The permeability of fractured rocks in pressurised volcanic and geothermal systems.

The connectivity of rocks' porous structure and the presence of fractures influence the transfer of fluids in the Earth's crust. Here, we employed laboratory experiments to measure the influence of macro-fractures and effective pressure on the permeability of volcanic rocks with a wide range of initial porosities (1-41 vol. %) comprised of both vesicles and micro-cracks. We used a hand-held permeameter and hydrostatic cell to measure the permeability of intact rock cores at effective pressures up to 30 MPa; we then induced a macro-fracture to each sample using Brazilian tensile tests and measured the permeability of these macro-fractured rocks again. We show that intact rock permeability increases non-linearly with increasing porosity and decreases with increasing effective pressure due to compactional closure of micro-fractures. Imparting a macro-fracture both increases the permeability of rocks and their sensitivity to effective pressure. The magnitude of permeability increase induced by the macro-fracture is more significant for dense rocks. We finally provide a general equation to estimate the permeability of intact and fractured rocks, forming a basis to constrain fluid flow in volcanic and geothermal systems.

Organisational culture: variation across hospitals and connection to patient safety climate.

CONTEXT: Bureaucratic organisational culture is less favourable to quality improvement, whereas organisations with group (teamwork) culture are better aligned for quality improvement. OBJECTIVE: To determine if an organisational group culture shows better alignment with patient safety climate. DESIGN: Cross-sectional administration of questionnaires. Setting 40 Hospital Corporation of America hospitals. PARTICIPANTS: 1406 nurses, ancillary staff, allied staff and physicians. MAIN OUTCOME MEASURES: Competing Values Measure of Organisational Culture, Safety Attitudes Questionnaire (SAQ), Safety Climate Survey (SCSc) and Information and Analysis (IA). RESULTS: The Cronbach alpha was 0.81 for the group culture scale and 0.72 for the hierarchical culture scale. Group culture was positively correlated with SAQ and its subscales (from correlation coefficient r = 0.44 to 0.55, except situational recognition), ScSc (r = 0.47) and IA (r = 0.33). Hierarchical culture was negatively correlated with the SAQ scales, SCSc and IA. Among the 40 hospitals, 37.5% had a hierarchical dominant culture, 37.5% a dominant group culture and 25% a balanced culture. Group culture hospitals had significantly higher safety climate scores than hierarchical culture hospitals. The magnitude of these relationships was not affected after adjusting for provider job type and hospital characteristics. CONCLUSIONS: Hospitals vary in organisational culture, and the type of culture relates to the safety climate within the hospital. In combination with prior studies, these results suggest that a healthcare organisation's culture is a critical factor in the development of its patient safety climate and in the successful implementation of quality improvement initiatives.

Fate of lysostaphin in milk from individual cows through pasteurization and cheesemaking.

Transgenic cows secreting over 3 microg of lysostaphin/ mL of milk are protected against mastitis caused by Staphylococcus aureus, but it is unknown if active lysostaphin persists through dairy processing procedures or affects the production of fermented dairy foods. The objective of this study was to determine the fate of lysostaphin as milk was pasteurized and then processed into cheese. Raw milk from transgenic cows was heat treated at 63 degrees C for 30 min, 72 degrees C for 15 s (high temperature, short time), or 140 degrees C for 2 s (UHT). Portions of the high temperature, short-time milk were manufactured into semi-hard cheeses. Aliquots taken at each processing step were assayed to determine the quantity (ELISA) and activity (ability to inhibit S. aureus growth) of lysostaphin. Results indicated that most of the lysostaphin was present in the aqueous portion of the milk and was not affected by pasteurization, although UHT treatment reduced enzyme concentration by 60%. The quantity and activity of the lysostaphin decreased during cheesemaking. Based on the amount of lysostaphin present in the starting cheesemilk, 10 to 15% of the lysostaphin was recovered in the whey, 21 to 55% in the cheese curd at d 1, and 21 to 36% in cheese stored at 4 degrees C for 90 d. Enough of the lysostaphin secreted into milk by transgenic cows survived typical dairy processing conditions to impart potential value as a bioprotective agent against staphylococci in dairy foods.

Are animal models as good as we think?

Models have been a tool of science at least since the 18th century and serve a variety of purposes from focusing abstract thoughts to representing scaled down version of things for study. Generally, animal models are needed when it is impractical or unethical to study the target animal. Biologists have taken modeling by analogy beyond most other disciplines, deriving the relationship between model and target through evolution. The "unity in diversity" concept suggests that homology between model and target foretells functional similarities. Animal model studies have been invaluable for elucidating general strategies, pathways, processes and guiding the development of hypotheses to test in target animals. The vast majority of animals used as models are used in biomedical preclinical trials. The predictive value of those animal studies is carefully monitored, thus providing an ideal dataset for evaluating the efficacy of animal models. On average, the extrapolated results from studies using tens of millions of animals fail to accurately predict human responses. Inadequacies in experimental designs may account for some of the failure. However, recent discoveries of unexpected variation in genome organization and regulation may reveal a heretofore unknown lack of homology between model animals and target animals that could account for a significant proportion of the weakness in predictive ability. A better understanding of the mechanisms of gene regulation may provide needed insight to improve the predictability of animal models.

In vivo evaluation of changes in body composition of transgenic mice expressing the myostatin pro domain using dual energy X-ray absorptiometry.

Over expression of the pro domain of myostatin (MLC-pro) interferes with myostatin function, thus promoting muscle growth. The purpose of this study was to use dual energy X-ray absorptiometry (DXA) to monitor, in vivo, the course of changes in body composition of control and MLC-pro transgenic (TG) mice between 10 and 91 days of age. MLC-pro TG (n = 32) and littermate control (n = 28) mice were produced by mating G-3 male TG mice with non-TG females. At days 10, 20 and weekly thereafter to day 62, and finally at day 91, the mice were anesthetized and scanned by DXA. By day 34, the body weight of the male TG mice was more than that of the control mice and was accompanied by a larger lean mass (LM) and a lower percentage of fat (%F) (P < 0.05). At day 91, the male TG mice had 15.6% greater body weight, 19.4% more LM, 22.4% lower %F, 11.5% more bone mineral, and 4.4% higher bone density (P < 0.05). The lower %F in the TG mice was due mainly to an increase in LM, rather than reduced FM. Measurements of the TG female mice were not different (P > 0.05) from those of control female mice. A region-of-interest analysis was used to provide a separate measure of the hind limb. By using DXA, this study determined the onset and degree of differences in body composition of MLC-pro TG and littermate control mice.

Regulation of animal biotechnology: research needs.

Livestock that result from biotechnology have been a part of agricultural science for over 30 years but have not entered the market place as food or fiber. Two biotechnologies are at the forefront as challenges to the world's systems for regulating the market place: animal clones and transgenic animals. Both technologies have come before the Food and Drug Administration in the United States and it appears that action is imminent for clones. The FDA has asserted principles for evaluation of clones and asserts that "... remaining hazard(s) from cloning are likely to be subtle in nature." The science-based principles recognize that in some areas related to developmental biology and gene expression in clones, additional scientific information would be useful. The role of science then is to use the genomic tools that we have available to answer questions about epigenetic regulation of development and reprogramming of genes to the state found in germ cells. Transgenics pose additional challenges to regulators. If the transgenics are produced using cloning from modified cells then the additional scientific information needed will be related to the effects of insertion and expression of the transgenes. Other approaches such as retrovirally vectored transgenesis will elicit additional questions. These questions will be challenging because the science will have to be related to the expression and function of each gene or class of genes. For the promises of animal biotechnology to be fulfilled, scientists will have to resolve many questions for regulators and the public but tools to answer those questions are rapidly becoming available.

Major advances associated with the biosynthesis of milk.

The mammary gland has an incredible level of organization and a remarkable ability to convert circulating nutrients into milk components. This review highlights four areas of high interest in the biology of milk synthesis where advances over the last quarter-century have resulted in new understanding or revealed new opportunities. First, advances in our understanding of the mechanisms of milk secretion has led to a substantial increase in our knowledge of the intracellular origin of lipid droplets and the identity and potential function of milk fat globule membrane proteins in milk-lipid secretion. Second, recent breakthroughs have advanced our understanding of the nutritional regulation of milk fat and highlighted the interrelations between dietary components, digestive processes in the rumen, and the regulation of mammary synthesis of milk fat. Third, nutritional quality is becoming increasingly important in food choices because of consumer awareness of the links between diet and health. The traditional nutritional value of milk and dairy products is well established, but recent discoveries have identified a number of "bioactive" components in milk with potential to improve human health. Finally, the concept of genetic engineering and the use of animals as "bioreactors" and the "pharming" of proteins not normally found in milk have gained recognition, with the dairy industry ideally suited to take advantage of advances in these areas.

Using real time process measurements to reduce catheter related bloodstream infections in the intensive care unit.

PROBLEM: Measuring a process of care in real time is essential for continuous quality improvement (CQI). Our inability to measure the process of central venous catheter (CVC) care in real time prevented CQI efforts aimed at reducing catheter related bloodstream infections (CR-BSIs) from these devices. DESIGN: A system was developed for measuring the process of CVC care in real time. We used these new process measurements to continuously monitor the system, guide CQI activities, and deliver performance feedback to providers. SETTING: Adult medical intensive care unit (MICU). KEY MEASURES FOR IMPROVEMENT: Measured process of CVC care in real time; CR-BSI rate and time between CR-BSI events; and performance feedback to staff. STRATEGIES FOR CHANGE: An interdisciplinary team developed a standardized, user friendly nursing checklist for CVC insertion. Infection control practitioners scanned the completed checklists into a computerized database, thereby generating real time measurements for the process of CVC insertion. Armed with these new process measurements, the team optimized the impact of a multifaceted intervention aimed at reducing CR-BSIs. EFFECTS OF CHANGE: The new checklist immediately provided real time measurements for the process of CVC insertion. These process measures allowed the team to directly monitor adherence to evidence-based guidelines. Through continuous process measurement, the team successfully overcame barriers to change, reduced the CR-BSI rate, and improved patient safety. Two years after the introduction of the checklist the CR-BSI rate remained at a historic low. LESSONS LEARNT: Measuring the process of CVC care in real time is feasible in the ICU. When trying to improve care, real time process measurements are an excellent tool for overcoming barriers to change and enhancing the sustainability of efforts. To continually improve patient safety, healthcare organizations should continually measure their key clinical processes in real time.

Follicular expression of a human beta-cell leukaemia/lymphoma-2 (Bcl-2) transgene does not decrease atresia or increase ovulation rate in swine.

Transgenic (TG) gilts carrying a human Bcl-2 cDNA transgene driven by mouse inhibin-alpha subunit promoter were produced and evaluated to determine if ectopic expression of Bcl-2 in the ovaries would decrease the frequency of atresia in antral follicles and increase ovulation rate. Immunohistochemical analysis showed that the Bcl-2 transgene protein was expressed in granulosa and theca cells, in 86% of healthy and 54% of atretic follicles analysed in TG prepubertal and Day 50 pregnant gilts combined (n = 24). In contrast, Bcl-2 transgene protein was expressed in only 1.4% of healthy and 0% of atretic follicles in non-TG littermates (n = 13). Real-time reverse transcription-polymerase chain reaction analysis confirmed that human Bcl-2 was expressed in follicles of TG gilts. The atresia rate for the TG and non-TG groups did not differ (P > 0.05) for prepubertal (45 v. 59%) and Day 50 pregnant gilts (53 v. 52%) respectively. The mean +/- s.e.m. ovulation rate did not differ (P > 0.5) between TG (15.9 +/- 0.8, n = 12) and non-TG (16.4 +/- 0.6, n = 7) Day 50 pregnant gilts. The molecular basis of the failure of ectopic Bcl-2 expression to increase the ratio of healthy to atretic follicles is unknown, but it is possible that the activity of the mitochondrial-dependent cell death pathway was not neutralized by ectopic expression of human Bcl-2 or that other cell death pathways compensated for the decreased mitochondrial-dependent cell death.

Growth and tissue accretion rates of swine expressing an insulin-like growth factor I transgene.

The goal of this research was to determine whether directing expression of an insulin-like growth factor I (IGF-I) transgene specifically to striated muscle would alter the growth characteristics in swine. Transgenic pigs were produced with a fusion gene composed of avian skeletal alpha-actin regulatory sequences and a cDNA encoding human IGF-I. Six founder transgenic pigs were mated to nontransgenic pigs to produce 11 litters of G1 transgenic and sibling control progeny. Birth weight, weaning weight, and proportion of pig survival did not differ between transgenic and control pigs. The ADG of pigs as they grew incrementally from 20 to 60 kg, 60 to 90 kg, and 90 to 120 kg, respectively, did not significantly differ between transgenic and control pigs. Efficiency of feed utilization (gain:feed) was also similar for transgenic and control pigs. Plasma IGF-I and porcine growth hormone (pGH) concentrations were determined at 60, 90, and 120 kg body weight. Plasma IGF-I concentrations were 19% higher in transgenic gilts than control gilts and 11.1% higher in transgenic boars than control boars (P=0.0005). Plasma IGF-I concentrations for boars were also higher than for gilts (P=0.0001). At 60, 90, and 120 kg body weight each pig was scanned by dual energy X-ray absorptiometry (DXA) to derive comparative estimates of carcass fat, lean, bone content of the live animal. Control pigs had more fat and less lean tissue than transgenic pigs at each of the scanning periods and the difference became more pronounced as the pigs grew heavier (P<0.005 at each weight). Transgenic pigs also had a slightly lower percentage of bone than control pigs (P<0.05 at each weight). While daily rates of lean tissue accretion did not differ for transgenic and control pigs, daily rates of fat accretion were lower in transgenic pigs than in control pigs (P<0.05). Based on these results we conclude that expression of IGF-I in the skeletal muscles gradually altered body composition as pigs became older but did not have a major affect on growth performance.

Cell donor influences success of producing cattle by somatic cell nuclear transfer.

To assess sources of variation in nuclear transfer efficiency, bovine fetal fibroblasts (BFF), harvested from six Jersey fetuses, were cultured under various conditions. After transfection, frozen-thawed lung or muscle BFF donor cells were initially cultured in DMEM in 5% CO(2) and air and some were transferred to MEM, with 5% or 20% O(2) or 0.5% or 10% serum and G418 for 2-3 wk. Selected clonal transfected fibroblasts were fused to enucleated oocytes. Fused couplets (n = 4007), activated with ionomycin and 6-dimethylaminopurine, yielded 927 blastocysts, and 650 were transferred to 330 recipients. Fusion rate was influenced by oxygen tension in a fetus-dependent manner (P < 0.001). Blastocyst development was influenced in a number of ways. Hip fibroblast generated more blastocysts when cultured in MEM (P < 0.001). The influence of serum concentration was fetus dependent (P < 0.001) and exposing fibroblast to low oxygen was detrimental to blastocyst development (P < 0.001). Cells from two of the six fetuses produced embryos that maintained pregnancies to term, resulting in eight viable calves. Pregnancy rates 56 days after transfer for the two productive donor fetuses, was at least double that of other recipients and may provide a fitness indicator of BFF cell sources for nuclear transfer. We conclude that a significant component in determining somatic cell nuclear transfer success is the source of the nuclear donor cells.

New gene transfer methods.

The intentional introduction of recombinant DNA molecules into a living organism can be achieved in many ways. Viruses have been making a living by practicing gene transfer for millennia. Recently, man has gotten into the act. The paradigm employed is fairly straightforward. First, a way must be found to move genetic information across biological membrane barriers. Then, presumably, DNA repair mechanisms do the rest. The array of methods available to move DNA into the nucleus provides the flexibility necessary to transfer genes into cells as physically diverse as sperm and eggs. Some of the more promising alternative strategies such as sperm-mediated gene transfer, restriction enzyme-mediated integration, metaphase II transgenesis, and a new twist on retrovirus-mediated gene transfer will be discussed, among other methods.

Protocol-driven care in the intensive care unit: a tool for quality.

Advances in organization and patient management in the intensive care unit (ICU) have led to reductions in the morbidity and mortality suffered by critically ill patients. Two such advances include multidisciplinary teams (MDTs) and the development of clinical protocols. The use of protocols and MDTs does not necessarily guarantee instant improvement in the quality of care, but it does offer useful tools for the pursuit of such objectives. As ICU physicians increasingly assume leadership roles in the pursuit of higher quality ICU care, their knowledge and skills in the discipline of quality improvement will become essential.

Expression of myostatin pro domain results in muscular transgenic mice.

Myostatin, a member of the TGF-beta family, negatively regulates skeletal muscle development. Depression of myostatin activity leads to increased muscle growth and carcass lean yield. In an attempt to down-regulate myostatin, transgenic mice were produced with a ribozyme-based construct or a myostatin pro domain construct. Though the expression of the ribozyme was detected, muscle development was not altered by the ribozyme transgene. However, a dramatic muscling phenotype was observed in transgenic mice carrying the myostatin pro domain gene. Expression of the pro domain transgene at 5% of beta-actin mRNA levels resulted in a 17-30% increase in body weight (P < 0.001). The carcass weight of the transgenic mice showed a 22-44% increase compared with nontransgenic littermates at 9 weeks of age (16.05 +/- 0.67 vs. 11.16 +/- 0.28 g in males; 9.99 +/- 0.38 vs. 8.19 +/- 0.19 g in females, P < 0.001). Extreme muscling was present throughout the whole carcass of transgenic mice as hind and fore limbs and trunk weights, all increased significantly (P < 0.001). Epididymal fat pad weight, an indicator of body fat, was significantly decreased in pro domain transgenic mice (P < 0.001). Analysis of muscle morphology indicated that cross-sectional areas of fast-glycolytic fibers (gastrocnemius) and fast-oxidative glycolytic fibers (tibialis) were larger in pro domain transgenic mice than in their controls (P < 0.01), whereas fiber number (gastrocnemius) was not different (P > 0.05). Thus, the muscular phenotype is attributable to myofiber hypertrophy rather than hyperplasia. The results of this study suggest that the over-expression of myostatin pro domain may provide an alternative to myostatin knockouts as a means of increasing muscle mass in other mammals.

Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

Pronuclear microinjection.

Last year marked the 20th anniversary of the invention of the term "transgenic" and the development of pronuclear microinjection, a straightforward technique designed to transfer genetic information from nearly any living organism to mammals. After two decades of use, pronuclear microinjection protocols have changed little from the reliable, if not efficient, method described by Gordon and Ruddle. Experience has taught us that once microinjection skills are perfected there are only a few parameters one needs to be concerned about to successfully produce transgenic animals. Those parameters will be discussed, as will some new innovations that promise to finally increase efficiency of pronuclear microinjection methodology.

DNA preparation method can influence outcome of transgenic animal experiments.

In our continuing quest to improve the efficiency of producing transgenic animals, we have compared the influence of two transgene purification techniques on the efficiency of creating transgenic sheep and mice. Three hundred eighty-seven sheep zygotes and 2,737 mouse zygotes were microinjected with one of four transgenes. Transgenes were isolated from plasmid sequences either by agarose gel electrophoresis followed by gel extraction or by a single step sodium chloride gradient fractionation technique. Four transgenic sheep and 61 transgenic mice were produced. Both sheep and mice embryos responded similarly to transgene preparation methods. Overall, pregnancy rate was higher for recipients that received embryos injected with NaCl purified DNA (mean +/- SEM: 64 +/- 7% vs. 38 +/- 7%). Furthermore, offspring per zygote transferred (NaCl, 22 +/- 3% vs. Gel, 12 +/- 3%) and transgenics born per zygote transferred (NaCl, 3.9 +/- 0.6% vs. Gel, 1.5 +/- 0.6%) were higher when the NaCl purified DNA was used. However, the proportion of offspring born that were identified as transgenic did not differ between transgene purification methods. Transgenes responded differently to methods of preparation. One of the four genes yielded a significantly higher proportion of transgenics when the transgene was prepared by NaCl purification. These data suggest that on average the NaCl gradient purification technique results in a higher embryo survival rate to term for both sheep and mice, but the technique has no influence on rate of transgene integration.

Fertility of bull sperm frozen and stored in clarified egg yolk-Tris-glycerol extender.

Egg yolk-Tris-glycerol extender, which is widely used in commercial artificial insemination (AI), was modified by replacing the 20% egg yolk (vol/vol) with a supernatant from egg yolk centrifuged at 50,000 x g for 2 h and then used to cryopreserve bull sperm. Preliminary studies showed that 20% egg yolk interfered with biochemical assays, which could be overcome by centrifugation. In Experiment 1, semen from 4 Holstein bulls was frozen in the experimental Tris extender and compared with the whole milk-glycerol control. A total of 2256 first services resulted in 72.6% 60- to 90-d nonreturns for the control and 71.0% for the Tris extender. In Experiment 2, semen from 10 Holstein bulls was frozen in the experimental Tris extender. Half of the semen was used immediately and half was stored in liquid nitrogen for 1 yr before distribution. The nonreturn rates based on 8878 first services for 7 bulls that completed both parts of the trial were 70.9% initially and 71.6% 1 yr later. This time trend difference of 0.7% was comparable to 0.6% for the AI mean for Holstein sires used at the same time. These fertility results and previous laboratory studies indicate that the conventional egg yolk-Tris-glycerol might be simplified for cryopreserving bull sperm. The experimental Tris extender also was suitable for making biochemical measurements.

Codon optimization, genetic insulation, and an rtTA reporter improve performance of the tetracycline switch.

The objective of this work was to further develop a tetracycline repressor (TetR) protein system that allows control of transgene expression. First, to circumvent the need for a binary approach, a single plasmid design was constructed and tested in tissue culture. To indirectly assay integrations that express the synthetic transcription factor (rtTA), a bicistronic gene was built which included an internal ribosome entry site (IRES) and a green fluorescent protein coding region (GFP) on the same expression cassette as the coding region of rtTA (pTetGREEN). This construct did not produce fluorescent colonies when stably integrated and provided minimal expression of GFP in the face of adequate expression of rtTA. The coding region for TetR was then altered by introducing 156 silent point mutations to simulate mammalian genes. Replacement of wild-type TetR gene (tetR) in pTetGREEN with 'mammalianized' tetR provided GFP expression. Adjustment of codon usage in the tetR region of rtTA nearly doubled the expression level of functional rtTA. To increase the number of rtTA expressing lines, the chicken egg-white lysozyme matrix attachment region (MAR) was introduced into the single plasmid design just upstream of the tetracycline operators (tetO). Inclusion of the MAR doubled the number of colonies that expressed rtTA (44% vs 88%). With the modifications described here, the number of lines that express rtTA and provide induction from a single plasmid design can be increased by the inclusion of a MAR and the level of rtTA expression can be further increased by adjusting the base composition of the TetR coding region. The MAR also insulates the inducible gene from the promoter driving rtTA.