Petter Mostad

Large-scale identification of genes implicated in kidney glomerulus development and function

To advance our understanding of development, function and diseases in the kidney glomerulus, we have established and large‐scale sequenced cDNA libraries from mouse glomeruli at different stages of development, resulting in a catalogue of 6053 different genes. The glomerular cDNA clones were arrayed and hybridized against a series of labeled targets from isolated glomeruli, non‐glomerular kidney tissue, FACS‐sorted podocytes and brain capillaries, which identified over 300 glomerular cell‐enriched transcripts, some of which were further sublocalized to podocytes, mesangial cells and juxtaglomerular cells by in situ hybridization. For the earliest podocyte marker identified, Foxc2, knockout mice were used to analyze the role of this protein during glomerular development. We show that Foxc2 controls the expression of a distinct set of podocyte genes involved in podocyte differentiation and glomerular basement membrane maturation. The primary podocyte defects also cause abnormal differentiation and organization of the glomerular vascular cells. We surmise that studies on the other novel glomerulus‐enriched transcripts identified in this study will provide new insight into glomerular development and pathomechanisms of disease.

Quantitative Real-Time PCR Method for Detection of B-Lymphocyte Monoclonality by Comparison of κ and λ Immunoglobulin Light Chain Expression

Background: An abnormal IgL:IgL ratio has long been used as a clinical criterion for non-Hodgkin B-cell lymphomas. As a first step toward a quantitative realtime PCR-based multimarker diagnostic analysis of lymphomas, we have developed a method for determination of IgL:IgL ratio in clinical samples. Methods: Light-up probe-based real-time PCR was used to quantify IgL and IgL cDNA from 32 clinical samples. The samples were also investigated by routine immunohistochemical analysis and flow cytometry. Results: Of 32 suspected non-Hodgkin lymphoma samples analyzed, 28 were correctly assigned from real-time PCR measurements assuming invariant PCR efficiencies in the biological samples. Four samples were false negatives. One was a T-cell lymphoma, one was a diffuse large B-cell lymphoma, and one was reanalyzed and found lymphoma-positive by in situ calibration, which takes into account sample-specific PCR inhibition. Twelve of the samples were fine-needle aspirates, and these were all correctly assigned. Conclusions: This work is a first step toward analyzing clinical samples by quantitative light-up probe-based real-time PCR. Quantitative real-time PCR appears suitable for high-throughput testing of cancers by measuring expression of tumor markers in fine-needle aspirates. The rapid expansion in knowledge of the human genome and the development of techniques for analysis of nucleic acids have opened new possibilities for diagnostics. In our first attempt to use quantitative real-time PCR (Q-PCR) 5 for detection of malignant tumors, we have focused on the analysis of non-Hodgkin lymphomas (NHLs).

A phylogenetic comparative method for studying multivariate adaptation

Phylogenetic comparative methods have been limited in the way they model adaptation. Although some progress has been made, there are still no methods that can fully account for coadaptation between traits. Based on Ornstein-Uhlenbeck (OU) models of adaptive evolution, we present a method, with R implementation, in which multiple traits evolve both in response to each other and, as in previous OU models, to fixed or randomly evolving predictor variables. We present the interpretation of the model parameters in terms of evolutionary and optimal regressions enabling the study of allometric and adaptive relationships between traits. To illustrate the method we reanalyze a data set of antler and body-size evolution in deer (Cervidae).

Design of improved membrane protein production experiments: Quantitation of the host response

Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial‐and‐error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high‐performance bioreactors under tightly‐defined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly‐controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology.

Analysis of linkage and linkage disequilibrium for eight X-STR markers

X-chromosomal short tandem repeats (X-STR) have proven to be informative and useful in complex relationship testing. The main feature of X-STR markers, compared to autosomal forensic markers, is that all loci are located on the same chromosome. Thus, linkage and linkage disequilibrium may occur. The aim of this work was to study population genetic parameters of eight X-STR markers, located in four linkage groups. We present haplotype frequencies, based on 718 Swedish males, for the four linkage groups included in the Argus X-8 kit. Forensic efficiency parameters have been calculated as well as the allelic association between the tested markers for detection of linkage disequilibrium. To study the occurrences of recombination between the loci, both Swedish and Somali families were typed. A mathematical model for the estimation of recombination frequencies is presented and applied on the family samples. Our study showed that the tested markers all have highly informative forensic values and that there is a significant degree of linkage disequilibrium between the STR markers within the four linkage groups. Furthermore, based on the tested families, we also demonstrated that two of the linkage groups are partially linked. A consequence of these findings is that both linkage and linkage disequilibrium should be accounted for when producing likelihood ratios in relationship testing with X-STR markers.

Predictive screening for regulators of conserved functional gene modules (gene batteries) in mammals

BackgroundThe expression of gene batteries, genomic units of functionally linked genes which are activated by similar sets of cis- and trans-acting regulators, has been proposed as a major determinant of cell specialization in metazoans. We developed a predictive procedure to screen the mouse and human genomes and transcriptomes for cases of gene-battery-like regulation.ResultsIn a screen that covered ~40 per cent of all annotated protein-coding genes, we identified 21 co-expressed gene clusters with statistically supported sharing of cis-regulatory sequence elements. 66 predicted cases of over-represented transcription factor binding motifs were validated against the literature and fell into three categories: (i) previously described cases of gene battery-like regulation, (ii) previously unreported cases of gene battery-like regulation with some support in a limited number of genes, and (iii) predicted cases that currently lack experimental support. The novel predictions include for example Sox 17 and RFX transcription factor binding sites that were detected in ~10% of all testis specific genes, and HNF-1 and 4 binding sites that were detected in ~30% of all kidney specific genes respectively. The results are publicly available at http://www.wlab.gu.se/lindahl/genebatteries.Conclusion21 co-expressed gene clusters were enriched for a total of 66 shared cis-regulatory sequence elements. A majority of these predictions represent novel cases of potential co-regulation of functionally coupled proteins. Critical technical parameters were evaluated, and the results and the methods provide a valuable resource for future experimental design.

Stochastic Structural Modeling

A consistent stochastic model for faults and horizons is described. The faults are represented as a parametric invertible deformation operator. The faults may truncate each other. The horizons are modeled as correlated Gaussian fields and are represented in a grid. Petrophysical variables may be modeled in a reservoir before faulting in order to describe the juxtaposition effect of the faulting. It is possible to condition the realization on petrophysics, horizons, and fault plane observations in wells in addition to seismic data. The transmissibility in the fault plane may also be included in the model. Four different methods to integrate the fault and horizon models in a common model is described. The method is illustrated on an example from a real petroleum field with 18 interpreted faults that are handled stochastically.

Estimating the number of contributors to a DNA profile

The broad topic of this paper is the evaluation of DNA evidence in criminal cases. More specifically, we deal with mixture evidence which refers to cases where there are, or could be, several contributors to a biological stain based on, e.g., blood or semen. The present paper adresses DNA mixtures based on single nucleotide polymorphism (SNP) markers, i.e., diallelic markers. Based on STR analysis, it is in most cases easy to identify the presence of a mixture since three or four bands will show up with a high probability for at least one locus. Obviously, this will not be the case for diallelic markers and interpreting mixtures will be a great challenge. We address this problem by first approaching the more general problem of estimating the number of contributors to a stain. In addition we discuss how the markers should be selected and how many are required.

Using X-chromosomal markers in relationship testing: Calculation of likelihood ratios taking both linkage and linkage disequilibrium into account

X-chromosomal markers in forensic genetics have become more widely used during recent years, particularly for relationship testing. Linkage and linkage disequilibrium (LD) must typically be accounted for when using close X-chromosomal markers. Thus, when producing the weight-of-evidence, given by a DNA-analysis with markers that are linked, the normally used product rule is invalid. Here we present an implementation of an efficient model for calculating likelihood ratios (LRs) with markers on the X-chromosome which are linked and in LD. Furthermore, the model was applied on several cases based on data from the eight X-chromosomal loci included in the Mentype(®) Argus X-8 (Biotype). Using a simulation approach we showed that the use of X-chromosome data can offer valuable information for choosing between the alternatives in each of the cases we studied, and that the LR can be high in several cases. We demonstrated that when linkage and LD were disregarded, as opposed to taken into account, the difference in calculated LRs could be considerable. When these differences were large, the estimated haplotype frequencies often had a strong impact and we present a method to estimate haplotype frequencies. Our conclusion is that linkage and LD should be accounted for when using the tested set of markers, and the used model is an efficient way of doing so.

Havana — a fault modeling tool

Improved knowledge on faults and hydrocarbon seal put pressure on geologists and reservoir engineers doing reservoir modeling. All geo-knowledge must be built into the reservoir models to assure that it is taken into account in the decision processes. The need for advanced modeling tools is increasing. This paper describes the development of a fault modeling tool, the methodology behind it and examples of fault modeling studies. The general focus is on the uncertainty related to faults. The tool can be used for sensitivity analysis of fault effects, including studies of the flow effects of all faults scales, adding faults to simulation grids, and studies of the geometric uncertainty of the faults. The work started out as a development of a tool for stochastic modeling of sub-seismic scale faults. The faults can be added to a flow simulation grid as both displacement and seal. The current tool has been designed to operate together with the Eclipse flow simulator and the IRAP RMS program package. IRAP RMS is the main tool for visualizing output and Eclipse is used to examine the effect of the faults on hydrocarbon recovery. The techniques for modeling of fault seal, outputting results in a format that Eclipse can directly utilize, and the possibility for displacing simulation grids has proved useful also to seismic scale faults. This has led to further development, more detailed fault models and improvements of the general fault modeling capabilities. Examples of fault modeling, including three field examples, Statfjord, Heidrun and Sleipner, are presented to illustrate ways of including fault modeling as part of the reservoir modeling workflow.