Replacing the Burden of the Gluten Free Diet: Then,Now, and the Future

Without a doubt, a majority of diseases are food-pattern-related. However, one disease stands out as an increasingly more common autoimmune-mediated enteropathy triggered by the ingestion of gluten. Celiac disease (CD) is an old disease, with changing clinical patterns, affecting any age, including infancy and adolescence, and becoming more frequent among the elderly. The gluten-free diet (GFD) has been the sole provider of clinical, serological, and histological improvement for patients with CD for more than seven decades. Nowadays, complete avoidance of dietary gluten is rarely possible because of the wide availability of wheat and other processed foods that contain even more gluten, to the detriment of gluten-free products. Undeniably, there is a definite need for replacing the burdensome GFD. An add-on therapy that could control the dietary transgressions and inadvertent gluten consumption that can possibly lead to overt CD should be considered while on GFD. Nevertheless, future drugs should be able to provide patients some freedom to self-manage CD and increase food independence, while actively reducing exposure and mucosal damage and alleviating GI symptoms. Numerous clinical trials assessing different molecules have already been performed with favorable outcomes, and hopefully they will soon be available for patient use.     

Insights into the Structure of the Highly Glycosylated Ffase from Rhodotorula dairenensis Enhance Its Biotechnological Potential

Rhodotorula dairenensis β-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives.     

Dual Mode of Mitochondrial ROS Action during Reprogramming to Pluripotency

Essential changes in cell metabolism and redox signaling occur during the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). In this paper, using genetic and pharmacological approaches, we have investigated the role of electron transport chain (ETC) complex-I (CI) of mitochondria in the process of cell reprogramming to pluripotency. Knockdown of NADH-ubiquinone oxidoreductase core subunits S1 (Ndufs1) or subunit B10 (Ndufb10) of the CI or inhibition of this complex with rotenone during mouse embryonic fibroblast (MEF) reprogramming resulted in a significantly decreased number of induced pluripotent stem cells (iPSCs). We have found that mitochondria and ROS levels due course of the reprogramming tightly correlate with each other, both reaching peak by day 3 and significantly declining by day 10 of the process. The transient augmentation of mitochondrial reactive oxygen species (ROS) could be attenuated by antioxidant treatment, which ameliorated overall reprogramming. However, ROS scavenging after day 3 or during the entire course of reprogramming was suppressive for iPSC formation. The ROS scavenging within the CI-deficient iPSC-precursors did not improve, but further suppressed the reprogramming. Our data therefore point to distinct modes of mitochondrial ROS action during the early versus mid and late stages of reprogramming. The data further substantiate the paradigm that balanced levels of oxidative phosphorylation have to be maintained on the route to pluripotency.     

Partial Reduction in BRCA1 Gene Dose Modulates DNA Replication Stress Level and Thereby Contributes to Sensitivity or Resistance

BRCA1 is a well-known breast cancer risk gene, involved in DNA damage repair via homologous recombination (HR) and replication fork protection. Therapy resistance was linked to loss and amplification of the BRCA1 gene causing inferior survival of breast cancer patients. Most studies have focused on the analysis of complete loss or mutations in functional domains of BRCA1. How mutations in non-functional domains contribute to resistance mechanisms remains elusive and was the focus of this study. Therefore, clones of the breast cancer cell line MCF7 with indels in BRCA1 exon 9 and 14 were generated using CRISPR/Cas9. Clones with successful introduced BRCA1 mutations were evaluated regarding their capacity to perform HR, how they handle DNA replication stress (RS), and the consequences on the sensitivity to MMC, PARP1 inhibition, and ionizing radiation. Unexpectedly, BRCA1 mutations resulted in both increased sensitivity and resistance to exogenous DNA damage, despite a reduction of HR capacity in all clones. Resistance was associated with improved DNA double-strand break repair and reduction in replication stress (RS). Lower RS was accompanied by increased activation and interaction of proteins essential for the S phase-specific DNA damage response consisting of HR proteins, FANCD2, and CHK1.     

Differences in Synovial Cytokine Profile Associated with Long-Term Clinical Outcomes in Patients with Knee Osteoarthritis Undergoing Corrective Osteotomy with Platelet-Rich Plasma or Stromal Vascular Fraction Post-Treatments

Functional outcomes and synovial fluid (SF) cytokine concentrations in response to platelet-rich plasma (PRP) or stromal vascular fraction (SVF) post-treatments following open wedge high tibial osteotomy (HTO) in 20 patients with knee osteoarthritis (OA) were examined. Six weeks after surgery, the knees of 10 patients were injected with autologous PRP (PRP subgroup), while another 10 patients were injected with autologous SVF (SVF subgroup) and monitored for 1.5 years. Pain assessment (VAS score) and functional activity (KOOS, KSS, Outerbridge, and Koshino scores) were applied. PRP subgroup performed better compared with the SVF subgroup according to KOOS, KSS, and VAS scores, while the SVF subgroup demonstrated better results according to Outerbridge and Koshino testing and produced more pronounced cartilage regeneration in the medial condyle and slowed down cartilage destruction in its lateral counterpart. SF was collected before and one week after PRP or SVF injections and tested for concentrations of 41 cytokines (Multiplex Assay). In the PRP subgroup, a significant decrease in IL-6 and CXCL10 synovial concentrations was accompanied by an increase in IL-15, sCD40L, and PDGF-AB/BB amounts. The SVF subgroup demonstrated a significant decrease in synovial TNFα, FLT-3L, MIP-1β, RANTES, and VEGF concentrations while SF concentrations of MCP-1 and FGF2 increased. Both post-treatments have a potential for increased tissue regeneration, presumably due to the downregulation of inflammation and augmentation of synovial growth factor concentrations.     

Tolerance,Adaptation, and Cell Response Elicited by Micromonospora sp. Facing Tellurite Toxicity: A Biological and Physical-Chemical Characterization

The intense use of tellurium (Te) in industrial applications, along with the improper disposal of Te-derivatives, is causing their accumulation in the environment, where oxyanion tellurite (TeO₃²⁻) is the most soluble, bioavailable, and toxic Te-species. On the other hand, tellurium is a rare metalloid element whose natural supply will end shortly with possible economic and technological effects. Thus, Te-containing waste represents the source from which Te should be recycled and recovered. Among the explored strategies, the microbial TeO₃²⁻ biotransformation into less toxic Te-species is the most appropriate concerning the circular economy. Actinomycetes are ideal candidates in environmental biotechnology. However, their exploration in TeO₃²⁻ biotransformation is scarce due to limited knowledge regarding oxyanion microbial processing. Here, this gap was filled by investigating the cell tolerance, adaptation, and response to TeO₃²⁻ of a Micromonospora strain isolated from a metal(loid)-rich environment. To this aim, an integrated biological, physical-chemical, and statistical approach combining physiological and biochemical assays with confocal or scanning electron (SEM) microscopy and Fourier-transform infrared spectroscopy in attenuated total reflectance mode (ATR-FTIR) was designed. Micromonospora cells exposed to TeO₃²⁻ under different physiological states revealed a series of striking cell responses, such as cell morphology changes, extracellular polymeric substance production, cell membrane damages and modifications, oxidative stress burst, protein aggregation and phosphorylation, and superoxide dismutase induction. These results highlight this Micromonospora strain as an asset for biotechnological purposes.     

Regulation of the Key Epithelial Cancer Suppressor miR-124 Function by Competing Endogenous RNAs

A decrease in the miR-124 expression was observed in various epithelial cancers. Like a classical suppressor, miR-124 can inhibit the translation of multiple oncogenic proteins. Epigenetic mechanisms play a significant role in the regulation of miR-124 expression and involve hypermethylation of the MIR-124-1/-2/-3 genes and the effects of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) according to the model of competing endogenous RNAs (ceRNAs). More than 40 interactomes (lncRNA/miR-124/mRNA) based on competition between lncRNAs and mRNAs for miR-124 binding have been identified in various epithelial cancers. LncRNAs MALAT1, NEAT1, HOXA11-AS, and XIST are the most represented in these axes. Fourteen axes (e.g., SND1-IT1/miR-124/COL4A1) are involved in EMT and/or metastasis. Moreover, eight axes (e.g., OIP5-AS1/miR-124-5p/IDH2) are involved in key pathways, such as Wnt/b-catenin, E2F1, TGF-β, SMAD, ERK/MAPK, HIF-1α, Notch, PI3K/Akt signaling, and cancer cell stemness. Additionally, 15 axes impaired patient survival and three axes reduced chemo- or radiosensitivity. To date, 14 cases of miR-124 regulation by circRNAs have been identified. Half of them involve circHIPK3, which belongs to the exonic ecircRNAs and stimulates cell proliferation, EMT, autophagy, angiogenesis, and multidrug resistance. Thus, miR-124 and its interacting partners may be considered promising targets for cancer therapy.     

Highly Charged Ru(II) Polypyridyl Complexes as Photosensitizer Agents in Photodynamic Therapy of Epithelial Ovarian Cancer Cells

Ovarian cancer recurrence is frequent and associated with chemoresistance, leading to extremely poor prognosis. Herein, we explored the potential anti-cancer effect of a series of highly charged Ru(II)-polypyridyl complexes as photosensitizers in photodynamic therapy (PDT), which were able to efficiently sensitize the formation of singlet oxygen upon irradiation (Ru1²⁺ and Ru2²⁺) and to produce reactive oxygen species (ROS) in their corresponding dinuclear metal complexes with the Fenton active Cu(II) ion/s ([CuRu1]⁴⁺ and [Cu₂Ru2]⁶⁺). Their cytotoxic and anti-tumor effects were evaluated on human ovarian cancer A2780 cells both in the absence or presence of photoirradiation, respectively. All the compounds tested were well tolerated under dark conditions, whereas they switched to exert anti-tumor activity following photoirradiation. The specific effect was mediated by the onset of programed cell death, but only in the case of Ru1²⁺ and Ru2²⁺ was preceded by the loss of mitochondrial membrane potential soon after photoactivation and ROS production, thus supporting the occurrence of apoptosis via type II photochemical reactions. Thus, Ru(II)-polypyridyl-based photosensitizers represent challenging tools to be further investigated in the identification of new therapeutic approaches to overcome the innate chemoresistance to platinum derivatives of some ovarian epithelial cancers and to find innovative drugs for recurrent ovarian cancer.     

Conjugates of Methylene Blue with Cycloalkaneindoles as New Multifunctional Agents for Potential Treatment of Neurodegenerative Disease

The development of multi-target-directed ligands (MTDLs) would provide effective therapy of neurodegenerative diseases (ND) with complex and nonclear pathogenesis. A promising method to create such potential drugs is combining neuroactive pharmacophoric groups acting on different biotargets involved in the pathogenesis of ND. We developed a synthetic algorithm for the conjugation of indole derivatives and methylene blue (MB), which are pharmacophoric ligands that act on the key stages of pathogenesis. We synthesized hybrid structures and performed a comprehensive screening for a specific set of biotargets participating in the pathogenesis of ND (i.e., cholinesterases, NMDA receptor, mitochondria, and microtubules assembly). The results of the screening study enabled us to find two lead compounds (4h and 4i) which effectively inhibited cholinesterases and bound to the AChE PAS, possessed antioxidant activity, and stimulated the assembly of microtubules. One of them (4i) exhibited activity as a ligand for the ifenprodil-specific site of the NMDA receptor. In addition, this lead compound was able to bypass the inhibition of complex I and prevent calcium-induced mitochondrial depolarization, suggesting a neuroprotective property that was confirmed using a cellular calcium overload model of neurodegeneration. Thus, these new MB-cycloalkaneindole conjugates constitute a promising class of compounds for the development of multitarget neuroprotective drugs which simultaneously act on several targets, thereby providing cognitive stimulating, neuroprotective, and disease-modifying effects.