Evaluating Surgery Resident Technical Skills: Intestinal Anastomosis in a Porcine Model.

Because work hour restrictions and technological developments such as staplers change the surgical landscape, efficient resident training methods are necessary to ensure surgical quality. This study evaluates efficacy of a porcine skills laboratory for teaching surgery residents to perform handsewn intestinal anastomoses based on a validated subjective tool and novel objective measurements. We hypothesized that resident performance would improve postintervention; junior residents would improve more than the seniors would. This prospective study was completed over a period of four months in 2015. Participants performed standardized two-layer, handsewn, end-to-end small intestine anastomosis in a live porcine model before (pretest) and after (posttest) an educational intervention. The intervention consisted of an instructional module and skills laboratory teaching session by attending surgeons. Participants were evaluated based on objective measurements of the anastomosis and blinded video evaluations using objective structured assessment of technical skills. Twenty-eight residents in a six-year general surgery program started and completed the study. The objective structured assessment of technical skills ratings demonstrated that the whole resident cohort had statistically significant improvement in pre- to posttest scores, 11.16 to 24.59 (P < 0.001). Junior and senior residents improved independently, 9.59 versus 22.53 (P < 0.001) and 13.59 versus 27.77 (P < 0.001), respectively. Finally, the cohort significantly improved in number of full-thickness Lembert sutures (2.36 vs 0.93, P = 0.001) and time to completion (31.28 vs 28.2 minutes, P = 0.046). Anastomotic leak pressure, anastomotic narrowing, and anastomotic tensile strength all trended toward improvement. A structured educational intervention, teaching intestinal anastomosis in a live porcine model produced significant improvement in residents' technical skills.

Characterization and serologic analysis of the Treponema pallidum proteome.

Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease characterized by widespread tissue dissemination and chronic infection. In this study, we analyzed the proteome of T. pallidum by the isoelectric focusing (IEF) and nonequilibrating pH gel electrophoresis (NEPHGE) forms of two-dimensional gel electrophoresis (2DGE), coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis. We determined the identity of 148 T. pallidum protein spots, representing 88 T. pallidum polypeptides; 63 of these polypeptides had not been identified previously at the protein level. To examine which of these proteins are important in the antibody response to syphilis, we performed immunoblot analysis using infected rabbit sera or human sera from patients at different stages of syphilis infection. Twenty-nine previously described antigens (predominantly lipoproteins) were detected, as were a number of previously unidentified antigens. The reactivity patterns obtained with sera from infected rabbits and humans were similar; these patterns included a subset of antigens reactive with all serum samples tested, including CfpA, MglB-2, TmpA, TmpB, flagellins, and the 47-kDa, 17-kDa, and 15-kDa lipoproteins. A unique group of antigens specifically reactive with infected human serum was also identified and included the previously described antigen TpF1 and the hypothetical proteins TP0584, TP0608, and TP0965. This combined proteomic and serologic analysis further delineates the antigens potentially useful as vaccine candidates or diagnostic markers and may provide insight into the host-pathogen interactions that occur during T. pallidum infection.

Histone acetyltransferase Hbo1: catalytic activity, cellular abundance, and links to primary cancers.

In addition to the well-characterized proteins that comprise the pre-replicative complex, recent studies suggest that chromatin structure plays an important role in DNA replication initiation. One of these chromatin factors is the histone acetyltransferase (HAT) Hbo1 which is unique among HAT enzymes in that it serves as a positive regulator of DNA replication. However, several of the basic properties of Hbo1 have not been previously examined, including its intrinsic catalytic activity, its molecular abundance in cells, and its pattern of expression in primary cancer cells. Here we show that recombinant Hbo1 can acetylate nucleosomal histone H4 in vitro, with a preference for lysines 5 and 12. Using semi-quantitative western blot analysis, we find that Hbo1 is approximately equimolar with the number of active replication origins in normal human fibroblasts but is an order of magnitude more abundant in both MCF7 and Saos-2 established cancer cell lines. Immunohistochemistry for Hbo1 in 11 primary human tumor types revealed strong Hbo1 protein expression in carcinomas of the testis, ovary, breast, stomach/esophagus, and bladder.

Cathepsin L proteolytically processes histone H3 during mouse embryonic stem cell differentiation.

Chromatin undergoes developmentally-regulated structural and chemical changes as cells differentiate, which subsequently lead to differences in cellular function by altering patterns of gene expression. To gain insight into chromatin alterations that occur during mammalian differentiation, we turned to a mouse embryonic stem cell (ESC) model. Here we show that histone H3 is proteolytically cleaved at its N-terminus during ESC differentiation. We map the sites of H3 cleavage and identify Cathepsin L as a protease responsible for proteolytically processing the N-terminal H3 tail. In addition, our data suggest that H3 cleavage may be regulated by covalent modifications present on the histone tail itself. Our studies underscore the intriguing possibility that histone proteolysis, brought about by Cathepsin L and potentially other family members, plays a role in development and differentiation that was not previously recognized.

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

Clostridium difficile lacks detectable superantigen activity.

Clostridium difficile colitis causes striking leukocytosis. We examined the possibility that toxins A or B, or other nontoxin products of C. difficile, act as superantigens, thereby stimulating leukocytosis. Our results failed to show major histocompatibility complex class II-dependent T lymphocyte proliferation, the hallmark of superantigen activity. Elevated white blood cell counts in C. difficile colitis are probably due to increased generation of cytokines such as interleukin-6 (IL-6) or IL-8.

Modifications of H3 and H4 during chromatin replication, nucleosome assembly, and histone exchange.

Histone posttranslational modifications that accompany DNA replication, nucleosome assembly, and H2A/H2B exchange were examined in human tissue culture cells. Through microsequencing analysis and chromatin immunoprecipitation, it was found that a subset of newly synthesized H3.2/H3.3 is modified by acetylation and methylation at sites that correlate with transcriptional competence. Immunoprecipitation experiments suggest that cytosolic predeposition complexes purified from cells expressing FLAG-H4 contain H3/H4 dimers, not tetramers. Studies of the deposition of newly synthesized H2A/H2B onto replicating and nonreplicating chromatin demonstrated that H2A/H2B exchange takes place in chromatin regions that contain acetylated H4; however, there is no single pattern of H4 acetylation that accompanies exchange. H2A/H2B exchange is also largely independent of the deposition of replacement histone variant, H3.3. Finally, immunoprecipitation of nucleosomes replicated in the absence of de novo nucleosome assembly showed that histone modifications do not prevent the transfer of parental histones to newly replicated DNA and thus have the potential to serve as means of epigenetic inheritance. Our experiments provide an in-depth analysis of the "histone code" associated with chromatin replication and dynamic histone exchange in human cells.

A chondroitin sulfate chain attached to the bone dentin matrix protein 1 NH2-terminal fragment.

Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein shown by gene ablations to be critical for the proper mineralization of bone and dentin. In the extracellular matrix of these tissues DMP1 is present as fragments representing the NH2-terminal (37 kDa) and COOH-terminal (57 kDa) portions of the cDNA-deduced amino acid sequence. During our separation of bone noncollagenous proteins, we observed a high molecular weight, DMP1-related component (designated DMP1-PG). We purified DMP1-PG with a monoclonal anti-DMP1 antibody affinity column. Amino acid analysis and Edman degradation of tryptic peptides proved that the core protein for DMP1-PG is the 37-kDa fragment of DMP1. Chondroitinase treatments demonstrated that the slower migration rate of DMP1-PG is due to the presence of glycosaminoglycan. Quantitative disaccharide analysis indicated that the glycosaminoglycan is made predominantly of chondroitin 4-sulfate. Further analysis on tryptic peptides led us to conclude that a single glycosaminoglycan chain is linked to the core protein via Ser74, located in the Ser74-Gly75 dipeptide, an amino acid sequence specific for the attachment of glycosaminoglycans. Our findings show that in addition to its existence as a phosphoprotein, the NH2-terminal fragment from DMP1 occurs as a proteoglycan. Amino acid sequence alignment analysis showed that the Ser74-Gly75 dipeptide and its flanking regions are highly conserved among a wide range of species from caiman to the Homo sapiens, indicating that this glycosaminoglycan attachment domain has survived an extremely long period of evolution pressure, suggesting that the glycosaminoglycan may be critical for the basic biological functions of DMP1.

MHC class Ib molecules bridge innate and acquired immunity.

Our understanding of the classical MHC class I molecules (MHC class Ia molecules) has long focused on their extreme polymorphism. These molecules present peptides to T cells and are central to discrimination between self and non-self. By contrast, the functions of the non-polymorphic MHC class I molecules (MHC class Ib molecules) have been elusive, but emerging evidence reveals that, in addition to antigen presentation, MHC class Ib molecules are involved in immunoregulation. As we discuss here, the subset of MHC class Ib molecules that presents peptides to T cells bridges innate and acquired immunity, and this provides insights into the origins of acquired immunity.

Human PAD4 regulates histone arginine methylation levels via demethylimination.

Methylation of arginine (Arg) and lysine residues in histones has been correlated with epigenetic forms of gene regulation. Although histone methyltransferases are known, enzymes that demethylate histones have not been identified. Here, we demonstrate that human peptidylarginine deiminase 4 (PAD4) regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. PAD4 targets multiple sites in histones H3 and H4, including those sites methylated by coactivators CARM1 (H3 Arg17) and PRMT1 (H4 Arg3). A decrease of histone Arg methylation, with a concomitant increase of citrullination, requires PAD4 activity in human HL-60 granulocytes. Moreover, PAD4 activity is linked with the transcriptional regulation of estrogen-responsive genes in MCF-7 cells. These data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones.

Structural requirements for signal transducer and activator of transcription 3 binding to phosphotyrosine ligands containing the YXXQ motif.

Stat3 is an Src homology (SH)2-containing protein constitutively activated in a wide variety of human cancers following its recruitment to YXXQ-containing motifs, which results in resistance to apoptosis. Despite resolution of the crystal structure of Stat3 homodimer bound to DNA, the structural basis for the unique specificity of Stat3 SH2 for YXXQ-containing phosphopeptides remains unresolved. We tested three models of this interaction based on computational analysis of available structures and sequence alignments, two of which assumed an extended peptide configuration and one in which the peptide had a beta-turn. By using peptide immunoblot affinity assays and mirror resonance affinity analysis, we demonstrated that only phosphotyrosine (Tyr(P)) peptides containing +3 Gln (not Leu, Met, Glu, or Arg) bound to wild type Stat3. Examination of a series of wild type and mutant Stat3 proteins demonstrated loss of binding to pYXXQ-containing peptides only in Stat3 mutated at Lys-591 or Arg-609, whose side chains interact with the Tyr(P) residue, and Stat3 mutated at Glu-638, whose amide hydrogen bonds with oxygen within the +3 Gln side chain when the peptide ligand assumes a beta-turn. These findings support a model for Stat3 SH2 interactions that could form the basis for anticancer drugs that specifically target Stat3.

Cotton rat Sihi-M3 is a minimally oligomorphic Mhc I-b molecule that binds the chemotactic peptide fMLF under stringent conditions. Evidence that positive selection drives inter-species diversity of residues interacting with the termini of short peptides.

The leading model for class I-b evolution suggests non-polymorphic I-b genes evolve by gene duplication from polymorphic I-a genes. We recently found N-formyl peptide-specific orthologs of the class I-b gene H2-M3 in the rodent subfamily Sigmodontinae. To test if sigmodont M3 is a I-b gene, we sequenced M3 from wild cotton rats ( Sigmodon hispidus) diverse at the class II locus, Sihi-DQA. These haplotypes carry a single allele of M3 that closely resembles H2-M3. However, peptide-binding assays showed that cotton rat M3 bound the chemotactic N-formylpeptide fMLF better than did rat or mouse M3. The Ala116-->Lys substitution in cotton rat M3 might enhance binding of fMLF and is one of eight residues of M3 that interact with ligand residues P3 and P4 and that are positively selected, with a d(N) /d(S) ratio of 1.8. Thus, M3 is a class I-b gene in both sigmodontine and murine murids, but positive selection operates on a small subset of residues in the traditionally defined antigen recognition site.

Hyperconservation of the N-formyl peptide binding site of M3: evidence that M3 is an old eutherian molecule with conserved recognition of a pathogen-associated molecular pattern.

The mouse MHC class I-b molecule H2-M3 has unique specificity for N-formyl peptides, derived from bacteria (and mitochondria), and is thus a pathogen-associated molecular pattern recognition receptor (PRR). To test whether M3 was selected for this PRR function, we studied M3 sequences from diverse murid species of murine genera Mus, Rattus, Apodemus, Diplothrix, Hybomys, Mastomys, and Tokudaia and of sigmodontine genera Sigmodon and PEROMYSCUS: We found that M3 is highly conserved, and the 10 residues coordinating the N-formyl group are almost invariant. The ratio of nonsynonymous and synonymous substitution rates suggests the Ag recognition site of M3, unlike the Ag recognition site of class I-a molecules, is under strong negative (purifying) selection and has been for at least 50-65 million years. Consistent with this, M3 alpha1alpha2 domains from Rattus norvegicus and Sigmodon hispidus and from the "null" allele H2-M3(b) specifically bound N-formyl peptides. The pattern of nucleotide substitution in M3 suggests M3 arose rapidly from murid I-a precursors by an evolutionary leap ("saltation"), perhaps involving intense selective pressure from bacterial pathogens. Alternatively, M3 arose more slowly but prior to the radiation of eutherian (placental) mammals. Older dates for the emergence of M3, and the accepted antiquity of CD1, suggest that primordial class I MHC molecules could have evolved originally as monomorphic PRR, presenting pathogen-associated molecular patterns. Such MHC PRR molecules could have been preadaptations for the evolution of acquired immunity during the early vertebrate radiation.

Identification and characterization of signal transducer and activator of transcription 3 recruitment sites within the epidermal growth factor receptor.

The oncogene signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a wide variety of human cancers, including squamous cell carcinoma of the head and neck. In squamous cell carcinoma of the head and neck, Stat3 activation is mediated by up-regulation of the autocrine ligand-receptor pair, tumor growth factor alpha and epidermal growth factor receptor (EGFR), resulting in cell growth and resistance to apoptosis. The initiating molecular event in Stat3 activation is recruitment to specific phosphotyrosine motifs within signaling complexes. Stat3 activation by the EGFR has been mapped to the COOH-terminal region of the EGFR between amino acid residues 1061 and 1123, which contains Y1068 and Y1086. However, it is not known if Stat3 binds directly to the EGFR or if either of these tyrosines is involved in this interaction. In this study, we demonstrated in stably transfected NIH-3T3 cells that activation of Stat3 by EGFR was eliminated by mutation of all five EGFR tyrosines to phenylalanine and that activation was restored with return of two of the mutated tyrosine sites, Y1068 and Y1086, to wild-type. Stat3 was detected in the activated EGFR complex, and its presence within the complex was dependent on Y1068 and/or Y1086. Phosphododecapeptides spanning Y1068 and Y1086 were able to pull down Stat3 with Y1068 being more effective than Y1086 in this regard. Real-time mirror resonance affinity analysis revealed Stat3 bound to phosphododecapeptide Y1068 with a K(D) of 135 +/- 20 nM and to phosphododecapeptide Y1086 with a K(D) of 243 +/- 36 nM (P = 0.044), consistent with the results of the pull-down assays. The lower K(D) of Y1068 was completely attributable to slower dissociation of Stat3 bound to Y1068 versus Y1086. Each phosphododecapeptide was capable of destabilizing Stat3 homodimers in vitro. When delivered into squamous carcinoma cells, phosphopeptides spanning Y1068 and Y1086 were able to inhibit EGFR-stimulated Stat3 DNA binding activity and cell proliferation.

Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites.

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173-Asp174, Ser180-Asp181, Ser217-Asp218, and Gln221-Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.

ABRF-ESRG'03: analysis of a PVDF-bound known protein with a homogeneous amino-terminus.

The Association of Biomolecular Resource Facilities 2003 Edman Sequencing Research Group (ABRF-ESRG'03) sample is the 15th in a series of studies designed to allow participating members to evaluate their abilities to analyze the N-terminus of a protein or peptide using automated Edman degradation chemistry. It is a follow-up study to the ESRG'02 sample, which was a single protein with a heterogeneous N-terminus. Both the 2002 and 2003 samples were obtained from the same protein complex and were resolved by SDS-PAGE followed by electrophoretic transfer to PVDF membrane. The ABRF-ESRG'03 sample had an apparent molecular weight of 49 kDa and a single N-terminus, with initial yields of approximately 2 pmol. Participants were asked to sequence 25 residues and return their results to the ESRG for analysis along with two completed surveys and an area/pmol table for repetitive and initial yield calculations. Data for 46 responses are presented which include initial yields, repetitive yields, sequencer performance, and ability to identify the protein.

Hyperconservation of the putative antigen recognition site of the MHC class I-b molecule TL in the subfamily Murinae: evidence that thymus leukemia antigen is an ancient mammalian gene.

"Classical" MHC class I (I-a) genes are extraordinarily polymorphic, but "nonclassical" MHC class I (I-b) genes are monomorphic or oligomorphic. Although diversifying (positive) Darwinian selection is thought to explain the origin and maintenance of MHC class I-a polymorphisms, genetic mechanisms underlying MHC class I-b evolution are uncertain. In one extreme model, MHC class I-b loci are derived by gene duplication from MHC class I-a alleles but rapidly drift into functional obsolescence and are eventually deleted. In this model, extant MHC class I-b genes are relatively young, tend to be dysfunctional or pseudogenic, and orthologies are restricted to close taxa. An alternative model proposed that the mouse MHC class I-b gene thymus leukemia Ag (TL) arose approximately 100 million years ago, near the time of the mammalian radiation. To determine the mode of evolution of TL, we cloned TL from genomic DNA of 11 species of subfamily Murinae: Every sample we tested contained TL, suggesting this molecule has been maintained throughout murine evolution. The sequence similarity of TL orthologs ranged from 85-99% and was inversely proportional to taxonomic distance. The sequences showed high conservation throughout the entire extracellular domains with exceptional conservation in the putative Ag recognition site. Our results strengthen the hypotheses that TL has evolved a specialized function and represents an ancient MHC class I-b gene.

The novel SLIK histone acetyltransferase complex functions in the yeast retrograde response pathway.

The SAGA complex is a conserved histone acetyltransferase-coactivator that regulates gene expression in Saccharomyces cerevisiae. SAGA contains a number of subunits known to function in transcription including Spt and Ada proteins, the Gcn5 acetyltransferase, a subset of TATA-binding-protein-associated factors (TAF(II)s), and Tra1. Here we report the identification of SLIK (SAGA-like), a complex related in composition to SAGA. Notably SLIK uniquely contains the protein Rtg2, linking the function of SLIK to the retrograde response pathway. Yeast harboring mutations in both SAGA and SLIK complexes displays synthetic phenotypes more severe than those of yeast with mutation of either complex alone. We present data indicating that distinct forms of the SAGA complex may regulate specific subsets of genes and that SAGA and SLIK have multiple partly overlapping activities, which play a critical role in transcription by RNA polymerase II.

Set2 is a nucleosomal histone H3-selective methyltransferase that mediates transcriptional repression.

Recent studies of histone methylation have yielded fundamental new insights pertaining to the role of this modification in gene activation as well as in gene silencing. While a number of methylation sites are known to occur on histones, only limited information exists regarding the relevant enzymes that mediate these methylation events. We thus sought to identify native histone methyltransferase (HMT) activities from Saccharomyces cerevisiae. Here, we describe the biochemical purification and characterization of Set2, a novel HMT that is site-specific for lysine 36 (Lys36) of the H3 tail. Using an antiserum directed against Lys36 methylation in H3, we show that Set2, via its SET domain, is responsible for methylation at this site in vivo. Tethering of Set2 to a heterologous promoter reveals that Set2 represses transcription, and part of this repression is mediated through the HMT activity of the SET domain. These results suggest that Set2 and methylation at H3 Lys36 play a role in the repression of gene transcription.