Impact of Sample Preparation Methods on Single-Cell X-ray Microscopy and Light Elemental Analysis Evaluated by Combined Low Energy X-ray Fluorescence, STXM and AFM

Background: Although X-ray fluorescence microscopy is becoming a widely used technique for single-cell analysis, sample preparation for this microscopy remains one of the main challenges in obtaining optimal conditions for the measurements in the X-ray regime. The information available to researchers on sample treatment is inadequate and unclear, sometimes leading to wasted time and jeopardizing the experiment's success. Many cell fixation methods have been described, but none of them have been systematically tested and declared the most suitable for synchrotron X-ray microscopy. Methods: The HEC-1-A endometrial cells, human spermatozoa, and human embryonic kidney (HEK-293) cells were fixed with organic solvents and cross-linking methods: 70% ethanol, 3.7%, and 2% paraformaldehyde; in addition, HEK-293 cells were subjected to methanol/ C3H6O treatment and cryofixation. Fixation methods were compared by coupling low-energy X-ray fluorescence with scanning transmission X-ray microscopy and atomic force microscopy. Results: Organic solvents lead to greater dehydration of cells, which has the most significant effect on the distribution and depletion of diffusion elements. Paraformaldehyde provides robust and reproducible data. Finally, the cryofixed cells provide the best morphology and element content results. Conclusion: Although cryofixation seems to be the most appropriate method as it allows for keeping cells closer to physiological conditions, it has some technical limitations. Paraformaldehyde, when used at the average concentration of 3.7%, is also an excellent alternative for X-ray microscopy

Unexpected silicon localization in calcium carbonate exoskeleton of cultured and fossil coccolithophores

Coccolithophores, marine calcifying phytoplankton, are important primary producers impacting the global carbon cycle at different timescales. Their biomineral structures, the calcite containing coccoliths, are among the most elaborate hard parts of any organism. Understanding the morphogenesis of coccoliths is not only relevant in the context of coccolithophore eco-physiology but will also inform biomineralization and crystal design research more generally. The recent discovery of a silicon (Si) requirement for crystal shaping in some coccolithophores has opened up a new avenue of biomineralization research. In order to develop a mechanistic understanding of the role of Si, the presence and localization of this chemical element in coccoliths needs to be known. Here, we document for the first time the uneven Si distribution in Helicosphaera carteri coccoliths through three synchrotron-based techniques employing X-ray Fluorescence and Infrared Spectromicroscopy. The enrichment of Si in specific areas of the coccoliths point to a targeted role of this element in the coccolith formation. Our findings mark a key step in biomineralization research because it opens the door for a detailed mechanistic understanding of the role Si plays in shaping coccolith crystals

X-ray Structures of the Signal Recognition Particle Receptor Reveal Targeting Cycle Intermediates

The signal recognition particle (SRP) and its conjugate receptor (SR) mediate cotranslational targeting of a subclass of proteins destined for secretion to the endoplasmic reticulum membrane in eukaryotes or to the plasma membrane in prokaryotes. Conserved active site residues in the GTPase domains of both SRP and SR mediate discrete conformational changes during formation and dissociation of the SRP·SR complex. Here, we describe structures of the prokaryotic SR, FtsY, as an apo protein and in two different complexes with a non-hydrolysable GTP analog (GMPPNP). These structures reveal intermediate conformations of FtsY containing GMPPNP and explain how the conserved active site residues position the nucleotide into a non-catalytic conformation. The basis for the lower specificity of binding of nucleotide in FtsY prior to heterodimerization with the SRP conjugate Ffh is also shown. We propose that these structural changes represent discrete conformational states assumed by FtsY during targeting complex formation and dissociation

Structures of the Signal Recognition Particle Receptor from the Archaeon Pyrococcus furiosus: Implications for the Targeting Step at the Membrane

In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP•magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP•SR targeting complexes

Numerical Simulation of HTPB Combustion in a 2D Hybrid Slab Combustor

A code for the numerical simulation of combustion processes in hybrid rockets, developed at the Space Propulsion Laboratory of Politecnico di Milano (SPLab), is presented. The code deals with Navier-Stokes equations solved with RANS approach, blowing effect, combustion kinetics and radiation. The equations are closed with k-epsilon turbulence model and well stirred reactor model. The P1 model, a simplification of the PN radiation model, is adopted. Specific simulation tools were developed using OpenFOAM (R) open source technology. The computational domain is 2D and split in two subdomains, simulating the reacting gas mixture on one side and the solid fuel grain on the other. The interface between the two regions plays a key role as the solid grain pyrolysis comes from a straight solution of the model without shortcuts. A propellant combination with polybutadiene and gaseous oxygen has been chosen and a reduced kinetic model for combustion of butadiene, considered as the major gaseous constituent coming from polybutadiene pyrolysis, has been developed for reactions occurring in oxygen atmosphere. The computational domain tries to replicate the real experimental setup and is split into three areas: pre-chamber, slab zone and post-chamber. High speed camera visualizations of the combustion processes allow to compare the flame height, obtained by the code and by experimental tests, along the grain for given boundary conditions