Toward genome editing in X-linked RP-development of a mouse model with specific treatment relevant features.

Genome editing represents a powerful tool to treat inherited disorders. Highly specific endonucleases induce a DNA double strand break near the mutant site, which is subsequently repaired by cellular DNA repair mechanisms that involve the presence of a wild type template DNA. In vivo applications of this strategy are still rare, in part due to the absence of appropriate animal models carrying human disease mutations and knowledge of the efficient targeting of endonucleases. Here we report the generation and characterization of a new mouse model for X-linked retinitis pigmentosa (XLRP) carrying a point mutation in the mutational hotspot exon ORF15 of the RPGR gene as well as a recognition site for the homing endonuclease I-SceI. Presence of the genomic modifications was verified at the RNA and protein levels. The mutant protein was observed at low levels. Optical coherence tomography studies revealed a slowly progressive retinal degeneration with photoreceptor loss starting at 9 months of age, paralleling the onset of functional deficits as seen in the electroretinogram. Early changes to the outer retinal bands can be used as biomarker during treatment applications. We further show for the first time efficient targeting using the I-SceI enzyme at the genomic locus in a proof of concept in photoreceptors following adeno-associated virus mediated gene transfer in vivo. Taken together, our studies not only provide a human-XLRP disease model but also act as a platform to design genome editing technology for retinal degenerative diseases using the currently available endonucleases.

A study of oxidative stress in neonates delivered through meconium-stained amniotic fluid.

To estimate the levels of malondialdehyde (MDA) and 8-hydroxy-2-deoxyguanosine (8-OH-dG) in cord blood plasma of newborns born through meconium-stained amniotic fluid (MSAF) and also to find out the correlation between their levels with birth weight and gestation, we measured the cord blood plasma levels of MDA and 8-OH-dG in 59 newborns born through MSAF and 50 newborns born through clear liquor. The levels of cord blood plasma MDA and 8-OH-dG were significantly higher in full-term and late-preterm newborns born through MSAF. On further comparison, it was found that both full-term and late-preterm intrauterine growth restricted (IUGR) neonates had higher levels of these markers as compared to babies born as appropriate for gestational age (AGA) through MSAF. Plasma levels of MDA and 8-OH-dG were significantly correlated with birth weight even after controlling the relationship with gestational age for all cases as well as all full-term cases. These markers are also significantly correlated to each other. CONCLUSIONS: The present study suggest that the neonates born through MSAF experience higher degrees of oxidative stress, as evidenced by increased levels of cord blood plasma MDA and 8-OH-dG. What is known: • Aspirated meconium has been found to induce free radical generation and cellular damage in animal studies. • Its role in free radical generation and oxidative damage in human neonates is scarce. What is new: • Neonates born through meconium-stained amniotic fluid experience significant oxidative stress.

An Assessment of Oxidative Damage and Non-Enzymatic Antioxidants Status Alteration in Relation to Disease Progression in Breast Diseases.

The present study was aimed to evaluate the levels of oxidative stress markers in breast diseases by measuring the 8-hydroxy-2-deoxyguanosine (8-OHdG), vitamin A, vitamin C, vitamin E, and total antioxidant status (TAS) alterations in relation to cell proliferation activity and disease progression. Significant increases in the level of the oxidative damage marker 8-OHdG and cell proliferation activity were observed in breast carcinoma patients in comparison to benign and normal controls, which were accompanied by a significant decrease in non-enzymatic antioxidants and TAS concentrations (p < 0.05). 8-OHdG and cell proliferation levels were negatively correlated with non-enzymatic antioxidants, namely, vitamin A, vitamin C, and vitamin E levels and total antioxidant activity. Altered levels of biomarkers of oxidative stress and cell proliferation activity among the malignant, the benign, and the controls suggest a correlation of increased oxidative stress and cell proliferation activity in the progression of disease in breast carcinoma patients. In conclusion, our results showed that the characterized biomarkers (i.e., low levels of vitamin A, C and D, and the TAS status; and high levels of 8-OHdG) could be used as a suitable method for detecting subjects with malignant and benign breast diseases.

Lipid peroxidation, DNA damage and total antioxidant status in neonatal hyperbilirubinemia.

OBJECTIVE: Lipid peroxidation, DNA damage and total antioxidant status (TAS) were assessed in neonates with unconjugated hyperbilirubinemia (UCH). STUDY DESIGN: Plasma malondialdehyde (MDA), 8-hydroxy-2-deoxy-guanosine (8-OH-dG) and TAS levels were compared between 64 term newborns with idiopathic UCH and 30 age-matched healthy controls. RESULT: Compared with controls, an overall increase in mean plasma MDA and 8-OH-dG levels and a decrease in TAS level were noted in the UCH group. Within the UCH group, mean plasma MDA level was found to be low in infants with lower bilirubin levels, but a progressive increase was documented above the bilirubin level of 20 mg dl(-1). A significant increase in 8-OH-dG level was documented even at lower bilirubin levels, and a decrease i plasma TAS level was found at bilirubin levels above 16 mg dl(-1). MDA and 8-OH-dG levels were significantly higher, whereas TAS level was significantly lower in five neonates who developed features of acute bilirubin encephalopathy compared with those with normal outcome. Alteration of MDA, 8-OH-dG and TAS levels showed high predictive accuracy for poor outcome. CONCLUSION: Moderate-to-severe UCH was associated with higher oxidative stress and lower antioxidant defense. Alteration of oxidative stress parameters may be utilized as early predictors for poor outcome. High DNA damage even at lower bilirubin levels suggests possible genotoxic effect of bilirubin in UCH.

NF-κB p65 subunit DNA-binding activity: association with depleted antioxidant levels in breast carcinoma patients.

NF-κB is recognized as a redox-sensitive transcription factor and has been implicated in cellular response to oxidative stress. The study was designed to correlate the changes in antioxidant status with the levels of nuclear factor-κB (NF-κB) p65 subunit DNA-binding activity in relation to lymph node involvement, tumor size, and staging in breast carcinoma patients. Case control study comprised of 40 breast carcinoma patients along with 40 age- and sex-matched healthy subjects as controls. Levels of enzymatic/nonenzymatic antioxidants along with the trace elements were measured to study the antioxidant status in the study subjects. Levels of NF-κB p65 subunit DNA-binding activity was estimated by ELISA assay. The levels of enzymatic, nonenzymatic antioxidants, and trace elements were found to be significantly depleted in breast carcinoma patients in comparison to healthy controls suggesting significantly decreased levels of antioxidant activity in the breast carcinoma patients. Also, these results indicate that antioxidant levels decrease progressively with the advancement of stage and subsequent progression of disease. DNA-binding activity of NF-κB p65 subunit was higher in breast cancer patients in comparison to normal healthy controls, and the activity was found to increase with the advancement of disease. Significant correlation was observed between the DNA-binding activity of NF-κB p65 subunit and antioxidant status in the patients. The logistic regression analysis revealed decreased levels of antioxidants and increased level of DNA-binding activity of NF-κB p65 subunit were significantly associated with incidence of breast carcinoma. Depleted antioxidant status and increased level of DNA-binding activity of NF-κB p65 subunit thus point clearly of an association in relation to disease progression, clinical stage, and cytological grade in the pathophysiology of breast carcinoma.

Oxidative damage markers as possible discriminatory biomarkers in breast carcinoma.

The study was designed to evaluate the markers of oxidative damage and to establish their diagnostic utility in breast carcinoma patients. Levels of 8-hydroxy-2-deoxyguanosine (8-OH-dG), protein carbonyl (PC), and malondialdehyde (MDA) along with total antioxidant status (TAS) were measured in breast carcinoma patients and controls. Receiver operating characteristic (ROC) analysis was done to study the diagnostic potential of the oxidative damage markers. Significant increases in oxidative damage markers were observed in breast carcinoma patients compared with the normal controls, which were accompanied by significant decrease in TAS. The logistic regression analysis revealed higher levels of oxidative stress marker and reduced level of TAS were significantly associated with breast cancer. ROC curves analysis demonstrates that 8-OHdG and PC are better indicators for distinguishing cancer patients from controls, followed by MDA and TAS. Our results indicate increased oxidative damage is associated with malignancy in breast cancer patients. High accuracy of oxidative stress markers in indicating cancer presence can be used as discriminatory makers for efficient diagnosis.

Evaluation of biomarkers of oxidative stress and antioxidant capacity in the cord blood of preterm low birth weight neonates.

OBJECTIVE: The objective of the study is to investigate the association between oxidative stress markers and enzymatic / non-enzymatic antioxidants (marker of the resistance in body to oxidative damage) in the cord blood of preterm low birth weight (LBW) neonates. METHODS: Malondialdehyde (MDA), carbonyl proteins, total antioxidant capacity and Vitamin A, E and C levels in the cord blood were determined by spectrophotometry. RESULTS: Increased lipid peroxidation, protein oxidation with decreased values of vitamin A, E, C and total antioxidant capacity were observed in the preterm LBW newborns. Observations of negative correlation between MDA and protein carbonyl with antioxidants vitamin A, E and C and total antioxidant status points towards the existence of oxidative stress in the preterm LBW newborns. CONCLUSIONS: Poor fetal growth affects the development of antioxidant defenses of preterm LBW babies, predisposing them to higher oxidative stress, which in turn may partly account for increased morbidity and mortality in these infants. The presence of an association between oxidative stress biomarkers and enzymatic /non-enzymatic antioxidants in the cord blood of preterm LBW neonates suggest that increased oxidative stress may be the result of changes in the levels of certain enzymatic and non-enzymatic antioxidants due to the cause or the effect of oxidative damage occurring at the molecular level.

Long-term follow-up of a family with dominant X-linked retinitis pigmentosa.

PURPOSE: To document the progression of disease in male and female members of a previously described family with X-linked dominant retinitis pigmentosa (RP) caused by a de novo insertion after nucleotide 173 in exon ORF15 of RPGR. METHODS: The clinical records of 19 members of family UTAD054 were reviewed. Their evaluations consisted of confirmation of family history, standardised electroretinograms (ERGs), Goldmann visual fields, and periodic ophthalmological examinations over a 23-year period. RESULTS: Male members of family UTAD054 had non-recordable to barely recordable ERGs from early childhood. The males showed contracted central fields and developed more severe retinopathy than the females. The female members showed a disease onset delayed to teenage years, recordable but diminishing photopic and scotopic ERG amplitudes in a cone-rod pattern, progressive loss and often asymmetric visual fields, and diffuse atrophic retinopathy with fewer pigment deposits compared with males. CONCLUSIONS: This insertion mutation in the RPGR exon ORF15 is associated with a RP phenotype that severely affects males early and females by 30 years of age, and is highly penetrant in female members. Families with dominant-acting RPGR mutations may be mistaken to have an autosomal mode of inheritance resulting in an incorrect prediction of recurrence risk and prognosis. Broader recognition of X-linked RP forms with dominant inheritance is necessary to facilitate appropriate counselling of these patients.

Indirect evaluation of corneal apoptosis in contact lens wearers by estimation of nitric oxide and antioxidant enzymes in tears.

BACKGROUND: Contact lens induced trauma to the corneal epithelium results in increased release of inflammatory mediators. The keratocyte apoptosis is directly related to epithelial injury and has been correlated with increased production of nitric oxide. Potent antioxidant enzymes protect cells from oxidative damage by inactivating reactive oxygen species and thus inhibiting apoptosis. This study aims at determination of total nitric oxide and antioxidant enzymes in tears which will be an indirect criteria for assessing apoptosis. MATERIALS AND METHODS: Nitric oxide and antioxidant enzymes were estimated in tears of 25 soft contact lens wearers and compared with 25 age and sex matched controls. RESULTS: Statistically significant increase of nitric oxide (P<0.001), superoxide dismutase (P<0.001) and glutathione peroxidase (P<0.001) levels was seen in tears of contact lens wearers as compared to controls. There was also statistically significant increase in the levels of antioxidant enzymes, superoxide dismutase (P<0.05) and glutathione peroxidase (P<0.01), with increase in the total duration of contact lens wear in years. CONCLUSIONS: Increase in the level of nitric oxide and antioxidant enzymes in tears of contact lens wearers suggested that contact lens wear suppresses the process of apoptosis. However, it was also postulated that the increased levels of nitric oxide balances the anti-apoptotic activities of increased levels of antioxidant enzymes by its pro-apoptotic activity leading to protective outcomes in contact lens wearers.

Evaluation of wound healing activity of extracts of plantain banana (Musa sapientum var. paradisiaca) in rats.

Plantain banana (M. sapientum var. paradisiaca, MS) has been shown to possess ulcer healing activity. The present work with plantain banana was undertaken with the premise that the drug promoting ulcer healing could have effect on wound healing also. Wound healing activity of MS was studied in terms of (i) percent wound contraction, epithelization period and scar area; (ii) wound breaking strength and (iii) on granulation tissue antioxidant status [estimation of superoxide dismutase (SOD) and reduced glutathione (GSH), free radical (lipid peroxidation, an indicator of tissue damage) and connective tissue formation and maturation (hexuronic acid, hydroxyproline and hexosamine levels)] in excision, incision and dead space wound models respectively. The rats were given graded doses (50-200 mg/kg/day) of aqueous (MSW) and methanolic (MSE) extracts of MS orally for a period of 10-21 days depending upon the type of study. Both extracts (100 mg/kg) when studied for incision and dead space wounds parameters, increased wound breaking strength and levels of hydroxyproline, hexuronic acid, hexosamine, superoxide dismutase, reduced glutathione in the granulation tissue and decreased percentage of wound area, scar area and lipid peroxidation when compared with the control group. Both the extracts showed good safety profile. Plantain banana thus, favoured wound healing which could be due to its antioxidant effect and on various wound healing biochemical parameters.

Antioxidant levels in cord blood of low birth weight newborns.

We evaluated the antioxidant status of 82 healthy term low birth weight (LBW) newborns and equal number of gestation and sex matched controls weighing <2500 g by measuring vitamin A and E, superoxide dismutase, catalase and glutathione peroxidase in cord serum. Levels of vitamin A and E, superoxide dismutase and catalase were significantly lower and glutathione peroxidase significantly higher in LBW babies compared to controls, with the lowest levels found in babies showing more severe growth restriction (<2000 g). We conclude that LBW newborns are deficient in several important antioxidants which may predispose them to higher oxidative stress.

Oxidative stress in hypertension: association with antihypertensive treatment.

There is growing evidence that oxidative stress contributes to the pathogenesis of hypertension. Our aim was to measure oxidative stress in hypertensive subjects, and assess the potential confounding influences of antihypertensive therapy. Serum malondialdehyde and antioxidant levels were estimated in patients at the time of presentation and also after a antihypertensive therapy for 3 months. During the period of study no antioxidant/s was given to the patients and control subjects. Mean blood pressure values were altered in the hypertensive patients following antihypertensive therapy from their respective values observed at the time of presentation. Serum malondialdehyde levels were significantly higher in the hypertensive patients in comparison to control cases. The antioxidant activity of enzymes super oxide dismutase, glutathione and non enzymatic antioxidant levels of vitamins E and C were significantly lower in patients compared to controls. After 3 months of antihypertensive treatment all the above parameters showed reversal in the respective levels of serum malondialdehyde and antioxidant activity. Antihypertensive medications lower the blood pressure and thereby results in reduced oxidative stress which indicates that oxidative stress is not the cause, but rather a consequence, of hypertension.

Oxidative stress in neonatal hyperbilirubinemia.

We investigated the role of bilirubin as an antioxidant in neonatal hyperbilirubinemia (NNH) by measuring malondialdehyde (MDA) levels, a marker of oxidative stress and key antioxidant enzymes viz., superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) in otherwise healthy 70 term newborns with NNH and 20 control newborns without jaundice. Jaundiced newborns had significantly lower MDA but higher SOD, catalase and GPx levels. Furthermore, plasma bilirubin showed significant negative correlation with MDA but positive correlation with antioxidant enzyme activities. It is concluded that NNH is associated with lower oxidative stress.

RPGR ORF15 isoform co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin.

The ORF15 isoform of RPGR (RPGR(ORF15)) and RPGR interacting protein 1 (RPGRIP1) are mutated in a variety of retinal dystrophies but their functions are poorly understood. Here, we show that in cultured mammalian cells both RPGR(ORF15) and RPGRIP1 localize to centrioles. These localizations are resistant to the microtubule destabilizing drug nocodazole and persist throughout the cell cycle. RPGR and RPGRIP1 also co-localize at basal bodies in cells with primary cilia. The C-terminal (C2) domain of RPGR(ORF15) (ORF15(C2)) is highly conserved across 13 mammalian species, suggesting that it is a functionally important domain. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we show that this domain interacts with a 40 kDa shuttling protein nucleophosmin (NPM). The RPGR(ORF15)-NPM interaction was confirmed by (i) yeast two-hybrid analyses; (ii) binding of both recombinant and native HeLa cell NPM to RPGR(ORF15) fusion proteins in vitro; (iii) co-immunoprecipitation of native NPM, RPGR(ORF15) and RPGRIP1 from bovine retinal extracts and of native HeLa cell NPM and transfected RPGR(ORF15) from cultured cells and (iv) co-localization of NPM and RPGR(ORF15) at metaphase centrosomes in cultured cells. NPM is a multifunctional protein chaperone that shuttles between the nucleoli and the cytoplasm and has been associated with licensing of centrosomal division. RPGR and RPGRIP1 join a growing number of centrosomal proteins involved in human disease.

Oxidative stress in patients with essential hypertension.

BACKGROUND: Free oxygen radicals react with membrane lipids to form lipid hydroperoxides, a destructive process known as lipid peroxidation. Lipid hydroperoxides decompose to form a variety of products including malondialdehyde, which is used as an indicator of the oxidative damage of cells and tissues. Endogenous antioxidant enzymes such as superoxide dismutase counteract the oxidative damage from oxidative stress. There is increasing evidence that free radicals are involved in the pathogenesis of hypertension by altering endothelial function. We evaluated the oxidative stress and endogenous enzymatic antioxidant status in patients with essential hypertension before and 3 months after treatment with antihypertensives. METHODS: Fifty patients with essential hypertension attending the outpatient services of the Department of Medicine, Institute of Medical Sciences, Banaras Hindu University and 20 age- and sex-matched healthy controls were studied. The serum malondialdehyde and superoxide dismutase levels were measured in patients at the time of presentation and after 3 months of antihypertensive treatment. No antioxidants were given to the patients during the period of the study. RESULTS: The mean (SD) serum malondialdehyde level was found to be significantly higher (0.33 [0.07] mmol/L) in patients with hypertension compared with controls (0.21 [0.05] mmol/L; p < 0.001). This showed a significant decrease following antihypertensive therapy (0.23 [0.06] mmol/L; p < 0.001) compared with pre-treatment values. The serum superoxide dismutase activity was significantly lower in patients (6.93 [1.35] mg protein/ml of serum) compared with controls (20.12 [3.65] mg protein/ml serum; p < 0.001) at the time of presentation and, compared with the pre-treatment values, increased significantly after 3 months of treatment (10.66 [2.91] mg protein/ml of serum; p < 0.001). CONCLUSION: Our study shows that essential hypertension is associated with increased oxidative stress and reduced antioxidant status. Adequate control of blood pressure with antihypertensive therapy decreases oxidative stress and improves the antioxidant status in these patients.

Lipid peroxidation and antioxidant enzyme status in oral carcinoma patients.

OBJECTIVE: To measure the lipid peroxidation and endogenous antioxidant enzyme status in oral carcinoma and the protective role of exogenous antioxidants. MATERIAL AND METHODS: 20 new cases of histologically proven oral squamous cell carcinoma, 20 of leukoplakia and 20 age and sex matched healthy conrols were included. Intra oral pH of patients and controlled were measured by quantitative litmus paper test and serum was analysed for malonialdehyde (MDA), super oxide bismutase (SOD), catalase and glutathione peroxidase (GP). Patients with leukoplakia were treated with exogenous antioxidants for 3 months and the same were reassessed. RESULTS: Oral pH of oral cancer patients was neutral (PH-7) but that of leukoplakia and controls were mildly acidic (6.64 and 6.58 respectively). Serum malonialdehyde levels were highest in oral cancer group. With antioxidant enzymes super oxide bismutase, catalase and glutathione peroxidase different pattern was noticed. Antioxidant enzymes remained almost the same (P > 0.005 each) in patients with leukoplakia after 3 months of vitamin A,C and E. but there was marginal increase in catalase level (P<0.05). CONCLUSION: This study shows the positive benefit of vitamin (A,C,E) and nutrition supplementation on the antioxidant enzyme defense system hence prevention of oral carcinogenesis in patients with leukoplakia.

Oxidative stress and antioxidants status in peptic ulcer and gastric carcinoma.

Oxidative stress is believed to initiate and aggravate many diseases including peptic ulcers and gastric carcinoma. We observed an increase in rat gastric mucosal lipid peroxidation (LPO) and superoxide dismutase (SOD) and a decrease in catalase (CAT) levels in cold restraint stress-induced gastric ulceration while, in clinical peptic ulceration and gastric carcinoma patients, an increase in serum LPO and a tendency to decrease in SOD and CAT levels were observed. The result thus, indicated a positive correlation between free radical-induced oxidative stress both in gastric and duodenal ulcers and gastric carcinoma.

Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium.

Immature embryo-derived calli of spring wheat (Triticum aestivum L.) cv Veery5 were transformed using Agrobacterium tumefaciens strain LBA4404 carrying either binary vector pHK22 or superbinary vector pHK21, the latter carrying an extra set of vir genes--vir B, -C and -G. In both cases, transient beta-glucuronidase ( GUS) expression ranging from 35-63% was observed 3 days after co-cultivation, but 587 calli infected with pHK22/LBA4404 failed to produce a single stably transformed plant, whereas 658 calli infected with pHK21/LBA4404 gave rise to 17 transformants carrying both the GUS and bar genes. Regeneration media supplemented with 0.1 M spermidine improved the recovery of transformants from pHK21/LBA4404-infected calli from 7% to 24.2%, resulting in an increase in the overall transformation frequency from 1.2% to 3.9%. The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are (1) the use of superbinary vectors and (2) modification of the polyamine ratio in the regeneration medium. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Of the transformed plants, 35% showed single-copy insertion of the transgene as shown by both Southern analysis and the segregation ratios.

Elite Indica transgenic rice plants expressing modified Cry1Ac endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borer (Scirpophaga incertulas).

Bt-transgenics of elite indica rice breeding lines (IR-64, Pusa Basmati-1 and Karnal Local) were generated through biolistic or Agrobacterium-mediated approaches. A synthetic cry1Ac gene, codon optimised for rice and driven by the maize ubiquitin-1 promoter, was used. Over 200 putative transformants of IR-64 and Pusa Basmati-1 and 26 of the Karnal Local were regenerated following use of the hpt (hygromycin phosphotransferase) selection system. Initial transformation frequency was in the range of 1 to 2% for particle bombardment while it was comparatively higher (approximately 9%) for Agrobacterium. An improved selection procedure, involving longer selection on the antibiotic-supplemented medium, enhanced the frequency of Bt-transformants and reduced the number of escapes. Molecular evaluation revealed multiple transgene insertions in transformants, whether generated through biolistic or Agrobacterium. In the latter case, it was also observed that all genes on the T-DNA do not necessarily get transferred as an intact insert. Selected Bt-lines of IR-64 and Pusa Basmati-1, having Bt-titers of 0.1% (of total soluble protein) and above were evaluated for resistance against manual infestation of freshly hatched neonate larvae of yellow stem borers collected from a hot spot stem borer infested area in northern India. Several Bt-lines were identified showing 100% mortality of larvae, within 4-days of infestation, in cut-stem as well as vegetative stage whole plant assays. However, there was an occasional white head even among such plants when assayed at the reproductive stage. Results are discussed in the light of resistance management strategies for deployment of Bt-rice.