Evidence for Myelin Sheath Remodeling in the CNS Revealed by In Vivo Imaging

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Synthesis and comparative assessment of a labeled RGD peptide bearing two different ⁹⁹mTc-tricarbonyl chelators for potential use as targeted radiopharmaceutical

During the past decade radiolabeled RGD-peptides have been extensively studied to develop site-directed targeting vectors for integrins. Integrins are heterodimeric cell-surface adhesion receptors, which are upregulated in cancer cells and neovasculature during tumor angiogenesis and recognize the RGD aminoacid sequence. In the present study, we report the synthesis and development of two derivatives of the Nε-Lys derivatized cyclic Arg-Gly-Asp-D-Phe-Lys peptide, namely of cRGDfKHis and cRGDfK-CPA (CPA: 3-L-Cysteine Propionic Acid), radiolabeled via the [(99m)Tc(H(2)O)(3)(CO)(3)](+) metal aquaion at a high yield even at low concentrations of 10-5M (>87%) for cRGDfK-10-5M (>93%) for cRGDfK-CPA. Radiolabeled peptides were characterized with regard to their stability in saline, in His/Cys solutions, as well as in plasma, serum and tissue homogenates and were found to be practically stable. Internalization and efflux assays using αvβ3-receptor-positive MDA-MB 435 breast cancer cells showed a good percentage of quick internalization (29.1 ± 9.8% for (99m)Tc-HiscRGDfK and 37.0 ± 0.7% for (99m)Tc-CPA-cRGDfK at 15 min) and no retention of radioactivity for both derivatives. Their in vivo behavior was assessed in normal mice and pathological SCID mice bearing MDA-MB 435 ανβ3 positive breast tumors. Both presented fast blood clearance and elimination via both the urinary and hepatobiliary systems, with (99m)Tc-His-cRGDfK remaining for a longer time than (99m)Tc-CPA-cRGDfK in all organs examined. Tumor uptake 30 min pi was higher for (99m)Tc-CPAcRGDfK (4.2 ± 1.5% ID/g) than for (99m)Tc-His-cRGDfK (2.8 ± 1.5% ID/g). Dynamic scintigraphic studies showed that the tumor could be visualized better between 15 and 45 min pi for both radiolabeled compounds but low delineation occurred due to high abdominal background. It was finally noticed that the accumulated activity on the tumor site was depended on the size of the experimental tumor; the smaller the size, the higher was the radioactivity concentration.     

Influence of precipitating agents on thermodynamic parameters of protein crystallization solutions

X‐ray crystallography is the most powerful method for determining three‐dimensional structures of proteins to (near‐)atomic resolution, but protein crystallization is a poorly explained and often intractable phenomenon. Differential Scanning Calorimetry was used to measure the thermodynamic parameters (ΔG, ΔH, ΔS) of temperature‐driven unfolding of two globular proteins, lysozyme, and ribonuclease A, in various salt solutions. The mixtures were categorized into those that were conducive to crystallization of the protein and those that were not. It was found that even fairly low salt concentrations had very large effects on thermodynamic parameters. High concentrations of salts conducive to crystallization stabilized the native folded forms of proteins, whereas high concentrations of salts that did not crystallize them tended to destabilize them. Considering the ΔH and TΔS contributions to the ΔG of unfolding separately, high concentrations of crystallizing salts were found to enthalpically stabilize and entropically destabilize the protein, and vice‐versa for the noncrystallizing salts. These observations suggest an explanation, in terms of protein stability and entropy of hydration, of why some salts are good crystallization agents for a given protein and others are not. This in turn provides theoretical insight into the process of protein crystallization, suggesting ways of predicting and controlling it. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 642–652, 2016.     

Enhanced Zone II Flexor Tendon Repair through a New Half Hitch Loop Suture Configuration

This study evaluated the impact of a new half hitch loop suture configuration on flexor tendon repair mechanics. Cadaver canine flexor digitorum profundus tendons were repaired with 4- or 8-strands, 4–0 or 3–0 suture, with and without half hitch loops. An additional group underwent repair with half hitch loops but without the terminal knot. Half hitch loops improved the strength of 8-strand repairs by 21% when 4–0, and 33% when 3–0 suture was used, and caused a shift in failure mode from suture pullout to suture breakage. 8-strand repairs with half hitch loops but without a terminal knot produced equivalent mechanical properties to those without half hitch loops but with a terminal knot. 4-strand repairs were limited by the strength of the suture in all groups and, as a result, the presence of half hitch loops did not alter the mechanical properties. Overall, half hitch loops improved repair mechanics, allowing failure strength to reach the full capability of suture strength. Improving the mechanical properties of flexor tendon repair with half hitch loops has the potential to reduce the postoperative risk of gap formation and catastrophic rupture in the early postoperative period.     

Structural and thermodynamic characterization of metal ion binding in Streptococcus suis Dpr

The use of protein cages for the creation of novel inorganic nanomaterials has attracted considerable attention in recent years. Ferritins are among the most commonly used protein cages in nanoscience. Accordingly, the binding of various metals to ferritins has been studied extensively. Dps (DNA-binding protein from starved cells)-like proteins belong to the ferritin superfamily. In contrast to ferritins, Dps-like proteins form 12-mers instead of 24-mers, have a different ferroxidase center, and are able to store a smaller amount of iron atoms in a hollow cavity (up to ∼ 500, instead of the ∼ 4500 iron atoms found in ferritins). With the exception of iron, the binding of other metal cations to Dps proteins has not been studied in detail. Here, the binding of six divalent metal ions (Zn²⁺, Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, and Mg²⁺) to Streptococcus suisDps-like peroxide resistance protein (SsDpr) was characterized by X-ray crystallography and isothermal titration calorimetry (ITC). All metal cations, except for Mg²⁺, were found to bind to the ferroxidase center similarly to Fe²⁺, with moderate affinity (binding constants between 0.1 × 10⁵ M^− 1 and 5 × 10⁵ M^− 1). The stoichiometry of binding, as deduced by ITC data, suggested the presence of a dication ferroxidase site. No other metal binding sites were identified in the protein. The results presented here demonstrate the ability of SsDpr to bind various metals as substitutes for iron and will help in better understanding protein-metal interactions in the Dps family of proteins as potential metal nanocontainers.     

Saccharomyces boulardii inhibits ERK1/2 mitogen-activated protein kinase activation both in vitro and in vivo and protects against Clostridium difficile toxin A-induced enteritis

Saccharomyces boulardii (Sb), a probiotic yeast, protects against intestinal injury and inflammation caused by a wide variety of enteric pathogens, including Clostridium difficile. Given the broad range of protective effects of Sb in multiple gastrointestinal disorders, we hypothesize that Sb modulates host signaling pathways involved in intestinal inflammatory responses. In this study, we found that Sb culture supernatant (SbS) inhibits interleukin-8 production induced by C. difficile toxin A or IL-1beta in human colonocyte NCM460 cells in a dose-dependent fashion. Furthermore, SbS inhibited IL-1beta and toxin A induced Erk1/2 and JNK/SAPK but not p38 activation in NCM460 cells. To test whether this inhibition also occurs in vivo, we used a previously established mouse ileal loop model. On its own, SbS had no significant effect on basal fluid secretion or intestinal histology. However, Erk1/2 activation was significantly inhibited by SbS in toxin A exposed mouse ileal mucosa. In control loops, toxin A increased fluid secretion (2.2-fold), histological score (3.3-fold), and levels of the chemokine KC (4.5-fold). SbS pretreatment completely normalized toxin A mediated fluid secretion (p < 0.01), and histopathologic changes (p < 0.01) and substantially inhibited toxin A-associated KC increases (p < 0.001). In summary, the probiotic yeast S. boulardii inhibits C. difficile toxin A-associated enteritis by blocking the activation of Erk1/2 MAP kinases. This study indicates a new mechanism whereby Sb protects against intestinal inflammation and supports the hypothesis that Sb modulates host inflammatory signaling pathways to exert its beneficial effects.     

Hyaluronan signaling during ozone-induced lung injury requires TLR4, MyD88, and TIRAP

Ozone exposure is associated with exacerbation of reactive airways disease. We have previously reported that the damage-associated molecular pattern, hyaluronan, is required for the complete biological response to ambient ozone and that hyaluronan fragments signal through toll-like receptor 4 (TLR4). In this study, we further investigated the role of TLR4 adaptors in ozone-induced airway hyperresponsiveness (AHR) and the direct response to hyaluronan fragments (HA). Using a murine model of AHR, C57BL/6J, TLR4-/-, MyD88-/-, and TIRAP-/- mice were characterized for AHR after exposure to either ozone (1 ppm × 3 h) or HA fragments. Animals were characterized for AHR with methacholine challenge, cellular inflammation, lung injury, and production of pro-inflammatory cytokines. Ozone-exposed C57BL/6J mice developed cellular inflammation, lung injury, pro-inflammatory cytokines, and AHR, while mice deficient in TLR4, MyD88 or TIRAP demonstrated both reduced AHR and reduced levels of pro-inflammatory cytokines including TNFα, IL-1β, MCP-1, IL-6 and KC. The level of hyaluronan was increased after inhalation of ozone in each strain of mice. Direct challenge of mice to hyaluronan resulted in AHR in C57BL/6J mice, but not in TLR4-/-, MyD88-/-, or TIRAP-/- mice. HA-induced cytokine production in wild-type mice was significantly reduced in TLR4-/-, MyD88-/-, or TIRAP-/- mice. In conclusion, our findings support that ozone-induced airway hyperresponsiveness is dependent on the HA-TLR4-MyD88-TIRAP signaling pathway.