A new genetic disorder in mitochondrial fatty acid beta-oxidation: ACAD9 deficiency

The acyl-CoA dehydrogenases are a family of multimeric flavoenzymes that catalyze the alpha,beta -dehydrogenation of acyl-CoA esters in fatty acid beta -oxidation and amino acid catabolism. Genetic defects have been identified in most of the acyl-CoA dehydrogenases in humans. Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified acyl-CoA dehydrogenase that demonstrates maximum activity with unsaturated long-chain acyl-CoAs. We now report three cases of ACAD9 deficiency. Patient 1 was a 14-year-old, previously healthy boy who died of a Reye-like episode and cerebellar stroke triggered by a mild viral illness and ingestion of aspirin. Patient 2 was a 10-year-old girl who first presented at age 4 mo with recurrent episodes of acute liver dysfunction and hypoglycemia, with otherwise minor illnesses. Patient 3 was a 4.5-year-old girl who died of cardiomyopathy and whose sibling also died of cardiomyopathy at age 21 mo. Mild chronic neurologic dysfunction was reported in all three patients. Defects in ACAD9 mRNA were identified in the first two patients, and all patients manifested marked defects in ACAD9 protein. Despite a significant overlap of substrate specificity, it appears that ACAD9 and very-long-chain acyl-CoA dehydrogenase are unable to compensate for each other in patients with either deficiency. Studies of the tissue distribution and gene regulation of ACAD9 and very-long-chain acyl-CoA dehydrogenase identify the presence of two independently regulated functional pathways for long-chain fat metabolism, indicating that these two enzymes are likely to be involved in different physiological functions.     

A collagen-based interface construct for the assessment of cell-dependent mechanical integration of tissue surfaces

The interface between any newly engineered tissue and pre-existing tissue is of great importance to tissue engineering; however, this process has so far been largely ignored, with few reports regarding the mechanical strength of newly integrated connective tissues surfaces. A new model system has been developed to generate a well-defined interface between two collagen lattices: one pre-contracted by resident fibroblasts and the other a cell-free wrapping gel. This construct can be cultured for prolonged periods (>2 weeks) and can also be fitted onto a mechanical testing system to measure the interface adhesive strength at the end of the culture time. Interface adhesive strength shows a six-fold increase after 1 week in culture, compared with the time-zero baseline. Observations of cell migration across the interface suggest that cell translocation in the three-dimensional matrix might play an important role in the integration process. In this new controlled geometry, normal and shear stresses at the interface can be analysed by finite element modelling and the areas at which debonding starts can be defined. The current experimental design permits solid multiple (homogeneous or heterogeneous) interface formation in vitro with a well-defined geometry and the possibility of measuring mechanical linkage. This design should enable many other factors affecting cell-driven interface strengthening to be investigated. This study was supported by the Fifth Framework Programme of the European Commission, “Biomechanical Interactions in Tissue Engineering and Surgical Repair (BITES)”.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip® array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.     

Utility of multiple chemical techniques in archaeological residential mobility studies: case studies from Tiwanaku- and Chiribaya-affiliated sites in the Andes

In the south central Andes, archaeologists have long debated the extent of Tiwanaku colonization during the Middle Horizon (AD 500-1000). We tested the hypotheses regarding the nature of Tiwanaku influence using strontium isotope, trace element concentration, and oxygen isotope data from archaeological human tooth enamel and bone from Tiwanaku- and Chiribaya-affiliated sites in the south central Andes. Strontium isotope analysis of 25 individuals buried at the Tiwanaku-affiliated Moquegua Valley site of Chen Chen demonstrates that it was likely a Tiwanaku colony. In contrast, no immigrants from the Lake Titicaca Basin were present in 27 individuals analyzed from the San Pedro de Atacama cemeteries of Coyo Oriental, Coyo-3, and Solcor-3; it is likely that these sites represent economic and religious alliances, but not colonies. However, strontium isotope analysis alone cannot distinguish movement between the Tiwanaku- and Chiribaya-affiliated sites in the Moquegua and Ilo Valleys of southern Peru. Analyzing oxygen isotope and trace element concentration data and comparing it with strontium isotope data from the same individuals provides a more detailed picture of residential mobility in the Tiwanaku and Chiribaya polities. In addition to monitoring diagenetic contamination, trace element concentration data identified movement during adulthood for certain individuals. However, these data could not distinguish movement between the Moquegua and Ilo Valleys. While oxygen isotope data could clearly distinguish the high-altitude sites from others, more data is needed to characterize the local oxygen isotope ratios of these regions. These data demonstrate the potential for archaeological reconstruction of residential mobility through multiple lines of evidence.     

Oxidized cholesterol metabolites found in human atherosclerotic lesions promote apolipoprotein C-II amyloid fibril formation

Apolipoprotein amyloid deposits and lipid oxidation products are colocalized in human atherosclerotic tissue. In this study we show that the primary ozonolysis product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (KA), rapidly promotes human apolipoprotein (apo) C-II amyloid fibril formation in vitro. Previous studies show that hydrophobic aldehydes, including KA, modify proteins by the formation of a Schiff base with the lysine epsilon-amino group or N-terminal amino group. High-performance liquid chromatography, mass spectrometry, and proteolysis of KA-modified apoC-II revealed that KA randomly modified six different lysine residues, with primarily one KA attached per apoC-II molecule. Competition experiments showed that an aldehyde scavenging compound partially inhibited the ability of KA to hasten apoC-II fibril formation. Conversely, the acid derivative of KA, lacking the ability to form a Schiff base, accelerated apoC-II fibril formation, albeit to a lesser extent, suggesting that amyloidogenesis triggered by KA involves both covalent and noncovalent mechanisms. The viability of a noncovalent mechanism mediated by KA has been observed previously with alpha-synuclein aggregation, implicated in Parkinson's disease. Electron microscopy demonstrated that fibrils formed in the presence of KA had a similar morphology to native fibrils; however, the isolated KA-apoC-II covalent adducts in the absence of unmodified apoC-II formed fibrillar structures with altered ropelike morphologies. KA-mediated fibril formation by apoC-II was inhibited by the addition of the amine-containing compound hydralazine and the lipid-binding protein apoA-I. These in vitro studies suggest that the oxidized small molecule pool could trigger or hasten the aggregation of apoC-II to form amyloid deposits.     

The oxidative stress metabolite 4-hydroxynonenal promotes Alzheimer protofibril formation

4-Hydroxynonenal (4-HNE), formed as a consequence of oxidative stress, exists at increased concentrations in Alzheimer's disease (AD) patients and is found in amyloid beta peptide (Abeta) plaques associated with AD. Although it remains an open question as to whether oxidative stress is a causative factor or a consequence of AD, we show here that 4-HNE, putatively resulting from the peroxidation of lipids, covalently modifies Abeta, triggering its aggregation. These Abeta modifications result from 1,4 conjugate addition and/or Schiff base formation, they occur at multiple locations on a single Abeta peptide, and they result in covalent cross-linking of Abeta peptides. The consequence of these reactions is that 4-HNE accelerates the formation of Abeta protofibrils while inhibiting the production of straight, mature fibrils. Recent studies implicating Abeta oligomers and protofibrils in the neurotoxic process that ultimately leads to AD suggest that the Abeta aggregates induced by 4-HNE may be important in the pathogenesis of AD. These results provide further incentive to understand the role of oxidative stress and small-molecule Abeta modifications in sporadic AD.     

An association of early puberty with disordered eating and anxiety in a population of undergraduate women and men

Eating and anxiety disorders are more prevalent in females, increase during adolescence, and are associated with early pubertal development. This study examined whether timing of puberty onset is associated with disordered eating and anxiety in a large sample of postpubertal male and female undergraduate students. Self-report questionnaires assessed timing of puberty, disordered eating, anxiety, alcohol use, personality, and sensation seeking. Females scored significantly higher on measures of disordered eating (binge eating, dietary restraint, eating concerns, and weight and shape concerns) and anxiety (state and trait anxiety) than did males. In addition, early maturing women and men scored significantly higher on measures of disordered eating and anxiety than on time or late maturing women and men. Measures of alcohol use, sensation seeking, and personality characteristics differed in males and females but did not vary with pubertal timing. Findings suggest that early puberty is associated with disordered eating and anxiety, and this association may be due to an organizational effect of pubertal hormones. Despite important differences in body fat composition, both males and females experiencing early puberty had an increased incidence of disordered eating. The fact that early puberty was associated with increased eating and anxiety symptoms in both sexes suggests that puberty may influence these symptoms through both biological and psychosocial mechanisms.     

Evaluation of the Trophic Diatom Index for assessing water quality in River Gharasou,western Iran

Water quality, diatom species composition and biomass estimates were performed in the Gharasou River in western Iran. Five sites along the River Gharasou were sampled every month from April to September 2005. Physical and chemical factors (pH, NO₃–N, PO₄–P, dissolved oxygen, total suspend solids, total dissolved solids, conductivity, turbidity, chemical oxygen demand and biological oxygen demand) were measured along with biological properties of the periphyton including biomass, ash-free dry mass, chlorophyll-a concentration and the taxonomic composition diatom assemblages. Information from the diatom assemblage was used to calculate the Trophic Diatom Index and biovolume. The TDI was significantly correlated with measures of human disturbance at the sites (e.g. PO₄–P, NO₃–N and dissolved oxygen) as well as to biomass measures (chlorophyll a, ash-free dry mass and biovolume). The sensitivity of the TDI and its component metrics to environmental stressors supports the use of this index for monitoring ecological conditions in streams in Iran and to aid diagnosis of the cause of their impairment. Handling editor: L. Naselli-Flores     

IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus

Ticks are ectoparasitic blood-feeders and important vectors for pathogens including arboviruses, rickettsiae, spirochetes and protozoa. As obligate blood-feeders, one possible strategy to retard disease transmission is disruption of the parasite's ability to digest host proteins. However, the constituent peptidases in the parasite gut and their potential interplay in the digestion of the blood meal are poorly understood. We have characterised a novel asparaginyl endopeptidase (legumain) from the hard tick Ixodes ricinus (termed IrAE), which we believe is the first such characterisation of a clan CD family C13 cysteine peptidase (protease) in arthropods. By RT-PCR of different tissues, IrAE mRNA was only expressed in the tick gut. Indirect immunofluorescence and EM localised IrAE in the digestive vesicles of gut cells and within the peritrophic matrix. IrAE was functionally expressed in Pichia pastoris and reacted with a specific peptidyl fluorogenic substrate, and acyloxymethyl ketone and aza-asparagine Michael acceptor inhibitors. IrAE activity was unstable at pH > or = 6.0 and was shown to have a strict specificity for asparagine at P1 using a positional scanning synthetic combinatorial library. The enzyme hydrolyzed protein substrates with a pH optimum of 4.5, consistent with the pH of gut cell digestive vesicles. Thus, IrAE cleaved the major protein of the blood meal, hemoglobin, to a predominant peptide of 4kDa. Also, IrAE trans-processed and activated the zymogen form of Schistosoma mansoni cathepsin B1 -- an enzyme contributing to hemoglobin digestion in the gut of that bloodfluke. The possible functions of IrAE in the gut digestive processes of I. ricinus are compared with those suggested for other hematophagous parasites.