 80% of the dead had sustained type B/C fractures. CONCLUSIONS: The present study confirms the actuality of traditional trauma algorithms with initial massive fluid resuscitation in the recent therapy of multiple trauma patients with pelvic disruptions. Low-volume resuscitation seems not yet to be accepted in practice in managing this special patient entity. Mechanically unstable pelvic-ring fractures type B/C (according to the Tile/OTA classification) form a distinct entity that must be considered notably in future trauma algorithms.

Balloon tibioplasty: a useful tool for reduction of tibial plateau depression fractures.

Reduction of the articular surface in displaced tibial plateau fractures is still challenging and may result in joint incongruence, leading to posttraumatic arthrosis. Conventional techniques use bone tamps and similar instruments, which can increase the surgical trauma due to their size. "Balloon tibioplasty" is a novel minimally invasive technique for the reduction of depressed tibial plateau fractures. We successfully applied an inflatable balloon, commercially available from kyphoplasty, to elevate the depressed articular fragments. This technique allowed for reduction of the depressed tibial plateau fragment without classic fenestration of the tibia, thereby minimizing surgical trauma. Furthermore, under fluoroscopic control, optimal centering of the expanding tibioplasty balloon allows a widespread and continuously increasing reduction force to the fracture area. After fluoroscopy or arthroscopic confirmation of reduction of the articular surface, the cavity resulting from tibioplasty was filled with ceramic bone cement through small incisions and fractures were fixed with a small fragment locking T-plate (3.5 mm). Balloon tibioplasty was applied in 5 patients with displaced tibial plateau fractures (OTA type B2/3). No intra- or postoperative complications were observed. This new technique may be a useful tool to facilitate the reduction of select depressed tibial fractures in the future.

Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.

It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. Here we show that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, we are able to alter the shape of the Fgf8 gradient. Our results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism.

Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms.

Analysis of receptor-ligand interactions in vivo is key to biology but poses a considerable challenge to quantitative microscopy. Here we combine static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy to solve this task at cellular resolution in complex biological environments. We quantified the mobility of fibroblast growth factor receptors Fgfr1 and Fgfr4 in cell membranes of living zebrafish embryos and determined their in vivo binding affinities to their ligand Fgf8.

Bilateral pneumothoraces, pneumomediastinum, pneumoperitoneum, pneumoretroperitoneum, and subcutaneous emphysema after thoracoscopic anterior fracture stabilization.

STUDY DESIGN: Case report and clinical discussion. OBJECTIVE: A rare case of air passage into multiple body compartments after thoracoscopic minimally invasive spine surgery is described. SUMMARY OF BACKGROUND DATA: In recent years, there is growing interest in thoracoscopic minimally invasive spine surgery for the treatment of thoracic and lumbar spine fractures. Severe complications due to the operative procedure are rare. METHODS: We present a case of a 73-year-old woman who developed bilateral pneumothoraces, pneumomediastinum, pneumoperitoneum, pneumoretroperitoneum, and subcutaneous emphysema after thoracoscopic anterior stabilization of a Th12 fracture. RESULTS: The operative procedure was completed without any obvious intraoperative complications. Routine made postoperative radiograph of the chest revealed a pneumothorax on the right side, bilateral subphrenic free air, and bilateral supraclavicular air. Subsequently, a CT scan showed bilateral pneumothoraces, pneumomediastinum, pneumoperitoneum, pneumoretroperitoneum and a supraclavicular subcutaneous emphysema. Bronchoscopy, esophagogastroduodenoscopy, and laryngoscopy showed no hollow organ injury or any other pathologic changes. Intraabdominal free air and pneumothoraces could not be detected on thoracic radiographs after 2 days. The patient remained cardiopulmonary stable throughout the hospital course. CONCLUSION: This report documents a rare case of air passage into multiple body compartments after thoracoscopic-assisted treatment of a spinal fracture, which has not yet been described previously. After exclusion of a tracheo-bronchial and hollow organ injury the process was self-limiting. To avoid this complication, special care should be taken to evacuate all intrathoracal air at the end of the endoscopic procedure.

Macroscopic properties and microstructure of HSA based organogels: sensitivity to polar additives.

Organogels can be formed by a large variety of different low molecular weight gelators. In our investigations we employed 12-hydroxy stearic acid (HSA) and studied its ability to gel various organic solvents, such as dodecane, paraffin oil, triglycerides, octyl palmitate, and silicone oil. Gelation typically occurs above a concentration of 0.1-0.8 wt% and the gelation concentration is related to the polarity of the oil. In a second part we investigated the influence of polar additives which can affect the gelation process due to their polar character. By doing so we varied largely the polarity conditions under which the gelation of the HSA occurs. The macroscopic gel properties were characterised by means of oscillatory rheology experiments, differential scanning calorimetry (DSC), and optical microscopy. The microstructure of the samples was studied by means of small-angle neutron scattering (SANS). The gelation of HSA mainly depends on the availability of free acidic and hydroxy groups for the formation of H-bonds. Accordingly the elastic properties and finally the gelation process become suppressed by the addition of polar additives such as alcohols. This effect depends just on the molar ratio of added alcohol and HSA, being independent of the length of the added alcohol. Evidently the added alcohol is competing for the H-bonding with the HSA and thereby effectively destroying the H-bonding necessary for the formation of the network-forming fibres. This finding is of importance for potential applications of such molecules as rheological modifiers in oil-based systems.

Water-soluble interpolyelectrolyte complexes of polyisobutylene-block-poly(methacrylic acid) micelles: formation and properties.

We report on interpolyelectrolyte complexes (IPECs) formed by micelles of ionic amphiphilic diblock copolymers with polyisobutylene (PIB) and poly(sodium methacrylate) (PMANa) blocks interacting with quaternized poly(4-vinylpyridine) (P4VPQ). The interpolyelectrolyte complexation was followed by turbidimetry and small angle neutron scattering (SANS). The data obtained by means of a combination of SANS, dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM) provide evidence on the core-shell-corona structure of the complex species with the shell assembled from fragments of electrostatically bound PMANa and quaternized P4VPQ fragments, original PIBx-b-PMAAy micelles apparently playing a lyophilizing part. The complex formation is followed by potentiometric titration as well. This process is initially kinetically controlled. In the second step larger aggregates rearrange in favor of smaller complexes with core-shell-corona structure, which are thermodynamically more stable. An increase in ionic strength of the solution results in dissociation of the complex species as proven by SANS and analytical ultracentrifugation (AUC). This process begins at the certain threshold ionic strength and proceeds via a salt-induced gradual release of chains of the cationic polyectrolyte from the complex species.

Polyisobutylene-block-poly(methacrylic acid) diblock copolymers: self-assembly in aqueous media.

Ionic amphiphilic diblock copolymer polyisobutylene-block-poly(methacrylic acid) (PIBx-b-PMAAy), with various lengths of nonpolar (x=25-75) and polyelectrolyte (y=170-2600) blocks, spontaneously dissolve in aqueous media at pH>4, generating macromolecular assemblies, the aggregation number of which depends on external stimuli (pH and ionic strength). Spherical micellar morphology with a compact core formed by the PIB blocks and a swollen corona built up from the PMAA blocks was deduced by cryogenic transmission electron microscopy. The micelles were further characterized by means of dynamic and static light scattering as well as small-angle neutron scattering. The critical micellization concentration, estimated by means of fluorescence spectroscopy with the use of pyrene as a polarity probe, is decisively determined by the length of the PIB block and is insensitive to changes in the length of the PMAA block.

Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is carried out with an electron multiplying CCD (EMCCD). This new strategy is compared to standard detection by an avalanche photo diode showing good agreement with respect to the resulting autocorrelation curves. Applying different readout modes, a time resolution of 20 micros can be achieved, which is sufficient to resolve the diffusion of free dye in solution. The advantages of implementing EMCCD cameras in wide-field ultra low light imaging, as well as in multi-spot confocal laser scanning microscopy, can consequently also be exploited for spatially resolved FCS. First proof-of-principle FCS measurements with two excitation volumes demonstrate the advantage of the flexible CCD area detection.

Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform.

We have developed a filterless multicolor detection unit for fluorescence correlation spectroscopy (FCS). This grating-based setup is continuously tunable for multicolor separation and is thus a powerful alternative to the classical cascade of dichroic mirrors and filters. Our tailored platform allows for accommodation of up to 15 detection channels covering the entire visible spectral range. As a proof of principle, we successfully demonstrate simultaneous FCS of four distinct fluorescent quantum dot species being mixed in aqueous solution. Grating-based detection allows for spectral high-resolution FCS in a stable and compact setup and is a feasible tool for quantitative investigation of complexbiomolecular dynamics on a single molecule level.