Three-dimensional Structural Analysis Research Articles

Novel homozygous mutation in the FBXL4 gene is associated with mitochondria DNA depletion syndrome-13

BackgroundMitochondrial DNA depletion syndrome-13 (MTDPS13) is caused by mutations in FBXL4 (F-box and leucine-rich repeat protein 4), a nuclear gene encoding an F-box protein that plays a role in maintaining mtDNA integrity and stability. MethodsWe identified a novel homozygous FBXL4 gene mutation, c.993dupA (p.L332Tfs*3), in a 1-year-old girl of Han Chinese descent. We performed three-dimensional protein structural analysis and targeted mtDNA next-generation sequencing. We analysed FBXL4 expression and mitochondrial DNA level, and reviewed mutations reported in FBXL4-related literature. ResultsThis mutation resulted in premature termination of translation and loss of 288 amino acids from C-terminus. A three-dimensional structural analysis revealed that conserved LRR domains were lost in mutant FBXL4 protein, which likely affected its ability to form protein-protein interactions. There were no differences in FBXL4 mRNA expression levels between the patient and her parents. There were no mtDNA mutations in either the patient or her parents. However, ND1/GAPDH ratio in lymphocytes and urine, which represents mtDNA/nuclear DNA ratio, showed that the number of mitochondrial genomes was significantly lower in the patient than in her parents or wild-type subjects. ConclusionHomozygous FBXL4 gene mutation, c.993dupA, can cause mitochondrial dysfunction, and LRR region is especially important for FBXL4 protein function.

The Evaluation of tLyP-1-Bound Mda-7/IL-24 Killing Activity on a Liver Tumor Cell Line.

Background: The melanoma differentiation-associated gene-7 (Mda-7)/interleukin-24 (IL-24) is a tumor killing cytokine, the bystander effect of which can be enhanced through tethering to tumor homing peptides (THPs). Materials and Methods: After fusing tLyP-1, RGR, and buforin as THPs to Mda-7/IL-24, enzyme-linked immunosorbent assay (ELISA) was used to determine the secretion potency of the recombinant proteins. The killing potency of plasmids expressing IL-24, IL-24.tLyP1, IL-24.RGR, and buf.IL-24 were assessed, using MTT, Annexin/PI staining assays, as well as measuring the expression level of GADD-153 and BCL2-associated X (BAX) on Huh-7 cells. Three-dimensional structural analysis and protein-receptor interaction were also evaluated by modeling. Results: The ELISA result showed that contrary to IL-24.RGR and buf.IL-24, IL-24.tLyP-1 retained the secretion potency, similar to the native form. The viability assessments showed that IL-24 and IL-24.tLyP-1 had the most growth suppressive effects in comparison with the control group (p < 0.0001). Furthermore, IL-24 and IL-24.tLyP-1 had the highest apoptotic activity and significant upregulatory effect on the GADD-153 and BAX genes (p < 0.0003). The modeling showed that peptide modifications left no detrimental effect on IL-24 attachment to the cognate receptor. Conclusion: IL-24 can tolerate tLyP-1 peptide modification by retaining its secretion potency. Tethering tLyP-1 to IL-24 can induce more apoptosis than its modified versions by RGR or buforin.

Hybrid nearly singular integration for three-dimensional isogeometric boundary element analysis of coatings and other thin structures

The isogeometric boundary element method (IGABEM) has great potential for the simulation of elasticity problems because of its exact geometric representation and good approximation properties. These advantages can be extended to thin structures, including coatings, but the development of an accurate and efficient method to deal with the large number of nearly singular integrals existing in the IGABEM presents a great challenge for very thin sections. In this paper, we propose a new sinh+ scheme for weakly, strongly and hyper near-singular integrals arising in 3D IGABEM for thermoelastic problems, based on the sinh transformation method and adaptive integral method. The presented scheme is efficient, since it combines the advantages of both methods: (1) when the thickness δ of coatings/thin structures is moderately small, an accurate and efficient integral result will be obtained by the adaptive integral method; (2) when δ is very small, the nearly singular integrals are computed by the sinh+ scheme efficiently. With the introduction of NURBS in IGABEM, truncation errors arising in the Taylor expansion cannot be ignored. Based on the values of these errors, the computed knot spans are further divided into several sub-knot spans and different methods will be used to evaluate the integral over each sub-knot span in the new scheme. In addition, based on the analytical extension of the NURBS surface, an adaptation of the sinh transformation method is proposed which can evaluate the near-singular integrals accurately for cases in which the projection point lies outside of the considered knot span. Several numerical examples are presented to validate the accuracy and efficiency of the 3D IGABEM based on the sinh+ scheme in the analysis of thermoelastic problems.

Light-Sheet Microscopy for Surface Topography Measurements and Quantitative Analysis.

A novel light-sheet microscopy (LSM) system that uses the laser triangulation method to quantitatively calculate and analyze the surface topography of opaque samples is discussed. A spatial resolution of at least 10 μm in z-direction, 10 μm in x-direction and 25 μm in y-direction with a large field-of-view (FOV) is achieved. A set of sample measurements that verify the system′s functionality in various applications are presented. The system has a simple mechanical structure, such that the spatial resolution is easily improved by replacement of the objective, and a linear calibration formula, which enables convenient system calibration. As implemented, the system has strong potential for, e.g., industrial sample line inspections, however, since the method utilizes reflected/scattered light, it also has the potential for three-dimensional analysis of translucent and layered structures.

Glutathione S-transferase (GST) genes from marine copepods Acartia tonsa: cDNA cloning and mRNA expression in response to 1,2-dimethylnaphthalene

The calanoid copepod, Acartia tonsa, is relatively sensitive to marine pollution. Glutathione S-transferase (GST) multifunctional enzyme, as a biomarker, play an important role in detoxification metabolism of exogenous substances. In the present study, GST-theta and GST-mu class homology genes (designated as AtGSTT1 and AtGSTM2) were identified and characterized from A. tonsa. The coding sequence of AtGSTT1 comprised 726 bp and encoded a putative protein of 241 amino acid residues. AtGSTM2 contained an open reading frame of 678 bp that encoded a putative 227 amino acid polypeptide. Both proteins contained a conserved GST-N domain and a GST-C domain. Structural analysis revealed the characteristic N-terminal G-site. Three-dimensional structure analysis showed that AtGSTT1 and AtGSTM2 have two typical domains of GST family: The βαβαββα topology structure at the N- terminus and the superhelical structure at the C- terminus. Subsequently, the expression levels of the two GST genes were detected in A. tonsa using real-time quantitative PCR after exposure to 1,2-dimethylnaphthalene (C2-NAPH) at different concentrations (0.574, 5.736 and 57.358 μg/L) for 24, 48, 72, and 96 h. AtGSTT1 mRNA expression was significantly up-regulated in a time-dependent manner and the highest mRNA expression occurred at 5.736 μg/L C2-NAPH exposure for 96 h. AtGSTM2 mRNA expression peaked at 72 h in 0.574 μg/L and 5.736 μg/L dose groups. The expression level of AtGSTM2 showed an increasing trend in a time-dependent manner at 57.358 μg/L of C2-NAPH. These results suggested that GST genes may play an important role in protecting A. tonsa from C2-NAPH pollution, and provide a theoretical basis for further study on the molecular mechanism of polycyclic aromatic hydrocarbon (PAHs) pollution on zooplankton.

Inhibition of biofilm and biofilm-associated virulence factor production in methicillin-resistant Staphylococcus aureus by docosanol

Antimicrobial resistance is a major public health concern in infection control. Hence, a multi-pronged approach is necessary to curb the severity of infections. The present study entails the identification of docosanol (fatty alcohol) from Streptomyces as a novel antibiofilm agent which can target the virulence factors of MRSA. Results showed that docosanol as a potent antibiofilm agent and found to inhibit several virulence factors of MRSA. The antibiofilm efficacy of docosanol analyzed through light and scanning electron microscopy showed a significant reduction in adherent cells. Moreover, analysis of three-dimensional structure of biofilm matrix by confocal laser scanning microscope demonstrated effective antibiofilm potential of docosanol. In addition, docosanol reduced the survival rate of MRSA in healthy human blood and enhanced the neutrophil-mediated killing by interfering with hemolysin production. RT-qPCR analysis revealed the down regulation of several virulence genes, possibly by affecting the expression of the accessory gene regulator (agr) system and transcriptional regulator sarA. These findings suggest that docosanol could effectively reduce the biofilm phenotype and virulence production, and thus becomes a promising candidate to treat MRSA infections.

Identification of six important amino acid residues of MenA from Bacillus subtilis natto for enzyme activity and formation of menaquinone

The enzyme 1, 4-dihydroxy-2-naphthoic acid (DHNA) prenyltransferase (MenA) is a critical player in determining the efficiency of the menaquinone (MK) synthesis pathway and is an attractive target for the development of novel chemotherapeutics against pathogenic Gram-positive bacteria. However, there has been no report on structural properties or active region of MenA. To solve this challenge, we predicted the three-dimensiona structure and critical amino acid sites of MenA by bioinformatics analysis. Six amino acid sites were chosen by alligning the amino acid sequence of MenA from Bacillus subtilis natto with 4-hydroxybenzoate octaprenyl transferase (UbiA) from Escherichia coli, Aeropyrum pernix and Archaeoglobus fulgidus. Among them, four Asp sites located in two Asp-rich motifs (D78XXXXXD84 and D208XXXD212) were found to be indispensable amino acid residues in maintaining MenA activity. Site-directed mutagenesis of two other sites (Q67th, N74th) positively affected the catalytic activity of MenA and the MK titer. Q67R resulted in more than a 5-fold increase in specific 2-demethylmenaquinone (DMK) content (YP1/x) compared to wild-type, and the hydrophobic interaction between Cys63 and Arg67 could be the main reason according to the three-dimensional structure analysis. Moreover, a dramatic increase in specific MK content (YP2/x) was realized by co-expressing menG in EcMenA (Q67R). The results obtained could be useful not only in developing novel chemotherapeutics to combat potentially pathogenic Gram-positive bacteria, but also in regulating and optimizating E. coli mutant cultures for the efficient production of MK metabolites.

Mitochondrial morphology and function: two for the price of one!

Mitochondrial shape and function are known to be linked; therefore, there is a need to combine three-dimensional EM structural analysis with functional analysis. Cytochrome c oxidase labelling is one approach to examine mitochondrial function at the EM level. However, previous efforts to apply this method have had several issues including inconsistent results, disruption to mitochondrial ultrastructure, and a lack of optimisation for volume EM methods. We have used short fixation and microwave processing to address these issues. We show that our method gives consistent cytochrome c oxidase labelling and improves labelling penetration across tissue volume. We also quantify mitochondrial morphology metrics, including in volume EM, to show that ultrastructure is unaltered by the processing. This work represents a technical advance that allows the correlation of mitochondrial function and morphology with greater resolution and volume than has previously been feasible. LAY SUMMARY: Transmission electron microscopy (TEM) is a high-resolution technique used for the study of cells and their components, such as mitochondria. However, the two-dimensional nature of TEM means that quantification of these structures is difficult without making assumptions about their shape; a problem that was solved by the advent of three-dimensional EM approaches. Mitochondrial shape and function are known to be linked therefore there is a need to combine three-dimensional EM structural analysis with functional analysis. To do this we used electron microscopy to visualise a reaction that assesses the activity of cytochrome c oxidase in the mitochondrial respiratory chain. The reaction deposits a dark staining on mitochondrial cristae where cytochrome c oxidase is functioning and a lack of staining where it is not. We first optimised this technique for TEM, showing that the tissue was evenly stained and exhibited no effect on mitochondrial shape when compared to conventionally processed tissue. We then demonstrated that this was also true of a sample processed for three-dimensional EM imaging. This work presents an advance in three-dimensional EM imaging that allows us to look at both mitochondrial function and shape and to detect subtle changes in shape.

Differences and controlling factors of composite hydrocarbon accumulations in the Tazhong uplift, Tarim Basin, NW China

Abstract Based on three-dimensional seismic interpretation, structural and sedimentary feature analysis, and examination of fluid properties and production dynamics, the regularity and main controlling factors of hydrocarbon accumulation in the Tazhong uplift, Tarim Basin are investigated. The results show that the oil and gas in the Tazhong uplift has the characteristics of complex accumulation mainly controlled by faults, and more than 80% of the oil and gas reserves are enriched along fault zones. There are large thrust and strike-slip faults in the Tazhong uplift, and the coupling relationship between the formation and evolution of the faults and accumulation determine the difference in complex oil and gas accumulations. The active scale and stage of faults determine the fullness of the traps and the balance of the phase, that is, the blocking of the transport system, the insufficient filling of oil and gas, and the unsteady state of fluid accumulation are dependent on the faults. The multi-period tectonic sedimentary evolution controls the differences of trap conditions in the fault zones, and the multi-phase hydrocarbon migration and accumulation causes the differences of fluid distribution in the fault zones. The theory of differential oil and gas accumulation controlled by fault is the key to the overall evaluation, three-dimensional development and discovery of new reserves in the Tazhong uplift.

Editorial: Biographical Memoirs , Volume 68

Editorial: <i>Biographical Memoirs</i> , Volume 68

Mutational effect of human CFAP43 splice-site variant causing multiple morphological abnormalities of the sperm flagella.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a rare disease associated with male infertility. In our previous study, we identified a homozygous CFAP43 splice-site variant, c.3661-2delA, in a patient with MMAF. However, the mutational effect of this variant was unknown. Here, using a minigene assay, we demonstrated that the c.3661-2delA variant may cause exon-30 to be skipped, thus generating the p.E1221_K1256del protein. By secondary and three-dimensional structural biology prediction analysis, we found that the mutant protein became 'tighter' in comparison with the wild-type protein, resulting in amino acid rearrangements in CFAP43 protein structure. We elucidated the molecular mechanism of the c.3661-2delA splice-site variant causing MMAF in the current study.

Guy Dodson. 13 January 1937—24 December 2012

Guy Dodson's scientific reputation is based on his seminal contributions to linking chemistry and biology through analyses of three-dimensional structures elucidated by crystallography. He inspired and supported the development of new methods to determine structure, to relate structure to chemical mechanism and to embed structural insights into the lexicon of biological research. He began his career working with Dorothy Hodgkin, at the University of Oxford, and in later life he set up two very successful structural biology laboratories, at the University of York and then at the National Institute of Medical Research, Mill Hill. He is particularly remembered for his research into improving insulin therapy, based on modifications suggested by structural insights. He showed early recognition of how to build synergy in industrial collaborations that led both to fundamental scientific discoveries and to the development of new medicines and products to benefit society. He is also remembered for the enthusiasm he brought to these studies, for the pleasure he got, and gave, from the successes of others, and his generosity of spirit.

Structural Strength Analysis and Fabrication of a Straight Blade of an H-Darrieus Wind Turbine

Small H-Darrieus wind turbines have become popular in the wind power market because of their many advantages, which include simplicity of design, low construction costs, and they are thought to represent an adequate solution even in unconventional installation regions. The blade is generally considered as the most important component of the wind turbine system because it controls the efficiency of the turbine. The blade structure must be designed to support the difficult environmental conditions (e.g., wind, and snow) encountered during the operational life of the wind turbine. This current study uses three-dimensional (3D) modeling and structural strength analysis to fabricate two straight blades (aluminum and galvanized steel) for a small H-Darrieus wind turbine. The 3D modeling of the blade structure is performed using SolidWorks, a computer-aided design (CAD) software package, and the structural strength analysis uses the Finite Element Analysis (FEA) technique to identify the stiffness, resistance, and reliability of the blade structure. The simulation results obtained indicate that no structural failures are predicted for either of the two structures tested because the factors of safety are larger than one, and the all maximum deflections are within the allowable deformation limits for the materials. Manufacturing processes for the two structures are described.

A consistent strain-based beam element with quaternion representation of rotations

We present a novel consistent singularity-free strain-based finite element formulation for the analysis of three-dimensional frame-like structures. Our model is based on a geometrically exact finite-strain beam theory, quaternion parametrization of spatial rotations, assumption that the strain measures are constant along the length of the element and a proper choice of basis for the translational strain vector representation. As it is common for strain-based elements, the present formulation does not suffer from shear locking. A comparison of our results with the results from the literature and a commercial finite element analysis software demonstrates the advantages of the proposed formulation, especially when the structure is subjected to larger shear deformations. This stems from the fact that our model ensures a mathematically consistent updating procedure for all the quantities describing the beam. This aspect is often overlooked, since most of the numerical cases from other studies on this topic engage rather small-shear strains for which the consistent update is not crucial as the number of elements is increased.

Use of Microct Scanner to Characterize the Histotechnological Processing of Bone using Different Tissue Fixatives: Relationship to DNA Preservation (Immunohistochemistry)

Micro-computed tomography (micro-CT) scanners allow for the three-dimensional analysis of bone structure to be visualized and numerous bone structural parameters to be quantified with a high degree of accuracy. Nucleic acid-based immunohistochemistry (IHC) allows for characterization of the presence, amounts and different types of DNA in paraffin-embedded fixed tissue sections. Micro-CT scanners (i.e., Bruker SkyScan 1173 µCT system), along with immunohistochemically processed tissue sections were used to examine a variety of different fixatives in order to characterize the effects of fixation on bone morphology (i.e., adult chicken), and preservation of different types of intact, non-denatured nucleic acids (e.g., B-DNA, Z-DNA). Several different types of bone (e.g., femur, tibia, and vertebra) were characterized. Fixation was performed using 10% Neutral Buffered Formalin (NBF), and Formalin-Alcohol-Acetic Acid (FAA) for 24, 48, 72 and 96 hours at room temperature. Both decalcified and non-decalcified bone tissues were processed. Molecular biological grade fixatives and water were used in order to ensure the best histotechnological results. Tissues processed in FAA resulted in hardening of tissue, but good fixation of intact B-DNA and Z-DNA. Tissues processed in 10% NBF resulted in softer tissue, but poor fixation of intact DNA. Tissues processed in 10% NBF required antigen retrieval. Our group is correlating morphological differences observed with different fixatives using a microCT scanner, relative to DNA identification employing immunohistochemical staining of bone tissue sections with anti-B-DNA and anti-Z-DNA monoclonal antibodies. Our data demonstrates that FAA results in easier histotechnological processing of bone tissue, better morphological results and superior IHC staining. Research project was supported by an NSF grant, Award ID No: 1828305. .

Post-transcriptional regulation of BRG1 by FIR\u0394exon2 in gastric cancer

Brahma-related gene 1 (BRG1), an ATPase subunit of the SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex controls multipotent neural crest formation by regulating epithelial-mesenchymal transition (EMT)-related genes with adenosine triphosphate-dependent chromodomain-helicase DNA-binding protein 7 (CHD7). The expression of BRG1 engages in pre-mRNA splicing through interacting RNPs in cancers; however, the detailed molecular pathology of how BRG1and CHD7 relate to cancer development remains largely unveiled. This study demonstrated novel post-transcriptional regulation of BRG1 in EMT and relationship with FIRΔexon2, which is a splicing variant of the far-upstream element-binding protein (FUBP) 1-interacting repressor (FIR) lacking exon 2, which fails to repress c-myc transcription in cancers. Previously, we have reported that FIR complete knockout mice (FIR−/−) was embryonic lethal before E9.5, suggesting FIR is crucial for development. FIRΔexon2 acetylated H3K27 on promoter of BRG1 by CHIP-sequence and suppressed BRG1 expression post-transcriptionally; herein BRG1 suppressed Snai1 that is a transcriptional suppressor of E-cadherin that prevents cancer invasion and metastasis. Ribosomal proteins, hnRNPs, splicing-related factors, poly (A) binding proteins, mRNA-binding proteins, tRNA, DEAD box, and WD-repeat proteins were identified as co-immunoprecipitated proteins with FIR and FIRΔexon2 by redoing exhaustive mass spectrometry analysis. Furthermore, the effect of FIRΔexon2 on FGF8 mRNA splicing was examined as an indicator of neural development due to impaired CHD7 revealed in CHARGE syndrome. Expectedly, siRNA of FIRΔexon2 altered FGF8 pre-mRNA splicing, indicated close molecular interaction among FIRΔexon2, BRG1 and CHD7. FIRΔexon2 mRNA was elevated in human gastric cancers but not in non-invasive gastric tumors in FIR+/ mice (K19-Wnt1/C2mE x FIR+/−). The levels of FIR family (FIR, FIRΔexon2 and PUF60), BRG1, Snai1, FBW7, E-cadherin, c-Myc, cyclin-E, and SAP155 increased in the gastric tumors in FIR+/− mice compared to those expressed in wild-type mice. FIR family, Snai1, cyclin-E, BRG1, and c-Myc showed trends toward higher expression in larger tumors than in smaller tumors in Gan-mice (K19-Wnt1/C2mE). The expressions of BRG1 and Snai1 were positively correlated in the gastric tumors of the Gan-mice. Finally, BRG1 is a candidate substrate of F-box and WD-repeat domain-containing 7 (FBW7) revealed by three-dimensional crystal structure analysis that the U2AF-homology motif (UHM) of FIRΔexon2 interacted with tryptophan-425 and asparate-399 (WD)-like motif in the degron pocket of FBW7 as a UHM-ligand motif. Together, FIRΔexon2 engages in multi-step post-transcriptional regulation of BRG1, affecting EMT through the BRG1/Snai1/E-cadherin pathway and promoting tumor proliferation and invasion of gastric cancers.

Investigation of PRDM7 and PRDM12 expression pattern during mouse embryonic development by using a modified passive clearing technique

Recent developments in tissue clearing methods such as CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue hYdrogel) have allowed for the three-dimensional analysis of biological structures in whole, intact tissue, providing greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, preventing the wide application of these techniques to fragile tissues such as developing embryos. Here, we present optimized passive clearing techniques, mPACT-A, that improve tissue rigidity without the expense of optical transparency. We also present a further modified mPACT-A protocol that is specifically optimized for handling mouse embryos, which are small and fragile, such that they easily dismantle when processed via established tissue clearing methods. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PRDM proteins, PRDM7 and PRDM12, in intact cleared mouse embryos at various stages of development. We observed strong PRDM7 and PRDM12 expression in the developing mouse nervous system, suggestive of potential roles in neural development that will be tested in future functional studies.

Analysis of Three-dimensional Structure and Formation Mechanism of Degenerate Pearlite in Eutectoid Steel

Analysis of Three-dimensional Structure and Formation Mechanism of Degenerate Pearlite in Eutectoid Steel

W(110)-O表面の光電子ホログラフィーによる立体構造解析

W(110)-O表面の光電子ホログラフィーによる立体構造解析

A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes.

Aberrantly truncated immature O-glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly associated with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immature O-glycans, no anti-MUC1 antibody recognises carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure analysis, epitope mapping and biochemical analysis. The X-ray crystal structure of the anti-MUC1 monoclonal antibody SN-101 complexed with the antigenic glycopeptide provides for the first time evidence that SN-101 recognises specifically the essential epitope by forming multiple hydrogen bonds both with the proximal peptide and GalNAc linked to the threonine residue, concurrently. Remarkably, the structure of the MUC1 glycopeptide in complex with SN-101 is identical to its solution NMR structure, an extended conformation induced by site-specific glycosylation. We demonstrate that this method accelerates dramatically the development of a new class of designated antibodies targeting a variety of “dynamic neoepitopes” elaborated by disease-specific O-glycosylation in the immunodominant mucin domains and mucin-like sequences found in intrinsically disordered regions of many proteins.