BACKGROUND

Radiation exposure poses a significant threat to public health. Hematopoietic injury is one of the major manifestations of acute radiation sickness. Protection and/or mitigation of hematopoietic stem cells (HSCs) from radiation injury is an important goal in the development of medical countermeasure agents (MCM). We recently identified thioredoxin (TXN) as a novel molecule that has marked protective and proliferative effects on HSCs. In the current study, we investigated the effectiveness of TXN in rescuing mice from a lethal dose of total body radiation (TBI) and in enhancing hematopoietic reconstitution following a lethal dose of irradiation.

METHODS

We used in-vivo and in-vitro methods to understand the biological and molecular mechanisms of TXN on radiation mitigation. BABL/c mice were used for the survival study and a flow cytometer was used to quantify the HSC population and cell senescence. A hematology analyzer was used for the peripheral blood cell count, including white blood cells (WBCs), red blood cells (RBCs), hemoglobin, and platelets. Colony forming unit (CFU) assay was used to study the colongenic function of HSCs. Hematoxylin and eosin staining was used to determine the bone marrow cellularity. Senescence-associated β-galactosidase assay was used for cell senescence. Western blot analysis was used to evaluate the DNA damage and senescence protein expression. Immunofluorescence staining was used to measure the expression of γ-H2AX foci for DNA damage.

RESULTS

We found that administration of TXN 24 h following irradiation significantly mitigates BALB/c mice from TBI-induced death: 70% of TXN-treated mice survived, whereas only 25% of saline-treated mice survived. TXN administration led to enhanced recovery of peripheral blood cell counts, bone marrow cellularity, and HSC population as measured by c-Kit+Sca-1+Lin- (KSL) cells, SLAM + KSL cells and CFUs. TXN treatment reduced cell senescence and radiation-induced double-strand DNA breaks in both murine bone marrow lineage-negative (Lin-) cells and primary fibroblasts. Furthermore, TXN decreased the expression of p16 and phosphorylated p38. Our data suggest that TXN modulates diverse cellular processes of HSCs.

CONCLUSIONS

Administration of TXN 24 h following irradiation mitigates radiation-induced lethality. To the best of our knowledge, this is the first report demonstrating that TXN reduces radiation-induced lethality. TXN shows potential utility in the mitigation of radiation-induced hematopoietic injury.

Oxidative stress has been implicated in the destruction of beta cells in type 1 diabetes (T1D). Thioredoxin has been shown to protect cells from oxidative stress and apoptosis. In this study, we screened for sequence variants of the human thioredoxin gene (TXN), and studied the association of the variants in persons with T1D in Japanese. The frequency of the A allele of the G/A SNP in the 3' flanking region was highest in T1D (8.4%), followed by type 2 diabetes (6.8%), and the lowest in the controls (5.9%), suggesting the contribution of TXN polymorphism to susceptibility to T1D.

Hydroperoxide metabolism in Crithidia fasciculata has recently been shown to be catalyzed by a cascade of three oxidoreductases comprising trypanothione reductase (TR), tryparedoxin (TXNTXNPx) (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). The existence of this metabolic system in the human pathogen Trypanosoma cruzi is supported here by immunohistochemistry. Epimastigotes of T. cruzi display strong immunoreactivity with antibodies raised against TXNTXNPx of C. fasciculata. In addition, a full-length open reading frame presumed to encode a peroxiredoxin-type protein in T. cruzi (Acc. Nr. AJ 012101) was heterologously expressed in Escherichia coli and shown to exhibit tryparedoxin peroxidase activity. With TXN, TXNPx, trypanothione and TR, T. cruzi possesses all components constituting the crithidial peroxidase system. It is concluded that the antioxidant defense of T. cruzi also depends on the NADPH-fuelled, trypanothione-mediated enzymatic hydroperoxide metabolism.

Within the mitochondrion of Leishmania infantum, hydroperoxide metabolism relies on the activity of tryparedoxin-dependent peroxidases (TXNPxs). Tryparedoxins (TXNs) are thioredoxin-related oxidoreductases, which in vitro are reduced by the trypanothione reductase/trypanothione [TR/T(SH)(2)] redox couple. Still, there is no evidence that this actually occurs in the mitochondrion. This communication addresses the question of how the mitochondrial TXN/TXNPx system is reduced. First, using a digitonin fractionation assay, we show that TR activity is absent from the L. infantum mitochondrion. The possibility that this organelle possesses alternative electron sources for TXN/TXNPx is then investigated. Biochemical assays performed with purified recombinant enzymes, revealed that TR and T(SH)(2) can be replaced, albeit less efficiently, by the dihydrolipoamide dehydrogenase/lipoamide redox system as TXN/TXNPx electron donor. This result challenges the classical view that T(SH)(2) is the only reductant for TXNs and add new prospects regarding the involvement of 2-oxo acid dehydrogenase complexes in L. infantum mitochondrial hydroperoxide metabolism.

High myopia is recognized as a risk factor for earlier onset of nuclear cataracts. One possible explanation for this is that lenses in highly myopic eyes are exposed to higher levels of oxygen than normal eyes owing to earlier vitreous liquefaction and, hence, are subjected to oxidative insults. Here, we first compared the methylation levels of six essential antioxidant genes (GSTP1, NRF2, OGG1, TXN, TXNRD1 and TXNRD2) between highly myopic cataract (HMC) and age-related cataract (ARC) lens epithelial samples via Sequenom MassARRAY. We found that specific CpG units in the promoters of GSTP1 and TXNRD2 were hypermethylated and that the expression levels of these two genes were lower in the HMC group than in the ARC group. A luciferase reporter assay confirmed the significance of differentially methylated fragments in the activation of transcription. The importance of GSTP1 and TXNRD2 in antioxidant capacity was confirmed by overexpression or knockdown experiments on cultured lens epithelial cells (LECs). In addition, the expression of DNA methyl transferase 1 (DNMT1) was higher in the lens epithelium of HMC patients than that of ARC patients, and the expression of GSTP1 and TXNRD2 was upregulated by use of a DNMT inhibitor in cultured LECs. Finally, we mimicked the intraocular environment of highly myopic eyes by treating LECs with hydrogen peroxide (H2O2) and observed both alterations in the methylation status of the GSTP1 and TXNRD2 promoters and time-dependent altered expression levels. Therefore, we propose that in an environment with high oxygen, in which lenses in highly myopic eyes are immersed, there exists a vicious cycle composed of increased oxidative stress and decreased enzymatic antioxidants via the hypermethylation of antioxidant genes.

Nickel (Ni)-induced oxidative damage is a serious problem that leads to reproductive system failure through testicular damage. The present investigation was carried out to determine the effect of troxerutin (Txn) on testicular toxicity induced by Ni in experimental rat testes. The oral administration of Txn (100 mg/kg body weight [bw]) showed a significant (p < 0.01) increase in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), reduced glutathione, ascorbate, total sulphydryl groups, and testis-organ weight. Subsequently, the administration of Txn also significantly reduced the accumulation of Ni, lipid peroxidation products, and protein carbonyl levels in Txn-treated animals. Testicular protection in the experimental animals by Txn is further substantiated by a remarkable reduction of Ni, which was revealed through testicular tissue histopathology. These studies suggest that Txn could prevent oxidative damage and testicular toxicity induced by Ni in experimental animals.

BACKGROUND

Abdominal obesity (AO) is a common modifiable risk factor for certain non-communicable diseases associated with enhanced oxidative stress (OS). The objective of this work was to investigate whether the interaction between antioxidant vitamin intake and OS-related polymorphisms modulates gene-associated anthropometry in a Spanish population.

METHODS

A total of 246 subjects with AO, and 492 age and gender matched non-AO subjects were included in the study. Anthropometric, biochemical, and OS parameters, and antioxidant dietary intake data were assessed using validated procedures. DNA from white blood cells was isolated and the genotype of seven polymorphisms from genes involved in OS (pro-oxidant and antioxidant) were analyzed using the SNPlex system. The effects of the c.-793T>> C polymorphism on promoter activity and thus thioredoxin (TXN) activity were examined using reporter assays.

RESULTS

The AO group had higher 8-Oxo-2'-deoxyguanosine levels and took in less vitamin A and vitamin E compared to the non-AO group. Logistic regression analysis revealed that the rs2301241 polymorphism in TXN and rs740603 in catechol-O-methyltransferase (COMT) were associated with waist circumference (WC) and AO. Moreover, these polymorphisms were more strongly associated with variations in WC in subjects with low vitamin E intakes. A promoter assay revealed that the T to C conversion at c.-793 (rs2301241) induced a more than two fold increase in reporter gene expression.

CONCLUSIONS

WC is associated both with dietary vitamin E intake and genetic variants of TXN and COMT suggesting that existence of a complex nutrigenetic pathway that involves regulation of AO.

The thioredoxin (Txn) system is the most crucial antioxidant defense mechanism in the myocardium, and hampering the Txn system may compromise cell survival. Calcium (Ca) imbalance is associated with a variety of cardiomyopathies, and dysregulation of Ca2+ homeostasis is often considered a critical starting point for heart disease. However, the roles of Txn and the Txn system in maintaining Ca2+ homeostasis in cardiomyocytes have been infrequently reported. Here, we examined the expression of genes associated with Ca2+ channels using a model of Txn suppression in cardiomyocyte cultures (siRNA and Txn inhibitor) and report that Txn knockdown can cause Ca2+ overload in the myocardial cytoplasm and release of endoplasmic reticulum (ER) Ca2+, which induces ER stress. Our results showed that Txn knockdown could lead to cytosolic Ca2+ overload through upregulated gene expression of Ca2+ channel-related genes in the cytoplasmic and ER membranes. Furthermore, we find that excessive Ca2+ concentrations in the cytoplasm may increase myocardial contraction, and heat shock proteins may play a protective role throughout the process. Our present study reveals a novel model of regulation for low Txn expression in myocardial injury.

The circadian clock, a biochemical oscillator, plays a fundamental role in health and diseases. Ferroptosis, a type of regulated cell death driven by oxidative stress, is a prominent feature in iron-induced tissue injury. However, whether an impaired circadian clock contributes to ferroptosis-induced sterile inflammation remains unknown. Here, we show that the circadian transcription factor ARNTL (also known as BMAL1) protects against experimental acute pancreatitis through blocking the ferroptosis-mediated release of HMGB1, a mediator of sterile inflammation. We utilized a Cre/LoxP system to generate mice with a specific depletion of Arntl in the pancreas (Pdx1-Cre;Arntl^flox/flox). These Arntl-deficient mice developed l-arginine-induced acute pancreatitis more rapidly than controls, with increased mortality, tissue injury, neutrophil infiltration, and HMGB1 release. In contrast, the administration of liproxstatin-1 (a ferroptosis inhibitor) or anti-HMGB1 neutralizing antibody attenuated the development of acute pancreatitis in the Arntl-deficient mice. Mechanistically, pancreatic ARNTL is a key regulator of the expression of multiple antioxidant or membrane repair systems (e.g., SLC7A11, GPX4, SOD1, TXN, NFE2L2, and CHMP5) to suppress ferroptotic tissue injury. Collectively, these findings uncover a novel link between the circadian clock and ferroptotic response in inflammation and pancreatic injury.

BACKGROUND

Leishmania contains a concatenated mitochondrial DNA, kDNA. Universal minicircle sequence binding protein (UMSBP), a mitochondrial protein, initiates kDNA replication by binding with a conserved universal minicircle sequence (UMS) of kDNA. Here, we describe first time in L. donovani the regulation of DNA binding activity of UMSBP and the role of UMSBP in virulence.

METHODS

Insilco and EMSA study were performed to show UMS-binding activity of UMSBP. Tryparedoxin(TXN)-tryparedoxin peroxidase(TXNPx) assay as well as co-overexpression of cytochrome-b5 reductase-like protein (CBRL) and tryparedoxin in L. donovani were done to know the regulation of DNA binding activity of UMSBP. Knockout and episomal-expression constructs of UMSBP were transfected in L. donovani. The cell viability assay and immunofluorescence study to know the status of kDNA were performed. Macrophages were infected with transfected parasites. mRNA level of cytochrome b, activity of complex-III, intracellular ATP level of both transfected promastigotes and amastigotes as well as ROS concentration and the level of apoptosis of transfected promastigotes were measured. Level of oxidative phosphorylation of both transfected and un-transfected amastigotes were compared. Burden of transfected amastigotes in both macrophages and BALB/c mice were measured.

RESULTS

L. donovani UMSBP is capable of binding with UMS, regulated by redox through mitochondrial enzymes, TXN, TXNPx and CBRL. Depletion of UMSBP (LdU(-/-)) caused kDNA loss, which decreased cytochrome-b expression [component of complex-III of electron transport chain (ETC)] and leads to the disruption of complex-III activity, decreased ATP generation, increased ROS level and promastigotes exhibited apoptosis like death. Interestingly, single knockout of UMSBP (LdU(-/+)) has no effect on promastigotes survival. However, single knockout in intracellular amastigotes demonstrate loss of mRNA level of cytochrome-b, disruption in the activity of complex-III and reduced production of ATP in amastigotes than wild type. This process interfere with the oxidative-phosphorylation and thereby completely inhibit the intracellular proliferation of LdU(-/+) amastigotes in human macrophages and in BALB/c mice. Amastigotes proliferation was restored as wild type after episomal expression of LdUMSBP in LdU(-/+) parasites (LdU(-/+)AB).

CONCLUSIONS

The LdUMSBP regulates leishmanial mitochondrial respiration and pathogenesis. So, LdUMSBP may be an attractive target for rational drug designing and LdU(-/+) parasites could be considered as a live attenuated vaccine candidate against visceral leishmaniasis.

Our aim was to assess the use of peripheral blood mononuclear cells (PBMC) as an in vivo cellular model to evaluate diet-induced changes in the oxidative stress status by analyzing the gene expression pattern of NADPH-oxidase subunits and antioxidant genes. A randomized, controlled trial assigned metabolic syndrome patients to 4 diets for 12 weeks each: (i) high-saturated fatty acid (HSFA), (ii) high-monounsaturated fatty acid, and (iii), (iv) two low-fat, high-complex carbohydrate diets supplemented with n-3 polyunsaturated fatty acids or placebo. A fat challenge reflecting the fatty acid composition as the original diets was conducted post-intervention. The mRNA levels of gp91(phox) (P<0.001), p22(phox) (P=0.005), p47(phox) (P=0.001) and p40(phox) (P<0.001) increased at 2h after the intake of the HSFA meal. The expression of SOD1, SOD2, GSR, GPx1, GPX4, TXN, TXNRD1 and Nrf2 increased after the HSFA meal (p<0.05). In contrast, the expression of these genes remained unaltered in response to the other dietary interventions. Our results suggest that the increased expression of antioxidant genes in PBMC seems to be due to the response to the postprandial oxidative stress generated mainly in adipose tissue after the consumption of an HSFA diet.

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) have been investigated as triple-negative breast cancer (TNBC) biomarkers. Reduced EGFR levels can be compensated by increases in HER3; thus, assaying EGFR and HER3 together may improve prognostic value. In a multi-institutional cohort of 510 TNBC patients, we analyzed the impact of HER3, EGFR, or combined HER3-EGFR protein expression in pre-treatment samples on breast cancer-specific and distant metastasis-free survival (BCSS and DMFS, respectively). A subset of 60 TNBC samples were RNA-sequenced using massive parallel sequencing. The combined HER3-EGFR score outperformed individual HER3 and EGFR scores, with high HER3-EGFR score independently predicting worse BCSS (Hazard Ratio [HR] = 2.30, p = 0.006) and DMFS (HR = 1.78, p = 0.041, respectively). TNBCs with high HER3-EGFR scores exhibited significantly suppressed ATM signaling and differential expression of a network predicted to be controlled by low TXN activity, resulting in activation of EGFR, PARP1, and caspases and inhibition of p53 and NFκB. Nuclear PARP1 protein levels were higher in HER3-EGFR-high TNBCs based on immunohistochemistry (p = 0.036). Assessing HER3 and EGFR protein expression in combination may identify which adjuvant chemotherapy-treated TNBC patients have a higher risk of treatment resistance and may benefit from a dual HER3-EGFR inhibitor and a PARP1 inhibitor.

Cerebrovascular diseases, including stroke and micro stroke, are the main causes of death in contemporary society. Hemorrhagic stroke is the fast emerging defficiency in the brain function resulting from disturbance of blood supply to the brain caused by the rapture of blood vessels (Lopez et al. in Proteomics Clin Appl 6:190-200, 2012). The influence of a model hemorrhagic stroke on white pigs with the change in the protein profile of their cortical samples 24 h and 2 months after the stroke was examined using mass-spectrometric analysis. Different proteins (n = 30) were identified, and their content was elevated. These proteins are involved in the mechanisms of neuroprotection, including compensation of oxidative stress (TXN, SNCA, PRDX6, ENO1), prevention of unwanted protein aggregation and apoptosis (PTMA, SNCA, SNCB), release of neurotransmitters (GAPDH, PEBP1) and assembly of the cytoskeleton (ACTA2, PTMA, TUBA4A, TUBA1D), etc. Also, a group of seven Ras family proteins involved in the regulation of cell proliferation and differentiation was found in the samples taken 24 h following the stroke. The relative concentrations of most of the proteins in the samples taken 2 months after the stroke demonstrate intermediate values between the control sample and the sample taken in 24 h, indicating the extinction of change in the protein profile with time. During the first 24 h after the stroke, there is an increase in protein fractions participating in exocytosis, synaptic plasticity/signaling, and support of neurotransmitter transport. Such shift in the weight of protein functional clusters can be attributed to activation of compensatory mechanisms in the body focused on neuroprotection.

Thioredoxin-interacting protein (TXNIP) is an α-arrestin that can bind to and inhibit the antioxidant protein thioredoxin (TXN). TXNIP expression is induced by glucose and promotes β-cell apoptosis in the pancreas, and deletion of its gene in mouse models protects against diabetes. TXNIP is currently studied as a potential new target for antidiabetic drug therapy. In this study, we describe a family with a mutation in the TXNIP gene leading to nondetectable expression of TXNIP protein. Symptoms of affected family members include lactic acidosis and low serum methionine levels. Using patient-derived TXNIP-deficient fibroblasts and myoblasts, we show that oxidative phosphorylation is impaired in these cells when given glucose and pyruvate but normalized with malate. Isolated mitochondria from these cells appear to have normal respiratory function. The cells also display a transcriptional pattern suggestive of a high basal activation of the Nrf2 transcription factor. We conclude that a complete lack of TXNIP in human is nonlethal and leads to specific metabolic distortions that are, at least in part, linked to a deficient respiration on pyruvate. The results give important insights into the impact of TXNIP in humans and thus help to further advance the development of antidiabetic drugs targeting this protein.

BACKGROUND

Oxidative stress occurs due to the excessive generation of cellular reactive oxygen species and antioxidant system dysfunction. The thioredoxin (TXN) system and TXN-domain-containing protein (TXNDC) family form networks maintaining the cellular reducing environment. Recently, the importance of these genes in the tumor environment has been emphasized.

OBJECTIVE

To investigate the clinical significance of TXNs and TXNDC family members in HCC.

METHODS

Genomic data from 367 hepatocellular carcinoma (HCC) patients who underwent hepatic resections were analyzed to determine genetic alterations in mRNA and protein levels between patients and healthy controls. In addition, functional enrichment and survival analyses were performed.

RESULTS

HCC patients were shown to have enhanced expression of TXN, TXNRD1, and TXNDC7/9/14 mRNA and protein compared with controls. In accordance with the survival analyses, strong associations were found that patients with TXN, TXNRD1, and TXNDC1/7/9 alterations were proven to have poor prognosis in overall survival. Moreover, gene set enrichment analysis and network analyses revealed that positive correlations were found in mRNA expression of TXN, TXNRD1, and TXNDC7/9 genes with upregulation of the tumor-promoting genes, specifically mTORC1, E2F targets, and Myc targets. On the other hand, elevated expressions of TXNIP and TXNDC11 genes were correlated with suppression of the above tumor-promoting genes.

CONCLUSIONS

TXN system and TXNDC family gene panel obtained from the resected tissue of the HCC patients could be used to predict survival prognosis of HCC, and these genes could be considered as potential therapeutic targets for improving HCC survival.

The earliest stages of embryo development are deeply influenced by reactive oxygen species (ROS), byproducts of the mitochondrial oxygen metabolism that play a key role as messengers in normal cell signal transduction and cell cycling. Despite its positive roles, the imbalance caused by the excess of ROS and an inefficient antioxidant system leads to oxidative stress, with negative consequences to the cell such as DNA damage, metabolic changes, mitochondrial stress and cell death. In the present work, crocetin - a natural antioxidant - was added to the culture media of bovine embryos to evaluate the efficiency of its antioxidant capability during embryo culture. Oocytes were in vitro matured (IVM) and fertilized according to standard protocols. Embryos were cultured at 38.5 °C under humidified air with 5% CO2, 7% O2, and 90% N2 in Synthetic Oviduct Fluid (SOF) medium supplemented with amino acids and either 5% of FBS (SOFaa) (control group) or SOFaa supplemented with 1 μM crocetin (crocetin group). After 5 days from the beginning of in vitro culture (IVC) (day 5 - D5), embryos were transferred to individual drops of culture media. At day 7 (D7), embryos were assessed by means of blastocyst rates, morphophysiological analyzes (total cell number, ROS and mitochondrial activity levels), transcript quantitation of 47 genes and metabolomic evaluation of the culture media by Raman spectroscopy. In the crocetin group blastocyst rates were higher and embryos had increased total cell number and decreased intracellular levels of ROS. These embryos also had upregulation of genes related with response to stress and lipid metabolism (ATF4, BAX, FOXO3, GADD45A, GPX1, GPX4, HSF1, SOD2, ACACA, SREBF1 and SREBF2). Raman spectroscopy corroborated these results indicating more active lipid and amino acid production in this group. The absence of crocetin in the culture media resulted in higher ROS level, as well as up regulation of genes related to DNA damage, stress response and energy metabolism (MORF4L2, SOD1, TXN, PFKP, PGK1 and PPARGC1A). In conclusion, crocetin supplementation during culture protects embryos from oxidative stress and influences the adaptive response to stress conditions, leading to an increase in both blastocyst yield and quality, as well as changes in transcriptomic and metabolic profile of in vitro produced bovine embryos.

Chronic graft-versus-host disease (GVHD) is the leading cause of long-term morbidity and mortality after allogeneic hematopoietic cell transplantation. To identify prognostic plasma proteins associated with de novo- or quiescent-onset chronic GVHD (cGVHD), we performed a discovery and validation proteomic study. The total study cohort included 167 consecutive patients who had no clinical evidence of GVHD under minimum glucocorticoid administration and had available plasma samples obtained at 80 ± 14 days after transplantation. We first used high-throughput mass spectrometry to screen pooled plasma using 20 cases with subsequent cGVHD and 20 controls without it, and we identified 20 candidate proteins. We then measured 12 of the 20 candidate proteins by ELISA on the same individual samples and identified 4 proteins for further verification (LGALS3BP, CD5L, CD163, and TXN for de novo onset, and LGALS3BP and CD5L for quiescent onset). The verification cohort included 127 remaining patients. The cumulative incidence of de novo-onset cGVHD was higher in patients with higher plasma soluble CD163 concentrations at day 80 than those with lower concentrations (75% versus 40%, P = .018). The cumulative incidence of de novo- or quiescent-onset cGVHD did not differ statistically according to concentrations of the 3 other proteins at day 80. CD163 is a macrophage scavenger receptor and is elevated in oxidative conditions. These results suggest that monocyte or macrophage activation or increased oxidative stress may contribute to the pathogenesis of cGVHD.

OBJECTIVE

Thioredoxin interacting protein (TXNIP), an inhibitor of antioxidant thioredoxin (Trx), is upregulated by hyperglycemia and implicated in pathogenesis of diabetes complications. We evaluated mRNA expressions of genes encoding TXNIP and Trx (TXN) in urinary sediment and peripheral blood mononuclear cells (PBMC) of type 1 diabetes (T1D) patients with different degrees of chronic complications.

METHODS

qPCR was employed to quantify target genes in urinary sediment (n = 55) and PBMC (n = 161) from patients sorted by presence or absence of diabetic nephropathy (DN), retinopathy, peripheral and cardiovascular neuropathy; 26 healthy controls and 13 patients presenting non-diabetic nephropathy (focal and segmental glomerulosclerosis, FSGS) were also included.

RESULTS

Regarding the urinary sediment, TXNIP (but not TXN) expression was higher in T1D (p = 0.0023) and FSGS (p = 0.0027) patients versus controls. Expressions of TXNIP and TXN were higher, respectively, in T1D patients with versus without DN (p = 0.032) and in those with estimated glomerular filtration rate (eGFR) < 60 versus ≥60 mL/min/1.73 m(2) (p = 0.008). eGFR negatively correlated with TXNIP (p = 0.04, r = -0.28) and TXN (p = 0.04, r = -0.30) expressions. T1D patients who lost ≥5 mL/min/1.73 m(2) yearly of eGFR presented higher basal TXNIP expression than those who lost <5 mL/min/1.73 m(2) yearly after median follow-up of 24 months. TXNIP (p < 0.0001) and TXN (p = 0.002) expressions in PBMC of T1D patients were significantly higher than in controls but no differences were observed between patients with or without chronic complications.

CONCLUSIONS

TXNIP and TXN are upregulated in urinary sediment of T1D patients with diabetic kidney disease (DKD), but only TXNIP expression is associated with magnitude of eGFR decline.

Trypanosoma cruzi, the causative agent of Chagas disease, possesses two tryparedoxins (TcTXNI and TcTXNII), belonging to the thioredoxin superfamily. TXNs are oxidoreductases which mediate electron transfer between trypanothione and peroxiredoxins. This constitutes a difference with the host cells, in which these activities are mediated by thioredoxins. These differences make TXNs an attractive target for drug development. In a previous work we characterized TcTXNI, including the redox interactome. In this work we extend the study to TcTXNII. We demonstrate that TcTXNII is a transmembrane protein anchored to the surface of the mitochondria and endoplasmic reticulum, with a cytoplasmatic orientation of the redox domain. It would be expressed during the metacyclogenesis process. In order to continue with the characterization of the redox interactome of T. cruzi, we designed an active site mutant TcTXNII lacking the resolving cysteine, and through the expression of this mutant protein and incubation with T. cruzi proteins, heterodisulfide complexes were isolated by affinity chromatography and identified by mass spectrometry. This allowed us to identify sixteen TcTXNII interacting proteins, which are involved in a wide range of cellular processes, indicating the relevance of TcTXNII, and contributing to our understanding of the redox interactome of T. cruzi.

UNASSIGNED

T. cruzi, the causative agent of Chagas disease, constitutes a major sanitary problem in Latin America. The number of estimated infected persons is ca. 8 million, 28 million people are at risk of infection and ~20,000 deaths occur per year in endemic regions. No vaccines are available at present, and most drugs currently in use were developed decades ago and show variable efficacy with undesirable side effects. The parasite is able to live and prolipherate inside macrophage phagosomes, where it is exposed to cytotoxic reactive oxygen and nitrogen species, derived from macrophage activation. Therefore, T. cruzi antioxidant mechanisms constitute an active field of investigation, since they could provide the basis for a rational drug development. Peroxide detoxification in this parasite is achieved by ascorbate peroxidase and different thiol-dependent peroxidases. Among them, both mitochondrial and cytosolic tryparedoxin peroxidases, typical two-cysteine peroxiredoxins, were found to be important for hydrogen peroxide and peroxynitrite detoxification and their expression levels correlated with parasite infectivity and virulence. In trypanosomes tryparedoxins and not thioredoxins act as peroxiredoxin reductases, suggesting that these enzymes substitute thioredoxins in these parasites. T. cruzi possesses two tryparedoxin genes, TcTXNI and TcTXN II. Since thioredoxins are proteins with several targets actively participating of complex redox networks, we have previously investigated if this is the case also for TcTXNI, for which we described relevant partners (J Proteomics. 2011;74(9):1683-92). In this manuscript we investigated the interactions of TcTXNII. We have designed an active site mutant tryparedoxin II lacking the resolving cysteine and, through the expression of this mutant protein and its incubation with T. cruzi proteins, hetero disulfide complexes were isolated by affinity chromatography purification and identified by electrophoresis separation and MS identification. This allowed us to identify sixteen TcTXNII interacting proteins which are involved in different and relevant cellular processes. Moreover, we demonstrate that TcTXNII is a transmembrane protein anchored to the surface of the mitochondria and endoplasmic reticulum.

The destruction of articular cartilage in osteoarthritis involves chondrocyte dysfunction and imbalanced extracellular matrix (ECM) homeostasis. Pro-inflammatory cytokines such as interleukin-1α (IL-1α) contribute to osteoarthritis pathophysiology, but the effects of IL-1α on chondrocytes within their tissue microenvironment have not been fully evaluated. To redress this we used label-free quantitative proteomics to analyze the chondrocyte response to IL-1α within a native cartilage ECM. Mouse femoral heads were cultured with and without IL-1α, and both the tissue proteome and proteins released into the media were analyzed. New elements of the chondrocyte response to IL-1α related to cellular stress included markers for protein misfolding (Armet, Creld2, and Hyou1), enzymes involved in glutathione biosynthesis and regeneration (Gstp1, Gsto1, and Gsr), and oxidative stress proteins (Prdx2, Txn, Atox1, Hmox1, and Vnn1). Other proteins previously not associated with the IL-1α response in cartilage included ECM components (Smoc2, Kera, and Crispld1) and cysteine proteases (cathepsin Z and legumain), while chondroadherin and cartilage-derived C-type lectin (Clec3a) were identified as novel products of IL-1α-induced cartilage degradation. This first proteome-level view of the cartilage IL-1α response identified candidate biomarkers of cartilage destruction and novel targets for therapeutic intervention in osteoarthritis.

Wilson disease (WD) is caused by mutations in the copper transporter ATP7B, leading to copper accumulation in the liver and brain. Excess copper inhibits S-adenosyl-L-homocysteine hydrolase, leading to variable WD phenotypes from widespread alterations in DNA methylation and gene expression. Previously, we demonstrated that maternal choline supplementation in the Jackson toxic milk (tx-j) mouse model of WD corrected higher thioredoxin 1 (TNX1) transcript levels in fetal liver. Here, we investigated the effect of maternal choline supplementation on genome-wide DNA methylation patterns in tx-j fetal liver by whole-genome bisulfite sequencing (WGBS). Tx-j Atp7b genotype-dependent differences in DNA methylation were corrected by choline for genes including, but not exclusive to, oxidative stress pathways. To examine phenotypic effects of postnatal choline supplementation, tx-j mice were randomized to one of six treatment groups: with or without maternal and/or continued choline supplementation, and with or without copper chelation with penicillamine (PCA) treatment. Hepatic transcript levels of TXNTXN) pathway were differentially methylated in human WD liver samples. In summary, Atp7b deficiency and choline supplementation have a genome-wide impact, including on TXN system-related genes, in tx-j mice. These findings could explain the variability of WD phenotype and suggest new complementary treatment options for WD.

Obesity has been shown to alter response to air pollution and smoking but underlying biological mechanisms are largely unknown and few studies have explored mechanisms by which obesity increases human sensitivity to environmental exposures.

Overall study goals were to investigate whole blood gene expression in smokers and non-smokers to examine associations between cigarette smoke and changes in gene expression by obesity status and test for effect modification.

Relative fold-change in mRNA expression levels of 84 genes were analyzed using a Toxicity and Stress PCR array among 50 21-54 year old adults. Data on smoking status was confirmed using urinary cotinine levels. Adjusted models included age, gender, white blood cell count and body-mass index.

Models comparing gene expression of smokers vs. non-smokers identified six differentially expressed genes associated with smoking after adjustments for covariates. Obesity was associated with 29 genes differentially expressed compared to non-obese. We also identified 9 genes with significant smoking/obesity interactions influencing mRNA levels in adjusted models comparing expression between smokers vs non-smokers for four DNA damage related genes (GADD45A, DDB2, RAD51 and P53), two oxidative stress genes (FTH1, TXN), two hypoxia response genes (BN1P3lL, ARNT), and one gene associated with unfolded protein response (ATF6B).

Findings suggest that obesity alters human sensitivity to smoke exposures through several biological pathways by modifying gene expression. Additional studies are needed to fully understand the clinical impact of these effects, but risk assessments should consider underlying phenotypes, such as obesity, that may modulate sensitivity of vulnerable populations to environmental exposures.

Xanthohumol (XN), a prenylated flavonoid found in hops, inhibits growth in a variety of cancer cell lines; however, its use raises concerns as gut microbiota and the host's hepatic cytochrome P450 enzymes metabolize it into the most potent phytoestrogen known, 8-prenylnaringenin (8-PN). The XN derivatives dihydroxanthohumol (DXN) and tetrahydroxanthohumol (TXN) are not metabolized into 8-PN and they show higher tissue concentrations in vivo compared with XN when orally administered to mice at the same dose. Here we show that DXN and TXN possess improved anti-proliferative activity compared with XN in two colon (HCT116, HT29) and two hepatocellular (HepG2, Huh7) carcinoma cell lines, as indicated by their respective IC₅₀ values. Furthermore, XN, DXN, and TXN induce extensive apoptosis in all these carcinoma cell lines. Finally, TXN induces G₀/G₁ cell cycle arrest in the colon carcinoma cell line HT29. Our findings suggest that DXN and TXN could show promise as therapeutic agents against colorectal and liver cancer in preclinical studies without the drawback of metabolism into a phytoestrogen.

Xanthohumol (XN), a prenylated flavonoid from hops, improves dysfunctional glucose and lipid metabolism in animal models of metabolic syndrome (MetS). However, its metabolic transformation into the estrogenic metabolite, 8-prenylnaringenin (8-PN), poses a potential health concern for its use in humans. To address this concern, we evaluated two hydrogenated derivatives, α,β-dihydro-XN (DXN) and tetrahydro-XN (TXN), which showed negligible affinity for estrogen receptors α and β, and which cannot be metabolically converted into 8-PN. We compared their effects to those of XN by feeding C57BL/6J mice a high-fat diet (HFD) containing XN, DXN, or TXN for 13 weeks. DXN and TXN were present at higher concentrations than XN in plasma, liver and muscle. Mice administered XN, DXN or TXN showed improvements of impaired glucose tolerance compared to the controls. DXN and TXN treatment resulted in a decrease of HOMA-IR and plasma leptin. C2C12 embryonic muscle cells treated with DXN or TXN exhibited higher rates of uncoupled mitochondrial respiration compared to XN and the control. Finally, XN, DXN, or TXN treatment ameliorated HFD-induced deficits in spatial learning and memory. Taken together, DXN and TXN could ameliorate the neurocognitive-metabolic impairments associated with HFD-induced obesity without risk of liver injury and adverse estrogenic effects.