Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

Development of a Topical Insulin Polymeric Nanoformulation for Skin Burn Regeneration: An Experimental Approach

Insulin is a peptide hormone with many physiological functions, besides its use in diabetes treatment. An important role of insulin is related to the wound healing process—however, insulin itself is too sensitive to the external environment requiring the protective of a nanocarrier. Polymer-based nanoparticles can protect, deliver, and retain the protein in the target area. This study aims to produce and characterize a topical treatment for wound healing consisting of insulin-loaded poly-DL-lactide/glycolide (PLGA) nanoparticles. Insulin-loaded nanoparticles present a mean size of approximately 500 nm and neutral surface charge. Spherical shaped nanoparticles are observed by scanning electron microscopy and confirmed by atomic force microscopy. SDS-PAGE and circular dichroism analysis demonstrated that insulin preserved its integrity and secondary structure after the encapsulation process. In vitro release studies suggested a controlled release profile. Safety of the formulation was confirmed using cell lines, and cell viability was concentration and time-dependent. Preliminary safety in vivo assays also revealed promising results.     

Cyanogenic,carotenoids and protein composition in leaves and roots across seven diverse population found in the world cassava germplasm collection at CIAT,Colombia

The objective of this study was to characterise the nutritional potential of leaves and identify a diversity centre with low cyanide and high nutrient content among 178 Latin American cassava genotypes. This field-based collection represents the seven diversity centres, held at The International Center for Tropical Agriculture (CIAT Palmira, Colombia) by the Cassava Program. The cyanide, all-trans-β-carotene and lutein concentrations in cassava leaves ranged from 346 to 7484 ppm dry basis (db), from 174–547 μg g⁻¹ db and 15–181 μg g⁻¹ db, respectively. Cassava leaves also showed significant levels of essential amino acids leucine, lysine, phenylalanine, valine and threonine, and average total protein content of 26.24 g 100 g⁻¹ db. Among seven diversity centres, South American rainforest group showed low cyanide and high carotene content in leaves. In addition, VEN77 and PAN51 genotypes stood out for having low cyanide in leaves and roots and high carotene in leaves. This genetic diversity can be used to select high potential progenitors for breeding purposes.     

A Potent,Selective CBX2 Chromodomain Ligand and Its Cellular Activity During Prostate Cancer Neuroendocrine Differentiation

Polycomb group (PcG) proteins are epigenetic regulators that facilitate both embryonic development and cancer progression. PcG proteins form Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC2 trimethylates histone H3 lysine 27 (H3K27me3), a histone mark recognized by the N-terminal chromodomain (ChD) of the CBX subunit of canonical PRC1. There are five PcG CBX paralogs in humans. CBX2 in particular is upregulated in a variety of cancers, particularly in advanced prostate cancers. Using CBX2 inhibitors to understand and target CBX2 in prostate cancer is highly desirable; however, high structural similarity among the CBX ChDs has been challenging for developing selective CBX ChD inhibitors. Here, we utilize selections of focused DNA encoded libraries (DELs) for the discovery of a selective CBX2 chromodomain probe, SW2_152F. SW2_152F binds to CBX2 ChD with a K_d of 80 nM and displays 24-1000-fold selectivity for CBX2 ChD over other CBX paralogs in vitro. SW2_152F is cell permeable, selectively inhibits CBX2 chromatin binding in cells, and blocks neuroendocrine differentiation of prostate cancer cell lines in response to androgen deprivation.     

Separation of Indium from Acid Sulfate-Containing Solutions by Ion Exchange with Impregnated Resins

Indium separation using ion exchange resins from acidic polymetallic and very diluted solutions are investigated. Since the selectivity of commercial ion exchange resins have proven to be too low for an effective separation from solutions with high content of other metals, Lewatit® TP 208 was impregnated with common extractants to enhance its properties. By resin impregnation with D2EHPA and Cyanex 272, not only the selective indium recovery was reached but also the resin capacity was increased approx. two times. The best loading and elution performance were shown by Cyanex 272-impregnated Lewatit® TP 208, increasing the indium purity in the eluate from 0.75 % to 85 %.     

Inhibition of Soluble Epoxide Hydrolase Ameliorates Phenotype and Cognitive Abilities in a Murine Model of Niemann Pick Type C Disease

Niemann–Pick type C (NPC) disease is a rare autosomal recessive inherited childhood neurodegenerative disease characterized by the accumulation of cholesterol and glycosphingolipids, involving the autophagy-lysosome system. Inhibition of soluble epoxide hydrolase (sEH), an enzyme that metabolizes epoxy fatty acids (EpFAs) to 12-diols, exerts beneficial effects in modulating inflammation and autophagy, critical features of the NPC disease. This study aims to evaluate the effects of UB-EV-52, an sEH inhibitor (sEHi), in an NPC mouse model (Npc) by administering it for 4 weeks (5 mg/kg/day). Behavioral and cognitive tests (open-field test (OF)), elevated plus maze (EPM), novel object recognition test (NORT) and object location test (OLT) demonstrated that the treatment produced an improvement in short- and long-term memory as well as in spatial memory. Furthermore, UB-EV-52 treatment increased body weight and lifespan by 25% and reduced gene expression of the inflammatory markers (i.e., Il-1β and Mcp1) and enhanced oxidative stress (OS) markers (iNOS and Hmox1) in the treated Npc mice group. As for autophagic markers, surprisingly, we found significantly reduced levels of LC3B-II/LC3B-I ratio and significantly reduced brain protein levels of lysosomal-associated membrane protein-1 (LAMP-1) in treated Npc mice group compared to untreated ones in hippocampal tissue. Lipid profile analysis showed a significant reduction of lipid storage in the liver and some slight changes in homogenated brain tissue in the treated NPC mice compared to the untreated groups. Therefore, our results suggest that pharmacological inhibition of sEH ameliorates most of the characteristic features of NPC mice, demonstrating that sEH can be considered a potential therapeutic target for this disease.     

Intergenerational Metabolomic Analysis of Mothers with a History of Gestational Diabetes Mellitus and Their Offspring

Shared metabolomic patterns at delivery have been suggested to underlie the mother-to-child transmission of adverse metabolic health. This study aimed to investigate whether mothers with gestational diabetes mellitus (GDM) and their offspring show similar metabolomic patterns several years postpartum. Targeted metabolomics (including 137 metabolites) was performed in plasma samples obtained during an oral glucose tolerance test from 48 mothers with GDM and their offspring at a cross-sectional study visit 8 years after delivery. Partial Pearson’s correlations between the area under the curve (AUC) of maternal and offspring metabolites were calculated, yielding so-called Gaussian graphical models. Spearman’s correlations were applied to investigate correlations of body mass index (BMI), Matsuda insulin sensitivity index (ISI-M), dietary intake, and physical activity between generations, and correlations of metabolite AUCs with lifestyle variables. This study revealed that BMI, ISI-M, and the AUC of six metabolites (carnitine, taurine, proline, SM(-OH) C14:1, creatinine, and PC ae C34:3) were significantly correlated between mothers and offspring several years postpartum. Intergenerational metabolite correlations were independent of shared BMI, ISI-M, age, sex, and all other metabolites. Furthermore, creatinine was correlated with physical activity in mothers. This study suggests that there is long-term metabolic programming in the offspring of mothers with GDM and informs us about targets that could be addressed by future intervention studies.     

Hydrothermal Carbonization of Sewage Sludge: Analysis of Process Severity and Solid Content

Hydrothermal carbonization of sewage sludge was carried out with the aim to evaluate the influence of process severity and initial solid content. Response surface methodology was applied to model yield and C yield responses. Enhanced dewaterability performance was recorded under mild processing conditions. The treatment promoted concentration and immobilization of Pb, Cd, Ni, Zn, and Cu. Variation of the solid content showed a stronger influence than severity on average yield and C yield. Higher heating values (HHVs) and energy retention efficiencies (EREs) of hydrochars obtained at the lowest solid content displayed the lowest values. Hence, the energy requirements of a first dewatering step should be compared with the related improvement in terms of HHV and ERE when sludge is used as feedstock.     

Stabilized β-Bi2O3 nanoparticles from (BiO)4CO3(OH)2 precursor and their photocatalytic properties under blue light

Stabilized tetragonal Bi₂O₃ nanoparticles (β-Bi₂O₃) were obtained by annealing treatments of amorphous Bi-based precursors, obtained by chemical precipitations, at temperatures between 350 and 450?°C. The formation of the stabilized β-Bi₂O₃ phase was possible by using (BiO)₄CO₃(OH)₂ while other precursors such as amorphous bismuth carbonate ((BiO)₂CO₃) and amorphous basic bismuth nitrate (Bi₆O₆(OH)₂(NO₃)₄·2H₂O) led to the formation of the thermodynamically stable monoclinic α-Bi₂O₃ and Bi₅O₇NO₃ phases. The Bi-based precursors were prepared by the chemical precipitation method at room temperature in ethylenediamine-solvent varying the HNO₃/Bi³⁺ molar ratio (10, 26 and 56). The physicochemical properties of the three as-prepared amorphous precursors and the formed-after-calcination β-Bi₂O₃, α-Bi₂O₃ and Bi₅O₇NO₃ phases were analyzed by X-ray diffraction, scanning electron microscopy, thermogravimetry, X-ray photoelectron spectroscopy (XPS), FTIR analysis, diffuse reflectance spectroscopy and surface area by BET method. The photocatalytic activity of all annealed solids containing the β-Bi₂O₃ phase was tested in the photodegradation of the indigo carmine (IC) dye under specific blue light. A schematic diagram of the Bi₂O₃ phases obtained as a function of the annealing conditions and initial amorphous precursor is proposed and explained in terms of the amount of CO₃^2-, NO₃^- and amine (ENH₂²⁺ ? ENH⁺) ions present in each bismuth precursor.