Dynamics and force generation of flagellum and pili in Caulobacter crescentus

Surface attachment of bacteria is the first step of biofilm formation and biofilms are associated with infections and bacterial resistance. Surface attachment of bacteria is often mediated by extracellular appendages, for example flagellum and pili. The flagellum is a cork-screw like structure used for swimming and surface sensing. Pili are filamentous structures and have a wide variety of functions, among them attachment on surfaces. Because of the small diameter of flagellum and pili, direct observations of flagellum and pili are challenging under physiological conditions. C. crescentus, a model organism for biofilm formation, has an asymmetric life cycle. The sessile and stalked mother cell produces a motile daughter cell that is equipped with a flagellum and pili at the free pole. In this work we investigated the dynamics and force generation of the flagellum and pili of C. crescentus under physiological conditions employing a label-free method

Bacteria-on-a-bead: probing the hydrodynamic interplay of dynamic cell appendages during cell separation

Surface attachment of bacteria is the first step of biofilm formation and is often mediated and coordinated by the extracellular appendages, flagellum and pili. The model organism Caulobacter crescentus undergoes an asymmetric division cycle, giving rise to a motile "swarmer cell" and a sessile "stalked cell", which is attached to the surface. In the highly polarized predivisional cell, pili and flagellum, which are assembled at the pole opposite the stalk, are both activated before and during the process of cell separation. We explored the interplay of flagellum and active pili by growing predivisional cells on colloidal beads, creating a bacteria-on-a-bead system. Using this set-up, we were able to simultaneously visualize the bacterial motility and analyze the dynamics of the flagellum and pili during cell separation. The observed activities of flagellum and pili at the new cell pole of the predivisional cell result in a cooperating interplay of the appendages during approaching and attaching to a surface. Even in presence of a functioning flagellum, pili are capable of surface attachment and keeping the cell in position. Moreover, while flagellar rotation decreases the average attachment time of a single pilus, it increases the overall attachment rate of pili in a synergetic manner

Tad pili play a dynamic role in Caulobacter crescentus surface colonization

Bacterial surface attachment is mediated by rotary flagella and filamentous appendages called pili. Here, we describe the role of Tad pili during surface colonization of Caulobacter crescentus. Using an optical trap and microfluidic controlled flow conditions as a mimic of natural environments, we demonstrate that Tad pili undergo repeated cycles of extension and retraction. Within seconds after establishing surface contact, pili reorient cells into an upright position promoting walking-like movements against the medium flow. Pili-mediated positioning of the flagellated pole close to the surface facilitates motor-mediated mechanical sensing and promotes anchoring of the holdfast, an adhesive substance that affords long-term attachment. We present evidence that the second messenger c-di-GMP regulates pili dynamics during surface encounter in distinct ways, promoting increased activity at intermediate levels and retraction of pili at peak concentrations. We propose a model, in which flagellum and Tad pili functionally interact and together impose a ratchet-like mechanism that progressively drives C. crescentus cells towards permanent surface attachment

Biocatalytic atom transfer radical polymerization in a protein cage nanoreactor

Incorporation of the ATRP-catalyzing enzyme horseradish peroxidase (HRP) into the cavities of the group II chaperonin thermosome is demonstrated. The resulting nanoreactor was used to polymerize an acrylate under ARGET ATRP conditions. The confined space within the protein cage results in poly(ethylene glycol) methyl ether acrylate (PEGA) with lower molecular weights (poly(styrene)-apparent M-n = 4400 g mol(-1)) as well as narrower molecular weight distributions (D = 1.08) compared to polymerizations with the free ATRPase (M-n = 43 700 g mol(-1) and a D of 1.23)

Live cell X-ray imaging of autophagic vacuoles formation and chromatin dynamics in fission yeast

Seeing physiological processes at the nanoscale in living organisms without labeling is an ultimate goal in life sciences. Using X-ray ptychography, we explored in situ the dynamics of unstained, living fission yeast Schizosaccharomyces pombe cells in natural, aqueous environment at the nanoscale. In contrast to previous X-ray imaging studies on biological matter, in this work the eukaryotic cells were alive even after several ptychographic X-ray scans, which allowed us to visualize the chromatin motion as well as the autophagic cell death induced by the ionizing radiation. The accumulated radiation of the sequential scans allowed for the determination of a characteristic dose of autophagic vacuole formation and the lethal dose for fission yeast. The presented results demonstrate a practical method that opens another way of looking at living biological specimens and processes in a time-resolved label-free setting

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Tad Pili Play a Dynamic Role in Caulobacter crescentus Surface Colonization

Bacteria are able to colonize surfaces in environmental, industrial, and medical settings, where they form resilient communities called biofilms. In order to control bacterial surface colonization, microbiologists need to gain a detailed understanding of the processes that bacteria use to live at the liquid-surface interface and that allow them to adhere to and move on surfaces and eventually grow and persist on solid media. To facilitate these processes, bacteria are equipped with adhesive structures such as flagella and pili and with matrix components such as exopolysaccharides. How these cellular organelles are coordinated to optimize surface processes is currently subject to intense investigations. Here we used the model organism Caulobacter crescentus to demonstrate that polar pili are highly dynamic structures that are functionally interconnected with the flagellar motor to mediate surface sensing, thereby enforcing rapid and permanent surface attachment. These studies provide an entry point for an in-depth molecular analysis of bacterial surface colonization.Bacterial surface attachment is mediated by filamentous appendages called pili. Here, we describe the role of Tad pili during surface colonization of Caulobacter crescentus. Using an optical trap and microfluidic controlled flow conditions to mimic natural environments, we demonstrated that Tad pili undergo repeated dynamic cycles of extension and retraction. Within seconds after establishing surface contact, pilus retraction reorients cells into an upright position, promoting walking-like movements against the medium flow. Pilus-mediated positioning of the flagellate pole close to the surface facilitates motor-mediated mechanical sensing and promotes anchoring of the holdfast, an adhesive substance that affords long-term attachment. We present evidence that the second messenger c-di-GMP regulates pilus dynamics during surface encounter in distinct ways, promoting increased activity at intermediate levels and retraction of pili at peak concentrations. We propose a model in which flagellum and Tad pili functionally interact and together impose a ratchet-like mechanism that progressively drives C. crescentus cells toward permanent surface attachment

Out-of-hospital cardiac arrests in Switzerland: Predictors for emergency department mortality in patients with ROSC or on-going CPR on admission to the emergency department.

BACKGROUND One of the leading causes of death is out-of-hospital cardiac arrest (OHCA) with an in-hospital mortality of about 70%. To identify predictors for the high mortality of OHCA patients and especially for women, that are considered at high risk for in-hospital mortality, we evaluated one specific setting of in-hospital treatment after OHCA: the emergency department (ED). METHODS Retrospective analysis of consecutive ED admissions with OHCA at the Inselspital Bern, Switzerland from 1st June 2012 to 31th Mai 2015. Demographic, preclinical and ED medical data were compared for patient groups with return of circulation (ROSC) and on-going resuscitation (CPR) on admission, as well as for subgroups with and without ED mortality. Predictors for ED mortality were investigated using univariate analysis with logistic regression. RESULTS In 354 patients (228 (64.4%) with ROSC; 126 (35.6%) with on-going CPR) we found an overall ED mortality of 28.5% (5.7% ROSC group; 69.8% on-going CPR group). Female gender (OR 7.053 (CI 95% 2.085; 24.853), p = 0.002) and greater age (OR 1.052 (95% CI 1.006-1.101), p = 0.029) were associated with ED mortality in the ROSC but not in the on-going CPR group. Ventricular fibrillation as initially monitored rhythm (OR 0.126 (95% CI 0.027-0.582), p = 0.008) and shorter CPR duration (OR 1.055 (95% CI 1.024;1.088), p = 0.001) were associated with ED survival in patients with ROSC but not in patients with on-going CPR on admission. In ROSC patients a higher lactate and lower pH were associated with mortality (pH: OR 0.009 (CI95% 0.000;0.420), p = 0.016; lactate: OR 1.183 (95% CI 1.037; 1.349), p = 0.013); similar in on-going CPR patients (pH 0.061 (95% CI 0.007, 0.558), p = 0.013, lactate: 1.146 (95% CI 1.041;1.261), p = 0.005). CONCLUSION Patients with ROSC who died during ED care were predominantly women and older patients, as well as patients with non-shockable initial heart rhythm and long CPR durations. In patients with on-going CPR on admission, no clinical or demographic predictors for ED mortality were found. Higher lactate and lower pH were predictors in both OHCA groups

openBEB: open biological experiment browser for correlative measurements

Background: New experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis. Results: We have developed openBEB (open Biological Experiment Browser), a software framework for data acquisition, coordination, annotation and synchronization with database solutions such as openBIS. OpenBEB consists of two main parts: A core program and a plug-in manager. Whereas the data-type independent core of openBEB maintains a local container of raw-data and metadata and provides annotation and data management tools, all data-specific tasks are performed by plug-ins. The open architecture of openBEB enables the fast integration of plug-ins, e.g., for data acquisition or visualization. A macro-interpreter allows the automation and coordination of the different modules. An update and deployment mechanism keeps the core program, the plug-ins and the metadata definition files in sync with a central repository. Conclusions: The versatility, the simple deployment and update mechanism, and the scalability in terms of module integration offered by openBEB make this software interesting for a large scientific community. OpenBEB targets three types of researcher, ideally working closely together: (i) Engineers and scientists developing new methods and instruments, e.g., for systems-biology, (ii) scientists performing biological experiments, (iii) theoreticians and mathematicians analyzing data. The design of openBEB enables the rapid development of plug-ins, which will inherently benefit from the “house keeping” abilities of the core program. We report the use of openBEB to combine live cell microscopy, microfluidic control and visual proteomics. In this example, measurements from diverse complementary techniques are combined and correlated

Baseline characteristics of OHCA patients according to ROSC or ongoing CPR on admission.

<p>Baseline characteristics of OHCA patients according to ROSC or ongoing CPR on admission.</p