Predictors of neonatal morbidity in fetuses with severe isolated congenital diaphragmatic hernia undergoing fetoscopic tracheal occlusion.

OBJECTIVES: To investigate neonatal morbidity in fetuses with severe congenital diaphragmatic hernia (CDH) treated with fetoscopic endoluminal tracheal occlusion (FETO) and compare it with historical controls with less severe forms of CDH that were managed expectantly. METHODS: This was a prospective, multicenter study on neonatal outcomes and prenatal predictors in 90 FETO survivors (78 left-sided, 12 right) and 41 controls from the antenatal CDH registry with either severe or moderate hypoplasia who were managed expectantly. We also investigated early neonatal morbidity indicators, including the need for patch repair, duration of mechanical ventilation and supplemental oxygen, age at full enteral feeding and incidence of pulmonary hypertension. RESULTS: Gestational age at delivery was predictive of duration of assisted ventilation (P = 0.046), days on supplemental oxygen (P = 0.019) and age at full enteral feeding (P = 0.020). When delivery took place after 34 weeks' gestation, neonatal morbidity of FETO cases was comparable with that of expectantly managed cases with moderate hypoplasia. CONCLUSIONS: Fetal intervention for severe CDH is associated with neonatal morbidity that is comparable with that of an expectantly managed group with less severe disease.

Gestational age-specific reference ranges for amniotic fluid assessment in monochorionic diamniotic twin pregnancies.

OBJECTIVES: To establish gestational age-specific reference ranges for amniotic fluid measurements in monochorionic diamniotic twin pregnancies, to compare them with previously reported singleton and twin reference ranges and to examine the rationale for using a gestational age-dependent cut-off to define polyhydramnios in twin-twin transfusion syndrome, as is the practice in most European centers. METHODS: We retrospectively evaluated amniotic fluid volume in 32 monochorionic diamniotic twin pregnancies that were followed longitudinally at 2-week intervals from the first trimester until birth. Amniotic fluid volume was assessed by measuring the deepest vertical pocket in both amniotic sacs. We used multilevel modeling to estimate the gestational age-specific reference ranges for deepest vertical pocket measurements. RESULTS: Based on 429 observations in 64 fetuses, we constructed gestational age-specific reference ranges from 11 weeks until term. The deepest pocket increased from the first trimester to reach a maximum at 26 weeks, followed by a gradual decrease towards term. Measurements between 18 and 28 weeks were comparable to those in singleton pregnancies. However, before 18 weeks values were higher, whereas after 28 weeks they were lower, as compared to singleton references. CONCLUSION: In monochorionic twin pregnancies, the deepest vertical pocket is a gestational age-dependent measurement. Therefore, a gestational age-dependent definition of polyhydramnios in twin-twin transfusion syndrome, as used by most European centers, seems a logical approach.

Maternal hyperoxygenation test in fetuses undergoing FETO for severe isolated congenital diaphragmatic hernia.

OBJECTIVES: To predict neonatal survival and pulmonary hypertension by measurement of fetal pulmonary artery reactivity to maternal hyperoxygenation in fetuses with severe congenital diaphragmatic hernia treated by fetoscopic endoluminal tracheal occlusion (FETO). METHODS: Thirty-eight fetuses underwent FETO at around 28 weeks' gestation and the balloon was removed at 34 weeks in most cases. We performed a hyperoxygenation test and measured the lung-to-head ratio of each fetus before and after each procedure. Outcome measures were neonatal survival, occurrence of pulmonary hypertension and its response to inhaled nitric oxide (iNO). RESULTS: Fetuses that survived had a larger increase in lung size and decrease of resistance in the first branch of the main pulmonary artery than did those that died. Both measures were also predictive of pulmonary hypertension unresponsive to iNO. The hyperoxygenation test and lung-to-head ratio were both best predictive for neonatal survival when measured following removal of the balloon (P < 0.002). Discriminant analysis confirmed that these two parameters are independent predictors of outcome. CONCLUSIONS: In fetuses undergoing FETO, pulmonary vascular reactivity in relation to oxygen and lung size are independent predictors of neonatal survival and pulmonary hypertension. The hyperoxygenation test merits further study in expectantly managed cases.

Accuracy of peritoneal fluid measurements by transvaginal ultrasonography.

OBJECTIVE: To assess the accuracy of the assessment of peritoneal fluid volumes of up to 1 L by transvaginal ultrasonography and to re-evaluate the formula used to calculate total volume from the dimensions of the largest pocket. METHODS: Patients (n = 13) enrolled for a minor laparoscopic procedure were prospectively recruited. At the end of the procedure, with the patient in the 30 degrees anti-Trendelenburg position, Ringer's lactate was instilled into the abdomen in discrete steps up to 1 L. Following equilibration the diameters of the single pocket of fluid were measured by transvaginal ultrasonography in order to calculate the volume, and regression models were used to determine the relationship between this and the instilled volume. The body mass index (BMI) of the patient was evaluated as a parameter for predicting the instilled volume more accurately. RESULTS: The intra-abdominal fluid volume could be calculated from the measured volume using a quadratic regression equation with an overall coefficient of variation of 19%. In individual patients, changes in volume could be assessed with a coefficient of variation of 7.3%. BMI was not found to be a significant parameter in relating the measured to the instilled volume. CONCLUSION: Transvaginal ultrasound in a standardized setting can accurately estimate the volume of peritoneal fluid, with the accuracy consistent for small and large volumes. Changes in peritoneal fluid volume over time in the same individual can be measured more accurately than the total volume present.

Reference ranges for middle cerebral artery peak systolic velocity in monochorionic diamniotic twins: a longitudinal study.

OBJECTIVES: The role of middle cerebral artery (MCA) peak systolic velocity (PSV) has become established in the management of fetal anemia. To date, singleton reference ranges have also been used in twin pregnancies. However, in monochorionic twin pregnancies, normal ranges for cerebral blood flow may differ from those in singletons owing to intertwin blood exchange. We aimed to establish gestational age-specific reference ranges for MCA-PSV in monochorionic diamniotic (MCDA) twin pregnancies, to compare them with previously reported singleton reference ranges, and to establish terms for calculating conditional reference intervals appropriate for individual serial measurements. METHODS: In a prospective longitudinal study we examined 50 uncomplicated MCDA pregnancies that were recruited between 11 and 14 weeks of gestation. Fetal MCA Doppler waveforms were assessed biweekly from 15 weeks of gestation onwards. Multilevel modeling was used to estimate gestational age-specific reference ranges for MCA-PSV and terms for conditional reference intervals were established. RESULTS: Based on 824 observations in 100 fetuses, normative ranges from 15 to 37 weeks of gestation were constructed. Median MCA-PSV increased with advancing gestational age. Measurements between 18 and 37 weeks were comparable to those in singletons. However, before 18 weeks MCA-PSV values were higher in MCDA twin pregnancies compared with singleton references. CONCLUSIONS: Between 18 and 37 weeks of gestation, reference ranges of singletons can be used to assess fetal anemia in MCDA twin pregnancies. Prior to 18 weeks the application of singleton references may lead to an increased number of false-positive diagnoses of presumed fetal anemia in MCDA twin pregnancies.

Validation of the fetal myocardial performance index in the second and third trimesters of gestation.

OBJECTIVES: To test the validity of the myocardial performance index (MPI) and its components against the more conventional methods of fetal cardiac function assessment: the ejection fraction (EF) for systolic function and the E/A index (ratio of transmitral flow during early (E) ventricular filling to flow during atrial (A) contraction) for diastolic function, both in a normal population and in a population at risk for cardiac failure because of volume overload (recipient fetuses in cases of twin-twin transfusion syndrome (TTTS)). METHODS: The MPI was measured prospectively in addition to more commonly used indices of systolic (EF) and diastolic (E/A index) cardiac function in 117 healthy fetuses (gestational age range, 20-36 weeks) and in 14 fetuses suspected of cardiac failure because of the presence of TTTS. Nomograms were constructed for all variables, and correlations between the MPI, EF and E/A index were assessed. The time taken to obtain the measurements as well as the interobserver and intraobserver variability were determined for the MPI and EF. RESULTS: In healthy fetuses, the MPI and EF were independent of gestational age, whereas the E/A index and isovolumetric relaxation time (IRT) increased with gestational age. The MPI correlated inversely with the EF (P<0.001). The IRT showed a trend towards an inverse correlation with the E/A index (P=0.10). The mean+/-SD time needed to measure the MPI and EF was 140+/-65 s and 185+/-187 s, respectively (P=0.43). Interobserver and intraobserver intraclass correlation coefficients for the MPI were 0.98 (95% CI, 0.85-0.99) and 0.82 (95% CI, 0.14-0.95), respectively; those for the EF were 0.58 (95% CI, -0.16 to 0.85) and 0.51 (95% CI, -0.46 to 0.83), respectively; and those for the E/A index were 0.97 (95% CI, 0.88-0.99) and 0.91 (95% CI, 0.66-0.98), respectively. All variables, except ejection time, were significantly different between normal fetuses and those with TTTS. CONCLUSIONS: The MPI is an indicator of the systolic component of fetal left ventricular function that can be easily acquired and reproduced. The MPI is strongly correlated with the EF but shows less interobserver and intraobserver variability.

Improving QSAR models for the biological activity of HIV Reverse Transcriptase inhibitors: Aspects of outlier detection and uninformative variable elimination.

The goal of this study is to derive a methodology for modeling the biological activity of non-nucleoside HIV Reverse Transcriptase (RT) inhibitors. The difficulties that were encountered during the modeling attempts are discussed, together with their origin and solutions. With the selected multivariate techniques: robust principal component analysis, partial least squares, robust partial least squares and uninformative variable elimination partial least squares, it is possible to explore and to model the contaminated data satisfactory. It is shown that these techniques are versatile and valuable tools in modeling and exploring biochemical data.

Classification and regression trees--studies of HIV reverse transcriptase inhibitors.

In this paper, the application of Classification And Regression Trees (CART) is presented for the analysis of biological activity of Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). The data consist of the biological activities, expressed as pIC50, of 208 NNRTIs against wild-type HIV virus (HIV-1) and four mutant strains (181C, 103N, 100I, 188L) and the computed interaction energies with the Reverse Transcriptase (RT) binding pocket. CART explains the observed biological activity of NNRTIs in terms of interactions with individual amino acids in the RT binding pocket, i.e., the original data variables.

Bioinformatic insight into the unity and diversity of cytochromes P450.

For the past few decades, cytochromes P450 (CYPs) have been the subject of extensive research, owing to the ability of these enzymes to serve as drug targets as well as to their active participation in drug metabolism. Other varieties and functions of CYPs have been discovered and this superfamily currently comprises over 2000 different protein species. In the present study, the protein sequences of CYPs were submitted to computer analysis for elucidation of the structural basis of their pronounced functional diversity. The basic local alignment search tool (BLAST) was used to demonstrate that CYP protein sequences share a certain general similarity; at the same time, it was shown that the CYP superfamily may be split into a number of groups of intimately related proteins. These groups, the families, were revealed by means of cluster analysis, which demonstrated a strong hierarchy among the animal, bacterial and fungal P450s, and the lack of such a hierarchy for plant enzymes. Multiple alignment and consensus sequence analysis were the approaches taken to find out which structural peculiarities of P450s are responsible for the deviations from the random picture. Proteins within each family were aligned and collapsed to the corresponding consensus sequences, the alignment of which produced the consensus for the whole superfamily. The latter consensus yielded a number of unity motifs (most of which being related to the heme-fixing assembly), while the cross-family comparison of consensus sequences enabled the retrieval of some diversity motifs. Three consensus sequences (for the CYP51 and CYP2 families and for the superfamily) were compared, to line up the unity and diversity motifs with the appropriate X-ray data.

Inhibition and substrate recognition--a computational approach applied to HIV protease.

We have developed a computational approach in which an inhibitor's strength is determined from its interaction energy with a limited set of amino acid residues of the inhibited protein. We applied this method to HIV protease. The method uses a consensus structure built from X-ray crystallographic data. All inhibitors are docked into the consensus structure. Given that not every ligand-protein interaction causes inhibition, we implemented a genetic algorithm to determine the relevant set of residues. The algorithm optimizes the q2 between the sum of interaction energies and the observed inhibition constants. The best possible predictive model resulting has a q2 of 0.63. External validation by examining the predictivity for compounds not used in derivation of the model leads to a prediction accuracy between 0.9 and 1.5 log10 unit. Out of 198 residues in the whole protein, the best internally predictive model defines a subset of 20 residues and the best externally predictive model one of 9 residues. These residues are distributed over the subsites of the enzyme. This approach provides insight in which interactions are important for inhibiting HIV protease and it allows for quantitative prediction of inhibitor strength.

Protein-protein interactions: mechanisms and modification by drugs.

Protein-protein interactions form the proteinaceous network, which plays a central role in numerous processes in the cell. This review highlights the main structures, properties of contact surfaces, and forces involved in protein-protein interactions. The properties of protein contact surfaces depend on their functions. The characteristics of contact surfaces of short-lived protein complexes share some similarities with the active sites of enzymes. The contact surfaces of permanent complexes resemble domain contacts or the protein core. It is reasonable to consider protein-protein complex formation as a continuation of protein folding. The contact surfaces of the protein complexes have unique structure and properties, so they represent prospective targets for a new generation of drugs. During the last decade, numerous investigations have been undertaken to find or design small molecules that block protein dimerization or protein(peptide)-receptor interaction, or on the other hand, induce protein dimerization.

Evolution of anti-HIV drug candidates. Part 2: Diaryltriazine (DATA) analogues.

A synthesis program directed toward improving the stability of imidoyl thiourea based non-nucleoside reverse transcriptase inhibitors (NNRTIs) led to the discovery of diaryltriazines (DATAs), a new class of potent NNRTIs. The synthesis and anti-HIV structure-activity relationship (SAR) studies of a series of DATA derivatives are described.

Evolution of anti-HIV drug candidates. Part 3: Diarylpyrimidine (DAPY) analogues.

The synthesis and anti-HIV-1 activity of a series of diarylpyrimidines (DAPYs) are described. Several members of this novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) are extremely potent against both wild-type and a panel of clinically significant single- and double-mutant strains of HIV-1.

The Lys103Asn mutation of HIV-1 RT: a novel mechanism of drug resistance.

Inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT) are widely used in the treatment of HIV infection. Loviride (an alpha-APA derivative) and HBY 097 (a quinoxaline derivative) are two potent non-nucleoside RT inhibitors (NNRTIs) that have been used in human clinical trials. A major problem for existing anti-retroviral therapy is the emergence of drug-resistant mutants with reduced susceptibility to the inhibitors. Amino acid residue 103 in the p66 subunit of HIV-1 RT is located near a putative entrance to a hydrophobic pocket that binds NNRTIs. Substitution of asparagine for lysine at position 103 of HIV-1 RT is associated with the development of resistance to NNRTIs; this mutation contributes to clinical failure of treatments employing NNRTIs. We have determined the structures of the unliganded form of the Lys103Asn mutant HIV-1 RT and in complexes with loviride and HBY 097. The structures of wild-type and Lys103Asn mutant HIV-1 RT in complexes with NNRTIs are quite similar overall as well as in the vicinity of the bound NNRTIs. Comparison of unliganded wild-type and Lys103Asn mutant HIV-1 RT structures reveals a network of hydrogen bonds in the Lys103Asn mutant that is not present in the wild-type enzyme. Hydrogen bonds in the unliganded Lys103Asn mutant but not in wild-type HIV-1 RT are observed between (1) the side-chains of Asn103 and Tyr188 and (2) well-ordered water molecules in the pocket and nearby pocket residues. The structural differences between unliganded wild-type and Lys103Asn mutant HIV-1 RT may correspond to stabilization of the closed-pocket form of the enzyme, which could interfere with the ability of inhibitors to bind to the enzyme. These results are consistent with kinetic data indicating that NNRTIs bind more slowly to Lys103Asn mutant than to wild-type HIV-1 RT. This novel drug-resistance mechanism explains the broad cross-resistance of Lys103Asn mutant HIV-1 RT to different classes of NNRTIs. Design of NNRTIs that make favorable interactions with the Asn103 side-chain should be relatively effective against the Lys103Asn drug-resistant mutant.

Using genetic algorithms for the construction of phylogenetic trees: application to G-protein coupled receptor sequences.

Many different phylogenetic clustering techniques are used currently. One approach is to first determine the topology with a common clustering method and then calculate the branch lengths of the tree. If the resulting tree is not optimal exchanging tree branches can make some local changes in the tree topology. The whole process can be iterated until a satisfactory result has been obtained. The efficiency of this method fully depends on the initially generated tree. Although local changes are made, the optimal tree will never be found if the initial tree is poorly chosen. In this article, genetic algorithms are applied such that the optimal tree can be found even with a bad initial tree topology. This tree generating method is tested by comparing its results with the results of the FITCH program in the PHYLIP software package. Two simulated data sets and a real data set are used.

Classification and identification of proteins by means of common and specific amino acid n-tuples in unaligned sequences.

Unaligned amino acid sequences can be characterized by their composition of amino acid n-tuples (i.e. doublets, triplets, quadruplets, etc.). In this study we investigated the performance of two statistics, termed commonality and specificity, that are derived from n-tuple counts using a set of G-protein coupled receptor (GPCR) sequences. The commonality of a tuple is defined as its relative occurrence in the sequences that belong to a given GPCR subtype. The specificity of a tuple is derived from its relative occurrence in the sequences of a given GPCR subtype and from its relative non-occurrence in the sequences that do not belong to this subtype. A graphical presentation, termed 'polygram', is described for the visualization of common and specific tuples. The method can be applied to the classification of unknown GPCR sequences. It can also be applied to the identification of fragments of GPCRs, such as may occur in chimeric receptors. The method is generally applicable to other protein families and other types of coding.

Human rhinovirus type 14:human immunodeficiency virus type 1 (HIV-1) V3 loop chimeras from a combinatorial library induce potent neutralizing antibody responses against HIV-1.

In an effort to develop a useful AIDS vaccine or vaccine component, we have generated a combinatorial library of chimeric viruses in which the sequence IGPGRAFYTTKN from the V3 loop of the MN strain of human immunodeficiency virus type 1 (HIV-1) is displayed in many conformations on the surface of human rhinovirus 14 (HRV14). The V3 loop sequence was inserted into a naturally immunogenic site of the cold-causing HRV14, bridged by linkers consisting of zero to three randomized amino acids on each side. The library of chimeric viruses obtained was subjected to a variety of immunoselection schemes to isolate viruses that provided the most useful presentations of the V3 loop sequence for potential use in a vaccine against HIV. The utility of the presentations was assessed by measures of antigenicity and immunogenicity. Most of the immunoselected chimeras examined were potently neutralized by each of the four different monoclonal anti-V3 loop antibodies tested. Seven of eight chimeric viruses were able to elicit neutralizing antibody responses in guinea pigs against the MN and ALA-1 strains of HIV-1. Three of the chimeras elicited HIV neutralization titers that exceeded those of all but a small number of previously described HIV immunogens. These results indicate that HRV14:HIV-1 chimeras may serve as useful immunogens for stimulating immunity against HIV-1. This method can be used to flexibly reconstruct varied immunogens on the surface of a safe and immunogenic vaccine vehicle.

Study of the dynamics of neutralization escape mutants in a chimpanzee naturally infected with the simian immunodeficiency virus SIVcpz-ant.

Here we report on the use of spectral map analysis of time-paired sequential neutralization data of 11 serum samples of a chimpanzee naturally infected with a simian immunodeficiency virus (SIVcpz-ant) and 8 primary consecutive SIVcpz-ant isolates, taken at about 4-month intervals. The analysis reveals the existence of three SIVcpz-ant isolate and serum neutralization clusters. Each cluster groups virus isolates and/or sera based on similarities of their neutralization spectra. On average, neutralization escape mutants emerged after 15 months and mounted a neutralization response approximately 8 months later. The entire gp160 regions of eight consecutive isolates were sequenced and analyzed by a new statistical method called polygram, which allowed the deduction of amino acid sequence motifs of gp160 which were specific for SIVcpz-ant isolates belonging to the same isolate neutralization clusters. Changes in specific amino acid quadruplets in V1, V2, C3, V4, V5, and CD4 domains of gp120 and gp40 were seen to correlate with the neutralization clusters with most of the specific changes occurring in the V4 region. This method of analysis may facilitate an understanding of the study of the dynamic interplay between human immunodeficiency virus (HIV) and host neutralization responses as well as providing possible insights into mechanisms of persistence of HIV-1-related lentiviruses in their natural hosts.

The alpha and omega of G-protein coupled receptors: a novel method for classification. Part 2. Bin classification.

A novel way of classification of G-protein coupled receptors is presented that is only based on receptor sequence information by counting of amino acid residues. It involves the number of amino acid residues between the Asn residue in TM1 and the residue Cys in the loop between TM4 and TM5, the number of residues between the latter Cys residue and Pro residue in TM6, and the number of residues between the latter Pro and the last amino acid residue (called omega) in the sequence. The classification of 131 sequences, covering biogenic amine, opioid and somatostatin receptors, is visualized by means of a diagram which is referred to as a bin map. Each bin in the diagram encloses all the sequences that belong to one and only one receptor type or subtype. This so-called bin classification was obtained by means of the genetic algorithm methodology, which offers new opportunities for classifying proteins.