Classification of cryo-electron sub-tomograms using constrained correlation

Cryo-electron tomography (CET) is currently the only three-dimensional imaging technique capable of visualizing macromolecules in their cellular context at close-to-native conditions with a resolution in the nanometer range. An important component for the analysis of the data is their classification, which should discriminate among various macromolecules, conformational changes and interaction partners. Missing structure factors, typically in a wedge-shaped region in Fourier space if single-axis tilting is performed, hamper classification of cryo-electron tomographic data. Here, we describe a classification method for three-dimensional (3D) sub-tomograms extracted from cryo-electron tomograms, which takes the missing wedge into account and provides reliable results. The similarity of the individually aligned sub-tomograms is scored by constrained correlation. Subsequently, they are clustered based on their pairwise correlation values. In order to demonstrate the feasibility of this approach, we apply the proposed method to simulated tomographic data of the chaperone thermosome in different conformations. By comparison of the principal components of the resulting matrix we show that the proposed metric is significantly less prone to the orientation of the missing wedge compared to the unconstrained correlation. Moreover, we apply our classification method to an experimental dataset of GroEL with and without GroES, where we achieve a distinct discrimination between the putative GroEL and GroEL/GroES complexes.     

Shear acoustic wave biosensor for detecting DNA intrinsic viscosity and conformation: a study with QCM-D

Direct biosensors are devices operating by monitoring the amount of surface-bound analyte. In this work a new approach is presented where a label-free acoustic biosensor, based on a QCM-D device, and solution viscosity theory, are used to study DNA intrinsic viscosity. The latter is quantitatively related to the DNA conformation and specifically the molecule's shape and size, in a manner that is independent of the amount of bound DNA mass. It is shown that acoustic measurements can clearly distinguish between ds-DNA of same shape (straight rod) but various sizes (from 20 to 198bp (base pairs)) and same mass and size (90bp) but various shapes ("straight", "bent", "triangle"). These results are discussed in the broader context of "coil" and sphere-like molecules detected on surfaces. A mathematical formula is presented relating the length of straight, surface-protruding DNA to the acoustic ratio DeltaD/Deltaf. The development of real-time rapid techniques for the characterization of DNA intrinsic curvature as well as DNA conformational changes upon interaction with proteins is of significance to analytical biotechnology due to the large number of DNA sequences and potential DNA bending proteins involved in genome analysis and drug screening.     

Reference Growth Curves for Cypriot Children 6 to 17 Years of Age

Objective: The purpose of the study was to present smoothed percentiles for body weight and height, waist circumference, and body mass index (BMI) in Cypriot children and to compare their BMI 85th and 95th percentiles with those of children in other countries. Research Methods and Procedures: The study was a cross‐sectional study, including a representative sample of 2472 healthy children (49.1% boys) in Cyprus ages 6 to 17 years, who were evaluated during the 1999–2000 school year. Body weight and height and waist circumference were measured using standard procedures. BMI was calculated as weight in kilograms per height in square meters. Smoothed, sex‐specific percentiles for these variables were calculated using polynomial regression models. Crude weight, height, waist, and BMI percentile values are presented in sex‐specific tables and smoothed percentile curves are presented in charts. The 85th and 95th percentiles for BMI were compared with measurements from other countries, because of the concern of the upper limits of BMI in respect to the evaluation of obesity. Results: The 85th and 95th BMI percentile values are higher in Cypriot boys than in Swedish and Iranian boys through all ages and in girls ages 6 to 15 years, whereas after the age of 15 years, both Swedish and Iranian girls’ percentiles are equalized with their Cypriot peers. Discussion: Weight, height, waist circumference, and BMI values and charts are presented for the first time for Cypriot children and adolescents. Much concern should be addressed to the observation that for the majority of the Cypriot sample, the upper BMI limits are higher than the peers of developing and developed countries.     

Crystal structure of a thwarted mismatch glycosylase DNA repair complex

The bacterial mismatch-specific uracil-DNA glycosylase (MUG) and eukaryotic thymine-DNA glycosylase (TDG) enzymes form a homologous family of DNA glycosylases that initiate base-excision repair of G:U/T mismatches. Despite low sequence homology, the MUG/TDG enzymes are structurally related to the uracil-DNA glycosylase enzymes, but have a very different mechanism for substrate recognition. We have now determined the crystal structure of the Escherichia coli MUG enzyme complexed with an oligonucleotide containing a non-hydrolysable deoxyuridine analogue mismatched with guanine, providing the first structure of an intact substrate-nucleotide productively bound to a hydrolytic DNA glycosylase. The structure of this complex explains the preference for G:U over G:T mispairs, and reveals an essentially non-specific pyrimidine-binding pocket that allows MUG/TDG enzymes to excise the alkylated base, 3, N(4)-ethenocytosine. Together with structures for the free enzyme and for an abasic-DNA product complex, the MUG-substrate analogue complex reveals the conformational changes accompanying the catalytic cycle of substrate binding, base excision and product release.     

Measuring outcomes in aesthetic surgery: a comprehensive review of the literature

Outcomes research examines the end results of medical interventions, taking into account patients' experiences, preferences, and values. The purpose of assessing outcomes is to provide evidence on which to base clinical decisions. The assessment of outcomes in aesthetic surgery is especially pertinent because patient satisfaction is the predominant factor in determining success. In cosmetic surgery, various scales have been used to assess outcomes. Unfortunately, none of these methods has achieved widespread use. The adoption of broadly accepted, relevant scales to measure outcomes would be advantageous, because this would allow the comparison of techniques, quantification of positive effects, and identification of patients unlikely to benefit from surgery. The purpose of this study was to critically review the present literature to identify the appropriate instruments to assess outcomes in aesthetic surgery. After a comprehensive review of aesthetic surgery outcome instruments, the authors identified body-image and quality-of-life measures to be of the greatest value in determining aesthetic surgery outcomes. These conclusions were based on a critical evaluation of the feasibility, validity, reliability, and sensitivity to change of these measures. The Multidimensional Body-States Relations Questionnaire (MBSRQ), a psychological assessment of body image, was selected as a potential candidate for further study. Two additional body-image assessment instruments, the Facial Appearance Sorting Test (FAST) and the Breast Chest Ratings Scale (BCRS), may be useful in the assessment of rhinoplasty and breast surgery, respectively. The Derriford Scale (DAS59), an instrument that assesses appearance-related quality of life, was also selected. In addition, the authors recommend the use of a generic, utility-based quality-of-life instrument, such as the Health Utilities Index (HUI) or the EuroQol (EQ-5D).     

Pig vitreous gel: macromolecular composition with particular reference to hyaluronan-binding proteoglycans

The aim of this study was to examine the macromolecular composition of pig vitreous body with particular emphasis on hyaluronan-binding proteoglycans. The whole pig vitreous gel was found to contain 76 microg of hyaluronan-derived uronic acid, 700 microg of total protein and 150 microg of collagen per ml of gel. The contents of neutral hexoses and sialic acids were 80 and 22 microg/ml of vitreous gel, but only a minor proportion of them were found to be associated with the proteoglycan fraction. As estimated by gel chromatography on Sepharose CL-2B, hyaluronan presents a polydisperse hydrodynamic behavior with a lower molecular mass (M(r)) value of 220 kDa. The existence of low amounts of a hyaluronan-binding proteoglycan population with structural and immunological characteristics similar to a member of the hyalectan family, versican, has also been demonstrated. The concentration of this versican-like proteoglycan in whole vitreous accounts for 50 microg proteoglycan protein per ml of vitreous gel and represents a minor proportion (about 7%) of the total protein content. The proteoglycan has an average M(r) of 360 kDa and is substituted by chondroitin sulphate (CS) side chains. Study of the CS sulphation pattern showed that the chains were composed of both type 4- and 6-sulphated disaccharide units.     

Human colon adenocarcinoma is associated with specific post-translational modifications of versican and decorin

In this study, the amounts and the fine structural characteristics of versican and decorin present in human colon adenocarcinomas (HCC) were investigated and compared with those in human normal colon (HNC). HCC is characterized by significant increase in the amounts of versican and decorin (13- and 8-fold in terms of protein, respectively). These two proteoglycans (PGs) were the predominant in HCC (86% of total uronic acid). In HNC, versican and decorin contained both chondroitin sulfate/dermatan sulfate chains (CS/DS), with DS to be the predominant one (90-93%). The molecular sizes (M(r)s) estimated for DS and CS chains were 25-28 and 21-28 kDa, respectively. In CS/DS chains isolated from both versican and decorin, 4-sulfated disaccharides accounted for 79-86% of total disaccharide units, respectively, whereas lower amounts of 6- and non-sulfated units were also recorded. In contrast, the tumor-associated versican and decorin were of smaller hydrodynamic size with lower glycosaminoglycan (GAG) content per PG molecule as compared with those found in HNC. In HCC, both PGs contained mainly CS chains (up to 86%) and the M(r)s of CS and DS chains were also found to be of smaller size (12 and 16 kDa, respectively). The sulfation patterns of CS/DS chains from both PGs were also significantly different. They were composed mainly of 6-sulfated disaccharides (63-70%), whereas 4-sulfated units accounted for 23-31%. A significant increase in the proportion of non-sulfated disaccharides was also recorded. These findings indicate that the colon adenocarcinoma is characterized by a remarkable increase in the concentration of versican and decorin. Furthermore, these PGs are significantly modified at the post-translational level, i.e. the type, length and the sulfation pattern of their GAG chains. These specific structural alterations of versican and decorin may influence the biology of cancer cells in HCC.     

Glycosaminoglycans in early chick embryo

The developmental profile of glycosaminoglycans (GAGs) were examined by cellulose acetate electrophoresis and high performance liquid chromatography in the early chick embryo from late blastula (stage XIII+) to early somite developmental stages (stage HH7-9). Sulphated GAGs were present from the earliest stages. They were more abundant than the non-sulphated forms and showed stage-related changes. Chondroitin sulphate and especially dermatan sulphate appeared to be the predominant GAGs in embryos at stage XIII+. Dermatan sulphate was about three times as abundant as chondroitin sulphate at stage XII+. In contrast, embryos at the definitive streak stage (stage HH4) produced about twice as much chondroitin sulphate as dermatan sulphate. At the head process stage (stage HH5), the level of chondroitin sulphate was reduced and its relative content in the embryo was about the same as dermatan sulphate. Levels of dermatan sulphate were more than five times those of heparan sulphate from stage XIII through to stage HH5 and three times more at stage HH7-9. The 4- and 6- sulphation of chondroitin sulphate increased 14- and 10-fold respectively, from stage XIII+ to stage HH 7-9. The sulphation pattern of chondroitin sulphate had a delta(di)-4S:delta(di)-6S molar ratio ranging from 4 to 8:1 and a delta(di)-4S:delta(di)-OS molar ratio ranging from 9 to 16:1 and was developmentally regulated. Thus, chondroitin sulphate in the early chick embryo was sulphated predominately in the 4-position in all stages studied. The presence of both 4- and 6-sulphated disaccharides in chondroitin sulphate indicated that both 4 and 6 sulfotransferases were active in the early embryo. Hyaluronate and sulphated GAG content increased markedly at gastrulation when the first major cellular migrations and tissue interactions begin.     

Effect of spacer attachment sites and pH-sensitive headgroup expansion on cationic lipid-mediated gene delivery of three novel myristoyl derivatives

The transfection activity and physicochemical properties of the dimyristoyl derivatives from three novel series of double-chained tertiary cationic lipids were compared. Two of the derivatives were constructed as isomers with different linkages of the same bis-(2-dimethylaminoethane) polar headgroup and hydrophobic chains to the diaminopropanol backbone, while the third was designed with a hydrophilic region containing only a single ionizable amine group. Such systematic molecular changes offer a great opportunity to delineate factors critical for transfection activity, which in this work include the intramolecular distance between the hydrophobic chains and pH-expandability of the polar headgroup. The physical studies comprised a variety of techniques, including pKa determination, Langmuir monolayer studies, fluorescence anisotropy, gel electrophoresis mobility shift assay, ethidium bromide displacement assay, particle size distribution, and zeta potential. These studies are crucial in the development of lipid-based gene delivery systems with improved efficacy. Physicochemical characterization revealed that a symmetric bivalent pH-expandable polar headgroup in combination with greater intramolecular space between the hydrophobic chains provide for high transfection activity through efficient binding and compaction of pDNA, increased acyl chain fluidity, and high molecular elasticity.