An improved protocol for the isolation of total genomic DNA from Labyrinthulomycetes

Many protocols have been used for extraction of DNA from Thraustochytrids. These generally involve the use of CTAB, phenol/chloroform and ethanol. They also feature mechanical grinding, sonication, N2 freezing or bead beating. However, the resulting chemical and physical damage to extracted DNA reduces its quality. The methods are also unsuitable for large numbers of samples. Commercially-available DNA extraction kits give better quality and yields but are expensive. Therefore, an optimized DNA extraction protocol was developed which is suitable for Thraustochytrids to both minimise expensive and time-consuming steps prior to DNA extraction and also to improve the yield. The most effective method is a combination of single bead in TissueLyser (Qiagen) and Proteinase K. Results were conclusive: both the quality and the yield of extracted DNA were higher than with any other method giving an average yield of 8.5 µg/100 mg biomass.

Adult total wellness: group differences based on sitting time and physical activity level

Background An increasing body of evidence associates a high level of sitting time with poor health outcomes. The benefits of moderate to vigorous-intensity physical activities to various aspects of health are now well documented; however, individuals may engage in moderate-intensity physical activity for at least 30 minutes on five or more days of the week and still exhibit a high level of sitting time. This purpose of this study was to examine differences in total wellness among adults relative to high/low levels of sitting time combined with insufficient/sufficient physical activity (PA). The construct of total wellness incorporates a holistic approach to the body, mind and spirit components of life, an approach which may be more encompassing than some definitions of health. Methods Data were obtained from 226 adult respondents (27 ± 6 years), including 116 (51%) males and 110 (49%) females. Total PA and total sitting time were assessed with the International Physical Activity Questionnaire (IPAQ) (short-version). The Wellness Evaluation of Lifestyle Inventory was used to assess total wellness. An analysis of covariance (ANCOVA) was utilised to assess the effects of the sitting time/physical activity group on total wellness. A covariate was included to partial out the effects of age, sex and work status (student or employed). Cross-tabulations were used to show associations between the IPAQ derived high/low levels of sitting time with insufficient/sufficient PA and the three total wellness groups (i.e. high level of wellness, moderate wellness and wellness development needed). Results The majority of the participants were located in the high total sitting time and sufficient PA group. There were statistical differences among the IPAQ groups for total wellness [F (2,220) = 32.5 (p <0.001)]. A Chi-square test revealed a significant difference in the distribution of the IPAQ categories within the classification of wellness [χ2 (N = 226) = 54.5, p < .001]. One-hundred percent (100%) of participants who self-rated as high total sitting time/insufficient PA were found in the wellness development needed group. In contrast, 72% of participants who were located in the low total sitting time/sufficient PA group were situated in the moderate wellness group. Conclusion Many participants who meet the physical activity guidelines, in this sample, sit for longer periods of time than the median Australian sitting time. An understanding of the effects of the enhanced PA and reduced sitting time on total wellness can add to the development of public health initiatives. Keywords: IPAQ; The Wellness Evaluation of Lifestyle (WEL); Sedentary lifestyle

Influence of posture and frequency modes in total body water estimation using bioelectrical impedance spectroscopy in boys and adult males

The aim of the study was to examine differences in total body water (TBW) measured using single-frequency (SF) and multi-frequency (MF) modes of bioelectrical impedance spectroscopy (BIS) in children and adults measured in different postures using the deuterium (2H) dilution technique as the reference. Twenty-three boys and 26 adult males underwent assessment of TBW using the dilution technique and BIS measured in supine and standing positions using two frequencies of the SF mode (50 kHz and 100 kHz) and the MF mode. While TBW estimated from the MF mode was comparable, extra-cellular fluid (ECF) and intra-cellular fluid (ICF) values differed significantly (p < 0.01) between the different postures in both groups. In addition, while estimated TBW in adult males using the MF mode was significantly (p < 0.01) greater than the result from the dilution technique, TBW estimated using the SF mode and prediction equation was significantly (p < 0.01) lower in boys. Measurement posture may not affect estimation of TBW in boys and adult males, however, body fluid shifts may still occur. In addition, technical factors, including selection of prediction equation, may be important when TBW is estimated from measured impedance.

Centile reference charts for total energy expenditure in infants from 1 to 12 months

Background: A knowledge of energy expenditure in infancy is required for the estimation of recommended daily amounts of food energy, for designing artificial infant feeds, and as a reference standard for studies of energy metabolism in disease states. Objectives: The objectives of this study were to construct centile reference charts for total energy expenditure (TEE) in infants across the first year of life. Methods: Repeated measures of TEE using the doubly labeled water technique were made in 162 infants at 1.5, 3, 6, 9 and 12 months. In total, 322 TEE measurements were obtained. The LMS method with maximum penalized likelihood was used to construct the centile reference charts. Centiles were constructed for TEE expressed as MJ/day and also expressed relative to body weight (BW) and fat-free mass (FFM). Results: TEE increased with age and was 1.40,1.86, 2.64, 3.07 and 3.65 MJ/day at 1.5, 3, 6, 9 and 12 months, respectively. The standard deviations were 0.43, 0.47, 0.52,0.66 and 0.88, respectively. TEE in MJ/kg increased from 0.29 to 0.36 and in MJ/day/kg FFM from 0.36 to 0.48. Conclusions: We have presented centile reference charts for TEE expressed as MJ/day and expressed relative to BW and FFM in infants across the first year of life. There was a wide variation or biological scatter in TEE values seen at all ages. We suggest that these centile charts may be used to assess and possibly quantify abnormal energy metabolism in disease states in infants.

Measured and predicted total body water in children with myelomeningocele

Objective: Children with myelomeningocele (MMC) have an altered body composition and an atypical distribution of total body water (TBW). The aim of the present study was to determine the accuracy of current predictive equations, based on bioelectrical impedance analysis (BIA), in determining TBW when compared with measured TBW using deuterium dilution. Methods: Fourteen children with MMC were measured for whole body BIA and TBW (using deuterium dilution and the Plateau method). Total body water was predicted using equations based on the resistance and characteristic frequency from BIA measurements and heights of subjects. Results: The mean measured TBW was 15.46 ± 8.28 L and the mean predictions for TBW using equations based on the resistance and characteristic frequency from BIA measurements and heights of subjects were 18.29 ± 8.41 L, 17.72 ± 11.42 L and 12.51 ± 7.59 L, respectively. The best correlation was found using characteristic frequency. The limits of agreement between measured and predicted TBW values using Bland-Altman analysis were large. Conclusions: The present study suggests that the prediction of TBW in children with MMC can be made accurately using the equation of Cornish et al. based on BIA measurements of characteristic frequency.

Changes in body composition in adolescents with anorexia nervosa. Comparison of bioelectrical impedance analysis and total body potassium

Patients with anorexia nervosa (AN) have low body weight, depleted fat stores, and reduced muscle mass. Both total body potassium (TBK) and bioelectrical impedance analysis (BIA) have been used to measure the body composition of these patients.1–4 Whereas TBK accurately measures body cell mass, the metabolically active compartment of the body, whole body potassium counters are expensive and not readily available. The purpose of this study was to investigate the potential of multiple frequency BIA (MFBIA) to monitor changes in body compartments in patients with AN.

Value of total body potassium in assessing the nutritional status of children with end-stage liver disease

Malnutrition is a common problem in children with end-stage liver disease (ESLD), and accurate assessment of nutritional status is essential in managing these children. In a retrospective study, we compared nutritional assessment by anthropometry with that by body composition. We analyzed all consecutive measurements of total body potassium (TBK, n = 186) of children less than 3 years old with ESLD awaiting transplantation found in our database. The TBK values obtained by whole body counting of 40K were compared with reference TRK values of healthy children. The prevalence of malnutrition, as assessed by weight (weight Z score < -2) was 28%, which was significantly lower (chi-square test, p < 0.0001) than the prevalence of malnutrition (76%) assessed by TBK (< 90% of expected TRK for age). These results demonstrated that body weight underestimated the nutritional deficit and stressed the importance of measuring body composition as part of assessing nutritional status of children with ESLD.

Nutritional status of children with cystic fibrosis measured by total body potassium as a marker of body cell mass: Lack of sensitivity of anthropometric measures

Objective: To investigate measures aimed at defining the nutritional status of cystic fibrosis (CF) populations, this study compared standard anthropometric measurements and total body potassium (TBK) as indicators of malnutrition. Methods: Height, weight, and TBK measurements of 226 children with CF from Royal Children's Hospital, Brisbane, Australia, were analyzed. Z scores for height for age, weight for age, and weight for height were analyzed by means of the National Centre for Health Statistics reference. TBK was measured by means of whole body counting and compared with predicted TBK for age. Two criteria were evaluated with respect to malnutrition: (1) a z score < -2.0 and (2) a TBK for age <80% of predicted. Results: Males and females with CF had lower mean height-for-age and weight-for-age z scores than the National Centre for Health Statistics reference (P < .01), but mean weight-for-height z score was not significantly different. There were no significant gender differences. According to anthropometry, only 7.5% of this population were underweight and 7.6% were stunted. However, with TBK as an indicator of nutritional status, 29.9% of males and 22.0% of females were malnourished. Conclusion: There are large differences in the percentage of patients with CF identified as malnourished depending on whether anthropometry or body composition data are used as the nutritional indicator. At an individual level, weight-based indicators are not sensitive indicators of suboptimal nutritional status in CF, significantly underestimating the extent of malnutrition. Current recommendations in which anthropometry is used as the indicator of malnutrition in CF should be revised.

Enantiospecific first total synthesis of (-)-4-thiocyanatoneopupukeanane

The first total synthesis of (-)-4-thiocyanatoneopupukeanane starting from (R)-carvone has been achieved, establishing the relative as well as absolute structure of the natural product.

Synthesis based on cyclohexadienes. Part 25. Total synthesis of (+/-)-allo-cedrol (khusiol)

The first stereoselective total synthesis of (+/-)-allo-cedrol 20, an enantiomer of khusiol and a complex sesquiterpene having a novel tricyclo[5.2.2.0(1,5)]undecane framework, is reported from 8-methoxytricyclo[6.2.2.0(1,6)]dodec-6-en-9-one 6c. The methodology involves preparation of 9-methoxytricyclo[7.2.1.0(1,6)]dodec-6-en-8-one 12 from 6c and its conversion through the compounds 8-benzyloxy-7,7-dimethyl-9-methoxytricyclo[7.2.1.0(1,6)]dodec-5-ene 38, 7-benzyloxy-8-methoxy-2,6,6-trimethyltricyclo[6.2.1.0(1,5)]undecane 48 into 8-methoxy-2,6,6-trimethyltricyclo[6.2.1.0(1,5)]undecan-7-one 49. Wittig reaction of 49 affords the olefin 50 which has been smoothly rearranged into khusione 51. Metal-ammonia reduction of khusione under specific conditions affords (+/-)-allo-cedrol. Thus, bridgehead substitution of a methoxy group by a methyl group is the key reaction in this synthesis. In an alternative strategy, attempted conversion of 8-methoxy-2-methyltricyclo[6.2.1.0(1,5)]undec-5-en-7-one 16 into khusione 37 results in an inseparable mixture of the isomers. A notable observation in this synthesis is the unusual formation of a gamma-alkylated product 27 during Woodward methylation of 16.

Formal total synthesis of 2-pupukeanone via radical mediated cyclisation of an enyne

Diels-Alder reaction of the dienone 12, obtained by C-alkylation of sodium 2,6-dimethylphenoxide, with acrylonitrile and phenyl vinyl sulfones generate the enynes 14 and 17. Tributyltin radical addition to the terminal acetylene in 14 and 17 lead to the vinylstannanes 15 and 18 via 5-exo trig cyclisation of the resulting vinyl radical, which on oxidative cleavage furnishes the isotwistane-diones 16 and 19. Reductive desulfonylation of the diketosulfone 19 furnishes the dione 11, constituting a formal total synthesis of 2-pupukeanone 5 and 2-isocyanopupukeanone 3.

Variations in the levels of aminoacylation and modified nucleotide content between total tRNAs from chloroplasts and etioplasts in cucumber cotyledons

Total tRNAs isolated from chloroplasts and etioplasts of cucumber cotyledons were compared with respect toamino acid acceptance, isoacceptor distribution and extent of modification. Aminoacylation of the tRNAs with nine different amino acids studied indicated that the relative acceptor activities of chloroplast total tRNAs for four amino acids are significantly higher than etioplast total tRNAs. Two dimensional polyacrylamide gel electrophoresis(2D-PAGE) of chloroplast total tRNAs separated at least 32 spots, while approximately 41 spots were resolved from etioplast total tRNAs. Comparison of the reversed-phase chromatography (RPC-5) profiles of chloroplast and etioplast leucyl-, lysyl-, phenylalanyl-, and valyl-tRNA species showed no qualitative differences in the elution profiles. However, leucyl-, lysyl- and valyl-tRNA species showed quantitative differences in the relative amounts of the isoaccepting species present in chloroplasts and etioplasts. The analysis of modified nucleotides of total tRNAs from the two plastid types indicated that total tRNA from etioplasts was undermodified with respect to ribothymidine, isopentenyladenosine/hydroxy-isopentenyladenosine, 1 -methylguanosine and 2-o-methylguanosine. This indicates that illumination may cause de novo synthesis of chloroplast tRNAmodifying enzymes encoded for by nuclear genes leading to the formation of highly modified tRNAs in chloroplasts. Based on these results, we speculate that the observed decrease in levels of aminoacylation, variations in the relative amounts of certain isoacceptors, and differences in the electrophoretic mobilities of some extra tRNA spots in the etioplast total tRNAs as compared to chloroplast total tRNAs could be due to some partially undermodified etioplast tRNAs. Taken together, the data suggested that the light-induced transformation of etioplasts into chloroplasts is accompanied by increases in the relative levels of some functional chloroplast tRNAs by post transcriptional nucleotide modifications.

Enantiospecific total synthesis of ent-10, 11-thapsan-10-ol

First enantiospecific total synthesis of optical antipode of the sesquiterpene 10,11-epoxythapsan-10-ol has been described. (R)-Carvone has been employed as the chiral starting material and a combination of intramolecular alkylation and Criegee fragmentation are employed for intramolecular stereospecific transfer of the chirality. An intramolecular diazoketone cyclopropanation and regioselective cyclopropane ring cleavage reactions have been employed for the creation of the three requisite contiguous quaternary carbon atoms.

Statistical modelling and power analysis for detecting trends in total suspended sediment loads

The export of sediments from coastal catchments can have detrimental impacts on estuaries and near shore reef ecosystems such as the Great Barrier Reef. Catchment management approaches aimed at reducing sediment loads require monitoring to evaluate their effectiveness in reducing loads over time. However, load estimation is not a trivial task due to the complex behaviour of constituents in natural streams, the variability of water flows and often a limited amount of data. Regression is commonly used for load estimation and provides a fundamental tool for trend estimation by standardising the other time specific covariates such as flow. This study investigates whether load estimates and resultant power to detect trends can be enhanced by (i) modelling the error structure so that temporal correlation can be better quantified, (ii) making use of predictive variables, and (iii) by identifying an efficient and feasible sampling strategy that may be used to reduce sampling error. To achieve this, we propose a new regression model that includes an innovative compounding errors model structure and uses two additional predictive variables (average discounted flow and turbidity). By combining this modelling approach with a new, regularly optimised, sampling strategy, which adds uniformity to the event sampling strategy, the predictive power was increased to 90%. Using the enhanced regression model proposed here, it was possible to detect a trend of 20% over 20 years. This result is in stark contrast to previous conclusions presented in the literature. (C) 2014 Elsevier B.V. All rights reserved.

Total body potassium in cystic fibrosis

To further evaluate the nature of malnutrition, define at-risk groups, and confirm the efficacy of nutritional supplementation on body composition in cystic fibrosis (CF), we have conducted longitudinal and cross-sectional studies of total body potassium (TBK) in 161 unselected CF subjects aged 1 month to 17 years. TBK was determined by measurement of40K in a whole body counter, reflecting body cell mass (BCM), the vital work-performing and growing cellular component of the body. Compared with normal TBK data for age and sex from pooled measurements of 1,629 healthy children aged 1 week to 17 years, CF infants (n = 12) diagnosed by newborn screening were depleted in TBK at diagnosis and showed catch-up with therapy by 1 year; CF children aged 2-17 years (n = 140) showed a tendency for inadequate accretion of TBK (and thus BCM) with increasing age, although the normal correlation between TBK and weight and height was maintained in the majority suggesting a pattern of nutritional stunting of growth; and malnourished CF children (n = 9) showed significant catchup in TBK with long-term nutritional rehabilitation (85-98% of TBK predicted for weight and height). These studies suggest that potentially serious, but possibly correctable deficits, in the growth of the body cell mass as measured by TBK occur commonly in CF. These deficits may be established very early in life and if not corrected lead to progressive nutritional growth retardation with increasing age. © 1989 Raven Press, Ltd., New York.