Background and Objectives Imaging monitored cryosurgery is emerging as an important minimally invasive surgical technique for treatment of cancer. Although imaging allows excellent control over the process of freezing itself, recent studies show that at high subzero temperatures cells survive freezing. Antifreeze proteins (AFP) are chemical compounds that modify ice crystals to needle‐like shapes that can destroy cells in cellular suspensions. The goal of this study was to determine whether these antifreeze proteins can also destroy cells in frozen tissue and serve as chemical adjuvants to cryosurgery. Methods Livers from six rats were excised, perfused with solutions of either phosphate‐buffered saline (PBS) or PBS with 10 mg/ml AFP‐I, and frozen with a special cryosurgery apparatus. Lobes were frozen with one or two freeze‐thaw cycles and the cell viability was examined with a two stain fluorescent dye test and histological assessment. Results A significant percentage of hepatocytes survive freezing on the margin of a frozen cryolesion. AFP significantly increase cellular destruction in that region apparently through formation of intracellular ice. Conclusions This preliminary study demonstrates that antifreeze proteins may be effective chemical adjuvants to cryosurgery. J. Surg. Oncol. 1997;66:114–121. © 1997 Wiley‐Liss, Inc.

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A number of structurally diverse classes of "antifreeze" proteins that allow fish to survive in sub-zero ice-laden waters have been isolated from the blood plasma of cold water teleosts. However, despite receiving a great deal of attention, the one or more mechanisms through which these proteins act are not fully understood. In this report we have synthesized a type I antifreeze polypeptide (AFP) from the shorthorn sculpin Myoxocephalus scorpius using recombinant methods. Construction of a synthetic gene with optimized codon usage and expression as a glutathione S-transferase fusion protein followed by purification yielded milligram amounts of polypeptide with two extra residues appended to the N terminus. Circular dichroism and NMR experiments, including residual dipolar coupling measurements on a 15 N-labeled recombinant polypeptide, show that the polypeptides are ␣-helical with the first four residues being more flexible than the remainder of the sequence. Both the recombinant and synthetic polypeptides modify ice growth, forming facetted crystals just below the freezing point, but display negligible thermal hysteresis. Acetylation of Lys-10, Lys-20, and Lys-21 as well as the N terminus of the recombinant polypeptide gave a derivative that displays both thermal hysteresis (0.4°C at 15 mg/ml) and ice crystal faceting. These results confirm that the N terminus of wild-type polypeptide is functionally important and support our previously proposed mechanism for all type I proteins, in which the hydrophobic face is oriented toward the ice at the ice/water interface.The type I AFPs 1 found in the blood of cold water teleosts (1-3) have attracted significant interest, due to the potential applications of such compounds in biotechnology and medicine (4 -9). These compounds are alanine-rich ␣-helical proteins of 33-47 residues in length (for reviews see Refs. 10 -13). Although 14 type I proteins have been identified in nature (summarized in Ref. 12), almost all studies to date have centered on HPLC6, the 37-residue protein from the winter flounder (see Table I below) (14). This protein is characterized as an AFP, because it modifies both the rate and shape of ice crystal growth and displays thermal hysteresis, i.e. a positive difference between the ice growth temperature and the equilibrium melting temperature of ice.During the last decade, significant progress has been made in elucidating the structural features of HPLC6 that are required to give antifreeze activity. Structure-activity studies have identified the importance of the Thr residues at positions 2, 13, 24, and 35 plus surrounding residues, for ice growth inhibition activity. Although the Thr residues were assumed to be involved in hydrogen-bonding interactions with ice for many years (15-19), more recent mutations have identified the hydrophobicity provided by the ␥-methyl group of Thr as a key factor related to the ability to inhibit ice growth (20 -24). Hydrogen bonding and other roles for the surrounding residues have also been considered (24 -29). Howe...

Background Accurate and timely injury identification is critical but difficult to achieve in trauma patients who die shortly after arrival to the hospital. Autopsy has historically been used to detect injuries, but few undergo formal autopsy. This study investigates the utility of post-mortem computed tomography (PMCT) for injury identification in a diverse trauma population. Methods Cross-sectional study of adult trauma patients who died within 24 hours of arrival to a Level I trauma center were included. Among patients with PMCT, injury severity score (ISS) and number of injuries (NOI) were calculated either from physical exam alone (pre-PMCT) or exam and imaging (post-PMCT). ISS and NOI before and after PMCT were compared. A cause of death analysis was performed for patients who underwent comprehensive (ie, head, neck, and torso) PMCT. Non-parametric repeated measures tests were used, as appropriate. Results 7.3% (N = 28) of patients received PMCT. Compared to pre-PMCT, median ISS (21 vs 3.5) and NOI (5 vs 2) were greater post-PMCT ( P < .001, respectively). Autopsy rate was 13.2% overall; 82.5% of autopsies were due to a penetrating mechanism, and median time to autopsy reporting was 38.5 days. Among 17 patients who received comprehensive PMCT, 64.7% had a single cause of death identified, and the remaining were classified as either multiple potential contributors or unknown. Discussion PMCT is a readily available method to identify injuries in trauma patients who expire shortly upon presentation. Given the low autopsy rate for blunt trauma and delay in reporting, PMCT is an important adjunct for trauma providers.

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