Improved testing inference in mixed linear models

Mixed linear models are commonly used in repeated measures studies. They account for the dependence amongst observations obtained from the same experimental unit. Often, the number of observations is small, and it is thus important to use inference strategies that incorporate small sample corrections. In this paper, we develop modified versions of the likelihood ratio test for fixed effects inference in mixed linear models. In particular, we derive a Bartlett correction to such a test, and also to a test obtained from a modified profile likelihood function. Our results generalize those in [Zucker, D.M., Lieberman, O., Manor, O., 2000. Improved small sample inference in the mixed linear model: Bartlett correction and adjusted likelihood. Journal of the Royal Statistical Society B, 62,827-838] by allowing the parameter of interest to be vector-valued. Additionally, our Bartlett corrections allow for random effects nonlinear covariance matrix structure. We report simulation results which show that the proposed tests display superior finite sample behavior relative to the standard likelihood ratio test. An application is also presented and discussed. (C) 2008 Elsevier B.V. All rights reserved.

Skovgaard`s adjustment to likelihood ratio tests in exponential family nonlinear models

Likelihood ratio tests can be substantially size distorted in small- and moderate-sized samples. In this paper, we apply Skovgaard`s [Skovgaard, I.M., 2001. Likelihood asymptotics. Scandinavian journal of Statistics 28, 3-321] adjusted likelihood ratio statistic to exponential family nonlinear models. We show that the adjustment term has a simple compact form that can be easily implemented from standard statistical software. The adjusted statistic is approximately distributed as X(2) with high degree of accuracy. It is applicable in wide generality since it allows both the parameter of interest and the nuisance parameter to be vector-valued. Unlike the modified profile likelihood ratio statistic obtained from Cox and Reid [Cox, D.R., Reid, N., 1987. Parameter orthogonality and approximate conditional inference. journal of the Royal Statistical Society B49, 1-39], the adjusted statistic proposed here does not require an orthogonal parameterization. Numerical comparison of likelihood-based tests of varying dispersion favors the test we propose and a Bartlett-corrected version of the modified profile likelihood ratio test recently obtained by Cysneiros and Ferrari [Cysneiros, A.H.M.A., Ferrari, S.L.P., 2006. An improved likelihood ratio test for varying dispersion in exponential family nonlinear models. Statistics and Probability Letters 76 (3), 255-265]. (C) 2008 Elsevier B.V. All rights reserved.

Useful Strategies for Algal Volatile Analysis

The production of volatile organic compounds (VOC) by plants is well known. However, few scientific groups have studied VOC produced by green, brown and red algae. Headspace collection of volatiles and solid phase microextraction, as well as the traditional extraction by hydrodistillation combined with analytical chromatographic techniques (i.e., GC-MS), have significantly improved the investigation of VOC from plants and algae. The major volatile compounds found in seaweeds are hydrocarbons, terpenes, phenols, alcohols, aldehydes, ketones, esters, fatty acids and halogen or sulfur-containing compounds. This article presents an overview of VOC isolated from and identified in marine macro-algae. Focus is given to non-halogenated and non-sulfur volatile compounds, as well as strategies to analyze and identify algal VOC by GC-MS.

Biochemical composition of two red seaweed species grown on the Brazilian coast

BACKGROUND: Algae species have been used as an important source of food because they are highly nutritive considering their vitamin, protein, mineral, fiber, essential fatty acid and carbohydrate contents. However, a large number of seaweeds have been poorly studied, especially Brazilian species. Two red macroalgae species from the Brazilian coast (Plocamium brasiliense and Ochtodes secundiramea) were assessed with respect to their total lipid, fatty acid, total nitrogen, protein, amino acid and total carbohydrate contents. RESULTS: The total lipid contents (dry weight) were 36.3 and 35.4 g kg(-1); fatty acid contents were 9.3 and 12.1 g kg(-1); total nitrogen contents were 37.4 and 24.9 g kg(-1); protein contents were 157.2 and 101.0 g kg(-1); amino acid contents were 127.5 and 91.4 g kg(-1); and total carbohydrate contents were 520.3 and 450.7 g kg(-1) for P. brasiliense and O. secundiramea, respectively. CONCLUSION: Considering these compositions, both algae species were determined to have sources of protein, essential amino acids and carbohydrates similar to the edible seaweeds Laminaria japonica and Palmaria palmata. (C) 2011 Society of Chemical Industry

A theoretical and mass spectrometry study of the fragmentation of mycosporine-like amino acids

In the present study, the mycosporine-like amino acids (MAAs) were isolated from the marine red alga Gracilaria tenuistipitata and analysed by high-resolution accurate-mass sequential mass spectrometry (MSn). In addition to the proposed fragmentation mechanism based on the MSn analysis, it is clearly demonstrated that the elimination of mass 15 is a radical processes taking place at the methoxyl substituent of the double bond. This characteristic loss of a methyl radical was studied by theoretical calculations and the homolytic cleavage of the O-C bond is suggested to be dependent on the bond weakening. The protonation site of the MAAs was indicated by analysis of the Fukui functions and the relative Gibbs energies of the several possible protonated forms. (C) 2008 Elsevier B.V. All rights reserved.

Co-stressors chilling and high light increase photooxidative stress in diuron-treated red alga Kappaphycus alvarezii but with lower involvement of H(2)O(2)

Diuron is one of the most commonly found N-phenylurea herbicides in marine/estuarine waters that promotes toxic effects by inhibiting photosynthesis and affecting the production of reactive oxygen species (ROS) in autotrophs. Since photo- and thermoacclimation are also ROS-mediated processes, this work evaluates a hypothetical additive effect of high light (HL) and chilling (12 degrees C) on 50 nM diuron toxicity to the highly-photosynthetically active apices of the red alga Kappaphycus alvarezii. Additive inhibition of photosynthesis was mainly evidenced by significant decreases of quantum yield of photosystem II and electron transfer rates upon co-stressors exposure to diuron-treated algae. Under extreme 12 degrees C/HL/diuron conditions, unexpected lower correlations between H(2)O(2) concentrations in seawater and radical-sensitive protein thiols were concomitantly measured with the highest indexes of photoinhibition (parameter beta). Altogether, these data support the hypothesis that co-stressors chilling/HL additively inhibit photosynthesis in diuron-exposed K. alvarezii but with less involvement of H(2)O(2) in injury effects than with only chilling or HL. (C) 2010 Elsevier Inc. All rights reserved.

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with ophthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C(18) stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L(-1) phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L-1 phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, SO:SO and 65:35. At a flow rate of 10 mu L s(-1) a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 mu L s(-1) such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 mu mol L(-1) for Tyr to 0.51 mu mol L(-1) for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%. (C) 2008 Elsevier B.V. All rights reserved.

Metabolomic assessment with CE-MS of the nutraceutical effect of Cystoseira spp extracts in an animal model

There is a need of scientific evidence of claimed nutraceutical effects, but also there is a social movement towards the use of natural products and among them algae are seen as rich resources. Within this scenario, the development of methodology for rapid and reliable assessment of markers of efficiency and security of these extracts is necessary. The rat treated with streptozotocin has been proposed as the most appropriate model of systemic oxidative stress for studying antioxidant therapies. Cystoseira is a brown alga containing fucoxanthin and other carothenes whose pressure-assisted extracts were assayed to discover a possible beneficial effect on complications related to diabetes evolution in an acute but short-term model. Urine was selected as the sample and CE-TOF-MS as the analytical technique to obtain the fingerprints in a non-target metabolomic approach. Multivariate data analysis revealed a good clustering of the groups and permitted the putative assignment of compounds statistically significant in the classification. Interestingly a group of compounds associated to lysine glycation and cleavage from proteins was found to be increased in diabetic animals receiving vehicle as compared to control animals receiving vehicle (N6, N6, N6-trimethyl-L-lysine, N-methylnicotinamide, galactosylhydroxylysine, L-carnitine, N6-acetyl-N6-hydroxylysine, fructose-lysine, pipecolic acid, urocanic acid, amino-isobutanoate, formylisoglutamine. Fructoselysine significantly decreased after the treatment changing from a 24% increase to a 19% decrease. CE-MS fingerprinting of urine has provided a group of compounds different to those detected with other techniques and therefore proves the necessity of a cross-platform analysis to obtain a broad view of biological samples.

Testing for Seasonal Unit Roots when Residuals Contain Serial Correlations under HEGY Test Framework

This paper introduces a corrected test statistic for testing seasonal unit roots when residuals contain serial correlations, based on the HEGY test proposed by Hylleberg,Engle, Granger and Yoo (1990). The serial correlations in the residuals of test regressionare accommodated by making corrections to the commonly used HEGY t statistics. Theasymptotic distributions of the corrected t statistics are free from nuisance parameters.The size and power properties of the corrected statistics for quarterly and montly data are investigated. Based on our simulations, the corrected statistics for monthly data havemore power compared with the commonly used HEGY test statistics, but they also have size distortions when there are strong negative seasonal correlations in the residuals.

Purification and analysis of protein synthetic initiation factors from Volvox Carteri

Volvox carteri, a multi-celled green algae, can grow synchronously given a sixteen hour light period followed by an eight hour dark period, a cycle which is repeated for a 48 hour growth cycle total. Near the end of each light period, reproductive cells divide rapidly resulting in the differentiation of ceIls. When the dark period begins, this differentiation stops and the cells remain dormant with little protein synthesis or differentiation occurring. Immediately after the lights come back on, however, the cells again undergo rapid protein synthesis and complete their differentiation. Previous studies have concluded that Volvox carteri discontinue protein synthesis during the dark phase due to regulation at the translational level and not the transcriptional level. Therefore, the inhibition of protein synthesis does not lie in the transfer of the protein coding sequence from DNA to mRNA, but rather in the transfer of this information from the mRNA to the ribosomes. My research examined this translational regulation to determine the factor(s) causing the discontinuation of protein synthesis during the dark phase. Evidence from other research further suggests that the control of translation lies in the initiation step rather than the elongation step. Eukaryotic initiation factors aid in the binding of the ribosomal subunits to the mRNA to initiate protein synthesis. It is known that initiation factors can be modified by phosphorylation, regulating their activity. Therefore, my study focused upon isolating some of these initiation factors in order to determine whether or not such modifications are responsible for the inhibition of dark phase protein synthesis in Volvox carteri.

Efeitos de variáveis abióticas na composição química de Gelidium Crinale (Gelidiaceae, Rhodophyta) em cultivo unialgal

Os efeitos individuais e interativos dos parâmetros ambientais físicos e químicos, como temperatura, intensidade luminosa, salinidade e concentração de fósforo inorgânico dissolvido na água do mar, na produção de proteínas, carboidratos, acúmulo de fósforo tecidual e taxa de absorção do fósforo inorgânico disponível no meio de cultura em Gelidium crinale (Turner) Lamouroux, foram investigados durante um período de sete dias de cultivo laboratorial, em condições controladas. A ação dos parâmetros abióticos foi analisada de três maneiras diferentes. A primeira avaliação integrou a ação de temperatura, intensidade luminosa e fósforo inorgânico dissolvido, mantendo-se fixa a salinidade em 25 ups, onde se constatou que em todos os componentes químicos algais ocorreram interações de terceira ordem. O incremento de 2,28 a 2,67 % nos teores de proteínas foram obtidos à temperatura de 25 °C e 12 μmol m-2 s-1 de intensidade luminosa, diminuindo com a elevação da intensidade luminosa para 40 μmol m-2 s-1. Para carboidratos, ocorreram interações significativas entre os três parâmetros, com um aumento de 6,85 % sendo registrado a 25 °C de temperatura, 24 μmol m-2 s-1 de intensidade luminosa e 10,0 μM de fósforo inorgânico. O aumento máximo na taxa de fósforo tecidual (0,56 %) ocorreu em talos cultivados nas menores temperatura e intensidade luminosa e na maior concentração de fósforo inorgânico dissolvido. Com relação à intensidade luminosa, foi observada uma correlação negativa entre proteínas e carboidratos. A segunda avaliação estabeleceu a ação independente e sinérgica de temperatura, salinidade e fósforo inorgânico disponível no meio de cultivo, fixando-se a intensidade luminosa em 24 μmol m-2s-1. A maior produção de proteínas ocorreu em cultivos onde a temperatura foi de 25 °C, com uma concentração de 5,0 e 10,0 μM de fósforo inorgânico dissolvido e salinidade entre 15 e 20 ups, cujos valores médios do incremento variaram entre 2,62 a 2,83 % peso seco de alga, resultando em uma interação de terceira ordem altamente significativa. Para carboidratos a elevação de 6,85 % em sua concentração está associada à maior temperatura (25 °C), maior salinidade (25 ups) e maior quantidade de fósforo inorgânico disponível no meio de cultivo (10,0 μM). Contudo, não foi observada uma interação de terceira ordem através da análise estatística. Para esta biomolécula observaram-se interações de segunda ordem altamente significativa (P < 0,005) entre temperatura e diferentes concentrações de fósforo inorgânico e entre temperatura e salinidade (P < 0,000). O acúmulo de fósforo nos talos da alga foi menor durante os cultivos em que a salinidade foi de 25 ups,nas temperaturas de 20 e 25 °C e concentração de fósforo disponível de 2,5 μM, com percentuais entre 0,08 a 0,11 % em peso de cinzas. O maior incremento ocorreu na menor temperatura, associada à baixa salinidade e alta concentração de fósforo inorgânico no meio. O coeficiente de correlação de Pearson revelou correlações positivas, altamente significativas (P < 0,001) entre teor de proteína, temperatura e disponibilidade de fósforo inorgânico no meio de cultivo. Para carboidratos, as correlações foram positivas com os três parâmetros abióticos. Para fósforo tecidual somente com o fósforo inorgânico disponível no cultivo foi que ocorreu uma relação positiva; com os outros dois parâmetros esta correlação foi negativa. Entre os componentes químicos encontrados nas algas, proteínas e carboidratos apresentaram uma relação positiva, porém fósforo tecidual apresentou uma correlação negativa com ambos, embora com proteínas esta relação não tenha sido significativa. A terceira avaliação estudou a ação individual e o sinergismo entre os parâmetros ambientais, temperatura, intensidade luminosa e salinidade, a uma concentração fixa de fósforo inorgânico disponível no meio de cultivo (10,0 μM), sobre a composição química, bem como na taxa de absorção de fósforo inorgânico disponível. Observou-se a ocorrência de interações de terceira ordem em todos as variáveis estudadas. O teor de proteínas apresentou um aumento de 3,72 % durante o período de cultivo, passando de 20,63 % antes do cultivo, para 24,35 % após o término do experimento, principalmente nas condições de 25 °C de temperatura, 12 μmol m-2s-1 de intensidade luminosa e 15 ups de salinidade. Para carboidratos, nas condições de baixa intensidade luminosa (12 μmol m-2s- 1), a uma temperatura de 20 °C e salinidades de 10 e 15 ups, foram registrados valores inferiores à amostra controle, caracterizando um consumo desta biomolécula por parte das algas. Nestas mesmas condições ambientais, foram registrados os maiores teores de fósforo tecidual, variando entre 0,86 a 1,09 % do peso das cinzas. As maiores taxas de absorção do fósforo do meio ocorreram na salinidade de 25 ups e 25 °C de temperatura, diminuindo da intensidade luminosa de 12 μmol m-2s-1 para 40 μmol m-2s-1. As maiores concentrações de fósforo inorgânico residual na água do meio de cultivo ocorreram nas salinidades de 10 e 15 ups, em todas as intensidade luminosas e temperaturas estudadas. Através do coeficiente de correlação de Pearson, observou-se que os teores de proteínas apresentaram uma forte correlação negativa com a intensidade luminosa e positiva com a temperatura e salinidade, embora com esta última não tenha sido significativa. Para carboidratos, as correlações com os parâmetros abióticos foram todas positivas. Correlações negativa e positiva, não significativas, foram observadas entre esta biomolécula e o teor de proteínas e a taxa de absorção de fósforo disponível no meio, respectivamente. Por outro lado, com fósforo tecidual, ocorreu uma correlação negativa, altamente significativa. Este estudo mostra o estado fisiológico de Gelidium crinale e contribui para o estabelecimento das melhores condições de cultivo para produção de proteína, carboidrato e fósforo tecidual e indicação do uso racional de nutrientes, fornecendo informações para a otimização de processos de maricultura, tanto em termos de cultivo bem sucedido de algas, quanto de redução no impacto sobre o ambiente.

Semiparametric estimation and inference using doubly robust moment conditions

We study semiparametric two-step estimators which have the same structure as parametric doubly robust estimators in their second step. The key difference is that we do not impose any parametric restriction on the nuisance functions that are estimated in a first stage, but retain a fully nonparametric model instead. We call these estimators semiparametric doubly robust estimators (SDREs), and show that they possess superior theoretical and practical properties compared to generic semiparametric two-step estimators. In particular, our estimators have substantially smaller first-order bias, allow for a wider range of nonparametric first-stage estimates, rate-optimal choices of smoothing parameters and data-driven estimates thereof, and their stochastic behavior can be well-approximated by classical first-order asymptotics. SDREs exist for a wide range of parameters of interest, particularly in semiparametric missing data and causal inference models. We illustrate our method with a simulation exercise.

Estudo comparativo de dois tipos de cultivo: monocultivo (camarões) versus cultivo integrado (algas/camarões)

The integrated culture of seaweed and aquatic animals is an ancient practice in Asian countries. The expansion of this practice to western countries is consequence of the recognition of this system as a sustainable alternative that allows economical diversification and mitigation of environmental impacts generated by effluents of aquaculture. This study evaluated the growth of the seaweed Gracilaria caudata and of the shrimp Litopenaeus vannamei in monoculture (shrimps) and integrated culture (shrimps and algae) systems, and accessed the effect of the seaweed in the water quality. There were two treatments in the experiment: monoculture (shrimps) and integrated culture (shrimps/ algae). The organisms were cultured in 6 aquaria (10L) filled with seawater (35.0±0.0 PSU and 28.1±0.4°C) for 28 days. The nutrients of water (PO43-, NH4+, NO2-, NO3- and DIN), the biomass and the relative growth rate (RGR, % day-1) of seaweed and shrimps were measured weekly. The parameters pH, temperature, salinity and dissolved oxygen were measured daily. The concentration of NH4+ in integrated culture (62.8±25.2µM) was lower (Mann-Whitney p<0.001) than in monoculture (85.6±24.3µM). The mean of PO4- in monoculture (10.4±4.6µM) was markedly higher (Mann-Whitney; p=0.024) than that in integrated culture (8.7±4.1µM). The level of dissolved oxygen in integrated culture (6.0±0.6mg/L) was higher (t-Student; P=0.014) than that in shrimp monoculture (5.8±0.6mg/L). The mean values of the parameters pH, NO2-, NO3- and DIN were 7.5±0.2, 10.1±12.2µM, 24.5±3.2µM and 120.17±30.76µM in monoculture, and 7.5±0.2, 10.5±13.2µM, 27.4±3.5µM and 100.76±49.59µM in integrated culture. There were not differences in these parameters between treatments. The biomass and RGR of seaweed reached 15.0±1.9g and 7.4±2.8% day-1 at the end of the experiment. The performance of shrimp was favorable in monoculture (1.5±0.8g; 5.7±1.6% dia-1) and in integrated culture (1.5±0.7g; 5.2±1.2% dia-1), and the rate of survival was 100% in both treatments. The tolerance and favorable performance of Gracilaria caudata suggest that this seaweed might be integrated into shrimp (Litopenaeus vannamei) culture systems

Influência da profundidade e dos parâmetros ambientais sobre o desenvolvimento de Gracilaria birdiae (Rhodophyta, Gracilariales) em viveiro de camarão

This study evaluates the influence of depth and environmental parameters on the development of Gracilaria birdiae Plastino & Oliveira (Gracilariaceae Rhodophyta) in an organic shrimp pound (Litopenaeus vannamei) under euthrophical conditions. PVC structures (module) witch four ropes laden with 150 g of macroalgae each, were kept during 35 days at three different depths (surface, 10 and 20 cm depth). Wet biomass weighing and environmental parameters (temperature, salinity, turbidity, pH, transparence, precipitation, evaporation, insolation, accumulated solar radiation, nitrite, nitrate, ammonium and orthophosphate) were measured weekly. At all three proposed depths, the macroalgae displayed a higher biomass at the end of experiment than at the initial inoculations. The module kept at a 10 cm depth presented the greatest average biomass (186,3), followed by that kept at 20 cm (180,4 g) and the surface module (169,9 g). Biomass variations showed algae to suffer the direct effects of depths. Biomass loss was associated with the factors that influence light penetration, such as sediment deposits above the thallus, rate of evaporation and precipitation. The smallest loses occurred in the algae kept on surface (0,16%), followed by the algae kept at 20 cm (0,20%) and 10 cm (0,22%). The specific growth rate (SGR) of G. birdiae showed no significant difference between the three depths nor the sample periods. Nevertheless, the modules kept at 10 and 20 cm depths presented similar growth evolution, both growing 0,38%·per day-1, while the module kept on surface had an average SGR of 0,36%·day-1. The models related to growth rate demonstrated temperature, salinity, pH, orthophosphate, ammonium, precipitation and turbidity as the principal environmental parameters influencing the development of G. birdiae

Dieta do ouriço-branco, Tripneustes ventricosus (Lamarck, 1816), no Arquipélago de Fernando de Noronha, PE

Sea urchins are benthic macroinvertebrates that inhabit shallow coastal waters in tropical and temperate zones. Urchins are usually classified as generalists or omnivores as they can adjust their diet according to the food resources available in the environment. Due to the strong grazing pressure they may exert, urchins have an important role in marine ecosystems, occupying different trophic levels and stimulating the intensification of the dynamics of communities where they occur. In 2004, a monitoring program focused on the population dynamics of the white sea urchin, Tripneustes ventricosus, has been initiated in the Fernando de Noronha Archipelago. At the same time, a surprisingly lack of information on the species biology has been noted, despite their wide geographical distribution and economic importance in many parts of its range. Hence, this work was developed to provide information on the feeding habits of T. ventricosus in the archipelago. Ten specimens were collected between December 2006 and July 2007 at two sites of the archipelago, Air France and Sueste Bay for biometrics and analysis of gut contents. Test diameters ranged from 9.19 cm (± 1.1) to 10.08 cm (± 0.58). Calculated stomach repletion index (IRE) was higher (p <0.05) in the Air France site and also during January and July. The IRE was not correlated to the gonad index. Fifteen different species of algae were detected in a total of 120 stomachs examined: 4 Chlorophytas, 4 Phaeophytas and 6 Rhodophytas. Food diversity (p <0.05) was higher in December 2006 and January 2007. Although several items had a high frequency of occurrence, they were low represented in terms of weight, and consequently, had a low level of relative importance. The brown algae Dictyopteris spp and Dictyota spp, followed by the green algae Caulerpa verticillata accounted for the greatest importance in T. ventricosus diet, comprising about 90% of the consumed items