Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH₂), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.     

Effect of microfragmented adipose tissue on osteoarthritic synovial macrophage factors

Cell-based therapies using adipose-derived mesenchymal stromal cells (ADMSCs) have shown promising results for the treatment of osteoarthritis (OA). In fact, ADMSCs are now indicated as one of the most powerful cell sources through their immunomodulatory and anti-inflammatory activities. Recently, an innovative one-step closed device was developed to obtain microfragmented adipose tissue (MF) to avoid the need for good manufacturing practices for ADMSCs expansion while maintaining their regenerative potential. The aim of this study was to assess the mechanisms of action of MF and ADMSCs from MF (MF-ADMSCs) on an inflammatory cell model of OA synoviocytes. We found that MF produced low levels of inflammatory factors such as interleukin 6 (IL-6), CC-chemokine ligand 5/receptor-activated normal T-cell expressed and secreted (CCL5/RANTES), CC-chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1), and CC-chemokine ligand 3/macrophage inflammatory protein-1α (CCL3/MIP-1α), and a higher level only of CXC-chemokine ligand 8/interleukin 8 compared with MF-ADMSCs. Matrix metalloproteinase 9 (MMP-9) degradative factor but released a lower level of its inhibitor tissue inhibitor of the metalloproteinase (TIMP-1). MF in coculture with synoviocytes significantly induced both the metabolic activity and the release of IL-6. In contrast, MF, not MF-ADMSCs, partially decreased CCL5/RANTES. Moreover, MF reduced the release of both macrophage-specific chemokines (CCL2/MCP-1 and CCL3/MIP-1α) and degradative marker MMP-9. Interestingly, MF increased TIMP-1 (the MMP-9 inhibitor) and down-modulated toll-like receptor (TLR4) receptor and key molecules of NFκB pathways. These data evidenced different effects of MF versus MF-ADMSCs on inflamed synoviocytes. MF reduced typical macrophages markers and its potentiality by switching off macrophages activity was strictly dependent on TLR4 and NFκB signaling.     

The small molecule SI113 hinders epithelial-to-mesenchymal transition and subverts cytoskeletal organization in human cancer cells

The small molecule SI113 is an inhibitor of the kinase activity of SGK1, a key biological regulator acting on the PI3K/mTOR signal transduction pathway. Several studies demonstrate that this compound is able to strongly restrain cancer growth in vitro and in vivo, alone or in associative antineoplastic treatments, being able to elicit an autophagic response, either cytotoxic or cytoprotective. To elucidate more exhaustively the molecular mechanisms targeted by SI113, we performed activity-based protein profiling (ABPP) proteomic analysis using a kinase enrichment procedure. This technique allowed the identification via mass spectrometry of novel targets of this compound, most of them involved in functions concerning cell motility and cytoskeletal architecture. Using a glioblastoma multiforme, hepatocarcinoma and colorectal carcinoma cell line, we recognized an inhibitory effect of SI113 on cell migration, invading, and epithelial-to-mesenchymal transition. In addition, these cancer cells, when exposed to this compound, showed a remarkable subversion of the cytoskeletal architecture characterized by F-actin destabilization, phospho-FAK delocalization, and tubulin depolimerization. These results were definitely concordant in attributing to SI113 a key role in hindering cancer cell malignancy and, due to its negligible in vivo toxicity, can sustain performing a Phase I clinical trial to employ this drug in associative cancer therapy.     

Romina: A powerful strawberry with in vitro efficacy against uterine leiomyoma cells

Uterine leiom yomas are benign tumors highly prevalent in reproductive women. In thecurrent study, initially, we aimed to screen five different strawberry cultivars (Alba, Clery, Portola, Tecla, and Romina) to identify efficient cultivars in terms of phytochemical characterization and biological properties by measuring phenolic and anthocyanin content as well as antioxidant capacity, and by measuring apoptotic rate and reactive oxygen species (ROS) production in uterine leiomyoma cells. Next, we focused on the most efficient ones, cultivar Alba (A) and Romina (R) as well as Romina anthocyanin (RA) fraction for their ability to regulate oxidative phosphorylation (oxygen consumption rate [OCR]) glycolysis (extracellular acidification rate [ECAR]), and also fibrosis. Leiomyoma and myometrial cells were treated with a methanolic extract of A and R (250 μg/ml) or with RA (50 μg/ml) for 48 hr to measure OCR and ECAR, as well as gene expression associated with fibrosis. In the leiomyoma cells, RA was more effective in inducing apoptosis and increasing intracellular ROS levels, followed by R and A. In myometrial cells, all strawberry treatments increased the cellular viability and decreased ROS concentrations. Leiomyoma cells showed also a significant decrease in ECAR, especially after RA treatment, while OCR was slightly increased in both myometrial and leiomyoma cells. R and RA treatment significantly decreased collagen 1A1, fibronectin, versican, and activin A messenger RNA expression in leiomyoma cells. In conclusion, this study suggests that Romina, or its anthocyanin fraction, can be developed as a therapeutic and/or preventive agent for uterine leiomyomas, confirming the healthy effects exerted by these fruits and their bioactive compounds.     

Belowground community responses to fire: meta‐analysis reveals contrasting responses of soil microorganisms and mesofauna

Global fire regimes are shifting due to climate and land use changes. Understanding the responses of belowground communities to fire is key to predicting changes in the ecosystem processes they regulate. We conducted a comprehensive meta‐analysis of 1634 observations from 131 empirical studies to investigate the effect of fire on soil microorganisms and mesofauna. Fire had a strong negative effect on soil biota biomass, abundance, richness, evenness, and diversity. Fire reduced microorganism biomass and abundance by up to 96%. Bacteria were more resistant to fire than fungi. Fire reduced nematode abundance by 88% but had no significant effect on soil arthropods. Fire reduced richness, evenness and diversity of soil microorganisms and mesofauna by up to 99%. We found little evidence of temporal trends towards recovery within 10 years post‐disturbance suggesting little resilience of the soil community to fire. Interactions between biome, fire type, and depth explained few of these negative trends. Future research at the intersection of fire ecology and soil biology should aim to integrate soil community structure with the ecosystem processes they mediate under changing global fire regimes.     

BAmSA: Visualising transmembrane regions in protein complexes using biotinylated amphipols and electron microscopy

Membrane protein (MP) complexes play key roles in all living cells. Their structural characterisation is hampered by difficulties in purifying and crystallising them. Recent progress in electron microscopy (EM) have revolutionised the field, not only by providing higher-resolution structures for previously characterised MPs but also by yielding first glimpses into the structure of larger and more challenging complexes, such as bacterial secretion systems. However, the resolution of pioneering EM structures may be difficult and their interpretation requires clues regarding the overall organisation of the complexes. In this context, we present BAmSA, a new method for localising transmembrane (TM) regions in MP complexes, using a general procedure that allows tagging them without resorting to neither genetic nor chemical modification. Labels bound to TM regions can be visualised directly on raw negative-stain EM images, on class averages, or on three-dimensional reconstructions, providing a novel strategy to explore the organisation of MP complexes.     

Silk fibroin hydrogels for potential applications in photodynamic therapy

In this study, we prepared translucid hydrogels with different concentrations of silk fibroin, extracted from raw silk fibers, and used them as a matrix to incorporate the photosensitizer 5-(4-aminophenyl)-10,15,20-tris-(4-sulphonatophenyl) porphyrin trisodium for application in photodynamic therapy (PDT). The hydrogels obtained were characterized by rheology, spectrophotometry, and scattering techniques to elucidate the factors involved in the formation of the hydrogel, and to characterize the behavior of silk fibroin (SF) after incorporating of the porphyrin to the matrix. The rheology results demonstrated that the SF hydrogels had a shear thinning behavior. In addition, we were able to verify that the structure of the material was able to be recovered over time after shear deformation. The encapsulation of porphyrins in hydrogels leads to the formation of self-assembled peptide nanostructures that prevent porphyrin aggregation, thereby greatly increasing the generation of singlet oxygen. Also, our findings suggest that porphyrin can diffuse out of the hydrogel and permeate the outer skin layers. This evidence suggests that SF hydrogels could be used as porphyrin encapsulation and as a drug carrier for the sustained release of photosensitizers for PDT.     

Preclinical three-dimensional colorectal cancer model: The next generation of in vitro drug efficacy evaluation

Colorectal cancer (CRC), the third most common cancer diagnosed in both men and women in the United States, shows a highly ineffective therapeutic management. In these years neither substantial improvements nor new therapeutic approaches have been provided to patients. Performing the early lead discovery phases of new cancer drugs in cellular models, resembling as far as possible the real in vivo tumor environment, may be more effective in predicting their future success in the later clinical phases. In this review, we critically describe the most representative bioengineered models for anticancer drug screening in CRC from the conventional two-dimensional models to the new-generation three-dimensional scaffold-based ones. The scaffold aims to replace the extracellular matrix, thus influencing the biomechanical, biochemical, and biological properties of cells and tissues. In this scenario, we believe that reconstitution of tumor condition is mandatory for an alternative in vitro methods to study cancer development and therapeutic strategies.     

Priming xylem for stress recovery depends on coordinated activity of sugar metabolic pathways and changes in xylem sap pH

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar‐proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.