Cytoprotective Processes Research Articles

Proteostasis Decline and Redox Imbalance in Age-Related Diseases: The Therapeutic Potential of NRF2

Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of cellular homeostasis, overseeing the expression of a wide array of genes involved in cytoprotective processes such as antioxidant and proteostasis control, mitochondrial function, inflammation, and the metabolism of lipids and glucose. The accumulation of misfolded proteins triggers the release, stabilization, and nuclear translocation of NRF2, which in turn enhances the expression of critical components of both the proteasomal and lysosomal degradation pathways. This process facilitates the clearance of toxic protein aggregates, thereby actively maintaining cellular proteostasis. As we age, the efficiency of the NRF2 pathway declines due to several factors including increased activity of its repressors, impaired NRF2-mediated antioxidant and cytoprotective gene expression, and potential epigenetic changes, though the precise mechanisms remain unclear. This leads to diminished antioxidant defenses, increased oxidative damage, and exacerbated metabolic dysregulation and inflammation—key contributors to age-related diseases. Given NRF2’s role in mitigating proteotoxic stress, the pharmacological modulation of NRF2 has emerged as a promising therapeutic strategy, even in aged preclinical models. By inducing NRF2, it is possible to mitigate the damaging effects of oxidative stress, metabolic dysfunction, and inflammation, thus reducing protein misfolding. The review highlights NRF2’s therapeutic implications for neurodegenerative diseases and cardiovascular conditions, emphasizing its role in improving proteostasis and redox homeostasis Additionally, it summarizes current research into NRF2 as a therapeutic target, offering hope for innovative treatments to counteract the effects of aging and associated diseases.

Therapeutic applications of local injection of hsa-miR-634 into canine spontaneous malignant melanoma tumors.

Malignant melanoma (MM) is one of the most common tumors in both dogs and humans. As canine MM (CMM) and human MM (HMM) have similar clinical characteristics, CMM appears to be a good clinical model for HMM. We previously demonstrated that the introduction of a synthetic double-strand-microRNA-634 (miR-634) mimic triggered apoptotic cell death by directly targeting the genes associated with cytoprotective processes in various human cancer cell lines, including those of HMM. This study aimed to investigate the antitumor effects of the local administration of miR-634 on spontaneous CMMs to provide a basis for future applications of miR-634 formulations in HMM treatment. We found that miR-634 administration induced apoptosis in CMM cell lines in vitro via downregulation of Asct2, Nrf2, and survivin expression, similar to the mechanisms in HMM cell lines. Furthermore, intratumoral miR-634 administration induced antitumor effects in four of seven spontaneous CMM cases, with no adverse effects. Local administration of miR-634 to lung metastasis under ultrasound guidance induced tumor shrinkage. These results confirm the antitumor effect of the local administration of miR-634 in spontaneous CMM, a model for spontaneous HMM, thereby providing a novel treatment strategy for HMM.

Flavonoids as new regulators of mitochondrial potassium channels: contribution to cardioprotection.

Acute myocardial ischemia is one of the major causes of illness in western society. Reduced coronary blood supply leads to cell death and loss of cardiomyocyte population, resulting in serious and often irreversible consequences on myocardial function. Mitochondrial potassium (mitoK) channels have been identified as fine regulators of mitochondrial function and, consequently, in the metabolism of the whole cell, and in the mechanisms underlying the cardioprotection. Interestingly, mitoK channels represent a novel putative target for treating cardiovascular diseases, particularly myocardial infarction, and their modulators represent an interesting tool for pharmacological intervention. In this review, we took up the challenge of selecting flavonoids that show cardioprotective properties through the activation of mitoK channels. A brief overview of the main information on mitoK channels and their participation in the induction of cytoprotective processes was provided. Then, naringenin, quercetin, morin, theaflavin, baicalein, epigallocatechin gallate, genistein, puerarin, luteolin and proanthocyanidins demonstrated to be effective modulators of mitoK channels activity, mediating many beneficial effects. The pathophysiological role of mitoK channels has been investigated as well as the impact of flavonoids on this target with particular attention to their potential role in the prevention of cardiovascular disorders.

The Role of Gasotransmitter-Dependent Signaling Mechanisms in Apoptotic Cell Death in Cardiovascular, Rheumatic, Kidney, and Neurodegenerative Diseases and Mental Disorders.

Cardiovascular, rheumatic, kidney, and neurodegenerative diseases and mental disorders are a common cause of deterioration in the quality of life up to severe disability and death worldwide. Many pathological conditions, including this group of diseases, are based on increased cell death through apoptosis. It is known that this process is associated with signaling pathways controlled by a group of gaseous signaling molecules called gasotransmitters. They are unique messengers that can control the process of apoptosis at different stages of its implementation. However, their role in the regulation of apoptotic signaling in these pathological conditions is often controversial and not completely clear. This review analyzes the role of nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H2S), and sulfur dioxide (SO2) in apoptotic cell death in cardiovascular, rheumatic, kidney, and neurodegenerative diseases. The signaling processes involved in apoptosis in schizophrenia, bipolar, depressive, and anxiety disorders are also considered. The role of gasotransmitters in apoptosis in these diseases is largely determined by cell specificity and concentration. NO has the greatest dualism; scales are more prone to apoptosis. At the same time, CO, H2S, and SO2 are more involved in cytoprotective processes.

Advanced Bioinformatics Analysis and Genetic Technologies for Targeting Autophagy in Glioblastoma Multiforme.

As the most malignant primary brain tumor in adults, a diagnosis of glioblastoma multiforme (GBM) continues to carry a poor prognosis. GBM is characterized by cytoprotective homeostatic processes such as the activation of autophagy, capability to confer therapeutic resistance, evasion of apoptosis, and survival strategy even in the hypoxic and nutrient-deprived tumor microenvironment. The current gold standard of therapy, which involves radiotherapy and concomitant and adjuvant chemotherapy with temozolomide (TMZ), has been a game-changer for patients with GBM, relatively improving both overall survival (OS) and progression-free survival (PFS); however, TMZ is now well-known to upregulate undesirable cytoprotective autophagy, limiting its therapeutic efficacy for induction of apoptosis in GBM cells. The identification of targets utilizing bioinformatics-driven approaches, advancement of modern molecular biology technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas9) or CRISPR-Cas9 genome editing, and usage of microRNA (miRNA)-mediated regulation of gene expression led to the selection of many novel targets for new therapeutic development and the creation of promising combination therapies. This review explores the current state of advanced bioinformatics analysis and genetic technologies and their utilization for synergistic combination with TMZ in the context of inhibition of autophagy for controlling the growth of GBM.

A New Phase of Networking: The Molecular Composition and Regulatory Dynamics of Mammalian Stress Granules.

Stress granules (SGs) are cytosolic biomolecular condensates that form in response to cellular stress. Weak, multivalent interactions between their protein and RNA constituents drive their rapid, dynamic assembly through phase separation coupled to percolation. Though a consensus model of SG function has yet to be determined, their perceived implication in cytoprotective processes (e.g., antiviral responses and inhibition of apoptosis) and possible role in the pathogenesis of various neurodegenerative diseases (e.g., amyotrophic lateral sclerosis and frontotemporal dementia) have drawn great interest. Consequently, new studies using numerous cell biological, genetic, and proteomic methods have been performed to unravel the mechanisms underlying SG formation, organization, and function and, with them, a more clearly defined SG proteome. Here, we provide a consensus SG proteome through literature curation and an update of the user-friendly database RNAgranuleDB to version 2.0 (http://rnagranuledb.lunenfeld.ca/). With this updated SG proteome, we use next-generation phase separation prediction tools to assess the predisposition of SG proteins for phase separation and aggregation. Next, we analyze the primary sequence features of intrinsically disordered regions (IDRs) within SG-resident proteins. Finally, we review the protein- and RNA-level determinants, including post-translational modifications (PTMs), that regulate SG composition and assembly/disassembly dynamics.

Potential Effect of Medicinal Plants on the Prevention of Gastric Ulcer: Mechanism of Actions

Medicinal plants have been widely studied to identify plant-based anti-gastric ulcer medicines. The mechanism of gastroprotective action is important to discover the potential lead compounds for drug development. All relevant articles between 2011 and 2021 focusing on Malaysian plants were collected and analyzed to understand the underlying pathways. Keywords include peptic ulcer, gastric ulcer, NSAIDs (Non-Steroidal Anti-Inflammatory Drugs), Helicobacterpylori, medicinal plant, gastroprotection, antiulcer, acid secretion, cytoprotective, and digestion processes were applied in the search engines. Twenty-two of the plants had been reported based on the collected data. The review concludes that Malaysian plants could protect the gastric wall against necrotizing agents like ethanol and NSAIDs. This is mainly due to four critical defensive mechanisms: cytoprotective barriers, regulation of heat-shock protein 70 (HSP70) and pro-apoptotic protein (BAX), gastric acid secretion, and antioxidant capability. The mechanisms have been illustrated in the schematic diagrams for better understanding.

Potential for reversing miR-634-mediated cytoprotective processes to improve efficacy of chemotherapy against oral squamous cell carcinoma

For advanced oral squamous cell carcinoma (OSCC), increasing sensitivity to chemotherapy is a major challenge in improving treatment outcomes, and targeting cytoprotective processes that lead to the chemotherapy resistance of cancer cells may be therapeutically promising. Tumor-suppressive microRNAs (miRNAs) can target multiple cancer-promoting genes concurrently and are thus expected to be useful seeds for cancer therapeutics. We revealed that miR-634-mediated targeting of multiple cytoprotective process-related genes, including cellular inhibitor of apoptosis protein 1 (cIAP1), can effectively increase cisplatin (CDDP)-induced cytotoxicity and overcome CDDP resistance in OSCC cells. The combination of topical treatment with miR-634 ointment and administration of CDDP was synergistically effective against OSCC tumor growth in a xenograft mouse model. Furthermore, the expression of miR-634 target genes is frequently upregulated in primary OSCC tumors. Our study suggests that reversing miR-634-mediated cytoprotective processes activated in cancer cells is a potentially useful strategy to improve CDDP efficacy against advanced OSCC.

Citrate-capped gold nanoparticles with a diameter of 14 nm alter the expression of genes associated with stress response, cytoprotection and lipid metabolism in CaCo-2 cells

Advancements in nanotechnology have provided insight into the unique opportunities for the application of nanomaterials such as gold nanoparticles (AuNPs) in medicine due to their remarkable properties, which includes low toxicity, large surface area, and the ease of synthesis and conjugation to other molecules. Therefore, AuNPs are often preferred for bio-applications. Citrate-capped AuNPs (cAuNPs) have been reported to be non-cytotoxic and are used in numerous studies as drug delivery vehicles to treat various diseases. However, the limitations of bioassays often used to assess the toxicity of AuNPs have been well documented. Herein, we investigate the cytotoxicity of 14 nm cAuNPs in the human colorectal adenocarcinoma (Caco-2) cell line. Treatment conditions (i.e. dose and exposure time) that were established to be non-toxic to Caco-2 cells were used to investigate the effect of cAuNPs on the expression of a Qiagen panel of 86 genes involved in cytotoxicity. Out of 86 studied, 23 genes were differentially expressed. Genes involved in oxidative stress and antioxidant response, endoplasmic reticulum (ER) stress and unfolded protein response, heat shock response, and lipid metabolism were more affected than others. While low concentrations of 14 nm cAuNPs was not cytotoxic and did not cause cell death, cells treated with these nanoparticles experienced ER and oxidative stress, resulting in the activation of cytoprotective cellular processes. Additionally, several genes involved in lipid metabolism were also affected. Therefore, 14 nm cAuNPs can safely be used as drug delivery vehicles at low doses.

A FRET-based ratiometric fluorescent probe for hydrogen polysulfide detection in living cells and zebrafish

Hydrogen polysulfide (H2Sn, n > 1) is an important active sulfur molecule (RSS) in organisms, which have been considered to be involved in redox signaling and cytoprotective processes. In this work, in order to quickly and accurately detect H2Sn in biosystems, 2-fluoro-5-nitrobenzoic ester was used as the response moiety for H2Sn, and the FRET strategy was adopted to effectively connect the donor (6-hydroxy-2-naphthoic acid) and acceptor (4-substituted-1,8-naphthalimide) to construct a new ratiometric H2Sn fluorescent probe NPNA-H2Sn. NPNA-H2Sn exhibited a more than ∼ 8.0-fold ratio enhancement towards H2Sn at I450/I526 and a very high sensitivity with a very low detection limit of 40.3 nM. Impressive, NPNA-H2Sn was further used for fluorescence imaging of H2Sn in living cells and zebrafish, which showed high-clear ratiometric images. Therefore, we have demonstrated that NPNA-H2Sn could be applied for ratiometric images of endogenous H2Sn in living biosystems and provide a powerful molecular tool for evaluating the physiological and pathological functions of H2Sn.

Photoactivated Self-Disassembly of Multifunctional DNA Nanoflower Enables Amplified Autophagy Suppression for Low-Dose Photodynamic Therapy.

Low-dose photodynamic therapy (PDT) holds great promise for reducing undesired patient photosensitivity in cancer treatment. Yet, its therapeutic effect is significantly affected by intracellular cytoprotective processes, such as autophagy. Here, an efficient autophagy suppressor is developed, which is a multifunctional DNA nanoflower (DNF) consisted of tumor-targeting aptamers and DNAzymes for silencing autophagy-related genes, with surface modification of low-dose photosensitizer (Ce6). It is found that the multifunctional DNF can specifically target tumor cells and generate reactive oxygen species (ROS) under light irradiation to trigger self-disassembly of DNF, enhancing the bioavailability of encoded DNAzymes, leading to amplified autophagy suppression. As a facile spatiotemporally programmable photogene therapy platform, the designed DNF is able to suppress tumor growth in vivo with a very low injection dose of Ce6 (18µg kg-1 , around 100 times lower than the generally applied dose), representing a promising strategy for cancer therapy with safely low-dose PDT.

Molecular dynamics, quantum mechanics and docking studies of some Keap1 inhibitors – An insight into the atomistic mechanisms of their antioxidant potential

Inhibitors of Keap1 would disrupt the covalent interaction between Keap1 and Nrf2 to unleash Nrf2 transcriptional machinery that orchestrates its cellular antioxidant, cytoprotective and detoxification processes thereby, protecting the cells against oxidative stress mediated diseases. In this in silico research, we investigated the Keap1 inhibiting potential of fifty (50) antioxidants using pharmacokinetic ADMET profiling, bioactivity assessment, physicochemical studies, molecular docking investigation, molecular dynamics and Quantum mechanical-based Density Functional Theory (DFT) studies using Keap1 as the apoprotein control. Out of these 50 antioxidants, Maslinic acid (MASA), 18-alpha-glycyrrhetinic acid (18-AGA) and resveratrol stand out by passing the RO5 (Lipinski rule of 5) for the physicochemical properties and ADMET studies. These three compounds also show high binding affinity of -10.6 kJ/mol, -10.4 kJ/mol and -7.8 kJ/mol at the kelch pocket of Keap1 respectively. Analysis of the 20ns trajectories using RMSD, RMSF, ROG and h-bond parameters revealed the stability of these compounds after comparing them with Keap1 apoprotein. Furthermore, the electron donating and accepting potentials of these compounds was used to investigate their reactivity using Density Functional Theory (HOMO and LUMO) and it was revealed that resveratrol had the highest stability based on its low energy gap. Our results predict that the three compounds are potential drug candidates with domiciled therapeutic functions against oxidative stress-mediated diseases. However, resveratrol stands out as the compound with the best stability and therefore, could be the best candidate with the best therapeutic efficacy.

Augmentation of lenvatinib efficacy by topical treatment of miR-634 ointment in anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is one of the most lethal types of human tumors. Lenvatinib can improve the disease control and prognosis in patients with ATC. However, there is an unmet need to develop a therapeutically safer and non-invasive strategy that improves the efficacy of lenvatinib for advanced ATC tumors, which grow larger close to the skin. We previously demonstrated that the topical application of an ointment incorporating tumor suppressive microRNA (TS-miR), miR-634, is a useful strategy as a TS-miR therapeutics. Here, we found that the overexpression of miR-634 synergistically increased lenvatinib-induced cytotoxicity by concurrently downregulating multiple genes related to cytoprotective processes, including ASCT2, a glutamine transporter, in ATC cell lines. Furthermore, the topical application of a miR-634 ointment on subcutaneous tumors effectively augmented the anti-tumor effects of lenvatinib in an ATC xenograft mouse model. Thus, we propose topical treatment of a miR-634 ointment as a rational strategy for improving lenvatinib-based therapy for ATC.

Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons.

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) through the activation of specialized sensors. UPR attempts first to restore homeostasis; but if damage persists the signaling induces apoptosis. There is increasing evidence that sustained and unresolved ER stress contributes to many pathological conditions including neurodegenerative diseases. Because the UPR controls cell fate by switching between cytoprotective and apoptotic processes, it is essential to understand the events defining this transition, as well as the elements involved in its modulation. Recently, we demonstrated that abnormal GM2 ganglioside accumulation causes depletion of ER Ca2+ content, which in turn activates PERK (PKR-like-ER kinase), one of the UPR sensors. Furthermore, PERK signaling participates in the neurite atrophy and apoptosis induced by GM2 accumulation. In this respect, we have established an experimental system that allows us to molecularly modulate the expression of downstream PERK components and thus change vulnerability of neurons to undergo neuritic atrophy. We performed knockdown of calcineurin (cytoprotective) and CHOP (pro-apoptotic) expression in rat cortical neuronal cultures. Cells were infected with lentivirus-delivered specific shRNA and then treated with GM2 at different times, fixed and immunostained with anti-MAP2 (microtube-associated protein 2) antibody. Later, cell images were recorded using a fluorescence microscope and total neurite outgrowth was evaluated by using the public domain image processing software ImageJ. The inhibition of expression of those PERK signaling components clearly made it possible to either accelerate or delay the neuritic atrophy induced by ER stress. This approach might be used in cell system models of ER stress to evaluate the vulnerability of neurons to neurite atrophy.

Dependence of the Viability of Ras-Expressing Cells on Damage to Mitochondria Caused by Antitumor Agents

Tumors with ocogenic Ras protein expression are hard to cure, since they are able to restore viability after treatment with antitumor agents by activating cytoprotective processes. The search for new targets for more effective therapy has shown that mitochondria can serve as such a target, since Ras-expressing cells receive ATP primarily through oxidative phosphorylation, rather than glycolysis, like many other types of tumor cells. The study investigated the death of tumor cells expressing oncogenic Ras after X-ray irradiation in comparison with inhibitor of mitochondrial integrity ABT199 inhibitor (venetoclax) of mitochondrial integrity. It was shown that cells show different response in time to the action of damaging agents. In the early stages (2–24 h after of treatment), autophagy is activated, which eliminates damaged mitochondria and increases cell viability, while the appearance of senescent cells after 72 h is part of a late response that suppresses proliferation. Inhibition of autophagy and senescence increases cell death under the action of damaging agents, which indicates that these processes play a key role in inhibition of cell death after mitochondrial damage. Thus, inhibition of autophagy and senescence can increase the efficiency of tumor cell elimination by antitumor agents.

Improving the Efficacy of EGFR Inhibitors by Topical Treatment of Cutaneous Squamous Cell Carcinoma with miR-634 Ointment

For cutaneous squamous cell carcinoma (cSCC), topical treatment is an essential option for patients who are not candidates for, or who refuse, surgery. Epidermal growth factor receptor (EGFR) plays a key role in the development of cSCC, but EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib, have shown only partial clinical benefit in this disease. Thus, there is an unmet need to develop novel strategies for improving the efficacy of TKIs in cSCC. We previously demonstrated that the tumor-suppressive microRNA (miRNA) miR-634 functions as a negative modulator of the cytoprotective cancer cell survival processes and is a useful anticancer therapeutic agent. In the present study, we found that topical application of an ointment containing miR-634 inhibited in vivo tumor growth without toxicity in a cSCC xenograft mouse model and a 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced papilloma mouse model. Functional validation revealed that miR-634 overexpression reduced glutaminolysis by directly targeting ASCT2, a glutamine transporter. Furthermore, overexpression of miR-634 synergistically enhanced TKI-induced cytotoxicity by triggering severe energetic stress in vitro and in vivo. Thus, we propose that topical treatment with miR-634 ointment is a useful strategy for improving for EGFR TKI-based therapy for cSCC.

Pro- and anti-inflammatory responses of common carp Cyprinus carpio head kidney leukocytes to E.coli LPS as modified by different dietary plant oils

Dietary lipids could modify fatty acid (FA) composition in fish tissues. Long chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic acid (ARA), eicosapentaneoic acid (EPA) and docosahexaenoic acid (DHA) are able to modulate the immune status in fish through an inflammatory process but their availability may be limited when fish are exclusively fed plant oils. This study was conducted to evaluate how to maximise the utilisation of dietary plant oil for an efficient inflammatory response in common carp head kidney leukocytes (HKLs) exposed to a gram-negative bacterial endotoxin, Escherichia coli lipopolysaccharides (LPS). HKLs were isolated from fish fed cod liver oil (CLO), linseed oil (LO), sesame oil (SO) a blend of SO and LO (SLO, v:v 1:1), and these plant oil diets supplemented with DHA (SO + DHA, SOD) or ARA (LO + ARA, LOA) for 6 weeks. Cells were then exposed to LPS at a dose of 10 μg/mL for 4 and 24 h. Peroxidase activity, total Ig, and NO levels were measured in the culture medium, while cells were used for expression analyses of candidate genes in pattern recognition (tlr-4), eicosanoid metabolism (pge2, 5-lox), pro-inflammatory (il-1, il-6, il-8, tnf-α, nf-kb, inos, cxc), anti-inflammatory (il-10, nf-kbi, tgf-β1) responses, and cytoprotective (gpx-1, prdx-3) processes. Results showed that LPS induced significantly inflammatory responses, evidenced by a high level of almost all the targeted humoral immune parameters and/or gene expression. Expression of inflammatory cytokines and other inflammatory mediators was upregulated after 4 h-LPS exposure and reverted to basal levels after 24 h. HKLs from fish fed SLO, LOA, or SOD diet exhibited a more efficient regulation of acute inflammatory processes than those fed CLO diet. The results indicate that the immune competence of fish fed plant oil mixture was comparable to the one of fish fed fish oil diet. Moreover, the supplementation of ARA or DHA induced similar immunomodulation in common carp.

Abnormalities in metabolic pathways in celiac disease investigated by the metabolic profiling of small intestinal mucosa, blood plasma and urine by NMR spectroscopy.

Celiac disease (CeD) is an autoimmune enteropathy caused by gluten intake in genetically predisposed individuals. We investigated the metabolism of CeD by metabolic profiling of intestinal mucosa, blood plasma and urine using NMR spectroscopy and multivariate analysis. The metabolic profile of the small intestinal mucosa was compared between patients with CeD (n = 64) and disease controls (DCs, n = 30). The blood plasma and urinary metabolomes of CeD patients were compared with healthy controls (HCs, n = 39). Twelve metabolites (proline (Pro), arginine (Arg), glycine (Gly), histidine (His), glutamate (Glu), aspartate, tryptophan (Trp), fumarate, formate, succinate (Succ), glycerophosphocholine (GPC) and allantoin (Alln)) of intestinal mucosa differentiated CeD from controls. The metabolome of blood plasma with 18 metabolites (Pro, Arg, Gly, alanine, Glu, glutamine, glucose (Glc), lactate (Lac), acetate (Ace), acetoacetate (AcAc), β-hydroxybutyrate (β-OHB), pyruvate (Pyr), Succ, citrate (Cit), choline (Cho), creatine (Cr), phosphocreatine (PCr) and creatinine) and 9 metabolites of urine (Pro, Trp, β-OHB, Pyr, Succ, N-methylnicotinamide (NMN), aminohippurate (AHA), indoxyl sulfate (IS) and Alln) distinguished CeD from HCs. Our data demonstrated changes in nine metabolic pathways. The altered metabolites were associated with increased oxidative stress (Alln), impaired healing and repair mechanisms (Pro, Arg), compromised anti-inflammatory and cytoprotective processes (Gly, His, NMN), altered energy metabolism (Glc, Lac, β-OHB, Ace, AcAc, Pyr, Succ, Cit, Cho, Cr and PCr), impaired membrane metabolism (GPC and Cho) and intestinal dysbiosis (AHA and IS). An orthogonal partial least square discriminant analysis model provided clear differentiation between patients with CeD and controls in all three specimens. A classification model built by combining the distinguishing metabolites of blood plasma and urine samples gave an AUC of 0.99 with 97.7% sensitivity, 93.3% specificity and a predictive accuracy of 95.1%, which was higher than for the models built separately using small intestinal mucosa, blood plasma and urine. In conclusion, a panel of metabolic biomarkers in intestinal biopsies, plasma and urine samples has potential to differentiate CeD from controls and may complement traditional tests to improve the diagnosis of CeD.

Manganese Porphyrin-Based SOD Mimetics Produce Polysulfides from Hydrogen Sulfide.

Manganese-centered porphyrins (MnPs), MnTE-2-PyP5+ (MnTE), MnTnHex-2-PyP5+ (MnTnHex), and MnTnBuOE-2-PyP5+ (MnTnBuOE) have received considerable attention because of their ability to serve as superoxide dismutase (SOD) mimetics thereby producing hydrogen peroxide (H2O2), and oxidants of ascorbate and simple aminothiols or protein thiols. MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+ are now in five Phase II clinical trials warranting further exploration of their rich redox-based biology. Previously, we reported that SOD is also a sulfide oxidase catalyzing the oxidation of hydrogen sulfide (H2S) to hydrogen persulfide (H2S2) and longer-chain polysulfides (H2Sn, n = 3–7). We hypothesized that MnPs may have similar actions on sulfide metabolism. H2S and polysulfides were monitored in fluorimetric assays with 7-azido-4-methylcoumarin (AzMC) and 3′,6′-di(O-thiosalicyl)fluorescein (SSP4), respectively, and specific polysulfides were further identified by mass spectrometry. MnPs concentration-dependently consumed H2S and produced H2S2 and subsequently longer-chain polysulfides. This reaction appeared to be O2-dependent. MnP absorbance spectra exhibited wavelength shifts in the Soret and Q bands characteristic of sulfide-mediated reduction of Mn. Taken together, our results suggest that MnPs can become efficacious activators of a variety of cytoprotective processes by acting as sulfide oxidation catalysts generating per/polysulfides.

The Novel Role That Nrf2 Plays in Erythropoiesis during Aging

Erythropoiesis is a dynamic and multistep process, resulting in generation of red cells from hematopoietic stem cells. Aging is characterized by increased oxidation and reduced efficiency of cytoprotective mechanisms. Nrf2 is a key transcription factor that participates in acute response to oxidative stress and controls the expression of anti-oxidant and cytoprotective systems. We previously documented activation of Nrf2 during β-thalassemic erythropoiesis. In addition, a previous study reported a mild chronic hemolytic anemia as a result of increased erythrophagocytosis in mice genetically lacking Nrf2 (PNAS, 2004). Here, we show an age-dependent worsening of anemia in Nrf2-/- mice characterized by accelerated senescence of circulating erythrocytes in association with reticulocytopenia, suggesting a perturbation of erythroblast maturation. Indeed, we found ineffective erythropoiesis in 12 months-old Nrf2-/- mice with extramedullary erythropoiesis, increased ROS levels, reduction in the expression of Nrf2 related anti-oxidant proteins and apoptosis of erythroid precursor cells. In agreement, we observed an age dependent sensitivity of Nrf2-/- mice to stress erythropoiesis induced by either phenyhydrazine (PHZ) or doxorubicine. In 12-month-old mice we observed (i) activation of the unfoldeld protein response system (UPR) due to endosomal reticulum stress and (ii) impaired autophagy. The cytoprotective processes were unable to fully counteract oxidation re-directing cells towards apoptosis as supported by the increased activity of caspase-3. To understand the impact of oxidation in this model of accelerated senescence, we treated Nrf2-/- mice with astaxanthin, a powerful anti-oxidant but with low oral bioavailability, by administrating it in the form of PLGA loaded nanoparticles (ATS-NP). Treatment of Nrf2-/- mice with ATS-NP ameliorated the age-dependent anemia and decreased ineffective erythropoiesis through (i) inactivation of UPR system; (ii) improvement of autophagy and (iii) reduction in caspase-3 activity. In future studies, we plan to evaluate the impact of ATS-NP administration in other models of pathologic erythropoiesis. In summary, we propose that Nrf2, a key transcriptional factor plays a key protective role in regulating aged related oxidation and ensures normal erythroid maturation and growth. Disclosures No relevant conflicts of interest to declare.