Novel method for proliferation of oral keratinocyte stem cells.

BACKGROUND AND OBJECTIVE: Stem cell-based tissue engineering offers clear advantages over conventional normal cell approaches. Owing to their specific characteristics, oral keratinocyte stem cells represent an attractive solution for therapeutic applications. However, when cultured in vitro, these cells lose their unique properties, acquiring a limited capacity for self-renewal, and differentiate rapidly into normal functional keratinocytes. The main aim of the present study was to develop an in-vitro method for the expansion of oral keratinocyte stem cells using a biomaterial approach. MATERIAL AND METHODS: Oral keratinocyte stem cells were isolated based on the identification of two surface markers - integrin α6ß4 and CD71 - using a magnetic method. The cells were cultured on specific substrates formed from blends of polymers: poly(lactide-co-glycolide) (PLGA); poly(lactide-co-glycolide) + polyurethane (PLGA + PU); and poly(lactide-co-glycolide) + extracellular matrix (PLGA + ECM). The polymers were deposited using a laser-based technique - matrix-assisted pulsed laser evaporation. The cells were analyzed for cell size, cell proliferation, colony-forming efficiency, cell adhesion markers (such as E-cadherin and beta 1 integrin), keratinocyte stem cells and differentiation markers. The methods included ELISAs, immunofluorescence and atomic force microscopy imaging. RESULTS: After 14 d in culture, cells seeded on PLGA + PU stained positive for p63, cd44H, cytokeratin 19 and integrin α6ß4 and negative for involucrin, cytokeratin 14 and cytokeratin 10. The levels of adhesion molecules were significantly increased in cells grown on PLGA + PU: at 14 d the E-cadherin levels were 5.4 ± 0.2 ng/mL (for cells grown on PLGA + PU) vs. 4.1 ± 0.4 ng/mL (for cells grown on control medium) (n = 5, p < 0.05 Bonferroni). Oral keratinocyte stem cells grown on PLGA + PU had the highest colony-forming efficiency and proliferation rate, together with the smallest cell size, compared with cells grown on control medium or other polymeric substrates. CONCLUSION: The present study demonstrates that by culturing oral keratinocyte stem cells on PLGA blended with PU it is possible to preserve their phenotype in vitro and to guide their short-term expansion and proliferation. Certain stem-cell characteristics are preserved and their short-term expansion may be enhanced.

Application of the finite element method in dental implant research.

This article provides a review of the achievements and advancements in dental technology brought about by computer-aided design and the all powerful finite element method (FEM) of analysis. The scope of the review covers dental implants, jawbone surrounding the implant and the biomechanical implant and jawbone interaction. Prevailing assumptions made in the published finite element analysis (FEA) and their limitations are discussed in some detail which helps identify the gaps in research as well as future research direction.

Automated detection of point mutations using fluorescent sequence trace subtraction.

The final step in the detection of mutations is to determine the sequence of the suspected mutant and to compare it with that of the wild-type, and for this fluorescence-based sequencing instruments are widely used. We describe some simple algorithms forcomparing sequence traces which, as part of our sequence assembly and analysis package, are proving useful for the discovery of mutations and which may also help to identify misplaced readings in sequence assembly projects. The mutations can be detected automatically by a new program called TRACE_DIFF and new types of trace display in our program GAP4 greatly simplify visual checking of the assigned changes. To assess the accuracy of the automatic mutation detection algorithm we analysed 214 sequence readings from hypermutating DNA comprising a total of 108 497 bases. After the readings were assembled there were 1232 base differences, including 392 Ns and 166 alignment characters. Visual inspection of the traces established that of the 1232 differences, 353 were real mutations while the rest were due to base calling errors. The TRACE_DIFF algorithm automatically identified all but 36, with 28 false positives. Further information about the software can be obtained from http://www.mrc-lmb.cam.ac.uk/pubseq/

Both DNA strands of antibody genes are hypermutation targets.

During the maturation of the immune response, antibody genes are subjected to localized hypermutation. Mutations are not evenly distributed along the V gene; intrinsic hot spots exist that are correlated with primary sequence motifs. Although the mechanism of hypermutation remains unknown, it has been proposed to exhibit DNA strand polarity because purine residues on the coding strand are more frequently targeted for mutation than pyrimidines. However, this polarity may not be an intrinsic property of the hypermutation mechanism but a consequence of evolutionary-selected peculiarities of V gene sequences. Furthermore, the possibility that both strands are hypermutation targets has received little attention. To discriminate between these possibilities, we have analyzed the average frequency of mutations of each of the three bases of all nucleotide triplets by using large databases taken from both V and non-V mutation targets. We also have reassessed the sequence motifs associated with hot spots. We find that even in non-Ig sequences, A mutates more than T, consistent with a strand-dependent component to targeting. However, the mutation biases of triplets and of their inverted complements are correlated, demonstrating that there is a sequence-specific but strand-independent component to mutational targeting. Thus, there are two aspects of the hypermutation process that are sensitive to local DNA sequences, one that is DNA strand-dependent and the other that is not.

An oligo-screening strategy to fill gaps found during shotgun sequencing projects.

During the course of projects to sequence human and nematode cosmids we encountered great difficulties generating contiguous sequence in regions with repetitive DNA (Alu repeats in humans and tandem or inverted repeats in the nematode). We have developed a simple and efficient strategy to fill gaps. By screening M13, plasmid or phagemid libraries with oligonucleotides flanking the gap, clones are identified that contiguate the cosmid sequence. Our method has been integrated into the GAP4 sequence assembly program. The strategy reduces both time and costs in large scale sequencing projects.

The Staden sequence analysis package.

I describe the current version of the sequence analysis package developed at the MRC Laboratory of Molecular Biology, which has come to be known as the "Staden Package." The package covers most of the standard sequence analysis tasks such as restriction site searching, translation, pattern searching, comparison, gene finding, and secondary structure prediction, and provides powerful tools for DNA sequence determination. Currently the programs are only available for computers running the UNIX operating system. Detailed information about the package is available from our WWW site: http:@www.mrc-lmb.cam.ac.uk/pubseq/.

Experiment files and their application during large-scale sequencing projects.

The data for large scale sequencing projects are passed through several processing steps prior to assembly, and post-assembly processing generally requires knowledge of more than just the sequence of each reading. We address here the problem of providing data to individual programs and of combining all the tasks into a single process. The solution comprises two components: a file format (experiment file format) that stores information about readings, and a script (PREGAP) that controls the creation and use of experiment files by the processing programs. PREGAP can take a batch of data from a variety of sequencing instruments, gather information about each reading, and then scan the reading to select the 3' end of the good quality data, mark sequencing vector, other cloning vector sequences, and Alu segments. The results of all these operations are added to the experiment file for each reading, ready for processing by the assembly program. Experiment files also provide a mechanism for using alternative assembly engines with our package.

A new DNA sequence assembly program.

We describe the Genome Assembly Program (GAP), a new program for DNA sequence assembly. The program is suitable for large and small projects, a variety of strategies and can handle data from a range of sequencing instruments. It retains the useful components of our previous work, but includes many novel ideas and methods. Many of these methods have been made possible by the program's completely new, and highly interactive, graphical user interface. The program provides many visual clues to the current state of a sequencing project and allows users to interact in intuitive and graphical ways with their data. The program has tools to display and manipulate the various types of data that help to solve and check difficult assemblies, particularly those in repetitive genomes. We have introduced the following new displays: the Contig Selector, the Contig Comparator, the Template Display, the Restriction Enzyme Map and the Stop Codon Map. We have also made it possible to have any number of Contig Editors and Contig Joining Editors running simultaneously even on the same contig. The program also includes a new 'Directed Assembly' algorithm and routines for automatically detecting unfinished segments of sequence, to which it suggests experimental solutions.

The application of numerical estimates of base calling accuracy to DNA sequencing projects.

During DNA sequencing projects one of the most labour intensive and highly skilled tasks is to view the original trace descriptions of gels and to adjudicate between conflicting readings. Given the current methods of calculating a consensus, the majority of the time employed in viewing traces and editing readings is actually devoted to making the poorer data fit the good data. We propose new consensus calculation algorithms that employ numerical estimates of base calling accuracy and which when used in conjunction with an automatic detector of contradictory data should greatly reduce the time spent checking and editing readings and hence improve DNA sequencing productivity.

[Results of long-term intensive care in 223 patients]. / Resultaten van langdurige intensieve zorg bij 223 patiënten.

OBJECTIVE: To determine survival rates of patients treated for more than 30 days in an intensive care unit (ICU). DESIGN: Retrospective, descriptive. SETTING: Intensive care unit of the St. Antonius Hospital in Nieuwegein, the Netherlands. METHODS: All patients who required more than 30 consecutive days ICU treatment between January 1985 and January 1992 were included. With the aid of a computerised data base the medical records of all patients were analysed. If discharged, their family doctor was contacted for information about survival and quality of life. Kaplan-Meier survival curves were calculated. RESULTS: Among a total of 18,126 ICU admissions, 223 patients required more than 30 days ICU treatment; 25% died in the ICU; 14% died after discharge from the ICU, but still in the hospital; 31% of the patients were discharged to another hospital or nursing home. Of all patients 50% eventually reached home. Two months after ICU discharge 75% were alive, after 1 year 50%. Mean survival time was 36 months (SD: 3). Patients under 60 years of age and those who were discharged directly home had the best prognosis. 30% of the protracted IC patients could ultimately function independently at home. CONCLUSIONS: Patients who needed more than 30 days ICU treatment had a high ICU mortality; 2 months after discharge 75% were alive.