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1.

Aim

To attack a widespread myth.

Location

World‐wide.

Methods

Simple mathematical logical and empirical examples.

Results

As both species and area are finite and non‐negative, the species–area relationship is limited at both ends. The log species–log area relationship is normally effectively linear on scales from about 1 ha to 107 km2. There are no asymptotes. At the intercontinental scale it may get steeper; at small scales it may in different cases get steeper or shallower or maintain its slope.

Main conclusion

The species–area relationship does not have an asymptote.
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2.

Aim

Large ‐ scale diversity patterns are generated by different but not mutually exclusive mechanisms. However, understanding of multiple facets of diversity and their determinants in the freshwater realm remains limited. Here, we characterized the geographical gradients, hotspots and spatial congruence of three facets of freshwater molluscan diversity and evaluated the relative importance of three different underlying mechanisms related to the energy, area/environmental heterogeneity and dispersal/historical hypotheses.

Location

China.

Methods

Species richness (SR), functional richness (FR) and taxonomic distinctness (TD, a proxy of phylogenetic diversity) were calculated for 212 drainage basins with a total of 313 molluscan species. Spatial congruence between the diversity facets was evaluated with Pearson correlation coefficient and overlap among hotspots. Multiple linear regression models and variation partitioning were used to assess the relative importance of different mechanisms.

Results

Hotspots of SR and FR were mainly concentrated in the Yangtze River and Huai River basins, while high TD values were patchily distributed across China. We found extremely low spatial congruence between TD and both SR and FR, while there was relatively high concordance between SR and FR. All diversity facets were best explained by the dispersal/historical hypothesis with strong unique effects, followed by the factors related to the energy hypothesis. The area/ environmental heterogeneity hypothesis was only weakly supported.

Main conclusions

We found a potentially strong influence of dispersal limitation and evolutionary history on the geographical diversity gradients of Chinese molluscs. This finding contrasts with the general finding that energy‐related factors are the strongest correlates of diversity patterns at large spatial scales. Moreover, our results do not support the idea that using any one diversity component as a surrogate of the others in developing conservation strategies. Instead, an integrative approach embracing multiple facets of diversity should be adopted in the conservation of freshwater biodiversity.
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3.

Aim

The primary objective of our study was to examine the factors affecting the distribution of vascular plants, springtails, butterflies and birds on small tropical islands to understand how different groups of organisms with distinct biological traits respond to biogeographical variables, such as island area.

Location

The Republic of Singapore (103°50′E, 1°20′N) located at the southern tip of Peninsular Malaysia.

Methods

Seventeen islands were surveyed for vascular plants, springtails, butterflies and birds. Correlation analysis, simple linear and multiple regression analyses and the nestedness index were used to test the hypotheses that (1) area is the best predictor of species/genus richness at both the community and specific/generic levels; (2) there is no correlation between population density and island area; and (3) species/genera are distributed as nested subsets.

Results

Area was the most significant factor in determining the island distribution of springtails, butterflies and birds at both the community and specific/generic levels, although there were disparate responses to the biogeographical variables between the three taxonomic groups, as well as between common species within each group. Individual species displayed disparate responses to biogeographical variables, suggesting that patterns of distribution at the community level may not be a good indicator of the population dynamics of individual species/genera. Plant species richness did not show any correlation with any of the tested variables. Population densities of springtails, butterflies and birds were positively correlated with area, contradicting the assumption of the equilibrium theory of island biogeography that population density of island species is independent of area. Population densities of plants showed no correlation with any of the tested biogeographical variables. Vascular plant, springtail, butterfly and bird communities on the islands showed significant patterns of nestedness, indicating there may be species/genus‐specific responses to biogeographical variables.

Main conclusions

We conclude that although area was the most important factor affecting the island distribution of springtails, butterflies and birds, conservation planning must take into consideration how target taxonomic groups respond to biogeographical variables, instead of relying on general principles (e.g. those derived from the equilibrium theory). On a local scale, in order to preserve the island biodiversity of Singapore, the highest priority should be given to preserving the larger islands (e.g. Pulau Ubin) which not only have higher numbers of species, but also species that are absent on smaller islands.
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4.

Background

The recently developed heterologous macrolide‐ (E.REX system) and streptogramin‐ (PIP system) responsive gene regulation systems show significant differences in their regulation performance in diverse cell lines.

Methods

In order to provide optimal regulation modalities for a wide variety of mammalian cell lines, we have performed a detailed analysis of E.REX and PIP systems modified in (i) the transactivation domains of the antibiotic‐dependent transactivators, (ii) the type of minimal promoter used, and (iii) the spacing between the operator module and the minimal promoter.

Results

These novel E.REX and PIP regulation components showed not only dramatically improved regulation performance in some cell types, but also enabled their use in cell lines which had previously been inaccessible to regulated transgene expression.

Conclusions

Due to their modular set‐up the novel E.REX and PIP regulation systems presented here are most versatile and ready for future upgrades using different cell‐specific key regulation components. Copyright © 2002 John Wiley & Sons, Ltd.
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5.

Aim

To demonstrate a new and more general model of the species–area relationship that builds on traditional models, but includes the provision that richness may vary independently of island area on relatively small islands (the small island effect).

Location

We analysed species–area patterns for a broad diversity of insular biotas from aquatic and terrestrial archipelagoes.

Methods

We used breakpoint or piecewise regression methods by adding an additional term (the breakpoint transformation) to traditional species–area models. The resultant, more general, species–area model has three readily interpretable, biologically relevant parameters: (1) the upper limit of the small island effect (SIE), (2) an estimate of richness for relatively small islands and (3) the slope of the species–area relationship (in semi‐log or log–log space) for relatively large islands.

Results

The SIE, albeit of varying magnitude depending on the biotas in question, appeared to be a relatively common feature of the data sets we studied. The upper limit of the SIE tended to be highest for species groups with relatively high resource requirements and low dispersal abilities, and for biotas of more isolated archipelagoes.

Main conclusions

The breakpoint species–area model can be used to test for the significance, and to explore patterns of variation in small island effects, and to estimate slopes of the species–area (semi‐log or log–log) relationship after adjusting for SIE. Moreover, the breakpoint species–area model can be expanded to investigate three fundamentally different realms of the species–area relationship: (1) small islands where species richness varies independent of area, but with idiosyncratic differences among islands and with catastrophic events such as hurricanes, (2) islands beyond the upper limit of SIE where richness varies in a more deterministic and predictable manner with island area and associated, ecological factors and (3) islands large enough to provide the internal geographical isolation (large rivers, mountains and other barriers within islands) necessary for in situ speciation.
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6.

Background

Twenty years ago this year was the first publication describing a region of neural crest cells necessary for normal cardiovascular development. Ablation of this region in chick resulted in persistent truncus arteriosus, mispatterning of the great vessels, outflow malalignments, and hypoplasia or aplasia of the pharyngeal glands.

Methods

We begin with a historical perspective and then review the progress that has been made in the ensuing 20 years in determining the direct and indirect contributions of the neural crest cells, now termed cardiac neural crest cells, in cardiovascular and pharyngeal arch development. Many of the molecular pathways that are now known to influence the specification, migration, patterning and final targeting of the cardiac neural crest cells are also reviewed.

Results

Although much knowledge has been gained by using many genetic manipulations to understand the cardiac neural crest cells' role in cardiovascular development, most models fail to explain the phenotypes seen in syndromic and non‐syndromic human congenital heart defects, such as the DiGeorge syndrome.

Conclusions

We propose that the cardiac neural crest exists as part of a larger cardiocraniofacial morphogenetic field and describe several human syndromes that result from abnormal development of this field. Birth Defects Research (Part C) 69:2–13, 2003. © 2003 Wiley‐Liss, Inc.
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7.

Background

Materno‐fetal transfer of intravenously administered liposome‐plasmid DNA complexes has been demonstrated only in mice. Studies on its materno‐fetal transfer in the pregnant monkey model is needed because of critical differences in placental structure between primates including humans and rodents.

Methods

The reporter plasmid pEGFP‐C1 was formulated in cationic lipid containing polybrene and vesicular stomatitis virus G protein. The fusogenic liposome‐plasmid DNA complexes were intradermally injected into pregnant common marmosets (N=2), a New World monkey, near term. DNA extracted from fetal tissues was subjected to PCR for detection of the egfp gene. Confocal microscopy and immunostaining were performed to determine the sites of transgene expression in the fetal organs.

Results

The egfp gene was detected in fetal blood and major organs (heart, liver, lung). The encoded protein was mainly produced in the endothelial cells of blood vessels in the fetal lungs.

Conclusions

This is the first report on materno‐fetal transfer of intradermally administered fusogenic liposome‐plasmid DNA complexes and fetal expression of a transgene in primates. Copyright © 2002 John Wiley & Sons, Ltd.
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8.

Objective:

Estrogen‐related receptors (ERRs) are important regulators of energy metabolism. Here we investigated the hypothesis that ERRγ impacts on differentiation and function of brown adipocytes.

Design and Methods:

We characterize the expression of ERRγ in adipose tissues and cell models and investigate the effects of modulating ERR? activity on UCP1 gene expression and metabolic features of brown and white adipocytes.

Results:

ERRγ was preferentially expressed in brown compared to white fat depots, and ERRγ was induced during cold‐induced browning of subcutaneous white adipose tissue and brown adipogenesis. Overexpression of ERRγ positively regulated uncoupling protein 1 (UCP1) expression levels during brown adipogenesis. This ERRγ‐induced augmentation of UCP1 expression was independent of the presence of peroxisome proliferator‐activated receptor coactivator‐1 (PGC‐1α) but was associated with increased rates of fatty acid oxidation in adrenergically stimulated cells. ERR? did not influence mitochondrial biogenesis, and its reduced expression in white adipocytes could not explain their low expression level of UCP1.

Conclusions:

Through its augmenting effect on expression of UCP1, ERRγ may physiologically be involved in increasing the potential for energy expenditure in brown adipocytes, a function that is becoming of therapeutic interest.
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9.

Background

Aberration in the pattern of DNA methylation is one of the hallmarks of cancer. We present data suggesting that dysregulation of MBD2, a recently characterized member of a novel family of methylated DNA binding proteins, is involved in tumorigenesis. Two functions were ascribed to MBD2, DNA demethylase activity and repression of methylated genes.

Methods

Multiple antisense expression and delivery systems, transfection, electrotransfer and adenoviral were employed to demonstrate that MBD2 is essential in tumorigenesis, both ex vivo and in vivo.

Results

Inhibition of MBD2 by antisense expression resulted in inhibition of anchorage‐independent growth of antisense transfected cancer cells or cells infected with an adenoviral vector expressing MBD2 antisense. Xenograft tumors treated with an adenoviral vector expressing MBD2 antisense or xenografts treated with electrotransferred plasmids expressing MBD2 antisense showed reduced growth.

Conclusions

These results support the hypothesis that one or both of the functions described for MBD2 are critical in tumorigenesis and that MBD2 is a potential anticancer target. Copyright © 2002 John Wiley & Sons, Ltd.
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10.
11.

Background

Gene therapy has been used to treat a variety of health problems, but transfection inefficiency and the lack of safe vectors have limited clinical progress. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapy. The present study aimed to synthesize chitosan‐alginate nanoparticles that can be used as carriers of the pAcGFP1‐C1 plasmid and to use these nanoparticles with an ultrasound protocol to achieve high efficiency gene transfection.

Methods

Chitosan was complexed with alginate and the pAcGFP1‐C1 plasmid at different charge ratios to create chitosan‐alginate‐DNA nanoparticles (CADNs). The average particle size and loading efficiency were measured. Plasmid DNA retardation and integrity were analysed on 1% agarose gels. The effect of CADNs and ultrasound on the efficiency of transfection of cells and subcutaneous tumors was evaluated.

Results

In the CADNs, the average size of incorporated plasmid DNA was 600–650 nm and the loading efficiency was greater than 90%. On the basis of the results of the plasmid DNA protection test, CADNs could protect the transgene from DNase I degradation. The transgene product expression could be enhanced efficiently if cells or tumor tissues were first given CADNs and then treated with ultrasound.

Conclusions

The use of CADNs combined with an ultrasound regimen is a promising method for safe and effective gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.
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12.

Background

The pig lung, given its gross anatomical, histological and physiological similarities to the human lung, may be useful as a large animal model, in addition to rodents, in which to assess the potential of vectors for pulmonary airway gene transfer. The aim of this study was to assess the utility of the pig lung as a model of gene transfer to the human lung with a synthetic vector system.

Methods

The LID vector system consists of a complex of lipofectin (L), integrin‐binding peptide (I) and plasmid DNA (D). LID complexes containing a β‐galactosidase reporter gene under a CMV promoter or a control plasmid at1 mg/3 ml PBS, or 3 ml buffer, was administered to the right lower lobe ofthe pig lung through a bronchoscope. Pigs were culled at 48 h and lung sections prepared for immunohistochemical and histological analysis. Bronchoalveolar lavage fluid was collected and analysed for TNF‐α by ELISA.

Results

Immunohistochemical staining for the β‐galactosidase reporter gene indicated high efficiency of gene transfer by the LID vector to pig bronchial epithelium with 46% of large bronchi staining positively. There was no evidence for vector‐specific inflammation assessed by leukocytosis and cytokine production.

Conclusions

This study demonstrates the use of the pig for studies of gene transfer in the lung and confirms in a second species the potential of the LID vector for gene therapy of pulmonary diseases such as cystic fibrosis. Copyright © 2002 John Wiley & Sons, Ltd.
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13.

Background

Somatic gene therapy requires safe and efficient techniques for the gene transfer procedure. The ovine mammary gland is described as a model system for the evaluation of somatic gene transfer methods.

Methods

Different gene delivery formulations were retrogradely injected into the mammary gland of lactating sheep. The efficiency of the gene transfer was subsequently measured by the detection of the secreted transgene products in the milk. To counteract the milk flow in the lactating gland caused by the permanent milk production, a newly developed pretreatment of the mammary gland with hyperosmotic solutions was applied. In addition, in vivo electroporation of DNA into the mammary gland is described.

Results

Gene transfer using naked DNA or simple complexes of DNA with polycations did not result in traceable amounts of reporter gene products. However, utilizing the complex cationic lipid DOSPER, a peak expression of about 400 ng/ml was observed 6 days after transfection. Maximum expression rates of more than 1 µg/ml were obtained by combining hyperosmotic pretreatment and receptor‐mediated gene transfer. For the in vivo electroporation, the proof of principle for this technique in the mammary gland is reported.

Conclusions

The ovine mammary gland turned out to be a very well suited as a model system for evaluation and optimization of various gene transfer protocols. Copyright © 2002 John Wiley & Sons, Ltd.
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14.

Objectives:

Nonalcoholic fatty liver disease (NAFLD) is increasingly an indication for liver transplantation in adults. While severe obesity (SO, BMI ≥40 kg m?2) in adults is long standing, it is recent in duration in adolescents. With adolescent obesity on the rise, NAFLD is becoming the most frequent liver disease in adolescents. The hypothesis that SO adolescents and adults have different severity of NAFLD because of longer duration of obesity in SO adults was tested.

Design and Methods:

Preoperative clinical data, NAFLD activity and NASH (Nonalcoholic steatohepatitis) scores from intraoperative liver biopsies were extracted from a prospective database of consecutively operated SO adolescents and adults (n = 24 each). Fasting preoperative serum inflammatory mediators were evaluated by ELISA.

Results:

Other than age, baseline BMI, ethnicity and gender distribution, the incidence and extent of dyslipidemia, hypertension, and metabolic syndrome were comparable between groups. Histologic scores for steatosis and inflammation were similar. Adolescents have significantly higher NASH incidence, hepatocyte injury scores and fibrosis. This was associated with higher serum C‐reactive protein and sCD14 levels.

Conclusion:

For comparable BMI and metabolic profile, SO adolescents have more advanced liver damage, more severe systemic inflammation, suggesting differences in NAFLD etiologies and more aggressive disease progression in the young obese population.
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15.

Background

Gene correction is an alternative approach to replacement gene therapy. By correcting mutations within the genome, some of the barriers to effective gene therapy are avoided. Homologous nucleic acid sequences can correct mutations by inducing recombination or mismatch repair. Recently, encouraging data have been presented using both short DNAfragments (SDFs) and RNA–DNA oligonucleotides (RDOs) in experimental strategies to realize clinical gene correction.

Methods

The delivery of labelled SDFs and RDOs to a variety of cell lines was tested using both FACS analysis and confocal microscopy. A GFP‐based reporter system was constructed, containing a nonsense mutation, to allow quantitation of gene correction in living cells. This reporter was used to compare efficiencies of functional gene correction using SDFs and RDOs in arange of mammalian cell lines.

Results

The delivery experiments highlight the inefficient delivery of SDFs and RDOs to the nucleus using polyethylenimine (PEI) transfection. This study compared the episomal correction efficiency of the reporter plasmid mediated by SDFs and RDOs within different cell types; low levels of functional correction were detected in cell culture.

Conclusions

Whilst delivery of PEI‐complexed SDFs or RDOs to the cell is highly effective, nuclear entry appears to be a limiting factor. SDFs elicited episomal GFP correction across a range of cell lines, whereas RDOs only corrected the reporter in a cell line that overexpresses RAD51. Copyright © 2002 John Wiley & Sons, Ltd.
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16.

Background

Mucopolysaccharidosis VI (MPS VI), due to recessively inherited 4‐sulfatase (4S) deficiency, results in lysosomal storage of dermatan sulfate in numerous tissues. Retinal involvement is limited to the retinal pigment epithelium (RPE). This study aimed to determine whether recombinant adeno‐associated virus (AAV)‐mediated delivery of 4S would reverse the RPE pathology seen in MPS VI cats.

Methods

AAV.f4S, containing the feline 4S cDNA, was delivered unilaterally to eyes of affected cats by subretinal or intravitreal injection. Contralateral eyes received AAV with the green fluorescent protein (GFP) reporter gene as control. At 2–11 months post‐injection, the cats were sacrificed and the treatment effects were evaluated histologically.

Results

By ophthalmoscopy and histological analyses, GFP was evident as early as 4 weeks and persisted through the latest time point (11 months). Untreated and AAV.GFP‐treated diseased retinas contained massively hypertrophied RPE cells secondary to accumulation of dilated lysosomal inclusions containing dermatan sulfate. MPS VI eyes treated subretinally with AAV.f4S had minimal RPE cell inclusions and, consequently, were not hypertrophied.

Conclusions

AAV‐mediated subretinal delivery of f4S provided correction of the disease phenotype in RPE cells of feline MPS VI, supporting the utility of AAV as a vector for the treatment of RPE‐specific as well as lysosomal storage diseases. Copyright © 2002 John Wiley & Sons, Ltd.
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17.

Objective:

Energy density (ED) and eating rate (ER) influence energy intake; their combined effects on intake and on postprandial pancreatic and gut hormone responses are undetermined. To determine the combined effects of ED and ER manipulation on voluntary food intake, subjective appetite, and postprandial pancreatic and gut hormone responses.

Design and Methods:

Twenty nonobese volunteers each consumed high (1.6 kcal g?1; HED) and low (1.2 kcal g?1; LED) ED breakfasts slowly (20 g min?1; SR) and quickly (80 g min?1; FR) ad libitum to satiation. Appetite, and pancreatic and gut hormone concentrations were measured periodically over 3 h. Ad libitum energy intake during the subsequent lunch was then measured.

Results:

Main effects of ED and ER on energy intake and a main effect of ER, but not ED, on mass of food consumed were observed, FR and HED being associated with increased intake (P < 0.05). Across all conditions, energy intake was highest during FR‐HED (P ≤ 0.01). Area under the curve (AUC) of appetite ratings was not different between meals. Main effects of ED and ER on insulin, peptide‐YY, and glucagon‐like peptide‐1 AUC (P < 0.05) were observed, FR and HED being associated with larger AUC. No effects on active or total ghrelin AUC were documented. Total energy intake over both meals was highest during the FR‐HED trial with the greatest difference between FR‐HED and SR‐LED trials (P ≤ 0.01).

Conclusion:

Consuming an energy dense meal quickly compounds independent effects of ER and ED on energy intake. Energy compensation at the following meal may not occur despite altered gut hormone responses.
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18.

Background

The low efficiency and toxicity of transfection in a primary culture of hepatocytes using cationic lipids remains a limiting step to the study of gene function and the setting up of non‐viral gene therapy.

Methods

A novel class of cationic lipids (GBs) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolysable peptide and ester bonds, a structure designed to reduce cytotoxicity, was used to improve transfection efficiency in a primary culture of rat hepatocytes. The relationship between lipid structure, lipoplex formulation and transfection efficiency was studied using six GBs (12‐14‐16, 22‐24‐26) varying in their spacer and acyl chains.

Results

GB12, characterized by short [(CH2)10] acyl chains and spacer, allowed plasmid uptake in all cells and reporter gene expression in up to 40% of hepatocytes with a low cytotoxicity, a much higher efficiency compared with transfections using other reagents including Fugene6? and Lipofectin?. We also showed that numerous cells accumulated high amounts of plasmids demonstrating that GB12 promoted a very efficient DNA transfer through plasma membrane leading to an increase in nuclear plasmid translocation, allowing a much higher gene expression. Moreover, GB12‐transfected hepatocytes survived to injection in normal livers and were found to express the LacZ reporter gene.

Conclusions

The non‐toxic GB12 formulation is a powerful vehicle for plasmid delivery in cultured hepatocytes with relevance in liver gene therapy. Copyright © 2002 John Wiley & Sons, Ltd.
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19.

Objective:

This study was designed to determine how gastric bypass affects the sympathetically‐mediated component of resting energy expenditure (REE) and muscle sympathetic nerve activity (MSNA).

Design and Methods:

We measured REE before and after beta‐blockade in seventeen female subjects approximately three years post‐gastric bypass surgery and in nineteen female obese individuals for comparison. We also measured MSNA in a subset of these subjects.

Results:

The gastric bypass subjects had no change in REE after systemic beta‐blockade, reflecting a lack of sympathetic support of REE, in contrast to obese subjects where REE was reduced by beta‐blockade by approximately 5% (P < 0.05). The gastric bypass subjects, while still overweight (BMI = 29.3 vs 38.0 kg·m?2 for obese subjects, P < 0.05), also had significantly lower MSNA compared to obese subjects (10.9 ± 2.3 vs. 21.9 ± 4.1 bursts·min?1, P < 0.05). The reasons for low MSNA and a lack of sympathetically mediated support of REE after gastric bypass are likely multifactorial and may be related to changes in insulin sensitivity, body composition, and leptin, among other factors.

Conclusions:

These findings may have important consequences for the maintenance of weight loss after gastric bypass. Longitudinal studies are needed to further explore the changes in sympathetic support of REE and if changes in MSNA or tissue responsiveness are related to the sympathetic support of REE.
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20.

Objective:

To investigate whether a combination of a selected but limited number of anthropometric measurements predicts visceral adipose tissue (VAT) better than other anthropometric measurements, without resort to medical imaging.

Hypothesis:

Abdominal anthropometric measurements are total abdominal adipose tissue indicators and global measures of VAT and SAAT (subcutaneous abdominal adipose tissue). Therefore, subtracting the anthropometric measurement the more correlated possible with SAAT while being the least correlated possible with VAT, from the most correlated abdominal anthropometric measurement with VAT while being highly correlated with TAAT, may better predict VAT.

Design and Methods:

BMI participants' range was from 16.3 to 52.9 kg m?2. Anthropometric and abdominal adipose tissues data by computed tomography (CT‐Scan) were available in 253 patients (18‐78 years) (CHU Nord, Marseille) and used to develop the anthropometric VAT prediction models.

Results:

Subtraction of proximal thigh circumference from waist circumference, adjusted to age and/or BMI, predicts better VAT (Women: VAT = 2.15 × Waist C ? 3.63 × Proximal Thigh C + 1.46 × Age + 6.22 × BMI ? 92.713; R2 = 0.836. Men: VAT = 6 × Waist C ? 4.41 × proximal thigh C + 1.19 × Age ? 213.65; R2 = 0.803) than the best single anthropometric measurement or the association of two anthropometric measurements highly correlated with VAT. Both multivariate models showed no collinearity problem. Selected models demonstrate high sensitivity (97.7% in women, 100% in men). Similar predictive abilities were observed in the validation sample (Women: R2 = 76%; Men: R2 = 70%). Bland and Altman method showed no systematic estimation error of VAT.

Conclusion:

Validated in a large range of age and BMI, our results suggest the usefulness of the anthropometric selected models to predict VAT in Europides (South of France).
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