Pedicle De-epithelialization in Reduction Mammoplasty: A Systematic Review of the Literature.

BACKGROUND: Reduction mammoplasty is among the most commonly performed plastic surgery procedures. The reduction pedicle is traditionally de-epithelialized. Many practitioners propose that preservation of the subdermal blood supply results in improved circulation for the nipple-areolar complex. However, this is a time-consuming step of the procedure. Presumed benefits have not been definitively demonstrated. OBJECTIVE: To evaluate current evidence regarding pedicle de-epithelialization in inferior pedicle reduction mammoplasty. METHODS: The MEDLINE database was searched for studies evaluating pedicle de-epithelialization in reduction mammoplasty surgery. Studies reporting outcomes after reduction mammoplasty with pedicle de-epithelialization and deskinning were included. Additionally, reports detailing novel techniques or modification for pedicle de-epithelialization were evaluated and included. RESULTS: One hundred and thirty-eight articles were identified on a systematic review. Thirty-six articles met the study criteria. This includes 23 reporting outcomes after reduction mammoplasty procedures and 13 describing novel techniques for pedicle de-epithelialization. Of the 23 studies reporting outcomes, 6 studies evaluated deskinning of the pedicle. Two of the six studies directly compared deskinning and de-epithelialization. They reported no significant difference in outcomes. The remaining 17 articles described outcomes of inferior pedicle reduction mammoplasty with pedicle de-epithelialization. Studies evaluating deskinning reported ischemic nipple-areolar complex complication rates between 0 and 1.4%. Studies evaluating de-epithelialization reported ischemic nipple-areolar complex complication rates between 0 and 11.1%. CONCLUSIONS: Pedicle de-epithelialization is commonly performed despite limited definitive evidence evaluating its surgical necessity or benefits. Available evidence suggests deskinning may yield acceptable results; however, further investigation is necessary. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

An Automated Miniaturized Method to Perform and Analyze Antimicrobial Drug Synergy Assays.

In the light of emerging antibiotic resistance mechanisms found in bacteria throughout the world, discovery of drugs that potentiate the effect of currently available antibiotics remains an important aspect of pharmaceutical research in the 21st century. Well-established clinical tests exist to determine synergy in vitro, but these are only optimal for low-throughput experimentation while leaving analysis of results and interpretation of high-throughput microscale assays poorly standardized. Here, we describe a miniaturized broth microdilution checkerboard assay and data analysis method in 384-well plate format that conforms to the Clinical Laboratory and Standards Institute (CLSI) methods. This method has been automated and developed to rapidly determine the synergism of current antibiotics with various beta-lactamase inhibitors emerging from our antimicrobial research efforts. This technique increases test throughput and integrity of results, and saves test compound and labor. We facilitated the interpretation of results with an automated analysis tool allowing us to rapidly qualify inter- and intraplate robustness, determine efficacy of multiple antibiotics at the same time, and standardize the results of synergy interpretation. This procedure should enhance high-throughput antimicrobial drug discovery and supersedes former techniques.

Comparative analysis of metazoan chromatin organization.

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

The CLAMP protein links the MSL complex to the X chromosome during Drosophila dosage compensation.

The Drosophila male-specific lethal (MSL) dosage compensation complex increases transcript levels on the single male X chromosome to equal the transcript levels in XX females. However, it is not known how the MSL complex is linked to its DNA recognition elements, the critical first step in dosage compensation. Here, we demonstrate that a previously uncharacterized zinc finger protein, CLAMP (chromatin-linked adaptor for MSL proteins), functions as the first link between the MSL complex and the X chromosome. CLAMP directly binds to the MSL complex DNA recognition elements and is required for the recruitment of the MSL complex. The discovery of CLAMP identifies a key factor required for the chromosome-specific targeting of dosage compensation, providing new insights into how subnuclear domains of coordinate gene regulation are formed within metazoan genomes.

Identification of chromatin-associated regulators of MSL complex targeting in Drosophila dosage compensation.

Sex chromosome dosage compensation in Drosophila provides a model for understanding how chromatin organization can modulate coordinate gene regulation. Male Drosophila increase the transcript levels of genes on the single male X approximately two-fold to equal the gene expression in females, which have two X-chromosomes. Dosage compensation is mediated by the Male-Specific Lethal (MSL) histone acetyltransferase complex. Five core components of the MSL complex were identified by genetic screens for genes that are specifically required for male viability and are dispensable for females. However, because dosage compensation must interface with the general transcriptional machinery, it is likely that identifying additional regulators that are not strictly male-specific will be key to understanding the process at a mechanistic level. Such regulators would not have been recovered from previous male-specific lethal screening strategies. Therefore, we have performed a cell culture-based, genome-wide RNAi screen to search for factors required for MSL targeting or function. Here we focus on the discovery of proteins that function to promote MSL complex recruitment to "chromatin entry sites," which are proposed to be the initial sites of MSL targeting. We find that components of the NSL (Non-specific lethal) complex, and a previously unstudied zinc-finger protein, facilitate MSL targeting and display a striking enrichment at MSL entry sites. Identification of these factors provides new insight into how MSL complex establishes the specialized hyperactive chromatin required for dosage compensation in Drosophila.

A map of minor groove shape and electrostatic potential from hydroxyl radical cleavage patterns of DNA.

DNA shape variation and the associated variation in minor groove electrostatic potential are widely exploited by proteins for DNA recognition. Here we show that the hydroxyl radical cleavage pattern is a quantitative measure of DNA backbone solvent accessibility, minor groove width, and minor groove electrostatic potential, at single nucleotide resolution. We introduce maps of DNA shape and electrostatic potential as tools for understanding how proteins recognize binding sites in a genome. These maps reveal periodic structural signals in yeast and Drosophila genomic DNA sequences that are associated with positioned nucleosomes.

X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila.

The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes. The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3' ends. However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. Here we use global run-on sequencing (GRO-seq) to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome.

ChIP-chip versus ChIP-seq: lessons for experimental design and data analysis.

BACKGROUND: Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq) allows genome-wide discovery of protein-DNA interactions such as transcription factor bindings and histone modifications. Previous reports only compared a small number of profiles, and little has been done to compare histone modification profiles generated by the two technologies or to assess the impact of input DNA libraries in ChIP-seq analysis. Here, we performed a systematic analysis of a modENCODE dataset consisting of 31 pairs of ChIP-chip/ChIP-seq profiles of the coactivator CBP, RNA polymerase II (RNA PolII), and six histone modifications across four developmental stages of Drosophila melanogaster. RESULTS: Both technologies produce highly reproducible profiles within each platform, ChIP-seq generally produces profiles with a better signal-to-noise ratio, and allows detection of more peaks and narrower peaks. The set of peaks identified by the two technologies can be significantly different, but the extent to which they differ varies depending on the factor and the analysis algorithm. Importantly, we found that there is a significant variation among multiple sequencing profiles of input DNA libraries and that this variation most likely arises from both differences in experimental condition and sequencing depth. We further show that using an inappropriate input DNA profile can impact the average signal profiles around genomic features and peak calling results, highlighting the importance of having high quality input DNA data for normalization in ChIP-seq analysis. CONCLUSIONS: Our findings highlight the biases present in each of the platforms, show the variability that can arise from both technology and analysis methods, and emphasize the importance of obtaining high quality and deeply sequenced input DNA libraries for ChIP-seq analysis.

Expression profile analysis of the inflammatory response regulated by hepatocyte nuclear factor 4α.

BACKGROUND: Hepatocyte nuclear factor 4α (HNF4α), a liver-specific transcription factor, plays a significant role in liver-specific functions. However, its functions are poorly understood in the regulation of the inflammatory response. In order to obtain a genomic view of HNF4α in this context, microarray analysis was used to probe the expression profile of an inflammatory response induced by cytokine stimulation in a model of HNF4α knock-down in HepG2 cells. RESULTS: The expression of over five thousand genes in HepG2 cells is significantly changed with the dramatic reduction of HNF4α concentration compared to the cells with native levels of HNF4α. Over two thirds (71%) of genes that exhibit differential expression in response to cytokine treatment also reveal differential expression in response to HNF4α knock-down. In addition, we found that a number of HNF4α target genes may be indirectly mediated by an ETS-domain transcription factor ELK1, a nuclear target of mitogen-activated protein kinase (MAPK). CONCLUSION: The results indicate that HNF4α has an extensive impact on the regulation of a large number of the liver-specific genes. HNF4α may play a role in regulating the cytokine-induced inflammatory response. This study presents a novel function for HNF4α, acting not only as a global player in many cellular processes, but also as one of the components of inflammatory response in the liver.

Evaluation of a synthetic peptide as a replacement for the recombinant fusion protein of respiratory syncytial virus in a potency ELISA.

This report describes the development of a potency ELISA using a peptide derived from the motavizumab binding epitope of respiratory syncytial virus (RSV) F-protein. Motavizumab is an antibody therapeutic studied for the prevention of RSV disease. It binds to the RSV glycoprotein F (F-protein), blocking the ability of RSV to fuse with target cells. This binding is the basis for a potency ELISA, however, due to inefficient F-protein production, development of an alternative ligand for the potency ELISA was investigated. A series of synthetic peptides spanning the motavizumab epitope on F-protein were evaluated for motavizumab binding activity. A 26-mer peptide was identified with desirable motavizumab binding kinetics, as shown by ELISA and surface plasmon resonance. The peptide corresponds to a portion of the motavizumab binding domain on the F-protein, and is referred to as F-peptide. The binding of motavizumab to the F-peptide is used in a new motavizumab potency ELISA, which was shown to be robust and statistically comparable to the F-protein ELISA. In addition, based on a qualitative observation, this new ELISA may be able to detect motavizumab degradation with greater sensitivity compared to the F-protein ELISA.

Comprehensive analysis of the chromatin landscape in Drosophila melanogaster.

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.

Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus.

We have identified a single tryptophan (Trp) residue responsible for loss of binding and biological activity upon ultraviolet (UV) light irradiation in MEDI-493, a humanized monoclonal antibody (MAb) against respiratory syncytial virus (RSV). This finding provides a better understanding of structure-function relationship in a 150-kDa protein. Irradiation of MEDI-493 with UV light resulted in spectral changes typical of Trp photoproducts and in a progressive loss of MEDI-493 binding and biological activity as measured by ELISA, Biacore, and cell-based assays. Mass spectrometric characterization of the proteolytic peptides generated from the UV irradiated MEDI-493 confirmed that most methionine (Met) and a few Trp residues were oxidized to various extents upon exposure to UV light. Among Trp residues, only Trp-105, containing the most solvent-exposed indole moiety in MEDI-493 and residing in a complementary-determining region (CDR) of the heavy chain, was significantly oxidized. When bound to a synthetic antigenic peptide, MEDI-493 showed significant resistance toward binding activity loss during UV irradiation. A second MAb (MEDI-524) with Trp-105 replaced by phenylalanine (Phe) showed a similar pattern of Met oxidation, but no loss of binding and biological activity following irradiation. Treatment of both MAbs with Met- and Trp-specific oxidizing reagents showed that oxidation of Trp-105 correlated with the activity loss, whereas Met oxidation did not affect the activity. These results demonstrate that Trp-105 in MEDI-493 is responsible for the UV light-induced effects.