Comparing two approaches of miR-34a target identification, biotinylated-miRNA pulldown vs miRNA overexpression

<p>microRNAs (miRNAs) are critical regulators of gene expression. For elucidating functional roles of miRNAs, it is critical to identify their direct targets. There are debates about whether pulldown of biotinylated miRNA mimics can be used to identify miRNA targets or not. Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays. In contrast to the approach of Bio-miR pulldown, RNA-seq of miR-34a overexpression samples had limited value in identifying direct targets of miR-34a. It seems that pulldown of 3′-Biotin-tagged miRNA can identify <i>bona fide</i> microRNA targets at least for miR-34a.</p

Density Functional Theory Study on the Enhancement Mechanism of the Photocatalytic Properties of the g‑C₃N₄/BiOBr(001) Heterostructure

The van der Waals heterostructures fabricated in two semiconductors are currently attracting considerable attention in various research fields. Our study uses density functional theory calculations within the Heyd–Scuseria–Ernzerhof hybrid functional to analyze the geometric structure and electronic structure of the g-C3N4/BiOBr(001) heterojunction in order to gain a better understanding of its photocatalytic properties. The calculated band alignments show that g-C3N4/BiOBr can function as a type-II heterojunction. In this heterojunction, the electrons and holes can effectively be separated at the interface. Moreover, we find that the electronic structure and band alignment of g-C3N4/BiOBr(001) can be tuned using external electric fields. It is also noteworthy that the optical absorption peak in the visible region is enhanced under the action of the electric field. The electric field may even improve the optical properties of the g-C3N4/BiOBr(001) heterostructure. Given the results of our calculations, it seems that g-C3N4/BiOBr(001) may be significantly superior to visible light photocatalysis

Correlation between the life span and fertility of <i>C</i>. <i>elegans</i> infected with STEC strains.

<p>The STEC (E15, E18 and E22) strains showed an increase in colonisation, which correlates with a decrease in longevity and fertility of the worm (<i>C</i>. <i>elegans</i>). This is considered evidence of STEC pathogenicity in the <i>C</i>. <i>elegans</i> model.</p

<i>In vivo</i> screening platform for shiga toxin-producing <i>Escherichia coli</i> (STEC) using <i>Caenorhabditis elegans</i> as a model

<div><p>Shiga toxin-producing <i>Escherichia coli</i> (STEC) strains are the main cause of bacillary dysentery, although STEC strains generally induce milder disease symptoms compared to <i>Shigella</i> specie<i>s</i>. This study aimed to determine the virulence of STEC using the nematode <i>Caenorhabditis elegans</i> as a model host. Worm killing, fertility and bacterial colonisation assays were performed to examine the potential difference in the virulence of STEC strains compared to that of the control <i>E</i>. <i>coli</i> OP50 strains on which worms were fed. A statistically significant difference in the survival rates of <i>C</i>. <i>elegans</i> was observed in that the STEC strains caused death in 8–10 days and the <i>E</i>. <i>coli</i> OP50 strains caused death in 15 days. STEC strains severely reduced the fertility of the worms. The intestinal load of bacteria in the adult stage nematodes harbouring the <i>E</i>. <i>coli</i> OP50 strains was found to be 3.5 log CFU mL^-1. In contrast, the STEC strains E15, E18 and E22 harboured 4.1, 4.2 and 4.7 log CFU ml⁻¹ per nematode, respectively. The heat-killed STEC strains significantly increased the longevity of the worms compared to the non-heated STEC strains. In addition, PCR-based genomic profiling of shiga toxin genes, viz., stx1 and stx2, identified in selected STEC strains revealed that these toxins may be associated with the virulence of the STEC strains. This study demonstrated that <i>C</i>. <i>elegans</i> is an effective model to examine and compare the pathogenicity and virulence variation of STEC strains to that of <i>E</i>. <i>coli</i> OP50 strains.</p></div

Fecundity of STEC strains in <i>C</i>. <i>elegans</i> compared to that of the <i>E</i>. <i>coli</i> OP50 strain.

<p>Brood sizes of <i>C</i>. <i>elegans</i> were examined at 20°C. n>10 in each case.</p

Bacterial (<i>E</i>. <i>coli)</i> strains used in the study and their respective sources.

<p>Bacterial (<i>E</i>. <i>coli)</i> strains used in the study and their respective sources.</p

Life span assay for <i>C</i>. <i>elegans</i> infected with STEC strains compared to that for <i>C</i>. <i>elegans</i> infected with the control <i>E</i>. <i>coli</i> OP50 strain.

<p><i>C</i>. <i>elegans</i> grown on STEC plates were significantly short-lived compared to those grown on <i>E</i>. <i>coli</i> OP50 plates. The <i>E</i>. <i>coli</i> OP50 strain was compared to 24 different STEC strains (A-L).A) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared to that of <i>C</i>. <i>elegans</i> grown on the E1 and E2 strains. <i>C</i>. <i>elegans</i> grown on both E1 andE2 exhibited a decreased life span at the end of 10 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50 survived for 15 days. B) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared to that of <i>C</i>. <i>elegans</i> grown on the E3 and E4 strains. <i>C</i>. <i>elegans</i> grown on both E3 and E4 exhibited a decreased life span, but when compared to <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50, <i>C</i>. <i>elegans</i> grown on E3 survived for 8 days, and <i>C</i>. <i>elegans</i> grown on E4 survived for 10 days. C) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared to that of <i>C</i>. <i>elegans</i> grown on the E5 and E6 strains. <i>C</i>. <i>elegans</i> grown on both E5 and E6 exhibited a decreased life span at the end of 10 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50 survived for 15 days. D) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared to that of <i>C</i>. <i>elegans</i> grown on the E7 and E8 strains. <i>C</i>. <i>elegans</i> grown on both E7 and E8 exhibited a decreased life span at the end of 10 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50 survived for 15 days. E) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared to that of <i>C</i>. <i>elegans</i> grown on the E9 and E10 strains. <i>C</i>. <i>elegans</i> grown on both E9 and E10exhibited a decreased life span at the end of 10 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50 survived for 15 days. F) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared to that of <i>C</i>. <i>elegans</i> grown on the E11 and E12 strains. <i>C</i>. <i>elegans</i> grown on both E11 and E12 showed a decreased life span at the end of 10 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50 survived for 15 days. G) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared that of <i>C</i>. <i>elegans</i> grown on the E13 and E14 strains. <i>C</i>. <i>elegans</i> grown on both E13 and E14 exhibited a decreased life span at the end of 10 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50 survived for 15 days. H) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared to that of <i>C</i>. <i>elegans</i> grown on the E15 and E16 strains. <i>C</i>. <i>elegans</i> grown on both E15 and E16 exhibited a decreased life span at the end of 9 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50 survived for 15 days. I) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared to that of <i>C</i>. <i>elegans</i> grown on the E17 and E18 strains. <i>C</i>. <i>elegans</i> grown on E17 exhibited a decreased life span at the end of 10 days, and <i>C</i>. <i>elegans</i> grown on E18 surprisingly survived for 6 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50 survived for 15 days. J) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared that of <i>C</i>. <i>elegans</i> grown on the E19 and E20 strains. <i>C</i>. <i>elegans</i> grown on both E19 and E20 exhibited a decreased life span at the end of 9 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i>OP50 survived for 15 days. K) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared to that of <i>C</i>. <i>elegans</i> grown on the E21 and E22 strains. <i>C</i>. <i>elegans</i> grown on E21 showed a decreased life span at the end of 10 days, and in contrast, <i>C</i>. <i>elegans</i> grown on E22 survived for 7 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50 survived for 15 days. L) Longevity of <i>C</i>. <i>elegans</i> grown on the <i>E</i>. <i>coli</i> OP50 strain compared to that of <i>C</i>. <i>elegans</i> grown on the E23 and E24 strains. <i>C</i>. <i>elegans</i> grown on E23 showed a decreased life span at the end of 11 days, and in contrast, <i>C</i>. <i>elegans</i> grown on E24 survived for 9 days, but <i>C</i>. <i>elegans</i> grown on <i>E</i>. <i>coli</i> OP50 survived for 15 days.</p

Conventional PCR and colonisation assay.

<p><b>Conventional PCR</b>: Based on the results of the life span assay, 6 STEC strains (L1—E1; L2—E3; L3—E12; L4—E15; L5—E18; L6—E22; L7 –OP50) out of the 24 strains were selected for further testing for the presence of toxin genes (stx1, stx2 and groEL) using 3 primer pairs (STX1-F/R-O157; STX2-F/R-O157; GroEL-F/R-O157) targeting the genes specific for toxin production(stx1, stx2 and groEL). <b>Colonisation assay</b>: The STEC strains colonised in the intestinal tract of L2 worms (log CFU/worm). p<0.001 was obtained for comparison with the <i>E</i>. <i>coli</i> OP50 control group by the t-test. The total numbers of worms (<i>C</i>. <i>elegans</i>) tested in each group are indicated by n.</p

Molecular-Level Insights into Orientation-Dependent Changes in the Thermal Stability of Enzymes Covalently Immobilized on Surfaces

Surface-immobilized enzymes are important for a wide range of technological applications, including industrial catalysis, drug delivery, medical diagnosis, and biosensors; however, our understanding of how enzymes and proteins interact with abiological surfaces on the molecular level remains extremely limited. We have compared the structure, activity, and thermal stability of two variants of a β-galactosidase attached to a chemically well-defined maleimide-terminated self-assembled monolayer surface through a unique cysteinyl residue. In one case the enzyme is attached through an α helix and in the other case through an adjacent loop. Both enzymes exhibit similar specific activities and adopt similar orientations with respect to the surface normal, as determined by sum-frequency generation and attenuated total reflectance FT-IR spectroscopies. Surprisingly, however, the loop-tethered enzyme exhibits a thermal stability 10 °C lower than the helix-tethered enzyme and 13 °C lower than the enzyme in free solution. Using coarse-grain models, molecular dynamics simulations of the thermal unfolding of the surface-tethered enzymes were able to reproduce these differences in stability. Thus, revealing that tethering through the more flexible loop position provides more opportunity for surface residues on the protein to interact with the surface and undergo surface-induced unfolding. These observations point to the importance of the location of the attachment point in determining the performance of surface-supported biocatalysts and suggest strategies for optimizing their activity and thermal stability through molecular simulations

Interfacial Behaviors of Antimicrobial Peptide Cecropin P1 Immobilized on Different Self-Assembled Monolayers

Surfaces immobilized with biological molecules such as peptides and proteins are widely used in many important applications including biosensors, medical devices, and food packaging. It was found that the structures of surface-immobilized peptides control their surface properties. In this study, we investigated interfacial behaviors of antimicrobial peptide cecropin P1 (CP1) immobilized onto a maleimide-terminated self-assembled monolayer (Mal SAM) and a mixed SAM (Mal-OH SAM, a mixture of maleimide-terminated SAM and hydroxyl-terminated SAM) surface via C-terminus modified cysteine (CP1c). The surface coverage, secondary structure, orientation, and antimicrobial activity of immobilized CP1c were investigated using surface plasmon resonance (SPR), circular dichroism (CD), sum frequency generation (SFG) vibrational spectroscopy, dynamic contact antimicrobial assay, and coarse grained molecular dynamics (MD) simulations. It was found that the surface coverages of CP1c on the Mal SAM and the mixed Mal-OH SAM are similar. CP1c on Mal SAM possessed a dominant helical structure with a single orientation of ∼32° versus surface normal. CP1c on Mal-OH mixed SAM surface also possessed a dominant helical structure but with multiple orientations. MD simulation results can be correlated to the experimental data: the simulation results indicate a narrow distribution of orientations of CP1c immobilized on Mal SAM, but multiple orientations are sampled on the more hydrophilic Mal-OH SAM. Even though the surface orientations of CP1c immobilized on the two SAM surfaces are different, activity against both Gram-negative and Gram-positive bacteria (Escherichia coli and Staphylococcus aureus) exhibited similar results for CP1c immobilized on both SAM surfaces. We believe that this is because the antimicrobial activity of the surface-immobilized peptides is mainly affected by the electrostatic interactions between the strong basic N-terminal residues and the negatively charged bacteria cell wall/cell membrane