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Complementary Metagenomic Approaches Improve Reconstruction of Microbial Diversity in a Forest Soil.
Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this "uncultivated majority" remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages.IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism
Effects of different culture conditions (photoautotrophic, photomixotrophic) and the auxin indole-butyric acid on the in vitro acclimatization of papaya (Carica papaya L. var. Red Maradol) plants using zeolite as support
Plant regeneration of papaya via organogenesis and somatic embryogenesis has been successful; however, the biggest problem of in vitro culture of this species is the acclimatization of regenerated plants, where over 70% of the plants are lost before being planted in the field. Decreasing the relative humidity inside the culture vessel and thus increasing the ventilation, appears to have a greater effect on the adaptation of papaya plants, strengthening the function of the stomata and with this, allowing better control of water loss from the leaves. The aim of this study was to determine the effects of different concentrations of sucrose and indole-butyric acid (IBA) on rooting and in vitro acclimatization of plants using sterile zeolite as support and culture vessels with increased ventilation. Three concentrations of sucrose (0, 10 and 20 g L-1) were studied with and without auxin and as the control treatment, the rooting culture medium with agar during 17, 27 and 37 culture days. The highest percentage of rooting was recorded at 37 culture days in the treatment without sucrose and IBA with 80.0% and zeolite as support. The best photosynthetic values were achieved when in vitro shoots were grown in culture medium with auxin and different concentrations of sucrose, even though they were also high in the treatment without the presence of IBA and without sucrose at 17 days of culture. The combined effect of the zeolite, auxin (IBA), without sucrose in the culture medium and increased ventilation allowed  photoautotrophic culture conditions which had effect of the increasing plant survival under ex vitro acclimatization conditions.Key words: Carica papaya, photosynthesis, roots formation
Harmonic Analysis of Boolean Networks: Determinative Power and Perturbations
Consider a large Boolean network with a feed forward structure. Given a
probability distribution on the inputs, can one find, possibly small,
collections of input nodes that determine the states of most other nodes in the
network? To answer this question, a notion that quantifies the determinative
power of an input over the states of the nodes in the network is needed. We
argue that the mutual information (MI) between a given subset of the inputs X =
{X_1, ..., X_n} of some node i and its associated function f_i(X) quantifies
the determinative power of this set of inputs over node i. We compare the
determinative power of a set of inputs to the sensitivity to perturbations to
these inputs, and find that, maybe surprisingly, an input that has large
sensitivity to perturbations does not necessarily have large determinative
power. However, for unate functions, which play an important role in genetic
regulatory networks, we find a direct relation between MI and sensitivity to
perturbations. As an application of our results, we analyze the large-scale
regulatory network of Escherichia coli. We identify the most determinative
nodes and show that a small subset of those reduces the overall uncertainty of
the network state significantly. Furthermore, the network is found to be
tolerant to perturbations of its inputs
Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: A multicenter study
The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1ÎČ and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1ÎČ and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by KruskalâWallis analysis of variance with Dunnâs multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1ÎČ determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface
Siglecg Limits the Size of B1a B Cell Lineage by Down-Regulating NFÎșB Activation
BACKGROUND: B1 B cells are believed to be a unique lineage with a distinct developmental pathway, function and activation requirement. How this lineage is genetically determined remained largely obscure. METHODS AND PRINCIPAL FINDINGS: Using the Siglecg-deficient mice with a knockin of green-fluorescent protein encoding sequence, we show here that, although the Siglecg gene is broadly expressed at high levels in all stages and/or lineages of B cells tested and at lower levels in other lineages, its deletion selectively expanded the B1a B cell lineages, including the frequency of the B1 cell progenitor in the bone marrow and the number of B1a cells in the peritoneal cavity, by postnatal expansion. The expansion of B1a B cells in the peritoneal correlated with enhanced activation of NFkappaB and was ablated by an IKK inhibitor. CONCLUSION AND SIGNIFICANCE: Our data revealed a critical role for Siglec G-NFkappaB pathway in regulating B1a B cell lineage. These data lead to a novel model of B1a lineage development that explains a large array of genetic data in this field
The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission
1Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (Îł-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that Îł-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcsâ parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcsâ parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito
Diets containing sea cucumber (Isostichopus badionotus) meals are hypocholesterolemic in young rats
Peer reviewedPublisher PD
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