Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: Its gene structure, mRNA expression and recombinant protein

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Two unique and highly compartmentalized bay scallop Argopecten irradians superoxide dismutases: MnSOD and ecCuZnSOD, have been molecularly characterized in our previous study. To complete characterize the SOD family in A. irradians, a novel intracellular copper/zinc SOD from the A. irradians (Ai-icCuZnSOD) was obtained and characterized. The full-length cDNA of Ai-icCuZnSOD was 1047 bp with a 459 bp open reading frame encoding 152 amino acids. The genomic length of the Ai-icCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region containing many putative transcription factor binding sites were analyzed. Furthermore, quantitative reverse transcriptase real-time PCR (qRT-PCR) analysis indicated that the highest expression of the Ai-icCuZnSOD was detected in gill and the expression profiles in hemocytes of bay scallops challenged with bacteria Vibrio anguillarum and lipopolysaccharide (LPS) were different. The result presented an increased expression after injection with LPS whereas no significant changes were observed after V. anguillarum injection. A fusion protein containing Ai-icCuZnSOD was produced in vitro. The rAi-icCuZnSOD is a stable enzyme, retaining more than 80% of its activity between 10 and 60 degrees C and keeping above 88% of its activity at pH values between 5.8 and 9. Ai-icCuZnSOD is more stable under alkaline than acidic conditions. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

Effects of effective population size on the F-2 growth and survival of bay scallop Argopecten irradians irradians (Lamarck)

In 2002, six cohorts of broodstock bay scallop Argopecten irradians irradians (Ne=1, 2, 10, 30, 50 and control) were randomly chosen from a population of bay scallop to produce offspring. After one year rearing, with the progeny matured, the similar experiment was done to produce the F-2 generation. To determine the magnitude of Ne effects, the growth and survival rates in larvae and adult of six F2 groups were compared. Results showed that inbreeding depression existed not only in the Ne=1 group but also in the Ne=2 group. The growth and survival rates of the two groups were significantly lower than those of the other groups (Ne=10, 30, 50, control), and there were no significant differences among the latter (P>0.05). At the same time, the amount of depression in the Ne=1 group was significantly higher than that of the Ne=2 group (P<0.05). These results indicated that the low effective population size (Ne), which increases the possibility of inbreeding, could lead to some harmful effects on the offspring. So it is essential to maintain a high level of Ne in commercial seed production. Furthermore, as the high fecundity of bay scallop might lead to increased inbreeding, selecting broodstock from different growout sites is recommended.

Effects of starvation on larval growth, survival and metamorphosis of Ivory shell Babylonia formosae habei Altena et al., 1981 (Neogastropoda : Buccinidae)

The impact of starvation on larvae of Ivory shell Babylonia formosae habei was studied in a laboratory experiment. Newly hatched veligers showed considerable tolerance to starvation due to their endogenous yolk material, and time to the point-of-no-return (PNR; the threshold point during starvation after which larvae can longer metamorphose even if food is provided) was calculated to be 104.5 h. However, starvation still affected larval growth, survival, and metamorphosis. Mean shell length of larvae increased 49.77 mum day(-1) for nonstarved, but only 11.13 mum day (-1) for larvae starved for 108 h. After larvae began feeding, their growth rates rapidly recovered to the level of the nonstarved following short periods of starvation (less than 48 h), but were inhibited and unable to ever reach the level of the nonstarved when being starved beyond 48 h. Percent metamorphosis was 53.75% for the nonstarved, but all larvae died before 10 days for those starved for 108 h. Starvation not only affected larval time to reach metamorphosis, but also caused the delay in the time to metamorphosis. For the nonstarved, larvae took only 11.5 days to reach spontaneous metamorphosis, but they took 20 days to reach spontaneous metamorphosis when starved for 96 h, and this duration of delayed metamorphosis reached 8.5 days. Furthermore, the importance of yolk material for maintaining larval survival of B. formosae habei during starvation periods is also discussed. (C) 2004 Elsevier B.V. All rights reserved.

Necessity of dietary lecithin and eicosapentaenoic acid for growth, survival, stress resistance and lipoprotein formation in gilthead sea bream Sparus aurata

The substitution of dietary docosahexaenoic acid (DHA) with eicosapentaenoic acid (EPA) reduces larval growth in gilthead sea bream. However, the value of EPA when dietary DHA is able to meet the requirements of the larvae has not been sufficiently studied. Dietary phosphoacylgliceride levels also affect fish growth and it has been suggested that they enhance lipid transport in developing larvae. The present experiment was carried out to further study the effect of dietary lecithin and eicosapentaenoic acid on growth, survival, stress resistance,. larval fatty acid composition and lipid transport, when DHA is present in the microdiets of gilthead:sea bream. Eighteen thousand gilt-head sea bream larvae of 4.99+/-0.53 mm total length were fed three microdiets tested by triplicate: a control diet [2% soybean lecithin (SBL) and 2.89% EPA], a low EPA diet,(2% SBL and 1.63% EPA) and a no SBL diet (0% SBL and 2.71% EPA). Handling, temperature and salinity tests determined larval resistance to stress. The results show that when dietary DHA levels are high, but dietary arachidonic acid (ARA) levels are about 0.2%, EPA is necessary to improve larval growth, and survival. Larval EPA content, but not DHA or ARA, was affected by dietary EPA levels. Increased dietary EPA improved larval stress resistance to handling and temperature tests, which could be related to its possible role as a regulator of cortisol production whereas it did not affect stress resistance after salinity shock. Larvae fed the no SBL diet showed a lower lipid content characterized by a low proportion of saturated and monounsaturated fatty acids, together with a significant reduction in the appearance of lipoprotein particles in the lamina propria and in the size of such particles, denoting a critical reduction in dietary lipid transport and utilization, and lower larval growth and survival rates.

Growth and survival of reciprocal crosses between two bay scallops, Argopecten irradians concentricus Say and A-irradians irradians Lamarck

A complete diallel cross between two bay scallop populations, Argopecten irradians concentricus Say (M) and A. irradians irradians Lamarck (C), was carried out. Growth and survival were compared among hybrids and pure populations. No significant difference in the shell length was found among the four groups on the first day of D-larvae. On day 10, shell lengths of the two reciprocal crosses (CM, MC)(female x male ) were significantly greater than those of the CC (141.97 mu m) and MM (146.20 mu m) groups, with the growth rate of the MC (156.14 mu m) cross greater than that of the CM (155.35 mu m) cross. Also, heterosis for survival was significantly larger than that for growth. Both maternal origin and mating strategy had significant effects on growth and survival throughout the whole larval stage. Heterosis was also observed in later spat and adult stages. On day 170, the mean shell length, shell height and total weight of the CM cross were significantly larger than those of the other crosses (P<0.05). The results from this study indicate that hybridization between A. irradians concentricus and A. irradians irradians may be a promising way for genetic improvement of existing bay scallop brood stocks in China. (c) 2007 Elsevier B.V. All rights reserved.

Effects of diet, stocking density, and environmental factors on growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum larvae

A series of experiments was conducted to evaluate the effects of diet, stocking density, and environmental factors on growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum larvae. These experiments examined the following factors: diet (Isochrysts spp., Chlorella spp., and a mixture of Isochrysis spp. and Chlorella spp. [ 1: 1 w/w]), stocking density (5, 10, 15, and 20 larvae ml(-1)), light intensity (un-shaded, partially shaded, and fully shaded), water filtration (unfiltered and sand-filtered), water exchange (50% and 100% once every other day, 25%, 50%, and 100% once daily; 50% and 100% twice daily), and the use of substrate (with and without sand as the substrate). Results indicated that Chlorella spp. could replace 50% of Isochrysis spp. as a food source for the Manila clam larvae without affecting growth, survival, and metamorphosis. Larval growth decreased significantly with increasing stocking density. A density of 5-10 larvae ml(-1) appeared to be optimal for normal growth of Manila clam larvae. Neither diet nor stocking density used in the study had a significant effect on larval survival. Under partially shaded (light intensity = 1000-5000 lx) and fully shaded (light intensity <500 lx) conditions, larval growth was significantly faster than under direct sunlight (un-shaded). A water exchange rate of 50% twice daily provided optimum larval growth. Larvae grew significantly faster in the unfiltered water than in the sand-filtered water. Using sand as the substrate in the culture system significantly depressed the metamorphosis rate. The type and particle size of sand used as the substrate did not significantly affect growth and metamorphosis rates of the larvae. (C) 2005 Published by Elsevier B.V.

Effect of Tannic Acid on the Growth and Survival of Root Vles (Micortus oeconomus)

The objective of this study was to investigate the effects of tannic acid on the gworth and survival of small mammalian berbirvores. Measurements were conducted with weaned root voles fed with 3% or 6% tannic acid and 10% or 20% protein in their diets. The results indicated that the effect of tannic acid on growth rate of weaned root voles was greater when given a lower protein diet than a higher protein diet. After 20 d, with 10% protein diets, mean growth rates of the weaned voles fed with 3% or 6% tannic acid were -0.135 g/d and -0.25 g/d, respectively. When given 20% protein diet, mean growth rates of weaned root voles fed with 3% and 6% tannic acid for 20 d were 0.134 g/d and -0.116 g/d, respectively. Food utilization efficiencies of the voles fed with 3% and 6% tannic acid diets were significantly lower than that of the control diet at the level of 10% protein. When given the 20% protein diet, food utilization efficiencies of weaned voles fed with 6% tannic acid were significantly lower than that of the voles fed with 3% tannic acid diet or the control diet with the 10% protein diets, the average survival days of the weaned voles fed with 3% and 6% tannic acid diets decreased 26.23% and 49.36% compared to controls at the end of trial period, respectively. With 20% protein diets, the average survival of weaned voles given 6% tannic acid diet decreased 39.41% compared to controls at the end of trial period, although weaned voles given 3% tannic acid had a slight decrease of average survival days. The results of study suggested that tannins could substanitially affect the individual performance of weaned root voles.

Factors affecting the survival of sheep embryos after transfer within a MOET program

F. Baria, M. Khalid, W. Haresign, A. Murray and B. Merrell (2003). Factors affecting the survival of sheep embryos after transfer within a MOET program. Theriogenology, 59 (5/6), 1265-1275. Sponsorship: DEFRA RAE2008

Rapid compensatory evolution promotes the survival of conjugative plasmids

Conjugative plasmids play a vital role in bacterial adaptation through horizontal gene transfer. Explaining how plasmids persist in host populations however is difficult, given the high costs often associated with plasmid carriage. Compensatory evolution to ameliorate this cost can rescue plasmids from extinction. In a recently published study we showed that compensatory evolution repeatedly targeted the same bacterial regulatory system, GacA/GacS, in populations of plasmid-carrying bacteria evolving across a range of selective environments. Mutations in these genes arose rapidly and completely eliminated the cost of plasmid carriage. Here we extend our analysis using an individual based model to explore the dynamics of compensatory evolution in this system. We show that mutations which ameliorate the cost of plasmid carriage can prevent both the loss of plasmids from the population and the fixation of accessory traits on the bacterial chromosome. We discuss how dependent the outcome of compensatory evolution is on the strength and availability of such mutations and the rate at which beneficial accessory traits integrate on the host chromosome.

Autofagia

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

The analysis of Bcr-Abl—Nox signalling in chronic myeloid leukaemia

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterised by increased proliferation of haematopoietic stem cells. CML results following generation of the chimeric protein Bcr-Abl, a constitutively active tyrosine kinase which induces oncogenesis in part by promoting increased cell survival and proliferation. Since the development of Bcr-Abl-specific tyrosine kinase inhibitors (TKIs) there has been a substantial improvement in the clinical treatment of CML. Unfortunately, residual disease and the development of TKI resistance has become an ever growing concern, resulting in the need for a greater understanding of the disease in order to develop new treatment strategies. Interestingly, constitutive expression of the Bcr-Abl in CML is known to produce elevated levels of Reactive Oxygen Species (ROS) which are known to influence a variety of cellular processes. Previous studies have demonstrated that NADPH oxidase (Nox) activity contributes to intracellular-ROS levels in Bcr-Abl-positive cells, enhancing survival signalling. The objective of this study was to elucidate how Nox protein activity was influenced downstream of Bcr-Abl while examining how Nox-derived ROS influenced CML disease phenotype to identify the potential in targeting these proteins to improve CML treatment. These studies demonstrated that inhibition of Bcr-Abl signalling, led to a significant reduction in ROS levels which was concurrent with the GSK-3dependent, post-translational down-regulation of the small membrane-bound protein p22phox, an essential component of the Nox complex. siRNA knockdown of p22phox identified it to have a significant role in cellular proliferation and cell viability, demonstrating the importance of Nox protein activity in CML disease phenotype. Furthermore, removal of p22phox was demonstrated to make cells significantly more susceptible to Bcr-Abl-specific TKI treatment, while pharmacological silencing of Nox activity in combination with TKIs was demonstrated to produce substantial, synergistic increases in cell death through augmentation of apoptosis, demonstrating the therapeutic potential of targeting Nox proteins in combination with Bcr-Abl inhibition.

Survival analysis of adult tuberculosis disease

Background: We conducted a survival analysis of all the confirmed cases of Adult Tuberculosis (TB) patients treated in Cork-City, Ireland. The aim of this study was to estimate Survival time (ST), including median time of survival and to assess the association and impact of covariates (TB risk factors) to event status and ST. The outcome of the survival analysis is reported in this paper. Methods: We used a retrospective cohort study research design to review data of 647 bacteriologically confirmed TB patients from the medical record of two teaching hospitals. Mean age 49 years (Range 18–112). We collected information on potential risk factors of all confirmed cases of TB treated between 2008–2012. For the survival analysis, the outcome of interest was ‘treatment failure’ or ‘death’ (whichever came first). A univariate descriptive statistics analysis was conducted using a non- parametric procedure, Kaplan -Meier (KM) method to estimate overall survival (OS), while the Cox proportional hazard model was used for the multivariate analysis to determine possible association of predictor variables and to obtain adjusted hazard ratio. P value was set at <0.05, log likelihood ratio test at >0.10. Data were analysed using SPSS version 15.0. Results: There was no significant difference in the survival curves of male and female patients. (Log rank statistic = 0.194, df = 1, p = 0.66) and among different age group (Log rank statistic = 1.337, df = 3, p = 0.72). The mean overall survival (OS) was 209 days (95%CI: 92–346) while the median was 51 days (95% CI: 35.7–66). The mean ST for women was 385 days (95%CI: 76.6–694) and for men was 69 days (95%CI: 48.8–88.5). Multivariate Cox regression showed that patient who had history of drug misuse had 2.2 times hazard than those who do not have drug misuse. Smokers and alcohol drinkers had hazard of 1.8 while patients born in country of high endemicity (BICHE) had hazard of 6.3 and HIV co-infection hazard was 1.2. Conclusion: There was no significant difference in survival curves of male and female and among age group. Women had a higher ST compared to men. But men had a higher hazard rate compared to women. Anti-TNF, immunosuppressive medication and diabetes were found to be associated with longer ST, while alcohol, smoking, RICHE, BICHE was associated with shorter ST.

Cancer associated malnutrition: prevalence of cachexia, sarcopenia and impact on treatment outcomes, survival and health related quality of life

Malnutrition, sarcopenia and cancer cachexia (CC) are prevalent among cancer patients and can have detrimental effects on clinical outcomes such as quality of life (QoL) and overall survival. Cachexia is associated with lower tolerance for chemotherapy, which limits the total dose that can be delivered, the number of symptomatic responses and any survival advantage that might be accrued. Moreover, for the majority who do not respond, cachexia may be exacerbated by systemic chemotherapy, thus increasing the net symptom burden experienced by patients. The multitude of interactions between cancer location, treatments, nutritional status and QoL has never been thoroughly explored in an Irish cancer cohort. The objectives of this thesis were to further understand nutritional status, especially body composition in ambulatory cancer patients and determine the relationship between nutritional status using different assessment criteria and QoL, chemotherapy toxicity and survival among cancer patients undergoing chemotherapy. Results aimed to identify baseline factors that may be predictive of poor outcome, toxicities to chemotherapy and disease-free and overall survival. This thesis broadly divides into two sections. The first section (Chapters 3 & 4) focuses on improving our knowledge of the nutritional status of Irish cancer outpatients using a cross sectional study design. A study of 517 patients referred for chemotherapy was conducted using computed tomography (CT) imaging (body composition) and a survey that documented oncologic data, weight loss (WL) data and QoL data. We revealed that a significant proportion of Irish cancer patients undergoing chemotherapy experience unintentional WL over the previous 6 months (62%), sarcopenia (45%) and CC (43%), and the distribution of WL and nutritional risk were associated with site of primary tumour and treatment intent. Patients that had sarcopenia, nutritional risk, or CC had significantly reduced functional abilities, more symptoms and adverse global QoL. In the second section of this thesis (Chapters 5 & 6) the potential link between developing toxicity to antineoplastic regimens in patients with sarcopenia was conducted by way of retrospective studies. A retrospective serial CT analysis defined the prevalence of sarcopenia in patients with metastatic renal cell carcinoma (mRCC) and metastatic castrate resistant prostate cancer (mCRPC), which was then correlated with dose limiting toxicities of sunitinib and docetaxel respectively. Sarcopenia was prevalent in patients with mRCC and mCRPC, was an occult condition in patients with normal/high BMI, was associated with less treatment days, was a significant predictor of DLT in patients receiving sunitinib and a significant predictor of neutropenia and neurosensory toxicities in patients receiving docetaxel. This thesis attempted to address the underlying research deficiencies in Irish oncology nutritional data at national level. The findings from this thesis have implications for the planning of cancer care interventions and indicate that further research is required to improve nutritional screening, in particular for CC and sarcopenia, in the hope that timely intervention can improve both patient-centered and oncologic outcomes.

Evaluation of tumor response, disease control, progression-free survival, and time to progression as potential surrogate end points in metastatic breast cancer.

PURPOSE: Overall survival (OS) can be observed only after prolonged follow-up, and any potential effect of first-line therapies on OS may be confounded by the effects of subsequent therapy. We investigated whether tumor response, disease control, progression-free survival (PFS), or time to progression (TTP) could be considered a valid surrogate for OS to assess the benefits of first-line therapies for patients with metastatic breast cancer. PATIENTS AND METHODS: Individual patient data were collected on 3,953 patients in 11 randomized trials that compared an anthracycline (alone or in combination) with a taxane (alone or in combination with an anthracycline). Surrogacy was assessed through the correlation between the end points as well as through the correlation between the treatment effects on the end points. RESULTS: Tumor response (survival odds ratio [OR], 6.2; 95% CI, 5.3 to 7.0) and disease control (survival OR, 5.5; 95% CI, 4.8 to 6.3) were strongly associated with OS. PFS (rank correlation coefficient, 0.688; 95% CI, 0.686 to 0.690) and TTP (rank correlation coefficient, 0.682; 95% CI, 0.680 to 0.684) were moderately associated with OS. Response log ORs were strongly correlated with PFS log hazard ratios (linear coefficient [rho], 0.96; 95% CI, 0.73 to 1.19). Response and disease control log ORs and PFS and TTP log hazard ratios were poorly correlated with log hazard ratios for OS, but the confidence limits of rho were too wide to be informative. CONCLUSION: No end point could be demonstrated as a good surrogate for OS in these trials. Tumor response may be an acceptable surrogate for PFS.