Differences between Ca2+ and Mg2+ in DNA binding and release by the SfiI restriction endonuclease: implications for DNA looping

Many enzymes acting on DNA require Mg²⁺ ions not only for catalysis but also to bind DNA. Binding studies often employ Ca²⁺ as a substitute for Mg²⁺, to promote DNA binding whilst disallowing catalysis. The SfiI endonuclease requires divalent metal ions to bind DNA but, in contrast to many systems where Ca²⁺ mimics Mg²⁺, Ca²⁺ causes SfiI to bind DNA almost irreversibly. Equilibrium binding by wild-type SfiI cannot be conducted with Mg²⁺ present as the DNA is cleaved so, to study the effect of Mg²⁺ on DNA binding, two catalytically-inactive mutants were constructed. The mutants bound DNA in the presence of either Ca²⁺ or Mg²⁺ but, unlike wild-type SfiI with Ca²⁺, the binding was reversible. With both mutants, dissociation was slow with Ca²⁺ but was in one case much faster with Mg²⁺. Hence, Ca²⁺ can affect DNA binding differently from Mg²⁺. Moreover, SfiI is an archetypal system for DNA looping; on DNA with two recognition sites, it binds to both sites and loops out the intervening DNA. While the dynamics of looping cannot be measured with wild-type SfiI and Ca²⁺, it becomes accessible with the mutant and Mg²⁺.     

miR‐516a‐3p inhibits breast cancer cell growth and EMT by blocking the Pygo2/Wnt signalling pathway

miR‐516a‐3p has been reported to play a suppressive role in several types of human tumours. However, the expression level, biological function and fundamental mechanisms of miR‐516a‐3p in breast cancer remain unclear. In the present study, we found that miR‐516a‐3p expression was down‐regulated and Pygopus2 (Pygo2) expression was up‐regulated in human breast cancer tissues and cells. Through analysing the clinicopathological characteristics, we demonstrated that low miR‐516a‐3p expression or positive Pygo2 expression was a predictor of poor prognosis for patients with breast cancer. The results of a dual luciferase reporter assay and Western blot analysis indicated that Pygo2 was a target gene of miR‐516a‐3p. Moreover, overexpression of miR‐516a‐3p inhibited cell growth, migration and invasion as well as epithelial‐mesenchymal transition (EMT) of breast cancer cells, whereas reduced miR‐516a‐3p expression promoted breast cancer cell growth, migration, invasion and EMT. Furthermore, we showed that miR‐516a‐3p suppressed cell proliferation, metastasis and EMT of breast cancer cells by inhibiting Pygo2 expression. We confirmed that miR‐516a‐3p exerted an anti‐tumour effect by inhibiting the activation of the Wnt/β‐catenin pathway. Finally, xenograft tumour models were used to show that miR‐516a‐3p inhibited breast cancer cell growth and EMT via suppressing the Pygo2/Wnt signalling pathway. Taken together, these results show that miR‐516a‐3p inhibits breast cancer cell growth, metastasis and EMT by blocking the Pygo2/ Wnt/β‐catenin pathway.     

Tuning a polar molecule for selective cytoplasmic delivery by a pH (Low) insertion peptide

Drug molecules are typically hydrophobic and small in order to traverse membranes to reach cytoplasmic targets, but we have discovered that more polar molecules can be delivered across membranes using water-soluble, moderately hydrophobic membrane peptides of the pHLIP (pH low insertion peptide) family. Delivery of polar cargo molecules could expand the chemical landscape for pharmacological agents that have useful activity but are too polar by normal drug criteria. The spontaneous insertion and folding of the pHLIP peptide across a lipid bilayer seeks a free energy minimum, and insertion is accompanied by a release of energy that can be used to translocate cell-impermeable cargo molecules. In this study, we report our first attempt to tune the hydrophobicity of a polar cargo, phallacidin, in a systematic manner. We present the design, synthesis, and characterization of three phallacidin cargoes, where the hydrophobicity of the cargo was tuned by the attachment of diamines of various lengths of hydrophobic chains. The phallacidin cargoes were conjugated to pHLIP and shown to selectively inhibit the proliferation of cancer cells in a concentration-dependent manner at low pH.     

Spatial variability in nutrient concentration and biofilm nutrient limitation in an urban watershed

Nutrient enrichment threatens river ecosystem health in urban watersheds, but the influence of urbanization on spatial variation in nutrient concentrations and nutrient limitation of biofilm activity are infrequently measured simultaneously. In summer 2009, we used synoptic sampling to measure spatial patterns of nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), and soluble reactive phosphorus (SRP) concentration, flux, and instantaneous yield throughout the Bronx River watershed within New York City and adjacent suburbs. We also quantified biofilm response to addition of NO₃ ⁻, phosphate (PO₄ ³⁻), and NO₃ ⁻ + PO₄ ³⁻ on organic and inorganic surfaces in the river mainstem and tributaries. Longitudinal variation in NO₃ ⁻ was low and related to impervious surface cover across sub-watersheds, but spatial variation in NH₄ ⁺ and SRP was higher and unrelated to sub-watershed land-use. Biofilm respiration on organic surfaces was frequently limited by PO₄ ³⁻ or NO₃ ⁻ + PO₄ ³⁻, while primary production on organic and inorganic surfaces was nutrient-limited at just one site. Infrequent NO₃ ⁻ limitation and low spatial variability of NO₃ ⁻ throughout the watershed suggested saturation of biological N demand. For P, both higher biological demand and point-sources contributed to greater spatial variability. Finally, a comparison of our data to synoptic studies of forested, temperate watersheds showed lower spatial variation of N and P in urban watersheds. Reduced spatial variation in nutrients as a result of biological saturation may represent an overlooked effect of urbanization on watershed ecology, and may influence urban stream biota and downstream environments.     

Roles of carboxyl groups in the transmembrane insertion of peptides

We have used pHLIP® [pH (low) insertion peptide] to study the roles of carboxyl groups in transmembrane (TM) peptide insertion. pHLIP binds to the surface of a lipid bilayer as a disordered peptide at neutral pH; when the pH is lowered, it inserts across the membrane to form a TM helix. Peptide insertion is reversed when the pH is raised above the characteristic pK_a (6.0). A key event that facilitates membrane insertion is the protonation of aspartic acid (Asp) and/or glutamic acid (Glu) residues, since their negatively charged side chains hinder membrane insertion at neutral pH. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ∼ pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. A further pH drop leads to the protonation of His residues in the TM part of the peptide, which induces peptide exit from the bilayer. We also find that the number of ionizable residues that undergo a change in protonation during membrane insertion correlates with the pH-dependent insertion into the lipid bilayer and exit from the lipid bilayer, and that cooperativity increases with their number. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP.     

Function‐based assessment of structural similarity measurements using metal co‐factor orientation

Structure comparison is widely used to quantify protein relationships. Although there are several approaches to calculate structural similarity, specifying significance thresholds for similarity metrics is difficult due to the inherent likeness of common secondary structure elements. In this study, metal co‐factor location is used to assess the biological relevance of structural alignments. The distance between the centroids of bound co‐factors adds a chemical and function‐relevant constraint to the structural superimposition of two proteins. This additional dimension can be used to define cut‐off values for discriminating valid and spurious alignments in large alignment sets. The hypothesis underlying our approach is that metal coordination sites constrain structural evolution, thus revealing functional relationships between distantly related proteins. A comparison of three related nitrogenases shows the sequence and fold constraints imposed on the protein structures up to 18 Å away from the centers of their bound metal clusters. Proteins 2014; 82:648–656. © 2013 Wiley Periodicals, Inc.     

Motility and Chemotaxis Mediate the Preferential Colonization of Gastric Injury Sites by Helicobacter pylori

Helicobacter pylori (H. pylori) is a pathogen contributing to peptic inflammation, ulceration, and cancer. A crucial step in the pathogenic sequence is when the bacterium first interacts with gastric tissue, an event that is poorly understood in vivo. We have shown that the luminal space adjacent to gastric epithelial damage is a microenvironment, and we hypothesized that this microenvironment might enhance H. pylori colonization. Inoculation with 10⁶ H. pylori (wild-type Sydney Strain 1, SS1) significantly delayed healing of acetic-acid induced ulcers at Day 1, 7 and 30 post-inoculation, and wild-type SS1 preferentially colonized the ulcerated area compared to uninjured gastric tissue in the same animal at all time points. Gastric resident Lactobacillus spp. did not preferentially colonize ulcerated tissue. To determine whether bacterial motility and chemotaxis are important to ulcer healing and colonization, we analyzed isogenic H. pylori mutants defective in motility (ΔmotB) or chemotaxis (ΔcheY). ΔmotB (10⁶) failed to colonize ulcerated or healthy stomach tissue. ΔcheY (10⁶) colonized both tissues, but without preferential colonization of ulcerated tissue. However, ΔcheY did modestly delay ulcer healing, suggesting that chemotaxis is not required for this process. We used two-photon microscopy to induce microscopic epithelial lesions in vivo, and evaluated accumulation of fluorescently labeled H. pylori at gastric damage sites in the time frame of minutes instead of days. By 5 min after inducing damage, H. pylori SS1 preferentially accumulated at the site of damage and inhibited gastric epithelial restitution. H. pylori ΔcheY modestly accumulated at the gastric surface and inhibited restitution, but did not preferentially accumulate at the injury site. H. pylori ΔmotB neither accumulated at the surface nor inhibited restitution. We conclude that bacterial chemosensing and motility rapidly promote H. pylori colonization of injury sites, and thereby biases the injured tissue towards sustained gastric damage.     

The Influence of Nanocrystal Aggregates on Photovoltaic Performance in Nanocrystal–Polymer Bulk Heterojunction Solar Cells

CdSe nanocrystals (NCs) can be used as an electron acceptor in solar cells, employing organic ligands to passivate their surface and make them processable from solution. The nature and abundance of impurities present after NC ligand exchange from oleic acid to n‐butylamine are identified. A further purification step using hexane as a selective solvent is described, which excludes NC aggregates from solution. The influence of NC aggregates on photovoltaic device performance is studied in a CdSe:poly[2‐methoxy‐5‐(3′,7′‐dimethyloctyloxy)‐1,4‐phenylene vinylene] (MDMO‐PPV) bulk heterojunction solar cell. The exclusion of NC aggregates leads to a four‐fold increase in device power conversion efficiency (PCE) in optimized devices. A superior blend morphology leading to improved charge generation and a better NC percolation network is identified as the main causes of this increased solar cell performance.