Three-year weight change in successful weight losers who lost weight on a low-carbohydrate diet

Objective: The purpose of this study was to evaluate long‐term weight loss and eating and exercise behaviors of successful weight losers who lost weight using a low‐carbohydrate diet. Research Methods and Procedures: This study examined 3‐year changes in weight, diet, and physical activity in 891 subjects (96 low‐carbohydrate dieters and 795 others) who enrolled in the National Weight Control Registry between 1998 and 2001 and reported ≥30‐lb weight loss and ≥1 year weight loss maintenance. Results: Only 10.8% of participants reported losing weight after a low‐carbohydrate diet. At entry into the study, low‐carbohydrate diet users reported consuming more kcal/d (mean ± SD, 1895 ± 452 vs. 1398 ± 574); fewer calories in weekly physical activity (1595 ± 2499 vs. 2542 ± 2301); more calories from fat (64.0 ± 7.9% vs. 30.9 ± 13.1%), saturated fat (23.8 ± 4.1 vs. 10.5 ± 5.2), monounsaturated fat (24.4 ± 3.7 vs. 11.0 ± 5.1), and polyunsaturated fat (8.6 ± 2.7 vs. 5.5 ± 2.9); and less dietary restraint (10.8 ± 2.9 vs. 14.9 ± 3.9) compared with other Registry members. These differences persisted over time. No differences in 3‐year weight regain were observed between low‐carbohydrate dieters and other Registry members in intent‐to‐treat analyses (7.0 ± 7.1 vs. 5.7 ± 8.7 kg). Discussion: It is possible to achieve and maintain long‐term weight loss using a low‐carbohydrate diet. The long‐term health effects of weight loss associated with a high‐fat diet and low activity level merits further investigation.     

Effects of a reimbursement incentive on enrollment in a weight control program

Objective: The objective was to examine the effect of offering a reimbursement incentive on the percentage of inquirers who enrolled in a weight control program and on weight loss and program attendance among enrollees. Research Methods and Procedures: We used a sequential control‐intervention design to observe how inquirers of the University of Alabama at Birmingham EatRight Lifestyle Program responded to an enrollment incentive for potential 50% ($150) reimbursement of the total program fee if they attended 10 of 12 classes and lost at least 6% of their current body weight. Inquirers had to be adults with a BMI ≥30 kg/m², seeking information about a weight control program, and informed of the program cost. Outcomes included proportion of inquirers enrolled, overall number of classes attended, and weight loss. Results: Of the 401 people who inquired during the study periods, 24.5% and 25.0% enrolled in the intervention and control periods, respectively. There was a trend toward higher attendance in the intervention group, compared with the control group; there were no differences in percentage of weight loss. The odds of attending ≥10 classes were 2.4 times as high, and both losing >6% body weight and attending ≥10 classes were three times as high in the intervention subjects compared with controls, although non‐significant. Discussion: The potential of earning a performance‐based reimbursement incentive did not affect enrollment in the EatRight Lifestyle Program. Performance‐based incentives may be an ideal mechanism for extending coverage of weight‐loss interventions by insurers because of limited financial risk and improved adherence.     

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.     

Manufacturing flexibility and performance: bridging the gap between theory and practice

How firms scan and interpret their environments has implications for the flexibility strategy that they choose, as well as for the performance of that strategy. We extend Daft and Weick’s (Acad Manage Rev 9(2):284–295, 1984) model of firms as interpretation systems into a theoretical model of flexibility performance through extended iterations between observations of a failed flexibility initiative and relevant literature. We test the model using well-known teaching cases. We argue that the use of an iterative process that involves cases and theory both stimulates creativity in integrating theory and lays an initial foundation for evidence-based practice.     

Cloning,Expression, Purification,and Characterization of Biologically Active Recombinant Hemagglutinin-33, Type A Botulinum Neurotoxin Associated Protein

Botulinum neurotoxin type A, the most toxic substance known to mankind, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycistronic expression of a clustered group of genes. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs, which is resistant to protease digestion, a feature likely to be involved in the protection of the botulinum neurotoxin from proteolysis. In order to fully understand the function of Hn-33, large amounts of Hn-33 will be needed without dealing with biosafety risks to grow large cultures of C. botulinum. There are difficulties to clone the genes with the high A + T contents produced by C. botulinum. We report here for the first time using the Gateway technology to clone functional Hn-33 that has been expressed in E. coli. The yield of the recombinant Hn-33 was about 12 mg per liter of E. coli culture. The recombinant Hn-33 folds well in aqueous solution as shown with circular dichroism spectra, resists temperature-denaturation, is totally resistant to trypsin proteolysis despite the presence of cleavage sites on the molecular surface, and maintains its biological activities comparable to the native Hn-33 hemagglutination.     

Identification and validation of colorectal neoplasia-specific methylation markers for accurate classification of disease

Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis.     

Two optimized combination assays to examine apoptosis pathways in clinical samples.

BACKGROUND: A consequence of a number of diseases is an alteration in apoptosis. Currently, there is no single assay that measures the main stages of apoptosis, requiring that multiple assays be performed. This hinders studies on clinical samples that have limited cell numbers. Our objective was to combine and optimize assays that target specific stages of apoptosis for use in a typical clinical blood sample. METHODS: Two flow cytometric assays were developed for use on peripheral blood mononuclear cells (PBMC) collected in two 8-ml tubes from a single draw. One measures caspase-12 activity, the level of active caspase-3 and DNA fragmentation. The second assesses depolarization of the mitochondria and phosphatidylserine externalization. Cell populations present within the samples were determined by flow cytometry. Apoptosis was validated by ELISA. RESULTS: Each assay was optimized for use with cell numbers and sample volumes typical of clinical blood samples. Each combination assay effectively distinguished apoptotic from nonapoptotic blood cells. CONCLUSIONS: This combined optimized method comprised of two independent assays makes it possible to assay the major pathways of apoptosis in addition to determining the blood cell subsets that are affected.     

Genetics and selective breeding of variation in wing truncation in a flightless aphid control agent

Augmentative biological control by predaceous ladybird beetles can be improved by using flightless morphs, which have longer residence times on the host plants. The two‐spot ladybird beetle, Adalia bipunctata (L.) (Coleoptera: Coccinellidae), is used for the biological control of aphids in greenhouses and on urban trees. Flightlessness due to truncated wings occurs at very low frequency in some natural populations of A. bipunctata. Pure‐breeding strains of this 'wingless' genotype of A. bipunctata can easily be obtained in the laboratory. Such strains have not been commercialized yet due to concerns about their reduced fitness compared to wild‐type strains, which renders mass production more expensive. Wingless strains exhibit, however, wide intra‐population phenotypic variation in the extent of wing truncation which is related to fitness traits. We here use classical quantitative genetic techniques to study the heritability and genetic architecture of variation in wing truncation in a wingless strain of A. bipunctata. Split‐families reared at one of two temperatures revealed strong family‐by‐temperature interaction: heritability was estimated as 0.64 ± 0.09 at 19 °C and 0.29 ± 0.06 at 29 °C. Artificial selection in opposite directions at 21 °C demonstrated that the degree of wing truncation can be altered within a few generations resulting in wingless phenotypes without any wing tissue (realized h² = 0.72), as well as those with minimal truncations (realized h² = 0.61) in two replicates. The latter lines produced more than twice as many individuals. This indicates that selective breeding of wing truncation may be exploited to improve mass rearing of flightless strains of A. bipunctata for commercial biological control. Our work illustrates that cryptic variation can also be a source for the selective breeding of natural enemies.