Arsenite and arsenate impact the oxidative status and antioxidant responses in Ocimum tenuiflorum L.

Biochemical responses of Ocimum tenuiflorum plants were studied upon exposure to arsenite (AsIII) and arsenate (AsV) for 1 to 10 d. Plants accumulated significant amounts of As in leaves (662 µg g(-1) dry weight; DW and 412 µg g(-1) DW in response to 100 µM AsIII and AsV exposure, respectively after 10 d). Consequently, fresh weight and growth of plants declined in a concentration dependent manner. Further, total chlorophyll and carotenoid contents also declined while oxidative stress markers increased, particularly on longer durations. Various antioxidant enzymes and thiols (cysteine and glutathione; GSH) showed significant and variable increases upon exposure to AsV and AsIII with the response being comparatively better in response to AsV. Proline increased significantly upon exposure to both AsIII and AsV. Plants thus tolerated high As concentrations through induced antioxidant machinery.

Analysis of arsenic induced physiological and biochemical responses in a medicinal plant, Withania somnifera.

Withania somnifera has been an important herb in the Ayurvedic and indigenous medical systems for centuries in India. However, these grow as weeds mostly in the wastelands, which receive contaminated water from municipal and industrial sources. In the present investigation, plants of Withania somnifera were exposed to various concentrations of arsenate (AsV) and arsenite (AsIII) (0, 10, 25, 50, 100 µM) for 10 days and analysed for accumulation of arsenic (As) and physiological and biochemical changes. Plants showed more As accumulation upon exposure to AsIII (320 µg g(-1) DW in roots and 161 µg g(-1) DW in leaves) than to AsV (173 µg g(-1) DW in roots and 100 µg g(-1) DW in leaves) after 10 days of treatment. Consequently, AsIII exposure caused more toxicity to plants as compared to that AsV, as evaluated in terms of the level of photosynthetic pigments and oxidative stress parameters (superoxide, hydrogen peroxide and lipid peroxidation), particularly at higher concentrations and on longer durations. Plants could tolerate low concentrations (variable for AsIII and AsV) until longer durations (10 days) and high concentrations for shorter durations (1-5 days) through increase in antioxidant enzymes and by augmented synthesis of thiols. In conclusion, As tolerance potential of Withania plants on one hand advocates its prospective use for remediation under proper supervision and on the other demonstrates possible threat of As entry into humans due to medicinal uses.

Effect of cadmium on growth, bacoside A, and bacopaside I of Bacopa monnieri (L.), a memory enhancing herb.

Bacopa monnieri (L.) is an important medicinal plant mainly known as a memory enhancing herb. It is important to see the effect of metal pollution on its active constituents. In this context, efforts have been made to observe the effect of Cd on the triterpenoid saponins bacoside A and bacopaside I in this plant. The influence of the metal on growth parameters like protein, chlorophyll content, and biomass has also been observed. It is interesting to note that the bacoside A and bacopaside I gradually increased by the Cd treatment up to 10 µM and then decreased at higher concentrations, that is, 50 and 100 µM, but the concentration of these components was more in all the treated plants as compared to control. On the contrary, protein, chlorophyll content, and biomass decreased with the increase in metal concentration and exposure duration due to metal toxicity.

Genetic diversity of pinus roxburghii sarg. Collected from different himalayan regions of India assessed by random amplified polymorphic DNA analysis.

Present study was aimed at molecular genetic fingerprint profile of 15 genotypes of three populations of Pinus roxburghii Sarg. from Himalayan regions of India using random amplified polymorphic DNA (RAPD) based markers. Needles of Pinus roxburghii Sarg. were collected from Dharamshala, Himachal Pradesh (HP), Nainital, Uttarakhand (UK) and Darjeeling, West Bengal (WB) regions of India. The samples were subjected to DNA extraction and RAPD analysis using oligonucleotide purification cartridge (OPC) primers. Out of 15 primers tested, nine primers gave scorable bands. Altogether 48 bands were obtained, out of which 43 were found to be polymorphic. Number of amplified fragments with RAPD primers ranged from four to eight with the size of amplicon ranging from 500 to 7,000bp. Investigation of natural diversity at intraspecies level was performed with 15 genotypes. Forty-eight amplification products were scored by RAPD and showed 89.58% polymorphism with a mean intrapopulation genetic diversity (Hpop) of 0.2754. A significant inter- and intrapopulation diversity was observed, with the percentage of polymorphic loci (Pp) ranging from 50.09 to 70.83%, Shannon's information index (I) from 0.3262 to 0.4689 and Nei's gene diversity (h) from 0.2032 to 0.3335 with mean Nei's gene diversity 0.377 and the overall estimate of gene flow being (Nm) 1.3555. Unweighted pair-group method with arithmetic average (UPGMA) analysis based Dendrogram showed single cluster. The variation amongst the samples of the three ecological regions can be attributed to varied climatic conditions and may help in conservation/future cultivation of these species.

Alemtuzumab vs. interferon beta-1a in early multiple sclerosis.

BACKGROUND: Alemtuzumab, a humanized monoclonal antibody that targets CD52 on lymphocytes and monocytes, may be an effective treatment for early multiple sclerosis. METHODS: In this phase 2, randomized, blinded trial involving previously untreated, early, relapsing-remitting multiple sclerosis, we assigned 334 patients with scores of 3.0 or less on the Expanded Disability Status Scale and a disease duration of 3 years or less to receive either subcutaneous interferon beta-1a (at a dose of 44 microg) three times per week or annual intravenous cycles of alemtuzumab (at a dose of either 12 mg or 24 mg per day) for 36 months. In September 2005, alemtuzumab therapy was suspended after immune thrombocytopenic purpura developed in three patients, one of whom died. Treatment with interferon beta-1a continued throughout the study. RESULTS: Alemtuzumab significantly reduced the rate of sustained accumulation of disability, as compared with interferon beta-1a (9.0% vs. 26.2%; hazard ratio, 0.29; 95% confidence interval [CI], 0.16 to 0.54; P<0.001) and the annualized rate of relapse (0.10 vs. 0.36; hazard ratio, 0.26; 95% CI, 0.16 to 0.41; P<0.001). The mean disability score on a 10-point scale improved by 0.39 point in the alemtuzumab group and worsened by 0.38 point in the interferon beta-1a group (P<0.001). In the alemtuzumab group, the lesion burden (as seen on T(2)-weighted magnetic resonance imaging) was reduced, as compared with that in the interferon beta-1a group (P=0.005). From month 12 to month 36, brain volume (as seen on T(1)-weighted magnetic resonance imaging) increased in the alemtuzumab group but decreased in the interferon beta-1a group (P=0.02). Adverse events in the alemtuzumab group, as compared with the interferon beta-1a group, included autoimmunity (thyroid disorders [23% vs. 3%] and immune thrombocytopenic purpura [3% vs. 1%]) and infections (66% vs. 47%). There were no significant differences in outcomes between the 12-mg dose and the 24-mg dose of alemtuzumab. CONCLUSIONS: In patients with early, relapsing-remitting multiple sclerosis, alemtuzumab was more effective than interferon beta-1a but was associated with autoimmunity, most seriously manifesting as immune thrombocytopenic purpura. The study was not powered to identify uncommon adverse events. (ClinicalTrials.gov number, NCT00050778.)

The multi-domain responder index: a novel analysis tool to capture a broader assessment of clinical benefit in heterogeneous complex rare diseases.

In traditional clinical trial design, efficacy is typically assessed using a single primary endpoint in a randomized controlled trial to detect an expected treatment effect of a therapy in a narrowly selected patient population. This accepted paradigm is based on clinical evaluations that may not actually capture the breadth of the impact of a disease, which is especially true in the setting of complex, multisystem, rare diseases with small, extremely heterogeneous patient populations. The multi-domain responder index (MDRI) is a novel approach that accommodates complex and heterogeneous disease manifestations and evaluates a broad array of clinical disease without impairing the power or rigor of a study to fully understand a treatment. The MDRI sums the scores corresponding to clinically significant thresholds of change for each component domain in each individual patient, capturing the mean clinically meaningful change across multiple domains within individuals. This novel approach combines and then sums the results of independent domain endpoint responder analyses into one responder score to provide a broad basis for the assessment of efficacy. The impact of a treatment across multiple, physiologically independent domains, can be assessed clinically, reducing the adverse impact of heterogeneity on trial outcomes and allowing eligibility criteria to enroll a wider range of patients, ultimately resulting in efficacy and safety assessments of a therapy across a broad group of heterogeneous patients in rare disease programs.Trial registration The following studies are referenced within this manuscript (CLINICALTRIALS.GOV registration numbers): NCT00912925; NCT00146770; NCT00067470; NCT00104234; NCT00069641; NCT02230566; NCT02377921; NCT02432144.

Placebo response in Parkinson's disease: comparisons among 11 trials covering medical and surgical interventions.

Placebo-associated improvements have been previously documented in small series of Parkinson's disease (PD) patients. Using a strict definition of placebo-associated improvement, we examined rates and timing of placebo responses to identify patient- and study-based characteristics, predicting positive placebo response in several PD clinical trials. We collected individual patient data from the placebo groups of 11 medical and surgical treatment trials involving PD patients with differing PD severities and placebo-assignment likelihoods. We defined a positive placebo response as > or = 50% improvement in total Unified Parkinson's Disease Rating Scale motor (UPDRSm) score or a decrease by > or = 2 points on at least two UPDRSm items compared to baseline. We calculated positive placebo response rates at early (3-7 weeks), mid (8-18 weeks), and late (23-35 weeks) stages of follow-up. Odds ratios for patient- and study-based characteristics were obtained from a model fitted using generalized estimating equations. There were 858 patients on placebo who met inclusion criteria for analysis. Three studies involved patients without need of symptomatic treatment, two involved patients without motor fluctuations needing symptomatic treatment, and six (three medical and three surgical) involved patients with motor fluctuations. The overall placebo response rate was 16% (range: 0-55%). Patients with higher baseline UPDRSm scores and studies that focused on PD with motor fluctuations, surgical interventions, or those with a higher probability of placebo assignment showed increased odds of positive placebo response. Placebo responses were temporally distributed similarly during early, mid, and late phases of follow-up. Placebo-related improvements occur in most PD clinical trials and are similarly distributed across all 6 months of follow-up. Recognition of factors that impact placebo response rates should be incorporated into individual study designs for PD clinical trials.

Clinical and prognostic significance of serum magnesium concentration in patients with severe chronic congestive heart failure: the PROMISE Study.

OBJECTIVES: The aim of this study was to determine the prognostic significance of alterations in serum magnesium in patients with moderate to severe congestive heart failure. BACKGROUND: Reductions in serum magnesium have been postulated to play a role in promoting arrhythmias and to have an adverse impact on survival in congestive heart failure, although support for this postulate is lacking. METHODS: Serum magnesium levels were measured in 1,068 patients enrolled in a survival study of class III or IV heart failure at the time of double-blind randomization to milrinone, a phosphodiesterase inhibitor, or placebo. All patients received conventional therapy with digoxin, diuretic drugs and a converting enzyme inhibitor throughout the trial. The median follow-up period was 6.1 months (range 1 day to 20 months). RESULTS: Patients with high serum magnesium (defined as > or = 1.9 mEq/liter, n = 242) were less likely to survive than were patients with a normal magnesium level (n = 627) (p < 0.05, risk ratio = 1.41). Patients with a low magnesium level (defined as < or = 1.5 mEq/liter, n = 199) had no difference in survival compared with the group with a normal magnesium level (p = NS, risk ratio = 0.89). At baseline, the patients in the high magnesium group were older and had more severe functional and renal impairment. An analysis after adjustment for these variables demonstrated no difference in survival comparing the low, normal and high magnesium groups. Although the three groups had no difference in frequency of ventricular tachycardia, length of longest run or frequency of ventricular premature beats on baseline Holter monitoring, the group with hypomagnesemia had more frequent ventricular couplets. CONCLUSIONS: Serum magnesium does not appear to be an independent risk factor for either sudden death or death due to all causes in patients with moderate to severe heart failure. Hypomagnesemia is associated with an increase in the frequency of certain forms of ventricular ectopic activity, but this is not associated with an increase in clinical events. The higher mortality rate among the patients with hypermagnesemia is attributable to older age, more advanced heart failure and renal insufficiency.

Recommendations for the development of rare disease drugs using the accelerated approval pathway and for qualifying biomarkers as primary endpoints.

For rare serious and life-threatening disorders, there is a tremendous challenge of transforming scientific discoveries into new drug treatments. This challenge has been recognized by all stakeholders who endorse the need for flexibility in the regulatory review process for novel therapeutics to treat rare diseases. In the United States, the best expression of this flexibility was the creation of the Accelerated Approval (AA) pathway. The AA pathway is critically important for the development of treatments for diseases with high unmet medical need and has been used extensively for drugs used to treat cancer and infectious diseases like HIV.In 2012, the AA provisions were amended to enhance the application of the AA pathway to expedite the development of drugs for rare disorders under the Food and Drug Administration Safety and Innovation Act (FDASIA). FDASIA, among many provisions, requires the development of a more relevant FDA guidance on the types of evidence that may be acceptable in support of using a novel surrogate endpoint. The application of AA to rare diseases requires more predictability to drive greater access to appropriate use of AA for more rare disease treatments that might not be developed otherwise.This white paper proposes a scientific framework for assessing biomarker endpoints to enhance the development of novel therapeutics for rare and devastating diseases currently without adequate treatment and is based on the opinions of experts in drug development and rare disease patient groups. Specific recommendations include: 1) Establishing regulatory rationale for increased AA access in rare disease programs; 2) Implementing a Biomarker Qualification Request Process to provide the opportunity for an early determination of biomarker acceptance; and 3) A proposed scientific framework for qualifying biomarkers as primary endpoints. The paper's final section highlights case studies of successful examples that have incorporated biomarker endpoints into FDA approvals for rare disease therapies. The focus of this paper is on the situation in the Unites States, but the recommendations are reasonably applicable to any jurisdiction.

Analysis of quality of life data from a randomized, placebo-controlled heart-failure trial.

In a randomized, double-blind, placebo-controlled, 3-months trial involving 111 congestive heart failure patients, one non-validated and three validated Quality of Life (QL) instruments were administered. Two randomized treatment groups were evaluated, one with 62 patients who continued on standard therapy and the other with 49 patients whose standard therapy was replaced by placebo. The data from Patient's Self-rating Scale (a non-validated instrument) and Spitzer's QL index showed a significant difference between the two treatment groups for an overall effect. There were no significant differences between two treatment groups for Sickness Impact Profile (SIP) and Quality of Well-Being (QWB). For analyzing the multiple components in a QL instrument, the global statistics as suggested by O'Brien were applied to compare the two treatment groups. Univariate statistics complemented the global methods. The general use of global statistics in analyzing QL data is recommended.

Face mask application as a tool to diminish the particulate matter mediated heavy metal exposure among citizens of Lucknow, India.

Traffic related fine particulate emissions, enriched in metal contents, are directly linked to respiratory disorders in human subjects. In view of the growing traffic related emissions in India, the present study was undertaken to estimate the heavy metal exposure among non-occupationally exposed two vehicle riders of Lucknow City and related health effects via application of face masks (FMs) fitted with cellulose nitrate filters and measuring the peak respiratory flow rate (PEFR). Carefully selected 200 volunteers (asymptomatic n=154 and symptomatic n=46) were advised to use FMs during their deriving time for 30 days and PEFR test was conducted on each subject at the beginning, i.e. 0 day, and at end of the study period, i.e. 30 days. On completion of the prescribed study period, filters from the used FMs were collected, acid leached and analyzed for Fe, Mn, Cu, Zn, Pb, Ni, Cr and Cd. Asymptomatic and symptomatic subject groups were further divided into two age groups of 15-40 years and 41-68. Pb, Cu and Cd were significantly higher in lower age group (15-40) of symptomatic group and Cr was in asymptomatic group. Negative associations were observed between metals viz. Pb (r=-0.39, p<0.001), Cd (r=-0.26, p<0.001), Fe (r=-0.37, p<0.001), Mn (r=-0.15, p<0.05) and the lung functioning. 30 days PEFR of all subjects were higher by nearly 10% than 0 day in all 200 samples irrespective of age and symptomatic nature of the subject. The improvement could also be due to metals and other organic species, not analyzed herein. Nevertheless the results indicate that FM usage has a role to play for immediate, if not ultimate, improvement in public health and need further studies.

Phytochelatins and antioxidant systems respond differentially during arsenite and arsenate stress in Hydrilla verticillata (L.f.) Royle.

Serious contamination of aquatic systems by arsenic (As) in different parts of the world calls for the development of an in situ cost-effective phytoremediation technology. In the present investigation, plants of Hydrilla verticillata (L.f.) Royle were exposed to various concentrations of arsenate (As(V)) (0-250 microM) and arsenite (AsIII) (0-25 microM) and analyzed for accumulation responses vis-à-vis biochemical changes. Total As accumulation was found to be higher in plants exposed to AsIII (315 microg g(-1) dw at 25 microM) compared to As(V) (205 microg g(-1) dw at 250 microM) after 7 d of treatment. Plants tolerated low concentrations of As(III) and As(V) by detoxifying the metalloid through augmented synthesis of thiols such as phytochelatins and through increased activity of antioxidant enzymes. While As(V) predominantly stimulated antioxidant enzyme activity, As(III) primarily caused enhanced levels of thiols. The maximum amount of As chelated by PCs was found to be about 39% in plants exposed to As(III) (at 10 microM) and 35% in As(V) exposed plants (at 50 microM) after 4 d. Only the respective highest concentrations of As(III) (25 microM) and As(V) (250 microM) proved toxic for normal plant growth after prolonged treatment. Thus, H. verticillata forms a promising candidate for the phytoremediation of As contaminated water.

Applications of global statistics in analysing quality of life data.

Quality of life (QOL) instruments usually consist of a number of components, each of which deals specifically with a particular functionally related dysfunction. In a clinical trial whose primary aim is the evaluation of the treatment by means of QOL instruments, analysis of each of the components usually consists of either univariate analysis of variance (ANOVA) or some non-parametric methods. This multiple testing approach can produce an increase in false positive findings. One attempt to correct for this is the Bonferroni adjustment. Another approach is to apply global statistics (parametric or non-parametric) for the null hypothesis of no treatment difference versus the alternative hypothesis that one treatment is uniformly better than the other for QOL instruments as a whole. Data from a randomized double-blind trial of 111 congestive heart failure patients, which involved four QOL instruments, were analysed with univariate ANOVA, Bonferroni adjustment, parametric and non-parametric global statistics. The global statistics complemented the univariate methods and made the presentation of QOL data very effective. I recommend the general use of global statistics in analysis of QOL data.

Application of new two-sample tests to data from a randomized placebo-controlled heart-failure trial.

In a randomized placebo-controlled double-blind trial of 230 congestive heart failure patients, four treatments were evaluated for efficacy, with exercise tolerance time (ETT) as the primary outcome. Various two-sample tests were applied to the analysis of ETT data. It is shown in this paper that the conventional two-sample tests (t and rank-sum) are insensitive to situations where the effect of the experimental therapy is not consistent across a patient population. Tests recommended by O'Brien are more appropriate for these data. It is also shown that the application of the O'Brien tests led to the identification of sub-groups where the observed effect of the experimental therapy was most pronounced.