Medical sequencing of candidate genes for nonsyndromic cleft lip and palate

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations.     

Allele-specific amplification in cancer revealed by SNP array analysis

Amplification, deletion, and loss of heterozygosity of genomic DNA are hallmarks of cancer. In recent years a variety of studies have emerged measuring total chromosomal copy number at increasingly high resolution. Similarly, loss-of-heterozygosity events have been finely mapped using high-throughput genotyping technologies. We have developed a probe-level allele-specific quantitation procedure that extracts both copy number and allelotype information from single nucleotide polymorphism (SNP) array data to arrive at allele-specific copy number across the genome. Our approach applies an expectation-maximization algorithm to a model derived from a novel classification of SNP array probes. This method is the first to our knowledge that is able to (a) determine the generalized genotype of aberrant samples at each SNP site (e.g., CCCCT at an amplified site), and (b) infer the copy number of each parental chromosome across the genome. With this method, we are able to determine not just where amplifications and deletions occur, but also the haplotype of the region being amplified or deleted. The merit of our model and general approach is demonstrated by very precise genotyping of normal samples, and our allele-specific copy number inferences are validated using PCR experiments. Applying our method to a collection of lung cancer samples, we are able to conclude that amplification is essentially monoallelic, as would be expected under the mechanisms currently believed responsible for gene amplification. This suggests that a specific parental chromosome may be targeted for amplification, whether because of germ line or somatic variation. An R software package containing the methods described in this paper is freely available at http://genome.dfci.harvard.edu/~tlaframb/PLASQ.     

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.     

Dynamics of production of sexual forms in aphids: theoretical and experimental evidence for adaptive "coin-flipping" plasticity

The best strategy for an organism to deal with unpredictable environmental conditions is a stochastic one, but it is not easy to distinguish it from nonadaptive randomness in phenotype production, and its convincing demonstrations are lacking. Here we describe a new method for detection of adaptive stochastic polyphenism and apply it to the following problem. In fall, each female of the bird cherry-oat aphid, Rhopalosiphum padi, faces a decision either to produce sexuals, which mate and lay cold-tolerant eggs, or to continue production of cold-sensitive parthenogenetic females, which potentially yields a higher population growth rate but is risky because a cold winter can kill all of her descendants. Using a simulation model, we show that global investment in sexual reproduction should be proportional to winter severity and that variance in the peak date of production of sexual individuals should depend on climate predictability. Both predictions are validated against standardized trap data on aphid flight accompanied by meteorological data, and the predictions support adaptive phenotypic plasticity.     

Heat shock modulates phosphorylation status and activity of nucleoside diphosphate kinase in cultured sugarcane cells

Nucleoside diphosphate kinase (NDPK) is involved in the regeneration of nucleoside triphosphates (NTPs) through its phosphotransferase activity via an autophosphorylating histidine residue. Additionally, autophosphorylation of serine and/or threonine residues is documented for NDPKs from various organisms. However, the metabolic significance of serine/threonine phosphorylation has not been well characterized. In this study we report the cloning and characterization of NDPKI from cultured sugarcane (Saccharum officinarum L. line H50-7209) cells, and modulation of serine autophosphorylation of NDPK1 in response to heat-shock (HS). Heat-shock treatment at 40°C for 2 h resulted in a 40% reduction in labeled phosphoserine in NDPK1. This dephosphorylation was accompanied by an increase in NDPK enzyme activity. In contrast, NDPK1 in cultured tobacco (cv. W-38) cells did not show changes in autophosphorylation or increased enzyme activity in response to HS. The mRNA or protein level of NDPK1 did not increase in response to HS. Sugarcane cells sustain the constitutive protein synthesis in addition to heat-shock protein synthesis during HS, while constitutive protein synthesis is significantly reduced in tobacco cells during HS. Thus, HS modulation of NDPK1 activity and serine dephosphorylation in sugarcane cells may represent an important physiological role in maintaining cellular metabolic functions during heat stress.     

Immunoregulatory effects of freeze injured whole tumour cells on human dendritic cells using an in vitro cryotherapy model

Tumour cryotherapy has been described as both immunostimulatory and immunoinhibitory in previous studies. However, previous studies have not accurately reproduced the precise conditions of current clinical cryotherapy. The objective of this study is to assess the immunological effects of cryotreated whole tumour cells on dendritic cells (DC) maturation and function using an in vitro model. Prostate cancer cells were cooled using Endocare cryo-system to mimic temperatures achieved during clinical cryotherapy. Human DC were prepared from cluster of differentiation (CD) 14 monocytes and matured with lipopolysaccharide (LPS). Cryotreated cancer cells were added to DC on day 3. On day 7, DC were harvested and phenotyped. Cytokine gene expression was assessed using real time quantitative polymerase chain reaction (RT-PCR). Functional activity of DC was assessed in allogenic mixed lymphocyte reaction (MLR) and the molecular changes using gene microarray technology. There was statistically significant upregulation of costimulatory molecules and maturation markers (CD86, CD83, CD80 and CL II) in DC loaded with cryotreated whole tumour cells compared to both control DC and DC matured with LPS (P < 0.001). There was a significant increase in stimulatory cytokines gene expression (IL-2, IL-12, IL-15, IL-18 and IFN-γ). However, IL-10 and TGF-β expression reduced significantly. The effect of different freezing temperature was equal. cDNA microarray analysis showed upregulation of interleukin 1 (IL-1) and cycline dependent kinase inhibitor 1A (CDKN1A (p21) and downregulation of Caspase 8 and BCL2. Overall, our findings suggest that the effect of cryotherapy is generally stimulatory to DC which may enhance anti-tumour effects. Therefore, the combination of cryotherapy and DC vaccine may represent a novel method to increase the efficacy of cryotherapy especially at the peripheral zones of the prostate where cells are exposed to sub-lethal temperature.     

Changes in Oregon white oak (Quercus garryana Dougl. ex Hook.) following release from overtopping conifers

Oregon white oak or Garry oak (Quercus garryana Dougl. ex Hook.) is a shade-intolerant, deciduous species that has been overtopped by conifers during the past century in parts of its range due to an altered disturbance regime. We examined the response of suppressed Oregon white oak trees in western Washington, USA, to three levels of release from overtopping Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). We treated individual oak trees with either full release from competition, partial (“half”) release from competition, or a stand-level thinning of Douglas-fir not directed toward release (control). Five years after treatment, oak trees had suffered no mortality or windthrow. Stem diameter growth was 194% greater in the full-release treatment relative to the control. Acorn production varied widely by year, but in years of higher production, acorn production was significantly greater in both release treatments than in the control. Frequency of epicormic branch formation was significantly increased for years 1 and 2 by the full release; the greatest response occurred between 2 and 6 m above ground level. The greatest number of epicormic branches formed on trees on which the majority of original limbs had died back prior to treatment. Trees with relatively less crown dieback at the time of treatment generally had greater stem growth and acorn production responses to release treatments. Our findings indicate that these released Oregon white oak trees are beginning to recover after an extended period of suppression.     

Modular, self-assembling peptide linkers for stable and regenerable carbon nanotube biosensor interfaces

As part of an effort to develop nanoelectronic sensors for biological targets, we tested the potential to incorporate coiled coils as metallized, self-assembling, site-specific molecular linkers on carbon nanotubes (CNTs). Based on a previously conceived modular anchor-probe approach, a system was designed in which hydrophobic residues (valines and leucines) form the interface between the two helical peptide components. Charged residues (glutamates and arginines) on the borders of the hydrophobic interface increase peptide solubility, and provide stability and specificity for anchor-probe assembly. Two histidine residues oriented on the exposed hydrophilic exterior of each peptide were included as chelating sites for metal ions such as cobalt. Cysteines were incorporated at the peptide termini for oriented, thiol-mediated coupling to surface plasmon resonance (SPR) biosensor surfaces, gold nanoparticles or CNT substrates. The two peptides were produced by solid phase peptide synthesis using Fmoc chemistry: an acidic 42-residue peptide E42C, and its counterpart in the heterodimer, a basic 39-residue peptide R39C. The ability of E42C and R39C to bind cobalt was demonstrated by immobilized metal affinity chromatography and isothermal titration calorimetry. SPR biosensor kinetic analysis of dimer assembly revealed apparent sub-nanomolar affinities in buffers with and without 1 mM CoCl2 using two different reference surfaces. For device-oriented CNT immobilization, R39C was covalently anchored to CNT tips via a C-terminal cysteine residue. Scanning electron microscopy was used to visualize the assembly of probe peptide (E42C) N-terminally labeled with 15 nm gold nanoparticles, when added to the R39C-CNT surface. The results obtained open the way to develop CNT tip-directed recognition surfaces, using recombinant and chemically synthesized chimeras containing binding epitopes fused to the E42C sequence domain.     

Bactericidal and cyclooxygenase inhibitory diterpenes from Eremophila sturtii

Two serrulatane diterpenes, 3,8-dihydroxyserrulatic acid (1) and serrulatic acid (2), have been isolated from Eremophila sturtii through bioassay-guided fractionation. These compounds inhibit the inflammation pathway enzymes cyclooxygenase 1 and 2, and exhibit bactericidal activity against Staphylococcus aureus.