High Resolution Crystal Structures of the Cerebratulus lacteus Mini-Hb in the Unligated and Carbomonoxy States

The nerve tissue mini-hemoglobin from Cerebratulus lacteus (CerHb) displays an essential globin fold hosting a protein matrix tunnel held to allow traffic of small ligands to and from the heme. CerHb heme pocket hosts the distal TyrB10/GlnE7 pair, normally linked to low rates of O(2) dissociation and ultra-high O(2) affinity. However, CerHb affinity for O(2) is similar to that of mammalian myoglobins, due to a dynamic equilibrium between high and low affinity states driven by the ability of ThrE11 to orient the TyrB10 OH group relative to the heme ligand. We present here the high resolution crystal structures of CerHb in the unligated and carbomonoxy states. Although CO binds to the heme with an orientation different from the O(2) ligand, the overall binding schemes for CO and O(2) are essentially the same, both ligands being stabilized through a network of hydrogen bonds based on TyrB10, GlnE7, and ThrE11. No dramatic protein structural changes are needed to support binding of the ligands, which can freely reach the heme distal site through the apolar tunnel. A lack of main conformational changes between the heme-unligated and -ligated states grants stability to the folded mini-Hb and is a prerequisite for fast ligand diffusion to/from the heme.     

Lactic fermentation of cooked navy beans by Lactobacillus paracasei CBA L74 aimed at a potential production of functional legume-based foods

In recent years, scientific interest in the development of non-dairy-based functional foods is increasing progressively and the fermentation of cereals, legumes, fruits and vegetable-based foods is becoming an important scientific research topic for the production of new probiotic products. In particular, legumes represent a possible alternative to protein foods from animal origins and an adequate fermentation substrate as they contain high amount of nutrients, such as proteins, carbohydrates, fibres, vitamins, and minerals, which are all useful to the growth and metabolic activity of certain microorganisms. This work focuses on the feasibility of developing a dry legume-based functional product using a fermentation process carried out on a 10% w/v navy bean suspension, in a lab-scale stirred batch reactor. After soaking and cooking dried navy beans, the fermentation tests performed on the resulting medium using Lactobacillus paracasei CBA L74 showed a maximum bacterial count of 10⁹ CFU/mL after 20 hours and a maximum lactic acid concentration of 1.9 g/L after 16 hours of process time. A freeze-drying process was performed on the fermented bean suspension, showing a 2-log microbial reduction and a bacterial viability in the resulting probiotic powder of 3.7 × 10⁸ CFU/g.     

Discovery of New Antiproliferative Imidazopyrazole Acylhydrazones Able To Interact with Microtubule Systems

Even though immunotherapy has radically changed the search for anticancer therapies, there are still many different pathways that are open to intervention with traditional small molecules. To expand our investigation in the anticancer field, we report here a new series of compounds in which our previous pyrazole and imidazopyrazole scaffolds are linked to a differently decorated phenyl ring through an acylhydrazone linker. Preliminary tests on the library were performed at the National Cancer Institute (USA) against the full NCI 60 cell panel. The best compounds among the imidazopyrazole series were then tested by immunofluorescence staining for their inhibition of cell proliferation, apoptosis induction, and their effect on the cell cycle and on microtubules. Two compounds, in particular 4-benzyloxy-3-methoxybenzyliden imidazopyrazole-7-carbohydrazide showed good growth inhibition, with IC₅₀ values in the low-micromolar range, and induced apoptosis. Both compounds altered the cell-cycle phases with the appearance of polyploid cells. Immunofluorescence analysis evidenced microtubules alterations; tubulin polymerization assays and docking studies suggested the tubulin system to be the possible, although not exclusive, target of the new acylhydrazone series reported here.     

The Anti-Inflammatory Properties of Mesenchymal Stem Cells in Epilepsy: Possible Treatments and Future Perspectives

Mesenchymal stem cells (MSCs) are multipotent adult cells with self-renewing capacities. MSCs display specific properties, such as the ability to repair damaged tissues, resulting in optimal candidates for cell therapy against degenerative diseases. In addition to the reparative functions of MSCs, growing evidence shows that these cells have potent immunomodulatory and anti-inflammatory properties. Therefore, MSCs are potential tools for treating inflammation-related neurological diseases, including epilepsy. In this regard, over the last decades, epilepsy has no longer been considered a purely neuronal pathology, since inflammatory events underlying the genesis of epilepsy have been demonstrated. This review assessed current knowledge on the use of MSCs in the treatment of epilepsy. Mostly, attention will be focused on the anti-inflammatory and immunological skills of MSCs. Understanding the mechanisms by which MSCs might modulate the severity of the disease will contribute to the development of new potential alternatives for both prophylaxis and treatment against epilepsy.     

NMR Profiling of Exhaled Breath Condensate Defines Different Metabolic Phenotypes of Non-Cystic Fibrosis Bronchiectasis

Nuclear-magnetic-resonance (NMR) profiling of exhaled breath condensate (EBC) provides insights into the pathophysiology of bronchiectasis by identifying specific biomarkers. We evaluated whether NMR-based metabolomics discriminates the EBC-derived metabolic phenotypes (“metabotypes”) of 41 patients with non-cystic fibrosis (nCF) bronchiectasis of various etiology [24 subjects with Primary Ciliary Dyskinesia (PCD); 17 patients with bronchiectasis not associated with PCD (nCF/nPCD)], who were compared to 17 healthy subjects (HS). NMR was used for EBC profiling, and Orthogonal Projections to Latent Structures with partial least-squares discriminant analysis (OPLS-DA) was used as a classifier. The results were validated by using the EBC from 17 PCD patients not included in the primary analysis. Different statistical models were built, which compared nCF/nPCD and HS, PCD and HS, all classes (nCF/nPCD-PCD-HS), and, finally, PCD and nCF/nPCD. In the PCD-nCF/nPCD model, four statistically significant metabolites were able to discriminate between the two groups, with only a minor reduction of the quality parameters. In particular, for nCF/nPCD, acetone/acetoin and methanol increased by 21% and 18%, respectively. In PCD patients, ethanol and lactate increased by 25% and 28%, respectively. They are all related to lung inflammation as methanol is found in the exhaled breath of lung cancer patients, acetone/acetoin produce toxic ROS that damage lung tissue in CF, and lactate is observed in acute inflammation. Interestingly, a high concentration of ethanol hampers cilia beating and can be associated with the genetic defect of PCD. Model validation with 17 PCD samples not included in the primary analysis correctly predicted all samples. Our results indicate that NMR of EBC discriminates nCF/nPCD and PCD bronchiectasis patients from HS, and patients with nCF/nPCD from those with PCD. The metabolites responsible for between-group separation identified specific metabotypes, which characterize bronchiectasis of a different etiology.     

Unpatterned Bioactive Poly(Butylene 1,4-Cyclohexanedicarboxylate)-Based Film Fast Induced Neuronal-Like Differentiation of Human Bone Marrow-Mesenchymal Stem Cells

Herein, we present poly(butylene 1,4-cyclohexanedicarboxylate) (PBCE) films characterized by an unpatterned microstructure and a specific hydrophobicity, capable of boosting a drastic cytoskeleton architecture remodeling, culminating with the neuronal-like differentiation of human bone marrow-mesenchymal stem cells (hBM-MSCs). We have used two different filming procedures to prepare the films, solvent casting (PBCE) and compression-moulding (PBCE*). PBCE film had a rough and porous surface with spherulite-like aggregations (Ø = 10–20 μm) and was characterized by a water contact angle = 100°. PBCE* showed a smooth and continuous surface without voids and visible spherulite-like aggregations and was more hydrophobic (WCA = 110°). Both surface characteristics were modulated through the copolymerization of different amounts of ether-oxygen-containing co-units into PBCE chemical structure. We showed that only the surface characteristics of PBCE-solvent-casted films steered hBM-MSCs toward a neuronal-like differentiation. hBM-MSCs lost their canonical mesenchymal morphology, acquired a neuronal polarized shape with a long cell protrusion (≥150 μm), expressed neuron-specific class III β-tubulin and microtubule-associated protein 2 neuronal markers, while nestin, a marker of uncommitted stem cells, was drastically silenced. These events were observed as early as 2-days after cell seeding. Of note, the phenomenon was totally absent on PBCE* film, as hBM-MSCs maintained the mesenchymal shape and behavior and did not express neuronal/glial markers.     

Dufour Gland Contents of Ants of the Cataglyphis bicolor Group

The species of desert-dwelling ants of the Cataglyphis bicolor (Hymenoptera: Formicidae) group are difficult to distinguish by morphological features. Analysis of the secretion from the Dufour glands of workers of a number of colonies was undertaken to see if it provided a clear test of species. Linked 6c-ms showed in all samples straight and branched-chain alkanes, linear alkenes, ketones, aldehydes, acetates, and a group of C₂₂ to C₂₈ esters not previously identified in this genus. Contents of the Dufour glands of C. savignyi from Tunisia and Egypt were similar, and comprised straight and branched-chain alkanes, alkenes and small amounts of esters. C. bicolor from Tunisia contained compounds similar to C. savignyi but was distinguished from the latter by larger amounts of the esters. The major compound in the glands of C. viaticus was tridecane, in contrast to the pentadecane of other species. It also contained a branched alkane, 3-methyltridecane as a major component. Branched-chain esters and a wide variety of acetates were also found in this species. C. diehlii had a limited range of compounds, with branched alkanes almost completely absent and high proportions of pentadecene and dodecyl acetate. C. bombycinus, a sympatric species, but recognized as not belonging to the bicolor group by its different mandibular gland substances, was notable in having butanoate esters in its Dufour glands. Despite these differences among species, both the great variability of individuals from a single colony and the among between conspecific colonies make species diagnosis from a few individuals difficult, in contrast with postpharyngeal glands, which, as recently reported, give a clearer indication of species.     

Pharmacogenetics Biomarkers and Their Specific Role in Neoadjuvant Chemoradiotherapy Treatments: An Exploratory Study on Rectal Cancer Patients

Background: Pathological complete response (pCR) to neoadjuvant chemoradiotherapy (CRT) in locally advanced rectal cancer (LARC) is still ascribed to a minority of patients. A pathway based-approach could highlight the predictive role of germline single nucleotide polymorphisms (SNPs). The primary aim of this study was to define new predictive biomarkers considering treatment specificities. Secondary aim was to determine new potential predictive biomarkers independent from radiotherapy (RT) dosage and cotreatment with oxaliplatin. Methods: Thirty germ-line SNPs in twenty-one genes were selected according to a pathway-based approach. Genetic analyses were performed on 280 LARC patients who underwent fluoropyrimidine-based CRT. The potential predictive role of these SNPs in determining pathological tumor response was tested in Group 1 (94 patients undergoing also oxaliplatin), Group 2 (73 patients treated with high RT dosage), Group 3 (113 patients treated with standard RT dosage), and in the pooled population (280 patients). Results: Nine new predictive biomarkers were identified in the three groups. The most promising one was rs3136228-MSH6 (p = 0.004) arising from Group 3. In the pooled population, rs1801133-MTHFR showed only a trend (p = 0.073). Conclusion: This exploratory study highlighted new potential predictive biomarkers of neoadjuvant CRT and underlined the importance to strictly define treatment peculiarities in pharmacogenetic analyses.     

Co-Expression and Co-Localization of Cartilage Glycoproteins CHI3L1 and Lubricin in Osteoarthritic Cartilage: Morphological,Immunohistochemical and Gene Expression Profiles

Osteoarthritis is the most common human arthritis characterized by degeneration of articular cartilage. Several studies reported that levels of human cartilage glycoprotein chitinase 3-like-1 (CHI3L1) are known as a potential marker for the activation of chondrocytes and the progression of Osteoarthritis (OA), whereas lubricin appears to be chondroprotective. The aim of this study was to investigate the co-expression and co-localization of CHI3L1 and lubricin in normal and osteoarthritic rat articular cartilage to correlate their modified expression to a specific grade of OA. Samples of normal and osteoarthritic rat articular cartilage were analyzed by the Kellgren–Lawrence OA severity scores, the Kraus’ modified Mankin score and the Histopathology Osteoarthritis Research Society International (OARSI) system for histomorphometric evaluations, and through CHI3L1 and lubricin gene expression, immunohistochemistry and double immuno-staining analysis. The immunoexpression and the mRNA levels of lubricin increased in normal cartilage and decreased in OA cartilage (normal vs. OA, p < 0.01). By contrast, the immunoexpression and the mRNA levels of CHI3L1 increased in OA cartilage and decreased in normal cartilage (normal vs. OA, p < 0.01). Our findings are consistent with reports suggesting that these two glycoproteins are functionally associated with the development of OA and in particular with grade 2/3 of OA, suggesting that in the future they could be helpful to stage the severity and progression of the disease.