Thoracic epidural-based Enhanced Recovery After Surgery (ERAS) pathway for Nuss repair of pectus excavatum shortened length of stay and decreased rescue intravenous opiate use.

BACKGROUND: PCA- and block-based enhanced recovery after surgery (ERAS) pathways have been shown to decrease hospital length of stay (HLOS) and opiate use following Nuss Repair for Pectus Excavatum (NRPE). No thoracic epidural-based ERAS pathway has demonstrated similar benefits. METHODS: In this pre-post single-center study, data were retrospectively collected for patients ≤ 21 years undergoing NRPE from May 2015 to August 2019. Univariate and multivariate methods were used to evaluate whether implementation of a thoracic epidural-based ERAS in April 2017 was associated with HLOS, opiate use, or pain scores. RESULTS: There were 110 patients: 35 pre- and 75 post-ERAS. HLOS decreased from median 4.8 (1.1) to 3.3 (0.6) days with ERAS (p < 0.001). Use of rescue intravenous opiates decreased from 35.3% pre- to 9.3% with ERAS (p = 0.013). When adjusted for baseline characteristics, ERAS was associated with a 1.3 ± 0.2 day decrease in HLOS and 0.188 times the odds of rescue intravenous opiate use (p = 0.011). CONCLUSIONS: Pain scores, ED visits, and readmissions did not change with ERAS (p > 0.05). Implementation of a thoracic epidural-based ERAS following NRPE was associated with decreased HLOS and need for any rescue intravenous opiates without a change in pain scores, ED visits, or readmission.

Carotid artery mobilization prior to pharyngeal flap inset for patients with 22q11.2 deletion syndrome.

The management of velopharyngeal insufficiency (VPI) in patients with 22q11.2 deletion syndrome (22q11DS) poses a significant clinical challenge due to presence of a large velopharyngeal gap and a relatively high rate of internal carotid artery (ICA) medialization. To our knowledge, we are the first group to have successfully managed VPI in a series of seven pediatric patients with 22q11DS with medialized ICAs via a novel surgical technique involving carotid artery mobilization followed by pharyngeal flap insertion. Thus far, we have found this technique to be reliably safe with no significant morbidity and caregivers have reported postoperative improvement in speech, swallowing and nasal regurgitation symptoms. Herein, we provide a detailed description of our novel surgical approach, including an instructional video, for correction of VPI in patients with medialized ICAs, who have previously had limited management options.

CHD4 slides nucleosomes by decoupling entry- and exit-side DNA translocation.

Chromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and it is unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family. We demonstrate that the binding energy for CHD4-nucleosome complex formation-even in the absence of nucleotide-triggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4-6 base-pair steps. This decoupling of entry- and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. Based on our work and previous studies, we propose a mechanism for nucleosome sliding.

The interplay between H2A.Z and H3K9 methylation in regulating HP1α binding to linker histone-containing chromatin.

One of the most intensively studied chromatin binding factors is HP1α. HP1α is associated with silenced, heterochromatic regions of the genome and binds to H3K9me3. While H3K9me3 is necessary for HP1α recruitment to heterochromatin, it is becoming apparent that it is not sufficient suggesting that additional factors are involved. One candidate proposed as a potential regulator of HP1α recruitment is the linker histone H1.4. Changes to the underlying make-up of chromatin, such as the incorporation of the histone variant H2A.Z, has also been linked with regulating HP1 binding to chromatin. Here, we rigorously dissected the effects of H1.4, H2A.Z and H3K9me3 on the nucleosome binding activity of HP1α in vitro employing arrays, mononucleosomes and nucleosome core particles. Unexpectedly, histone H1.4 impedes the binding of HP1α but strikingly, this inhibition is partially relieved by the incorporation of both H2A.Z and H3K9me3 but only in the context of arrays or nucleosome core particles. Our data suggests that there are two modes of interaction of HP1α with nucleosomes. The first primary mode is through interactions with linker DNA. However, when linker DNA is missing or occluded by linker histones, HP1α directly interacts with the nucleosome core and this interaction is enhanced by H2A.Z with H3K9me3.

Surgical options in the treatment of ulcerative colitis.

Children and young adults with ulcerative colitis tend to present with more extensive colonic disease than an adult population. The need for surgical intervention in the pediatric population with ulcerative colitis occurs earlier after diagnosis and has a greater incidence than a comparably matched adult population with an estimated need for colectomy at 5 years following diagnosis of 14-20%. Perhaps, even more than the adult population, there is a desire to restore intestinal continuity for the pediatric patient to achieve as healthy and normal quality of life as possible. With surgery playing such a prominent role in the treatment of ulcerative colitis in this age group, an understanding of the surgical treatment options that are available is important. The surgeon's awareness of the complexities of the different operations associated with proctocolectomy and reestablishing intestinal continuity may help to avoid early complications and minimize the risk of less than ideal long-term outcomes.

Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes.

The yeast Chd1 protein acts to position nucleosomes across genomes. Here, we model the structure of the Chd1 protein in solution and when bound to nucleosomes. In the apo state, the DNA-binding domain contacts the edge of the nucleosome while in the presence of the non-hydrolyzable ATP analog, ADP-beryllium fluoride, we observe additional interactions between the ATPase domain and the adjacent DNA gyre 1.5 helical turns from the dyad axis of symmetry. Binding in this conformation involves unravelling the outer turn of nucleosomal DNA and requires substantial reorientation of the DNA-binding domain with respect to the ATPase domains. The orientation of the DNA-binding domain is mediated by sequences in the N-terminus and mutations to this part of the protein have positive and negative effects on Chd1 activity. These observations indicate that the unfavorable alignment of C-terminal DNA-binding region in solution contributes to an auto-inhibited state.

The Chromatin Remodelling Protein CHD1 Contains a Previously Unrecognised C-Terminal Helical Domain.

The packaging of eukaryotic DNA into nucleosomes, and the organisation of these nucleosomes into chromatin, plays a critical role in regulating all DNA-associated processes. Chromodomain helicase DNA-binding protein 1 (CHD1) is an ATP-dependent chromatin remodelling protein that is conserved throughout eukaryotes and has an ability to assemble and organise nucleosomes both in vitro and in vivo. This activity is involved in the regulation of transcription and is implicated in mammalian development and stem cell biology. CHD1 is classically depicted as possessing a pair of tandem chromodomains that directly precede a core catalytic helicase-like domain that is then followed by a SANT-SLIDE DNA-binding domain. Here, we have identified an additional conserved domain C-terminal to the SANT-SLIDE domain and determined its structure by multidimensional heteronuclear NMR spectroscopy. We have termed this domain the CHD1 helical C-terminal (CHCT) domain as it is comprised of five α-helices arranged in a variant helical bundle topology. CHCT has a conserved, positively charged surface and is able to bind DNA and nucleosomes. In addition, we have identified another group of proteins, the as yet uncharacterised C17orf64 proteins, as also containing a conserved CHCT domain. Our data provide new structural insights into the CHD1 enzyme family.

CHD4 Is a Peripheral Component of the Nucleosome Remodeling and Deacetylase Complex.

Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that in general comprise a central ATP-dependent Snf2 family helicase that is decorated with a variable number of regulatory subunits. The nucleosome remodeling and deacetylase (NuRD) complex, which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises ∼10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD family remodeling enzyme, most commonly CHD4 (chromodomain helicase DNA-binding protein 4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is a peripheral component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species. The addition of recombinant CHD4 to this nucleosome deacetylase complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data contribute to our understanding of the architecture of the NuRD complex.

A dual affinity-tag strategy for the expression and purification of human linker histone H1.4 in Escherichia coli.

Linker histones are an abundant and critical component of the eukaryotic chromatin landscape. They play key roles in regulating the higher order structure of chromatin and many genetic processes. Higher eukaryotes possess a number of different linker histone subtypes and new data are consistently emerging that indicate these subtypes are functionally distinct. We were interested in studying one of the most abundant human linker histone subtypes, H1.4. We have produced recombinant full-length H1.4 in Escherichia coli. An N-terminal Glutathione-S-Transferase tag was used to promote soluble expression and was combined with a C-terminal hexahistidine tag to facilitate a simple non-denaturing two-step affinity chromatography procedure that results in highly pure full-length H1.4. The purified H1.4 was shown to be functional via in vitro chromatin assembly experiments and remains active after extended storage at -80 °C.

The N-terminal Region of Chromodomain Helicase DNA-binding Protein 4 (CHD4) Is Essential for Activity and Contains a High Mobility Group (HMG) Box-like-domain That Can Bind Poly(ADP-ribose).

Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA-damage responses through its N-terminal region in a poly(ADP-ribose) polymerase-dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this N-terminal region. The-fold consists of a four-α-helix bundle with structural similarity to the high mobility group box, a domain that is well known as a DNA binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4-NuRD acts to regulate gene expression and participates in the DNA-damage response.

1H, 13C and 15N resonance assignments of a C-terminal domain of human CHD1.

Chromatin remodelling proteins are an essential family of eukaryotic proteins. They harness the energy from ATP hydrolysis and apply it to alter chromatin structure in order to regulate all aspects of genome biology. Chromodomain helicase DNA-binding protein 1 (CHD1) is one such remodelling protein that has specialised nucleosome organising abilities and is conserved across eukaryotes. CHD1 possesses a pair of tandem chromodomains that directly precede the core catalytic Snf2 helicase-like domain, and a C-terminal SANT-SLIDE DNA-binding domain. We have identified an additional conserved domain in the C-terminal region of CHD1. Here, we report the backbone and side chain resonance assignments for this domain from human CHD1 at pH 6.5 and 25 °C (BMRB No. 25638).

A peptide affinity reagent for isolating an intact and catalytically active multi-protein complex from mammalian cells.

We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1-15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST-FOG1(1-45).

Management of congenital tracheal anomalies and laryngotracheoesophageal clefts.

Congenital obstructions and anomalies of the pediatric airway are rare problems that may be associated with mild symptoms or critical stenoses that may be life threatening in the first few days of life. This review provides an overview of the embryologic development of the airway, different congenital anomalies associated with airway development, and surgical correction that may be associated with good long-term outcome.

Histone variants at the transcription start-site.

The function of a eukaryotic cell crucially depends on accurate gene transcription to ensure the right genes are expressed whereas unrequired genes are repressed. Therefore, arguably, one of the most important regions in the genome is the transcription start-site (TSS) of protein-coding and non-coding genes. Until recently, understanding the mechanisms that define the location of the TSS and how it is created has largely focused on the role of DNA sequence-specific transcription factors. However, within the nucleus of a eukaryotic cell, transcription occurs in a highly compacted nucleosomal environment, and it is becoming clear that accessibility of the TSS is a key controlling step in transcriptional regulation. It has traditionally been thought that transcription can only proceed once the nucleosomes at the TSS have been evicted. New work suggests otherwise, however, and the focus of this review is to challenge this belief.

Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription.

Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001.

Primitive myxoid mesenchymal tumor of infancy with rosettes: a new finding and literature review.

Primitive myxoid mesenchymal tumor of infancy (PMMTI) is a relatively recently described tumor arising in infants and demonstrating a unique histomorphology. We present an unusual case of PMMTI with rosettes, a hitherto undescribed finding in the reported cases. We also present the cytogenetic and ultrastructural findings of this tumor and review the literature. As awareness of PMMTI increases, additional clinical data and histopathologic findings will aid in the morphologic and behavioral characterization of this neoplasm.