Split Variant Research Articles

Smart stacking for import containers using customer information at automated container terminals

Motivated by the practical import free-flow program that aims to expedite the container retrieval process, we conceptualize a new container stacking strategy, termed Smart Stacking (SS) strategy. The SS strategy aims to create relocation-free stacks (smart stacks) by utilizing customer information. The Storage Location Assignment Problem (SLAP) under the SS strategy is addressed. The problem is to determine the smart customers/containers, and the number and locations of smart stacks, when assigning a batch of import containers to a yard block at an automated container terminal to minimize the total retrieval time. Two variants of SLAP are investigated under the non-split policy and the split policy, depending on whether the containers from the same customer are allowed to be split between smart stacks and non-smart stacks. For the non-split variant, a mixed-integer programming (MIP) model is formulated first. By analyzing the properties of the optimal solution, an improved formulation with enhanced computational performance is then proposed. Based on the structure of the model, a divide-and-conquer heuristic is designed to solve the non-split variant more efficiently. For the split variant, a MIP model under the optimal partitions of the non-split model is developed. We theoretically prove that the split variant yields better results than the non-split variant. Extensive experiments are carried out to illustrate the effectiveness of smart stacking. It is found that customer information and yard utilization rate have a significant influence on the effectiveness of smart stacking.

Crystal structure and superconducting properties of Sc5Ir6Sn18

Sc5Ir6Sn18 crystallizes with a split variant of the Tb5Rh6Sn18 structure type (space group I41/acd, , ). DFT calculations confirmed the instability of the structural arrangement with the fully occupied and unsplit crystallographic sites. High quality single crystals were grown from a Sn melt. Sc5Ir6Sn18 is a diamagnetic metal showing a superconducting transition at a critical temperature K. The relatively low critical magnetic field 3.2 T as well as the obtained values of the specific heat ratio and energy-gap ratio suggest this system to be a weakly coupled BCS-like superconductor.

Nonparametric tests for equality of psychometric functions.

Many empirical studies measure psychometric functions (curves describing how observers' performance varies with stimulus magnitude) because these functions capture the effects of experimental conditions. To assess these effects, parametric curves are often fitted to the data and comparisons are carried out by testing for equality of mean parameter estimates across conditions. This approach is parametric and, thus, vulnerable to violations of the implied assumptions. Furthermore, testing for equality of means of parameters may be misleading: Psychometric functions may vary meaningfully across conditions on an observer-by-observer basis with no effect on the mean values of the estimated parameters. Alternative approaches to assess equality of psychometric functions per se are thus needed. This paper compares three nonparametric tests that are applicable in all situations of interest: The existing generalized Mantel-Haenszel test, a generalization of the Berry-Mielke test that was developed here, and a split variant of the generalized Mantel-Haenszel test also developed here. Their statistical properties (accuracy and power) are studied via simulation and the results show that all tests are indistinguishable as to accuracy but they differ non-uniformly as to power. Empirical use of the tests is illustrated via analyses of published data sets and practical recommendations are given. The computer code in MATLAB and R to conduct these tests is available as Electronic Supplemental Material.

Bimolecular fluorescence complementation based on the red fluorescent protein FusionRed

The aim of this work is to construct split variants of the red fluorescent protein FusionRed, each of which consists of two separate polypeptides, nonfluorescent parts of FusionRed, that can form functional fluorescent proteins upon reassociation. At the first stage, various circularly permuted FusionRed variants have been created (in circular permutants the protein polypeptide chain is divided into two parts, which change places so that the C-terminal part is followed by the N-terminal part). Two variants with the highest rate of chromophore maturation (fluorescence development) have been selected out of 23 tested permutation points. These proteins called cpFR76-73 and cpFR189-188 (the first number indicates the last amino acid residue of the N-terminal part; the second number, the first residue of the C-terminal part) are spectrally similar to parental FusionRed but possess lower fluorescence quantum yields. Split variants corresponding to these two circular permutants have been tested in mammalian cells. For reassembly of the fluorescent protein fragments, heterodimerizing leucine zippers have been used. It has been shown that split variant FR189-188 matures at 37°C and possesses fluorescence brightness similar to that of FusionRed. Consequently, FR189-188 is potentially suitable for a wide range of applications, for example, the study of protein–protein interactions or visualization of cell populations, in which two target gene promoters are simultaneously active.

Abstract 4096: A pilot study was initiated to determine if the immunomodulatory protein, the progesterone-induced blocking factor (PIBF), is present in higher quantity in the sera of patients with gynecologic cancer as compared to controls without cancer

Abstract PIBF is a unique protein that is secreted by gamma/delta T cells and precipitously rises in the sera following progesterone (P) exposure. The 34 kDa protein protects the fetal semi-allograft from immune surveillance by reducing cytoxicity of natural killer (NK) cells by stabilizing perforin granules. The parent 90 kDa form occupies a centrosomal position close to BRCA-1. PIBF can be found in most rapidly dividing cells. One study found an over-expression of the mRNA for the PIBF protein in all 29 leukemia cell lines tested. The P receptor antagonist mifepristone was found to down-regulate PIBF protein. Mifepristone provided significant palliative benefit to a large variety of spontaneous murine and human cancers. P could also be responsible for converting the 90 kDa parent form to a 34-36 kDa split variant in the cytoplasm which may be the immunosuppressive form in cancer cells. Alternatively, some P-like secretion by the tumor could influence gamma/delta T cells in the tumor microenvironment to secrete PIBF and possibly spill over to the sera. The aim of this study was to determine if there is any increase in serum PIBF in women with gynecologic cancer as opposed to controls. Serum was obtained from women about to have surgery for gynecologic problems including malignant and benign disorders. The samples would then be measured for PIBF using a new non-commercial enzyme linked immunoabsorbent assay (ELISA) for PIBF and serum progesterone. The PIBF levels (ng/mL) from lowest to highest in women with various gynecologic cancers (in all cases serum progesterone (P) <2ng/mL) were 10.06, 17.35, 32.59, 35.62, 54,7, and 57.17. The average serum PIBF was 34.6 ng/mL. There were 3 women with benign gynecologic tumors and the serum PIBF was 14.76, 15.7, and 36.64 their average serum PIBF was 22.5 ng/mL). There were 2 women with no tumors having gynecologic surgery and their serum PIBF levels (ng/mL) were 9.56 and 35.27 (with an average of 22.4 ng/mL). At least for gynecologic cancers if PIBF is conferring immune protection it is more likely operating through its intracytoplasmic presence rather than working through sera levels. In women or men exposed to exogenous or endogenous P for just 6 days it is not unusual to see sera PIBF rise to 300 to >800ng/mL. Citation Format: Jerome H. Check, Mojirayo Sarumi, Ann DiAntonio, Krystal Hunter, Gunda Simpkins, Marie Duroseau. A pilot study was initiated to determine if the immunomodulatory protein, the progesterone-induced blocking factor (PIBF), is present in higher quantity in the sera of patients with gynecologic cancer as compared to controls without cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4096. doi:10.1158/1538-7445.AM2014-4096

Probing Intein-Catalyzed Thioester Formation by Unnatural Amino Acid Substitutions in the Active Site

Inteins are single-turnover catalysts that splice themselves out of a precursor polypeptide chain. For most inteins, the first step of protein splicing is the formation of a thioester through an N-S acyl shift at the upstream splice junction. However, the mechanism by which this reaction is achieved and the impact of mutations in and close to the active site remain unclear on the atomic level. To investigate these questions, we have further explored a split variant of the Ssp DnaB intein by introducing substitutions with unnatural amino acids within the short synthetic N-terminal fragment. A previously reported collapse of the oxythiazolidine anion intermediate into a thiazoline ring was found to be specificially dependent on the methyl side chain of the flanking Ala(-1). The stereoisomer d-Ala and the constitutional isomers β-Ala and sarcosine did not lead to this side reaction but rather supported splicing. Substitution of the catalytic Cys1 with homocysteine strongly inhibited protein splicing; however, thioester formation was not impaired. These results argue against the requirement of a base to deprotonate the catalytic thiol group prior to the N-S acyl shift, because it should be misaligned for optimal proton abstraction. A previously described mutant intein evolved for more general splicing in different sequence contexts could even rather efficiently splice with this homocysteine. Our findings show the large impact of some subtle structural changes on the protein splicing pathway, but also the remarkable tolerance toward other changes. Such insights will also be important for the biotechnological exploitation of inteins.

Establishment of a functional cell line expressing both subunits of H1a and H2c of human hepatocyte surface molecule ASGPR

To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.

Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site-specific chemical modification

Naturally split DnaE intein from Nostoc punctiforme ( Npu) has robust protein trans-splicing activity and high tolerance of sequence variations at the splicing junctions. We determined the solution structure of a single chain variant of NpuDnaE intein by NMR spectroscopy. Based on the NMR structure and the backbone dynamics of the single chain NpuDnaE intein, we designed a functional split variant of the NpuDnaE intein having a short C-terminal half (C-intein) composed of six residues. In vivo and in vitro protein ligation of model proteins by the newly designed split intein were demonstrated.

Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI.

The general structural fold of the LAGLIDADG endonuclease family consists of two similar alpha/beta domains (alphabetabetaalphabetabetaalpha) that assemble either as homodimers or monomers with the domains related by pseudo-two-fold symmetry. At the center of this symmetry is the closely packed LAGLIDADG two-helix bundle that forms the main inter- or intra-molecular contact region between the domains of single- or double-motif proteins, respectively. In this work, we further examine the role of the LAGLIDADG residues involved in the helix-helix interaction. The interchangeability of the LAGLIDADG helix interaction was explored by grafting interfacial residues from the homodimeric I-CreI into the corresponding positions in the monomeric I-DmoI. The resulting LAGLIDADG exchange mutant is partially active, preferring to nick dsDNA rather than making the customary double-strand break. A series of partial revertants within the mutated LAGLIDADG region are shown to restore cleavage activity to varying degrees resulting in one I-DmoI mutant that is more active than wild-type I-DmoI. The phenotype of some of these mutants was reconciled on the basis of similarity to the GxxxG helix interaction found in transmembrane proteins. Additionally, a split variant of I-DmoI was created, demonstrating that the LAGLIDADG helices of I-DmoI are capable of forming and maintaining the protein-protein interface in trans to create an active heterodimer.

Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS)

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICB Glc subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICB Glc are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICB Glc and IIA Glc were produced in vivo by RecA intein-mediated trans-splicing. Purified, cyclized IIA Glc and IICB Glc had 100% and 30% of wild-type glucose phosphotransferase activity, respectively. Cyclized IIA Glc displayed increased stability against temperature and GuHCl-induced unfolding.

Expression of CD44 variant proteins in adenocarcinoma of Barrett's esophagus and its relation to prognosis.

None of the commonly used staging criteria accurately determine the prognosis of a patient with adenocarcinoma of Barrett's esophagus. The authors therefore assessed the expression pattern and prognostic impact of CD44 standard and CD44 isoforms CD44v4, v5,v6,v7, and v10 in adenocarcinoma of Barrett's esophagus. Specimens from 41 patients with adenocarcinoma of Barrett's esophagus who underwent esophageal resection were embedded in paraffin and studied immunohistochemically to determine the expression of CD44 splice variants. Histomorphologic parameters and survival time were not known at the time of the investigation. Correlations between favorable clinical or histomorphologic parameters and CD44s or any of the split variants could not be established. Down-regulation of CD44s and the split variant v10 was significantly correlated with pT classification. Furthermore, down-regulation of CD44v10 and up-regulation of CD44v7 were significantly correlated with ploidy. There was a significant correlation between CD44s and split variants in tumorous and nontumorous tissue from the same patient. Down-regulation of CD44s and CD44v4 had a significant influence on prognosis in that it was associated with shortened life expectancy. Multivariate analysis revealed that the expression of CD44v4 was an independent factor in prognosis. The results obtained for this small patient sample suggest that CD44v4 is a new independent prognostic parameter for adenocarcinoma of Barrett's esophagus that can be determined preoperatively by biopsy. It may therefore be helpful in planning therapy by allowing the identification of patients who may benefit from esophageal resection as well as those who are at high risk for morbidity and mortality even when the tumor is otherwise resectable. Further studies of larger patient samples are required to validate the results of the current study.

Die struktur des BaPtSn 3, BaNiSn 3 und SrNiSn 3 und ihre verwandtschaft zum ThCr 2Si 2-strukturtyp

The new compounds BaPtSn 3, BaNiSn 3 and SrNiSn 3 have been prepared and their crystal structures determined. They crystallize in the tetragonal system ( I4 mm) with 2 formula units in the cell with lattice constants: BaPtSn 3, a = 485 ± 1 pm, c = 1107 ± 2 pm, c a = 2.28 ; BaNiSn 3, a = 482 ± 1 pm, c = 1093 ± 2 pm, c a = 2.27 ; SrNiSn 3, a = 474 ± 1 pm, c = 1088 ± 2 pm, c a = 2.30 . The structure can be described as a split variant of the ThCr 2Si 2 structure type.