Four-Component Relativistic State-Specific Multireference Perturbation Theory with a Simplified Treatment of Static Correlation

The relativistic multireference (MR) perturbative approach is one of the most successful tools for the description of computationally demanding molecular systems of heavy elements. We present here the ground state dissociation energy surfaces, equilibrium bond lengths, harmonic frequencies, and dissociation energies of Ag₂, Cu₂, Au₂, and I₂ computed using the four-component (4c) relativistic spinors based state-specific MR perturbation theory (SSMRPT) with improved virtual orbital complete active space configuration interaction (IVO-CASCI) functions. The IVO-CASCI method is a simple, robust, useful and lower cost alternative to the complete active space self-consistent field approach for treating quasidegenerate situations. The redeeming features of the resulting method, termed as 4c-IVO-SSMRPT, lies in (i) manifestly size-extensivity, (ii) exemption from intruder problems, (iii) the freedom of convenient multipartitionings of the Hamiltonian, (iv) flexibility of the relaxed and unrelaxed descriptions of the reference coefficients, and (v) manageable cost/accuracy ratio. The present method delivers accurate descriptions of dissociation processes of heavy element systems. Close agreement with reference values has been found for the calculated molecular constants indicating that our 4c-IVOSSMRPT provides a robust and economic protocol for determining the structural properties for the ground state of heavy element molecules with eloquent MR character as it treats correlation and relativity on equal footing

Additional file 2: Figure S2. of Human hyaluronic acid synthase-1 promotes malignant transformation via epithelial-to-mesenchymal transition, micronucleation and centrosome abnormalities

Effect of HAS1 expression on mitotic index and cell growth. (A) Lower mitotic index was observed in HAS1 expressing MCF10A cells in comparison to LMA2-expressing of mock transfected cells. MCF10A cells transfected with the indicated cDNA in pCDNA3. The selected populations were seeded onto 8-chamber glass slides, incubated overnight, and then fixed and DAPI-stained to count mitotic/non-mitotic nuclei based on the chromatin / nucleus structure. HE-HAS1: HAS1 in pCDNA3 with N-terminal hemagglutinin fusion-tag, A2-HAS1: HAS1 in pCDNA3 with N-terminal A2 fusion-tag, LMA2: unrelated protozoa gene in pCDNA3 with C-terminal A2 fusion tag and Mock: transfection without any plasmid and not selected with any antibiotic. (B) HAS1 expressing cells showed the slower growth after induction with Dox. HeLa cells engineered and selected for Tetracycline-on inducible HAS1 or GFP expressing plasmids. The cell populations were subjected to growth analysis to test the effect of inducible expression of genes (GFP and HAS1) on growth for 13-days with Dox at different concentrations. The results are presented as fold increase of viable cells compared to seeded cells at Day 0. The growth of all HAS1-expressing cells was slower than the GFP-puromycin-vector controls, may be due to background synthesis (leakiness) of intracellular-HA by HAS1 even at 0 μg/ml Dox induction. At higher concentrations of Dox (6 μg/ml) the growth cease beyond 10th day for HAS1 but not for control GFP. (PDF 12 kb

Additional file 1: Figure S1. of Human hyaluronic acid synthase-1 promotes malignant transformation via epithelial-to-mesenchymal transition, micronucleation and centrosome abnormalities

Expression of HAS1. (A) Lung primary cells were transiently transfected with pCDNA3-A2-HAS1 or empty vector (pCDNA3-A2) and subjected to HA fluorescence staining (green) after 72 h. Nucleus was stained with DAPI. (B) HeLa cells were engineered and selected for Tetracycline-on inducible HAS1 expression. Cells were grown in tetracycline-free media in 8-well chamber slides for 16 h followed by with or without doxycycline (Dox) treatment for 40 h, and then HA fluorescence staining (white) and nuclear staining with DAPI (blue). (C) DLD1 cells were transfected and selected for Tet-inducible HAS1 expression. The cells were grown in tetracycline-free media followed by induced with doxycycline (Dox) treatment for 40 h. The cells were stained for HA localization using bHABP (Green). DLD1-pTET cells served as negative control. (PDF 150 kb

Inhibition and Degradation of Amyloid Beta (Aβ40) Fibrillation by Designed Small Peptide: A Combined Spectroscopy, Microscopy, and Cell Toxicity Study

A designed nontoxic, nonhemolytic 11-residue peptide, NF11 (NAVR­WSLM­RPF), not only inhibits the aggregation of amyloid beta (Aβ40) protein but also disaggregates the preformed oligomers and mature Aβ fibrils, thereby reducing associated-toxicity. NMR experiments provide evidence of NF11’s ability to inhibit fibril formation, primarily through interaction with the N-terminus region as well as the central hydrophobic cluster of Aβ40. NF11 has micromolar binding affinity toward both monomeric and aggregated species for efficient clearance of toxic aggregates. From these in vitro results, the future development of a next generation peptidomimetic therapeutic agent for amyloid disease may be possible

Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in <i>Salmonella</i> Typhimurium

<div><p>In this study we adapted a Mu<i>d</i>-based delivery system to construct a random <i>yfp</i> reporter gene (encoding the yellow fluorescent protein) insertion library in the genome of <i>Salmonella</i> Typhimurium LT2, and used fluorescence activated cell sorting and fluorescence microscopy to screen for translational fusions that were able to clearly and specifically label the bacterial nucleoid. Two such fusions were obtained, corresponding to a translational <i>yfp</i> insertion in <i>iscR</i> and <i>iolR</i>, respectively. Both fusions were further validated, and the IscR::YFP fluorescent nucleoid reporter together with time-lapse fluorescence microscopy was subsequently used to monitor nucleoid dynamics in response to the filamentation imposed by growth of LT2 at high hydrostatic pressure (40–45 MPa). As such, we were able to reveal that upon decompression the apparently entangled LT2 chromosomes in filamentous cells rapidly and efficiently segregate, after which septation of the filament occurs. In the course of the latter process, however, cells with a “trilobed” nucleoid were regularly observed, indicative for an imbalance between septum formation and chromosome segregation.</p></div

Growth and nucleoid dynamics of HP stressed LT2.

<p>(A) Representative images showing cell growth and nucleoid dynamics of HP stressed LT2 <i>iscR</i>::<i>yfp</i> cells (grown overnight at 40–45 MPa and 37°C) at the indicated time points after pressure release. Merged phase contrast and YFP images are shown. (B) Representative images showing cell growth and nucleoid dynamics of a HP stressed LT2 <i>iscR</i>::<i>yfp</i> cell (grown overnight at 40–45 MPa and 37°C) at the indicated time points after pressure release in the presence of DAPI and intermittent UV excitation. Phase contrast (top panels), DAPI (middle panels) and YFP (lower panels) images are shown. Scale bars correspond to 5 μm.</p

STD NMR study of KH16p and KH16 with 14-3-3ζ.

<p><b>(A)</b> One-dimensional ¹H NMR reference spectrum of KH16p in the presence of 14-3-3ζ at 500:1 molar ratio. <b>(B)</b> The absence of STD signals in the aromatic region and the prominent STD signals in the aliphatic region demonstrate the close proximity of few residues of KH16p to the 14-3-3ζ protein. <b>(C)</b> One-dimensional ¹H NMR reference spectrum of KH16 in the presence of 14-3-3ζ at 500:1 molar ratio. <b>(D)</b> STD spectrum of KH16. The STD NMR experiments were carried out in aqueous solution on a Bruker AVANCE III 500 MHz spectrometer at 288 K.</p

Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein

<div><p>Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615–630) in the presence of 14-3-3ζ protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3ζ protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3ζ protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3ζ binding at an atomic resolution.</p></div

Overview of docked complex of the peptide KH16p oriented over 14-3-3ζ protein.

<p>Sphere representation of protons indicates their presence in close proximity of peptide atoms, in conjunction with data obtained from STD NMR. Relative orientation of negatively charged phosphate group (pS7) surrounded by positively charged residues like R56, R60, and R127 is shown.</p

Sequence of KH16p with structure and purification of 14-3-3ζ protein.

<p><b>(A)</b> Schematic presentation of C-Raf protein showing the sub-domains RBD (RAS-binding domain), ZF (Zinc Finger domain), Kinase domain, and KH16p (615–630 residues). pS621 is indicated in red color (denoted as KH16p), and the control peptide KH16 devoid of phosphorylarion at S621 is shown below. (<b>B)</b> Purification of 14-3-3ζ protein. Human 14-3-3ζ protein was cloned into pQE30 vector containing N-terminal His-tag. <i>E</i>. <i>Coli</i> M15 strain was transformed with this clone and 14-3-3ζ protein was expressed after induction with 0.5 mM IPTG for 3 hours. 14-3-3ζ protein was purified by Ni²⁺-NTA resin. 10 μg of purified 14-3-3ζ protein was loaded into SDS-PAGE and stained with Coomassie brilliant blue. (<b>C)</b> Crystal Structure of human 14-3-3ζ (PDB ID: 4IHL, Chain A) demonstrating α-helical structure.</p