Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies

The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10(6) reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs) and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially confound the interpretation of microbial community data.     

A biophysical model of Sugarcane growth

Scientists predict that global agricultural lands will expand over the next few decades due to increasing demands for food production and an exponential increase in crop‐based biofuel production. These changes in land use will greatly impact biogeochemical and biogeophysical cycles across the globe. It is therefore important to develop models that can accurately simulate the interactions between the atmosphere and important crops. In this study, we develop and validate a new process‐based sugarcane model (included as a module within the Agro‐IBIS dynamic agro‐ecosystem model) which can be applied at multiple spatial scales. At site level, the model systematically under/overestimated the daily sensible/latent heat flux (by ?10.5% and 14.8%, H and λE, respectively) when compared against the micrometeorological observations from southeast Brazil. The model underestimated ET (relative bias between ?10.1% and –12.5%) when compared against an agro‐meteorological field experiment from northeast Australia. At the regional level, the model accurately simulated average yield for the four largest mesoregions (clusters of municipalities) in the state of São Paulo, Brazil, over a period of 16 years, with a yield relative bias of ?0.68% to 1.08%. Finally, the simulated annual average sugarcane yield over 31 years for the state of Louisiana (US) had a low relative bias (?2.67%), but exhibited a lower interannual variability than the observed yields.     

Isolation and characterization of polymorphic microsatellite loci for the horn fly, Haematobia irritans (L.) (Diptera: Muscidae)

The horn fly, Haematobia irritans (L.) (Diptera: Muscidae), is a cosmopolitan livestock pest that has caused a great negative impact on the animal production sector throughout the world. Here, we describe 10 polymorphic microsatellite loci isolated from H. irritans. The number of alleles found ranged from two to eight per locus and the expected heterozygosity from 0.1421 to 0.7702. These loci are potentially useful for the fine-scale genetic characterization of horn fly populations and provide fundamental information for pest management and planning of control programs.     

Expression of estrogen receptor (ER) -α and -βtranscripts in the neonatal and adult rat cerebral cortex, cerebellum, and olfactory bulb

INTRODUCTIONEstrogen has been known to exert extensive effects via estrogen receptor (ER) on diverse physio-logical and develoPmental functions of the brain[1,2]. It has been observed that the distribution ofthe classical ER subtype-a (ERa) and the recentlycharacterized novel ER subtype--fi (ERg), and theirexpression patterns (ERcr/ERfi) vary greatly amongvarious brain regions[1, 3]. These evidences suggestthat each ER subtype may play a different role inestrogen's effects on the br…     

Reconstitution of a porcine submaxillary gland beta-D-galactoside alpha 2----3 sialyltransferase into liposomes

Form A of the beta-D-galactoside alpha 2----3 sialyltransferase from porcine submaxillary glands was incorporated into liposomes. Incorporation was achieved by gel filtration of the enzyme in the presence of octylglucoside-phospholipid micelles. As detergent was removed during gel filtration, liposomes (average diameter, 370 A) with bound enzyme were formed and emerged unretarded from the column. The recovery of enzyme activity in the liposomes was about 40% of the initial activity starting with as little as 9 micrograms of transferase. Chromatography on Sepharose CL6B and sucrose density gradient centrifugation confirmed the association of enzyme with liposomes. In contrast to Form A, Form B of the sialyltransferase, which lacks the proposed lipid-binding domain of Form A, cannot be incorporated into liposomes. Form A of the transferase was also incorporated into liposomes composed of phosphatidylcholine, cholesterol, and a mixture of phospholipids from the membranes of the Golgi apparatus from porcine submaxillary glands. Although the transferase was distributed about equally on the internal and external surface of the phosphatidylcholine liposomes, most of the transferase was on the external surface in liposomes containing cholesterol (72%) or in liposomes containing Golgi apparatus phospholipids (88%). The enzyme bound to phosphatidylcholine liposomes was shown by kinetic analysis to have the same activity as that found in the presence of activity-stimulating detergents such as Triton X-100. Enzyme incorporated into cholesterol-containing liposomes had the same activity. In contrast, enzyme bound to liposomes formed from the Golgi apparatus mixed phospholipids had a lower activity, but one similar to that of the transferase in Golgi apparatus membranes. These studies suggest that the composition of a biological membrane may well influence the orientation of the transferase in the membrane as well as modulate its enzymic activity.     

Ultrastructural Studies of Spermiogenesis of Conocephalus saltator (Saussure)

The acrosome of the spermatozoa is the organelle involved in its penetration through the ova membranes during the fertilization process. Several features of this process are considered to be related to the fertilization events e.g. some substance coats outside the membrane. During the maturation process the spermatozoa of Conocephalus saltator develop a coat of tubules and filaments which overly some membrane regions in a specific array. The mature spermatozoa are seen to adhere in these regions and form threads of 20 cells long and five to six wide. It is suggested that the external coat plays a role in this 'sticking' phenomenon.