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Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology. info:eu-repo/semantics/publishedVersion

Uterine leiomyomata (UL) pose apublic health problem given high prevalence (>70%) in women of reproductive age and being a major indication for hysterectomies. Genome-wide association studies for U...

In this report we describe a cDNA sequence, BS106, identified from Incyte Genomics LifeSeq Expressed Sequence Tag database. A multi-tissue mRNA expression array, northern blots, and RT-PCR assays demonstrate the expression of BS106 in mammary, salivary and prostate glands, but not in other tissue types. BS106 mRNA was detected in 90% of the breast tissues examined. The cDNA encodes a 90-amino acid protein characterized as a small, mucin-like protein based on amino acid composition, extensive O-linked glycosylation, and expression profile. BS106 protein was recombinantly expressed in human embryonic kidney 293 cells and the secreted product was purified from the culture media. Monoclonal antibodies were prepared and used for immunohistochemical analysis of early stage breast cancer. BS106 protein was detected in the vast majority of carcinomas (70-100%) and overexpressed in approximately 30% of the 22 specimens analyzed. BS106 protein was not detected in other solid tumor types including bladder carcinoma, colon carcinoma, endometrial carcinoma, gastric carcinoma, squamous cell lung carcinoma, adenocarcinoma of the lung, ovarian carcinoma, pancreatic and prostatic carcinoma.

The mammaglobin gene has been shown to be preferentially expressed in breast tissue. Few genes match its specificity. Mammaglobin has generated much interest, and studies are ongoing to develop diagnostic tests for breast cancer based on the detection of mammaglobin. While searching the Incyte Genomics Lifeseq database for tissue-specific markers, we observed a second secretoglobin, BU101, also known as lipophilin B. We report here that mammaglobin, in breast tissue, is found as a complex with BU101. The complex was isolated from breast cancer tissue and was characterized as the biologically relevant form of mammaglobin.

The recent publication of representative genomic sequences of GBV-C has permitted the selection of PCR primers for detection of GBV-C in clinical samples by PCR techniques. Traditional amplification methodologies which couple reverse transcription polymerase chain reaction (RT-PCR) and Southern blot detection are slow, cumbersome, and can be technique dependent. This has hampered studies to determine the clinical significance of GBV-C. We report the selection of highly conserved PCR primers and a probe useful for semi-automated RT-PCR using the Abbott LCx® system. This adaptation of the LCx® system expands its capabilities to include the detection of RNA by RT-PCR, in addition to DNA detection by ligase chain reaction (LCR).

We hypothesized that a new test of infant lung function, less affected by shifts in lung volume, might better detect bronchodilator effects. Using the raised volume forced expiration technique (RVFET), the effect of a bronchodilator on lung function was studied in 22 infants with a history of recurrent wheeze and five healthy infants. Forced expiratory volume in 0.75 s (FEV0.75), forced expiratory vital capacity (FVC), and forced expiratory flow at 75% of FVC (FEF75%) were measured by forcing expiration, using an inflatable jacket from a lung volume set by an inspiratory pressure of 20 cm H2O. A minimum of five measurements were made at baseline and following the administration of 500 μg of salbutamol from a metered dose inhaler via a small volume metal spacer. Changes in lung function in the group of 25 infants who received salbutamol were compared to seven infants who received placebo aerosol. No significant changes occurred in measures of lung function following salbutamol administration when compared to baseline or placebo despite a significant increase in heart rate. A shift in lung volume is unlikely the reason why infants do not demonstrate a change in forced expiration following bronchodilator administration. Pediatr Pulmonol. 1998; 26:35–41. © 1998 Wiley-Liss, Inc.

Infant pulmonary function tests (PFTs) have proven increasingly popular and useful for clinical and research purposes. Informed consent requires accurate information on side effects. Our aim was to quantify minor side effects from a parental point of view by means of a questionnaire. The parents of 97 infants attending for PFTs were asked to complete a simple questionnaire. Eighty-one parents (84%) returned the questionnaire. Forty-one percent felt that their infants were not troubled by the process of administering the sedative chloral hydrate, whereas 55% suffered mild to moderate distress. In contrast, 94% of infants were not distressed by the actual PFTs. Similarly, 46% of parents were not distressed by the administration of sedative to their infant, with 49% expressing distress to a mild or moderate degree. Although 73% of parents were not distressed by watching their infants undergo the PFTs, 27% were to a mild to moderate degree. Seventy-three percent of infants were untroubled on waking. Seventy percent of infants had a good nights sleep after the PFTs. The vast majority of parents (94%) were happy to recommend that others allow their infants to undergo similar testing. We noted that most problems caused by infant PFTs relate to the administration of the sedative. Most infants awake from the tests not distressed and sleep normally the following night.

Study objectives The aim of our study was to determine the in vitro delivery of salbutamol from a pressurized metered-dose inhaler (pMDI) containing hydrofluoroalkane (HFA) propellant through various delivery devices to four models of a pediatric lung. Design To determine the effect of electrostatic charge, delivery of salbutamol was initially assessed with a multistage liquid impinger (MSLI) through an inline nonchamber device (Baxter MDI Adapter) and a small (Aerochamber MV) and a large (Nebuhaler) inline chamber device. Following this, the delivery was assessed to four lung models appropriate for a child of 70 kg, 50 kg, 15 kg, and 4 kg, with the same three reduced static devices inserted directly into a pediatric ventilator circuit. Measurements and results Reduction of electrostatic charge improved small particle delivery through holding chambers to the MSLI by 12 to 14%. In the ventilator model, the mean delivery was between 1.9% and 5.4% for the nonchamber device, between 14.3% and 27.2% for the small holding chamber, and between 7.2% and 25.7% for the large holding chamber. Delivery was the least efficient in the 4-kg model compared to the 70-kg, 50-kg, and 15-kg models. Conclusions Salbutamol from an HFA pMDI is delivered efficiently through inline holding chambers with reduced static in pediatric ventilator settings. A large holding chamber has no advantage over a small holding chamber. In addition, salbutamol delivery is more efficient through a holding chamber than through a nonchamber device.

The raised-volume forced-expiration technique measures infant lung function over an extended volume range. To improve comparisons between individuals and populations, we investigated the influence of jacket pressure on outcome variables in 21 infants. To quantify pressure transmitted from the jacket to the pleural space at a given lung volume, the jacket was inflated against an occluded airway, and the increase in pressure at the mouth was measured. Flow-volume curves were recorded at transmitted pressure (Ptrans) values ranging from 0 to 41.9 cm H20. The effect of Ptrans on the FEV measures of FEV0.5, FEV0.75, and FVC, and on the forced expiratory flow measures of FEF25%, FEF50% and FEF75% was assessed. At Ptrans values between 0 to 20 cm H20, a significant positive relationship existed between transmitted pressure (Ptrans) and all outcome variables except FVC. At higher Ptrans values, all outcome variables demonstrated pressure independence, with the exception of FEF25% (which remained positive) and FVC (which was negative in a subgroup of wheezy infants). FEF75% values tended to decrease at Ptrans values25 cm H20. At Ptrans values between 20 and 25 cm H20, most outcome variables are pressure independent. This range is therefore the most suitable for use with the raised-volume forced expiration technique.

We previously studied low-frequency respiratory impedance (Zrs) data at an elevated lung volume to separate airway and tissue mechanical properties in normal infants (Am. I. Respir. Crit. Care Med. 1996; 154:161-166). The aim of the present study was to determine the volume dependence of the airway and tissue mechanics by extending Zrs measurements to lower lung volumes. Zrs spectra between 0.5 and 21 Hz were measured in supine sleeping infants (n = 8; 7 to 26 mo of age) at mean transrespiratory pressures (Ptr[mean]) of 20, 10, and 0 cm H2O, during periods of apnea induced by inflating the infants' lungs to a pressure of 20 cm H2O through a face mask. At each inflation pressure, a model containing airway resistance (Raw) and inertance (law) and tissue damping (G) and elastance (H) was fitted to Zrs data. At FRC, the values of Raw, law, G, and H were 20.6+/-4.9 (SD) cm H2O x s/L, 0.037+/-0.014 cm H2O x s2/L, 39.6+/-10.3 cm H2O/L, and 147+/-35 cm H2O/L, respectively. Increase of Ptr(mean) caused a monotonous decrease in Raw (42+/-7% of the value at FRC), while law remained constant. The tissue parameters were minimal at a Ptr(mean) of 10 cm H2O (68+/-10% and 78+/-6% in G and H, respectively) and significantly higher at both 0 and 20 cm H2O. Although Zrs measurements can be made in most infants at lung volumes as low as FRC, an inflation pressure of 20 cm H2O provides a higher success rate and is therefore a more suitable condition for general use.

Inhalation therapy for wheezy infants with either a nebulizer or a pressurized metered-dose inhaler (pMDI) through a spacer is common practice. The aim of our study was to compare aerosol delivery to wheezy infants from a nebulizer and from a pMDI via two small volume spacers. Twenty wheezy infants (aged 4–12 months) were recruited. They inhaled salbutamol from a Pari-Baby® nebulizer, from a detergent-coated Babyhaler®, and from a Nebuchamber® in random order. A filter was placed between the inhalation systems and the patients. The amount of salbutamol deposited on the filter was measured using an ultraviolet spectrophotometer and was expressed as a percentage of the total nebulized or actuated doses. The mean total nebulized dose for the Pari-Baby® (1030 μg) was higher (P < 0.001) than the mean actuated dove from a pMDI for the Babyhaler® (374 μg) and for the Nebuchamber® (378 μg). Mean drug deposition on the filter was 40.2% (150 μg) of the total actuated dose for the detergent-coated Babyhaler® and 40.7% (154 μg) of the total actuated dose for the Nebuchamber®. There was no significant difference in drug deposition on the filter between the two spacers. Mean drug deposition on the filter was 25.3% (260 μg) of the total nebulized dose for the Pari-Baby® nebulizer. There was no weight dependence in drug deposition on the filter for the two spacers, but, drug deposition increased with the subject's weight for the nebulizer. We have shown that aerosol delivery to wheezy infants from a pMDI through small volume spacers is effective and that a higher percentage of the total amount of salbutamol is delivered than from a nebulizer. The weight dependence in drug deposition for the nebulizer can be of clinical relevance. Pediatr Pulmonol. 1997; 23:212–216 © 1997 Wiley-Liss, Inc.

Low-frequency respiratory impedance (Zrs) data permit the separate estimation of the mechanical properties of the airways and the tissues, but they are difficult to collect in humans because of the need for apneic conditions. We exploited the apneic phase produced by invoking the Hering-Breuer reflex with end-inspiratory airway occlusion in five sedated infants aged 9 to 16 mo. A computer-controlled pump and solenoid valves were used to inflate the supine infants through a face mask to a transrespiratory pressure of 20 cm H2O and to affect the airway occlusion. A loudspeaker-in-box system was connected to the mask through a side-arm, and small-amplitude pseudorandom oscillations containing 23 frequency components between 0.5 and 20.75 Hz were applied for 6 s. Four consecutive measurements were made in each infant, and the averaged Zrs spectra were evaluated on the basis of a model containing the frequency-independent resistance (Raw) and inertance (law) of the airways, and the viscous damping (G) and elastance (H) parameters of the constant-phase compartment of the chest wall and parenchymal tissues. The measured Zrs values were consistent with the model up to 15 Hz, and the average fitting error was 0.89 +/- 0.11 (SD) cm H2O.s/L. The following parameter values were obtained: Raw = 10.0 +/- 2.1 cm H2O.s/L, law = 0.061 +/- 0.014 cm H2O.s2/L, G = 28.6 +/- 4.9 cm H2O/L, H = 141 +/- 55 cm H2O/L. The tissue hysteresivity (G/H) values were 0.218 +/- 0.061. Our results indicate that, in short apneic periods evoked by the Hering-Breuer reflex, reliable low-frequency Zrs data can be collected to partition the tissue and airway impedances in sedated infants.

Reduction of geminal proton relaxation within methyl groups is made possible by use of the dideuteriomethyl group (–CHD2), so that residual dipolar relaxation can reflect the steric environment of the methyl group. To search for any relationship between spin-lattice relaxation and steric environment, we have prepared a series of so-labelled molecules that are rigid except for methyl rotation. The importance of dipolar relaxation was demonstrated by nuclear Overhauser enhancements and by the temperature dependence of T1. The extent of motional anisotropy was assessed by measuring the 13C relaxation times as a function of position. Proton relaxation times were adjusted for molecular size by multiplying by the molecular weight. Comparison of the size-adjusted proton relaxation times showed qualitative trends with steric environment. The major factor vitiating a more quantitative relationship is probably the contribution from spin-rotation relaxation, with a minor contribution from anisotropic motion.

A first totally synthetic Escherichia coli plasmid has been designed, constructed and shown to be a functional, stable, high-copy cloning vector. The FokI method of gene synthesis [Mandecki and Bolling, Gene 68 (1988) 101-107] was used to assemble the plasmid from 30 oligodeoxyribonucleotides. The plasmid contains synthetic modules for the beta-lactamase-encoding gene (bla), replication origin, lacZ gene fragment and multicloning site. The plasmid is patterned after the pUC-type plasmids and has a copy number similar to that of pUC plasmids. The major changes introduced include the removal of nearly 50% of the restriction sites present in pUC plasmids, reduction of plasmid size to 2050 bp, and introduction of transcription terminators downstream from both the bla gene and lacZ fragment. The changes facilitate a number of techniques, such as cloning, mutagenesis, expression and restriction analysis.

Crash-resistant fuel systems (CRFS) have demonstrated close to 100% effectiveness in survivable crashes of Army helicopters, but the technology has been slow to transfer into the civil helicopter arena. Federal standards for civil helicopter CRFS are less stringent than those for military helicopters. A reduction in standards for CRFS in military helicopters is being considered.The goal of this study was to determine whether crashes of civil helicopters with CRFS are less likely to result in post-crash fire than crashes of those without.Crashes of civil helicopters during 1982-2004 were analyzed, comparing Bell 206 helicopters manufactured with CRFS with Aerospatial 350 helicopters manufactured during the same period (post-1981), but lacking CRFS. Bell 206 helicopters with CRFS were also compared with earlier models without CRFS.The highest proportion of crashes with post-crash fires (11.3%) was in AS-350s manufactured after 1981 (non-CRFS), and the lowest (3.7%) was in Bell 206s (with CRFS) [unadjusted risk ratio (RR) = 3.3, 95% confidence interval (CI) = 1.04, 10.50; adjusted for light and weather, RR = 2.81, Cl = 0.82, 9.69]. Earlier models of Bell 206s without CRFS had higher risk of post-crash fire than post-1981 models with CRFS (7.4% vs. 3.7%; adjusted RR = 2.11, Cl = 0.82, 5.45).The results of this study suggest a better performance, in terms of post-crash fire prevention, of CRFS-equipped civil helicopters as compared with those without CRFS. It is possible that CRFS in civil helicopters have not achieved the same degree of effectiveness as CRFS in military helicopters. CRFS should be used more widely in civil helicopters. The more stringent CRFS requirements for military helicopters should not be reduced without further research.

The potential of the low-frequency forced oscillation technique (FOT) to measure the response to inhaled salbutamol was studied in 13 infants with a history of recurrent wheeze and nine healthy infants. The input impedance of the respiratory system (Zrs) between 0.5 and 20 Hz was measured at a transrespiratory pressure of 20 cm H2O during a brief Hering-Breuer reflex-induced pause in breathing. Parameters representing the airway resistance (Raw) and inertance (law), and a constant-phase tissue damping (G) and elastance (H) were estimated from the Zrs spectra. Lung function was measured before and after the administration of 500 microg of salbutamol via a small-volume metal spacer. Six of these infants also received a placebo aerosol. A fall in Raw (13% for the entire group) occurred following treatment with salbutamol (p0.008) but not placebo. There was no significant difference in the response to salbutamol between the normal infants (7.65% +/- 5.49%) and those with recurrent wheeze (17.58% +/- 8.67%). On grouped data, the fall in G just failed to reach statistical significance (p = 0.05) after correcting the significance level for multiple tests. No significant change occurred in law or H. We conclude that the low-frequency FOT is a suitable methodology for studying bronchodilator responsiveness in infants.

Infant lung function can be assessed with the tidal volume "squeeze" technique or, over an extended volume range, with the newer raised volume forced expiration technique (RVFET). We assessed methacholine responsiveness in 11 infants, measuring both maximal expiratory flow at functional residual capacity (V.max,FRC)with the tidal volume technique, and forced expiratory volume/time (FEV(t)) with RVFET. We used a standard methodology for the former. FEV(t) was measured by inflating the infant's lungs to 20 cm H2O and forcing expiration using a jacket setup to transmit a pressure of 20 cm H2O to the airway. Lung function was measured at baseline and after methacholine inhalations, increasing from 0.1 g/L to 10 g/L in half log dosage increments (DI). The provocative concentrations (PC) of methacholine leading to a 40% fall in V.max,FRC and a 15 or 20% fall in FEV(t) were calculated. The mean provocative concentration of methacholine required to produce a 40% fall in V.max,FRC was less than that required to produce a 20% fall in FEV0.5 by 0.39 DI (95% CI, -0.60 to 1.38) and less than that required to produce a 20% fall in FEV0.75 by 0.42 DI (95%, CI, -0.54 to 1.39). Similarly, the provocative concentration of methacholine required to produce a 40% fall in V.max,FRC was less than that required to produce a 15% fall in FEV0.5 by 0.14 DI (95% CI, -0.99 to 1.28) or a 15% fall in FEV0.75 by 0.13 DI (95% CI, -0.80 to 1.08), but the differences were small and not significant. Despite these differences the agreement between the two methods was good, and bronchoconstriction was not attenuated by the forced inspiration delivered by the raised volume maneuver. We conclude that the raised volume forced expiration technique is able to detect methacholine-induced bronchoconstriction.

The abilities of the Escherichia coli lipoic acid auxotrophs W1485-lip2 and JRG33-lip9 to grow on succinate medium in the presence of octanoate, 8-mercaptooctanoate, or 6-mercaptooctanoate have been determined. Both organisms are mutated in lipA. Neither organism can use octanoate or 6-mercaptooctanoate for production of lipoate, but the lip2 allele can use 8-mercaptooctanoate. Chromosomal DNA from the auxotrophs was amplified by PCR using primers derived from the DNA sequence of wild-type lipA and then sequenced. Both mutants contain single G/C to A/T mutations in lipA, resulting in conversion of Ser307 into Phe in W1485-lip2 and Glu195 into Lys in JRG33-lip9. These results support the hypothesis that lipA is involved in the sulfur insertion step(s) of lipoate biosynthesis and indicate that it is possible to selectively block formation of the C8-S bond through suitable mutation in lipA.

Reduction of geminal proton relaxation within methyl groups is made possible by use of the dideuteriomethyl group (–CHD2), so that residual dipolar relaxation can reflect the steric environment of the methyl group. To search for any relationship between spin-lattice relaxation and steric environment, we have prepared a series of so-labelled molecules that are rigid except for methyl rotation. The importance of dipolar relaxation was demonstrated by nuclear Overhauser enhancements and by the temperature dependence of T1. The extent of motional anisotropy was assessed by measuring the 13C relaxation times as a function of position. Proton relaxation times were adjusted for molecular size by multiplying by the molecular weight. Comparison of the size-adjusted proton relaxation times showed qualitative trends with steric environment. The major factor vitiating a more quantitative relationship is probably the contribution from spin-rotation relaxation, with a minor contribution from anisotropic motion.

A rapid and simple method of gene synthesis is presented. A gene is constructed by serial additions of individual gene fragments in the 5'----3' direction. The vector used as the synthesis vehicle contains a unique Bsm I site at the amino terminus of the lacZ gene. Plasmid linearized with Bsm I is recircularized in vivo by oligonucleotide-directed double-strand break repair. The synthetic oligonucleotide used to "bridge" the double-strand break carries a 70- to 100-nucleotide insert which constitutes a portion of the gene along with a BsmI site at the 3' end that regenerates the site and allows for another consecutive round of mutagenesis to extend the gene sequence. The process is repeated until the entire gene is assembled. The method uses the beta-galactosidase color assay as a means of screening for correct insert lengths. The only in vitro enzymatic step necessary is a single Bsm I restriction digest of plasmid DNA. No ligation reactions are required. Only one strand of a gene sequence needs to be synthesized chemically. The gene synthesis method presented here was used to construct the human anaphylatoxin complement factor C3a gene.

We have found that 50 mM L-histidine pH 7.6 as a buffer for gel electrophoresis greatly improves the resolution of oligonucleotides less than 70 residues long on denaturing polyacrylamide gels. The histidine buffer increases spacing between DNA bands on the gel about twofold in comparison with a standard buffer (89 mM Tris-borate, 2 mM EDTA pH 8.3). In addition, low conductivity of the histidine buffer results in a threefold reduction of the electrophoresis time. Conditions for electrophoresis were optimized by varying both histidine and acrylamide concentrations. Other polycationic compounds, such as spermidine and ethylenediamine, were also tested for improved resolution of oligonucleotides. Several hypotheses as to the factors influencing the separation of DNA on gels are presented.

The isolation and genomic sequence of one of possibly four glyceraldehyde-3-phosphate dehydrogenase genes in the nematode, Caenorhabditis elegans is presented. The complete nucleotide sequence of the coding as well as the noncoding flanking regions of this gene has been determined. The deduced amino-acid sequence agrees with the sequence of typical glyceraldehyde-3-phosphate dehydrogenase enzymes and its molecular weight of 36 235 agrees with its size determined previously (Yarbrough, P. and Hecht, R. (1984) J. Biol. Chem. 259, 14711–14720). That this isolated gene encodes a nematode glyceraldehyde-3-phosphate dehydrogenase is additionally confirmed by demonstrating its immunoreactivity to an anti-nematode glyceraldehyde-3-phosphate dehydrogenase antibody after its expression as a fusion protein with dihydrofolate reductase. Codon utilization follows a pattern typical of other expressed nematode genes. The gene is split by two introns that are highly conserved in comparison to other introns observed in C. elegans. The placement of one of these introns is conserved with respect to the chicken glyceraldehyde-3-phosphate dehydrogenase gene. Within the 5′ flanking sequence homology to actin and the homology 2 block of the major myosin gene (unc-54) is noted. It is of interest that the 3′ flanking region contains a CAAAT box, followed by a TATAAT box, before an open reading frame of a closely linked gene that also contains a small AT-rich intron with the nematode consensus splice junction.