Microseed matrix-screening combined with random screens (rMMS) is a significant recent breakthrough in protein crystallization. In this study, a very reproducible assay for crystal seeds was set up that allowed the following recommendations to be made: (1) the suitability of a solution for suspending seed crystals can be predicted by incubating (uncrushed) crystals in it for one day and observing crystal stability. (2) For routine rMMS, seed crystals should be suspended in the crystallization cocktail that gave the original crystals. (3) Seed crystals can be suspended in PEG or NaCl solutions to reduce the prevalence of salt crystals. (4) Protein complexes can be seeded with seed crystals suspended in PEG. If necessary, seed crystals can also be suspended in the original crystallization cocktail with any individual ingredients that destabilize the complex removed. ( 5) "Preseeding" of the protein stock should not be used if rMMS is available, because it is less effective. ( 6) Seed crystals can be harvested from microfluidic devices. (7) Heterogeneous nucleants and cross-seeding are less effective than rMMS, but they can be used if seed crystals cannot be obtained. A theoretical case and practical suggestions are also put forward for producing crystals with different space groups.
The use of conventional ultrafiltration during cardiopulmonary bypass (CPB) has been well recognized as an efficient modality of therapy to reverse the effects of deliberate haemodilution. Routine use of the haemofilter was prospectively studied on 60 patients undergoing coronary artery bypass surgery. Group A consisted of 30 patients on whom the ultrafiltrator was used and compared to group B who did not receive the ultrafiltration technique. The COBE 1200 ultrafiltration device was used. The results of the study demonstrated that, in group A, the mean total amount of ultrafiltrate collected during bypass was 2510 +/- 804 ml per patient. The mean 24-h postoperative blood loss was 440 +/- 192 ml in group A and 451 +/- 136 ml in group B. The average bank blood transfused was 0.6 +/- 1.3 units per patient in group A and 0.75 +/- 1.5 units per patient in group B. Postoperative weight gain in group A averaged 3.5 +/- 3.45 lb per patient, compared to 4.8 +/- 3.7 lb per patient in group B. Postoperative length of stay averaged 6.4 +/- 1.5 days per patient in group A and 6.4 +/- 2.1 days per patient in group B. Overall patient charges averaged $33,706 +/- 8348 per patient in group A and $33,041 +/- 7674 per patient in group B. It was concluded that routine use of ultrafiltration during routine coronary artery bypass surgery with CPB offers no improvement in the quality of care nor does it decrease the patient's overall charges.
A new method of increasing the success rate in protein crystallization screening experiments by combining microseeding with counter-diffusion crystallization in capillaries (SCD) is presented. We have investigated the number of crystallization hits obtained with and without microseeding with 10 model proteins. For the cases studied, SCD generally increases the number of hits and is particularly useful when only relatively low protein concentration stocks are available, either because the stocks were prepared for, e.g., vapor diffusion experiments, or because the protein is poorly soluble. In either case, the addition of seeds becomes necessary to overcome the nucleation energy barrier so that crystal growth can take place even when the wave of supersaturation that passes along the capillary is insufficient to promote nucleation.
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