Resolution by Unassisted Top3 Points to Template Switch Recombination Intermediates during DNA Replication

The evolutionarily conserved Sgs1/Top3/Rmi1 (STR) complex plays vital roles in DNA replication and repair. One crucial activity of the complex is dissolution of toxic X-shaped recombination intermediates that accumulate during replication of damaged DNA. However, despite several years of study the nature of these X-shaped molecules remains debated. Here we use genetic approaches and two-dimensional gel electrophoresis of genomic DNA to show that Top3, unassisted by Sgs1 and Rmi1, has modest capacities to provide resistance to MMS and to resolve recombination-dependent X-shaped molecules. The X-shaped molecules have structural properties consistent with hemicatenane-related template switch recombination intermediates (Rec-Xs) but not Holliday junction (HJ) intermediates. Consistent with these findings, we demonstrate that purified Top3 can resolve a synthetic Rec-X but not a synthetic double HJ in vitro. We also find that unassisted Top3 does not affect crossing over during double strand break repair, which is known to involve double HJ intermediates, confirming that unassisted Top3 activities are restricted to substrates that are distinct from HJs. These data help illuminate the nature of the X-shaped molecules that accumulate during replication of damaged DNA templates, and also clarify the roles played by Top3 and the STR complex as a whole during the resolution of replication-associated recombination intermediates.     

Comparison of the Accuracy of Voxel Based Registration and Surface Based Registration for 3D Assessment of Surgical Change following Orthognathic Surgery

Purpose

Superimposition of two dimensional preoperative and postoperative facial images, including radiographs and photographs, are used to evaluate the surgical changes after orthognathic surgery. Recently, three dimensional (3D) imaging has been introduced allowing more accurate analysis of surgical changes. Surface based registration and voxel based registration are commonly used methods for 3D superimposition. The aim of this study was to evaluate and compare the accuracy of the two methods.

Materials and methods

Pre-operative and 6 months post-operative cone beam CT scan (CBCT) images of 31 patients were randomly selected from the orthognathic patient database at the Dental Hospital and School, University of Glasgow, UK. Voxel based registration was performed on the DICOM images (Digital Imaging Communication in Medicine) using Maxilim software (Medicim-Medical Image Computing, Belgium). Surface based registration was performed on the soft and hard tissue 3D models using VRMesh (VirtualGrid, Bellevue City, WA). The accuracy of the superimposition was evaluated by measuring the mean value of the absolute distance between the two 3D image surfaces. The results were statistically analysed using a paired Student t-test, ANOVA with post-hoc Duncan test, a one sample t-test and Pearson correlation coefficient test.

Results

The results showed no significant statistical difference between the two superimposition methods (p<0.05). However surface based registration showed a high variability in the mean distances between the corresponding surfaces compared to voxel based registration, especially for soft tissue. Within each method there was a significant difference between superimposition of the soft and hard tissue models.

Conclusions

There were no significant statistical differences between the two registration methods and it was unlikely to have any clinical significance. Voxel based registration was associated with less variability. Registering on the soft tissue in isolation from the hard tissue may not be a true reflection of the surgical change.     

3d haro-Noesy-ch3nh and caro-Noesy-ch3nh Experiments for Double Labeled Proteins

Precision in the determination of the 3D structures of proteins by NMR depends on obtaining an adequate number of NOE restraints. Ambiguity in the assignment of NOE cross peaks between aromatic and other protons is an impediment to high quality structure determination. Two pulse sequences, 3D H_aro-NOESY-CH₃NH and 3D C_aro-NOESY-CH₃NH, based on a modification of a technique for simultaneous detection of ¹³C-¹H (of CH₃) and ¹⁵N-¹H correlations in one measurement, are proposed in the present work. These 3D experiments, which are optimized for resolution in the ¹³C and ¹⁵N dimensions, provide NOE information between aromatic protons and methyl or amide protons. CH₂ moieties are filtered out and the CH groups in aromatic rings are selected, allowing their NOE cross peaks to be unambiguously assigned. Unambiguous NOEs connecting aromatic and methyl or amide protons will provide important restraints for protein structure calculations.     

Regularization strategies for hyperplane classifiers: application to cancer classification with gene expression data

Linear discrimination, from the point of view of numerical linear algebra, can be treated as solving an ill-posed system of linear equations. In order to generate a solution that is robust in the presence of noise, these problems require regularization. Here, we examine the ill-posedness involved in the linear discrimination of cancer gene expression data with respect to outcome and tumor subclasses. We show that a filter factor representation, based upon Singular Value Decomposition, yields insight into the numerical ill-posedness of the hyperplane-based separation when applied to gene expression data. We also show that this representation yields useful diagnostic tools for guiding the selection of classifier parameters, thus leading to improved performance.     

Registration of 6-DOFs electrogoniometry and CT medical imaging for 3D joint modeling

The paper describes a method in which two data-collecting systems, medical imaging and electrogoniometry, are combined to allow the accurate and simultaneous modeling of both the spatial kinematics and the morphological surface of a particular joint. The joint of interest (JOI) is attached to a Plexiglas jig that includes four metallic markers defining a local reference system (R(GONIO)) for the kinematics data. Volumetric data of the JOI and the R(GONIO) markers are collected from medical imaging. The spatial location and orientation of the markers in the global reference system (R(CT)) of the medical-imaging environment are obtained by applying object-recognition and classification methods on the image dataset. Segmentation and 3D isosurfacing of the JOI are performed to produce a 3D model including two anatomical objects-the proximal and distal JOI segments. After imaging, one end of a custom-made 3D electrogoniometer is attached to the distal segment of the JOI, and the other end is placed at the R(GONIO) origin; the JOI is displaced and the spatial kinematics data is recorded by the goniometer. After recording, data registration from R(GONIO) to R(CT) occurred prior to simulation. Data analysis was performed using both joint coordinate system (JCS) and instantaneous helical axis (IHA).Finally, the 3D joint model is simulated in real time using the experimental kinematics data. The system is integrated into a computer graphics interface, allowing free manipulation of the 3D scene.The overall accuracy of the method has been validated with two other kinematics data collection methods including a 3D digitizer and interpolation of the kinematics data from discrete positions obtained from medical imaging. Validation has been performed on both superior and inferior radio-ulna joints (i.e. prono-supination motion). Maximal RMS error was 1 degrees and 1.2mm on the helical axis rotation and translation, respectively. Prono-supination of the forearm showed a total rotation of 132 degrees for 0.8mm of translation. The method reproducibility using JCS parameters was in average 1 degrees (maximal deviation=2 degrees ) for rotation, and 1mm (maximal deviation=2mm) for translation. In vitro experiments have been performed on both knee joint and ankle joint. Averaged JCS parameters for the knee were 109 degrees, 17 degrees and 4 degrees for flexion, internal rotation and abduction, respectively. Averaged maximal translation values for the knee were 12, 3 and 4mm posteriorly, medially and proximally, respectively. Averaged JCS parameters for the ankle were 43 degrees, 9 degrees and 3 degrees for plantarflexion, adduction and internal rotation, respectively. Averaged maximal translation values for the ankle were 4, 2 and 1mm anteriorly, medially and proximally, respectively.     

Registration of drugs for treating cancer and HIV infection: a plea to carry out phase 3 trials before admission to the market.

Drugs for cancer and HIV infection tend to be admitted to the market on the basis of results from phase 2 trials. Assessing the benefit-risk balance with phase 2 trials often is difficult--the effect of the drug is usually temporary; the correlation between response or improvement of clinical measurements and the patient''s wellbeing is often poor; and the side effects of drugs for these fatal diseases are serious. Therefore, although sometimes difficult to conduct, comparative trials that use standard treatment, placebos, or best supportive care remain the cornerstone for reliably assessing the benefit-risk balance.     

An attempt to apply ligand field theory to positronium reactions with 3d complexes

Positronium, Ps, the bound state between an electron, e, and a positron, c⁺, may have two spin states: theortho,o-Ps, and thepara,p-Ps, states, which differ for the spin orientation of the two particles. The two types of Ps atoms may be inter-converted by collision with paramagnetic compounds, such as several3d complexes. By investigating about 90 complexes of V^II, Cr^II, Cr^III, Mn^II, Fe^II, Co^II and Ni^II as a function of temperature, it was found that the rate constants k_CR of theo-Ps intop-Ps conversion reactions, CR, are linearly correlated to the delocalization β of metal electrons caused by the ligands. Therefore, if β known, k_CR may be estimated andvice versa. This suggests a new method for the experimental determination of β. The rate constants of the Ps oxidation reactions by Co^III complexes were also investigated and discussed. The paper is preceded by four sections dealing with: 1) the positron and positronium formation; 2) the positron annihilation modes; 3) the methods for measuring the Ps rate constants and establishing the Ps reaction types; 4) the application of the Smoluchowski equation to Ps reactions. Moreover, an attempt is made to ascertain the standard electrochemical potential of Ps atoms. The positron reactions and the formation of positronides are also taken into consideration. Presentata nella seduta del 13 dicembre 2002 dal Socio F. Calderazzo.     

3D Reconstruction and Standardization of the Rat Vibrissal Cortex for Precise Registration of Single Neuron Morphology

The three-dimensional (3D) structure of neural circuits is commonly studied by reconstructing individual or small groups of neurons in separate preparations. Investigation of structural organization principles or quantification of dendritic and axonal innervation thus requires integration of many reconstructed morphologies into a common reference frame. Here we present a standardized 3D model of the rat vibrissal cortex and introduce an automated registration tool that allows for precise placement of single neuron reconstructions. We (1) developed an automated image processing pipeline to reconstruct 3D anatomical landmarks, i.e., the barrels in Layer 4, the pia and white matter surfaces and the blood vessel pattern from high-resolution images, (2) quantified these landmarks in 12 different rats, (3) generated an average 3D model of the vibrissal cortex and (4) used rigid transformations and stepwise linear scaling to register 94 neuron morphologies, reconstructed from in vivo stainings, to the standardized cortex model. We find that anatomical landmarks vary substantially across the vibrissal cortex within an individual rat. In contrast, the 3D layout of the entire vibrissal cortex remains remarkably preserved across animals. This allows for precise registration of individual neuron reconstructions with approximately 30 µm accuracy. Our approach could be used to reconstruct and standardize other anatomically defined brain areas and may ultimately lead to a precise digital reference atlas of the rat brain.     

A Fast Algorithm to Estimate the Deepest Points of Lakes for Regional Lake Registration

When conducting image registration in the U.S. state of Alaska, it is very difficult to locate satisfactory ground control points because ice, snow, and lakes cover much of the ground. However, GCPs can be located by seeking stable points from the extracted lake data. This paper defines a process to estimate the deepest points of lakes as the most stable ground control points for registration. We estimate the deepest point of a lake by computing the center point of the largest inner circle (LIC) of the polygon representing the lake. An LIC-seeking method based on Voronoi diagrams is proposed, and an algorithm based on medial axis simplification (MAS) is introduced. The proposed design also incorporates parallel data computing. A key issue of selecting a policy for partitioning vector data is carefully studied, the selected policy that equalize the algorithm complexity is proved the most optimized policy for vector parallel processing. Using several experimental applications, we conclude that the presented approach accurately estimates the deepest points in Alaskan lakes; furthermore, we gain perfect efficiency using MAS and a policy of algorithm complexity equalization.     

Spatio-Temporal Trends and Risk Factors for Shigella from 2001 to 2011 in Jiangsu Province,People's Republic of China

Objective

This study aimed to describe the spatial and temporal trends of Shigella incidence rates in Jiangsu Province, People''s Republic of China. It also intended to explore complex risk modes facilitating Shigella transmission.

Methods

County-level incidence rates were obtained for analysis using geographic information system (GIS) tools. Trend surface and incidence maps were established to describe geographic distributions. Spatio-temporal cluster analysis and autocorrelation analysis were used for detecting clusters. Based on the number of monthly Shigella cases, an autoregressive integrated moving average (ARIMA) model successfully established a time series model. A spatial correlation analysis and a case-control study were conducted to identify risk factors contributing to Shigella transmissions.

Results

The far southwestern and northwestern areas of Jiangsu were the most infected. A cluster was detected in southwestern Jiangsu (LLR = 11674.74, P<0.001). The time series model was established as ARIMA (1, 12, 0), which predicted well for cases from August to December, 2011. Highways and water sources potentially caused spatial variation in Shigella development in Jiangsu. The case-control study confirmed not washing hands before dinner (OR = 3.64) and not having access to a safe water source (OR = 2.04) as the main causes of Shigella in Jiangsu Province.

Conclusion

Improvement of sanitation and hygiene should be strengthened in economically developed counties, while access to a safe water supply in impoverished areas should be increased at the same time.     

Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21)

A vital role for complement in adaptive humoral immunity is now beyond dispute. The crucial interaction is that between B cell and follicular dendritic cell-resident complement receptor 2 (CR2, CD21) and its Ag-associated ligands iC3b and C3dg, where the latter have been deposited as a result of classical pathway activation. Despite the obvious importance of this interaction, the location of a CR2 binding site within C3d, a proteolytic limit fragment of C3dg retaining CR2 binding activity, has not been firmly established. The recently determined x-ray structure of human C3d suggested a candidate site that was remote from the site of covalent attachment to Ag and consisted of an acidic residue-lined depression, which accordingly displays a significant electronegative surface potential. These attributes were consistent with the known ionic strength dependence of the CR2-C3d interaction and with the fact that a significant electropositive surface was apparent in a modeled structure of the C3d-binding domains of CR2. Therefore, we have performed an alanine scan of all of the residues within and immediately adjacent to the acidic pocket in C3d. By testing the mutant iC3b molecules for their ability to bind CR2, we have identified two separate clusters of residues on opposite sides of the acidic pocket, specifically E37/E39 and E160/D163/I164/E166, as being important CR2-contacting residues in C3d. Within the second cluster even single mutations cause near total loss of CR2 binding activity. Consistent with the proposed oppositely charged nature of the interface, we have also found that removal of a positive charge immediately adjacent to the acidic pocket (mutant K162A) results in a 2-fold enhancement in CR2 binding activity.     

Inference for the Break Point in Segmented Regression with Application to Longitudinal Data

This paper considers inference for the break point in the segmented regression or piece‐wise regression model. Standard likelihood theory does not apply because the break point is absent under the null hypothesis. We use results by Davies for this type of non‐standard set‐up [Biometrika 64 (1977), 247–254 and 74 (1987), 33–43] to obtain a test for the null hypothesis of no break point. A confidence interval can be constructed provided replicate data are available. The methods are exemplified using two longitudinal datasets, the one from ecology, the other from pharmacology.     

Template location on the human ribosome: environment of the mRNA nucleotide adjacent to the A-site codon on the 3'-side

The 18S rRNA nucleotides close to the 80S ribosome template nucleotide adjacent to the A-site codon on the 3-end (i.e., the nucleotide in position +7 relative to the first nucleotide of the P-site codon) were identified using template-controlled chemical affinity ligation. For this purpose, used the photoreactive mRNA analogues with a perfluorophenylazido group attached through various linkers to the uridine C5,3'-terminal phosphate, or guanosine N7 were used. The position of the mRNA analogues on the ribosome was preset using tRNAPhe, which recognized the phenylalanine codon directed to the P-site. An analysis of the rRNAs isolated from the irradiated complexes of 80S ribosomes showed that all the analogues are almost equally ligated to the 18S rRNA nucleotides we attributed to the A-site codon environment: namely, to nucleotides A1823, A1824, and A1825 of the 3'-minidomain and to the 620-630 fragment of the 18S rRNA 5'-domain. In addition, we identified a new component of the mRNA binding site of human ribosomes, nucleotide C1698 belonging to the 18S rRNA 3-minidomain, using analogues bearing a perfluorophenylazido group on uridine and guanine residues. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.