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1.  A scale down process for the development of large volume cryopreservation☆ 
Cryobiology  2014;69(3):367-375.
The process of ice formation and propagation during cryopreservation impacts on the post-thaw outcome for a sample. Two processes, either network solidification or progressive solidification, can dominate the water–ice phase transition with network solidification typically present in small sample cryo-straws or cryo-vials. Progressive solidification is more often observed in larger volumes or environmental freezing. These different ice phase progressions could have a significant impact on cryopreservation in scale-up and larger volume cryo-banking protocols necessitating their study when considering cell therapy applications.
This study determines the impact of these different processes on alginate encapsulated liver spheroids (ELS) as a model system during cryopreservation, and develops a method to replicate these differences in an economical manner.
It was found in the current studies that progressive solidification resulted in fewer, but proportionally more viable cells 24 h post-thaw compared with network solidification. The differences between the groups diminished at later time points post-thaw as cells recovered the ability to undertake cell division, with no statistically significant differences seen by either 48 h or 72 h in recovery cultures.
Thus progressive solidification itself should not prove a significant hurdle in the search for successful cryopreservation in large volumes. However, some small but significant differences were noted in total viable cell recoveries and functional assessments between samples cooled with either progressive or network solidification, and these require further investigation.
doi:10.1016/j.cryobiol.2014.09.003
PMCID: PMC4271741  PMID: 25219980
ELS, encapsulated liver spheroids; PS, progressive solidification; NS, network solidification; BAL, bioartificial liver device; Large volume cryopreservation; Bioartificial liver; Undercooling; Network solidification; Progressive solidification
2.  Enteral Contrast in the Computed Tomography Diagnosis of Appendicitis 
Annals of surgery  2014;260(2):311-316.
Objective
Our goal was to perform a comparative effectiveness study of intravenous (IV)-only versus IV + enteral contrast in computed tomographic (CT) scans performed for patients undergoing appendectomy across a diverse group of hospitals.
Background
Small randomized trials from tertiary centers suggest that enteral contrast does not improve diagnostic performance of CT for suspected appendicitis, but generalizability has not been demonstrated. Eliminating enteral contrast may improve efficiency, patient comfort, and safety.
Methods
We analyzed data for adult patients who underwent nonelective appendectomy at 56 hospitals over a 2-year period. Data were obtained directly from patient charts by trained abstractors. Multivariate logistic regression was utilized to adjust for potential confounding. The main outcome measure was concordance between final radiology interpretation and final pathology report.
Results
A total of 9047 adults underwent appendectomy and 8089 (89.4%) underwent CT, 54.1% of these with IV contrast only and 28.5% with IV + enteral contrast. Pathology findings correlated with radiographic findings in 90.0% of patients who received IV + enteral contrast and 90.4% of patients scanned with IV contrast alone. Hospitals were categorized as rural or urban and by their teaching status. Regardless of hospital type, there was no difference in concordance between IV-only and IV + enteral contrast. After adjusting for age, sex, comorbid conditions, weight, hospital type, and perforation, odds ratio of concordance for IV + enteral contrast versus IV contrast alone was 0.95 (95% CI: 0.72–1.25).
Conclusions
Enteral contrast does not improve CT evaluation of appendicitis in patients undergoing appendectomy. These broadly generalizable results from a diverse group of hospitals suggest that enteral contrast can be eliminated in CT scans for suspected appendicitis.
doi:10.1097/SLA.0000000000000272
PMCID: PMC4208938  PMID: 24598250
appendicitis; comparative effectiveness; Computed tomography; diagnosis; enteral contrast; oral contrast
3.  Time to Appendectomy and Risk of Perforation in Acute Appendicitis 
JAMA surgery  2014;149(8):837-844.
IMPORTANCE
In the traditional model of acute appendicitis, time is the major driver of disease progression; luminal obstruction leads inexorably to perforation without timely intervention. This perceived association has long guided clinical behavior related to the timing of appendectomy.
OBJECTIVE
To evaluate whether there is an association between time and perforation after patients present to the hospital.
DESIGN, SETTING, AND PARTICIPANTS
Using data from the Washington State Surgical Care and Outcomes Assessment Program (SCOAP), we evaluated patterns of perforation among patients (≥18 years) who underwent appendectomy from January 1, 2010, to December 31, 2011. Patients were treated at 52 diverse hospitals including urban tertiary centers, a university hospital, small community and rural hospitals, and hospitals within multi-institutional organizations.
MAIN OUTCOMES AND MEASURES
The main outcome of interest was perforation as diagnosed on final pathology reports. The main predictor of interest was elapsed time as measured between presentation to the hospital and operating room (OR) start time. The relationship between in-hospital time and perforation was adjusted for potential confounding using multivariate logistic regression. Additional predictors of interest included sex, age, number of comorbid conditions, race and/or ethnicity, insurance status, and hospital characteristics such as community type and appendectomy volume.
RESULTS
A total of 9048 adults underwent appendectomy (15.8% perforated). Mean time from presentation to OR was the same (8.6 hours) for patients with perforated and nonperforated appendicitis. In multivariate analysis, increasing time to OR was not a predictor of perforation, either as a continuous variable (odds ratio = 1.0 [95% CI, 0.99-1.01]) or when considered as a categorical variable (patients ordered by elapsed time and divided into deciles). Factors associated with perforation were male sex, increasing age, 3 or more comorbid conditions, and lack of insurance.
CONCLUSIONS AND RELEVANCE
There was no association between perforation and in-hospital time prior to surgery among adults treated with appendectomy. These findings may reflect selection of those at higher risk of perforation for earlier intervention or the effect of antibiotics begun at diagnosis but they are also consistent with the hypothesis that perforation is most often a prehospital occurrence and/or not strictly a time-dependent phenomenon. These findings may also guide decisions regarding personnel and resource allocation when considering timing of nonelective appendectomy.
doi:10.1001/jamasurg.2014.77
PMCID: PMC4160117  PMID: 24990687
4.  Isolation of human skeletal muscle myosin heavy chain and actin for measurement of fractional synthesis rates 
The American journal of physiology  1998;275(6 0 1):E1092-E1099.
Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we have developed a simple method to isolate myosin heavy chain (MHC) and actin from small (60–80 mg) human skeletal muscle samples for the determination of their fractional synthesis rates. The amounts of MHC and actin isolated are adequate for the quantification of [13C]leucine abundance by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Fractional synthesis rates of mixed muscle protein (MMP), MHC, and actin were determined in six healthy young subjects (27 ± 1 yr) after they received a 14-h intravenous infusion (prime = 7.58 μmol/kg body wt, constant infusion = 7.58 μmol·kg body wt−1·h−1) of [1-13C]leucine. The fractional synthesis rates of MMP, MHC, and actin were found to be 0.0468 ± 0.0048, 0.0376 ± 0.0033, and 0.0754 ± 0.0078%/h, respectively. Overall, the synthesis rate of MHC was 20% lower (P = 0.012), and the synthesis rate of actin was 61% higher (P = 0.060, not significant) than the MMP synthesis rate. The isolation of these proteins for isotope abundance analysis by GC-C-IRMS provides important information about the synthesis rates of these specific contractile proteins, as opposed to the more general information provided by the determination of MMP synthesis rates.
PMCID: PMC4139966  PMID: 9843753
muscle protein synthesis; amino acid metabolism; protein metabolism; stable isotope tracers; mass spectrometry
5.  GMP Cryopreservation of Large Volumes of Cells for Regenerative Medicine: Active Control of the Freezing Process 
Cryopreservation protocols are increasingly required in regenerative medicine applications but must deliver functional products at clinical scale and comply with Good Manufacturing Process (GMP). While GMP cryopreservation is achievable on a small scale using a Stirling cryocooler-based controlled rate freezer (CRF) (EF600), successful large-scale GMP cryopreservation is more challenging due to heat transfer issues and control of ice nucleation, both complex events that impact success. We have developed a large-scale cryocooler-based CRF (VIA Freeze) that can process larger volumes and have evaluated it using alginate-encapsulated liver cell (HepG2) spheroids (ELS). It is anticipated that ELS will comprise the cellular component of a bioartificial liver and will be required in volumes of ∼2 L for clinical use. Sample temperatures and Stirling cryocooler power consumption was recorded throughout cooling runs for both small (500 μL) and large (200 mL) volume samples. ELS recoveries were assessed using viability (FDA/PI staining with image analysis), cell number (nuclei count), and function (protein secretion), along with cryoscanning electron microscopy and freeze substitution techniques to identify possible injury mechanisms. Slow cooling profiles were successfully applied to samples in both the EF600 and the VIA Freeze, and a number of cooling and warming profiles were evaluated. An optimized cooling protocol with a nonlinear cooling profile from ice nucleation to −60°C was implemented in both the EF600 and VIA Freeze. In the VIA Freeze the nucleation of ice is detected by the control software, allowing both noninvasive detection of the nucleation event for quality control purposes and the potential to modify the cooling profile following ice nucleation in an active manner. When processing 200 mL of ELS in the VIA Freeze—viabilities at 93.4%±7.4%, viable cell numbers at 14.3±1.7 million nuclei/mL alginate, and protein secretion at 10.5±1.7 μg/mL/24 h were obtained which, compared well with control ELS (viability −98.1%±0.9%; viable cell numbers −18.3±1.0 million nuclei/mL alginate; and protein secretion −18.7±1.8 μg/mL/24 h). Large volume GMP cryopreservation of ELS is possible with good functional recovery using the VIA Freeze and may also be applied to other regenerative medicine applications.
doi:10.1089/ten.tec.2013.0571
PMCID: PMC4152792  PMID: 24410575
6.  Toward an Understanding of Agonist Binding to Human Orexin-1 and Orexin-2 Receptors with G-Protein-Coupled Receptor Modeling and Site-Directed Mutagenesis 
Biochemistry  2013;52(46):8246-8260.
The class A G-protein-coupled receptors (GPCRs) Orexin-1 (OX1) and Orexin-2 (OX2) are located predominantly in the brain and are linked to a range of different physiological functions, including the control of feeding, energy metabolism, modulation of neuro-endocrine function, and regulation of the sleep–wake cycle. The natural agonists for OX1 and OX2 are two neuropeptides, Orexin-A and Orexin-B, which have activity at both receptors. Site-directed mutagenesis (SDM) has been reported on both the receptors and the peptides and has provided important insight into key features responsible for agonist activity. However, the structural interpretation of how these data are linked together is still lacking. In this work, we produced and used SDM data, homology modeling followed by MD simulation, and ensemble-flexible docking to generate binding poses of the Orexin peptides in the OX receptors to rationalize the SDM data. We also developed a protein pairwise similarity comparing method (ProS) and a GPCR-likeness assessment score (GLAS) to explore the structural data generated within a molecular dynamics simulation and to help distinguish between different GPCR substates. The results demonstrate how these newly developed methods of structural assessment for GPCRs can be used to provide a working model of neuropeptide–Orexin receptor interaction.
doi:10.1021/bi401119m
PMCID: PMC3880013  PMID: 24144388
7.  Progress in the Diagnosis of Appendicitis: A Report from Washington State’s Surgical Care and Outcomes Assessment Program (SCOAP) 
Annals of surgery  2012;256(4):586-594.
BACKGROUND and OBJECTIVES
Studies suggest that CT and US can effectively diagnose and rule-out appendicitis, safely reducing negative appendectomies (NA); however, some within the surgical community remain reluctant to add imaging to clinical evaluation of patients with suspected appendicitis. The Surgical Care and Outcomes Assessment Program (SCOAP) is a physician-led quality initiative that monitors performance by benchmarking processes of care and outcomes. Since 2006, accurate diagnosis of appendicitis has been a priority for SCOAP. The objective of this study was to evaluate the association between imaging and NA in the general community.
METHODS
Data were collected prospectively for consecutive appendectomy patients (age > 15) at nearly 60 hospitals. SCOAP data are obtained directly from clinical records, including radiology, operative, and pathology reports. Multivariate logistic regression models were used to examine the association between imaging and NA. Tests for trends over time were also conducted.
RESULTS
Among 19,327 patients (47.9% female) who underwent appendectomy, 5.4% had NA. Among patients who were imaged, frequency of NA was 4.5%, whereas among those who were not imaged, NA was 15.4% (p < 0.001). This association was consistent for males (3% vs. 10%, p < 0.001) and for reproductive-age females (6.9% vs. 24.7%, p < 0.001). In a multivariate model adjusted for age, sex, and WBC, odds of NA for patients not imaged were 3.7 times the odds for those who received imaging (95%CI 3.0 – 4.4). Among SCOAP hospitals, use of imaging increased and NA decreased significantly over time; frequency of perforation was unchanged.
CONCLUSIONS
Patients who were not imaged during work-up for suspected appendicitis had over three times the odds of NA as those who were imaged. Routine imaging in the evaluation of patients suspected to have appendicitis can safely reduce unnecessary operations. Programs such as SCOAP improve care through peer-led, benchmarked practice change.
doi:10.1097/SLA.0b013e31826a9602
PMCID: PMC3475492  PMID: 22964731
8.  A Low Temperature Limit for Life on Earth 
PLoS ONE  2013;8(6):e66207.
There is no generally accepted value for the lower temperature limit for life on Earth. We present empirical evidence that free-living microbial cells cooling in the presence of external ice will undergo freeze-induced desiccation and a glass transition (vitrification) at a temperature between −10°C and −26°C. In contrast to intracellular freezing, vitrification does not result in death and cells may survive very low temperatures once vitrified. The high internal viscosity following vitrification means that diffusion of oxygen and metabolites is slowed to such an extent that cellular metabolism ceases. The temperature range for intracellular vitrification makes this a process of fundamental ecological significance for free-living microbes. It is only where extracellular ice is not present that cells can continue to metabolise below these temperatures, and water droplets in clouds provide an important example of such a habitat. In multicellular organisms the cells are isolated from ice in the environment, and the major factor dictating how they respond to low temperature is the physical state of the extracellular fluid. Where this fluid freezes, then the cells will dehydrate and vitrify in a manner analogous to free-living microbes. Where the extracellular fluid undercools then cells can continue to metabolise, albeit slowly, to temperatures below the vitrification temperature of free-living microbes. Evidence suggests that these cells do also eventually vitrify, but at lower temperatures that may be below −50°C. Since cells must return to a fluid state to resume metabolism and complete their life cycle, and ice is almost universally present in environments at sub-zero temperatures, we propose that the vitrification temperature represents a general lower thermal limit to life on Earth, though its precise value differs between unicellular (typically above −20°C) and multicellular organisms (typically below −20°C). Few multicellular organisms can, however, complete their life cycle at temperatures below ∼−2°C.
doi:10.1371/journal.pone.0066207
PMCID: PMC3686811  PMID: 23840425
9.  Enhancement of the radiation response of EMT-6 tumours by a copper octabromotetracarboranylphenylporphyrin 
The British Journal of Radiology  2012;85(1012):443-450.
Objective
The carborane-containing porphyrin, copper (II) 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(3-[1,2-dicarba-closo-dodecaboranyl]methoxyphenyl)-porphyrin (CuTCPBr), was investigated as a potential radiation enhancing agent for X-ray radiotherapy (XRT) in a subcutaneously implanted EMT-6 murine carcinoma.
Method
The biodistribution and toxicological profile of this porphyrin has been shown to be favourable for another bimodal radiotherapy technique, boron neutron-capture therapy. For the XRT studies, CuTCPBr was formulated in either 9% Cremophor® (BASF Corporation, Ludwigschafen, Germany) EL and 18% propylene glycol (9% CRM) or a revised formulation comprising 1% Cremophor ELP, 2% Tween 80® (JT Baker, Mansfield, MA), 5% ethanol and 2.2% PEG 400 (CTEP formulation), which would be more clinically acceptable than the original 9% CRM formulation. Using the 9% CRM formulation of CuTCPBr, doses of 100, 210 or 400 mg kg−1 of body weight were used in combination with single doses of 25–35 Gy 100 kVp X-rays.
Results
While doses of 100 mg kg−1 and 210 mg kg−1 did not result in any significant enhancement of tumour response, the 400 mg kg−1 dose did. A dose modification factor of 1.20±0.10 was obtained based on the comparison of doses that produced a 50% local tumour control probability. With the CTEP formulation of CuTCPBr, doses of 83 and 170 mg kg−1 produced significant radiation enhancement, with dose modification factors based on the TCP50 of 1.29±0.15 and 1.84±0.24, respectively.
Conclusion
CuTCPBr significantly enhanced the efficacy of XRT in the treatment of EMT-6 carcinomas in mice. The CTEP formulation showed a marked improvement, with over 9% CRM being associated with higher dose modification factors. Moreover, the radiation response in the skin was not enhanced.
doi:10.1259/bjr/87260973
PMCID: PMC3486664  PMID: 22096223
10.  Physical activity among adult survivors of childhood lower extremity sarcoma 
INTRODUCTION
Adult survivors of childhood lower-extremity sarcoma are largely physically inactive, a behavior which potentially compounds their health burden. Altering this behavior requires understanding those factors that contribute to their physical inactivity. Therefore, this investigation sought to identify factors associated with inactivity in this subpopulation of cancer survivors.
METHODS
Demographic, personal, treatment and physical activity information from adult survivors of childhood lower-extremity sarcomas was obtained from the Childhood Cancer Survivor Study (CCSS) cohort. Generalized linear models were used to identify variables that best identified those individuals who were physically inactive.
RESULTS
Only 41% of survivors met Center for Disease Control (CDC) activity guidelines. Survivors were 1.20 (95% CI 1.11–1.30) more likely compared to CCSS sibling cohort and 1.12 (95% CI 1.10–1.15) times more likely than the general population to fail to meet CDC guidelines. Significant predictors of physical inactivity included female sex, hemipelvectomy surgery, and platinum and vinca alkaloid chemotherapy.
CONCLUSIONS
The primary findings of this study are that survivors of childhood onset lower-extremity sarcoma are 1) highly likely to be physically inactive and 2) less likely than their siblings or the general population to regularly exercise. This study has identified treatment related risk factors associated with inactivity that will help health and wellness practitioners develop successful exercise interventions to help these survivors achieve recommended levels of physical activity for health.
IMPLICATIONS FOR CANCER SURVIVORS
These results suggest that physical activity interventions for adult survivors of childhood lower-extremity sarcomas should be sex specific and responsive to unique physical late effects experienced by these survivors.
doi:10.1007/s11764-011-0187-5
PMCID: PMC3248971  PMID: 21681405
Childhood cancer; physical activity; exercise; late-effects; sedentary
11.  The Influence of Pain Distribution on Walking Velocity and Horizontal Ground Reaction Forces in Patients with Low Back Pain 
Pain Research and Treatment  2012;2012:214980.
Objective. The primary purpose of this paper was to evaluate the influence of pain distribution on gait characteristics in subjects with low back problems (LBP) during walking at preferred and fastest speeds. Design. Cross-sectional, observational study. Setting. Gait analysis laboratory in a health professions university. Participants. A convenience age- and gender-matched sample of 20 subjects with back pain only (BPO), 20 with referred leg pain due to back problems (LGP), and 20 pain-free individuals (CON). Methods and Measures. Subjects completed standardized self-reports on pain and disability and were videotaped as they walked at their preferred and fastest speeds along a walkway embedded with a force plate. Temporal and spatial gait characteristics were measured at the midsection of the walkway, and peak medial, lateral, anterior, and posterior components of horizontal ground reaction forces (hGRFs) were measured during the stance phase. Results. Patients with leg pain had higher levels of pain intensity and affect compared to those with back pain only (t = 4.91, P < .001 and t = 5.80, P < 0.001, resp.) and walking had an analgesic effect in the BPO group. Gait velocity was highest in the control group followed by the BPO and LGP group and differed between groups at both walking speeds (F2.57 = 13.62, P < .001 and F2.57 = 9.09, P < .001, for preferred and fastest speed condition, resp.). When normalized against gait velocity, the LGP group generated significantly less lateral force at the fastest walking speed (P = .005) and significantly less posterior force at both walking speeds (P ≤ .01) compared to the control group. Conclusions. Pain intensity and distribution differentially influence gait velocity and hGRFs during gait. Those with referred leg pain tend to utilize significantly altered gait strategies that are more apparent at faster walking speeds.
doi:10.1155/2012/214980
PMCID: PMC3325118  PMID: 22550576
12.  A pilot study evaluating predictors of postoperative outcomes after major abdominal surgery: physiological capacity compared with the ASA physical status classification system 
BJA: British Journal of Anaesthesia  2010;104(4):465-471.
Background
This pilot study compared the risk predictive value of preoperative physiological capacity (PC: defined by gas exchange measured during cardiopulmonary exercise testing) with the ASA physical status classification in the same patients (n=32) undergoing major abdominal cancer surgery.
Methods
Uni- and multivariate logistic regression models were fitted to measurements of PC and ASA rank data determining their predictive value for postoperative morbidity. Receiver operating characteristic (ROC) curves were used to discriminate between the predictive abilities, exploring trade-offs between sensitivity and specificity.
Results
Individual statistically significant predictors of postoperative morbidity included the ASA rank [P=0.038, area under the curve (AUC)=0.688, sensitivity=0.630, specificity=0.750] and three newly identified measures of PC: PAT (% predicted anaerobic threshold achieved, <75% vs ≥75%), ΔHR1 (heart rate response from rest to the anaerobic threshold), and HR3 (heart rate at the anaerobic threshold). A two-variable model of PC measurements (ΔHR1+PAT) was also shown to be statistically significant in the prediction of postoperative morbidity (P=0.023, AUC=0.826, sensitivity=0.813, specificity=0.688).
Conclusions
Three newly identified PC measures and the ASA rank were significantly associated with postoperative morbidity; none showed a statistically greater association compared with the others. PC appeared to improve predictive sensitivity. The potential for new unidentified measures of PC to predict postoperative outcomes remains unexplored.
doi:10.1093/bja/aeq034
PMCID: PMC2837548  PMID: 20190255
assessment, preanaesthetic; complications, morbidity; measurement techniques, gas exchange metabolic; metabolism, oxygen consumption; oxygen uptake; risk; surgery, postoperative
13.  A Reappraisal of the Role of Abscisic Acid and its Interaction with Auxin in Apical Dominance 
Annals of Botany  2006;98(4):891-897.
• Background and Aims Evidence from pea rms1, Arabidopsis max4 and petunia dad1 mutant studies suggest an unidentified carotenoid-derived/plastid-produced branching inhibitor which moves acropetally from the roots to the shoots and interacts with auxin in the control of apical dominance. Since the plant hormone, abscisic acid (ABA), known to inhibit some growth processes, is also carotenoid derived/plastid produced, and because there has been indirect evidence for its involvement with branching, a re-examination of the role of ABA in apical dominance is timely. Even though it has been determined that ABA probably is not the second messenger for auxin in apical dominance and is not the above-mentioned unidentified branching inhibitor, the similarity of their derivation suggests possible relationships and/or interactions.
• Methods The classic Thimann–Skoog auxin replacement test for apical dominance with auxin [0·5 % naphthalene acetic acid (NAA)] applied both apically and basally was combined in similar treatments with 1 % ABA in Ipomoea nil (Japanese Morning Glory), Solanum lycopersicum (Better Boy tomato) and Helianthus annuus (Mammoth Grey-striped Sunflower).
• Key Results Auxin, apically applied to the cut stem surface of decapitated shoots, strongly restored apical dominance in all three species, whereas the similar treatment with ABA did not. However, when ABA was applied basally, i.e. below the lateral bud of interest, there was a significant moderate repression of its outgrowth in Ipomoea and Solanum. There was also some additive repression when apical auxin and basal ABA treatments were combined in Ipomoea.
• Conclusion The finding that basally applied ABA is able partially to restore apical dominance via acropetal transport up the shoot suggests possible interactions between ABA, auxin and the unidentified carotenoid-derived branching inhibitor that justify further investigation.
doi:10.1093/aob/mcl173
PMCID: PMC2806172  PMID: 16882681
Abscisic acid; auxin; branching; apical dominance; branching inhibitor; decapitated shoot; Ipomoea nil; strain violet; Solanum lycopersicum; Helianthus annuus
14.  Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects 
Applied and Environmental Microbiology  2006;72(10):6474-6482.
The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at −196°C and −20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (−196°C and −80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at −20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.
doi:10.1128/AEM.00998-06
PMCID: PMC1610330  PMID: 17021195
15.  Hypoparathyroidism and 22q11 deletion syndrome 
Archives of Disease in Childhood  2003;88(6):520-522.
Aims: To investigate a population of individuals with 22q11 deletion syndrome for hypocalcaemia.
Methods: A detailed clinical history enquiring into symptoms of hypocalcaemia and blood sampling to assess for hypocalcaemia and hypoparathyroidism, of patients outside the neonatal period known to have the 22q11 microdeletion from fluorescent in situ hybridisation studies was taken.
Results: Sixty one individuals were identified, of whom 23 were untraceable and one was unable to give informed consent. Biochemical investigations were performed on 27 subjects. Ten subjects had review of notes only. Four subjects had previously identified hypoparathyroidism. A new case of hypoparathyroidism was identified. Three subjects had borderline hypocalcaemia.
Discussion: In this population of patients with 22q11 deletion syndrome, 13% of the total or 30% of those biochemically assessed had evidence of reduced serum calcium concentrations. It is likely that 13–30% of patients with 22q11 deletion syndrome have possible hypoparathyroidism outside the neonatal period. Reported symptoms of hypocalcaemia did not correlate with biochemical evidence of persisting hypocalcaemia. We have shown that previously undiagnosed asymptomatic hypoparathyroidism occurs in patients with 22q11 deletion syndrome and conclude that screening of this population should be considered.
doi:10.1136/adc.88.6.520
PMCID: PMC1763111  PMID: 12765920
17.  Global amplification of mRNA by template-switching PCR: linearity and application to microarray analysis 
Nucleic Acids Research  2003;31(22):e142.
Conventional approaches to target labelling for expression microarray analysis typically require relatively large amounts of total RNA, a serious limitation when the sample available is small. Here we explore the cycle-dependent amplification characteristics of Template-Switching PCR and validate its use for microarray target labelling. TS-PCR identifies up to 80% of the differentially expressed genes identified by direct labelling using 30-fold less input RNA for the amplification, with the equivalent of 1000-fold less starting material being used for each hybridisation. Moreover, the sensitivity of microarray experiments is increased considerably, allowing the identification of differentially expressed transcripts below the level of detection using targets prepared by direct labelling. We have also validated the fidelity of amplification and show that the amplified material faithfully represents the starting mRNA population. This method outperforms conventional labelling strategies, not only in terms of sensitivity and the identification of differentially expressed genes, but it is also faster and less labour intensive than other amplification protocols.
doi:10.1093/nar/gng142
PMCID: PMC275579  PMID: 14602935
18.  A Preliminary Investigation of the Role of Auxin and Cytokinin in Sylleptic Branching of Three Hybrid Poplar Clones Exhibiting Contrasting Degrees of Sylleptic Branching 
Annals of Botany  2002;90(3):417-421.
Sylleptic branches grow out from lateral buds during the same growing season in which the buds are formed. This type of branching is present in poplar and in many tropical species. It results in the production of more branches, more leaves and expanded photosynthetic capacity and is thought to assist in increasing the overall growth and biomass of the tree at a young age. However, very little is known about the physiology of sylleptic branching in poplar, which is an extremely important source of fibre and fuel. In the present study of three hybrid poplar clones (11‐11, 47‐174 and 49‐177) of Populus trichocarpa × P. deltoides exhibiting contrasting degrees of sylleptic branching, an analysis was carried out on parent shoot elongation and sylleptic branching, together with a preliminary comparison of the parent shoots’ sensitivity to auxin (naphthaleneacetic acid) as a repressor of lateral bud outgrowth, and cytokinin (benzyladenine) as a promoter. Suggestive evidence was found for an inverse correlation between parent shoot sensitivity to auxin and the degree of sylleptic branching, as well as a partially positive correlation with respect to sensitivity to cytokinin. The present data are consistent with the hypothesis that auxin and cytokinin may play repressive and promotive roles, respectively, in the sylleptic branching of hybrid poplar.
doi:10.1093/aob/mcf195
PMCID: PMC4240395  PMID: 12234154
Populus; hybrid poplar; sylleptic branching; auxin; cytokinin; apical dominance; lateral bud outgrowth; decapitated shoot
19.  Does the survival motor neuron protein (SMN) interact with Bcl-2? 
Journal of Medical Genetics  2000;37(7):536-539.
doi:10.1136/jmg.37.7.536
PMCID: PMC1734632  PMID: 10970187
20.  Boron microlocalization in oral mucosal tissue: implications for boron neutron capture therapy 
British Journal of Cancer  2000;82(11):1764-1771.
Clinical studies of the treatment of glioma and cutaneous melanoma using boron neutron capture therapy (BNCT) are currently taking place in the USA, Europe and Japan. New BNCT clinical facilities are under construction in Finland, Sweden, England and California. The observation of transient acute effects in the oral mucosa of a number of glioma patients involved in the American clinical trials, suggests that radiation damage of the oral mucosa could be a potential complication in future BNCT clinical protocols, involving higher doses and larger irradiation field sizes. The present investigation is the first to use a high resolution surface analytical technique to relate the microdistribution of boron-10 (10B) in the oral mucosa to the biological effectiveness of the 10B(n,α)7Li neutron capture reaction in this tissue. The two boron delivery agents used clinically in Europe/Japan and the USA, borocaptate sodium (BSH) and p-boronophenylalanine (BPA), respectively, were evaluated using a rat ventral tongue model. 10B concentrations in various regions of the tongue mucosa were estimated using ion microscopy. In the epithelium, levels of 10B were appreciably lower after the administration of BSH than was the case after BPA. The epithelium:blood 10B partition ratios were 0.2:1 and 1:1 for BSH and BPA respectively. The 10B content of the lamina propria was higher than that measured in the epithelium for both BSH and BPA. The difference was most marked for BSH, where 10B levels were a factor of six higher in the lamina propria than in the epithelium. The concentration of 10B was also measured in blood vessel walls where relatively low levels of accumulation of BSH, as compared with BPA, was demonstrated in blood vessel endothelial cells and muscle. Vessel wall:blood 10B partition ratios were 0.3:1 and 0.9:1 for BSH and BPA respectively. Evaluation of tongue mucosal response (ulceration) to BNC irradiation indicated a considerably reduced radiation sensitivity using BSH as the boron delivery agent relative to BPA. The compound biological effectiveness (CBE) factor for BSH was estimated at 0.29 ± 0.02. This compares with a previously published CBE factor for BPA of 4.87 ± 0.16. It was concluded that variations in the microdistribution profile of 10B, using the two boron delivery agents, had a significant effect on the response of oral mucosa to BNC irradiation. From a clinical perspective, based on the findings of the present study, it is probable that potential radiation-induced oral mucositis will be restricted to BNCT protocols involving BPA. However, a thorough high resolution analysis of 10B microdistribution in human oral mucosal tissue, using a technique such as ion microscopy, is a prerequisite for the use of experimentally derived CBE factors in clinical BNCT. © 2000 Cancer Research Campaign
doi:10.1054/bjoc.2000.1148
PMCID: PMC2363229  PMID: 10839288
borocaptate sodium; p-boronophenylalanine; rat ventral tongue mucosa; compound biological effectiveness factor; ion microscopy imaging
21.  Human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50 are admixtures of the human colon carcinoma cell line HCT 116 
British Journal of Cancer  2000;82(9):1510-1512.
In two recently described human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50, derived from one tumour, we detected identical E-cadherin and β-catenin gene mutations as in colon carcinoma cell line HCT 116. We demonstrate by HLA-typing, mutation analysis and microsatellite analysis that cell lines JROECL 47 and JROECL 50 are admixtures of the human colon adenocarcinoma cell line HCT 116. © 2000 Cancer Research Campaign
doi:10.1054/bjoc.1999.1170
PMCID: PMC2363398  PMID: 10789716
adenocarcinoma; oesophagus; colon; cell lines; admixtures
22.  Massive Idiosyncratic Exon Skipping Corrects the Nonsense Mutation in Dystrophic Mouse Muscle and Produces Functional Revertant Fibers by Clonal Expansion 
The Journal of Cell Biology  2000;148(5):985-996.
Conventionally, nonsense mutations within a gene preclude synthesis of a full-length functional protein. Obviation of such a blockage is seen in the mdx mouse, where despite a nonsense mutation in exon 23 of the dystrophin gene, occasional so-called revertant muscle fibers are seen to contain near-normal levels of its protein product. Here, we show that reversion of dystrophin expression in mdx mice muscle involves unprecedented massive loss of up to 30 exons. We detected several alternatively processed transcripts that could account for some of the revertant dystrophins and could not detect genomic deletion from the region commonly skipped in revertant dystrophin. This, together with exon skipping in two noncontiguous regions, favors aberrant splicing as the mechanism for the restoration of dystrophin, but is hard to reconcile with the clonal idiosyncrasy of revertant dystrophins. Revertant dystrophins retain functional domains and mediate plasmalemmal assembly of the dystrophin-associated glycoprotein complex. Physiological function of revertant fibers is demonstrated by the clonal growth of revertant clusters with age, suggesting that revertant dystrophin could be used as a guide to the construction of dystrophin expression vectors for individual gene therapy. The dystrophin gene in the mdx mouse provides a favored system for study of exon skipping associated with nonsense mutations.
PMCID: PMC2174546  PMID: 10704448
reversion; dystrophin; nonsense mutation; splicing; exon mapping
23.  Gastrointestinal transit and prolonged ambulatory colonic motility in health and faecal incontinence 
Gut  1997;41(3):381-389.
Background—Colonic motor function has not been studied in the ambulatory setting over a prolonged period in the unprepared state. Furthermore, the disturbance of this function in patients with faecal incontinence is unknown. 
Aim—To study colonic function over two to three days in the ambulatory, unprepared state in health and in patients with idiopathic faecal incontinence. 
Methods—Six healthy women and six women with faecal incontinence and a structurally intact anal sphincter ingested a dual radioisotope meal, and had a six sensor, solid state manometric probe colonoscopically inserted into the left colon. Scanning was performed until radioisotope left the gut and pressure was recorded for a median of 44hours. 
Results—Three of six patients showed abnormal gastric emptying. Patients showed no disturbance of colonic radioisotope transit. Controls had a median of 12, whereas patients had a median of 16, high amplitude propagated waves per 24 hours. In three patients urge incontinence was associated with high amplitude (up to 500 cm water) propagated waves which often reached the rectum. These high pressure waves were identical to those occuring in healthy subjects, the only difference being the lack of adequate sphincter response. Passive incontinence was not associated with colonic motor activity. Defaecation in all subjects was associated with identical propagated waves, and distal movement of 13% (median) of right colonic content and excretion of 32% from the left colon and rectum. The urge to defaecate was associated with either propagated waves (45%) or non-propagated contractions (55%). Rectal motor complexes were recorded in both groups of subjects, but similar rhythmic activity was also recorded in the sigmoid and descending colon. 
Conclusions—Normal colonic function consists of frequent high pressure propagated waves. Rhythmic activity occurs both proximal to and in the rectum. Defaecation is characterised by high pressure propagated waves associated with coordinated anal sphincter relaxation. Patients with faecal incontinence may have a widespread disturbance of gut function. Urge incontinence, an urge to defaecate, and defaecation can all be associated with identical high amplitude propagated pressure waves. 


Keywords: colonic motility; gastric emptying; faecal incontinence
PMCID: PMC1891481  PMID: 9378396
24.  An acutely painful leg. 
Postgraduate Medical Journal  1997;73(860):359-360.
Images
PMCID: PMC2431330  PMID: 9246342
25.  The integration of HPV-18 DNA in cervical carcinoma. 
Molecular Pathology  1999;52(5):275-282.
AIMS: Little information is available on the patterns of integration into the host chromosomal DNA of cervical carcinomas of human papillomavirus type 18 (HPV-18) DNA, which is associated with up to 20% of these carcinomas. Because integration of the viral genome may be extremely important in the pathogenesis of cervical carcinoma, the aim of this study was to investigate which regions of HPV-18 DNA are integrated into the cellular DNA of cervical carcinomas. METHODS: Southern analysis using four subgenomic probes covering the entire HPV-18 genome was used to map viral DNA integrated within cellular DNA. The polymerase chain reaction (PCR) was used to confirm the presence of specific regions of the viral genome. RESULTS: In all 11 carcinomas there was a single major HPV-18 DNA integrant, retaining approximately 4000 bp of HPV-18 DNA, indicating that approximately half of the virus genome had been lost upon integration. Southern analysis suggested strongly that the viral breakpoint was within the E1/E2 gene boundary, with concomitant loss of part or all of the E2 ORF (open reading frame), all of the E4, E5, and L2 ORFs and part of the L1 ORF. These data were supported by the PCR results, which confirmed that the region of integrated HPV-18 DNA from nucleotides 6558 to 162 was present in all the carcinoma samples studied. Assuming that no genomic rearrangements, deletions, or insertions had occurred, 4131 bp of integrated HPV-18 DNA could be accounted for in eight cervical carcinoma samples. The results of Southern analysis also suggested that integration of HPV-18 DNA may have occurred at a specific host chromosomal site. CONCLUSIONS: Broadly, the viral sequences retained upon HPV-18 integration resemble those found when HPV-16 is integrated. However, it appears that the HPV-18 E2 region is more consistently deleted.
PMCID: PMC395710  PMID: 10748877

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