Superoxide dismutase 1-modified dental pulp stem cells alleviate high-altitude pulmonary edema by inhibiting oxidative stress through the Nrf2/HO-1 pathway.

High-altitude pulmonary edema (HAPE) is a deadly form of altitude sickness, and there is no effective treatment for HAPE. Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell isolated from dental pulp tissues and possess various functions, such as anti-inflammatory and anti-oxidative stress. DPSCs have been used to treat a variety of diseases, but there are no studies on treating HAPE. In this study, Sprague-Dawley rats were exposed to acute low-pressure hypoxia to establish the HAPE model, and SOD1-modified DPSCs (DPSCsHiSOD1) were administered through the tail vein. Pulmonary arterial pressure, lung water content (LWC), total lung protein content of bronchoalveolar lavage fluid (BALF) and lung homogenates, oxidative stress, and inflammatory indicators were detected to evaluate the effects of DPSCsHiSOD1 on HAPE. Rat type II alveolar epithelial cells (RLE-6TN) were used to investigate the effects and mechanism of DPSCsHiSOD1 on hypoxia injury. We found that DPSCs could treat HAPE, and the effect was better than that of dexamethasone treatment. SOD1 modification could enhance the function of DPSCs in improving the structure of lung tissue, decreasing pulmonary arterial pressure and LWC, and reducing the total lung protein content of BALF and lung homogenates, through anti-oxidative stress and anti-inflammatory effects. Furthermore, we found that DPSCsHiSOD1 could protect RLE-6TN from hypoxic injury by reducing the accumulation of reactive oxygen species (ROS) and activating the Nrf2/HO-1 pathway. Our findings confirm that SOD1 modification could enhance the anti-oxidative stress ability of DPSCs through the Nrf2/HO-1 signalling pathway. DPSCs, especially DPSCsHiSOD1, could be a potential treatment for HAPE. Schematic diagram of the antioxidant stress mechanism of DPSCs in the treatment of high-altitude pulmonary edema. DPSCs can alleviate oxidative stress by releasing superoxide dismutase 1, thereby reducing ROS production and activating the Nrf2/HO-1 signalling pathway to ameliorate lung cell injury in HAPE.

Early deficits in GABA inhibition parallels an increase in L-type Ca2+ currents in the jaw motor neurons of SOD1G93A mouse model for ALS.

Amyotrophic Lateral Sclerosis (ALS) involves protracted pre-symptomatic periods of abnormal motor neuron (MN) excitability occurring in parallel with central and peripheral synaptic perturbations. Focusing on inhibitory control of MNs, we first compared longitudinal changes in pre-synaptic terminal proteins for GABA and glycine neurotransmitters around the soma of retrogradely identified trigeminal jaw closer (JC) MNs and ChAT-labeled midbrain extraocular (EO) MNs in the SOD1G93A mouse model for ALS. Fluorescence immunocytochemistry and confocal imaging were used to quantify GAD67 and GlyT2 synaptic bouton density (SBD) around MN soma at pre-symptomatic ages ∼P12 (postnatal), ∼P50 (adult) and near disease end-stage (∼P135) in SOD1G93A mice and age-matched wild-type (WT) controls. We noted reduced GAD67 innervation in the SOD1G93A trigeminal jaw closer MNs around P12, relative to age-matched WT and no significant difference around P50 and P135. In contrast, both GAD67 and GlyT2 innervation were elevated in the SOD1G93A EO MNs at the pre-symptomatic time points. Considering trigeminal MNs are vulnerable in ALS while EO MNs are spared, we suggest that upregulation of inhibition in the latter might be compensatory. Notable contrast also existed in the innate co-expression patterns of GAD67 and GlyT2 with higher mutual information (co-dependency) in EO MNs compared to JC in both SOD1G93A and WT mice, especially at adult stages (P50 and P135). Around P12 when GAD67 terminals expression was low in the mutant, we further tested for persistent GABA inhibition in those MNs using in vitro patch-clamp electrophysiology. Our results show that SOD1G93A JC MNs have reduced persistent GABA inhibition, relative to WT. Pharmacological blocking of an underlying tonically active GABA conductance using the GABA-α5 subunit inverse agonist, L-655-708, disinhibited WT JC MNs and lowered their recruitment threshold, suggesting its role in the control of intrinsic MN excitability. Quantitative RT-PCR in laser dissected JC MNs further supported a reduction in GABA-α5 subunit mRNA expression in the mutant. In light of our previous report that JC MNs forming putative fast motor units have lower input threshold in the SOD1G93A mice, we suggest that our present result on reduced GABA-α5 tonic inhibition provides for a mechanism contributing to such imbalance. In parallel with reduced GABA inhibition, we noted an increase in voltage-gated L-type Ca2+ currents in the mutant JC MNs around P12. Together these results support that, early modifications in intrinsic properties of vulnerable MNs could be an adaptive response to counter synaptic deficits.

Nanoliposomes Reduce Stroke Injury Following Middle Cerebral Artery Occlusion in Mice.

BACKGROUND AND PURPOSE: Neuroprotective strategies for stroke remain inadequate. Nanoliposomes comprised of phosphatidylcholine, cholesterol, and monosialogangliosides (nanoliposomes) induced an antioxidant protective response in endothelial cells exposed to amyloid insults. We tested the hypotheses that nanoliposomes will preserve human neuroblastoma (SH-SY5Y) and human brain microvascular endothelial cells viability following oxygen-glucose deprivation (OGD)-reoxygenation and will reduce injury in mice following middle cerebral artery occlusion. METHODS: SH-SY5Y and human brain microvascular endothelial cells were exposed to oxygen-glucose deprivation-reoxygenation (3 hours 0.5%-1% oxygen and glucose-free media followed by 20-hour ambient air/regular media) without or with nanoliposomes (300 µg/mL). Viability was measured (calcein-acetoxymethyl fluorescence) and protein expression of antioxidant proteins HO-1 (heme oxygenase-1), NQO1 (NAD[P]H quinone dehydrogenase 1), and SOD1 (superoxide dismutase 1) were measured by Western blot. C57BL/6J mice were treated with saline (n=8) or nanoliposomes (10 mg/mL lipid, 200 µL, n=7) while undergoing 60-minute middle cerebral artery occlusion followed by reperfusion. Day 2 postinjury neurological impairment score and infarction size were compared. RESULTS: SH-SY5Y and human brain microvascular endothelial cells showed reduced viability post-oxygen-glucose deprivation-reoxygenation that was reversed by nanoliposomes. Nanoliposomes increased protein expressions of HO-1, NQO1 in both cell types and SOD1 in human brain microvascular endothelial cells. Nanoliposomes-treated mice showed reduced neurological impairment and brain infarct size (18.8±2% versus 27.3±2.3%, P=0.017) versus controls. CONCLUSIONS: Nanoliposomes reduced stroke injury in mice subjected to middle cerebral artery occlusion likely through induction of an antioxidant protective response. Nanoliposome is a candidate novel agent for stroke.

An extracellular Cu/Zn superoxide dismutase from Neocaridina denticulata sinensis: cDNA cloning, mRNA expression and characterizations of recombinant protein.

Neocaridina denticulata sinensis possesses characters of rapid growth, tenacious vitality, short growth cycle, transparent, and easy feeding. Therefore, it is gradually being developed into an animal model for basic research on decapod crustaceans. Herein, a Cu/Zn superoxide dismutase (Cu/Zn-SOD), named as Nd-ecCu/Zn-SOD, was identified and characterized from N. denticulata sinensis. The full-length cDNA sequence of Nd-ecCu/Zn-SOD is 829 bp containing a 684 bp open reading frame, which encodes a protein of 227 amino acid residues with a typical Sod_Cu domain. The quantitative real-time PCR analysis showed that Nd-ecCu/Zn-SOD mRNA was expressed in all the tested tissues. Under challenge with copper, the mRNA expression of Nd-ecCu/Zn-SOD reached the maximum at 6 h, and decreased until 24 h. After 24 h of exposure, its expression was up-regulated significantly at 36 h. After then its expression sharply decreased with a comeback at 48 h. The result indicated that Nd-ecCu/Zn-SOD might play an important role in the stress response of N. denticulata sinensis. The expression of Nd-ecCu/Zn-SOD in gills challenged with Vibrio parahaemolyticus changed in a time-dependent manner. Nd-ecCu/Zn-SOD was lowly expressed in early developmental stages by RNA-Seq technology, yet it showed that a cyclical rise and fall occurred between middle stages and late stages. In addition, Nd-ecCu/Zn-SOD was recombinantly expressed using E. coli and the recombinant protein was purified as a single band on SDS-PAGE. The recombinant Nd-ecCu/Zn-SOD (rNd-ecCu/Zn-SOD) existed enzymatic activity under a wide range of temperature and pH. The exposure of metal ions was found that Zn2+, Mg2+, Ca2+, Ba2+, and Cu2+ could inhibit the enzymatic activity of rNd-ecCu/Zn-SOD, and Mn2+ increased the enzymatic activity of rNd-ecCu/Zn-SOD. These results indicate that Nd-ecCu/Zn-SOD may play a pivotal role in resistant against oxidative damage and act as a biomarker under stressful environment.

The Effects of Seven-Day Exposure to Silver Nanoparticles on Fertility and Homeostasis of Zebrafish (Danio rerio).

Silver nanoparticles (AgNPs) are found in open waters, but the effect of their low concentrations on an organism's homeostasis is not fully understood. The aim of the study was to determine the short-term exposure effects of AgNPs coated by PvP (polyvinylpyrrolidone) on the homeostasis of livers and gonads in zebrafish. Sexually mature zebrafish were exposed for seven days to silver ions (0.01 mg/dm3) or AgNPs (0.01; 0.05; 0.1; 0.5; 1.0 mg/dm3). On the last day, the liver, testes, and ovaries were subjected to a histology analysis. In the liver, we analyzed the expression of the cat, gpx1a, gsr, sod1, and cyp1a genes. On the last day of the experiment, the lowest survival rate was found in the AgNPs 0.05 mg/dm3 group. The histological analysis showed that AgNPs and silver ions cause an increase in the area of hepatocytes. The highest proliferation index of hepatocytes was found in the AgNP 0.05 mg/dm3 group. Furthermore, AgNPs were found to interfere with spermatogenesis and oogonesis as well as reduce the expression levels of the cat, gpx1a, and sod1 genes in the liver compared with the control group. Based on the results, it can be concluded that exposure to AgNPs causes cytotoxic changes in zebrafish, activates the immune system, negatively affects the process of meiosis in the gonads, and generates oxidative stress.

Dysregulated biodynamics in metabolic attractor systems precede the emergence of amyotrophic lateral sclerosis.

Evolutionarily conserved mechanisms maintain homeostasis of essential elements, and are believed to be highly time-variant. However, current approaches measure elemental biomarkers at a few discrete time-points, ignoring complex higher-order dynamical features. To study dynamical properties of elemental homeostasis, we apply laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) to tooth samples to generate 500 temporally sequential measurements of elemental concentrations from birth to 10 years. We applied dynamical system and Information Theory-based analyses to reveal the longest-known attractor system in mammalian biology underlying the metabolism of nutrient elements, and identify distinct and consistent transitions between stable and unstable states throughout development. Extending these dynamical features to disease prediction, we find that attractor topography of nutrient metabolism is altered in amyotrophic lateral sclerosis (ALS), as early as childhood, suggesting these pathways are involved in disease risk. Mechanistic analysis was undertaken in a transgenic mouse model of ALS, where we find similar marked disruptions in elemental attractor systems as in humans. Our results demonstrate the application of a phenomological analysis of dynamical systems underlying elemental metabolism, and emphasize the utility of these measures in characterizing risk of disease.

Circuit-Specific Early Impairment of Proprioceptive Sensory Neurons in the SOD1G93A Mouse Model for ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons degenerate, resulting in muscle atrophy, paralysis, and fatality. Studies using mouse models of ALS indicate a protracted period of disease development with progressive motor neuron pathology, evident as early as embryonic and postnatal stages. Key missing information includes concomitant alterations in the sensorimotor circuit essential for normal development and function of the neuromuscular system. Leveraging unique brainstem circuitry, we show in vitro evidence for reflex circuit-specific postnatal abnormalities in the jaw proprioceptive sensory neurons in the well-studied SOD1G93A mouse. These include impaired and arrhythmic action potential burst discharge associated with a deficit in Nav1.6 Na+ channels. However, the mechanoreceptive and nociceptive trigeminal ganglion neurons and the visual sensory retinal ganglion neurons were resistant to excitability changes in age-matched SOD1G93A mice. Computational modeling of the observed disruption in sensory patterns predicted asynchronous self-sustained motor neuron discharge suggestive of imminent reflexive defects, such as muscle fasciculations in ALS. These results demonstrate a novel reflex circuit-specific proprioceptive sensory abnormality in ALS.SIGNIFICANCE STATEMENT Neurodegenerative diseases have prolonged periods of disease development and progression. Identifying early markers of vulnerability can therefore help devise better diagnostic and treatment strategies. In this study, we examined postnatal abnormalities in the electrical excitability of muscle spindle afferent proprioceptive neurons in the well-studied SOD1G93A mouse model for neurodegenerative motor neuron disease, amyotrophic lateral sclerosis. Our findings suggest that these proprioceptive sensory neurons are exclusively afflicted early in the disease process relative to sensory neurons of other modalities. Moreover, they presented Nav1.6 Na+ channel deficiency, which contributed to arrhythmic burst discharge. Such sensory arrhythmia could initiate reflexive defects, such as muscle fasciculations in amyotrophic lateral sclerosis, as suggested by our computational model.

Poloxamer 188 decreases membrane toxicity of mutant SOD1 and ameliorates pathology observed in SOD1 mouse model for ALS.

Here we report a gain in function for mutant (mt) superoxide dismutase I (SOD1), a cause of familial amyotrophic lateral sclerosis (FALS), wherein small soluble oligomers of mtSOD1 acquire a membrane toxicity. Phosphatidylglycerol (PG) lipid domains are selectively targeted, which could result in membrane damage or "toxic channels" becoming active in the bilayer. This PG-selective SOD1-mediated membrane toxicity is largely reversible in vitro by a widely-available FDA-approved surfactant and membrane-stabilizer P188. Treatment of G93ASOD1 transgenic mice with P188 significantly delayed symptoms onset, extended survival and decreased motoneuron death. The use of P188 or an analogue, which targets mtSOD1 misfolding-induced membrane toxicity, may provide a new direction for ALS treatment.

Novel targets for sensitizing breast cancer cells to TRAIL-induced apoptosis with siRNA delivery.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in variety of cancer cells without affecting most normal cells, which makes it a promising agent for cancer therapy. However, TRAIL therapy is clinically not effective due to resistance induction. To identify novel regulators of TRAIL that can aid in therapy, protein targets whose silencing sensitized breast cancer cells against TRAIL were screened with an siRNA library against 446 human apoptosis-related proteins in MDA-231 cells. Using a cationic lipopolymer (PEI-αLA) for delivery of library members, 16 siRNAs were identified that sensitized the TRAIL-induced death in MDA-231 cells. The siRNAs targeting BCL2L12 and SOD1 were further evaluated based on the novelty and their ability to sensitize TRAIL induced cell death. Silencing both targets sensitized TRAIL-mediated cell death in MDA-231 cells as well as TRAIL resistant breast cancer cells, MCF-7. Combination of TRAIL and siRNA silencing BCL2L12 had no effect in normal human umbilical vein cells and human bone marrow stromal cell. The silencing of BCL2L12 and SOD1 enhanced TRAIL-mediated apoptosis in MDA-231 cells via synergistically activating capsase-3 activity. Hence, here we report siRNAs targeting BCL2L12 and SOD1 as a novel regulator of TRAIL-induced cell death in breast cancer cells, providing a new approach for enhancing TRAIL therapy for breast cancer. The combination of siRNA targeting BCL2L12 and TRAIL can be a highly effective synergistic pair in breast cancer cells with minimal effect on the non-transformed cells.

SALS-linked WT-SOD1 adopts a highly similar helical conformation as FALS-causing L126Z-SOD1 in a membrane environment.

So far >180 mutations have been identified within the 153-residue human SOD1 to cause familial amyotrophic lateral sclerosis (FALS), while wild-type (WT) SOD1 was intriguingly implicated in sporadic ALS (SALS). SOD1 mutations lead to ALS by a dominant gain of cytotoxicity but its mechanism still remains elusive. Previously functional studies have revealed that SOD1 mutants became unexpectedly associated with organelle membranes. Indeed we decoded that the ALS-causing truncation mutant L126Z-SOD1 with an elevated toxicity completely loses the ability to fold into the native ß-barrel structure but acquire a novel capacity to interact with membranes by forming helices over hydrophobic/amphiphilic segments. Very recently, the abnormal insertion of SOD1 mutants into ER membrane has been functionally characterized to trigger ER stress, an initial event of a cascade of cell-specific damages in ALS pathogenesis. Here we attempted to understand the mechanism for gain of cytotoxicity of the WT SOD1. We obtained atomic-resolution evidence that the nascent WT SOD1 without metalation and disulfide bridge is also highly disordered as L126Z. Most importantly, it owns the same capacity in interacting with membranes by forming very similar helices over the first 125 residues identical to L126Z-SOD1, plus an additional hydrophobic helix over Leu144-Ala152. Our study thus implies that the WT and mutant SOD1 indeed converge on a common mechanism for gain of cytotoxicity by abnormally interacting with membranes. Moreover, any genetic/environmental factors which can delay or impair its maturation might act to transform SOD1 into cytotoxic forms with the acquired capacity to abnormally interact with membranes.

Granulocyte Colony-Stimulating Factor Ameliorates Skeletal Muscle Dysfunction in Amyotrophic Lateral Sclerosis Mice and Improves Proliferation of SOD1-G93A Myoblasts in vitro.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) causes loss of upper and lower motor neurons as well as skeletal muscle (SKM) dysfunction and atrophy. SKM is one of the tissues involved in the development of ALS pathology, and studies in a SOD1-G93A mouse model of ALS have demonstrated alterations in SKM degeneration/regeneration marker expression in vivo and defective mutant myoblast proliferation in vitro. Granulocyte colony-stimulating factor (G-CSF) has been shown to alleviate SOD1-G93A pathology. However, it is unknown whether G-CSF may have a direct effect on SKM or derived myoblasts. OBJECTIVE: To investigate effects of G-CSF and its analog pegfilgrastim (PEGF) on SOD1-G93A- associated SKM markers in vivo and those of G-CSF on myoblast proliferation in vitro. METHODS: The effect of PEGF treatment on hematopoietic stem cell mobilization, survival, and motor function was determined. RNA expression of SKM markers associated with mutant SOD1 expression was quantified in response to PEGF treatment in vivo, and the effect of G-CSF on the proliferation of myoblasts derived from mutant and control muscles was determined in vitro. RESULTS: Positive effects of PEGF on hematopoietic stem cell mobilization, survival, and functional assays in SOD1-G93A animals were confirmed. In vivo PEGF treatment augmented the expression of its receptor Csf3r and alleviated typical markers for mutant SOD1 muscle. Additionally, G-CSF was found to directly increase the proliferation of SOD1-G93A, but not wild-type primary myoblasts in vitro. CONCLUSION: Our results support the beneficial role of the G-CSF analog PEGF in a SOD1-G93A model of ALS. Thus, G-CSF and its analogs may be directly beneficial in diseases where the SKM function is compromised.

The mRNA expression of genes encoding selected antioxidant enzymes and thioredoxin, and the concentrations of their protein products in gingival crevicular fluid and saliva during periodontitis.

BACKGROUND: The activity of antioxidant enzymes in periodontitis is reduced, but results vary between studies and are subject to bias. In turn, the expression of genes encoding antioxidant factors has not been examined yet. OBJECTIVES: This is the first study to evaluate the expression of genes encoding superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1) and thioredoxin 1 (TXN1) in the saliva and gingival tissue of patients with periodontitis. The activity of the antioxidant enzyme protein products in the unstimulated and stimulated saliva and the gingival crevicular fluid (GCF) of patients with periodontitis was also investigated. MATERIAL AND METHODS: The prospective study involved 65 patients with periodontitis, who were divided into groups depending on the disease stage, and a control group of 31 ageand gender-matched healthy patients. RESULTS: We demonstrated that the expression of genes encoding GPX1 and TXN1 in saliva was significantly higher, and the expression of genes encoding SOD1, GPX1 and TXN1 in the gingival tissue was significantly lower in periodontitis patients as compared to the control group. We noted a lower activity of GPX1 in unstimulated saliva, of SOD1 in stimulated saliva and of both antioxidant enzymes in GCF in patients with periodontitis. CONCLUSIONS: The GPX1 transcriptome and its activity in the salivary and GCF proteome appear to be dependent on the oxidative stress related to the destructive inflammatory changes in periodontitis.

Targeting SOD1 via RNAi with PEGylated graphene oxide nanoparticles in platinum-resistant ovarian cancer.

Acquired platinum resistance poses a significant therapeutic impediment to ovarian cancer patient care, accounting for more than 200,000 deaths annually worldwide. We previously identified that overexpression of the antioxidant superoxide dismutase 1 (SOD1) in ovarian cancer is associated with a platinum-resistant phenotype via conferring oxidative stress resistance against platinum compounds. We further demonstrated that enzymatic inhibition using small-molecule inhibitors or silencing of SOD1 via RNA interference (RNAi) increased cisplatin sensitivity and potency in vitro. We launched this study to explore the potential therapeutic applications of SOD1 silencing in vivo in order to reverse cisplatin resistance using a graphene-based siRNA delivery platform. PEGylated graphene oxide (GO) polyethyleneimine (GOPEI-mPEG) nanoparticle was complexed with SOD1 siRNA. GOPEI-mPEG-siSOD1 exhibited high biocompatibility, siRNA loading capacity, and serum stability, and showed potent downregulation of SOD1 mRNA and protein levels. We further observed that cisplatin and PEI elicited mitochondrial dysfunction and transcriptionally activated the mitochondrial unfolded protein response (UPRmt) used as a reporter for their respective cytotoxicities. SOD1 silencing was found to augment cisplatin-induced cytotoxicity resulting in considerable tumour growth inhibition in cisplatin-sensitive A2780 and cisplatin-resistant A2780DDP subcutaneous mouse xenografts. Our study highlights the potential therapeutic applicability of RNAi-mediated targeting of SOD1 as a chemosensitizer for platinum-resistant ovarian cancers.

Hereditary motor and sensory neuropathy with SOD1-mutant: A case report.

RATIONALE: Hereditary motor-sensory peripheral neuropathy, or Charot-Marie-Tooth (CMT) Charcot-Marie-Tooth disease is an inherited peripheral neuropathy characterized by progressive limb weakness and muscle atrophy. As the disease progresses, sensory and autonomic involvement may occur. We report a case of CMT associated with SOD1 gene mutation, in order to provide new ideas for clinical disease diagnosis. PATIENT CONCERNS: A 50-years-old female patient was admitted to the hospital with "progressive weakness of the right lower extremity for 5 years, aggravating, and weakness of the left lower extremity for 4 months". DIAGNOSIS: The patient was diagnosed CMT. INTERVENTION: Nerve nutrition and rehabilitation therapy were given, but the patient's condition still did not improve significantly. OUTCOMES: The improvement of symptoms was not obvious. LESSONS: The clinical manifestations and electromyography results of this patient are consistent with the characteristics of CMT. The peripheral nerve-related hereditary gene test found mutation in SOD1. It is possible that this mutation is linked to CMT. The disease is a neurodegenerative disease, that may be slowed by physical therapy and rehabilitation, but could not be healed.

miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis.

BACKGROUND: Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. OBJECTIVE: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and -149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. METHODOLOGY: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. RESULTS: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. CONCLUSION: The polymorphism -149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.

Breathing New Life into TRAIL for Breast Cancer Therapy: Co-Delivery of pTRAIL and Complementary siRNAs Using Lipopolymers.

Preclinical studies showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapy is safe and effective to combat cancers, but clinical outcomes have been less than optimal due to short half-life of TRAIL protein, insufficient induction of apoptosis, and TRAIL resistance displayed in many tumors. In this study, we explored co-delivery of a TRAIL expressing plasmid (pTRAIL) and complementary small interfering RNAs (siRNAs) (silencing Bcl2-like 12 [BCL2L12] and superoxide dismutase 1 [SOD1]) to improve the response of breast cancer cells against TRAIL therapy. It is desirable to co-deliver the pDNA along with siRNA using a single delivery agent, but this is challenging given different structures of long/flexible pDNA and short/rigid siRNA. Toward this goal, we identified an aliphatic lipid-grafted low-molecular weight polyethylenimine (PEI) that accommodated both pDNA and siRNA in a single complex. The co-delivery of pTRAIL with BCL2L12- or SOD1-specific siRNAs resulted more significant cell death in different breast cancer cells compared with separate delivery without affecting nonmalignant cells viability. Ternary complexes of lipopolymer with pTRAIL and BCL2L12 siRNA significantly retarded the growth of breast cancer xenografts in mice. The enhanced anticancer activity was attributed to increased in situ secretion of TRAIL and sensitization of breast cancer cells against TRAIL by the co-delivered siRNAs. The lipid-grafted PEIs capable of co-delivering multiple types of nucleic acids can serve as powerful carriers for more effective complementary therapeutics. Graphical Abstract [Figure: see text].

Association of pre-eclampsia with SOD2 Ala16Val polymorphism among mother-father-infant triads.

OBJECTIVE: To determine whether pre-eclampsia is associated with polymorphisms in superoxide dismutase (SOD) genes among mother-father-infant triads. METHODS: We did this follow-up cohort study at 17 urban hospitals in Canada between October 1, 2008, and September 30, 2010. We recruited Canadian participants who had participated in the International Trial of Antioxidant Supplementation for the Prevention of Pre-eclampsia. Saliva specimens were collected for DNA extraction. The SOD1 +35A/C (rs2234694) and SOD2 Ala16Val C/T (rs4880) single-nucleotide polymorphisms (SNPs) were genotyped. RESULTS: Dual presence of the SOD2 Ala16Val TT variant among mother-father pairs (n=657) was associated with an increased risk of pre-eclampsia when compared with the absence of the TT variant among the mother-father pairs (7/48 [14.6%] vs 11/339 [3.2%]; adjusted odds ratio 6.80, 95% confidence interval 2.32-19.95; P<0.001). By contrast, presence of a single T variant in mother-father pairs (16/270 [5.9%]) or mother-infant pairs (8/179 [4.5%]) was not associated with pre-eclampsia. The SOD1 +35A/CSNP was not associated with pre-eclampsia. CONCLUSION: The SOD2 Ala16Val SNP might be involved in paternal influence on the maternal predisposition to pre-eclampsia. Genotyping of mother-father pairs could be a promising strategy to identify pre-eclampsia genes.

Glycerolipid Headgroups Control Rate and Mechanism of Superoxide Dismutase-1 Aggregation and Accelerate Fibrillization of Slowly Aggregating Amyotrophic Lateral Sclerosis Mutants.

Interactions between superoxide dismutase-1 (SOD1) and lipid membranes might be directly involved in the toxicity and intercellular propagation of aggregated SOD1 in amyotrophic lateral sclerosis (ALS), but the chemical details of lipid-SOD1 interactions and their effects on SOD1 aggregation remain unclear. This paper determined the rate and mechanism of nucleation of fibrillar apo-SOD1 catalyzed by liposomal surfaces with identical hydrophobic chains (RCH2(O2C18H33)2), but headgroups of different net charge and hydrophobicity (i.e., R(CH2)N+(CH3)3, RPO4-(CH2)2N+(CH3)3, and RPO4-). Under semiquiescent conditions (within a 96 well microplate, without a gyrating bead), the aggregation of apo-SOD1 into thioflavin-T-positive (ThT(+)) amyloid fibrils did not occur over 120 h in the absence of liposomal surfaces. Anionic liposomes triggered aggregation of apo-SOD1 into ThT(+) amyloid fibrils; cationic liposomes catalyzed fibrillization but at slower rates and across a narrower lipid concentration; zwitterionic liposomes produced nonfibrillar (amorphous) aggregates. The inability of zwitterionic liposomes to catalyze fibrillization and the dependence of fibrillization rate on anionic lipid concentration suggests that membranes catalyze SOD1 fibrillization by a primary nucleation mechanism. Membrane-catalyzed fibrillization was also examined for eight ALS variants of apo-SOD1, including G37R, G93R, D90A, and E100G apo-SOD1 that nucleate slower than or equal to WT SOD1 in lipid-free, nonquiescent amyloid assays. All ALS variants (with one exception) nucleated faster than WT SOD1 in the presence of anionic liposomes, wherein the greatest acceleratory effects were observed among variants with lower net negative surface charge (G37R, G93R, D90A, E100G). The exception was H46R apo-SOD1, which did not form ThT(+) species.

SOD1 nanozyme with reduced toxicity and MPS accumulation.

We previously developed a "cage"-like nano-formulation (nanozyme) for copper/Zinc superoxide dismutase (SOD1) by polyion condensation with a conventional block copolymer poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) followed by chemical cross-linking. Herein we report a new SOD1 nanozyme based on PEG-b-poly(aspartate diethyltriamine) (PEG-PAsp(DET), or PEG-DET for short) engineered for chronic dosing. This new nanozyme was spherical (Rg/Rh=0.785), and hollow (60% water composition) nanoparticles with colloidal properties similar to PLL-based nanozyme. It was better tolerated by brain microvessel endothelial/neuronal cells, and accumulated less in the liver and spleen. This formulation reduced the infarct volumes by more than 50% in a mouse model of ischemic stroke. However, it was not effective at preventing neuromuscular junction denervation in a mutant SOD1(G93A) mouse model of amyotrophic lateral sclerosis (ALS). To our knowledge, this work is the first report of using PEG-DET for protein delivery and a direct comparison between two cationic block copolymers demonstrating the effect of polymer structure in modulating the mononuclear phagocyte system (MPS) accumulation of polyion complexes.