Comparison of iceball diameter and temperature distribution achieved with 3-mm accuprobe cryoprobes in porcine and human liver tissue and human colorectal liver metastases in vitro

We aimed to assess the thermal profile and size of iceballs produced by Accuprobe cryoprobes in fresh porcine and human liver and human colorectal cancer liver metastases in vitro to allow better planning of cryosurgical treatment of liver metastases. Iceballs were produced by a 20-min single freeze cycle using 8-mm cryoprobes in pig liver in a waterbath at 37 degrees C (n = 8) and 3-mm cryoprobes in pig liver (n = 8), human liver (n = 3), and human colorectal cancer liver metastases (n = 8). The iceball diameters and the temperatures at different distances from the cryoprobe were measured. Mean iceball diameters produced by 8-mm cryoprobes in pig liver were 56.3 mm and varied from 38.7 to 39.6 mm for 3-mm cryoprobes in the different tissues used. There was no significant difference in iceball size in the different tissues. The diameter of the zone of -40 degrees C or less was approximately 44 mm using 8-mm cryoprobes in porcine liver and between 27 and 31 mm using 3-mm cryoprobes in the different tissues examined. The results may allow better preoperative planning of the cryosurgical treatment of liver metastases with Accuprobe cryoprobes.     

Studying the performance of bifurcate cryoprobes based on shape factor of cryoablative zones

Conventional cryosurgical process employs extremely low temperatures to kill tumor cells within a closely defined region. However, its efficacy can be markedly compromised if the same treatment method is administrated for highly irregularly shaped tumors. Inadequate controls of freezing may induce tumor recurrence or undesirable over-freezing of surrounding healthy tissue. To address the cryosurgical complexity of irregularly shaped tumors, an analytical treatment on irregularly-shaped tumors has been performed and the degree of tumor irregularities is quantified. A novel cryoprobe coined the bifurcate cryoprobe with the capability to generate irregularly shaped cryo-lesions is proposed. The bifurcate cryoprobe, incorporating shape memory alloy functionality, enables the cryoprobe to regulate its physical configuration. To evaluate the probe’s performance, a bioheat transfer model has been developed and validated with in vitro data. We compared the ablative cryo-lesions induced by different bifurcate cryoprobes with those produced by conventional cryoprobes. Key results have indicated that the proposed bifurcate cryoprobes were able to significantly promote targeted tissue destruction while catering to the shape profiles of solid tumors. This study forms an on-going framework to provide clinicians with alternative versatile devices for the treatment of complex tumors.     

Numerical simulation for heat transfer in prostate cancer cryosurgery

A comprehensive computational framework to simulate heat transfer during the freezing process in prostate cancer cryosurgery is presented. Tissues are treated as nonideal materials wherein phase transition occurs over a temperature range, thermophysical properties are temperature dependent and heating due to blood flow and metabolism are included. Boundary conditions were determined at the surfaces of the commercially available cryoprobes and urethral warmer by experimental study of temperature combined with a mathematical optimization process. For simulations, a suitable computational geometry was designed based on MRI imaging data of a real prostate. An enthalpy formulation-based numerical solution was performed for a prescribed surgical protocol to mimic a clinical freezing process. This computational framework allows for the individual planning of cryosurgical procedures and objective assessment of the effectiveness of prostate cryosurgery.     

Cryosurgical societies: a historical note

Interest in cryosurgery developed quickly after modern cryosurgical apparatus became available early in the 1960s and a forum for the clinical reports was provided by the Society for Cryobiology at its annual meetings. The exchange of ideas made possible by interaction with the membership of the Society enhanced the scientific basis and expedited the subsequent evolution of the new cryosurgical techniques. In later years, especially in the 1970s, the increased use of cryosurgery in the diverse specialities of medicine stimulated the formation of cryosurgical societies on a national and international basis. The initial growth of these organizations was rapid, but recently the rate of acquisition of new members has slowed, though new national societies continue to form in areas where none previously existed. The progress in the development of cryosurgical techniques and apparatus has also slowed. To infuse some vitality in the specialty, collaborative interaction with cryobiologists and cryoengineers is essential. To provide this interaction, the efforts toward joint meetings of the disciplines merit renewed emphasis.     

Computerized planning for multiprobe cryosurgery using a force-field analogy

Cryosurgery is the destruction of undesired biological tissues by freezing. For internal organs, multiple cryoprobes are inserted into the tissue with the goal of maximizing cryoinjury within a predefined target region, while minimizing cryoinjury to the surrounding tissues. The objective of this study is to develop a computerized planning tool to determine the best locations to insert the cryoprobes, based on bioheat transfer simulations. This tool is general and suitable for all available cooling techniques and hardware. The planning procedure employs a novel iterative optimization technique based on a force-field analogy. In each iteration, a single transient bioheat transfer simulation of the cryoprocedure is computed. At the end of the simulation, regions of tissue that would have undesired temperatures apply "forces" to the cryoprobes directly moving them to better locations. This method is more efficient than traditional numerical optimization techniques, because it requires significantly fewer bioheat transfer simulations for each iteration of planning. For demonstration purposes, 2D examples on cross sections typical of prostate cryosurgery are given.     

Experimental cryogenic injury of the palate: Observations pertinent to cryosurgical destruction of tumors

In experiments using cryosurgical apparatus to freeze the canine palate in situ, observations were made on techniques of producing tissue destruction. Several time-temperature schedules of freezing were studied. The results showed the great tolerance of palatal tissues to extremely low temperatures for short time periods. Melanocytes were extraordinarily sensitive to cold injury. Tissue necrosis increased with duration of freezing, but repeated freezing was lethal and obviously critical for successful cryosurgical destruction. Thermocouples must be used in clinical cryosurgery to insure that lethal tissue temperatures (colder than ?50 °C) are attained. The incidence of sequestration in the canine palate showed the need for use of proper technique and suitable precautions in the cryosurgical treatment of human palatal tumors.     

A helium gas probe for use in cryosurgery

The design and testing of a prototype cryosurgical probe utilizing helium gas precooled with liquid nitrogen are described. An 8-mm-diameter probe produced an ice ball with a diameter of 28 mm after 10 min freezing using a helium gas flow rate of 42 liter/min. This indicated a surface heat transfer coefficient of 0.34 W/cm² °K and temperature of ?138 °C at the probe tip. Improved performance figures can be achieved using higher gas pressures and flow rates. A helium gas flow system schematic for use with this new type of cryoprobe is also presented. It is claimed that this system will overcome the problems of developing both multiple-tipped probes and small-diameter needle probes for use in cryoanalgesia.     

Rodent stereotaxic surgery and animal welfare outcome improvements for behavioral neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents(1-4), public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals(5)) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus(6).     

The Development and Use of a Flow-through Apparatus for Measuring Nitrogenase Activity and Photosynthesis in Field Crops

A flow-through apparatus for measuring nitrogenase activityin field-grown crops is described. The apparatus was used tomeasure nitrogenase activity in a sainfoin crop throughout agrowth season. A marked seasonal variation was observed whichwas partly the consequence of defoliation. The acetylene-basedestimate of the nitrogen fixed annually by sainfoin was less(146 kg N ha^–1) than the nitrogen accumulated in the shoots.Reasons for this difference are discussed with special emphasison the validity of the technique and alternative sources ofnitrogen. There were no treatment differences between the ratesof growth of either sainfoin or lucerne when grown with or withoutfertilizer nitrogen in the field. This result is in marked contrastwith results from a glasshouse experiment where sainfoin plantsutilizing nitrate-nitrogen exhibited faster growth rates thansimilar plants that were solely dependent on symbiotic nitrogenfixation. Key words: Nitrogenase, photosynthesis, field-technique, sainfoin, lucerne     

The bacterial cell cycle: DNA replication, nucleoid segregation, and cell division

Data on the bacterial cell cycle published in the last 10-15 years are considered, with a special stress on studies of nucleoid segregation between dividing cells. The degree of similarity between the eukaryotic mitotic apparatus and the apparatus performing nucleoid separation is discussed.     

中国大陆第四纪冰期潜在植物避难所研究进展

研究第四纪冰期潜在植物避难所,对了解当今植物分布格局和未来的演变具有重要意义,是全球变化生态学研究的热点问题。根据已发表的文献,本文综述了我国大陆第四纪冰期冰川的发生规模、范围和对植物分布的影响,探讨了冰期潜在植物避难所形成的特点,包括特殊地理位置的生物多样性和避难所残遗种分布等问题。本文还讨论了孢粉分析和分子生物技术的方法在确定冰期植物避难所方面的应用,以及我国科学家在该领域重要研究结果。同时,本文还介绍了国外在该领域的研究进展和我们存在的问题,以期促进我国第四纪冰期植物避难所研究工作的深入开展。     

催化抗体研究新进展

催化抗体也叫抗体酶,是具有催化活性的免疫球蛋白.由于它兼具抗体的高度选择性和酶的高效催化性,因而催化抗体制备技术的开发预示着可以人为生产适应各种用途的,特别是自然界不存在的高效催化剂,对生物学、化学和医学等多种学科有重要的理论意义和实用价值.综述了催化抗体研究的最新进展,讨论了该领域目前存在的问题,提出了解决这些问题的可能办法.     

Investigation of temperature fields around embedded cryoprobes

The temperature fields around cryoprobes were investigated analytically and experimentally. Two cryoprobes were employed: a spherically shaped general purpose probe utilizing liquid nitrogen and a cylindrical "glaucoma" probe utilizing the Joule-Thomson effect in gaseous CO2. Both probes were operated by commercial cryostats. The analytical solutions included a one-dimensional integral solution for the general purpose cryoprobe, and finite element solutions for both cryoprobes. Both solutions were based on the enthalpy method. Analytical and experimental results compared reasonably well. Deviations of these results are believed to be due, mainly, to the incomplete specification of the boundary conditions on the surface of the cryoprobe.     

Experimental measurement and theoretical calculations on muscle and liver tissue during cryosurgery. II. A theory on freezing of tissue.

A theory concerning growth of an iceball using round cryoprobes is presented. The equation for heat conduction is solved for homogeneous tissue, and mathematical equations are presented for the radius of the iceball at any freezing time and temperature. The agreement between theory and freezing experiments at T = ?196 °C is good.     

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

 Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway. Accepted: 26 January 1998     

Patents in combi-space: patent challenges in combinatorial chemistry

Patent protection of inventions relating to combinatorial chemistry is attended by special challenges. The "breakthrough" nature of the field together with the always complex and often arcane chemical manipulations, apparatus, and strategies which suffuse this field make it difficult to describe the inventions adequately. It can be a challenge to communicate effectively with official authorities charged with patent examination. Extraordinary effort is called for in clarifying such inventions such that their patentability can be appreciated. The utility of some types of inventions in this field may be open to question; clear statements of at least one acceptable utility-even if only a minor utility-is beneficial. Because a principal product of many aspects of combinatorial chemistry is information, e.g., the identification of a lead compound, offshore "piracy" is a risk. Domestic claim tie-ins may improve the ability to abate such piracy. Copyright 1998 John Wiley & Sons, Inc.     

The intracellular pathway and kinetics of digestive enzyme secretion in an insect midgut cell

The opaque zone cells of the midgut of the stablefly, Stomoxys calcitrans display a cyclical series of ultrastructural events in response to feeding, which it has been suggested are related to the synthesis and secretion of digestive enzymes. These cells have been studied in vivo using a combination of biochemical, morphometric and electron microscopical autoradiographic techniques. The cyclical nature, timing and relationship of the ultrastructural events to enzyme secretion has been confirmed. The autoradiographic data presented is in good agreement with the classical synthetic pathway for exported proteins. The kinetics of the cellular process have been described: transfer of newly synthesized product from the rough endoplasmic reticulum to Golgi apparatus begins ca. 13-14 min after labelling of the fly and from the Golgi apparatus to secretory granules after ca. 24-26 min. Secretion of this newly synthesized material begins before 60 min and possibly as early as 30 min after labelling of the fly. The data are discussed in relation to the comparable studies in other tissues.     

Controlled freezing of nonideal solutions with application to cryosurgical processes.

Success of a cryosurgical procedure, i.e., maximal cell destruction, requires that the cooling rate be controlled during the freezing process. Standard cryosurgical devices are not usually designed to perform the required controlled process. In this study, a new cryosurgical device was developed which facilitates the achievement of a specified cooling rate during freezing by accurately controlling the probe temperature variation with time. The new device has been experimentally tested by applying it to an aqueous solution of mashed potatoes. The temperature field in the freezing medium, whose thermal properties are similar to those of biological tissue, was measured. The cryoprobe temperature was controlled according to a desired time varying profile which was assumed to maximize necrosis. The tracking accuracy and the stability of the closed loop control system were investigated. It was found that for most of the time the tracking accuracy was excellent and the error between the measured probe temperature and the desired set point is within +/- 0.4 degrees C. However, noticeable deviations from the set point occurred due to the supercooling phenomenon or due to the instability of the liquid nitrogen boiling regime in the cryoprobe. The experimental results were compared to those obtained by a finite elements program and very good agreement was obtained. The deviation between the two data sets seems to be mainly due to errors in positioning of the thermocouple junctions in the medium.     

Cell-free transfer and sorting of membrane lipids in spinach

Summary The donor and acceptor specificity of cell-free transfer of radiolabeled membrane constituents, chiefly lipids, was examined using purified fractions of endoplasmic reticulum, Golgi apparatus, nuclei, plasma membrane, tonoplast, mitochondria, and chloroplasts prepared from green leaves of spinach. Donor membranes were radiolabeled with [¹⁴C]acetate. Acceptor membranes were unlabeled and immobilized on nitrocellulose filters. The assay was designed to measure membrane transfer resulting from ATP-and temperature-dependent formation of transfer vesicles by the donor fraction in solution and subsequent attachment and/or fusion of the transfer vesicles with the immobilized acceptor. When applied to the analysis of spinach fractions, significant ATP-dependent transfer in the presence of cytosol was observed only with endoplasmic reticulum as donor and Golgi apparatus as acceptor. Transfer in the reverse direction, from Golgi apparatus to endoplasmic reticulum, was only 0.2 to 0.3 that from endoplasmic reticulum to Golgi apparatus. ATP-dependent transfers also were indicated between nuclei and Golgi apparatus from regression analysis of transfer kinetics. Specific transfer between Golgi apparatus and plasma membrane and, to a lesser extent, from plasma membrane to Golgi apparatus was observed at 25°C compared to 4°C but was not ATP plus cytosol-dependent. All other combinations of organelles and membranes exhibited no ATP plus cytosol-dependent transfer and only small increments of specific transfer comparing transfer at 37°C to transfer at 4°C. Thus, the only combinations of membranes capable of significant cell-free transfer in vitro were those observed by electron microscopy of cells and tissues to be involved in vesicular transport in vivo (endoplasmic reticulum, Golgi apparatus, plasma membrane, nuclear envelope). Of these, only with endoplasmic reticulum (or nuclear envelope) and Golgi apparatus, where transfer in situ is via 50 to 70 nm transition vesicles, was temperature-and ATP-dependent transfer of acetatelabeled membrane reproduced in vitro. Lipids transferred included phospholipids, mono-and diacylglycerols, and sterols but not triacylglycerols or steryl esters, raising the possibility of lipid sorting or processing to exclude transfer of triacylglycerols and steryl esters at the endoplasmic reticulum to Golgi apparatus step.