Complete Molecule Research Articles

An Atomic Model for a Complete Myosin Molecule within a Native Thick Filament

We have obtained a 4.3A resolution 3-D image of the relaxed thick filament from Lethocerus indicus using IHRSR. The image resolution is variable being highest for the region where the myosin rods are packed and lowest for the blocked head within the interacting heads motif (IHM). Although the resolution is low for de novo atomic model building, by using coiled-coil constraints within the model building software Rosetta, we obtained a complete atomic model for the Lethocerus flight muscle myosin rod, its least understood domain. For the rod, we started with a homology model of the Skip 1 crystal structure (PDB 4XA1) and built outward to the N- and C-termini. The myosin rods assemble into “curved molecular crystalline layers” (ribbons) and not as helical subfilaments. The number of plausible salt bridges between coiled-coils within ribbons is 30% greater than the number between ribbons. For the IHM, we used flexible fitting starting with an homology model of the Lethocerus myosin sequence and the scallop transition state structure, PDB 1DFL. Notable features of the IHM atomic model include the following: (1) Hydrophobic content in the free head RLC interaction with the myosin rods. This is the best-ordered region of the IHM. (2) At the free head S1/S2 junction, 11 residues of the heavy chain α-helix are unwound. Density connecting the displaced parts of the heavy chain α-helix is clearly resolved. (3) The heavy chain α-helices bound by the free head ELC and RLC could not be connected without unwinding 10 residues of the heavy chain α-helix. The blocked head density is the lowest resolution region of the reconstruction and could be fit without the heavy chain helical alterations seen in the free head. Supported by NIH and AHA.

Di-n-butyl-bis-[N-(2-meth-oxy-eth-yl)-N-methyl-dithio-carbamato-κ2S,S']tin(IV): crystal structure and Hirshfeld surface analysis.

The complete mol-ecule of the title compound, [Sn(C4H9)2(C5H10NOS2)2], is generated by a crystallographic mirror plane, with the SnIV atom and the two inner methyl-ene C atoms of the butyl ligands lying on the mirror plane; statistical disorder is noted in the two terminal ethyl groups, which deviate from mirror symmetry. The di-thio-carbamate ligand coordinates to the metal atom in an asymmetric mode with the resulting C2S4 donor set defining a skew trapezoidal bipyramidal geometry; the n-butyl groups are disposed to lie over the longer Sn-S bonds. Supra-molecular chains aligned along the a-axis direction and sustained by methyl-ene-C-H⋯S(weakly coordinating) inter-actions feature in the mol-ecular packing. A Hirshfeld surface analysis reveals the dominance of H⋯H contacts in the crystal.

Leporinus elongatus (Characiformes, Anostomidae): complete mtDNA sequence of an economically important fish from the Paraná and La Plata river basins

Leporinus elongatus is an important commercial fish found in the La Plata and Paraná River basins. Next-generation sequencing was used to sequence the mitochondrial DNA (mtDNA) of L. elongatus. The mtDNA was assembled using the CLC Workbench software v. 9.0 and subsequently aligned to other 10 complete fish mitochondrial sequences to enable phylogenetic analysis using MEGA 7.0. The complete mtDNA molecule had 16,784 bp and its GC content was 43%. The mtDNA structure was similar to that of other vertebrates: two ribosomal RNA, 22 transfer RNA, 13 protein-coding genes, and a D-loop region containing 1115 bp. Phylogenetic analysis yielded a tree with high bootstrap value that was coherent with the current phylogeny proposed for Characiformes.

The involvement of the ceramide transporter in Alzheimer’s disease Investigating the binding properties of the ceramide transporter in the human brain by optimizing the splitubiquitin Yeast Two-Hybrid system

Background: Alzheimer’s disease (AD) is the most common form of dementia, characterized by aggregates of Amyloid-ß (Aβ) peptides. The ceramide transporter (CERTL ) exists in a complex with serum amyloid P component in the Aβ plaques of AD patients. The goal is to obtain more information about the protein binding activity of CERTL in the human brain. To be able to do so, the Yeast Two-Hybrid (Y2H) technique, that was used to confirm the results from a previously executed CERTL Y2H screening, had to be optimized. Technical problems were encountered with the plasmid purification of the yeast NMY51 using the EZNA plasmid DNA minikit I and with the transformation in competent E.coli cells. Initially it was believed that there was a problem with the EZNA plasmid DNA minikit I. In this report troubleshooting was performed to identify the problem of the plasmid purification technique that has been used. Methods: To examine the protein binding activity of CERTL a split-ubiquitin Y2H system was used. In this system proteins are attached to two parts of one complete ubiquitin molecule, called the split-ubiquitin. The middle region domain of CERTL (the bait) was attached to the C-terminal half of the split-ubiquitin and the transcription factor LexA-VP16. The possible interactor, a prey library containing human brain proteins was fused to the N-terminal half of the split-ubiquitin. The presence of an interaction between bait and prey will results in the re-assembly of the split-ubiquitin, which activates ubiquitin proteases. The transcription factor is released and reporter genes are activated.The results of a previously executed Y2H screening had to be confirmed by performing another Y2H screening. The new screening had to be carried out using glycerol stocks of the yeast NMY51, containing both bait and prey, from the earlier performed Y2H screening. To optimize prey plasmid purification from the yeast using the EZNA plasmid DNA minikit I, troubleshooting was performed on the technique. Next to this, other methods to isolate the plasmid DNA, a QIAprep spin miniprep kit protocol and a phenol extraction and ethanol precipitation, were performed. The failing results were possibly due to the yeast being stored as a glycerol stock. In order to correct for this, a freshly transformed yeast sample was made. Results: Extracting the plasmid from glycerol stocks from the yeast NMY51 did not succeed. Plasmid purification and transformation of the prey plasmid from the fresh transformed yeast in E.coli competent cells was successful. Conclusion: From the results it is now believed that storing the yeast NMY51 containing the prey plasmid in glycerol stocks causes the prey plasmid to integrate into the genomic DNA of the yeast. Performing a new Y2H screening would therefore only be possible on fresh transformed yeast NMY51 cells.

Dimethyl 4,4′-(dimethylsilanediyl)dibenzoate

The complete molecule of the title compound, C18H20O4Si, is generated by crystallographic twofold symmetry, with the Si atom lying on the rotation axis. The molecule adopts a V-shape: the dihedral angle between the benzene ring and it attached methyl formate unit is 9.3 (2)°, and the dihedral angle between the benzene rings is 68.8 (1)°. In the crystal, weak C—H...O hydrogen bonds link the molecules into [101] chains.

Development of Novel Human Anti-HLA-Monoclonal Antibodies for Clinical Applications Using Peripheral Blood B Cells Derived from Anti-HLA Antibody-Positive Donors

Introduction: The development of monoclonal antibodies (mAbs) such as anti-CD20 mAb (rituximab) and anti-CD38 mAb (daratumumab) has revolutionized the treatment of lymphoid malignancies. Potential efficacy of HLA allele-specific mAbs in treating malignant lymphoma has been shown by several studies. However, treatment of B-cell malignancies with humanized mAbs against HLA-DR alleles is associated with infusion-related toxicities (Lin et al., Leuk Lymphoma, 2009). In addition, previous attempts to develop mAbs specific to some HLA alleles (e.g., HLA-B61) by immunizing mice with recombinant HLA proteins have failed, likely because of a wide variety of polymorphisms of HLA molecules. To overcome these problems, we recently created a novel method to develop mAbs using microarray technology and human peripheral blood (PB) B lymphocytes derived from donors who are positive for anti-HLA antibodies. The process took approximately 1 month to produce human anti-HLA mAbs.Methods: Approximately 20 ml of PB was collected from anti-HLA antibody-positive donors, and mononuclear cells (MNCs) isolated using the Ficoll-Hypaque method were cultured in RPMI-1640 + 10% FCS containing 5 μg/ml R-848, 1000 IU/ml hIL2, 2.5 μg/ml CpG2006, 2.5 μg/ml anti-CD40 antibody, 100 ng/ml hIL21, 2 ng/ml hIL17, 10 ng/ml hIL4, and 100 ng/ml hBAFF for 6 days. CD138+ cells were then isolated using anti-CD138-antibody-conjugated microbeads. A microwell array chip was manufactured using micromachining techniques, as previously described (Jin et al., Nature Medicine, 2009). Microwells (diameter, 10 μm; depth, 15 μm) were formed on a silicon surface. The chip was coated with a PBS-containing purified HLA antigen (10 μg/ml) and incubated overnight at 4°C. After removing the antigen solution, 50 μl of the CD138+ cell suspension was added to the chip, and the mixture was incubated for 4 h at 37°C. After gentle washing with PBS, 2 μg/ml of anti-human IgG Fc-Cy3 solution was added to the microwells, and the plate was left at room temperature for 15 min. Finally, the cells were stained with 1 μM Oregon Green for 2 min. The microwells were screened for the antigen-specific antibodies released from single cells under a fluorescence microscope. Antigen-specific antibody-secreting cells (ASCs) from individual wells were isolated using a micromanipulator fitted with capillaries under the fluorescence microscope and were then expelled to microtubes for reverse transcription. The antibody cDNA fragments for VH and VL fragments were amplified using the single-cell 5′-RACE method and inserted into expression vectors containing the complete constant region of cDNA for heavy or light chains. Thereafter, CHO cells were transfected with both the heavy and light chain expression vectors to obtain a supernatant containing complete antibody molecules. The antigen specificity of the recombinant antibodies was examined using ELISA and flow cytometry (FCM). The frequency of ASCs in the PB-MNCs of donors was quantified using allele-specific oligonucleotide PCR. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of the mAbs were assessed using conventional methods.Results and Discussion: Two novel human mAbs specific to HLA-A24 and HLA-B61 were successfully generated. The antigen specificity of the mAbs was confirmed using ELISA and FCM. The mAbs bound to normal and malignant blood cells that expressed corresponding HLA alleles and showed CDC/ADCC activities against five B lymphoblastoid cell lines but not against lymphocytes/monocytes/granulocytes derived from five healthy donors, probably because of low expression levels of the target HLA alleles. The frequency of ASCs in PB-MNCs of donors was less than 0.001%.Conclusions: The present method enabled the generation of mAbs specific to HLA alleles, which can be used for detecting minimal residual diseases of hematological malignancies as well as for treating B-cell lymphoma, within 1 month. DisclosuresTakamatsu:Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria. Kawai:Wakunaga Pharmaceutical Co., Ltd.: Employment. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

Diethyl 2,2′-(trisulfane-1,3-diyl)dibenzoate

The title compound, C18H18O4S3, was synthesized in the presence of chloridoauric acid from 3H-1,2-benzodithiole-3-thione as starting material. The asymmetric unit comprises one half of the molecule, the complete molecule being generated by the application of twofold rotation symmetry. The two benzene rings are inclined by 81.0 (2)°. In the crystal, slipped π–π stacking interactions are observed between the benzene rings of neighbouring molecules.

Chloridobis[2-(pyridin-2-yl-κN)benzo[b][1,5]naphthyridine-κN 1]copper(II) perchlorate acetonitrile disolvate

The copper(II) ion in the title complex, [CuCl(C17H11N3)2]ClO4·2CH3CN, is coordinated by four N atoms from two pbn ligands and one Cl− ion in a distorted trigonal–bipyramidal geometry (τ = 0.84). The asymmetric unit comprises half of the cationic complex molecule, and complete molecules are generated by twofold rotation symmetry with the corresponding axis running through the Cu atom and the coordinating Cl atom. The perchlorate anion is also located on a twofold rotation axis (passing through the Cl atom). In the crystal, there are π–π stacking interactions between the benzonaphthyridine rings of the pbn ligand of neighbouring cations.

2-Methyl-N-(pyrazin-2-yl)propanamide–1,2,4,5-tetrafluoro-3,6-diiodobenzene (2/1)

In the title compound, C8H11N3O·0.5C6F4I2, molecules ofiPr-substituted pyrazine are co-crystallized with 1,4-diiodo-2,3,5,6-tetrafluorobenzene. The complete molecule of 1,4-diiodo-2,3,5,6-tetrafluorobenzene is generated by an inversion centre at the middle of the aromatic ring. Both molecules have normal geometry and theiPr acylamine group is disordered over two sets of sites with an occupancy ratio of 0.51:0.49. In the crystal, the components are linked by I...N halogen bonds [2.830 (2) Å] and C—H...F interactions are observed.

Vibrational spectroscopic analysis, molecular dynamics simulations and molecular docking study of 5-nitro-2-phenoxymethyl benzimidazole

FT-IR and FT-Raman spectra of 5-nitro-2-phenoxymethylbenzimidazole were recorded and analyzed theoretically and experimentally. The splitting of NH stretching mode in the IR spectrum with a red shift from the calculated value indicates the weakening of the NH bond. The theoretical calculations give the phenyl ring breathing modes at 999 cm−1 for mono substituted benzene ring and at 1040 cm−1 for tri-substituted benzene ring. The theoretical NMR chemical shifts are in agreement with the experimental chemical shifts. The most reactive sites for electrophilic and nucleophilic attack are predicted from the MEP analysis. HOMO of π nature is delocalized over the entire molecule whereas the LUMO is located over the complete molecule except mono-substituted phenyl ring and oxygen atom. Reactive sites of the title molecule have been located with the help of ALIE surfaces and Fukui functions. In order to determine locations prone to autoxidation and locations interesting for starting of degradation, bond dissociation energies have been calculated for all single acyclic bonds. For the determination of atoms with pronounced interactions with water we have calculated radial distribution functions obtained after molecular dynamics simulations. The calculated first hyperpolarizability of the title compound is 58.03 times that of standard nonlinear optical material urea. The substrate binding site interactions of the title compound with Topo II enzyme is reported by using molecular docking study. Biological activity studies show that the title compound can be leaded for developing new anticancer agents.

Isotypic MnII and FeII binuclear complexes of the ligand 5,6-bis-(pyridin-2-yl)-pyrazine-2,3-di-carb-oxy-lic acid.

The title isotypic complexes, bis-[μ-5,6-bis-(pyridin-2-yl)pyrazine-2,3-di-carboxylato]-κ4N1,O2,N6:O3;κ4O3:N1,O2,N6-bis-[di-aqua-manganese(II)] tetra-hydrate, [Mn2(C16H8N4O4)2(H2O)4]·4H2O, (I), and bis-[μ-5,6-bis-(pyridin-2-yl)pyrazine-2,3-di-carboxyl-ato]-κ4N1,O2,N6:O3;κ4O3:N1,O2,N6-bis-[di-aqua-iron(II)] tetrahydrate, [Fe2(C16H8N4O4)2(H2O)4]·4H2O, (II), are, respectively, the mangan-ese(II) and iron(II) complexes of the ligand 5,6-bis-(pyridin-2-yl)-pyrazine-2,3-di-carb-oxy-lic acid. The complete mol-ecule of each complex is generated by inversion symmetry. Each metal ion is coordinated by a pyrazine N atom, a pyridine N atom, two carboxyl-ate O atoms, one of which is bridging, and two water O atoms. The metal atoms have MN2O4 coordination geometries and the complexes have a cage-like structure. In the crystals of both compounds, the complexes are linked by O-H⋯O and O-H⋯N hydrogen bonds involving the coordinating water mol-ecules, forming chains along [100]. These chains are linked by O-H⋯O hydrogen bonds involving the non-coordinating water mol-ecules, forming layers parallel to (011). The layers are linked by pairs of C-H⋯O hydrogen bonds and offset π-π inter-actions, so forming a hydrogen-bonded three-dimensional framework.

1,4-Bis[3,11-dithiatricyclo[11.3.1.15,9]octadeca-1(17),5,7,9(18),13,15-hexaen-7-yl]buta-1,3-diyne

The complete molecule of the title compound, C36H30S4{common name: 1,4-[4-(9,17-dithia[3.3]metacyclophane)]-1,3-butadiyne}, is generated by a crystallographic inversion centre at the mid-point of the central C—C bond [1.367 (5) Å]. Both cyclophane units exist incisoidpseudo-boat–chair chair–boat conformations. In the crystal, the packing is controlled by van der Waals interactions.

N,N′-Bis(pyridin-2-yl)octanediamide

The complete molecule of the title compound, C18H22N4O2, is generated by crystallographic inversion symmetry. In the crystal, N—H...N hydrogen bonds connect the molecules into [010] chains, which featureR22(8) loops. The packing is consolidated by C—H...O interactions.

1,1′-Biphenyl-2,2′,5,5′-tetracarboxylic acid

In the title compound, C16H10O8or H4bptc, the dihedral angle between the planes of the phenyl rings is 51.90 (4)°. The asymmetric unit contains one half-molecule; complete molecules are generated by a twofold rotation axis. In the crystal, O—H...O and C—H...O hydrogen-bonding generate a two-dimensional supramolecular network. In addition, weak π–π interactions are also observed.

105. Enhanced Oncolytic Activity Mediated by a Novel Human Adenovirus Type 6 Based Vector

Most existing oncolytic adenoviruses (AdV) are based on human AdV type 5 (hAdV-5). Clinical efficacy of hAdV-5 based oncolytic viruses is limited by variable expression levels of coxsackie- and adenovirus receptor (CAR) in different tumor cells and insufficient replication rates. Additionally, high prevalence of neutralizing antibodies against hAdV-5 resulting in lower efficiency makes hAdV-5 a less suitable candidate for systemic application. Recent studies have highlighted human adenovirus type 6 (hAdV-6) as a promising candidate for oncolytic and vaccine vectors. Thus, development of novel oncolytic AdV based on hAdV-6 may help overcoming these limitations. We further hypothesised that oncolytic efficacy of the candidate virus can be augmented by expression of RNAi suppressor protein P19 (Lecellier et al., Nature 2005) as has been shown previously for hAdV-5 (Rauschhuber et al, Sci. Rep. 2013). Here we aim at evaluating a novel hAdV-6-based, p19-containing oncolytic AdV as novel candidate for oncolytic applications in different tumour cell lines. We cloned a P19-containing hAdV-6 based virus (hAdV-6FP19) by a novel seamless recombineering technique (Zhang et al, unpublished). In order to allow P19 expression from the adenoviral vector genome, the P19 cDNA was fused via an internal ribosome entry site (IRES) to the late fiber gene. After release of the respective recombinant adenoviral genomes from plasmids containing the complete DNA molecule, linearized viral DNA was transfected into HEK293 cells for virus reconstitution. After initial amplification steps which were monitored by virus specific PCRs upscaling and virus purification using cesium-chloride density-gradient ultracentrifugation was performed. Rescue and amplification efficiencies were comparable to commonly used hAdV-5 based vectors. Various cancer cell lines from different origin were used to perform oncolysis assays. This included A549 (lung carcinoma), HCT 116 (colon carcinoma), Hela (cervical carcinoma) and Huh7 (hepatocellular carcinoma) cells which were infected with hAdV-6FP19, hAdV-6 and hAdV-5 at various multiplicities of infection (MOI). 2-3 days after infection cells were fixed and stained with methylene blue. We observed significantly higher cell lysis (up to 100-fold) for hAdV-6FP19 infected cells as compared to hAdV-5 and 6 at identical MOIs. Higher cell lysis rates for hAdV-6FP19 compared to wildtype virus were present in all evaluated cell lines, suggesting significantly enhanced oncolytic potential for hAdV-6FP19. In total we believe that hAdV6-based vectors hold great promise for oncolytic applications and that their oncolytic effectiveness can be further improved by RNAi-suppression.

Characterization of the cytotoxic activity of [2]rotaxane (TRO-A0001), a novel supramolecular compound, in cancer cells.

Rotaxanes comprise a class of interlocked molecules containing a wheel threaded onto an axle with blocking groups on the ends to keep the wheel from sliding off. Here, we show that [2][bis(2-(3,5-dimethylphenylcarbonyloxy)ethyl) ammoniumtrifluoromethanesulfonate]-[dibenzo-24-crown-8] rotaxane (TRO-A0001), a rotaxane compound, exerted a growth inhibitory effect on several human cancer cell lines. An MTT assay revealed an IC50 of 14-830nM for TRO-A0001 in these cells. Neither the wheel nor the axle part alone inhibited tumor cell growth, suggesting that the complete rotaxane molecule with its unique "intramolecular mobility" is required to inhibit cell growth. Annexin-V/PI staining provided evidence of the induction of apoptosis, which was further confirmed by the observation of poly (ADP-ribose) polymerase cleavage. Furthermore, a cell cycle analysis using flow cytometry showed that TRO-A0001 treatment resulted in G1 arrest in glioblastoma T98G and melanoma G361 cells. An immunoblot analysis revealed that in both cell lines, TRO-A0001 treatment caused the induction of p21/Cip1, thereby down-regulating Cdks 2, 4 and 6 and reducing Cyclins D1 and E. The results presented in this study demonstrate cytotoxicity of the rotaxane compound and its potential as a lead compound for the development of a chemotherapeutic agent against cancer.

Crystal structure of 1,2-bis-[(1H-imidazol-2-yl)methylidene]hydrazine and its one-dimensional hydrogen-bonding network.

In the title compound, C8H8N6, two imidazolyl groups are separated by a zigzag -CH=N-N=CH- linkage. An inversion center is located at the mid-point of the N-N single bond and the complete molecule is generated by symmetry. In the crystal, each mol-ecule forms four N-H⋯N hydrogen bonds with two neighbouring mol-ecules to constitute a one-dimensional ladder-like structure propagating along the a-axis direction.

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library.

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.

Diethyl 2,2′-({[1,4-phenylenebis(azanediyl)]bis(methylene)}bis(1H-pyrrole-2,1-diyl))diacetate

The complete molecule of the title compound, C24H30N4O4, is generated by crystallographic inversion symmetry. The molecule is S-shaped and the pyrrole groups have anantiortransconfirmation with respect to the central benzene ring, to which they are inclined by 76.38 (9)°. In the crystal, molecules are linkedviaC—H...O hydrogen bonds, forming layers parallel to theacplane. Within the layers there are C—H...π interactions present. There are, however, no significant interactions between the layers.

Local structure of As2O3 glass from first principles simulations

We model As2O3 glass from the liquid state using ab initio molecular dynamics simulations and probe its atomic structure using various analyzing techniques. The model is almost free from coordination and chemical defects and has an average coordination number of 2.38. The AsO3 pyramids are randomly connected via corner sharing to form the glass state. The glass network presents six membered hexagonal-like rings similar to those formed in the As4O6 molecules but isolated or complete As4O6 molecules do not exist. The glass state mainly consists of layer-like and incomplete As4O6-like structures.