Search

Black soldier fly larvae are mass produced globally for use as livestock, poultry, and aquaculture feed. Efforts are continuously seeking processes optimising larval rate of growth, size, and waste conversion as a means to lower cost and increase output. Manipulating microbes in the larval substrate (i.e. fermentation or supplementation) has been demonstrated as a potential solution. However, identifying appropriate microbes for use in this process has been limited. The objective of this study was to determine whether supplementing black soldier fly larvae with the oleaginous microbe Rhodococcus rhodochrous would result in accelerated larval development, increased final larval body size, and increased conversion efficiency. Larvae fed a sterile, or non-sterile, diet treated with R. rhodochrous grew 3× faster than the control by the third day and were approximately 2× larger than the control by the conclusion of the experiment. Conversion rate was 2× greater for the treatments indicating less feed would be needed to achieve maximum weight gain. Protein composition of resulting larvae fed diet supplemented with R. rhodochrous, was significantly different than the control not receiving the microbial infusion. Larvae provided the microbe had 4.20 and 2.79% greater fatty acid composition and short-chained fatty acids, respectively, but lower monounsaturated fatty acids (1.60%) and polyunsaturated fatty acids (2.4%). Furthermore, larvae provided R. rhodochrous produced significantly more proteins related to energy production and storage, as well as muscle development and contraction, while those sans microbe, produced proteins related to stress responses (e.g. heat shock proteins). While, this study yielded positive results for the inclusion of R. rhodochrous as part of the black soldier fly larval diet, additional research is needed to optimise the dose at an industrial scale.

BACKGROUND: Concurrent chemotherapy and thoracic radiotherapy followed by prophylactic cranial irradiation (PCI) is the standard treatment in limited-disease small-cell lung cancer (LD-SCLC), with 5-year overall survival (OS) of only 25% to 33%. PATIENTS AND METHODS: STIMULI is a 1:1 randomised phase II trial aiming to demonstrate superiority of consolidation combination immunotherapy versus observation after chemo-radiotherapy plus PCI (protocol amendment-1). Consolidation immunotherapy consisted of four cycles of nivolumab [1 mg/kg, every three weeks (Q3W)] plus ipilimumab (3 mg/kg, Q3W), followed by nivolumab monotherapy (240 mg, Q2W) for up to 12 months. Patient recruitment closed prematurely due to slow accrual and the statistical analyses plan was updated to address progression-free survival (PFS) as the only primary endpoint. RESULTS: Of the 222 patients enrolled, 153 were randomised (78: experimental; 75: observation). Among the randomised patients, median age was 62 years, 60% males, 34%/65% current/former smokers, 31%/66% performance status (PS) 0/1. Up to 25 May 2020 (median follow-up 22.4 months), 40 PFS events were observed in the experimental arm, with median PFS 10.7 months [95% confidence interval (CI) 7.0-not estimable (NE)] versus 42 events and median 14.5 months (8.2-NE) in the observation, hazard ratio (HR)= 1.02 (0.66-1.58), two-sided P= 0.93. With updated follow-up (03 June 2021; median: 35 months), median OS was not reached in the experimental arm, while it was 32.1 months (26.1-NE) in observation, with HR= 0.95 (0.59-1.52), P= 0.82. In the experimental arm, median time-to-treatment-discontinuation was only 1.7 months. CTCAE v4 grade =3 adverse events were experienced by 62% of patients in the experimental and 25% in the observation arm, with 4 and 1 fatal, respectively. CONCLUSIONS: The STIMULI trial did not meet its primary endpoint of improving PFS with nivolumab-ipilimumab consolidation after chemo-radiotherapy in LD-SCLC. A short period on active treatment related to toxicity and treatment discontinuation likely affected the efficacy results.

In this first na tional survey of public hospitals in The Republic of Ireland, we found fracture liaison services (FLS) to be heterogeneous, limited in many cases and poorly supported. A national strategy is urgently needed to support the implementation and operation of an FLS, and thus help reduce the burden of fragility fractures for patients and the healthcare system.Fragility/low-trauma fractures are a global concern, whose incidence is rising as the population ages. Many are preventable, and people with a prior fragility fracture are at particularly high risk of further fractures. This patient group is the target of the International Osteoporosis Foundation (IOF) Capture the Fracture campaign, advocating global adoption of fracture liaison services (FLS), with the aim of preventing secondary fragility fractures. We wished to determine the current availability and standards of an FLS in Ireland, ahead of the launch of a National FLS database.We devised a questionnaire encompassing the thirteen IOF standards for an FLS and asked all 16 public hospitals with an orthopaedic trauma unit in Ireland, to complete for the calendar year 2019 in patients aged ≥ 50 years.All sites returned the questionnaire, i.e. 100% response rate. Nine hospitals stated that they have an FLS, additionally one non-trauma hospital running a FLS responded, and were included. These 10 FLS had identified and managed 3444 non-hip fractures in the year 2019. This figure represents 19% of the expected non-hip fragility fracture numbers occurring annually in Ireland. Implementation of the IOF standards was very variable. All sites reported being inadequately resourced to provide a high-quality service necessary to be effective.The existence and functioning of FLS in Ireland are heterogeneous and suboptimal. A national policy to support the implementation of this programme in line with international standards of patient care is urgently needed.

A total of 1379 beef bull pictures were surveyed to determine visibility of feet and legs from four American semen company websites. Five different breeds were represented: Angus, Red Angus, Hereford (polled and horned), Simmental, and Charolais. In addition to visibility, data on other variables were collected to establish frequencies and correlations. These included breed, color, material that obscured visibility, such as grass, picture taken at livestock show or outside, semen company, photographer, video, and age of bull. A foot and leg visibility score was given to each bull picture. Only 19.4% of the pictures had fully visible feet and legs. Both the hooves and dewclaws were hidden on 32.5% of the pictures. Correlation between bull’s birthdate and the first four visibility scores was statistically significant (P &lt, 0.0001). As age increased the feet and legs were more likely to be visible in the bull’s picture. This may possibly be due to greater availability of both photo editing software and digital photography. One positive finding was that 6% of the bulls had a video of the bull walking which completely showed his feet and legs.

In order to meet the F-35 Joint Strike Fighter’s strict radar cross-section and weight requirements, stealth coatings must be applied to extremely precise thickness tolerances. To meet these unprecedented tolerances, Lockheed Martin Aeronautics Company has implemented a unique Robotic Aircraft Finishing System (RAFS). This paper details the capabilities of RAFS hardware for precision coating application as compared to legacy systems. The iterative process of optimizing spray parameters and robot programs for coating thickness control on challenging F-35 geometry is also presented. Finally, results from the first coating of a production aircraft at RAFS are compared with previous hand-spray results. In keeping with Security and International Traffic in Arms Regulations, restricted information on coating materials and tolerances is not presented.

Developed recently, high resolution melting (HRM) analysis is an efficient, accurate and inexpensive method for distinguishing DNA polymorphisms. HRM has been used to identify mutations in human genes, and to detect SNPs, INDELs and microsatellites in plants. However, its capacity to discriminate DNA variants in the context of complex haplotypes involving INDEL as well as SNP variants has not been examined until now. In this study, we genotyped an almond (Prunus dulcis (Mill.) D. A. Webb, syn. Prunus amygdalus Batsch) pseudo-testcross mapping population that showed segregation of complex haplotypes associated with CYP79D16 promoter sequence. The 175 bp region in question included a 7 bp INDEL and 3 SNPs, and manifested as three different haplotypes in the parents. Thus, with one homozygous and one heterozygous parent, two relevant genotypes were identified in the mapping population. Although the population displayed monomorphism with respect to the INDEL and one of the SNPs, HRM was sufficiently sensitive to distinguish genotypes on the basis of the two informative SNPs, and the resulting data were used to map CYP79D16 to linkage group 6 of the almond genome. Thus the capacity of HRM to resolve genotypes arising from complex haplotypes has been demonstrated, and this has important implications for the design of efficient HRM markers for various genetic applications including mapping, population studies and biodiversity analyses.

To determine the chemical basis of almond flavour phenotypes, non-bitter, semi-bitter, and bitter kernels from 101 almond trees, all derived from a common maternal parent, were evaluated for flavou...

To dissect the genetic architecture of blood pressure and assess effects on target organ damage, we analyzed 128,272 SNPs from targeted and genome-wide arrays in 201,529 individuals of European ancestry, and genotypes from an additional 140,886 individuals were used for validation. We identified 66 blood pressure-associated loci, of which 17 were new; 15 harbored multiple distinct association signals. The 66 index SNPs were enriched for cis-regulatory elements, particularly in vascular endothelial cells, consistent with a primary role in blood pressure control through modulation of vascular tone across multiple tissues. The 66 index SNPs combined in a risk score showed comparable effects in 64,421 individuals of non-European descent. The 66-SNP blood pressure risk score was significantly associated with target organ damage in multiple tissues but with minor effects in the kidney. Our findings expand current knowledge of blood pressure-related pathways and highlight tissues beyond the classical renal system in blood pressure regulation. © 2016 Nature America, Inc. All rights reserved.

A multigenic trait (biosynthesis of the secondary metabolite, dhurrin cyanogenic glucoside) was engineered de novo in grapevine (Vitis vinifera L.). This follows a recent report of transfer of the same trait to Arabidopsis (Arabidopsis thaliana) using three genetic sequences from sorghum (Sorghum bicolor): two cytochrome P450-encoding cDNAs (CYP79A1 and CYP71E1) and a UDPG-glucosyltransferase-encoding cDNA (sbHMNGT). Here we describe the two-step process involving whole plant transformation followed by hairy root transformation, which was used to transfer the same three sorghum sequences to grapevine. Transgenic grapevine hairy root lines that accumulated transcript from none, one (sbHMNGT), two (CYP79A1 and CYP71E1) or all three transgenes were recovered and characterisation of these lines provided information about the requirements for dhurrin biosynthesis in grapevine. Only lines that accumulated transcripts from all three transgenes had significantly elevated cyanide potential (up to the equivalent of about 100 mg HCN kg(-1) fresh weight), and levels were highly variable. One dhurrin-positive line was tested and found to release cyanide upon maceration and can therefore be considered 'cyanogenic'. In in vitro dual co-culture of this cyanogenic hairy root line or an acyanogenic line with the specialist root-sucking, gall-forming, aphid-like insect, grapevine phylloxera (Daktulosphaira vitifoliae, Fitch), there was no evidence for protection of the cyanogenic plant tissue from infestation by the insect. Consistently high levels of dhurrin accumulation may be required for this to occur. The possibility that endogenous grapevine gene expression is modulated in response to engineered dhurrin biosynthesis was investigated using microarray analysis of 1225 grapevine ESTs, but differences in patterns of gene expression associated with dhurrin-positive and dhurrin-negative phenotypes were not identified.

A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 muM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.

Advanced electromagnetic sensor systems more commonly associated with the hightech military battlefield may be applied to remote surveillance of wildlife. The first comprehensive study of a wide global variety of Near Infra Red (NIR) and thermal wildlife portraits are presented with this technology: for mammals, birds and other animals. The paper illustrates the safety aspects afforded to zoo staff and personnel in the wild during the day and night from potentially lethal and aggressive animals, and those difficult to approach normally. Such remote sensing systems are non-invasive and provide minimal disruption and distress to animals both in captivity and in the wild. We present some of the veterinarian advantages of such all weather day and night systems to identify sickness and injuries at an early diagnostic stage, as well as age related effects and mammalian cancer. Animals have very different textured surfaces, reflective and emissive properties in the NIR and thermal bands than when compared with the visible spectrum. Some surface features may offer biomimetic materials design advantages.

Twelve grapevine (Vitis vinifera L.) cultivars were surveyed for 'cyanide potential' (i.e. the total cyanide measured in beta-glucosidase-treated crude, boiled tissue extract) in mature leaves. Two related cultivars (Carignan and Ruby Cabernet) had mean cyanide potential (equivalent to 110 mgHCNkg-1fr.wt) ca. 25-fold greater than that of the other 10 cultivars, and so the trait is polymorphic in the species. In boiled leaf extracts of Carignan and Ruby Cabernet, free cyanide constituted a negligible fraction of the total cyanide potential because beta-glucosidase treatment was required to liberate the major cyanide fraction - which is therefore bound in glucosylated cyanogenic compound(s) (or cyanogenic glucosides). In addition, cyanide was liberated from ground leaf tissue of Ruby Cabernet but not Sultana (a cultivar with low cyanide potential). Hence, the high cyanide potential in Ruby Cabernet leaves is coupled with endogenous beta-glucosidase(s) activity and this cultivar may be considered 'cyanogenic'. A method was developed to detect and identify cyanogenic glucosides using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). Two putative cyanogenic glucosides were found in extracts from leaves of Carignan and Ruby Cabernet and were identified as the epimers prunasin and sambunigrin. Cyanide potential measured at three times over the growing season in young and mature leaves, petioles, tendrils, flowers, berries, seeds and roots of Ruby Cabernet was substantially higher in the leaves compared with all other tissues. This characterisation of cyanogenic glucoside accumulation in grapevine provides a basis for gauging the involvement of the trait in interactions of the species with its pests and pathogens.

A simple system for in vitro dual culture of grapevine (Vitis spp.) plantlets and root-knot nematode (Meloidogyne javanica (Treub) Chitwood) is described. Based on the presence or absence of mature females, or the total number of nematodes in the roots after 36-day co-culture, the system reliably discriminated resistant (cv. Ramsey) and susceptible (cv. Chardonnay) grapevines. The system was sensitive enough to differentiate between infestation levels of cvs Börner and Chardonnay, both susceptible in the in vitro conditions. A modification of the system to use plantlets from rooted petioles has reduced labour and space requirements and would suit mass screening of grapevine genotypes in traditional or genetic engineering-based breeding programmes. In both systems, nematodes in roots of cv. Ramsey tended to be associated with brown tissue and, compared with those in roots of cv. Chardonnay, were more likely to be confined to tips rather than be distributed along the root after co-culture for 11 or 36 days.

This paper presents a systematic study of the MgO–CaO–Na2O–P2O5 glass system, which has great potential to be used as temporary hard and soft tissue implant materials. An overall study of solubility behavior of ternary and quaternary-based phosphate glass system have been carried out in order to understand the out-leaching progress of different ions and to determine their effect on cell proliferation. Originally, soluble phosphate based glasses within the ternary glass system of Na2O–CaO–P2O5 have been developed to create a simple baseline system. This paper, however, presents the development of this system by introducing magnesium oxide as a partial calcium oxide substitute and solubility behaviors as well as cell studies have been carried out to check the effect on magnesium ions. Glasses have been prepared via standard glass melting techniques and their solubility behavior has been tested in distilled water via simple weight loss, pH and ion measurements. The way the glasses dissolve is an inverse exponential behavior which is mirrored by the calcium ion release. Other ions show a less exponential behavior. The MTT test has been used to check preliminary in vitro studies on a human MG63 cell line and the result indicates that cell proliferation is increased for glasses with minimal CaO substitution.

Glasses from the quaternary glass system K2O-Na2O-CaO-P2O5 were produced by standard glass forming techniques. The compositions were limited by fixing the P2O5 at 45mol%, fixing the CaO content at either 20,24 or 28 mol%. The K2O and Na2O made up the residual varying from 0 to 25 mol % K2O. General trends showed that with increasing CaO content, the glasses showed a decrease in solubility as expected. For a single system of fixed CaO content, with increasing K2O content, there was an increase in solubility. This was seen at all three CaO contents. All the glasses showed an initial increase in pH followed by a gradual decrease with time and this was accounted for by the initial release of Na+ ions into solution. For the ion release curves, for all fixed CaO contents, the glass with 0mol% K2O showed the lowest Ca2+ release. This was accounted for as being due to the low solubility compared to the K2O containing glasses. The Na+ release appeared anomalous, as it was higher than all the K2O containing glasses. Even though the glasses with 0 mol% K2O showed the lowest solubility, the amount of Na+ contained in the glass was high, hence the high levels of release. (C) 2001 Elsevier Science Ltd. All rights reserved.

We have developed an Agrobacterium-mediated transformation system for a number of important grapevine cultivars used in wine production. Transgenic plants were obtained for the seven cultivars: Cabernet Sauvignon, Shiraz, Chardonnay, Riesling, Sauvignon Blanc, Chenin Blanc and Muscat Gordo Blanco. Embryogenic callus was initiated from anther filaments and genotypic differences were observed for initiation and subsequent proliferation with Chardonnay responding most favourably to culture conditions. The transformation system allowed the recovery of germinating transgenic embryos 10-12 weeks after Agrobacterium inoculation and plants within 18 weeks. Examination of the expression patterns of the green fluorescent protein gene under the control of the CAMV35S promoter in leaf tissue of transgenic plants showed that for up to 35% of plants the pattern was not uniform. The successful transformation of a genetically diverse group of wine grape cultivars indicates that the transformation system may have general application to an even wider range of Vitis vinifera cultivars.

We have previously reported cosegregation of bipolar affective disorder and Darier disease, a dominant skin disorder with a neuropsychiatric component. The gene for Darier disease was mapped to chromosome 12q23-q24.1 and linkage studies have subsequently implicated this region as harboring a susceptibility gene for bipolar affective disorder. We have genomically characterized the human homologue of murine Cux-2, a neuronal-specific transcription factor potentially involved in the regulation of neural cell adhesion molecule expression that maps to this region. Also, in a panel of 15 unrelated bipolar patients from multiply affected families showing increased allele sharing at markers in the 12q23-q24.1 region, we performed mutational screening of the CUX2 coding sequence, and 5' untranslated region (5' UTR). Resulting sequence were analyzed in a large sample of bipolar patients (n = 218) and control subjects (n = 218). No evidence was found for the involvement of variants within the CUX2 coding, or 5' UTR sequence in producing susceptibility to bipolar disorder.

Bipolar affective disorder is a genetically complex psychiatric disorder with a population prevalence of approximately 1%. We have previously reported cosegregation of bipolar affective disorder and Darier's disease, a dominant skin disorder with a neuropsychiatric component. The gene for Darier's disease was mapped to chromosome 12q23-q24.1 and linkage studies by us and others have subsequently implicated this region as harbouring a susceptibility gene for bipolar affective disorder. In this study we have investigated the Darier's disease gene ATP2A2, the calcium pumping ATPase SERCA2, as a potential susceptibility gene for bipolar disorder under the hypothesis that variations in SERCA2 have pleiotropic effects in brain. Support for this hypothesis comes from clinical evidence of neuropsychiatric abnormalities in Darier's disease, genetic data produced in our study showing non-random clustering of missense mutations in ATP2A2 in neuropsychiatric Darier patients, and functional data demonstrating the role of SERCA2 in intracellular calcium regulation. In a panel of 15 unrelated bipolar patients from multiply affected families showing increased allele sharing at markers in the 12q23-q24.1 region, we performed mutational screening of the ATP2A2 coding sequence, promoter regions, and 3' untranslated region and identified six sequence variations. These were analysed in a large sample of bipolar patients (n = 324) and control subjects (n = 327). Analysis of allele and genotype distributions for all six variations, and of haplotype frequencies showed no evidence for the involvement of ATP2A2 in producing susceptibility to bipolar disorder.

Molecular technology and genetic modification of grapevines, applied either to new varieties or to existing cultivars, offers the opportunity to directly address issues of fruit quality, disease resistance, cost of production and sustainability. The technology provides the means to selectively improve specific traits of cultivars of known performance and market acceptance whilst preserving their original identity. Progress in recent years has been significant with the development of transformation systems for both rootstocks and scions. Complementary investigation and isolation of genes and tissue-specific promoter regions will contribute to our ability to improve specific traits. It is expected that future research will provide an increasing range of possibilities for genetic manipulation of grapevines with advances in our understanding of plant processes and ability to isolate genes controlling plant development, primary and secondary metabolism and resistance to pathogens.

Soluble glasses are considered to be of potential clinical value in orthopaedic and dental surgery. However, the biological response to these materials is not well understood. To determine the effects of these glasses, two human osteoblast cell lines, MG63 and HOS (TE85), were incubated in vitro in the presence of increasing concentrations of extracts of the glasses. The effects of the extracts on cell growth was measured using the MTT assay and an ELISA assay was used to measure the expression of bone sialoprotein (BSP), osteonectin (ON) and fibronectin (FN), antigens which play a fundamental part in the integrity and function of hard connective tissue. The results showed that the proliferation of the cells was adversely affected only by the more soluble glasses, which also down-regulated the expression of the bone-associated proteins. In contrast, the extract of the glass with the lowest dissolution rate, which contains relatively elevated levels of Ca2+, was found to enhance bone cell growth and antigen expression. These findings suggest that the compositions of these glasses at least partly determine the response of cells and thus, that the glasses could be modified to elicit a more optimal biological response and clinical efficacy.

In this study soluble glasses have been developed for biomedical applications containing P2O5 as a network former and CaO and Na2O as modifiers. This study shows that as expected, the glasses have an inverse exponential relationship between solubility and CaO content. Furthermore, there is evidence for compositional related non-linearity in the dissolution of the glasses with time. This is thought to be due to either layer formation on the glass surface hindering ion diffusion, ion exchange process or change of ionic strength of the solution. Bioactivity of these glasses is indicated by the formation of a brushite precipitate, a precursor to apatite formation. Further evidence for bioactivity is also presented in the second part of this paper.

Evidence for the involvement of genetic factors in the pathogenesis of bipolar affective disorder is now well established. However, the mode of inheritance is non-mendelian and this makes the identification of susceptibility loci difficult. A short-cut to localisation of a disease gene for an oligogenic/multifactorial disorder such as bipolar disorder may come from observation of cosegregation with a monogenic trait. We have described a family (pedigree 324) in which there was cosegregation of major affective disorder and Darier's disease, a dominantly inherited skin disorder, and hypothesised that this reflects genetic linkage between genes involved in these disorders. Genetic mapping studies have placed the locus for Darier's disease on chromosome 12q23-q24. We conducted subsequent linkage studies (1995) upon 45 bipolar families (without Darier's disease). These results showed some evidence in favour of linkage with chromosome 12q markers with maximum evidence at a trinucleotide repeat marker within intron 1 of the phospholipase A2A (PLA2A) gene. Evidence for linkage was more significant when analysing the 22 families comprising the Cardiff centre sample, which were expected to be most genetically similar to pedigree 324.

The hypothesis that one or more genes containing expanded trinucleotide repeats contribute to the pathogenesis of bipolar disorder has received support from three independent studies demonstrating that bipolar patients tend to have larger CAG/CTG repeat expansion detection products than controls. In an attempt to identify the specific expanded CAG/CTG locus or loci which are associated with bipolar disorder, we determined repeat size at CAG/CTG loci mapping to candidate regions for bipolar disorder. Recent linkage studies suggest the existence of a bipolar susceptibility gene on chromosome 12q23-q24.1 in the region of the Darier's disease (DAR) gene. In this study we report our findings from 11 loci which map to chromosome 12, including CAG repeat polymorphisms within the genes SCA2 and ASH1. We conclude that all of these loci are excluded as candidates for CAG/CTG repeat expansion in bipolar disorder.

Different approaches to producing transgenic grapevines based on regeneration via embryogenesis were investigated. Embryogenic callus was initiated from anther tissue of Vitis vinifera cv. Sultana and three embryogenic culture types (embryogenic callus, tissue type I; proliferating embryos, tissue type II; and a suspension) were established. The three culture types were incolucaled with Agrobacterium tumefaciens harbouring a binary vector which contained a uidA reporter gene and either a hpt or nptII selectable marker gene or the cultures were bombarded with microprojectiles carrying a uidA/nptII binary vector. Transgenic plants were produced only from Agrobacterium transformation experiments. Transformed embryos were selected with kanamycin or hygromycin antibiotics and recovered with the highest efficiency from inoculated type I cultures. Southern analysis of genomic DNA extracted from ten transgenic plants showed that the number of T-DNA insertions in the genome ranged from 1 to at least 4. Evidence for methylation of the T-DNA at cytosine and adenine residues in transgenic plants was found by Southern analysis of DNA digested with two isoschizomer pairs of restriction endonucleases. No evidence for genotype alterations or somatic meiosis was found when DNA from 80 somatic embryos and seven plants regenerated from embryogenic culture were analysed at six sequence-tagged sites which are heterozygous in cv. Sultana. Expression of the uidA gene in in vitro grown leaves of transgenic plants was most often high and uniform but GUS staining was occasionally observed to be low and/or patchy. Transgenic plants and all plants regenerated from embryogenic culture produced red veined, lobed leaves which are uncharacteristic of the accepted ampelographic phenotype of Sultana. It is suggested that this phenotype may represent a juvenile growth stage.

A high copy, tandemly repeated, sequence (Bd49) specific to the B chromosome and located near the centromere in Brachycome dichromosomatica was used to identify lambda genomic clones from DNA of a 3B plant. Only one clone of those analysed was composed entirely of a tandem array of the B-specific repeat unit. In other clones, the Bd49 repeats were linked to, or interspersed with, sequences that are repetitious and distributed elsewhere on the A and B chromosomes. One such repetitious flanking sequence has similarity to retrotransposon sequences and a second is similar to chloroplast DNA sequences. Of the four separate junctions analysed of Bd49-like sequence with flanking sequence, three were associated with the same A/T-rich region in Bd49 and the fourth was close to a 25 bp imperfect dyadic sequence. No novel B-specific sequences were detected within the genomic clones.

We have investigated the ability of the nucleotides GTP, CTP, and 1-N6-etheno-2-aza-ATP (aza-ATP) to support contraction of chemically skinned rabbit psoas fibers. Working at 10 degrees C, millimolar concentrations of all nucleotides relaxed fibers in the absence of calcium. In active fibers, GTP served as a very poor substrate with isometric tension, isometric GTPase rate, and maximum shortening velocity (Vmax) all less than 10% of those obtained with ATP. Aza-ATP was only a slightly better substrate. CTP, on the other hand, was an effective substrate with mechanical parameters which were 65-100% those obtained with ATP, and with a hydrolysis rate that exceeded that of ATP. For all three ligands, Vmax followed Michaelis-Menten saturation behavior with values for Km which were from 2.5 to 12 times greater than that for ATP, showing that the analogs bound slowly to myosin in the fibers. Increasing concentrations of orthophosphate inhibited tension with CTP, to a lesser extent with aza-ATP, but not all with GTP. A combination of the mechanical data obtained in fibers with the kinetic data obtained in solution (White, H.D., Belknap, B., and Jiang, W. (1993) J. Biol. Chem. 268, 10039-10045) is used to better define the actomyosin interaction in fibers.

We have measured the isometric tension and force-velocity relationships of glycerinated rabbit slow-twitch semimembranosus muscle as a function of MgATP concentration ([MgATP]) and have compared the results with those obtained previously from fast-twitch psoas muscle. We find that isometric tension decreases as [MgATP] increases. The magnitude of the decrease is not as great as observed in psoas. Maximum shortening velocity (Vmax) exhibits classical Michaelian saturation behavior with respect to [MgATP] with a Michaelis constant (Km) for half-maximal velocity of 18 microM and a value at saturating [MgATP] of 0.6 muscle lengths/s. Similar values were observed in fibers from soleus, another slow-twitch muscle. The corresponding values in rabbit psoas muscle are 150 microM and 1.6 lengths/s. Compared with psoas, in semimembranosus muscle Km decreases by a factor of approximately 10, whereas Vmax decreases by about a factor of 3. Thus, although in a nonphysiological regime, at low [MgATP], a "fast" muscle actually has a lower shortening velocity than a "slow" muscle.

We have investigated the ability of the photoaffinity, nonnucleotide ATP analogues, 2-[(4-azido-2-nitrophenyl) amino] ethyl triphosphate (NANTP) and 2-[(4-azido-2-nitrophenyl) amino] propyl triphosphate (PrNANTP), to support active contraction in glycerinated rabbit psoas fibers. At millimolar concentrations, in the absence of calcium, both analogues relaxed fibers. In the presence of calcium, MgNANTP produced isometric tension and stiffness that were one-half to two-thirds the values obtained in MgATP. Maximum shortening velocity and the calcium-activated, myofibrillar catalyzed rate of hydrolysis were approximately the same for MgNANTP as for MgATP. With MgNANTP as the substrate, increasing concentrations of the diphosphate analogue, MgNANDP, inhibited shortening velocity but did not change isometric tension. The addition of increased concentrations of orthophosphate (P) decreased tension while shortening velocity increased. Thus, the effects of the hydrolysis products of NANTP were quite similar to those observed previously for ADP and P in the presence of MgATP. Taken together, these observations show that MgNANTP binds to, and functions in the active site of myosin in a manner quite analogous to MgATP. Thus, the aryl azido group should serve as a valid photoaffinity label for the purine portion of the active site. In contrast, MgPrNANTP, which differs from MgNANTP only in an extra CH2 spacer between the nitrophenyl ring and the triphosphate moiety did not support isometric tension or active shortening in the presence of calcium. Fiber stiffness increased in the presence of calcium and MgPrNANTP, with a calcium-activated, myofibrillar MgPrNANTPase which was about half that obtained with MgATP. Thus, in the presence of MgPrNANTP, cross-bridges appeared to be cycling through states that were attached to actin, but not producing force.

The secondary metabolite amygdalin is a cyanogenic diglucoside that at high concentrations is associated with intense bitterness in seeds of the Rosaceae, including kernels of almond (Prunus dulcis (Mill.), syn. Prunus amygdalus D. A. Webb Batsch). Amygdalin is a glucoside of prunasin, itself a glucoside of R-mandelonitrile (a cyanohydrin). Here we report the isolation of an almond enzyme (UGT85A19) that stereo-selectively glucosylates R-mandelonitrile to produce prunasin. In a survey of developing kernels from seven bitter and 11 non-bitter genotypes with polyclonal antibody raised to UGT85A19, the enzyme was found to accumulate to higher levels in the bitter types in later development. This differential accumulation of UGT85A19 is associated with more than three-fold greater mandelonitrile glucosyltransferase activity in bitter kernels compared with non-bitter types, and transcriptional regulation was demonstrated using quantitative-PCR analysis. UGT85A19 and its encoding transcript were most concentrated in the testa (seed coat) of the kernel compared with the embryo, and prunasin and amygdalin were differentially compartmentalised in these tissues. Prunasin was confined to the testa and amygdalin was confined to the embryo. These results are consistent with the seed coat being an important site of synthesis of prunasin as a precursor of amygdalin accumulation in the kernel. The presence of UGT85A19 in the kernel and other tissues of both bitter and non-bitter types indicates that its expression is unlikely to be a control point for amygdalin accumulation and suggests additional roles for the enzyme in almond metabolism.

Purpose To evaluate the potential occupational health hazards associated with scattered actinic ultraviolet (UV) laser radiation and broadband actinic UV plasma emissions during refractive surgery. Setting Center for Refractive Surgery, Walter Reed Army Medical Center, Washington, D.C., USA. Methods Intraoperative radiometric measurements were made with the Ophir Power/Energy Meter (LaserStar Model with silicon detector, Model PD-10) and the International Light Radiometer/Photometer (Model IL 1400 with actinic ultraviolet detector, Model SEL240) with and without UV blocking filters (BLK 270 and Schott types WG-280 and WG-230). Measurements made during laser calibration as well as laser in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) procedures were evaluated using a worst-case scenario and then compared with the American Conference of Governmental Industrial Hygeinists (ACGIH) Threshold Value Limits (TLV) to perform a risk/hazard analysis. Results Most optical emissions were between 193 nm and 280 nm, and approximately 25% of the measurement result was due to broadband emissions greater than 270 nm for calibration targets. About 25% of optical emissions during LASIK were beyond 230 nm. No emissions beyond 230 nm were observed during PRK. Ultraviolet scattered radiation level was similar between PRK and LASIK. Maximum measured values of 80 nJ/pulse at 14 cm for PRK and 45 nJ/pulse at 38 cm for LASIK were used as the absolute worst-case analysis for exposure. Assuming the worst-case exposure conditions are equal to the maximum measured value during these studies at a workload of 20 patients per day, the cumulative occupational exposure at close range of actinic UV radiation did not exceed the 8-hour occupational exposure limit of 3 mJ/cm 2 for any 24-hour period. Conclusions Scattered UV laser radiation did not exceed occupational exposure limits at distances greater than 30 cm from either laser calibration targets or patient treatments over a workday. Laser eye protection is not necessary to protect operating room personnel since exposure levels are very low even under a worst-case scenario.

This paper presents a systematic study of the MgO-CaO-Na(2)O-P(2)O(5) glass system, which has great potential to be used as temporary hard and soft tissue implant materials. An overall study of solubility behavior of ternary and quaternary-based phosphate glass system have been carried out in order to understand the out-leaching progress of different ions and to determine their effect on cell proliferation. Originally, soluble phosphate based glasses within the ternary glass system of Na(2)O-CaO-P(2)O(5) have been developed to create a simple baseline system. This paper, however, presents the development of this system by introducing magnesium oxide as a partial calcium oxide substitute and solubility behaviors as well as cell studies have been carried out to check the effect on magnesium ions. Glasses have been prepared via standard glass melting techniques and their solubility behavior has been tested in distilled water via simple weight loss, pH and ion measurements. The way the glasses dissolve is an inverse exponential behavior which is mirrored by the calcium ion release. Other ions show a less exponential behavior. The MTT test has been used to check preliminary in vitro studies on a human MG63 cell line and the result indicates that cell proliferation is increased for glasses with minimal CaO substitution.

In this study soluble glasses have been developed for biomedical applications containing P2O5 as a network former and CaO and Na2O as modifiers. This study shows that as expected, the glasses have an inverse exponential relationship between solubility and CaO content. Furthermore, there is evidence for compositional related non-linearity in the dissolution of the glasses with time. This is thought to be due to either layer formation on the glass surface hindering ion diffusion, ion exchange process or change of ionic strength of the solution. Bioactivity of these glasses is indicated by the formation of a brushite precipitate, a precursor to apatite formation. Further evidence for bioactivity is also presented in the second part of this paper.

Soluble glasses are considered to be of potential clinical value in orthopaedic and dental surgery. However, the biological response to these materials is not well understood. To determine the effects of these glasses, two human osteoblast cell lines, MG63 and HOS (TE85), were incubated in vitro in the presence of increasing concentrations of extracts of the glasses. The effects of the extracts on cell growth was measured using the MTT assay and an ELISA assay was used to measure the expression of bone sialoprotein (BSP), osteonectin (ON) and fibronectin (FN), antigens which play a fundamental part in the integrity and function of hard connective tissue. The results showed that the proliferation of the cells was adversely affected only by the more soluble glasses, which also down-regulated the expression of the bone-associated proteins. In contrast, the extract of the glass with the lowest dissolution rate, which contains relatively elevated levels of Ca2+, was found to enhance bone cell growth and antigen expression. These findings suggest that the compositions of these glasses at least partly determine the response of cells and thus, that the glasses could be modified to elicit a more optimal biological response and clinical efficacy.

In the course of DNA profiling of grapevine cultivars using microsatellite loci we have occasionally observed more than two alleles at a locus in some individuals and have identified periclinal chimerism as the source of such anomalies. This phenomenon in long-lived clonally propagated crops, such as grapevine, which contains historically ancient cultivars, may have a role in clonal differences and affect cultivar identification and pedigree analysis. Here we show that when the two cell layers of a periclinal chimera, Pinot Meunier, are separated by passage through somatic embryogenesis the regenerated plants not only have distinct DNA profiles which are different from those of the parent plant but also have novel phenotypes. Recovery of these phenotypes indicates that additional genetic differences can exist between the two cell layers and that the Pinot Meunier phenotype is due to the interaction of genetically distinct cell layers. It appears that grapevine chimerism can not only modify phenotype but can also impact on grapevine improvement as both genetic transformation and conventional breeding strategies separate mutations in the L1 and L2 cell layers.

This paper presents the results of a study of the thermal properties of a range of phosphate-based glasses in the system CaO-Na2O-P2O5. The glasses had a fixed P2O5 content of 45 mol% and the CaO:Na2O ratio was varied. The glasses were produced and ground and their thermal properties measured using a Setaram differential thermal analyser (DTA). Initial data showed that for the high Na2O containing glass. NaPO3 forms and a second unidentified phase that is probably calcium rich. In the middle compositional region. multiple phases precipitated out and were identified as NaPO3 and Na4Ca(PO3)(6). For the high CaO content glasses. Na4Ca(PO3)(6) was the only phase formed. Further studies were carried out to examine whether the phases were bulk or surface nucleating. by grinding the samples to different particle sizes. From this data, it is evident that the NaPO3 forms via a bulk nucleation mechanism and that Na4Ca(PO3)(6) forms via surface nucleation. The factors controlling this precipitation process are discussed and ionic radius as a controlling factor is hypothesised. (C) 2001 Elsevier Science Ltd. All rights reserved.