Superficial implantation in pigs is associated with decreased numbers and redistribution of endometrial NK-cell populations

PROBLEM: We evaluated implantation-associated quantitative changes in endometrial and peripheral natural killer (NK)-cell populations of pigs. METHOD OF STUDY: Natural killer cell populations were investigated in 10, 15, 20, 30 and 40 days pregnant and non-pregnant (NP) sows by flow cytometry, immunohistochemistry and morphometry. RESULTS: The number of endometrial CD16(+) NK cells significantly declined at attachment phase of implantation and remained relatively low over the course of implantation. The CD16(+) NK cells in situ showed implantation-phase dependent density and localization. Prior to implantation, they substantially resided in the subepithelial stroma. As implantation advances, the density of NK cells into subepithelial stroma decreased while that of NK cells into glandular layer increased, suggesting implantation-induced re-location far from the attached conceptus. The number of CD56(+) lymphocytes was the greatest at pre-attachment phase of implantation, dropped at the time of attachment and increased up to end of early pregnancy period. The CD3(-) CD8(+) NK-cell number decreased significantly when the definitive placenta is established. No significant differences in the numbers of peripheral blood CD16(+), CD56(+) and CD3(-) CD8(+) NK cells between pregnant and NP animals as well as relative to the implantation phase were observed. CONCLUSION: Superficial and adeciduate implantation of pigs is associated with decreased numbers of endometrial NK-cell populations and specific spatiotemporal profile of classical NK cells.     

Utility of the hybrid LTQ-FTMS for drug metabolism applications

Fourier Transform Ion Cyclotron Mass Spectrometry (FTMS) provides the highest mass accuracy and mass resolving power of the currently available mass spectrometers. One of the main drawbacks in its use for absorption, distribution, metabolism and excretion (ADME) applications has been its incompatibility with standard HPLC columns and flow rates. Hybrid instruments, such as the LTQ-FT, provide the much needed bridge between the excellent performance and capabilities of the FT mass spectrometers and the well-established, tested and validated features of quadrupoles and ion traps. The hybrid instruments are compatible with standard HPLC flow rates, have high-throughput and automation compatibility, and also provide data dependant MSn. The ability to maintain the fidelity of an externally calibrated accurate mass measurement across an HPLC peak, where the analyte concentrations are rapidly changing, is a significant advance for this technology, as is the ability to perform data dependent MS/MS experiments on the chromatographic time scale. The MSn and accurate mass capabilities are routinely utilized to rapidly confirm the identification of expected metabolites or to elucidate the structures of unusual or unexpected metabolites. The combination of traditional high-flow chromatography and robust, externally calibrated accurate mass determination for both parent and product ions makes the LTQ-FTMS a very powerful analytical tool for the characterization of metabolites, identification of metabolic soft-spots and for metabonomics studies.     

Efficacy of ganitumab (AMG 479), alone and in combination with rapamycin, in Ewing's and osteogenic sarcoma models

Ewing's and osteogenic sarcoma are two of the leading causes of cancer deaths in children and adolescents. Recent data suggest that sarcomas may depend on the insulin-like growth factor type 1 (IGF-1) receptor (IGF1R) and/or the insulin receptor (INSR) to drive tumor growth, survival, and resistance to mammalian target of rapamycin complex 1 (mTORC1) inhibitors. We evaluated the therapeutic value of ganitumab (AMG 479; C(6472)H(10028)N(1728)O(2020)S(42)), an anti-IGF1R, fully human monoclonal antibody, alone and in combination with rapamycin (mTORC1 inhibitor) in Ewing's (SK-ES-1 and A673) and osteogenic (SJSA-1) sarcoma models. IGF1R was activated by IGF-1 but not by insulin in each sarcoma model. INSR was also activated by IGF-1 in the SJSA-1 and SK-ES-1 models, but not in the A673 model where insulin was the preferred INSR ligand. Ganitumab significantly inhibited the growth of SJSA-1 and SK-ES-1 xenografts; inhibition was associated with decreased IGF1R and Akt phosphorylation, reduced total IGF1R and bromodeoxyuridine detection, and increased caspase-3 expression. Ganitumab inhibited rapamycin-induced IGF1R, Akt, and glycogen synthase kinase-3β hyperphosphorylation in each sarcoma model. However, ganitumab in combination with rapamycin also resulted in a marked increase in INSR expression and activity in the SJSA-1 and A673 models. The in vivo efficacy of ganitumab in the two ganitumab-sensitive models (SJSA-1 and SK-ES-1) was significantly enhanced in combination with rapamycin. Our results support studying ganitumab in combination with mTORC1 inhibitors for the treatment of sarcomas and suggest that INSR signaling is an important mechanism of resistance to IGF1R blockade.     

Synthesis and biological activity of potent HIV-1 protease inhibitors based on Phe-Pro dihydroxyethylene isosteres

Peptidomimetic inhibitors of HIV-1 PR are still a key resource in the fight against AIDS. Here we describe the synthesis and biological activity of HIV-1 PR inhibitors based on four novel dihydroxyethylene isosteres of the Phe-Pro and Pro-Pro dipeptides. The isosteres, containing four stereogenic centers, were synthesized in high yield and excellent stereoselectivity via the cyclization of epoxy amines derived from α-amino acids. The inhibitors were assembled by coupling the isosteres with suitable flanking groups and were screened against recombinant HIV PR showing activities in the subnanomolar to micromolar range. Two Phe-Pro-based inhibitors active at the nanomolar level were further investigated: both inhibitors combine the ability to suppress HIV-1 replication in infected MT-2 cells with low cytotoxicity against the same cells, thereby displaying a high therapeutic index. These results demonstrate the potential of the new Phe-Pro dihydroxyethylene isostere as a core unit of powerful HIV-1 PR inhibitors.     

HoliMAb: A holistic approach for Media-Adventitia border detection in intravascular ultrasound

We present a fully automatic methodology for the detection of the Media-Adventitia border (MAb) in human coronary artery in Intravascular Ultrasound (IVUS) images. A robust border detection is achieved by means of a holistic interpretation of the detection problem where the target object, i.e. the media layer, is considered as part of the whole vessel in the image and all the relationships between tissues are learnt. A fairly general framework exploiting multi-class tissue characterization as well as contextual information on the morphology and the appearance of the tissues is presented. The methodology is (i) validated through an exhaustive comparison with both Inter-observer variability on two challenging databases and (ii) compared with state-of-the-art methods for the detection of the MAb in IVUS. The obtained averaged values for the mean radial distance and the percentage of area difference are 0.211mm and 10.1%, respectively. The applicability of the proposed methodology to clinical practice is also discussed.     

Adsorption of azide-functionalized thiol linkers on zinc oxide surfaces

A comprehensive understanding of the interactions between organic molecules and a metal oxide surface is essential for an efficient surface modification and the formation of organic–inorganic hybrids with technological applications ranging from heterogeneous catalysis and biomedical templates up to functional nanoporous matrices. In this work, first-principles calculations supported by experiments are used to provide the microstructural characteristics of (10$\bar{1}$0) surfaces of zinc oxide single crystals modified by azide terminated hydrocarbons, which graft on the oxide through a thiol group. On the computational side, we evaluate the specific interactions between the surface and the molecules with the chemical formula N₃(CH₂)_nSH, with n = 1, 3, 6, 9. We demonstrate that the molecules chemisorb on the bridge site of ZnO(10$\bar{1}$0). Upon adsorption, the N₃(CH₂)_nSH molecules break the neutral (Zn^δ+–O^δ−) dimers on ZnO(10$\bar{1}$0) resulting in a structural distortion of the ZnO(10$\bar{1}$0) substrate. The energy decomposition analysis revealed that such structure distortion favors the adsorption of the molecules on the surface leading to a strong correlation between the surface distortion energy and the interaction energy of the molecule. An azide-terminated thiol with three methylene groups in the hydrocarbon chain N₃(CH₂)₃SH was synthesized, and the assembly of this linker on ZnO surfaces was confirmed through atomic force microscopy. The bonding to the inorganic surface was examined via X-ray photoelectron spectroscopy (XPS). Clear signatures of the organic components on the oxide substrates were observed underlying the successful realization of thiol-grafting on the metal oxide. Temperature-dependent and angle-resolved XPS were applied to examine the thermal stability and to determine the thickness of the grafted SAMs, respectively. We discuss the high potential of our hybrid materials in providing further functionalities towards heterocatalysis and medical applications.

Studying the interaction between organic molecules and metal oxide surfaces is key to the development and modification of organic–inorganic hybrids for application in heterogeneous catalysis, biomedical implants, and functional nanoporous matrices.