Pain Control after Laparoscopic Radical Prostatectomy: Comparison between Unilateral Transversus Abdominis Plane Block and Wound Infiltration.

BACKGROUND AND OBJECTIVES: To determine the efficacy of unilateral transversus abdominis plane (TAP) block versus wound local infiltration for postoperative pain following laparoscopic radical prostatectomy (LRP). METHODS: Data of consecutive patients who underwent extraperitoneal LRP and received either wound infiltration or unilateral TAP block for analgesia were retrospectively analyzed. The patients were divided into 2 groups based on the technique used. We compared pain intensity scores and on-demand analgesic use both during the hospital stay and post-discharge between the 2 groups. RESULTS: A total of 48 patients were included, 27 received unilateral TAP blocks (group 1) and 21 were managed with wound infiltration (group 2). The unilateral TAP block group showed lower median pain scores on postoperative days (POD) 1 with pain scores being 0.2 (0-4) and 0.8 (0-4), respectively (p < 0.05). On POD2, the median pain intensity was 0.9 (0-5) and 1.6 (0-6) in groups 1 and 2, respectively (p < 0.05). The median number of on-demand analgesic doses during the POD1 was 0.2 (0-2) and 0.4 (0-2) in groups 1 and 2, respectively (p = 0.19). On POD2, the patients received 0.5 (0-2) and 1.1 (0-3) on-demand doses in groups 1 and 2, respectively (p < 0.05). CONCLUSION: Unilateral TAP block might improve pain control more pronounced after LRP than wound infiltration.

Miniaturized sequencing gel system for quick analysis of DNA by hydroxyl radical cleavage.

We described a simple and quick miniaturized sequencing gel system for DNA analysis. Two major modifications were made to the previously reported miniaturized DNA sequencing gel system to achieve high-resolution hydroxyl radical cleavage analysis: including formamide in the miniaturized gel and providing uniform heating during electrophoresis. Our method enables one to reduce the cost for chemicals and to significantly reduce electrophoresis time. Furthermore, minimal gel handling simplifies the entire process. We show that the resolution of DNA fragments obtained by hydroxyl radical cleavage for the miniaturized gel is similar to that of a large conventional sequencing gel.

Affordable hands-on DNA sequencing and genotyping: an exercise for teaching DNA analysis to undergraduates.

In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C Sanger sequencing reactions. They prepare and run the gels, perform Southern blots (which require only 10 min), and detect sequencing ladders using a colorimetric detection system. Students enlarge their sequencing ladders from digital images of their small nylon membranes, and read the sequence manually. They compare their reads with the actual DNA sequence using BLAST2. After mastering the DNA sequencing system, students prepare their own DNA from a cheek swab, polymerase chain reaction-amplify a region of their DNA that encompasses a SNP of interest, and perform sequencing to determine their genotype at the SNP position. A family pedigree can also be constructed. The SNP chosen by the instructor was rs17822931, which is in the ABCC11 gene and is the determinant of human earwax type. Genotypes at the rs178229931 site vary in different ethnic populations.

Direct measurements of the nucleosome-forming preferences of periodic DNA motifs challenge established models.

Several periodic motifs have been implicated in facilitating the bending of DNA around the histone core of the nucleosome. For example, di-nucleotides AA/TT/TA and GC at ∼10-bp periods, but offset by 5 bp, are found with higher-than-expected occurrences in aligned nucleosomal DNAs in vitro and in vivo. Additionally, regularly oscillating period-10 trinucleotide motifs non-T, A/T, G and their complements have been implicated in the formation of regular nucleosome arrays. The effects of these periodic motifs on nucleosome formation have not been systematically tested directly by competitive reconstitution assays. We show that, in general, none of these period-10 motifs, except TA, in certain sequence contexts, facilitates nucleosome formation. The influence of periodic TAs on nucleosome formation is appreciable; with some of the 200-bp DNAs out-competing bulk nucleosomal DNA by more than 400-fold. Only the nucleotides immediately flanking TA influence its nucleosome-forming ability. Period-10 TA, when flanked by a pair of permissive nucleotides, facilitates DNA bending through compression of the minor groove. The free energy change for nucleosome formation decreases linearly with the number of consecutive TAs, up to eight. We suggest how these data can be reconciled with previous findings.

Are nucleosome positions in vivo primarily determined by histone-DNA sequence preferences?

Large-scale and genome-wide studies have concluded that approximately 80% of the yeast (Saccharomyces cerevisiae) genome is occupied by positioned nucleosomes. In vivo this nucleosome organization can result from a variety of mechanisms, including the intrinsic DNA sequence preferences for wrapping the DNA around the histone core. Recently, a genome-wide study was reported using massively parallel sequencing to directly compare in vivo and in vitro nucleosome positions. It was concluded that intrinsic DNA sequence preferences indeed have a dominant role in determining the in vivo nucleosome organization of the genome, consistent with a genomic code for nucleosome positioning. Some other studies disagree with this view. Using the large amount of data now available from several sources, we have attempted to clarify a fundamental question concerning the packaging of genomic DNA: to what extent are nucleosome positions in vivo determined by histone-DNA sequence preferences? We have analyzed data obtained from different laboratories in the same way, and have directly compared these data. We also identify possible problems with some of the experimental designs used and with the data analysis. Our findings suggest that DNA sequence preferences have only small effects on the positioning of individual nucleosomes throughout the genome in vivo.

Sequence information encoded in DNA that may influence long-range chromatin structure correlates with human chromosome functions.

Little is known about the possible function of the bulk of the human genome. We have recently shown that long-range regular oscillation in the motif non-T, A/T, G (VWG) existing at ten-nucleotide multiples influences large-scale nucleosome array formation. In this work, we have determined the locations of all 100 kb regions that are predicted to form distinctive chromatin structures throughout each human chromosome (except Y). Using these data, we found that a significantly greater fraction of 300 kb sequences lacked annotated transcripts in genomic DNA regions > or = 300 kb that contained nearly continuous chromatin organizing signals than in control regions. We also found a relationship between the meiotic recombination frequency and the presence of strong VWG chromatin organizing signals. Large (> or = 300 kb) genomic DNA regions having low average recombination frequency are enriched in chromatin organizing signals. As additional controls, we show using chromosome 1 that the VWG motif signals are not enriched in randomly selected DNA regions having the mean size of the recombination coldspots, and that non-VWG motif sets do not generate signals that are enriched in recombination coldspots. We also show that tandemly repeated alpha satellite DNA contains strong VWG signals for the formation of distinctive nucleosome arrays, consistent with the low recombination activity of centromeres. Our correlations cannot be explained simply by variations in the GC content. Our findings suggest that a specific set of periodic DNA motifs encoded in genomic DNA, which provide signals for chromatin organization, influence human chromosome function.

Detecting nucleosome ladders on unique DNA sequences in mouse liver nuclei.

Nucleosome arrangements and possibly chromatin higher order structures differ in different regions of genomic DNA, and these differences could be functionally important. Nucleosome arrangements are reflected by the nucleosome ladders they give rise to upon micrococcal nuclease digestion of the chromatin. Here we describe how Southern hybridization can be used to detect the nucleosome ladders arising from different regions of DNA in mouse liver nuclei.

Aspects of large-scale chromatin structures in mouse liver nuclei can be predicted from the DNA sequence.

The large amount of non-coding DNA present in mammalian genomes suggests that some of it may play a structural or functional role. We provide evidence that it is possible to predict computationally, from the DNA sequence, loci in mouse liver nuclei that possess distinctive nucleosome arrays. We tested the hypothesis that a 100 kb region of DNA possessing a strong, in-phase, dinucleosome period oscillation in the motif period-10 non-T, A/T, G, should generate a nucleosome array with a nucleosome repeat that is one-half of the dinucleosome oscillation period value, as computed by Fourier analysis of the sequence. Ten loci with short repeats, that would be readily distinguishable from the pervasive bulk repeat, were predicted computationally and then tested experimentally. We estimated experimentally that less than 20% of the chromatin in mouse liver nuclei has a nucleosome repeat length that is 15 bp, or more, shorter than the bulk repeat value of 195 +/- bp. All 10 computational predictions were confirmed experimentally with high statistical significance. Nucleosome repeats as short as 172 +/- 5 bp were observed for the first time in mouse liver chromatin. These findings may be useful for identifying distinctive chromatin structures computationally from the DNA sequence.

Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation.

Linker histone H1 plays an important role in chromatin folding in vitro. To study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. H1 depletion caused dramatic chromatin structure changes, including decreased global nucleosome spacing, reduced local chromatin compaction, and decreases in certain core histone modifications. Surprisingly, however, microarray analysis revealed that expression of only a small number of genes is affected. Many of the affected genes are imprinted or are on the X chromosome and are therefore normally regulated by DNA methylation. Although global DNA methylation is not changed, methylation of specific CpGs within the regulatory regions of some of the H1 regulated genes is reduced. These results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns.

Long-range oscillation in a periodic DNA sequence motif may influence nucleosome array formation.

We have experimentally examined the characteristics of nucleosome array formation in different regions of mouse liver chromatin, and have computationally analyzed the corresponding genomic DNA sequences. We have shown that the mouse adenosine deaminase (MADA) gene locus is packaged into an exceptionally regular nucleosome array with a shortened repeat, consistent with our computational prediction based on the DNA sequence. A survey of the mouse genome indicates that <10% of 70 kb windows possess a nucleosome-ordering signal, consisting of regular long-range oscillations in the period-10 triplet motif non-T, A/T, G (VWG), which is as strong as the signal in the MADA locus. A strong signal in the center of this locus, confirmed by in vitro chromatin assembly experiments, appears to cooperate with weaker, in-phase signals throughout the locus. In contrast, the mouse odorant receptor (MOR) locus, which lacks locus-wide signals, was representative of approximately 40% of the mouse genomic DNA surveyed. Within this locus, nucleosome arrays were similar to those of bulk chromatin. Genomic DNA sequences which were computationally similar to MADA or MOR resulted in MADA- or MOR-like nucleosome ladders experimentally. Overall, we provide evidence that computationally predictable information in the DNA sequence may affect nucleosome array formation in animal tissue.

Exploring the characteristics of sequence elements in proximal promoters of human genes.

Central to reconstruction of cis-regulatory networks is identification and classification of naturally occurring transcription factor-binding sites according to the genes that they control. We have examined salient characteristics of 9-mers that occur in various orders and combinations in the proximal promoters of human genes. In evaluations of a dataset derived with respect to experimentally defined transcription initiation sites, in some cases we observed a clear correspondence of highly ranked 9-mers with protein-binding sites in genomic DNA. Evaluations of the larger dataset, derived with respect to the 5' end of human ESTs, revealed that a subset of the highly ranked 9-mers corresponded to sites for several known transcription factor families (including CREB, ETS, EGR-1, SP1, KLF, MAZ, HIF-1, and STATs) that play important roles in the regulation of vertebrate genes. We identified several highly ranked CpG-containing 9-mers, defining sites for interactions with the CREB and ETS families of proteins, and identified potential target genes for these proteins. The results of the studies imply that the CpG-containing transcription factor-binding sites regulate the expression of genes with important roles in pathways leading to cell-type-specific gene expression and pathways controlled by the complex networks of signaling systems.

DNA sequence alterations affect nucleosome array formation of the chicken ovalbumin gene.

The role of the large amount (more than half of the genome) of noncoding DNA in higher organisms is not well understood. DNA evolved to function in the context of chromatin, and the possibility exists that some of the noncoding DNA serves to influence chromatin structure and function. In this age of genomics and bioinformatics, genomic DNA sequences are being searched for informational content beyond the known genetic code. The discovery that period-10 non-T, A/T, G (VWG) triplets are among the most abundant motifs in human genomic DNA suggests that they may serve some function in higher organisms. In this paper, we provide direct evidence that the regular oscillation of period-10 VWG that occurs in the chicken ovalbumin gene sequence with a dinucleosome-like period facilitates nucleosome array formation. Using a linker histone-dependent in vitro chromatin assembly system that spontaneously aligns nucleosomes into a physiological array, we show that nucleosomes tend to avoid DNA regions with low period-10 VWG counts. This avoidance leads to the formation of an array with a nucleosome repeat equal to half the period value of the oscillation in period-10 VWG, as determined by Fourier analysis. Two different half-period deletions in the wild-type DNA sequence altered the nucleosome array, as predicted computationally. In contrast, a full-period deletion had an insignificant effect on the nucleosome array formed, also consistent with the prediction. An inversion mutation, with no DNA sequences deleted, again altered the nucleosome array formed, as predicted computationally. Hence, a VWG dinucleosome signal is plausible.

Postoperative nausea and vomiting: comparison of the effect of postoperative meperidine or morphine in gynecologic surgery patients.

STUDY OBJECTIVE: To evaluate the incidence and severity of postoperative nausea and vomiting in women receiving postoperative intravenous morphine or meperidine following gynecologic surgery. DESIGN: Prospective, double-blind, randomized study. SETTING: Tertiary-care academic medical center. PATIENTS: 200 ASA physical status I, II, and III patients scheduled for elective gynecologic surgery. INTERVENTIONS: Patients received either postoperative IV morphine (n = 100) or meperidine (n = 100) following gynecologic surgery. MEASUREMENTS: We compared pain scores, sedation scores, nausea scores, well-being scores, vomiting rate, and patient satisfaction in both groups 15, 30, 60, and 120 minutes after arrival in the postoperative anesthesia care unit. MAIN RESULTS: The vomiting rate was 8/100 versus 7/100 (at 15 min), 4/100 versus 26/100 (at 30 min) (p < 0.05), 3/100 versus 23/100 (at 60 min) (p < 0.05), and 0/100 versus 0/100 (at 120 min) in the morphine or meperidine groups, respectively. The pain and sedation scores were similar in both groups. No major complications were noted in either group. CONCLUSION: Our study demonstrates an advantage of the use of morphine rather than meperidine for pain control in the immediate postoperative period following gynecologic surgery.