Karen Friis Henriksen

Biomineralization Within Vesicles: The Calcite of Coccoliths

Coccolithophores are a group of unicellular plant plankton, which produce exoskeletons of minute calcite plates, called coccoliths. Despite their small size (1–10 μm across), coccoliths are remarkably elaborate biomineral structures characterized by precise control of both nucleation and growth of calcite by the organic system. They are also of considerable environmental and applied geological importance. Coccolithophores occur in enormous abundances, forming a significant proportion of total marine primary production and carbon fixation, especially in open ocean environments. Their biogeochemical impact is magnified by export of coccoliths to the ocean floor, where coccoliths are the largest single component of deep-sea sediments, forming vast accumulations of calcareous oozes and chalks, including the Late Cretaceous chalks of Northwestern Europe. Moreover, coccoliths are extensively employed by geologists as marker fossils to determine the ages of sediments, especially from drill cores. More recently, satellite imaging of immense coccolithophore blooms has led to extensive multi-disciplinary research into their biology and impact on the marine ecosystem and global carbon cycle (e.g., Westbroek et al. 1993; Paasche 2002; Thierstein and Young in press). ### Biological affinities Coccolithophores belong to the algal division (or phylum) Haptophyta. Other haptophytes are non-calcifying and include the well-known genera Phaeocystis, Prymnesium, Pavlova and Chrysochromulina . They are all characterized by possessing golden-brown chloroplasts, two smooth flagella and a third flagellum-like organelle, the haptonema. The haptonema shows distinctive coiling behavior and has a quite different microtubular sub-structure from flagella. Molecular genetic data have shown that the haptophytes are a group discrete from other algal protists that probably originated during the Precambrian (>600 Ma) protist radiation and acquired chloroplasts subsequently (possibly in the Late Palaeozoic, ca. 300–400 Ma) as a result of secondary endosymbiosis (Medlin et al. 1997). Molecular genetic phylogenies of the haptophytes are still being developed, but all results to date (Edvardsen …

Biological control on calcite crystallization: AFM investigation of coccolith polysaccharide function

Abstract Calcite crystals grown by organisms can be elaborate and enigmatic. One of the best examples is the tiny calcite shields known as coccoliths that are produced by unicellular algae. Coccoliths consist of interlocking single crystals of highly modified morphology, and complex organic molecules called CAPs (coccolith associated polysaccharides) are known to be intimately associated with their formation. Here, we show how a CAP can regulate crystal morphology to enhance precipitation of specific faces, a crucial aspect of the biomineralization process. Using atomic force microscopy (AFM), we investigated the interaction of CAP from the species Emiliania huxleyi with the calcite surface during dissolution, precipitation, and dynamic equilibrium. We were able to see the polysaccharide adsorbed to the surface and observe its impact on mineral behavior. These experiments demonstrate that CAP preferentially interacts with surface sites defined by acute, rather than obtuse, angles and blocks acute sites during dissolution and growth. Therefore, CAP makes the calcite face that is most stable in the pure system, {101̄4}, extend preferentially on the obtuse edges, promoting development of faces with lower angles to c-axis. AFM images of E. huxleyi at micrometer and atomic scales established that this is precisely the type of faces that define the morphology of the coccolith crystals. Therefore, we propose that crystal shape regulation by CAP is a fundamental aspect of coccolith biomineralization, and that preferential growth inhibition by site-specific functional groups is the mechanism causing CAP functionality.

Corpus callosum abnormalities, intellectual disability, speech impairment, and autism in patients with haploinsufficiency of ARID1B

Halgren C, Kjaergaard S, Bak M, Hansen C, El‐Schich Z, Anderson CM, Henriksen KF, Hjalgrim H, Kirchhoff M, Bijlsma EK, Nielsen M, den Hollander NS, Ruivenkamp CAL, Isidor B, Le Caignec C, Zannolli R, Mucciolo M, Renieri A, Mari F, Anderlid B‐M, Andrieux J, Dieux A, Tommerup N, Bache I. Corpus callosum abnormalities, intellectual disability, speech impairment, and autism in patients with haploinsufficiency of ARID1B.

MECP2 mutations in Danish patients with Rett syndrome: High frequency of mutations but no consistent correlations with clinical severity or with the X chromosome inactivation pattern

Rett syndrome (RTT) is a neurodevelopmental disorder, which almost exclusively affects girls, who, after an initial period of apparently normal development, display gradual loss of speech and purposeful hand use, gait abnormalities and stereotypical hand movements. In the year 2000, mutations in the gene for the methyl CpG binding protein 2, MECP2, have been identified in 35–80% of the patients in three different studies. We have identified 15 different MECP2 mutations in 26 of 30 Danish RTT patients. The mutations included five novel mutations (one point mutation, three smaller deletions involving identical regions in the gene, and one duplication). In contrast to the point mutations and the duplication, which all affect the methyl binding domain or the transcriptional repressing domain, the three overlapping deletions are clustered in the 3′ end of the gene. We found no consistent correlation between the type of mutation and the clinical presentation of the patient or the X-inactivation pattern in peripheral blood. Our high mutation detection rate, compared to two of the previous studies, underscores the importance of the inclusion criteria of the patients and supports that MECP2 is the most important, if not the only, gene responsible for RTT.

Pierre Robin sequence may be caused by dysregulation of SOX9 and KCNJ2

Background: The Pierre Robin sequence (PRS), consisting of cleft palate, micrognathia and glossoptosis, can be seen as part of the phenotype in other Mendelian syndromes—for instance, campomelic dysplasia (CD) which is caused by SOX9 mutations—but the aetiology of non-syndromic PRS has not yet been unravelled. Objective: To gain more insight into the aetiology of PRS by studying patients with PRS using genetic and cytogenetic methods. Methods: 10 unrelated patients with PRS were investigated by chromosome analyses and bacterial artificial chromosome arrays. A balanced translocation was found in one patient, and the breakpoints were mapped with fluorescence in situ hybridisation and Southern blot analysis. All patients were screened for SOX9 and KCNJ2 mutations, and in five of the patients expression analysis of SOX9 and KCNJ2 was carried out by quantitative real-time PCR. Results: An abnormal balanced karyotype 46,XX, t(2;17)(q23.3;q24.3) was identified in one patient with PRS and the 17q breakpoint was mapped to 1.13 Mb upstream of the transcription factor SOX9 and 800 kb downstream of the gene KCNJ2. Furthermore, a significantly reduced SOX9 and KCNJ2 mRNA expression was observed in patients with PRS. Conclusion: Our findings suggest that non-syndromic PRS may be caused by both SOX9 and KCNJ2 dysregulation.

Tailoring calcite: Nanoscale AFM of coccolith biocrystals

Abstract Biomineralization produces crystals of elaborate shapes, never seen in inorganic mineralogy, with tightly regulated compositions and axis orientations. The calcite coccoliths produced by unicellular marine algae provide an example of such control at very tiny scales. Atomic force microscopy (AFM) of two species provided nanoscale images allowing us to define crystallographic orientation in the crystal elements and to establish the relationship between crystallographic orientation and coccolith morphology. Both species adopt the inorganically stable calcite rhomb, but differences in crystal orientation enable them to construct distinct architectures with properties tailored to suit the requirements of their ecological niche.

Molecular basis for nonphenylketonuria hyperphenylalaninemia

Nonphenylketonuria hyperphenylalaninemia (non-PKU HPA) is defined as phenylalanine hydroxylase (PAH) deficiency with blood phenylalanine levels below 600 mumol/liter (i.e., within the therapeutic range) on a normal dietary intake. Haplotype analysis at the PAH locus was performed in 17 Danish families with non-PKU HPA, revealing compound heterozygosity in all individuals. By allele-specific oligonucleotide (ASO) probing for common PKU mutations we found 12 of 17 non-PKU HPA children with a PKU allele on one chromosome. To identify molecular lesions in the second allele, individual exons were amplified by polymerase chain reaction and screened for mutations by single-strand conformation polymorphism. Two new missense mutations were identified. Three children had inherited a G-to-A transition at codon 415 in exon 12 of the PAH gene, resulting in the substitution of asparagine for aspartate, whereas one child possessed an A-to-G transition at codon 306 in exon 9, causing the replacement of an isoleucine by a valine in the enzyme. It is further demonstrated that the identified mutations have less impact on the heterozygotes ability to hydroxylate phenylalanine to tyrosine compared to the parents carrying a PKU mutation. The combined effect on PAH activity explains the non-PKU HPA phenotype of the child. The present observations that PKU mutations in combination with other mutations result in the non-PKU HPA phenotype and that particular mutation-restriction fragment length polymorphism haplotype combinations are associated with this phenotype offer the possibility of distinguishing PKU patients from non-PKU individuals by means of molecular analysis of the hyperphenylalaninemic neonate and, consequently, of determining whether a newborn child requires dietary treatment.

In vivo assessment of mutations in the phenylalanine hydroxylase gene by phenylalanine loading: Characterization of seven common mutations

AbstractMutations in the gene encoding phenylalanine hydroxylase (PAH) cause persistent hyperphenyl-alaninaemia. To date, more than 200 point mutations and microdeletions have been characterized. Each mutation has a particular quantitative effect on enzyme activity and recessive expression of different mutant alleles results in a marked interindividual heterogeneity of metabolic and clinical phenotypes. In this paper we demonstrate how a simple clinical test can be used to evaluate the correlation between mutation genotype and phenylalanine metabolism. In hyperphenylalaninaemic patients with known PAH mutation genotype, we have investigated phenylalanine turnover in vivo by measuring the ability to eliminate a test dose ofl-phenyl-alanine. All patients could be considered functionally hemizygous for one of their mutant alleles by carrying on the other allele a mutation that is known to completely abolish PAH activity and encode a peptide with no immunoreactivity. Seven mutations (R408W, IVS-12nt1, R261Q, G46S, Y414C, A104D, and D415N) were characterized by oral phenylalanine loading, each mutation being represented by at least three patients. The elimination profile determined for a 3-day period provides a measure to compare residual activity of the mutant proteins and to assign each mutation to a particular metabolic phenotype. The established relation between genotype and phenotype may enable prediction of the severity of the disease by genotype determination in the newborn period. This will aid in the management of hyperphenylalaninaemia and may improve prognosis.ConclusionThe possibility of predicting the residual enzyme activity by DNA analysis performed already in the newborn period allows the prompt implementation of a diet that is adjusted to the degree of PAH deficiency. This may improve management and prognosis of hyperphenylalaninaemia.

Image distortion in scanning probe microscopy

Abstract Scanning probe microscopy (SPM) has become a common tool in mineralogy but distortion of images complicates interpretation and often limits the amount of information one can extract. Image distortion arises from a discrepancy between the intended and actual scan area caused by relative movement between tip and sample that is additional the intended scanning motion. We present a mathematical model to describe distortion in SPM images and provide a simple algebraic correction method. It uses Fourier periodicities for correcting high-resolution images; for micrometer-scale images, it can use any three non-colinear points that define a feature with known geometry. Observed distortion can be accounted for by two components: drift, the vector that quantifies the shape change of the intended scan area from a square into a parallelogram, and scaling, a constant that describes an isotropic change in dimension of the resulting scan area. The correction restores angular relationships and distances. The method was tested on the mineral graphite. In order to define the most important parameters affecting distortion, we made a sensitivity analysis by systematically varying temperature, scan speed, and time lapsed after the microscope was powered on. Neither drift nor scaling were found to be temperature dependent as such. However, both do depend on the time lapse after imaging begins. After the instrument is powered on, an initial 40 minute period of erratic drift is observed, whereafter drift velocity decreases with time while scaling increases slightly. Temperature variations in the range of 23 to 43 °C have negligible influence on distortion whereas scan speed affects scaling.

Identification of novel genes regulated in the developing human ventral mesencephalon

In the human embryo, from approximately 6 weeks gestational age (GA), dopaminergic (DA) neurons can be found in the ventral mesencephalon (VM). More specifically, the post-mitotic neurons are located in the ventral part of the tegmentum (VT), whereas no mature DA neurons are found in the neighboring dorsal part. We used Affymetrix HG-U133 GeneChip technology to compare genome-wide expression profiles of ventral and dorsal tegmentum from 8 weeks GA human embryos, in order to identify genes involved in specification, differentiation, and survival of mesencephalic DA (mDA) neurons. Known mDA marker genes including ALDH1A1, DAT1, VMAT2, TH, CALB1, NURR1, FOXA1, GIRK2, PITX3, RET, and DRD2 topped the list of 96 genes from HG-U133A with higher expression in VT, validating the experimental set-up. In addition, 28 probes from HG-U133B were identified whereof most are annotated to UniGene clusters with no gene associated or to genes of unknown function. Of these, the fifteen most regulated transcripts, representing changes down to 56% could be verified by quantitative real-time PCR (Q-PCR) on a developmental series of subdissected human embryonic and fetal brain material, resulting in not only a regional but also a temporal expression profile. This revealed a distinct DA-associated profile for in particular a putative transcription factor (FLJ45455) and the uncharacterized transmembrane proteins KIAA1145 and SLC10A4. The data presented here may help to device cell replacement and regenerative therapies for Parkinsons disease (PD).