High-Resolution Differentiation of Enteric Bacteria in Premature Infant Fecal Microbiomes Using a Novel rRNA Amplicon.

Identifying and tracking microbial strains as microbiomes evolve are major challenges in the field of microbiome research. We utilized a new sequencing kit that combines DNA extraction with PCR amplification of a large region of the rRNA operon and downstream bioinformatic data analysis. Longitudinal microbiome samples of coadmitted twins from two different neonatal intensive care units (NICUs) were analyzed using an ∼2,500-base amplicon that spans the 16S and 23S rRNA genes and mapped to a new, custom 16S-23S rRNA database. Amplicon sequence variants (ASVs) inferred using DADA2 provided sufficient resolution for the differentiation of rRNA variants from closely related but not previously sequenced Klebsiella, Escherichia coli, and Enterobacter strains, among the first bacteria colonizing the gut of these infants after admission to the NICU. Distinct ASV groups (fingerprints) were monitored between coadmitted twins over time, demonstrating the potential to track the source and spread of both commensals and pathogens. The high-resolution taxonomy obtained from long amplicon sequencing enables the tracking of strains temporally and spatially as microbiomes are established in infants in the hospital environment.IMPORTANCE Achieving strain-level resolution is a major obstacle for source tracking and temporal studies of microbiomes. In this study, we describe a novel deep-sequencing approach that provides species- and strain-level resolution of the neonatal microbiome. Using Klebsiella, E. coli, and Enterobacter as examples, we could monitor their temporal dynamics after antibiotic treatment and in pairs of twins. The strain-level resolution, combined with the greater sequencing depth and decreased cost per read of PacBio Sequel 2, enables this advantageous source- and strain-tracking analysis method to be implemented widely across more complex microbiomes.

High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. RESULTS: SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. CONCLUSIONS: Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.

Fusion estrogen receptor proteins: toward the development of receptor-based agonists and antagonists.

Estrogen-induced signaling mediated by estrogen receptors (ERs) is also affected by aberrant ERs that act as constitutively active or dominant negative modulators. Variant ERs can contribute to carcinogenesis and to the loss of estrogen responsiveness, rendering antiestrogen therapy ineffective. Determining target gene response during co-synthesis of different ER species is difficult, because dimers formed in the presence of more than one ER species are a heterogenous population of homo- or heterodimers. We engineered a homofusion ERalpha as a prototype single-chain receptor by genetically conjugating two ER monomers into a covalently fused single-chain protein to obtain a homogeneous population. This permits analysis of symmetrical or asymmetrical mutations that simulate variant homo- and heterodimers. Although a monomer, the homofusion receptor exhibited similar biochemical and functional properties to the dimeric ERalpha. We used activation function-2 (AF2) defective mutants as a model in either one or both receptor domains for a dominant-negative phenotype by suppressing the reporter activity induced by the WT receptor. When co-expressed with ERalpha, the fusion variant deficient in both AF2 functions suppressed the reporter activity effectively induced by ERalpha. These results show the utility of fusion receptors as models for generation of receptor-based agonists and antagonists.

In vitro analysis of human immunodeficiency virus type 1 minus-strand strong-stop DNA synthesis and genomic RNA processing.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA. A detailed understanding of these activities is required to understand the process of reverse transcription. HIV-1 viral DNA is initiated from a tRNA(3)(Lys) primer bound to the viral genome at the primer binding site. The U3 and R regions of the RNA genome are the first sequences to be copied. The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation. Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR. In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization. If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities. After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization. Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion. RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5' end of the RNA that remained annealed to the 3' end of the minus-strand strong-stop DNA only if NC was present in the reaction.

Effects of spinal manipulative therapy on autonomic activity and the cardiovascular system: a case study using the electrocardiogram and arterial tonometry.

OBJECTIVE: To determine if there is alteration in the autonomic nervous and cardiovascular systems after chiropractic manipulative therapy (CMT). A novel approach was used to quantitatively probe for changes in the activity of the autonomic nervous system, in blood pressure, and in pressure pulse transmission time. This approach uses the electrocardiogram and arterial tonometry equipment. DESIGN: This case study involves 1 subject treated over a 6-week period (2 visits/week). Respiration, electrocardiogram, and left and right radial artery blood pressures were measured during the baseline (2 visits) and treatment (10 visits) phases. Measurements were obtained before (n = 3) and after (n = 3) a break period (baseline) or before and after CMT. High-velocity, low-amplitude CMT that produced joint cavitation was used. SETTING: The study was performed at the Parker College Research Institute in a temperature-controlled laboratory. MAIN OUTCOME MEASURES: Fourier analysis was performed on the electrocardiogram-determined rest-redistribution intervals. The low frequency power between 0.04 to 0.15 Hz and the high frequency power between 0.15 to 0.40 Hz represent the activity of the sympathetic and parasympathetic nervous systems, respectively. The main outcome measure was the sympathovagal index, which is determined from the ratio of low frequency to high frequency. The arterial pressure and the time for pressure pulses to travel from the heart to the radial artery recording sites (pressure pulse transmission time) were studied. Differences (average of 3 measurements after treatment minus measurements before treatment) for each variable were calculated. RESULTS: After the 1st CMT treatment, the difference between treatment and baseline decreased for both the low frequency/high frequency (-2.804 +/- 1.273) and low frequency power (-0.135 +/- 0.056). These findings indicated that the parasympathetic nervous system predominated the sympathetic nervous system. After the 3rd, 4th, 6th, and 9th treatment, the difference between treatment and baseline increased for low frequency/high frequency (0.908 +/- 0.338, 2.313 +/- 0.300, 2.776 +/- 1.102, and 0. 988 +/- 0.269, respectively) and indicated that the sympathetic nervous system predominated the parasympathetic nervous system. In addition, the difference between treatment and baseline for the pressure pulse transmission time decreased bilaterally after the 4th treatment (left, -13.52 +/- 3.70 ms; right, -9.75 +/- 3.76 ms) and 6th treatment (left, -9.53 +/- 3.60 ms; right, -9.24 +/- 3.50 ms), which indicated that arterial compliance had decreased. Furthermore, after the 6th treatment, the difference between treatment and baseline for the rest-redistribution interval time decreased (-0.084 +/- 0.014 s). The difference between treatment and baseline for the systolic, diastolic, and mean arterial pressure for the above-mentioned treatments was not considered significant. CONCLUSION: This case study is the first to attempt to use electrocardiogram and arterial tonometry data to study the effects of CMT on the autonomic nervous and cardiovascular systems over an extended period of time. These devices allowed a more in-depth study of the cardiovascular and autonomic changes associated with CMT. Although changes in the autonomic nervous and cardiovascular systems can be detected, further development of a reliable and reproducible experimental protocol is required before validating the effects of CMT on these systems.

Human immunodeficiency virus type 1 nucleocapsid protein can prevent self-priming of minus-strand strong stop DNA by promoting the annealing of short oligonucleotides to hairpin sequences.

Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (-sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with -sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic -sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3' end of the -sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.

An explanation for observed estrogen receptor binding to single-stranded estrogen-responsive element DNA.

Estrogen-inducible genes contain an enhancer called the estrogen response element (ERE), a double-stranded inverted repeat. The estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo.

Sequence requirements for estrogen receptor binding to estrogen response elements.

The estrogen receptor (ER) is a transcription factor that binds to a specific DNA sequence found in the regulatory regions of estrogen-responsive genes, called the estrogen response element (ERE). Many genes that contain EREs have been identified, and most of these EREs contain one or more changes from the core consensus sequence, a 13-nucleotide segment with 10 nucleotides forming an inverted repeat. A number of genes have multiple copies of these imperfect EREs. In order to understand why natural EREs have developed in this manner, we have attempted to define the basic sequence requirements for ER binding. To this end, we measured the binding of homodimeric ER to a variety of nonconsensus EREs. We discovered that an ERE containing even a single change from the consensus may be unable to bind ER. However, an ERE with two changes from the consensus may be capable of binding avidly to ER in the context of certain flanking sequences. We found that changes in the sequences flanking a nonconsensus ERE can greatly alter ER-ERE affinity, either positively or negatively. Careful study of sequences flanking a series of EREs made it possible to develop rules that predict whether ER binds to a given natural ERE and also to predict the relative amounts of binding when comparing two EREs.

Determination of appropriate recording force for non-invasive measurement of arterial pressure pulses.

1. Non-invasive recording techniques of the arterial pressure pulse will distort the arterial wall and may alter pulse wave measurements. We hypothesized that intersubject variability of these measurements would be reduced if recording forces were normalized to reflect individualized arterial occlusion forces. 2. In 10 normal male subjects (age 24 +/- 1 years), brachial, radial and finger arterial pressure pulses were recorded simultaneously using volume displacement pulse transducers (Fukuda TY-303) and a finger pressure monitoring system (Finapres, Ohmeda 2300) and were made at 2, 5 and 10-100% (10% increments) of the brachial arterial force associated with marked distortion of finger pulsations. Forces were applied at the brachial site in a randomized order while a constant 1.8 N force was applied at the radial artery site. Pressure pulses were analysed using the discrete fast Fourier transform. 3. Pulse amplitude, contour, wave velocity and relative transmission ratios remained relatively constant until the branchial artery recording force exceeded 59.9 +/- 0.3% of the largest recording force used in each subject (7.14 +/- 0.75 N). The finger pulse pressures (P < 0.0001), radial pulse amplitudes (P < 0.0001) and contours (harmonics 2-6, P < 0.003), pulse wave velocity (P < 0.021) and relative transmission ratios (harmonics 3-7, P < 0.01) then decreased with higher recording forces. 4. To avoid distortion, non-invasive recordings of arterial pressure pulse amplitude, contour, pressure wave velocity and relative transmission ratios along a peripheral arterial segment should use recording forces of less than 60% of the force associated with marked distortion of finger pulsations.

Arterial tonometry and assessment of cardiovascular alterations with chiropractic spinal manipulative therapy.

OBJECTIVES: To (a) introduce arterial tonometry, which may offer a greater opportunity to detect alterations of the cardiovascular system with spinal manipulative therapy, (b) discuss the limitations of standard blood pressure measurements and (c) discuss factors that govern the contour of arterial pressure pulses. DATA SOURCES: Information was obtained from peer-reviewed medical and chiropractic journal articles and medical textbooks written in English (mainly from 1980 to 1995). CONCLUSIONS: Arterial tonometry is a noninvasive method to continuously measure the contour of arterial pressure pulses. Pulses can be simultaneously measured from several arterial sites because vessel occlusion is not required. Arterial tonometry supplements standard blood pressure measurements that measure only the systolic and diastolic pressures by permitting the investigation of additional parameters of cardiovascular functions. Changes in large artery function can be inferred through alterations of pulse contour, wave velocity and harmonic transmission ratios. Analysis of the beat-to-beat fluctuations of arterial pressure and heart rate can be used to determine whether SMT alters the autonomic nervous system. Reductions of arterial pressure in central vessels may not be apparent from standard blood pressure measurements at peripheral vessels. The contour of a pressure pulse depends on the magnitude and timing between incident and reflected pressure waves from the upper and lower body, body shape and size, the duration and pattern of ventricular systole, and dispersion and attenuation. Therefore, arterial tonometry may improve the ability to detect alterations of the cardiovascular system in chiropractic research.

Chicken ovalbumin upstream promoter-transcription factor interacts with estrogen receptor, binds to estrogen response elements and half-sites, and inhibits estrogen-induced gene expression.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) was identified as a low abundance protein in bovine uterus that co-purified with estrogen receptor (ER) in a ligand-independent manner and was separated from the ER by its lower retention on estrogen response element (ERE)-Sepharose. In gel mobility shift assays, COUP-TF bound as an apparent dimer to ERE and ERE half-sites. COUP-TF bound to an ERE half-site with high affinity, Kd = 1.24 nM. In contrast, ER did not bind a single ERE half-site. None of the class II nuclear receptors analyzed, i.e. retinoic acid receptor, retinoid X receptor, thyroid receptor, peroxisome proliferator-activated receptor, or vitamin D receptor, were constituents of the COUP-TF.DNA binding complex detected in gel mobility shift assays. Direct interaction of COUP-TF with ER was indicated by GST "pull-down" and co-immunoprecipitation assays. The nature of the ER ligand influenced COUP-TF-ERE half-site binding. When ER was liganded by the antiestrogen 4-hydroxytamoxifen (4-OHT), COUP-TF-half-site interaction decreased. Conversely, COUP-TF transcribed and translated in vitro enhanced the ERE binding of purified estradiol (E2)-liganded ER but not 4-OHT-liganded ER. Co-transfection of ER-expressing MCF-7 human breast cancer cells with an expression vector for COUP-TFI resulted in a dose-dependent inhibition of E2-induced expression of a luciferase reporter gene under the control of three tandem copies of EREc38. The ability of COUP-TF to bind specifically to EREs and half-sites, to interact with ER, and to inhibit E2-induced gene expression suggests COUP-TF regulates ER action by both direct DNA binding competition and through protein-protein interactions.

Site-directed estrogen receptor antibodies stabilize 4-hydroxytamoxifen ligand, but not estradiol, and indicate ligand-specific differences in the recognition of estrogen response element DNA in vitro.

Conformational differences between type I antiestrogen-liganded estrogen receptor and estradiol (E2)-liganded estrogen receptor (ER) are thought to be responsible for differentiating agonist versus antagonist ER activity at individual genes. To examine the impact of ER ligand on estrogen-response element (ERE) binding kinetics and receptor conformation, we quantitated the effect of site-directed, ER-specific antibodies raised against synthetic peptides corresponding to the DNA-binding domain of human ER on ER-ERE binding in vitro. Although 4-hydroxytamoxifen-liganded-ER (4-OHT-ER) and E2-ER bind a consensus ERE with equal high affinity, the stoichiometry of 4-OHT-ER-ERE binding at saturation is approximately 50% lower than that of E2-ER binding to all ERE sequences tested. In contrast, the ERE binding stoichiometry of tamoxifen aziridine-liganded ER (TAz-ER) is identical to that of E2-ER: one receptor dimer bound per ERE. The difference in binding stoichiometry is caused by dissociation of one molecule of 4-OHT from the ER as the dimeric receptor binds DNA. Addition of low concentrations of ER-specific polyclonal antibodies AT3A or AT3B prevented 4-OHT ligand dissociation, yielding an increase in specific 4-OHT-ER-ERE binding to a level equal to that of E2-ER- or TAz-ER-ERE binding. However, higher amounts of AT3A or AT3B inhibited specific ERE binding of both 4-OHT- and E2-ER. We conclude that differences in ER conformation when liganded with 4-OHT versus E2 are revealed by these antibodies and that such differences in receptor conformation may influence subsequent interaction of the receptor with other proteins necessary for transactivation.

Footprint analysis of estrogen receptor binding to adjacent estrogen response elements.

Quantitative DNase I footprinting assays were employed to simultaneously measure the amount of estrogen receptor (ER) bound to each site in constructs containing multiple estrogen response elements (EREs). These assays revealed identical, high affinity ER-ERE binding, Kd of approximately 0.25 nM, for estradiol-liganded ER (E2-ER), 4-hydroxytamoxifen liganded ER (4-OHT-ER), tamoxifen aziridine liganded ER (TAz-ER), and unliganded dimeric ER, for each ERE in constructs containing up to four tandem EREs. Increasing concentrations of ER resulted in the same pattern of occupancy for each ERE, whether or not the site was located near other EREs. Similarly, the presence or absence of E2, 4-OHT, or TAz ligand did not change ER-ERE interaction. Since activated ER-ERE binding affinity is identical, whether ER is liganded or unliganded, ligand cannot regulate ER-ERE binding affinity. These results support the hypothesis that ligand-dependent conformational changes primarily determine how ER interacts with components of the transcription initiation complex that mediate gene transactivation. In addition, footprint assays revealed that, following ER binding, an AT-rich site adjacent to the ERE becomes hypersensitive to DNase I digestion. This sequence may be easily or intrinsically bent, assisting in recruiting ER to ERE sites.

Influence of applied brachial recording forces on pulse wave velocity and transmission in the brachio-radial arterial segment.

Arterial stiffness in hypertension and heart failure may increase afterload on the left ventricle. Pulse wave velocity and transmission ratio measurements are noninvasive methods to assess arterial stiffness. Since noninvasive pressure pulse recording requires sufficient applied force to distort the vessel wall, we hypothesized that the pulse wave velocity, transmission ratios, and distal pulse amplitudes and contours may be altered by the recording technique. Brachial and radial arterial pressure pulses were recorded simultaneously using a piezoelectric pulse transducer in 14 young, normal, male subjects using 10 brachial artery recording forces (0.35-3.58N, approximately equal to 0.36N increments) applied in a random order and a constant radial force (2.35N). Pulses were Fourier analyzed. One subject was excluded from analysis because of improper transducer positioning over the brachial artery. In 8 subjects, no significant changes occurred in any variable over all brachial recording forces. In the remaining 5 subjects, the measured variables remained constant until brachial artery recording forces exceeded 2.42 +/- 0.03N. The pulse wave velocity (p < 0.04), transmission ratios (harmonics 2-5, p < 0.0001), radial pulse amplitude (p < 0.0003), and relative powers (harmonics 2-5, p < 0.02) then decreased. In these subjects, brachial artery depths were less than the other subjects (5.9 +/- 0.4 vs. 7.7 +/- 0.4 mm, p < 0.05). The brachial and radial artery recording forces normally used during clinical measurements by 2 investigators were 1.43 +/- 0.01N (95% confidence intervals (CI) = 1.23N, 1.62N) and 1.88 +/- 0.11N (95% CI = 1.65N, 2.10N), respectively. Therefore, at forces normally used by clinical investigators, the pulse wave velocity, harmonic transmission ratios, and pulse amplitudes and contours obtained at the brachial and radial artery are not significantly influenced by forces applied at the brachial artery. However, these variables may be decreased in subjects with more superficial arteries when higher recording forces are used.

Applied recording force and noninvasive arterial pulses.

Arterial pressure pulses are often measured noninvasively, but the influence of external recording forces has not been well documented. Accordingly, the main goal was to delineate pulse contour and amplitude distortion with progressively compressive forces applied to an external transducer. Ten young normal male subjects were studied while supine. Pulses were recorded at the end of a normal expiration over a range of forces (0.45-4.29, 0.42N increments) applied in a randomized order over first the brachial and then the radial artery. Pulse contours were Fourier analyzed and harmonic powers were normalized to the peak power at the fundamental frequency. Brachial artery pulse amplitudes progressively decreased (p = 0.013), whereas, those at the radial artery peaked at a recording force of 1.79 +/- 0.01N (p < 0.001) and subsequently decreased parallel to the brachial data with larger forces. No significant pulse contour distortions occurred at either the brachial or radial artery with applied forces of up to 2.18 +/- 0.02 and 2.59 +/- 0.02N, respectively, as indicated by the similarity of the relative power for harmonics 2-7. Radial artery pulses were distorted at and beyond a force of 2.99 +/- 0.01N as indicated by the increased relative power of harmonics 3-7 (p < 0.05). Therefore, despite significant alterations in pulse amplitude, the relative shape of pulses remains similar over a large range of lower recording forces.