Novel ortho ester-based,pH-sensitive cationic lipid for gene delivery in vitro and in vivo

Context: Cationic lipoplexes are less toxic than viral gene vectors and more convenient to prepare but their efficiencies of gene delivery are generally lower.

Objective: To develop ortho ester-based, pH-sensitive lipoplexes for efficient gene delivery both in cultured cells and in vivo.

Materials and methods: A novel cationic and acid-labile lipid (DOC) containing a cationic headgroup and a cholesterol-derived lipid tail joined together by an acid-labile ortho ester linker was designed and synthesized. DOC was formulated into liposomes with the conical helper lipid DOPE, and then into lipoplexes with plasmid DNA encoding a luciferase reporter gene. The physicochemical properties of the lipoplexes (size, surface charge and pH-sensitivity) were characterized. Gene delivery by DOC/DOPE/DNA lipoplexes was also evaluated in CV-1 cells and in CD-1 mice following intratracheal injection. Lipoplexes consisting of the acid-stable cationic lipid DC-Chol were characterized as a control.

Results: DOC formed cationic lipoplexes with DOPE and DNA. After incubation at acidic pH 4.6, DOC/DOPE/DNA lipoplexes lost their positive charges and aggregated with one another as a result of DOC hydrolysis. Both in CV-1 cell culture and in CD-1 mice, DOC/DOPE/DNA lipoplexes increased the luciferase gene expression by 5- to 10-fold compared with the analogous but acid-stable DC-Chol/DOPE/DNA lipoplexes.

Discussion and conclusion: Incorporation of an acid-labile ortho ester linker into a cationic lipid is a viable approach to enhance gene delivery by the corresponding lipoplexes both in cultured cells and in vivo.     

Tumor delivery of liposomal doxorubicin prepared with poly-L-glutamic acid as a drug-trapping agent

Context: Poly-l-glutamic acid (PGA) is an anionic polymer with a large number of carboxyl groups that can interact electrostatically with cationic drugs such as doxorubicin (DOX).

Objective: For stable encapsulation of DOX into liposomes, we prepared triethylamine (TEA)-PGA-liposomes using PGA as an internal trapping agent.

Methods: We prepared TEA-PGA-liposomes by remote loading of DOX with a TEA gradient into preformed liposomes prepared with 1, 2, or 4?mg/mL PGA (molecular weights 4800, 9800, and 20 500), and evaluated their biodistribution and antitumor effects on Lewis lung carcinoma (LLC) tumor-bearing mice.

Results: TEA-PGA-liposomes using the higher the molecular weight or concentration of PGA showed a slower release of DOX from the liposomes. TEA-PGA-liposomes prepared with a high concentration of PGA could enhance DOX accumulation in tumors and prolonged DOX circulation in the serum, indicating that DOX may be retained stably in the liposomal interior by interaction with PGA. Furthermore, injection of TEA-PGA-liposomes prepared with 4?mg/mL of PGA₉₈₀₀ or 2?mg/mL PGA₂₀₅₀₀ strongly inhibited tumor growth in LLC tumor-bearing mice.

Conclusions: PGA may be a potential trapping agent for liposomal DOX for tumor drug delivery.     

Biomarker discovery for drug-induced phospholipidosis: phenylacetylglycine to hippuric acid ratio in urine and plasma as potential markers

Context: Drug-induced phospholipidosis is one of the significant concerns in drug development, especially in safety assessment and noninvasive diagnostic tool is highly desirable.

Objective: The objective of this study is to explored novel biomarkers for phospholipidosis using a metabolomic approach.

Method: NMR spectrometry and LC/MS/MS analyses were applied to urine and plasma of rats administrated cationic amphiphilic drugs.

Results: The phenylacetylglycine to hippuric acid ratio in plasma was increased in time and dose-dependent manners; and it was well correlated with histopathological observation.

Conclusion: The plasma phenylacetylglycine to hippuric acid ratio is a potential marker in monitoring drug-induced phospholipidosis.     

Inhibitory effect of Tanshinone IIA on inverted formin-2 protects HaCaT cells against oxidative injury via regulating mitochondrial stress

Context: Epidermal cells play an important role in regulating the regeneration of skin after burns and wounds.

Objective: The aim of our study is to explore the role of Tanshinone IIA (Tan IIA) in the apoptosis of epidermal HaCaT cells induced by H₂O₂, with a focus on mitochondrial homeostasis and inverted formin-2 (INF2).

Materials and methods: Cellular viability was determined using the MTT assay, TUNEL staining, western blot analysis and LDH release assay. Adenovirus-loaded INF2 was transfected into HaCaT cells to overexpress INF2 in the presence of Tan IIA treatment. Mitochondrial function was determined using JC-1 staining, mitochondrial ROS staining, immunofluorescence and western blotting.

Results: Oxidative stress promoted the death of HaCaT cells and this effect could be reversed by Tan IIA. At the molecular levels, Tan IIA treatment sustained mitochondrial energy metabolism, repressed mitochondrial ROS generation, stabilized mitochondrial potential, and blocked the mitochondrial apoptotic pathway. Furthermore, we demonstrated that Tan IIA modulated mitochondrial homeostasis via affecting INF2-related mitochondrial stress. Overexpression of INF2 could abolish the protective effects of Tan IIA on HaCaT cells viability and mitochondrial function. Besides, we also reported that Tan IIA regulated INF2 expression via the ERK pathway; inhibition of this pathway abrogated the beneficial effects of Tan IIA on HaCaT cells survival and mitochondrial homeostasis.

Conclusions: Overall, our results indicated that oxidative stress-mediated HaCaT cells apoptosis could be reversed by Tan IIA treatment via reducing INF2-related mitochondrial stress in a manner dependent on the ERK signaling pathway.     

Lipoprotein-associated phospholipase A2, and subsequent cardiovascular events and mortality among patients with coronary heart disease

Objective: The objective of this study is to evaluate the relevance of Lp-PLA₂ to risk prediction among coronary heart disease (CHD) patients.

Methods: Lp-PLA₂ activity was measured in 2538 CHD patients included in the Bezafibrate Infarction Prevention (BIP) study.

Results: Adjusting for patient characteristics and traditional risk factors, 1 standard deviation of Lp-PLA₂ was associated with a hazard ratio (HR) of 1.12 (95% confidence interval (CI): 1.00–1.25) for mortality and 1.03 (0.93–1.14) for cardiovascular events. Lp-PLA₂ did not significantly improve model discrimination, or calibration nor result in noteworthy reclassification.

Conclusions: Our results do not support added value of Lp-PLA₂ for predicting cardiovascular events or mortality among CHD patients beyond traditional risk factor.     

Downregulation of IDH2 exacerbates H2O2-mediated cell death and hypertrophy

Objectives: Reactive oxygen species-mediated cell death contributes to the pathophysiology of cardiovascular disease and myocardial dysfunction. We recently showed that mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) functions as an antioxidant and anti-apoptotic protein by supplying NADPH to antioxidant systems.

Methods: In the present study, we demonstrated that H₂O₂-induced apoptosis and hypertrophy of H9c2 cardiomyoblasts was markedly exacerbated by small interfering RNA (siRNA) specific for IDH2.

Results: Attenuated IDH2 expression resulted in the modulation of cellular and mitochondrial redox status, mitochondrial function, and cellular oxidative damage. MitoTEMPO, a mitochondria-targeted antioxidant, efficiently suppressed increased caspase-3 activity, increased cell size, and depletion of cellular GSH levels in IDH2 siRNA-transfected cells that were treated with H₂O₂.

Discussion: These results indicated that the disruption of cellular redox balance might be responsible for the enhanced H₂O₂-induced apoptosis and hypertrophy of cultured cardiomyocytes by the attenuated IDH2 expression.     

Serum adenosine deaminase,catalase, and carbonic anhydrase activities in patients with renal cell carcinoma

Objectives: To determine whether serum levels of adenosine deaminase (AD), catalase (CAT), and carbonic anhydrase (CA) enzymes may be useful biomarkers in the diagnosis of renal tumors and may lead to early diagnosis of renal tumors.

Material and methods: The study included 33 patients with renal cell carcinoma (RCC) and 31 healthy controls. The activity of serum AD, CA, and CAT was determined and analyzed using the Giusti spectrophotometric method, H₂O₂ substrate, and C0₂ hydration, respectively.

Results: Serum AD and CA activity were significantly higher in patients with RCC than in controls. However, serum CAT activity was significantly lower in patients with RCC than in controls.

Conclusion: These markers might be potentially important as an additional biochemical tool for diagnosing RCC. We believe multidisciplinary studies are needed to plan patients’ preoperative and postoperative treatment and to create follow-up protocols.     

Effect of different concentrations of oxygen on expression of sigma 1 receptor and superoxide dismutases in human colon adenocarcinoma cell lines

Context: Tumor cells due to distance from capillary vessels exist in different oxygenation conditions (anoxia, hypoxia, normoxia). Changes in cell oxygenation lead to reactive oxygen species production and oxidative stress. Sigma 1 receptor (Sig1R) is postulated to be stress responding agent and superoxide dismutases (SOD1 and SOD2) are key antioxidant enzymes. It is possible that they participate in tumor cells adaptation to different concentrations of oxygen.

Objective: Evaluation of Sig1R, SOD1, and SOD2 expression in different concentrations of oxygen (1%, 10%, 21%) in colon adenocarcinoma cell lines.

Materials and methods: SW480 (primary adenocarcinoma) and SW620 (metastatic) cell lines were cultured in standard conditions in Dulbecco’s modified Eagle’s medium for 5 days, and next cultured in Hypoxic Chamber in 1% O₂, 10% O₂, 21% O₂. Number of living cells was determined by trypan blue assay. Level of mRNA for Sig1R, SOD1, and SOD2 was determined by standard PCR method. Statistical analysis was conducted using Statistica 10.1 software.

Results: We observed significant changes in expression of Sig1R, SOD1, SOD2 due to different oxygen concentrations. ANOVA analysis revealed significant interactions between studied parameters mainly in hypoxia conditions in SW480 cells and between Sig1R and SOD2 in SW620 cells. It also showed that changes in expression of studied proteins depend significantly on type of the cell line.

Conclusion: Changes of Sig1R and SOD2 expression point to mitochondria as main organelle responsible for survival of tumor cells exposed to hypoxia or oxidative stress. Studied proteins are involved in intracellular response to stress related with different concentrations of oxygen.     

Vancomycin-sensitized photooxidation in the presence of the natural pigment vitamin B2: Interaction with excited states and photogenerated ROS

Sensitized photooxidation processes in the presence of natural pigments may provide an alternative to antibiotics degradation since these compounds are transparent to natural light irradiation, therefore, they can be degraded by the action of photosensitizers which absorb light and produce highly reactive species, especially those derived from molecular oxygen (ROS). Most antibiotics used currently belong to a group of pharmaceutical substances that have been considered a new type of contaminants due to their persistence and bioaccumulation in the environment.

Objective: In this context, we decided to investigate the kinetic and mechanistic aspects of Vancomycin (Vanco) photosensitized degradation in the presence of the natural pigment Riboflavin (Vitamin B2, Rf) and the artificial dye Rose Bengal (RB) for comparative purposes.

Methods: The study have been done by using Stationary photolysis, Laser flash photolysis, Time-resolved phosphorence detection of O₂(1Δg) experiments and Bactericidal activity evaluation. The experiments were carried out in aqueous solution at different pH values in order to establish relationships between the structure of the compound and its susceptibility to ROS-mediated photooxidation.

Results: Experimental evidence indicates that in the presence of Rf there is considerable contribution of the radical-mediated mechanism, while in the presence of RB the photooxidation process occurs exclusively through O₂(1Δg) and the reactivity to this excited species increases with increasing pH of the environment.

Discussion: The results obtained, have been shown that Rf can raise the photodegradation of Vanco by both the radical pathway and the O₂(¹Δ_g) mediated. Furthermore, the antibiotic is able to interact with the excited electronic states of Rf as well as O₂(¹Δ_g) generated by energy transfer between the excited triplet state of the photosensitizer and the oxygen ground state. The predominant mechanism for photodegradation of Vanco in the presence of the Rf is the radical via because of the considerable interaction with the excited triplet state of the photosensitizer demonstrated by laser flash photolysis experiments.

Microbiological test on Staphylococcus aureus ATCC25923 showed that the bactericidal activity of the antibiotic on the strain studied was affected by the sensitized photodegradation process, suggesting that photoproducts generated eventually do not retain the bactericidal properties of the original antibiotic.     

Mutation in the beta3 subunit of Guanine nucleotide-binding protein (GNB3) gene is not associated with Type II diabetes mellitus risk: a case–control study of a North Indian population

Context: Genetics play a major role in development and pathophysiology of Type 2 diabetes mellitus (T2DM).

Objective: To asses the association of Guanine nucleotide-binding protein (GNB3) (C825T) gene's polymorphism with T2DM.

Materials and methods: A case–control study including 400 North Indians was performed using Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) approach to analyze genetic polymorphism.

Results: No significant difference was observed in genotype and allele frequencies of GNB3 gene on comparing cases with controls.

Discussion: Our study is in agreement with studies on Polish, Japanese, Hispanic-American and Danish populations who observed no significant association between GNB3 (C825T) polymorphism and T2DM.

Conclusion: GNB3 (C825T) polymorphism is not associated with T2DM.     

Dynamic variation of histone H3 trimethyl Lys4 (H3K4me3) and heterochromatin protein 1 (HP1) with employment length in nickel smelting workers

Objective: To investigate the dynamic variation in H3K4me3 and HP1 with employment length in nickel smelting workers.

Methods: Blood samples were collected from 140 nickel smelting workers and 140 age-matched office workers to test for H3K4me3, and HP1 levels.

Results: H3K4me3 was statistically significantly different (p?<?0.05) between the two groups and positively correlated with employment length (r_s?=_?0.267). HP1 was not correlated with employment length (p?=?0.066) but was significantly different between the two groups.

Conclusions: Chronic exposure to nickel can induce oxidative damage, and increase H3K4me3 expression and inhibit HP1 expression.     

Solubilization and Plant Uptake of Zinc and Cadmium from Soils Treated with Elemental Sulfur

In growth chamber experiments we studied the potential use of elemental sulfur (S₈) as an acidifying agent to enhance the uptake of Cd and Zn from three different polluted soils by candidate phytoremediation plants (Brassica juncea, Helianthus annuus, Salix viminalis). Two of the three soils were calcareous, the other slightly acidic. One of the calcareous soils had been contaminated by dust emissions from a nearby brass smelter. The pollution of the other two soils had resulted from sewage sludge applications.

Sulfur was added to soils in quantities of 20 to 400 mmol sulfur kg^-1 soil. Plants were grown under fluorescent light in 1.5 l ($OS 13 cm) pots for 28 d.

Within 700 h soil pH decreased significantly in all soils, depending on S₈ dosage. In the acid soil, pH decreased from pH 6.5 to about 4 at the highest treatment level, while pH in one of the calcareous soils dropped even below pH 4. The effect was smaller in the second calcareous soil.

NaNO₃-extractable Cd and Zn increased up to 26-and 13-fold, respectively, in the acid soil, while in the calcareous soils, maximum increases were 9-and 11-fold, respectively.

Increased NaNO₃-extractable concentrations translated well into shoot concentrations (dry matter) in plants. Shoot Zn concentrations in H. annuus, for example, increased from 930 in the controls to 4300 mg kg^-1 in the highest S₈ treatment. However, effects observed in the plants were generally smaller than in the soils. In addition, in some variants growth was negatively affected, resulting in reduced metal removal from the soils.     

Assessment of titanium dioxide nanoparticles toxicity via oral exposure in mice: effect of dose and particle size

Objective: The aim of the present work is to evaluate the toxicity of titanium dioxide nanoparticles (TiO₂NPs) according to their doses and particle sizes.

Materials and methods: The effect of five days oral administration of TiO₂NPs (21 and 80?nm) with different doses (50, 250 and 500?mg/kg body weight) was assessed in mice via measurement of oxidative stress markers; glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and nitric oxide (NO), liver function indices; aspartate and alanine aminotransferases (AST and ALT), chromosomal aberrations and liver histopathological pattern.

Results: The results revealed drastic alterations in all the measured parameters and showed positive correlation with the gradual dose increment. In addition, the smaller particle size of TiO₂NPS (21?nm) had more adverse effect in all the selected biochemical parameters, genetic aberrations and histological investigations.

Conclusions: Toxicity of TiO₂NPs increases in a dose-dependent manner and vice versa with particles size. The evaluated biomarkers are good indicators for TiO₂NPs toxicity. More detailed studies are required before the recommendation of TiO₂NP_S as food additives.     

Aberrant methylation of nucleotide excision repair genes is associated with chronic arsenic poisoning

Objective: To define whether aberrant methylation of DNA repair genes is associated with chronic arsenic poisoning.

Methods: Hundred and two endemic arsenicosis patients and 36 healthy subjects were recruited. Methylight and bisulfite sequencing (BSP) assays were used to examine the methylation status of ERCC1, ERCC2 and XPC genes in peripheral blood lymphocytes (PBLs) and skin lesions of arsenicosis patients and NaAsO₂-treated HaCaT cells.

Results: Hypermethylation of ERCC1 and ERCC2 and suppressed gene expression were found in PBLs and skin lesions of arsenicosis patients and was correlated with the level of arsenic exposure. Particularly, the expression of ERCC1 and ERCC2 was associated with the severity of skin lesions. In vitro studies revealed an induction of ERCC2 hypermethylation and decreased mRNA expression in response to NaAsO₂ treatment.

Conclusion: Hypermethylation of ERCC1 and ERCC2 and concomitant suppression of gene expression might be served as the epigenetic marks associated with arsenic exposure and adverse health effects.     

Proteomics and phosphoproteomics study of LCMT1 overexpression and oxidative stress: overexpression of LCMT1 arrests H2O2-induced lose of cells viability

Objectives: Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, is also known to be a target of ROS. The methylation of PP2A can be catalyzed by leucine carboxyl methyltransferase-1 (LCMT1), which regulates PP2A activity and substrate specificity.

Methods: In the previous study, we have showed that LCMT1-dependent PP2Ac methylation arrests H₂O₂-induced cell oxidative stress damage. To explore the possible protective mechanism, we performed iTRAQ-based comparative quantitative proteomics and phosphoproteomics studies of H₂O₂-treated vector control and LCMT1-overexpressing cells.

Results: A total of 4480 non-redundant proteins and 3801 unique phosphopeptides were identified by this means. By comparing the H₂O₂-regulated proteins in LCMT1-overexpressing and vector control cells, we found that these differences were mainly related to protein phosphorylation, gene expression, protein maturation, the cytoskeleton and cell division. Further investigation of LCMT1 overexpression-specific regulated proteins under H₂O₂ treatment supported the idea that LCMT1 overexpression induced ageneral dephosphorylation of proteins and indicated increased expression of non-erythrocytic hemoglobin, inactivation of MAPK3 and regulation of proteins related to Rho signal transduction, which were known to be linked to the regulation of the cytoskeleton.

Discussion: These data provide proteomics and phosphoproteomics insights into the association of LCMT1-dependent PP2Ac methylation and oxidative stress and indirectly indicate that the methylation of PP2A plays an important role against oxidative stress.     

Circulating adiponectin as a biomarker in renal cell carcinoma: a systematic review and meta-analysis

Context: Metabolic imbalance in renal cell carcinoma (RCC) can lead to abnormal adiponectin levels.

Objective: To evaluate circulating adiponectin as a detection or predictive marker for RCC.

Methods: A comprehensive literature search and meta-analysis was performed on studies reporting circulating adiponectin levels and RCC. The meta-analysis was performed using RevMan.

Results: Seven studies compared the circulating adiponection levels between RCC cases and controls. Adiponectin level was significantly lower in RCC cases compared to controls at pre-diagnosis and pre-operative time-points. RCC stage, grade and subtype did not affect adiponectin levels.

Conclusion: Low circulating adiponectin could be a predictive or risk factor for RCC.     

Beta-2-glycoprotein-1 and alpha-1-antitrypsin as urinary markers of renal cancer in von Hippel–Lindau patients

Context: Von Hippel–Lindau disease (VHLD) is a rare inherited neoplastic syndrome. Among all the VHLD-associated tumors, clear cell renal cell carcinoma (ccRCC) is the major cause of death.

Objective: The aim of this paper is the discovery of new non-invasive biomarker for the monitoring of VHLD patients.

Materials and methods: We compared the urinary proteome of VHLD patients, ccRCC patients and healthy volunteers.

Results: Among all differentially expressed proteins, alpha-1-antitrypsin (A1AT) and APOH (beta-2-glycoprotein-1) are strongly over-abundant only in the urine of VHLD patients with a history of ccRCC.

Discussion and conclusion: A1AT and APOH could be promising non-invasive biomarkers.     

Trolox-equivalent antioxidant capacity and composition of five alpine plant species growing at different elevations on the Qinghai–Tibetan Plateau

Background: Alpine plants on the Qinghai–Tibetan Plateau are exposed to an extremely harsh environment, namely severe cold, strong ultraviolet radiation, hypoxia and low CO₂ partial pressure. These conditions are sources of oxidative stress, which increase in severity with increasing elevation.

Aims: To examine whether antioxidant capacity and chemical composition of alpine plants change with increasing elevation.

Methods: We measured the Trolox-equivalent antioxidant capacity (TEAC) and chemical composition of five alpine plant species at 3016, 3814 and 4621 m a.s.l.

Results: With increasing elevation: (1) the TEAC increased and total phenols and tannins tended to increase in two forb and two shrub species but not in a sedge species; (2) concentrations of protein and fat increased in all five plant species; (3) polyunsaturated fatty acids (PUFA) increased and (4) mineral concentrations tended to decrease, but trends were inconsistent.

Conclusions: We conclude that with increasing elevation, TEAC and total phenols and tannins increased which we interpreted as an adaptation to higher oxidative stress; and protein and fat contents increased to support high metabolic activity. The increase in PUFA and the trend for minerals to decrease with increasing elevation require further investigation.     

Determination of air pollution-related biomarkers of exposure in urine of travellers between Germany and China using liquid chromatographic and liquid chromatographic-mass spectrometric methods: a pilot study

Objective: The influence of different exposures to PM_2.5 (particulate matter with an aerodynamic diameter below 2.5?μm) on the concentrations of biomarkers of exposure and oxidative stress should be investigated. For this purpose, urine samples from individuals travelling from Germany to China were collected and analysed.

Materials: Robust LC and LC-MS/MS methods were established for the determination of biomarkers including 8-hydroxy-2′-deoxyguanosine, malondialdehyde, F_2α-isoprostanes and hydroxylated polycyclic aromatic hydrocarbons. As a pilot study, nine volunteers travelled from Germany (mean daily concentration of PM_2.5: 21?μg/m³) to China (mean daily concentration of PM_2.5: 108?μg/m³). Urine samples were collected before and after the trip.

Results: In samples collected after return to Germany, the median concentrations of oxidative stress biomarkers were observed to be higher than in samples collected before leaving Germany. Decreasing trends were observed in the sequences of samples collected after return in the following weeks. Correlations were found between exposure and oxidative stress biomarkers.

Conclusion: Travellers are ideal models for PM pollution-induced acute health effects study. Exposure to PM pollution can cause oxidative stress and damage.