Improved two‐stage group sequential procedures for testing a secondary endpoint after the primary endpoint achieves significance

In two‐stage group sequential trials with a primary and a secondary endpoint, the overall type I error rate for the primary endpoint is often controlled by an α‐level boundary, such as an O'Brien‐Fleming or Pocock boundary. Following a hierarchical testing sequence, the secondary endpoint is tested only if the primary endpoint achieves statistical significance either at an interim analysis or at the final analysis. To control the type I error rate for the secondary endpoint, this is tested using a Bonferroni procedure or any α‐level group sequential method. In comparison with marginal testing, there is an overall power loss for the test of the secondary endpoint since a claim of a positive result depends on the significance of the primary endpoint in the hierarchical testing sequence. We propose two group sequential testing procedures with improved secondary power: the improved Bonferroni procedure and the improved Pocock procedure. The proposed procedures use the correlation between the interim and final statistics for the secondary endpoint while applying graphical approaches to transfer the significance level from the primary endpoint to the secondary endpoint. The procedures control the familywise error rate (FWER) strongly by construction and this is confirmed via simulation. We also compare the proposed procedures with other commonly used group sequential procedures in terms of control of the FWER and the power of rejecting the secondary hypothesis. An example is provided to illustrate the procedures.     

Polyphasic characterization of Delftia acidovorans ESM-1, a facultative methylotrophic bacterium isolated from rhizosphere of Eruca sativa

In this study, one bacterial strain, ESM-1, was isolated from rhizosphere of Eruca sativa, growing in Al Hofouf, Saudia Arabia, after enrichment with methanol as a sole carbon and energy source in a batch culture. ESM-1 was characterized by a polyphasic approach. The strain was identified as Delftia acidovorans at similarity level of 99.9% of the 16S rRNA gene sequences. Results of the Biolog Gen III MicroPlate test system showed that strain ESM-1 reacted positively to 47 (50%) including the one-carbon compound formic acid, and partially positive to 6 (∼6.4%) out of the 94 different the traits examined. The total cellular fatty acids composition of the strain ESM-1 was (C_16:1ω7c/C_16:1ω6c) and C_16:0) and matched that of Delftia acidovorans at a similarity index of 0.9, providing a robustness to the ESM-1 identification. Furthermore, ESM-1 displayed a complex polar lipid profile consisting of phosphatidylethanolamine, phosphatidylglycerol, glycolipid, aminolipid, in addition to uncharacterized lipids. The DNA G+C content of the strain was 66.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain ESM1-1 was clearly clustered within the Delftia clade and constructed a monophyletic subcluster with Delftia acidovorans NBRC14950. The results addressed that ESM-1 is a facultative methylotrophic bacterium indigenous to Al Hofouf region and opens the door for potential biotechnological applications (e.g., bioremediation) of this strain, in future. Additionally, these findings assure that the total cellular fatty acid analysis and 16S rRNA gene are reliable tool for bacterial characterization and identification.     

Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties

Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA XII (a transmembrane, tumor-associated enzyme also involved in glaucoma-genesis). The new sulfonamides acted as medium potency inhibitors of hCA I (K_Is of 28.5–2954 nM), being highly effective as hCA II (K_Is in the range of 0.62–12.4 nM) and XII (K_Is of 0.54–7.11 nM) inhibitors. All substitution patterns present in these compounds (e.g., halogens, methyl and methoxy moieties, in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring) led to highly effective hCA II/XII inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of isoforms CA II and XII is dysregulated.     

Using dynamic sustainability indicators to assess environmental policy measures in Biosphere Reserves

The assessment of different policy options represents a major tool for decision-makers in Biosphere Reserves, to develop more-resilient strategies for sustainable development and to visualise unintended consequences of these policies.In this work we analyse eight measures proposed by different agents in order to meet the main objectives of environmental sustainability, included in the Action Plan of the Fuerteventura Biosphere Reserve (Spain). We quantified the effects of these measures in terms of the sustainability thresholds of 10 environmental indicators, also proposed by the Action Plan, which was integrated in the Fuerteventura Biosphere Reserve dynamic model. Their behaviours under these measures allow determination of whether the objectives will be met in the period 2012–2025. Although some indicators would improve under these measures, fitting certain objectives, some negative effects on other indicators confirm the existence of trade-offs among these objectives. For instance, grazing limitation would improve the proportion of high-quality vegetation but would negatively affect the Egyptian vulture population, which would even fall below its sustainability threshold. The definition of thresholds for each indicator allows decision-makers to establish a way to prioritise among the eight measures analysed. The results show that these measures are insufficient to meet the sustainability thresholds of four indicators (the landscape indicator, the proportion of renewable energy, the per capita primary energy consumption and carbon dioxide emissions). Focusing on the remaining six indicators and following the rule “Threshold out, measure out”, seven out of the eight measures would exceed some thresholds and should be avoided. Only one option, aimed at growing fodder to feed cattle on restored traditional agricultural lands, would not exceed any of these thresholds. However, this measure also presents certain negative effects regarding indicators related to flagship species (the houbara habitat and the Egyptian vulture population), which would require compensation measures.     

Determinants of Land Degradation and Fragmentation in Semiarid Vegetation at Landscape Scale

The objective of this paper was to investigate the sensitivity to degradation of semiarid plant communities in terms of plant cover and fragmentation, quantifying relationships between landscape characteristic (physical, socio-economical and historical) and vegetation degradation. The degradation of vegetation was measured as the degree of coverage of the two dominant vegetation types (i.e. tall arid brush and tall grass steppe), while fragmentation was measured as patch size and isolation. Data were obtained using GIS tools, and analyzed by logistic regression and linear multivariate regression. Results showed denser coverage at more elevated, gradual slopes that were not sea-oriented. Historical elements of the landscape had a significant effect on current natural vegetation. According to the fragmentation patterns, the vegetation is in fairly good condition (medium coverage had the largest patches but dense coverage was less isolated) but attention must be given to preserve vegetation, due to the relationships between fragmentation and human activities. Moreover, the protection plan under way in the area appeared to favour denser vegetation cover, while human activities had a measurable effect on vegetation degradation.     

Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Quality control of Utermöhl-based phytoplankton counting and biovolume estimates—an easy task or a Gordian knot?

The most suitable way for standardizing biovolume based assemblage analysis using the Utermöhl technique for counting combined with biomass conversion has not yet been found even though this method has been successfully used in plankton studies for many years. Due to the complexity of the approach easily applicable steps to initially meet primary standard end point or target counts for intra-laboratory standardization tested here seem promising. Examples from count validation and biometric data from the large datasets of three laboratories are given. The first two examples initially intended to quantify the taxon specific scatter of counts by (A) using identical replica of field samples combined with a half-chamber count (scan of every second transect) and the 30 random field approach, respectively, and (B) the replication of transect counts in one sample. Both examples identified relatively low minimum count thresholds to delimit counting errors for single cell counts. The third example identifies shape variability and allometric relationships of the main axes and shows a way to improve taxon specific biomass estimates with special reference to cell thickness. However improved precision of quantitative phytoplankton analysis requires optimization of combined counting strategy and biovolume assessment methods.     

Correction of Eye-estimation by Using Shrunken Predictors

The eye-estimation method is widely used in practice. Several agronomic and biological measures are currently estimated by this method. If a simple linear regression is the kernel model a shrinkage technique can be used for correcting the bias associated with this method. Two predictors of the population total are proposed and the corresponding model-based errors are deduced. A simulation study fixes the behaviour of the predictors.