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A procedure and double-chambered device for macromolecular crystal flash-cooling in different cryogenic liquids.

ABSTRACT: Flash-cooling of macromolecular crystals for X-ray diffraction analysis is usually performed in liquid nitrogen (LN2). Cryogens different than LN2 are used as well for this procedure but are highly underrepresented, e.g., liquid propane and liquid ethane. These two cryogens have significantly higher cooling rates compared with LN2 and may thus be beneficial for flash-cooling of macromolecular crystals. Flash-cooling in liquid propane or liquid ethane results in sample vitrification but is accompanied by solidification of these cryogens, which is not compatible with the robotic systems nowadays used for crystal mounting at most synchrotrons. Here we provide a detailed description of a new double-chambered device and procedure to flash-cool loop mounted macromolecular crystals in different cryogenic liquids. The usage of this device may result in specimens of better crystal- and optical quality in terms of mosaic spread and ice contamination. Furthermore, applying the described procedure with the new double-chambered device provides the possibility to screen for the best flash-cooling cryogen for macromolecular crystals on a routine basis, and, most importantly, the samples obtained allow the usage of state-of-the-art robotic sample-loading systems at synchrotrons.

SUBMITTER: Jeckelmann JM

PROVIDER: S-EPMC7518608 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A procedure and double-chambered device for macromolecular crystal flash-cooling in different cryogenic liquids.

Jeckelmann Jean-Marc JM Ilgü Hüseyin H Bosshart Patrick D PD Fotiadis Dimitrios D

PloS one 20200925 9

Flash-cooling of macromolecular crystals for X-ray diffraction analysis is usually performed in liquid nitrogen (LN2). Cryogens different than LN2 are used as well for this procedure but are highly underrepresented, e.g., liquid propane and liquid ethane. These two cryogens have significantly higher cooling rates compared with LN2 and may thus be beneficial for flash-cooling of macromolecular crystals. Flash-cooling in liquid propane or liquid ethane results in sample vitrification but is accomp ...[more]

PMID: 32976530

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Project description:BackgroundIt is necessity to maintain donor kidneys at a low temperature for laparoscopic orthotopic kidney transplantation (LOKT). We recently designed a controllable double-cycle cryogenic device (DCD) with a safer and more effective cooling setting. This study aimed to evaluate the safety and efficacy of this device in a swine model.MethodsA total of 20 pigs were procured for this study. We performed LOKT on 8 pigs using the controllable DCD for hypothermia, on another 8 pigs using the silicon tube cage (STC) for hypothermia, and on 4 pigs as the control group with no hypothermia measures (NH). The LOKTs were performed in autotransplantation mode in which the right kidney vein and artery were end-to-end anastomosed to the left kidney vein and artery in all three groups. The perioperative data, graft temperatures, and surgical complications were recorded, and histological examinations of kidney specimens were performed.ResultsThe average surface temperature in the DCD group (5±3.5 °C) was significantly lower than that in the STC (10±5.5 °C) and NH (28±3.0 °C) groups. In the DCD and STC groups, the temperature between the cooling setting and the adjacent peritoneum was 30±3.0 and 12±5.0 °C, respectively. All pigs survived for 7 days in the DCD group. In the STC group, 2 pigs died from liver lesions and other causes. A single pig had diabetes insipidus. No pigs survived for 7 days in the NH group. The specimens from pigs immediately after revascularization and from the surviving pigs on day 7 following transplantation showed that the morphology of the glomerular and renal tubules was largely normal in the DCD and STC groups. However, in the NH group, there was minimal interstitial and tubular oedema, and endothelial cell swelling occurred in some specimens immediately after revascularization. Pathological examination of the harvested graft specimens showed severe acute tubular necrosis in the NH group.ConclusionsThe newly designed controllable DCD can achieve the goal of cooling the kidney and warming the patient and may be a reliable, effective, inexpensive, and practical hypothermic maintenance system for LOKT in swine.

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A procedure and double-chambered device for macromolecular crystal flash-cooling in different cryogenic liquids.

Publications

A procedure and double-chambered device for macromolecular crystal flash-cooling in different cryogenic liquids.

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