Landscape phages and their fusion proteins targeted to breast cancer cells

Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific 'fusion pVIII proteins' (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage-cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1.     

Hydrogen Peroxide-Preconditioned Human Adipose-Derived Stem Cells Enhance the Recovery of Oligodendrocyte-Like Cells after Oxidative Stress-Induced Damage

Oxidative stress associated with neuroinflammation is a key process involved in the pathophysiology of neurodegenerative diseases, and therefore, has been proposed as a crucial target for new therapies. Recently, the therapeutic potential of human adipose-derived stem cells (hASCs) has been investigated as a novel strategy for neuroprotection. These cells can be preconditioned by exposing them to mild stress in order to improve their response to oxidative stress. In this study, we evaluate the therapeutic potential of hASCs preconditioned with low doses of H₂O₂ (called HC016 cells) to overcome the deleterious effect of oxidative stress in an in vitro model of oligodendrocyte-like cells (HOGd), through two strategies: i, the culture of oxidized HOGd with HC016 cell-conditioned medium (CM), and ii, the indirect co-culture of oxidized HOGd with HC016 cells, which had or had not been exposed to oxidative stress. The results demonstrated that both strategies had reparative effects, oxidized HC016 cell co-culture being the one associated with the greatest recovery of the damaged HOGd, increasing their viability, reducing their intracellular reactive oxygen species levels and promoting their antioxidant capacity. Taken together, these findings support the view that HC016 cells, given their reparative capacity, might be considered an important breakthrough in cell-based therapies.     

GOLPH3 Regulates EGFR in T98G Glioblastoma Cells by Modulating Its Glycosylation and Ubiquitylation

Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.     

The influence of different zeolitic supports on hydrogen production and waste degradation

The photocatalyst composition affects the chemical–physical properties and directly impacts photocatalytic activity, both in the hydrogen production and degradation of organic contaminants. In this work, the influence of zeolitic structures NaA, NaY, and ZSM-5 combined with a 10% active phase, TiO₂ catalyst doped with 1% copper, and cobalt cocatalysts was tested to mineralize the reactive blue dye (CI250) and to produce hydrogen by photocatalysis under ultraviolet radiation. The band gap energy was affected mainly by the cocatalyst, while the Brunauer-Emmett-Teller method (BET) area was affected by the zeolite structure as well as the X-ray diffraction (XRD). The most active catalyst was the Cu@TiO₂/NaY, which promoted a hydrogen production rate of 240 μmolH₂gcat⁻¹ using 10% ethanol (v/v) aqueous solution as a sacrificial agent and mineralization of 53% of the organic dye, followed by the catalysts impregnated on ZSM-5 zeolites, which had discolouration up to 50% and hydrogen evolution of 92.6 and 109.7 μmolH₂gcat⁻¹ for the catalyst doped with Cu and Co, respectively.     

Imbalance diets in preschool children: study in a day-care center in the Province of Buenos Aires,Argentina

The purpose of this study was to investigate the impact of low fat diets in children aged 2 to 5. Eighty two children (40 females and 42 males) attending a school cafeteria (Province of Buenos Aires, Argentina), in a cross sectional study, were evaluated. Body weight (W), height (H) and body composition (BC) by bioimpedance were recorded. The anthropometric raw data were processed as Z-score of the weight-for-age (WEZ) and of the height-for-age (HAZ). Serum insulin-like growth factor 1 (IGF-1) and Zinc/haemoglobin ratio (Zn/Hb) were also measured. Results showed that 73.2% of the children were adequate (A) according WEZ, 13.4% were lean (L) and 13.4% overweight (O). 8.5% presented simultaneously impairment in WEZ and HAZ. Body fat percentage and energy metabolism were higher in O than in L and A (p < 0.05). Serum IGF-1's children--aged 4 to 5 years--with HAZ deficit were low than adequate HAZ ones. No statistical differences in Zn/Hb ratio between A, L and O were found. This cross sectional study suggests metabolic disorders in young children attending school cafeterias. These conclusions will allow to design balanced diets in order to optimize the resources, promote optimal growth and development and prevent adult diseases through dietary practices in childhood.     

Chemical pathways for the polymerization of enines

AM1 calculations are reported for enines with aromatic substituents. It was found that the position of the unsaturated bonds in each molecule may account for the reactivity of each isomer via different polymerization pathways. The resonant structure of the charged form of one of the molecules allowed speculation about the observed inhibition of the polymerization reaction.     

Surface hydrolysis of filaments based on poly(trimethylene terephthalate) spun at high spinning speeds

The surface alkaline hydrolysis of fibers made from poly(trimethylene terephthalate) (PTT) was studied after extruding the polymer at high spinning speeds from 2000 to 6000 m/min and heat setting in the range of temperatures from 100 to 180°C. Fiber weight loss increased with an increasing heat‐setting temperature but it was also dependent on the spinning speed. Some of the partially hydrolyzed fibers had a well‐developed, hydrophilic surface, and pore size in the range of 0.69 to 1.20 μm. The optimum reaction and morphological conditions for increasing porosity in PTT fibers depends on spinning speed and heat‐setting temperature. A temperature of 180°C is the upper limit for heat‐setting PTT filaments but seems to be the most effective for making porous fibers. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 1724–1730, 2004     

Chitin characterization by SEM,FTIR, XRD,and 13C cross polarization/mass angle spinning NMR

The full characterization of chitin obtained from squid, shrimp, prawn, lobsters, and king crab is reported. Elemental analysis, including metals such as Ca, Mg, Zn, Cd, Hg, Cr, Mn, Cu, and Pb, was performed, which is quite relevant because the skeleton composition is slightly different for each species. The morphology was studied by means of TEM and their compositions were determined by energy‐dispersive X‐ray analysis. ¹³C cross polarization/magic angle spinning NMR was applied to determine the chemical shift of all the carbons and the difference between them. Chitin was isolated by using chemical methods, alternating hydrochloric acid and sodium hydroxide. The α‐chitin from shrimp, prawn, lobsters, and king crabs showed two signals at 73.7 and 75.6 ppm. Meanwhile, the β‐chitin from squid exhibited one signal at 75.2 ppm. FTIR studies were used to analyze α‐chitin from shrimp and β‐chitin from squid. The α‐chitin exhibited amide I vibration modes at 1660 and 1627 cm^?1, whereas the β‐chitin showed one band at 1656 cm^?1. X‐ray diffraction showed that α‐chitin is orthorhombic (a = 4.74 Å, b = 18.86 Å, and c = 10.32 Å) and β‐chitin had a monoclinic dihydrated form (a = 4.80 Å, b = 10.40 Å, c = 11.10 Å, and β = 97°). © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1876–1885, 2004     

Identification and Molecular Cloning of Putative Odorant-Binding Proteins from the American Palm Weevil, Rhynchophorus palmarum L.

We have identified and cloned the cDNAs encoding two odorant-binding proteins (OBPs) from the American palm weevil (APW) Rhynchophorus palmarum (Coleoptera, Curculionidae). Degenerate primers were designed from the N-terminal sequences and were used in polymerase chain reaction (PCR) in order to obtain full-length sequences in both males and females. In both sexes, two different cDNAs were obtained, encoding 123 and 115 amino acid-deduced sequences. Each sequence showed few amino acid differences between the sexes. The proteins were named RpalOBP2 and RpalOBP4 for male, RpalOBP2' and RpalOBP4' for female, with the types 2 and 4 presenting only 34% identities. These proteins shared high identity with previously described coleopteran OBPs. In native gels, RpalOBP2 clearly separated into two bands and RpalOBP4 into three bands, suggesting the presence of several conformational isomers. Thus, OBP diversity in this species may rely on both the presence of OBPs from different classes and the occurrence of isoforms for each OBP.     

Structure-function studies of an IGF-I analogue that can be chemically cleaved to a two-chain mini-IGF-I

The structure and biological activities of two disulphide isomersof a C-region deletion mutant of insulin-like growth factor-I(IGF-I) which has an Asn–Gly link engineered at the junctionof the A- and B-regions were studied before and after chemicalcleavage. Circular dichroism (CD) spectra and binding affinityto IGF binding protein 3 (IGFBP3) indicated that the treatmentwith hydroxylamine did not disrupt the overall tertiary foldof the hormones. Cleavage restored some binding affinity forthe IGF-I receptor in both isomers and weakly restored the abilityto stimulate incorporation of tritiated thymidine into DNA inNIH 3T3 fibroblasts transfected with the human IGF-I receptor.Cleavage also restored metabolic capacity, as measured by theability of the isomers to promote lipogenesis in isolated ratadipocytes through the insulin receptor. These results are consistentwith the theory that binding of IGF-I to the IGF-I receptorrequires a conformational change similar to that involved ininsulin binding the insulin receptor. The weak affinity forthe IGF-I receptor after cleavage is consistent with the beliefthat residues in the C-region interact with the IGF-I receptor.This structural difference between insulin and IGF-I gives eacha higher binding affinity for its own receptor.