Groundtruthing next-gen sequencing for microbial ecology-biases and errors in community structure estimates from PCR amplicon pyrosequencing

Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3-V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Evaluation of the viability and germination of tempisque [<i>Sideroxylon capiri</i> (A.DC.) Pittier Sapotaceae]

The tempisque (Sideroxylon capiri) is a tree native to Mexico used by the rural population for housing construction, poles and hedges, as fuel (wood) and also for fodder and ornamental purposes, among others. It is considered an endangered species. In order to contribute to its preservation and sustainable management, it was considered important to determine the proportion of viable seeds, the loss of viability due to storage period and the germination process by applying pregerminative treatments. We found that freshly collected seeds showed 100% viability, which decreased to 0% after 5 months of storage. According to the cumulative germination significant differences between treatments (p≤0.01) were found. It was observed that seeds can accelerate their time of germination with the previous exposure of 24 h in water at room temperature. The soaking treatment in water for 24 h at room temperature obtained final germination of 55%, while with the control 39% was reached. Soaking in hydrogen peroxide and scarification were the treatments with lower germination percentage (33 and 23%, respectively). To get a higher percentage of germinated seeds in a short time, it is necessary to give a soaking treatment in water for 24 h before sowing.     

Higher‐order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm‐to‐vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi‐scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X‐ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo‐EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher‐order chain structures that are broken upon interaction with the receptor Atg19 in vitro. The stoichiometry of these cargo–receptor complexes is key to maintaining the size of the Cvt aggregate in vivo. Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.     

An insert for the reorienting gradient rotor for virus extraction from infected plants

A device made of nylon and which is inserted in the bowl of the reorienting gradient rotor is described. This insert acts as a container for holding infected plant tissues for the purpose of separating virus by centrifugal force from intact and fresh plants, from frozen and thawed plants and from plant tissue disintegrated by mechanical means. The extracted fluid represents 80 to 90% of that present in the untreated plant tissue. Electron micrographs taken of concentrates of the viruses prepared by the centrifugation extraction procedure indicate that extraneous materials are reduced to a low level.     

An alternative to the diagnostic dilatation and curettage--endometrial cytology.

Endometrial aspiration cytology has been shown by multicentre prospective studies to be an acceptable and valuable method of assessing the endometrium. A retrospective study was undertaken over three years'' routine use of the Isaacs cell sampler. In 86% of the cases suitable endometrium was obtained, with experience of the technique being the important factor. When compared with histological findings, all 11 cases of malignancy were confirmed, including one ovarian adenocarcinoma. Of the cytological reports of endometrial hyperplasia, 78% were confirmed by the histological findings, with the remainder showing minor degrees of cystic hyperplasia or normal endometrium. In no case was a more abnormal lesion present on histological examination than had been suggested by cytological findings. The use of progestogens in reversing hyperplasia is seen to be effective, though the long term benefit remains uncertain. It is concluded that with an experienced cytologist, Isaacs endometrial aspiration should be used routinely for the primary investigation of dysfunctional uterine bleeding and postmenopausal bleeding.     

Purification of filamentous plant viruses by thin layer centrifugation.

The construction of a modified thin layer ultracentrifuge rotor is described. This rotor was used in the purification of five filamentous plant viruses, viz. TMV, SCMV, PVX, SGV and YMC. The purification and concentration of these viruses in their monomeric forms is hazardous when conventional "tube" rotors are used since they invariably result in dissociation and aggregation of the virus particles. Using the thin layer rotor these infective agents may be concentrated in volumes of fluid equal to approximately 1% of the starting suspension and not as pellets obtained after ultracentrifugation is conventional "tube" rotors. Electron microscopy revealed that the virus particles concentrated by thin layer centrifugation were not aggregated and that only few fragments of the virus filaments were present in the final preparations.     

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Optimization of a diagnostic platform for oxidation–reduction potential (ORP) measurement in human plasma

Objectives: Oxidation–reduction potential (ORP) measurement can demonstrate the extent of oxidative stress in patients with severe illness and/or injury. A novel ORP diagnostic platform using disposable sensors (RedoxSYS) has been validated by comparison to mass spectrometry, but the optimal methods of sample handling for best performance of the device have not been described.

Methods: We sought to optimize ORP measurement in human plasma under controlled conditions. We hypothesized that the anticoagulant, freeze–thawing, and storage duration would influence measured ORP levels.

Results: The platform was sensitive to exogenous oxidation with hydrogen peroxide and reduction with ascorbic acid. Plasma anticoagulated with heparin was more sensitive to differences in ORP than plasma prepared in citrate. ORP measurements decreased slightly after a freeze–thaw cycle, but once frozen, ORP was stable for up to one month.

Discussion: We confirm that ORP detects oxidative stress in plasma samples. Optimal measurement of plasma ORP requires blood collection in heparin anticoagulant tubes and immediate analysis without a freeze–thaw cycle.