Evidence-based decision-making in a climate of political expediency: insights from local government.

AIMS: Local authorities in England are responsible for public health and health promotion. This article sought to explore how research and decision-making co-exist in a local authority in England. METHODS: An Embedded Researcher was based within the local authority and used qualitative methodology to address the research aim. Interviews and focus groups were employed to ascertain a range of stakeholder views in the local authority. All transcripts were coded on NVivo 12 by the Embedded Researcher and two members of the research team cross-checked a sample for coding accuracy. Data were analysed using framework analysis. RESULTS: The data suggest several barriers to using research to inform decision-making in health promotion and public health. The study shows that research is valued in local authorities, but not always privileged - this is due to cultural factors and practical political reasons which often means that decisions need to be made expediently. Participants outlined a juxtaposition between academic credibility; timeliness to complete the research and the financial cost associated with it; against the independence and credibility that independent academics could bring. CONCLUSION: Policy formulation and delivery is an integral aspect of health promotion and critical to achieving improved population health and reductions in health inequalities. However, there exists tensions between gathering research evidence and making research-informed decisions. The article concludes by advocating the use of Embedded Researchers to fully understand how research is gathered and used to support public health and health promotion policymaking.

qSR: a quantitative super-resolution analysis tool reveals the cell-cycle dependent organization of RNA Polymerase I in live human cells.

We present qSR, an analytical tool for the quantitative analysis of single molecule based super-resolution data. The software is created as an open-source platform integrating multiple algorithms for rigorous spatial and temporal characterizations of protein clusters in super-resolution data of living cells. First, we illustrate qSR using a sample live cell data of RNA Polymerase II (Pol II) as an example of highly dynamic sub-diffractive clusters. Then we utilize qSR to investigate the organization and dynamics of endogenous RNA Polymerase I (Pol I) in live human cells, throughout the cell cycle. Our analysis reveals a previously uncharacterized transient clustering of Pol I. Both stable and transient populations of Pol I clusters co-exist in individual living cells, and their relative fraction vary during cell cycle, in a manner correlating with global gene expression. Thus, qSR serves to facilitate the study of protein organization and dynamics with very high spatial and temporal resolutions directly in live cell.

Diagnosis and management of McFarland fractures.

BACKGROUND: McFarland fracture is the eponym for a rare Salter Harris III or IV fracture involving the medial distal tibia. These fractures can be difficult to diagnose without a high index suspicion and appropriate radiographic imaging. These fractures may result in significant growth disturbances to the pediatric patient. When diagnosed and treated acutely, these fractures can be managed with cast immobilization and close follow up. If diagnoses in a delayed fashion they can result in significant morbidity including prolonged casting and possible surgical treatment. CASE REPORT: In this case report we discuss a pediatric patient with a delayed presentation McFarland fracture which was initially diagnosed and treated as an ankle sprain. He required a prolonged course of treatment and we describe his clinical progression. We review the literature regarding this fracture pattern including history, acute management and outcomes. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: McFarland fractures are rare Salter Harris III fractures of the medial malleolus. These ankle fractures affect patients nearing skeletal maturity and may be difficult to diagnose without the appropriate orthogonal X-ray imaging. Also, a missed diagnosis can lead to unnecessary morbidity to the patient. If diagnosed acutely, these fractures can be easily treated with immobilization but if allowed to become chronic they require prolonged periods of casting and possibly even surgical intervention. A patient with a specific constellation of symptoms and history should raise suspicion for these injuries and prompt a thorough workup.

doublesex is a mimicry supergene.

One of the most striking examples of sexual dimorphism is sex-limited mimicry in butterflies, a phenomenon in which one sex--usually the female--mimics a toxic model species, whereas the other sex displays a different wing pattern. Sex-limited mimicry is phylogenetically widespread in the swallowtail butterfly genus Papilio, in which it is often associated with female mimetic polymorphism. In multiple polymorphic species, the entire wing pattern phenotype is controlled by a single Mendelian 'supergene'. Although theoretical work has explored the evolutionary dynamics of supergene mimicry, there are almost no empirical data that address the critical issue of what a mimicry supergene actually is at a functional level. Using an integrative approach combining genetic and association mapping, transcriptome and genome sequencing, and gene expression analyses, we show that a single gene, doublesex, controls supergene mimicry in Papilio polytes. This is in contrast to the long-held view that supergenes are likely to be controlled by a tightly linked cluster of loci. Analysis of gene expression and DNA sequence variation indicates that isoform expression differences contribute to the functional differences between dsx mimicry alleles, and protein sequence evolution may also have a role. Our results combine elements from different hypotheses for the identity of supergenes, showing that a single gene can switch the entire wing pattern among mimicry phenotypes but may require multiple, tightly linked mutations to do so.

Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system.

STUDY QUESTION: What are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? SUMMARY ANSWER: The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. WHAT IS KNOWN ALREADY: For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers. STUDY DESIGN, SIZE, DURATION: Non-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm(3)) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 24/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A4), estradiol (E2)] production were analyzed weekly. MAIN RESULTS AND THE ROLE OF CHANCE: Dense stroma and intact pre-antral follicles were observed using VS containing 27% glycerol, 27% EG and 0.8% polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12%) and tissues vitrified with VEG (52 ± 2%) showed similar survival rates (all data: mean ± SEM; P > 0.05). For both groups, the initial follicle diameter was similar and increased (P < 0.05) by Week 3, but diameters in vitrified follicles were smaller (P < 0.05) by Week 6 (566 ± 27 µm) than those of the fresh follicles (757 ± 26 µm). Antrum formation rates were lower (P < 0.05) for vitrified (37 ± 6%) relative to fresh (64 ± 8%) follicles. There was no significant change in levels in culture media of E2, P4 and A4 between fresh and VEG groups at any time point during culture. LIMITATIONS, REASONS FOR CAUTION: Only in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues. WIDER IMPLICATIONS OF THE FINDINGS: This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined. STUDY FUNDING/COMPETING INTEREST(S): National Institutes of Health (NIH) Oncofertility Consortium UL1 RR024926 (1RL1-HD058293, HD058295, PL1 EB008542), the Eunice Kennedy Shriver NICHD/NIH (U54 HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.

Hybridization and speciation.

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near-instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky-Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock-on effects on speciation both within and outside regions of hybridization.

A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep.

Vitrification as a means of cryopreservation has become a standard approach for oocytes from livestock. This paradigm shift occurred primarily as a result of the demonstration in 1996 that bovine oocytes are extremely susceptible to chilling injury. Since that early work, numerous devices have been used as supports for oocytes during so-called "ultra-rapid cooling", and occasionally, trials involving the deposition of small volumes of media containing oocytes directly into liquid nitrogen to facilitate cooling have been reported. Results reporting blastocyst development exceeding 10% are common, but variability remains high, and a standard method for bovine oocytes remains to be established. Oocytes from pigs are particularly difficult to cryopreserve, even with the use of ultrarapid cooling approaches. Few reports have demonstrated blastocyst development exceeding 5%. The application of hydrostatic pressure before vitrification appears to impart stress tolerance to porcine oocytes, as the results of some treatments have shown development to blastocysts at proportions >10%. Work on sheep oocyte vitrification is relatively new, and a few articles have reported blastocyst development at 10% or more. Messenger RNA levels are reportedly altered in sheep oocytes as a result of vitrification, and damage to the cytoskeleton is common across species.

Absence epilepsies with widely variable onset are a key feature of familial GLUT1 deficiency.

BACKGROUND: Familial glucose transporter type 1 (GLUT1) deficiency due to autosomal dominant inheritance of SLC2A1 mutations is associated with paroxysmal exertional dyskinesia; epilepsy and intellectual disability occur in some family members. We recently demonstrated that GLUT1 deficiency occurs in over 10% of patients with early-onset absence epilepsy. METHODS: This family study analyses the phenotypes in 2 kindreds segregating SLC2A1 mutations identified through probands with early-onset absence epilepsy. One comprised 9 individuals with mutations over 3 generations; the other had 6 individuals over 2 generations. RESULTS: Of 15 subjects with SLC2A1 mutations, epilepsy occurred in 12. Absence seizures were the most prevalent seizure type (10/12), with onset from 3 to 34 years of age. Epilepsy phenotypes varied widely, including idiopathic generalized epilepsies (IGE) with absence (8/12), myoclonic-astatic epilepsy (2/12), and focal epilepsy (2/12). Paroxysmal exertional dyskinesia occurred in 7, and was subtle and universally undiagnosed prior to molecular diagnosis. There were 2 unaffected mutation carriers. CONCLUSIONS: GLUT1 deficiency is an important monogenic cause of absence epilepsies with onset from early childhood to adult life. Individual cases may be phenotypically indistinguishable from common forms of IGE. Although subtle paroxysmal exertional dyskinesia is a helpful diagnostic clue, it is far from universal. The phenotypic spectrum of GLUT1 deficiency is considerably greater than previously recognized. Diagnosis of GLUT1 deficiency has important treatment and genetic counseling implications.

A neurologist's guide to genome-wide association studies.

Genome-wide association studies are utilized for gene discovery in common diseases. Genotypes of large groups of unrelated patients are compared to controls. This has become feasible due to the recent technical advances in genomics and convincing positive results are now regularly being published. This review is an accessible introduction to the genetic and technical knowledge needed to interpret such studies. Genome-wide association studies are being applied to many neurologic diseases. Here we use idiopathic generalized epilepsy as an example to highlight the phenotyping, sample size, and statistical issues that must be addressed in such studies. These studies are likely to transform our understanding of complex neurologic diseases in the next few years.

Morphological and apoptotic comparison of primordial and primary follicles in cryopreserved human ovarian tissue.

There are few reports which were designed to compare the survival rate of human primary follicles with primordial follicles after cryopreservation. This study was designed to evaluate whether such a difference occurs. Human ovarian biopsies were cryopreserved using dimethylsulphoxide/sucrose as the cryoprotectants. Fresh and cryopreserved ovarian samples were evaluated for viability differences between the two types of follicles using the endpoints of histology, ultrastructure and DNA fragmentation. In comparison with fresh ovarian tissue (83.9%+/-10.0%), the percentage of morphologically normal primordial follicles was not significantly different in cryopreserved tissue (73.9%+/-17.2%). However, a lower percentage of primary follicles with normal morphology was seen in the cryopreserved group (43.3%+/-25.7% vs 74.8%+/-19.4% for the fresh group). Transmission electron microscopy revealed that the cryopreservation did not appear to affect the structural integrity of primordial follicles; however, varying ultrastructural damage to the cytoplasm was observed in the majority of the cryopreserved primary follicles. Using a DNA fragmentation assay, the percentage of apoptotic primordial and primary follicles in the unfrozen (26.3% and 20%) and frozen (23.3% and 25%) ovarian tissue was similar. A higher proportion of primary follicles, compared to primordial follicles, suffer histological damage after slow freezing.

Accuracy of space analysis with emodels and plaster models.

INTRODUCTION: The purposes of this study were to determine the accuracy and speed of measuring the overall arch length and the Bolton ratio, and the time to perform a Bolton analysis for each patient by using software (emodel, version 6.0, GeoDigm Corp, Chanhassen, Minn) compared with hand-held plaster models. METHODS: Models from 30 patients selected from the files of the Department of Orthodontics at West Virginia University were included in this study. The mesiodistal width of each tooth from first molar to first molar was measured to the nearest 0.1 mm with digital calipers, and the Bolton ratio was calculated for each patient. The times required to make the measurements and to perform the analysis were recorded in seconds by using a stopwatch. This process was repeated to record the digital measurements with the software. To evaluate whether there was any magnification in the emodels, quarter-inch ball bearings were mounted on a modified study model. Measurements of the greatest diameter were taken on each ball bearing by using digital calipers and the emodel software. The difference between the 2 methods was calculated, and a paired t test was used to analyze the data. RESULTS: There was no significant difference between the Bolton ratios calculated with the 2 methods. A significant difference in arch length calculations was found between the 2 methods, but it was within the range of error found in this study and was considered clinically insignificant. Significant differences were found in the time needed to make the measurements and the calculations between the 2 methods; the emodel software was an average of 65 seconds faster. The measurements on the ball-bearing mounted models were an average of .067 mm greater on the emodel software than direct measurements on the casts (range, 0 to -0.16 mm). The difference was significant (P <.0045). CONCLUSIONS: These results suggest that, when performing a Bolton analysis, the emodel can be as accurate as, and significantly faster than, the traditional method of digital calipers and plaster models. A clinician who has switched to using emodel software can be confident in his or her diagnoses using it.

Rapid evolution of sexual signals in sympatric Calopteryx damselflies: reinforcement or 'noisy-neighbour' ecological character displacement?

Enhanced prezygotic isolation in sympatry is one of the most intriguing patterns in evolutionary biology and has frequently been interpreted as evidence for reinforcement. However, the frequency with which reinforcement actually completes speciation remains unclear. The Jewelwing damselflies (Calopteryx aequabilis and C. maculata) have served as one of the few classic examples of speciation via reinforcement outside of Drosophila. Although evidence for wing pattern displacement and increased mate discrimination in this system have been demonstrated, the degree of hybridization and gene flow in nature are unknown. Here, we show that sympatric populations of these two species are the result of recent secondary contact, as predicted under a model of speciation via reinforcement. However, we found no phenotypic evidence of hybridization in natural populations and a complete association between species-specific haplotypes at two different loci (mitochondrial CO I and nuclear EF1-alpha), suggesting little or no contemporary gene flow. Moreover, genealogical and coalescent-based estimates of divergence times and migration rates indicate that, speciation occurred in the distant past. The rapid evolution of wing colour in sympatry is recent, therefore, relative to speciation and seems to be better explained by selection against wasting mating effort and/or interspecific aggression resulting from a 'noisy neighbour' signalling environment.

The effect of osmotic stress on the cell volume, metaphase II spindle and developmental potential of in vitro matured porcine oocytes.

Porcine animal models are used to advance our understanding of human physiology. Current research is also directed at methods to produce transgenic pigs. Cryobanking gametes and embryos can facilitate the preservation of valuable genotypes, yet cryopreserving oocytes from pigs has proven very challenging. The current study was designed to understand the effects of anisotonic solutions on in vitro matured porcine oocytes as a first step toward designing improved cryopreservation procedures. We hypothesized that the proportion of oocytes demonstrating a normal spindle apparatus and in vitro developmental potential would be proportional to the solution osmolality. Oocytes were incubated for 10 min at 38 degrees C in various hypo- or hypertonic solutions, and an isotonic control solution and then assessed for these two parameters. Our results support the hypothesis, with an increasing proportion of spindles showing a disrupted structure as the levels of anisotonic exposure diverge from isotonic. Only about half of the oocytes maintained developmental potential after exposure to anisotonic solutions compared to untreated controls. Oocyte volume displayed a linear response to anisotonic solutions as expected, with an estimated relative osmotically inactive cell volume of 0.178. The results from this study provide initial biophysical data to characterize porcine oocytes. The results from future experiments designed to determine the membrane permeability to various cryoprotectants will allow predictive modeling of optimal cryopreservation parameters and provide a basis for designing improved cryopreservation procedures.

Xenotransplantation of cryopreserved equine squamous cell carcinoma to athymic nude and SCID mice.

Cryopreserved equine ocular squamous cell carcinoma (SCC) was inoculated subcutaneously into 15 athymic nude and 15 SCID mice. Xenotransplantation resulted in tumor growth in two athymic nude mice and 1 SCID mouse. Histological appearance and immunohistochemical characterization using cytokeratin 5/6 markers and p53 markers of the tumor grown in mice was in full accord with the original equine tumors. No evidence of metastasis was noted in any mouse. This model may serve as a relevant in vivo model for studying the biology of equine ocular SCC and for the testing of new therapeutic modalities.

Ancient trans-Atlantic flight explains locust biogeography: molecular phylogenetics of Schistocerca.

The desert locust (Schistocerca gregaria) has been an important agricultural pest at least since biblical times. Although the ecology, physiology and behaviour of this insect species have been well characterized, its biogeographical origins and evolutionary history are more obscure. Schistocerca gregaria occurs throughout Africa, the Middle East and Western Asia, but all other species in the genus Schistocerca are found in the New World. Because S. gregaria has the capacity for extreme long-distance movement associated with swarming behaviour, dispersal may have played an important role in determining current distribution patterns. Some authors have argued that S. gregaria is the product of an eastward trans-Atlantic dispersal from North America to Africa; others consider it more likely that the New World taxa are the product of westward dispersal from Africa. Here, we present a mitochondrial DNA phylogeny of Schistocerca species that supports the monophyly of New World species (including the Galapagos endemic Halmenus) relative to S. gregaria. In concert with observed patterns of molecular divergence, and in contrast to previous morphological studies, our analysis indicates a single trans-Atlantic flight from Africa to South America, followed by extensive speciation and ecological divergence in the New World.

Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes.

Transient elevations of cytosolic Ca2+ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) plays an important role in terminating Ca2+ transients by returning cytosolic Ca2+ to intracellular stores. Stored Ca2+ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca2+ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na+/Ca2+ exchanger, indicating that intracellular store deficiency was compensated for by Ca2+ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na+/Ca2+ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca2+ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca2+ signaling dynamics and in cardiac development and hypertrophy.

The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation.

BACKGROUND: Knowing osmotic tolerance limits is important in the design of optimal cryopreservation procedures for cells. METHODS: Mature human oocytes were exposed to anisosmotic sucrose solutions at concentrations of 35, 75, 150, 600, 1200, or 2400 (+/-5) milliosmolal (mOsm) at 37 degrees C. A control treatment at 290 mOsm was also utilized. Oocytes were randomly allocated to each experimental treatment. After the treatment, the oocytes were cultured for 1 h, then fixed in cold methanol. Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II (MII) spindle. Logistic regression was used to determine if media osmolality had a significant effect on spindle structure. RESULTS: Osmolality was a significant predictor of spindle morphology. Hyposmotic effects at 35, 75, and 150 mOsm resulted in 100, 67, and 56% of oocytes having abnormal spindles, respectively. Hyperosmotic effects at 600, 1200, and 2400 mOsm resulted in 44, 44, and 100% of the spindles with abnormal structure, respectively. CONCLUSIONS: Anisosmotic conditions lead to disruption of the MII spindle in human oocytes. Applying this fundamental knowledge to human oocyte cryopreservation should result in increased numbers of cells maintaining viability.

Determination of bull sperm membrane permeability to water and cryoprotectants using a concentration-dependent self-quenching fluorophore.

The objective of this study was to determine the membrane permeability characteristics of bovine spermatozoa. These included the hydraulic conductivity (Lp), the permeability coefficients (Ps) of four common cryoprotective agents (CPAs) and the associated reflection coefficients (sigma). Stopped-flow fluorometry was applied in order to capture rapid cell volume changes under anisosmotic conditions in the absence or presence of permeant solutes (CPAs). This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute Lp in the absence of permeant solutes and Ps and Lp in the presence of permeating solutes (CPAs) at 22 degrees C. The hydraulic conductivity in the absence of permeating solutes was estimated to be 0.68+/-0.05 microm/min/atm (mean+/-SEM). Hydraulic conductivity (Lp) in the presence of CPAs was 0.91+/-0.27 (mean+/-SEM), 0.29+/-0.04, 0.42+/-0.05, and 0.39+/-0.03 microm/min/atm in the presence of dimethylsulfoxide (Me(2)SO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. The values for Ps were estimated to be 1.72+/-0.36, 1.75+/-0.03, 2.47+/-0.24, and 1.49+/-0.33 x 10(-3)cm/min for Me(2)SO, glycerol, PG, and EG, respectively. The data were used to simulate volume excursions during addition and removal of CPA, to predict the different effects of the four CPAs.

o-Phthalaldehyde activates the Ca(2+) release mechanism from skeletal muscle sarcoplasmic reticulum.

o-Phthalaldehyde (OPA) is a bifunctional reagent that forms an isoindole derivative by reacting with cysteine and lysine residues separated by approximately 0.3 nm. OPA inhibits sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity at low micromolar concentrations and induces Ca(2+) release from actively loaded SR vesicles by activating the ryanodine receptor from fast twitch skeletal muscle. Both ryanodine binding and single-channel activity show a biphasic concentration dependence. At low OPA concentrations (<100 microM), ryanodine binding and single channel activity are stimulated, while at higher concentrations, a time-dependent sequential activation and inhibition of receptor binding is observed. Activation is characterized by a Ca(2+)-independent increase in maximal receptor occupancy. Data are presented to support a model in which Ca(2+) channel and ryanodine binding activity are enhanced due to an intramolecular cross-linking of nearby lysine and nonhyperreactive cysteine residues. OPA complexation with endogenous lysine residue(s) is critical for receptor activation.

How covert are covertly manipulated diets?

OBJECTIVE: To quantitatively assess subjects' ability to detect hedonic (palatability), sensory and nutritional differences between covertly manipulated high-fat (HF) and low-fat (LF) diets. SUBJECTS AND DIETS: This study examined the response of 16 subjects (eight men, eight women) to 20 LF and 20 HF versions of manipulated foods. Average percentage protein:fat:carbohydrate (by energy) and energy density (ED) of the two diets were 13:25:62, 371 kJ/100 g and 13:50:37, 672 kJ/100 g, respectively. PROTOCOL: Subjects were asked to simultaneously assess the HF and LF versions of each food in terms of (i) subjective pleasantness of each food, (ii) perceived differences in appearance, smell, taste and texture of the foods, and (iii) for each sample to assess whether it was high or low in energy, protein, carbohydrate, fat, fibre, sugar and salt. ANALYSIS: Perceived pleasantness of HF and LF versions of the foods was compared by analysis of variance. Comparisons used chi-squared tests of independence to assess departure from the null hypothesis of no perceived difference in remaining parameters (ii-iii). RESULTS: On average, subjects exhibited no significant preference for LF or HF versions of the foods (no difference 15 foods, three HF foods more pleasant, two LF foods more pleasant (P<0.03)). On average there were no general differences in comparison of sensory attributes that were consistently ascribable to the LF or HF foods, although there were numerous significant differences for individual foods. Subjects appeared unable to distinguish the HF foods as being HF (66% of estimates) and guessed correctly 33% of the time. They were better able to categorize the LF foods correctly (53% correct). On aggregate 43% of HF and LF foods were correctly identified. Subjects appeared able to detect sensory differences between foods but not to relate them to energy or nutrient content of these foods. CONCLUSIONS: These data suggest that subjects are on average not able to perceive large differences in the fat content of diets manipulated in this manner. In general the assumption that the manipulation of such foods is covert appears to hold, but subjects were far better at correctly identifying certain food types (dairy-based) over others.