Protein crystal structure obtained at 2.9 ? resolution from injecting bacterial cells into an X-ray free-electron laser beam

It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.The advent of X-ray free-electron lasers (XFELs) has made it possible to obtain atomic resolution macromolecular structures from crystals with sizes approximating only 1/60th of the volume of a single red blood cell. Brief, intense pulses of coherent X-rays, focused on a spot of 3-μm diameter, have produced 1.9-Å-resolution diffraction data from a stream of lysozyme crystals, each crystal no bigger than 3 μm³ (1). A stream of crystals, not just one crystal, is required to collect the many tens of thousands of diffraction patterns that compose a complete data set. No single crystal can contribute more than one diffraction pattern because the XFEL beam is so intense and the crystals so small that the crystals are typically vaporized after a single pulse. Impressively, a photosystem I crystal no bigger than 10 unit cells (300 nm) on an edge produced observable subsidiary diffraction peaks between Bragg reflections, details which would be unobservable from conventionally sized crystals (2). With this new ability to collect diffraction patterns from crystals of unprecedentedly small dimensions, it is conceivable that high-resolution diffraction data could be collected from crystals in vivo. The structure obtained in this manner would be unaltered from that occurring naturally in a living cell, free from distortion that might otherwise potentially arise from nonphysiological conditions imposed by recrystallization. A practical advantage would also be gained by eliminating the need for a protein purification step, whether the in vivo grown crystals were naturally, or heterologously expressed (3).The nascent field of serial femtosecond crystallography (SFX) has published results on nine different macromolecular systems since its inception in 2009 (3, 9). The crystals for this study were not grown in artificial crystallization chambers as has been the protocol of conventional macromolecular crystallography since the 1950s. Instead, crystals were grown in cells. Specifically, they were grown in Sf9 insect cells, heterologously expressing Trypanosoma brucei cathepsin B. These in vivo-grown crystals were used for the XFEL diffraction experiment. To this end, the cells were lysed and the crystals were extracted before injecting them in the XFEL beam for data collection. This last purification step seems to be the only major departure from our goal of obtaining high-resolution structural information from crystal inclusions in vivo, without requiring the crystal to be extracted from the cell that assembled it. Here we attempt to go one step further than previous studies—to record diffraction from crystals within living cells.

Table 1.

SFX publications from XFEL sources to date

Extratemporal functional connectivity impairments at rest are related to memory performance in mesial temporal epilepsy

Mesial temporal lobe epilepsy (MTLE) is the most frequent form of focal epilepsy. At rest, there is evidence that brain abnormalities in MTLE are not limited to the epileptogenic region, but extend throughout the whole brain. It is also well established that MTLE patients suffer from episodic memory deficits. Thus, we investigated the relation between the functional connectivity seen at rest in fMRI and episodic memory impairments in MTLE. We focused on resting state BOLD activity and evaluated whether functional connectivity (FC) differences emerge from MTL seeds in left and right MTLE groups, compared with healthy controls. Results revealed significant FC reductions in both patient groups, localized in angular gyri, thalami, posterior cingulum and medial frontal cortex. We found that the FC between the left non‐pathologic MTL and the medial frontal cortex was positively correlated with the delayed recall score of a non‐verbal memory test in right MTLE patients, suggesting potential adaptive changes to preserve this memory function. In contrast, we observed a negative correlation between a verbal memory test and the FC between the left pathologic MTL and posterior cingulum in left MTLE patients, suggesting potential functional maladaptative changes in the pathologic hemisphere. Overall, the present study provides some indication that left MTLE may be more impairing than right MTLE patients to normative functional connectivity. Our data also indicates that the pattern of extra‐temporal FC may vary as a function of episodic memory material and each hemisphere's capacity for cognitive reorganization. Hum Brain Mapp 34:2202–2216, 2013. © 2012 Wiley Periodicals, Inc.     

Improving Participant Feedback to Continuing Medical Education Presenters in Internal Medicine: A Mixed-Methods Study

BACKGROUND  

Feedback is essential for improving the skills of continuing medical education (CME) presenters. However, there has been little research on improving the quality of feedback to CME presenters.     

Split or slip – passive generation of monodisperse double emulsions with cores of varying viscosity in microfluidic tandem step emulsification system

We investigate the role of viscosities on the formation of double emulsion in a microfluidic step emulsification system. Aqueous droplets of various viscosities and sizes were engulfed in fluorocarbon oil and subsequently transformed into double droplets in the microfluidic step emulsifying device. We identify two distinct regimes of double droplet formation: (i) core droplets split into multiple smaller droplets, or (ii) cores slip whole into the forming oil shell. We show that the viscosity ratio of the core and shell phases plays a crucial role in determining the mode of formation of the double emulsions. Finally, we demonstrate that high viscosity of the core droplet allows for generation of double emulsions with constant shell thickness for cores of various sizes.

We investigate the role of fluid viscosities on formation of double emulsion in a microfluidic step emulsification system. The ratio of fluid viscosities controls double droplet formation, leading to splitting of the core for low core-to-shell viscosity ratio.