Ligation Site Research Articles

A novel point mutation affecting Asn76 of dystrophin protein leads to dystrophinopathy

Mutations in the DMD gene lead to Duchenne and Becker muscular dystrophy (DMD/BMD). Missense mutations are rare cause of DMD/BMD. A six-month-old male patient presented with mild generalized muscle weakness, hypotonia, and delayed motor development. Dystrophinopathy was suspected because of highly elevated serum creatine kinase level (1497 U/L) and tiered DMD gene analysis was performed. Multiplex ligation-dependent probe amplification (MLPA) assay showed deletion of exon 4, which could not be confirmed by another method. Sequencing of exon 4 revealed a novel de novo point mutation (c.227A>T, p.Asn76Ile) in the N-terminal actin-binding domain (N-ABD) of dystrophin protein. The false positive MLPA result was explained by the fact that the affected nucleotide lies directly at the 3' ligation site of the MLPA probe. Sequencing of the whole coding region of DMD gene proved c.227A>T to be the sole variant being potentially pathogenic. According to in silico analyses the mutation was predicted to be highly destabilizing on N-ABD structure possibly leading to protein malfunction. Muscle biopsy was performed and dystrophin immunohistochemistry results were suggestive of BMD. Our results highlight the importance of confirmatory testing of single-exon deletions detected by MLPA and we describe a novel, destabilizing missense mutation in the DMD gene.

Thirteen decades of peptide synthesis: key developments in solid phase peptide synthesis and amide bond formation utilized in peptide ligation.

A historical overview of peptide chemistry from T. Curtius to E. Fischer to M. Bergmann and L. Zervas is first presented. Next, the fundamentals of peptide synthesis with a focus on solid phase peptide synthesis by R. B. Merrifield are described. Immobilization strategies to attach the first amino acid to the resin, coupling strategies in stepwise peptide chain elongation, and approaches to synthesize difficult peptide sequences are also shown. A brief comparison between tert-butyloxycarbonyl (Boc)/benzyl (Bzl) strategy and 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (t -Bu) strategy utilized in solid phase peptide synthesis is given with an emphasis on the latter. Finally, the review focuses on the discovery and development of peptide ligation and the latest advances in this field including native amide bond formation strategies, these include the native chemical ligation, α-ketoacid-hydroxylamine ligation, and serine/threonine ligation which are the most commonly used chemoselective ligation methods that provide amide bond at the ligation site. This review provides an overview of the literature concerning the most important advances in the chemical synthesis of proteins and peptides covering the period from 1882 to 2017.

Engineering peptide ligase specificity by proteomic identification of ligation sites.

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme–substrate specificity limits its utility. Here, we present an approach for comprehensive characterization of peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme–substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. These studies provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Anatomical and electrical remodeling with incomplete left atrial appendage ligation: Results from the LAALA-AF registry.

The anatomical, electrical, and clinical impact of incomplete Lariat left atrial appendage ligation remains unclear. We studied LAA anatomy pre- and postligation using contrast enhanced-computed tomography (CT) scans in 91 patients with atrial fibrillation (AF) who subsequently underwent catheter ablation (CA). Eleven patients had an incomplete exclusion (12%) with a central leak ranging from 1 to 5 mm. Despite incomplete ligation; the LAA volume were reduced by 67% postprocedurally when compared to preprocedure. In 7 patients with a leak between 1 and 3 mm, there was a 77% reduction in LAA volume beyond the ligation site suggestive of remodeling of the LAA. In 4 patients with larger (4-5 mm) leak the LAA remnants (LAARs) were slightly larger than those with smaller leaks on follow-up CT scan. Three out of the 4 demonstrated LAA electrical activity during CA and underwent isolation of the LAA ostium. Follow-up imaging showed two of these LAARs completely sealed with no communication with the left atrium. There was no significant difference in the AF recurrence rates between the patients who had a leak versus those with complete ligation (4 of 11 [36%] vs. 22 of 80 [27%]; P=0.6). Oral anticoagulation was discontinued in all patients with small leaks and 2 patients with large leaks that sealed completely upon follow-up imaging. There were no strokes or TIAs at 12 months. Despite incomplete LAA ligation by Lariat device there is significant anatomical and electrical remodeling that resulted in reduction in LAA size, volume, and electrical activity.

Native Chemical Ligation Directed by Photocleavable Peptide Nucleic Acid (PNA) Templates

A novel peptide-peptide ligation strategy is introduced that has the potential to provide peptide libraries of linearly or branched coupled fragments and will be suited to introduce simultaneous protein modifications at different ligation sites. Ligation is assisted by templating peptide nucleic acid (PNA) strands, and therefore, ligation specificity is solely encoded by the PNA sequence. PNA templating, in general, allows for various kinds of covalent ligation reactions. As a proof of principle, a native chemical ligation strategy was elaborated. This PNA-templated ligation includes easy on-resin procedures to couple linkers and PNA to the respective peptides, and a traceless photocleavage of the linker/PNA oligomer after the ligation step. A 4,5-dimethoxy-2-nitrobenzaldehyde-based linker that allowed the photocleavable linkage of two bio-oligomers was developed.

Cytochrome c as a Peroxidase: Activation of the Precatalytic Native State by H2O2-Induced Covalent Modifications.

In addition to serving as respiratory electron shuttle, ferri-cytochrome c (cyt c) acts as a peroxidase; i.e., it catalyzes the oxidation of organic substrates by H2O2. This peroxidase function plays a key role during apoptosis. Typical peroxidases have a five-coordinate heme with a vacant distal coordination site that permits the iron center to interact with H2O2. In contrast, native cyt c is six-coordinate, as the distal coordination site is occupied by Met80. It thus seems counterintuitive that native cyt c would exhibit peroxidase activity. The current work scrutinizes the origin of this structure-function mismatch. Cyt c-catalyzed peroxidase reactions show an initial lag phase that is consistent with the in situ conversion of a precatalyst to an active peroxidase. Using mass spectrometry, we demonstrate the occurrence of cyt c self-oxidation in the presence of H2O2. The newly generated oxidized proteoforms are shown to possess significantly enhanced peroxidase activity. H2O2-induced modifications commence with oxidation of Tyr67, followed by permanent displacement of Met80 from the heme iron. The actual peroxidase activation step corresponds to subsequent side chain carbonylation, likely at Lys72/73. The Tyr67-oxidized/carbonylated protein has a vacant distal ligation site, and it represents the true peroxidase-active structure of cyt c. Subsequent self-oxidation eventually causes deactivation. It appears that this is the first report that identifies H2O2-induced covalent modifications as an essential component for the peroxidase activity of "native" cyt c.

Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation

The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5'-azide and 3'-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.

Chemical Protein Synthesis with the α-Ketoacid-Hydroxylamine Ligation.

The coupling of an α-ketoacid and a hydroxylamine (KAHA ligation) affords amide bonds under aqueous, acidic conditions without the need for protecting groups or coupling agents. Translating this finding into a general approach to chemical protein synthesis required the identification of methods to incorporate the key functional groups into unprotected peptide segments-ideally using well-established Fmoc solid-phase peptide synthesis protocols. A decade of effort has now led to robust, convenient methods for preparing peptides bearing free or masked C-terminal α-ketoacids and N-terminal hydroxylamines. The facile synthesis of the segments and the aqueous, acidic conditions of the KAHA ligation make it ideal for the construction of small proteins (up to 200 residues), including SUMO and related modifier proteins, betatrophin and other protein hormones, nitrophorin 4, S100A4, and the cyclic protein AS-48. Key to the successful development of this protein synthesis platform was the identification and gram-scale synthesis of (S)-5-oxaproline. This hydroxylamine monomer is completely stable toward standard methods and practices of solid-phase peptide synthesis while still performing very well in the KAHA ligation. This reaction partner-in contrast to all others examined-affords esters rather than amides as the primary ligation product. The resulting depsipeptides often offer superior solubility and handling and have been key in the chemical synthesis of hydrophobic and ampiphilic proteins. Upon facile O-to-N acyl shift, peptides bearing a noncanonical homoserine residue at the ligation site are formed. With proper choice of the ligation site, the incorporation of this unnatural amino acid does not appear to affect the structure or biological activity of the protein targets. The development of the chemical methods for preparing and masking peptide α-ketoacids and hydroxyalmines, the preparation of several protein targets by convergent ligation strategies, and the synthesis of new hydroxylamine monomers affording either natural or unnatural residues at the ligation site are discussed. By operation under acidic conditions and with a distinct preference for the ligation site, these efforts establish KAHA ligation as a complementary method to the venerable native chemical ligation (NCL) for chemical protein synthesis. This Account documents both the state of the KAHA ligation and the challenges in identifying, inventing, and optimizing new reactions and building blocks needed to interface KAHA ligation with Fmoc solid-phase peptide chemistry. With these challenges largely addressed, peptide segments ready for ligation are formed directly upon resin cleavage, facilitating rapid assembly of four to five segments into proteins. This work sets the stage for applications of the KAHA ligation to chemical biology and protein therapeutics.

Influence of coronary architecture on the variability in myocardial infarction induced by coronary ligation in rats.

It has been shown that the size of myocardial infarction in rats created by coronary ligation technique is not uniform, varying from 4% to 65%. We hypothesized that infarct size variability induced by coronary artery ligation might be caused by coronary artery branching pattern. Coronary artery angiography was performed in 50 normal Lewis rats and in chronic myocardial infarction models in which coronary artery was ligated immediately below the left atrial appendage or 2mm distal to the left atrial appendage (n = 25 for each), followed by histological analysis. Unlike the human, the rats had a single major septal artery arising from the proximal part of the left coronary artery (n = 30) or right coronary artery (n = 20). There were three branching patterns of left circumflex artery (LCX): 33 (66%) had LCX branching peripherally from a long left main coronary artery (LMCA), while the remainder 17 (34%) had the LCX branching from the proximal part of the septal artery or a short LMCA. The rats with distal coronary ligation presented myocardial infarction localized to an anterior territory irrespective of LCX branching pattern. In the rats with proximal coronary ligation, 64% (n = 16) had broad myocardial infarction involving the anterior and lateral territories, while the remainder (36%, n = 9) had myocardial infarction localized to an anterior territory with the intact LCX arising proximally from a short LMCA. The interventricular septum was spared from infarction in all rats because of its anatomical location. Infarct size variations were caused not only by ligation site but also by varying LCX branching patterns. There are potential risks to create different sizes of myocardial infarction, particularly when targeting a broad range of myocardial infarction. The territory of the septal artery always appears to be spared from myocardial infarction induced by the coronary ligation technique.

Immobilized copper iodide on a porous organic polymer bearing P,N-ligation sites: A highly efficient heterogeneous catalyst for C[sbnd]O bond formation reaction

Immobilized copper iodide on a triazine-phosphite porous organic polymer (CuI-TP-POP) was introduced as a novel heterogeneous catalyst, to be used in CO band formation reaction. The CuI-TP-POP catalyst was synthesized by complexation of CuI with a triazine-phosphite porous organic polymer (TP-POP), incorporating P,N-ligation sites. The TP-POP substrate is a porous material in sheet morphology with cavities in the range of nanometer scale incorporating P and N atoms. These nano-cavities create appropriate ligation sites on the surface of TP-POP with chelation character to coordinate with CuI, leading to the formation of a heterogeneous copper (I) catalyst. The CuI-TP-POP was found to be an efficient heterogeneous catalyst in CO bond formation reaction between phenolic compounds and aryl halides toward synthesis of diaryl ethers in high isolated yields. This catalyst system was reusable at least for five times without significant decreasing in its catalytic activity.

Self-assembly of peptide boroxoles on cis-dihydroxylated oligoamide templates in water.

We develop templates that can be used to stabilize consistent oligomers of a bioactive peptide. In the present study, we synthesize oligomers of an antibody epitope from the amyloidogenic prion protein. Dynamic covalent chemistry is the basis for the spontaneous condensation of 2, 3, 4 or 6 peptides with qualified polyol templates presenting the required number of bioorthogonal ligation sites. To study this process in aqueous solution, the N-terminal amino acid of a 13-mer peptide is first acylated with 4-carboxy-benzoboroxole (1-hydroxy-1,3-dihydrobenzo[c][1,2] oxaborole-5-carboxylic acid) and then mixed with the template to obtain self-assembled miniamyloids of specified degree of oligomerization. The template is assembled from bicyclic dipeptides of alternating d- and l-stereochemistry. The cis-diol group of this dipeptide hot=Tap (hot: d-hydroxythreonine, Tap: l-thiaproline) has sufficiently high affinity for boroxoles in water. A single N3 -hot=Tap-OMe dipeptide template forms a 1:1 complex with 4-carboxy-benzoboroxole with excellent diastereoselectivity. The oligomeric template N3 -(hot=Tap)n -OMe (n=2, 3, 4 or 6) presents a regular pattern of 2, 3, 4 or 6 cis-diol groups for the spontaneous esterification with the same number of boronic acids. Nuclear magnetic resonance identifies the homogenous regioselectivity and stereoselectivity of this ligation process. The combination of electron-poor benzoboroxoles with this optimized cis-diol template allows for the complete ligation under high-dilution conditions in water with only 1.3 equivalents of peptide-boroxole per diol functionality. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Silver ions as em marker of congo red ligation sites in amyloids and amyloid-like aggregates.

Congo red (CR) is a known selective amyloid ligand. The focus of our work is identification (by EM imaging) of dye binding sites and their distribution in amyloids and amyloid-like aggregates formed in vitro. In order to produce the required contrast, CR has been indirectly combined with metal via including Titan yellow (TY) by intercalation which exhibits a relatively strong affinity for silver ions. The resulting combined ligand retains its ability to bind to proteins (which it owes to CR) and can easily be detected in EM studies thanks to TY. We have found, however, that in protein aggregates where unfolding is stabilized by aggregation and therefore is irreversible, TY alone may serve as both, the ligand and the metal carrier. The formation of ordered structures in amyloids was studied using IgG light chains with amyloidogenic properties, converted into amyloids by shaking. The resulting EM images were subjected to interpretation on the basis of the authors' earlier research on the CR/light chain complexation process. Our results indicate that dimeric light chains, which are the subject of our study, produce amyloids or amyloid-like complexes with chain-like properties and strong helicalization tendencies. Cursory analysis suggests that the edge polypeptide loops belonging to unstable light chains form intermolecular bridges which promote creation of loose gel deposits, or are otherwise engaged in the swapping processes leading to higher structural ordering.

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

2% of Healthy Newborns Reveal ETV6-RUNX1 Fusion By Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL)

Pediatric acute lymphoblastic leukemia (ALL) is characterized by preleukemic recurrent chromosomal translocations that emerge in utero. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell precursor patients. A previous study suggested that ETV6-RUNX1-positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1-positive ALL in children (1/10,000) by a factor of 100. This finding strongly indicated that secondary cooperating oncogenic hits were necessary for development of overt leukemia. However, later studies could not confirm this high frequency.To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR-based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based retrospective screening of 300 cord blood samples from Danish newborns.The GIPFEL method is capable of detecting the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In contrast to previously used RNA-based methods, it relies on DNA as sample material, which is more stable than RNA. In the case of ETV6-RUNX1-positive leukemia GIPFEL exploits the unique presence of a genomic fragment joining material from chromosome 12 and 21. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. The ligation site is independent of the translocation point within the individual DNA circle. Digestion of the breakpoint regions of the ETV6 and RUNX1 gene with the restriction enzyme SacI generates fragments smaller than 50 kb. Primer pairs amplify the complete set of theoretically predicted circularized fragments requiring 37 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from mononuclear cells from cord blood samples of 300 newborns that were cryopreserved within 24 h (median 12 h) from birth. After B cell enrichment and column purification of DNA, the DNA was subjected to SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. Samples that screened positive underwent one further demultiplexed PCR, agarose gelelectrophoresis and Sanger sequencing to validate the result and to identify the breakpoint region. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In previously published proof-of-principle blinded studies we tested 61 samples obtained from ETV6-RUNX1-positive ALL patients. Without any unspecific result, 64% for ETV6-RUNX1 fusion genes were detected in that sample set. The sensitivity of the technique was estimated to be 10-4, i.e. one translocation carrying cell within 10,000 normal cells can theoretically be detected.Within the analyzed cohort of 300 healthy newborns 6 screened positive for the ETV6-RUNX1 translocation (2%) (Table 1). Further 700 cord blood samples are currently screened.Table 1: 6 of 300 cord blood samples from healthy newborns screened positive for the ETV6-RUNX1 translocation using the GIPFEL technique (Fueller E*, Schaefer D* et al. PloS One 2014, 9(8): e104419). Number of the positively tested healthy newborn within the cohort, used primers, and introns of RUNX1 and ETV6 affected by the translocation are presented.Our results indicate that the actual incidence of ETV6-RUNX1-positive cells in healthy newborns might be even higher than previously assumed, potentially due to instability of the ETV6-RUNX1 RNA transcript in preserved cord blood samples. This would hint at a comparably low penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and further strengthen the importance of secondary environmentally caused or spontaneously occurring cooperating oncogenic lesions for ETV6-RUNX1-positive childhood leukemia to emerge. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel Multiplex NGS-Based Digital MLPA Assay for Copy Number Detection of 700 Target Sequences in Childhood Acute Lymphoblastic Leukemia

▪Background: Recurrent and clonal genetic alterations are characteristic of different subtypes of pediatric acute lymphoblastic leukemia (ALL) and several of them are strong independent predictors of patient outcome. Multiplex Ligation-dependent Probe Amplification (MLPA) has become one of the standard methods for detection of common copy number alterations (CNAs), including IKZF1 deletions, which are associated with a poor outcome. However, standard MLPA assays only accommodate a maximum of 60 MLPA probes. In order to increase the number of target sequences in one assay, a next generation sequencing (NGS)-based MLPA variant has been developed. This digitalMLPA assay is based on the well-known MLPA procedure but can include up to 1000 probes in a single reaction, uses minute amounts of DNA (≥20ng) and can be analysed on all Illumina NGS platforms.Methods: A digitalMLPA assay was designed and optimised to detect CNAs of 55 key target genes and regions in childhood ALL, including deletions of B-cell differentiation and cell cycle control genes (PAX5, IKZF1/2/3, EBF1, RB1, CDKN2A/B, ETV6, and BTG1), iAMP21 and rearrangements of the PAR1 region, T-cell ALL associated aberrations (STIL-TAL1, LEF1, CASP8AP2, MYB, EZH2, MLLT3, NUP214-ABL1, PTEN, LMO1/2, NF1, SUZ12, PTPN2, PHF6), ERG and TP53 deletions. Several genetic alterations of potential prognostic and/or therapeutic relevance in ALL have also been included (NOTCH1, CD200/BTLA, VPREB1, TBL1XR1, PDGFRB-EBF1, IGHM, NR3C1/2, CREBBP, CTCF, ADD3, EPHA1, FHIT, SPRED1 and TOX). All target genes are covered by at least 3 digitalMLPA probes (~450 probes in total). Moreover, a set of 200 probes was designed for genome-wide detection of gross ploidy changes (high hyper- or hypodiploidy) and to determine the extent of CNAs, while also acting as reference probes for data normalization. Performance of all probes has been extensively tested on genomic DNA from healthy individuals and positive cell lines. Only probes with a standard deviation <0.12 were included. All probes were also tested under various experimental conditions, such as different salt, probe and polymerase concentrations. A series of 76 pediatric ALL patient samples (including both B-ALL and T-ALL samples), previously characterized for specific genetic aberrations by array CGH (aCGH) and/or SNP array, has been analysed single-blinded using the digitalMLPA assay. Results were compared to those obtained from standard MLPA assays (P335 ALL-IKZF1 and P327 iAMP21-ERG), which contain probes with different ligation sites for the respective genes. A dilution series of three B-ALL patient samples was analysed to determine the detection limit for subclonal aberrations.Results: All aberrations previously identified by other methods in 67 pediatric ALL samples, were also identified using the digitalMLPA assay. These included whole chromosome gains and losses, whole gene deletions or gains, iAMP21, fusion genes (STIL-TAL1, NUP214-ABL1 and PDGFRB-EBF1) and intragenic gene deletions (IKZF1, ERG, CDKN2A/B, ETV6, PAX5 RUNX1, RB1, LEF1, NR3C1, RAG2, VPREB1, MLLT3, BTG1 and PTEN). Of interest was a case of high hyperdiploidy, which was correctly identified by digitalMLPA, while by aCGH analysis this case was misinterpreted as having multiple deletions. Among several other intragenic deletions, a heterozygous deletion of ERG exons 5-9 was observed in one patient. In addition, several cases of homozygous single-exon deletions were observed (e.g. IKZF1 exon 8, LEF1exon 3) and confirmed by aCGH. These findings should be further investigated with respect to their clinical impact. Results from the dilution series indicated that subclonal aberrations can be reliably detected by this digitalMLPA assay if present in at least 20- 30% of tumour cells.Conclusions: This study demonstrates that digitalMLPA is a reliable technique that can be used to genetically characterize clinical samples of ALL patients. Experiments can be performed on small amounts of DNA in a high-throughput and cost-effective fashion. Because of the targeted approach, data analysis will be much easier as compared to array or other sequencing platforms. These results merit further consideration of (digital)MLPA as a valuable alternative for genetic work-up of newly diagnosed ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Influences of inferior mesenteric artery types and Riolan artery arcade absence on the incidence of anastomotic leakage after laparoscopic resection of rectal cancer

To evaluate the influences of inferior mesenteric artery (IMA) types and Riolan artery arcade absence on the incidence of anastomotic leakage(AL) after laparoscopic resection of rectal cancer. Clinical data of 116 local advanced rectal cancer patients who underwent laparoscopic resection in The Sixth Affiliated Hospital of Sun Yat-sen University from January 2012 to December 2014 were analyzed retrospectively. IMA and Riolan artery arcade were examined by preoperative computed tomography angiography (CTA) reconstruction. The influences of IMA type, Riolan artery arcade absence and ligation site (high or low) on AL were analyzed by Logistic regression. The proportion of IMA types(I(-IIII() was 57.8%(67/116), 10.3%(12/116), 31.0%(36/116) and 0.9%(1/116), respectively. Riolan artery arcade absence was found in 60.3%(70/116). Eight (6.9%) patients suffered from AL. IMA type III( had significantly higher AL incidence as compared to other IMA types [19.4%(7/36) vs. 1.2%(1/80), P=0.001]. Meanwhile, patients with Riolan artery arcade absence also had significantly higher AL incidence[11.4%(8/70) vs. 0.0%(0/46), P=0.030]. However, the difference in AL incidence between high and Low IMA ligation was not statistically significant [8.0%(7/87) vs. 3.4%(1/29), P=0.531]. Seven of these 8 AL patients were found in IMA type III( with Riolan artery arcade absence and high ligation. Multivariate analysis showed that IMA type III( (P=0.001) and Riolan artery arcade absence (P=0.002) were independent risk factors of AL. IMA type III( with Riolan artery arcade absence increases AL incidence significantly in laparoscopic resection of rectal cancer. IMA type and Riolan aretry arcade absence or not contribute to the selection of IMA ligation site in the operation. For the colorectal cancer patients with IMA type III( and Riolan artery arcade absence, selective low IMA ligation with root lymph node dissection should be recommended.

Combination of Thiol-Additive-Free Native Chemical Ligation/Desulfurization and Intentional Replacement of Alanine with Cysteine.

We report a novel strategy for native chemical ligation (NCL). Alanines not located at a ligation site are temporarily replaced with cysteines, and this enables efficient thiol-additive-free NCL, with subsequent desulfurization to regenerate the target peptide. We synthesized stresscopin-related peptide and neuroendocrine regulatory peptide-2 (NERP-2) by this method. We confirmed that both conventional alkyl thioester and thioester-equivalent N-acyl-N'-methyl-benzimidazolinone (MeNbz) can be adopted as thioester components for thiol-additive-free NCL of multi-Cys-containing peptides.

Anatomical and radiographical studies on the arterial supply of the udder in goat and their surgical importance

The present study aimed to show the arterial blood supply of the udder of the Egyptian native breed of goat (Baladi goat) to be used as a guide during mastectomy and other surgical interferences. The study was carried out on the udder of twelve apparently healthy adult female Egyptian Baladi goats. Four goats were used for mastectomy, one specimen was used for radiography and the other specimens were subjected to gum-milk latex injection to clarify the origin, course and distribution of the main arteries supplying the udder. The results revealed that the udder of goat was supplied by the external pudendal artery and dorsal labial and mammary branch of the ventral perineal artery. The course of the external pudendal artery through the inguinal canal before reaching the base of the udder, as well as that of the dorsal labial and mammary branch of the ventral perineal artery in the perineal region, were briefly described to determine the appropriate site for ligation of these vessels before mastectomy. Moreover, the arterial interconnection between the two halves of the udder was emphasized to be highlighted during unilateral mastectomy. Vascular ligation of the main blood vessels supplying the udder on the basis of the anatomical description provided less traumatic surgery and reduced the severity of the blood loss.

OS 21-08 NOVEL REDUCED UTERINE PERFUSION PRESSURE (RUPP) MODEL OF PREECLAMPSIA IN MICE

Objective: Preeclampsia (PE) is a pregnancy-induced hypertension with proteinuria that typically develops after 20 weeks of gestation. A reduction in uterine blood flow causes placental ischemia and placental release of anti-angiogenic factors such as soluble fms-like tyrosine kinase (sFlt-1) followed by PE. Although the reduced uterine perfusion pressure (RUPP) model is widely used in rats, investigating the genetics of PE has been problematic because it has been difficult to make a useful RUPP model in mice. The aim of the present study is to establish a novel PE model using an improved RUPP method in mice. Design and Method: As shown in the Figure 1B, we bilaterally ligated ovarian vessels distal to ovarian branches, uterine vessels, or both in ICR-strain mice at 14.5 days post coitum (dpc). BP, renal phenotype and pregnancy outcome were analyzed. Results: Unlike the RUPP models described in the literature, this model did not constrict the abdominal aorta, which allowed BP to be measured with a tail cuff. RUPP in mice had elevated BP, increased urinary albumin excretion, severe endotheliosis, and mesangial expansion. They also had an increased incidence of miscarriage and premature delivery. Embryonic weight at 18.5 dpc was significantly lower than that in sham mice. The closer to the ligation site the embryos were, the higher the resorption rate and the lower the embryonic weight. The phenotype was more severe in the order of ligation at the ovarian vessels < uterine vessels < both. Conclusions: We developed a novel RUPP mouse model that recapitulates the phenotype of PE. This model is expected to be useful for investigating pathogenesis of PE in genetically engineered mice and for evaluating new therapies for PE.

Methods for Acute and Subacute Murine Hindlimb Ischemia.

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators' ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.