Novel Algorithms for Improved Sensitivity in Non-Invasive Prenatal Testing.

Non-invasive prenatal testing (NIPT) of cell-free DNA in maternal plasma, which is a mixture of maternal DNA and a low percentage of fetal DNA, can detect fetal aneuploidies using massively parallel sequencing. Because of the low percentage of fetal DNA, methods with high sensitivity and precision are required. However, sequencing variation lowers sensitivity and hampers detection of trisomy samples. Therefore, we have developed three algorithms to improve sensitivity and specificity: the chi-squared-based variation reduction (χ2VR), the regression-based Z-score (RBZ) and the Match QC score. The χ2VR reduces variability in sequence read counts per chromosome between samples, the RBZ allows for more precise trisomy prediction, and the Match QC score shows if the control group used is representative for a specific sample. We compared the performance of χ2VR to that of existing variation reduction algorithms (peak and GC correction) and that of RBZ to trisomy prediction algorithms (standard Z-score, normalized chromosome value and median-absolute-deviation-based Z-score). χ2VR and the RBZ both reduce variability more than existing methods, and thereby increase the sensitivity of the NIPT analysis. We found the optimal combination of algorithms was to use both GC correction and χ2VR for pre-processing and to use RBZ as the trisomy prediction method.

Genetic correlation between heart ratio and body weight as a function of ascites frequency in broilers split up into sex and health status.

Ascites or pulmonary hypertension syndrome is a metabolic disorder in broilers. Male broilers have a higher BW and are therefore expected to be more prone to developing ascites than females. As genetic parameters might be affected by the ascites incidence, genetic parameters might differ between male and female broilers. The aims of this study were to estimate the heritability for the ratio of right ventricular weight to total ventricular weight (RATIO) and BW in male and female broilers, the genetic correlation between RATIO and BW separately for male and female broilers, and the genetic correlations between BW for ascitic and nonascitic broilers. Data were available from 7,856 broilers (3,819 males and 4,037 females). The broilers in the experiment were kept under a cold temperature regimen and increased CO(2) levels. In this study, we showed that the incidence of ascites is higher in male than in female broilers. Heritability estimates for BW at 7 wk of age were higher for male (0.22) than for female (0.17) broilers, and for RATIO heritability, estimates were higher for female (0.44) than for male (0.32) broilers. The genetic correlations between RATIO and BW measured at different ages changed from slightly positive at 2 wk of age to moderately negative at 7 wk. The change in genetic correlation was more extreme for male (from 0.01 to -0.62) than for female (from 0.13 to -0.24) broilers. The difference in ascites incidence between male and female broilers is the most likely reason for the difference in genetic correlations. The genetic correlation between BW traits measured in broilers with fluid in the abdomen and without fluid in the abdomen decreased from 0.91 at 2 wk to 0.69 at 7 wk. We conclude that under circumstances with ascites, data from male and female broilers should be analyzed separately.

Molecular characterization of the leptin receptor gene as a candidate gene in the pulmonary hypertension syndrome in broiler chickens.

Leptin Receptor Gene (LEPR) is a candidate gene in understanding the genetic basis of the Pulmonary Hypertension Syndrome (PHS) in broilers. Identification and evaluation of genetic polymorphisms in LEPR may provide a link between traits like Body Weight (BW) and Total Ventricle weight (TV) to the development of PHS. In this study, primers were designed in exons, upstream and downstream sequences to identify mutations in the LEPR on four broilers selected with respect to the PHS-related traits. About 77% of the 11,820 bp of the LEPR gene covered by the primers were sequenced. No mutations were found between the chickens associating the traits to the occurrence of PHS. However, 42 single nucleotide polymorphisms and four Indels were found between the reference sequences of the red jungle fowl and the experimental population. Ten of these mutations were not previously reported in LEPR at the genomic and transcript sequences (NP_989654.1, ENSGALT00000018009). The 10 mutations include six SNPs in intron regions, two Indels and two non-synonymous SNPs. The two new non-synonymous SNPs; G301A and A1637G, led to amino acid change A89T and N534S, respectively.

Gene expression analysis of precision-cut human liver slices indicates stable expression of ADME-Tox related genes.

In the process of drug development it is of high importance to test the safety of new drugs with predictive value for human toxicity. A promising approach of toxicity testing is based on shifts in gene expression profiling of the liver. Toxicity screening based on animal liver cells cannot be directly extrapolated to humans due to species differences. The aim of this study was to evaluate precision-cut human liver slices as in vitro method for the prediction of human specific toxicity by toxicogenomics. The liver slices contain all cell types of the liver in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process. Previously we showed that toxicogenomic analysis of rat liver slices is highly predictive for rat in vivo toxicity. In this study we investigated the levels of gene expression during incubation up to 24 h with Affymetrix microarray technology. The analysis was focused on a broad spectrum of genes related to stress and toxicity, and on genes encoding for phase-I, -II and -III metabolizing enzymes and transporters. Observed changes in gene expression were associated with cytoskeleton remodeling, extracellular matrix and cell adhesion, but for the ADME-Tox related genes only minor changes were observed. PCA analysis showed that changes in gene expression were not associated with age, sex or source of the human livers. Slices treated with acetaminophen showed patterns of gene expression related to its toxicity. These results indicate that precision-cut human liver slices are relatively stable during 24h of incubation and represent a valuable model for human in vitro hepatotoxicity testing despite the human inter-individual variability.

The use of blood gas parameters to predict ascites susceptibility in juvenile broilers.

Ascites syndrome is a metabolic disorder found in modern broilers that have insufficient pulmonary vascular capacity. Commercial breeding programs have heavily focused on high growth rate, which led to fast-growing chickens, but as a negative consequence, the incidence of ascites syndrome increased. However, not all birds with a high growth rate will suffer from ascites syndrome, which might indicate a genetic susceptibility to ascites. Information on blood gas parameters measured early in life and their relation to ascites susceptibility is expected to contribute to identification on the cause of ascites syndrome. In this study, several physiological parameters, such as blood gas parameters [pH, partial pressure of CO(2) in venous blood (pvCO(2)), and partial pressure of O(2) in venous blood], hematocrit, electrolytes (Na(+), Ca(2+), and K(+)), metabolites (lactate and glucose), were measured at d 11 to 12 of age from 100 female and 100 male broilers. From d 14 onward, the birds were challenged to provoke the development of ascites syndrome. Our results showed that high pvCO(2) values together with low pH values (males) or high pH values (females) in the venous blood of juvenile broilers coincided with ascites. Therefore, blood pvCO(2) and pH in both juvenile male and female broilers seem to be critical factors in ascites pathophysiology and can be used as phenotypic traits to predict ascites susceptibility in juvenile broilers at d 11 to 12. A prediction model was built on a subpopulation of the broilers without any loss in sensitivity (0.52) and specificity (0.78) when applied to the validation population. The parameter sex was included in the prediction model because levels of pvCO(2) and pH that associated with ascites susceptibility are different between males and females. Commercial breeders can include these phenotypic traits in their genetic selection programs to reduce the incidence of ascites syndrome.

Microarray analysis in rat liver slices correctly predicts in vivo hepatotoxicity.

The microarray technology, developed for the simultaneous analysis of a large number of genes, may be useful for the detection of toxicity in an early stage of the development of new drugs. The effect of different hepatotoxins was analyzed at the gene expression level in the rat liver both in vivo and in vitro. As in vitro model system the precision-cut liver slice model was used, in which all liver cell types are present in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process involving not only hepatocytes but also other cell types such as Kupffer and stellate cells. As model toxic compounds lipopolysaccharide (LPS, inducing inflammation), paracetamol (necrosis), carbon tetrachloride (CCl(4), fibrosis and necrosis) and gliotoxin (apoptosis) were used. The aim of this study was to validate the rat liver slice system as in vitro model system for drug-induced toxicity studies. The results of the microarray studies show that the in vitro profiles of gene expression cluster per compound and incubation time, and when analyzed in a commercial gene expression database, can predict the toxicity and pathology observed in vivo. Each toxic compound induces a specific pattern of gene expression changes. In addition, some common genes were up- or down-regulated with all toxic compounds. These data show that the rat liver slice system can be an appropriate tool for the prediction of multi-cellular liver toxicity. The same experiments and analyses are currently performed for the prediction of human specific toxicity using human liver slices.

Coordinated induction of drug transporters and phase I and II metabolism in human liver slices.

Although regulation of phase I drug metabolism in human liver is relatively well studied, the regulation of phase II enzymes and of drug transporters is incompletely characterized. Therefore, we used human liver slices to investigate the PXR, CAR and AhR-mediated induction of drug transporters and phase I and II metabolic enzymes. Precision-cut human liver slices were incubated for 5 or 24h with prototypical inducers: phenobarbital (PB) (50 microM) for CAR, beta-naphthoflavone (BNF) (25 microM) for AhR, and rifampicin (RIF) (10 microM) for PXR, and gene expression of the phase I enzymes CYP1A1, 1A2, 3A4, 3A5, 2B6, 2A6, the phase II enzymes UGT1A1 and 1A6, and the transporters MRP2, MDR1, BSEP, NTCP and OATP8 was measured. BNF induced CYP1A1, UGT1A1 and UGT1A6 and MRP2, NTCP and MDR1. RIF induced CYP3A4, 3A5, 2B6, 2A6, UGT1A1, UGT1A6 and BSEP, MRP2 and MDR1 and slightly downregulated OATP8. PB induced CYP3A4, 3A5, 2B6 and 2A6, UGT1A1 and all transporters. Large interindividual differences were found with respect to the level of induction. Enzyme activity of CYP3A4, measured by testosterone metabolism, was increased after 24h by RIF. 7-Ethoxycoumarin O-deethylation activity, mediated predominantly by CYP 1A1/1A2 but also by other CYPs, was increased after 24h with PB. We have shown that regulation of all phases of the (in)activation of a drug via the CAR, AhR and the PXR pathways can be studied in human liver slices. The concomitant induction of metabolic enzymes and transporters shows that also in the human liver transporters and metabolic enzymes are regulated coordinately.

An ex vivo human model system to evaluate specificity of replicating and non-replicating gene therapy agents.

BACKGROUND: Inefficiency, aspecificity and toxicity of gene transfer vectors hamper gene therapy from showing its full potential. On this basis significant research currently focuses on developing vectors with improved infection and/or expression profiles. Screening assays with validity to the clinical context to determine improved characteristics of such agents are not readily available since this requires a close relationship to the human situation. We present a clinically relevant tissue slice technology to preclinically test improved vector characteristics. METHODS: Slices were prepared from rat, mouse and human liver samples and from tumor tissue. Specificity of gene expression and replication was determined by infecting target and non-target tissue slices with transcriptionally retargeted adenoviruses and oncolytic viruses. RESULTS: Using rat liver slices, we demonstrate efficient knob-mediated adenoviral infectivity. A favorable tumor-on/liver-off profile, resembling in vitro and mouse in vivo data, was shown for a tumor-specific transcriptionally retargeted adenovirus by infecting slices prepared from tumor or liver tissue. Similar liver-off data were found for mouse, rat and human samples (over 3-log lower activity of the tumor-specific promoter compared to cytomegalovirus (CMV)). More importantly, we show that this technology when applied to human livers is a powerful tool to determine aspecific replication of oncolytic viruses in liver tissue. A 2- to 6-log reduction in viral replication was observed for a tumor-specific oncolytic virus compared to the wild-type adenovirus. CONCLUSIONS: The precision-cut tissue slice technology is a powerful method to test specificity and efficiency of gene transfer as well as of viral replication using human tissue.

Cellobiose uptake in the hyperthermophilic archaeon Pyrococcus furiosus is mediated by an inducible, high-affinity ABC transporter.

The hyperthermophilic archaeon Pyrococcus furiosus can utilize different beta-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (K(m) = 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from P. furiosus membranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The cbtA gene was cloned and functionally expressed in Escherichia coli. cbtA belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters.

Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein-dependent ABC transporters.

The extreme thermoacidophilic archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 3 and uses a variety of sugars as sole carbon and energy source. Glucose transport in this organism is mediated by a high-affinity binding protein-dependent ATP-binding cassette (ABC) transporter. Sugar-binding studies revealed the presence of four additional membrane-bound binding proteins for arabinose, cellobiose, maltose and trehalose. These glycosylated binding proteins are subunits of ABC transporters that fall into two distinct groups: (i) monosaccharide transporters that are homologous to the sugar transport family containing a single ATPase and a periplasmic-binding protein that is processed at an unusual site at its amino-terminus; (ii) di- and oligosaccharide transporters, which are homologous to the family of oligo/dipeptide transporters that contain two different ATPases, and a binding protein that is synthesized with a typical bacterial signal sequence. The latter family has not been implicated in sugar transport before. These data indicate that binding protein-dependent transport is the predominant mechanism of transport for sugars in S. solfataricus.

Glucose transport in the extremely thermoacidophilic Sulfolobus solfataricus involves a high-affinity membrane-integrated binding protein.

The archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 2.5 to 3.5 on carbon sources such as yeast extracts, tryptone, and various sugars. Cells rapidly accumulate glucose. This transport activity involves a membrane-bound glucose-binding protein that interacts with its substrate with very high affinity (Kd of 0. 43 microM) and retains high glucose affinity at very low pH values (as low as pH 0.6). The binding protein was extracted with detergent and purified to homogeneity as a 65-kDa glycoprotein. The gene coding for the binding protein was identified in the S. solfataricus P2 genome by means of the amino-terminal amino acid sequence of the purified protein. Sequence analysis suggests that the protein is anchored to the membrane via an amino-terminal transmembrane segment. Neighboring genes encode two membrane proteins and an ATP-binding subunit that are transcribed in the reverse direction, whereas a homologous gene cluster in Pyrococcus horikoshii OT3 was found to be organized in an operon. These data indicate that S. solfataricus utilizes a binding-protein-dependent ATP-binding cassette transporter for the uptake of glucose.

Slow fusion of liposomes composed of membrane-spanning lipids.

The fusion characteristics of large unilamellar liposomes composed of bipolar tetraether lipids extracted from the thermophilic archaeon Sulfolobus acidocaldarius, was investigated. These lipids span the entire membrane and form single monolayer liposomes in aqueous media [Elferink, M.G.L., de Wit, J.G., Demel, R., Driessen, A.J.M. and Konings, W.N., (1992) J. Biol. Chem. 267, 1375-1381]. In the presence of calcium-phosphate, slow mixing of the aqueous liposome contents and membrane lipids occurred, demonstrating that these liposomes are fusion-competent. The fusion process was essentially nonleaky. The rate of fusion increased with the pH and the concentration of calcium and phosphate. Fusion resulted in an increase of the size of the liposomes. These data demonstrate that a monolayer organization of lipids in a membrane does not per se interfere with membrane fusion competence.

Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector.

We describe a transformation system for extremely thermophilic archaea of the genus Sulfolobus in the kingdom Crenarchaeota. We have constructed in vitro a recombinant derivative of the recently described conjugative plasmid pNOB8, containing a beta-galactosidase gene downstream of a strong promotor. Transformation of a beta-galactosidase negative mutant of Sulfolobus solfataricus with this construct resulted in its spreading through the culture containing the primary transformants and in efficient restoration of beta-galactosidase activity.

Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of bacteria and archaea.

Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions. In some thermophiles, sodium is the sole energy-coupling ion. To test whether sodium is the preferred coupling ion at high temperatures, the proton- and sodium permeability was determined in liposomes prepared from lipids isolated from various bacterial and archaeal species that differ in their optimal growth temperature. The proton permeability increased with the temperature and was comparable for most species at their respective growth temperatures. Liposomes of thermophilic bacteria are an exception in the sense that the proton permeability is already high at the growth temperature. In all liposomes, the sodium permeability was lower than the proton permeability and increased with the temperature. The results suggest that the proton permeability of the cytoplasmic membrane is an important parameter in determining the maximum growth temperature.

Thermostability of respiratory terminal oxidases in the lipid environment.

The effect of the lipid environment on the thermostability of three respiratory terminal oxidases was determined. Cytochrome-c oxidase from beef heart and Bacillus stearothermophilus were used as representative proteins from mesophilic and thermophilic origin, respectively. Quinol oxidase from the archaeon Sulfolobus acidocaldarius represented the model for a extreme thermoacidophilic enzyme. All three integral membrane proteins were tested for their thermal inactivation in detergent and after reconstitution in liposomes composed of phospholipids of Escherichia coli or tetraether lipids from S. acidocaldarius. When preincubated at 0 degrees C, all three enzymes exhibited biphasic thermal inactivation curves. Data could be analysed according to a two-state model that defines two conformations of the enzyme, differing in their thermostability. Monophasic inactivation curves were observed when the enzymes were preincubated at higher temperatures prior to thermal inactivation. Lipids rendered the beef-heart cytochrome-c oxidase and S. acidocaldarius quinol oxidase more thermostable as compared to detergent solution. In contrast, the B. stearothermophilus oxidase, an intrinsically thermostable enzyme, was as thermostable in detergent as in the reconstituted state. These data suggest that the lipid environment can be an important factor in the thermostability of membrane proteins.

Generation of proton-motive force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius.

The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius. In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol. The enzyme was functionally reconstituted into monolayer liposomes composed of archaeal tetraether lipids also derived from S. acidocaldarius. With the electron donor system ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine, the reconstituted enzyme was more active in the archaeal lipids as compared to lipids derived from Escherichia coli at temperatures above 50 degrees C. Due to the low proton permeability of the tetraether lipids, it was possible to generate a steady-state transmembrane electrical potential (delta psi, interior negative), and transmembrane pH gradient (delta pH, interior alkaline) at temperatures up to 70 degrees C. The successful functional reconstitution of the cytochrome aa3-type quinol oxidase from Sulfolobus identifies it as the key energy converter in the respiratory system of this hyperthermophilic archaeon.

Stability and proton-permeability of liposomes composed of archaeal tetraether lipids.

Liposomes composed of tetraether lipids originating from the thermoacidophilic archaeon Sulfolobus acidocaldarius were analyzed for their stability and proton permeability from 20 degrees C up to 80 degrees C. At room temperature, these liposomes are considerably more stable and have a much lower proton permeability than liposomes composed of diester lipids originating from the mesophilic bacterium Escherichia coli or the thermophilic bacterium Bacillus stearothermophilus. With increasing temperature, the stability decreased and the proton permeability increased for all liposomes. Liposomes composed from tetraether lipids, however, remain the most stable. These data suggest these liposomes retain the rigidity of the cytoplasmic membrane of S. acidocaldarius needed to endure extreme environmental growth conditions.

Energy-transducing properties of primary proton pumps reconstituted into archaeal bipolar lipid vesicles.

Archaeal lipids differ considerably from eubacterial and eukaryotic lipids in their structure and physical properties. From the membranes of the extreme thermophilic archaea Sulfolobus acidocaldarius a tetraether lipid fraction was isolated, which can form closed and stable monolayer liposomes in aqueous media. The function of three different primary proton pumps originating from archaeal, bacterial and eukaryotic lipid sources have been studied after reconstitution in these liposomes: bacteriorhodopsin from the archaea Halobacterium halobium; cytochrome-c oxidase from the thermophilic bacterium Bacillus stearothermophilus and cytochrome-c oxidase from beef heart mitochondria. Liposomes composed of tetraether lipids form a competent matrix for all three exogenous proton pumps. Bacteriorhodopsin was inserted inside-out in these liposomes, as normally observed in bilayer-forming lipid. The activities of the two oxidases were inhibited at high tetraether-lipid concentration, probably due to the low fluidity of these membranes. Only bacteriorhodopsin, which originates from diether archaeal lipids is fully functional in the tetraether membranes.

In vitro pore-forming activity of the lantibiotic nisin. Role of protonmotive force and lipid composition.

Nisin is a lantibiotic produced by some strains of Lactococcus lactis subsp. lactis. The target for nisin action is the cytoplasmic membrane of Gram-positive bacteria. Nisin dissipates the membrane potential (delta psi) and induces efflux of low-molecular-mass compounds. Evidence has been presented that a delta psi is needed for nisin action. The in vitro action of nisin was studied on liposomes loaded with the fluorophore carboxyfluorescein. Nisin-induced efflux of carboxyfluorescein was observed in the absence of a delta psi from liposomes composed of Escherichia coli lipids or dioleoylglycerophosphocholine (Ole2GroPCho) at low nisin/lipid ratios. The initial rate of carboxyfluorescein efflux is dependent on the nisin/lipid ratio and saturates at high ratios. Both delta psi (inside negative) and delta pH (inside alkaline) enhance the action of nisin, while nisin is more potent at acidic external pH values. Efficient carboxyfluorescein efflux is observed with the zwitterionic phospholipid Ole2GroPCho or mixtures of Ole2GroPCho with dioleoylglycerophosphoethanolamine and neutral glycolipids, while anionic phospholipids are strongly inhibitory. It is concluded that a delta psi is not essential, but that the total protonmotive force stimulates the action of nisin.

Reconstitution of the leucine transport system of Lactococcus lactis into liposomes composed of membrane-spanning lipids from Sulfolobus acidocaldarius.

The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated. Liposomes were prepared from mixtures of monolayer lipids and the bilayer lipid phosphatidylcholine (PC), analyzed on their miscibility, and fused with membrane vesicles from L. lactis. Freeze-fracture electron microscopy demonstrates that the bipolar lipids in the hybrid membranes adopted a monomolecular organization at high S. acidocaldarius lipid content. Leucine transport activity (i.e., delta mu H(+)-driven and counterflow uptake) increased with the content of S. acidocaldarius lipids and was optimal at a one-to-one (w/w) ratio of PC to S. acidocaldarius lipids. Membrane fluidity decreased with increasing S. acidocaldarius lipid content. These data suggest that transport proteins can be functionally reconstituted into membranes composed of membrane-spanning lipids provided that membrane viscosity is restricted.