From Genus to Phylum: Large-Subunit and Internal Transcribed Spacer rRNA Operon Regions Show Similar Classification Accuracies Influenced by Database Composition

We compared the classification accuracy of two sections of the fungal internal transcribed spacer (ITS) region, individually and combined, and the 5′ section (about 600 bp) of the large-subunit rRNA (LSU), using a naive Bayesian classifier and BLASTN. A hand-curated ITS-LSU training set of 1,091 sequences and a larger training set of 8,967 ITS region sequences were used. Of the factors evaluated, database composition and quality had the largest effect on classification accuracy, followed by fragment size and use of a bootstrap cutoff to improve classification confidence. The naive Bayesian classifier and BLASTN gave similar results at higher taxonomic levels, but the classifier was faster and more accurate at the genus level when a bootstrap cutoff was used. All of the ITS and LSU sections performed well (>97.7% accuracy) at higher taxonomic ranks from kingdom to family, and differences between them were small at the genus level (within 0.66 to 1.23%). When full-length sequence sections were used, the LSU outperformed the ITS1 and ITS2 fragments at the genus level, but the ITS1 and ITS2 showed higher accuracy when smaller fragment sizes of the same length and a 50% bootstrap cutoff were used. In a comparison using the larger ITS training set, ITS1 and ITS2 had very similar accuracy classification for fragments between 100 and 200 bp. Collectively, the results show that any of the ITS or LSU sections we tested provided comparable classification accuracy to the genus level and underscore the need for larger and more diverse classification training sets.     

Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy

The Ribosomal Database Project (RDP) Classifier, a na?ve Bayesian classifier, can rapidly and accurately classify bacterial 16S rRNA sequences into the new higher-order taxonomy proposed in Bergey's Taxonomic Outline of the Prokaryotes (2nd ed., release 5.0, Springer-Verlag, New York, NY, 2004). It provides taxonomic assignments from domain to genus, with confidence estimates for each assignment. The majority of classifications (98%) were of high estimated confidence (> or = 95%) and high accuracy (98%). In addition to being tested with the corpus of 5,014 type strain sequences from Bergey's outline, the RDP Classifier was tested with a corpus of 23,095 rRNA sequences as assigned by the NCBI into their alternative higher-order taxonomy. The results from leave-one-out testing on both corpora show that the overall accuracies at all levels of confidence for near-full-length and 400-base segments were 89% or above down to the genus level, and the majority of the classification errors appear to be due to anomalies in the current taxonomies. For shorter rRNA segments, such as those that might be generated by pyrosequencing, the error rate varied greatly over the length of the 16S rRNA gene, with segments around the V2 and V4 variable regions giving the lowest error rates. The RDP Classifier is suitable both for the analysis of single rRNA sequences and for the analysis of libraries of thousands of sequences. Another related tool, RDP Library Compare, was developed to facilitate microbial-community comparison based on 16S rRNA gene sequence libraries. It combines the RDP Classifier with a statistical test to flag taxa differentially represented between samples. The RDP Classifier and RDP Library Compare are available online at http://rdp.cme.msu.edu/.     

Using the RDP classifier to predict taxonomic novelty and reduce the search space for finding novel organisms

Background

Currently, the naïve Bayesian classifier provided by the Ribosomal Database Project (RDP) is one of the most widely used tools to classify 16S rRNA sequences, mainly collected from environmental samples. We show that RDP has 97+% assignment accuracy and is fast for 250 bp and longer reads when the read originates from a taxon known to the database. Because most environmental samples will contain organisms from taxa whose 16S rRNA genes have not been previously sequenced, we aim to benchmark how well the RDP classifier and other competing methods can discriminate these novel taxa from known taxa.

Principal Findings

Because each fragment is assigned a score (containing likelihood or confidence information such as the boostrap score in the RDP classifier), we “train” a threshold to discriminate between novel and known organisms and observe its performance on a test set. The threshold that we determine tends to be conservative (low sensitivity but high specificity) for naïve Bayesian methods. Nonetheless, our method performs better with the RDP classifier than the other methods tested, measured by the f-measure and the area-under-the-curve on the receiver operating characteristic of the test set. By constraining the database to well-represented genera, sensitivity improves 3–15%. Finally, we show that the detector is a good predictor to determine novel abundant taxa (especially for finer levels of taxonomy where novelty is more likely to be present).

Conclusions

We conclude that selecting a read-length appropriate RDP bootstrap score can significantly reduce the search space for identifying novel genera and higher levels in taxonomy. In addition, having a well-represented database significantly improves performance while having genera that are “highly” similar does not make a significant improvement. On a real dataset from an Amazon Terra Preta soil sample, we show that the detector can predict (or correlates to) whether novel sequences will be assigned to new taxa when the RDP database “doubles” in the future.     

A Statistical Framework for Accurate Taxonomic Assignment of Metagenomic Sequencing Reads

The advent of next-generation sequencing technologies has greatly promoted the field of metagenomics which studies genetic material recovered directly from an environment. Characterization of genomic composition of a metagenomic sample is essential for understanding the structure of the microbial community. Multiple genomes contained in a metagenomic sample can be identified and quantitated through homology searches of sequence reads with known sequences catalogued in reference databases. Traditionally, reads with multiple genomic hits are assigned to non-specific or high ranks of the taxonomy tree, thereby impacting on accurate estimates of relative abundance of multiple genomes present in a sample. Instead of assigning reads one by one to the taxonomy tree as many existing methods do, we propose a statistical framework to model the identified candidate genomes to which sequence reads have hits. After obtaining the estimated proportion of reads generated by each genome, sequence reads are assigned to the candidate genomes and the taxonomy tree based on the estimated probability by taking into account both sequence alignment scores and estimated genome abundance. The proposed method is comprehensively tested on both simulated datasets and two real datasets. It assigns reads to the low taxonomic ranks very accurately. Our statistical approach of taxonomic assignment of metagenomic reads, TAMER, is implemented in R and available at http://faculty.wcas.northwestern.edu/hji403/MetaR.htm.     

The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy

The Ribosomal Database Project-II (RDP-II) pro-vides data, tools and services related to ribosomal RNA sequences to the research community. Through its website (http://rdp.cme.msu.edu), RDP-II offers aligned and annotated rRNA sequence data, analysis services, and phylogenetic inferences (trees) derived from these data. RDP-II release 8.1 contains 16 277 prokaryotic, 5201 eukaryotic, and 1503 mitochondrial small subunit rRNA sequences in aligned and annotated format. The current public beta release of 9.0 debuts a new regularly updated alignment of over 50 000 annotated (eu)bacterial sequences. New analysis services include a sequence search and selection tool (Hierarchy Browser) and a phylogenetic tree building and visualization tool (Phylip Interface). A new interactive tutorial guides users through the basics of rRNA sequence analysis. Other services include probe checking, phylogenetic placement of user sequences, screening of users' sequences for chimeric rRNA sequences, automated alignment, production of similarity matrices, and services to plan and analyze terminal restriction fragment polymorphism (T-RFLP) experiments. The RDP-II email address for questions or comments is rdpstaff@msu.edu.     

Evaluating and optimizing the performance of software commonly used for the taxonomic classification of DNA metabarcoding sequence data

The taxonomic classification of DNA sequences has become a critical component of numerous ecological research applications; however, few studies have evaluated the strengths and weaknesses of commonly used sequence classification approaches. Further, the methods and software available for sequence classification are diverse, creating an environment in which it may be difficult to determine the best course of action and the trade‐offs made using different classification approaches. Here, we provide an in silico evaluation of three DNA sequence classifiers, the rdp Naïve Bayesian Classifier, rtax and utax . Further, we discuss the results, merits and limitations of both the classifiers and our method of classifier evaluation. Our methods of comparison are simple, yet robust, and will provide researchers a methodological and conceptual foundation for making such evaluations in a variety of research situations. Generally, we found a considerable trade‐off between accuracy and sensitivity for the classifiers tested, indicating a need for further improvement of sequence classification tools.     

Impact of training sets on classification of high-throughput bacterial 16s rRNA gene surveys

Taxonomic classification of the thousands–millions of 16S rRNA gene sequences generated in microbiome studies is often achieved using a naïve Bayesian classifier (for example, the Ribosomal Database Project II (RDP) classifier), due to favorable trade-offs among automation, speed and accuracy. The resulting classification depends on the reference sequences and taxonomic hierarchy used to train the model; although the influence of primer sets and classification algorithms have been explored in detail, the influence of training set has not been characterized. We compared classification results obtained using three different publicly available databases as training sets, applied to five different bacterial 16S rRNA gene pyrosequencing data sets generated (from human body, mouse gut, python gut, soil and anaerobic digester samples). We observed numerous advantages to using the largest, most diverse training set available, that we constructed from the Greengenes (GG) bacterial/archaeal 16S rRNA gene sequence database and the latest GG taxonomy. Phylogenetic clusters of previously unclassified experimental sequences were identified with notable improvements (for example, 50% reduction in reads unclassified at the phylum level in mouse gut, soil and anaerobic digester samples), especially for phylotypes belonging to specific phyla (Tenericutes, Chloroflexi, Synergistetes and Candidate phyla TM6, TM7). Trimming the reference sequences to the primer region resulted in systematic improvements in classification depth, and greatest gains at higher confidence thresholds. Phylotypes unclassified at the genus level represented a greater proportion of the total community variation than classified operational taxonomic units in mouse gut and anaerobic digester samples, underscoring the need for greater diversity in existing reference databases.     

Factors that affect large subunit ribosomal DNA amplicon sequencing studies of fungal communities: classification method, primer choice, and error

Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1) a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN); 2) a composition-based method (Ribosomal Database Project na?ve bayesian classifier, NBC); and, 3) a phylogeny-based method (Statistical Assignment Package, SAP). We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50-100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys.     

Systemic immune activation in HIV infection is associated with decreased MDC responsiveness to TLR ligand and inability to activate naive CD4 T-cells

Background

HIV infection is characterized by ineffective anti-viral T-cell responses and impaired dendritic cell (DC) functions, including response to Toll-Like Receptor (TLR) ligands. Because TLR responsiveness may affect a host''s response to virus, we examined TLR ligand induced Myeloid and Plasmacytoid DC (MDC and PDC) activation of naïve T-cells in HIV+ subjects.

Methods

Freshly purified MDC and PDC obtained from HIV+ subjects and healthy controls were cultured in the presence and absence of TLR ligands (poly I∶C or R-848). We evaluated indices of maturation/activation (CD83, CD86, and HLA-DR expression), cytokine secretion (IFN-alpha and IL-6), and ability to activate allogeneic naïve CD4 T-cells to secrete IFN-gamma and IL-2.

Results

MDC from HIV+ subjects had increased spontaneous IL-6 production and increased CD83 and CD86 expression when compared to MDC of controls. MDC IL-6 expression was associated with plasma HIV level. At the same time, poly I∶C induced HLA-DR up-regulation on MDC was reduced in HIV+ persons when compared to controls. The latter finding was associated with impaired ability of MDC from HIV+ subjects to activate allogeneic naïve CD4 T-cells. PDC from HIV+ persons had increased spontaneous and TLR ligand induced IL-6 expression, and increased HLA-DR expression at baseline. The latter was associated with an intact ability of HIV PDC to activate allogeneic naïve CD4 T-cells.

Conclusion

These results have implications for the ability of the HIV+ host to form innate and adaptive responses to HIV and other pathogens.     

The anti-CD74 humanized monoclonal antibody, milatuzumab, which targets the invariant chain of MHC II complexes, alters B-cell proliferation, migration, and adhesion molecule expression

Introduction

Targeting CD74 as the invariant chain of major histocompatibility complexes (MHC) became possible by the availability of a specific humanized monoclonal antibody, milatuzumab, which is under investigation in patients with hematological neoplasms. CD74 has been reported to regulate chemo-attractant migration of macrophages and dendritic cells, while the role of CD74 on peripheral naïve and memory B cells also expressing CD74 remains unknown. Therefore, the current study addressed the influence of milatuzumab on B-cell proliferation, chemo-attractant migration, and adhesion molecule expression.

Methods

Surface expression of CD74 on CD27^- naïve and CD27⁺ memory B cells as well as other peripheral blood mononuclear cells (PBMCs) obtained from normals, including the co-expression of CD44, CXCR4, and the adhesion molecules CD62L, β7-integrin, β1-integrin and CD9 were studied after binding of milatuzumab using multicolor flow cytometry. The influence of the antibody on B-cell proliferation and migration was analyzed in vitro in detail.

Results

In addition to monocytes, milatuzumab also specifically bound to human peripheral B cells, with a higher intensity on CD27⁺ memory versus CD27^- naïve B cells. The antibody reduced B-cell proliferation significantly but moderately, induced enhanced spontaneous and CXCL12-dependent migration together with changes in the expression of adhesion molecules, CD44, β7-integrin and CD62L, mainly of CD27^- naïve B cells. This was independent of macrophage migration-inhibitory factor as a ligand of CD74/CD44 complexes.

Conclusions

Milatuzumab leads to modestly reduced proliferation, alterations in migration, and adhesion molecule expression preferentially of CD27^- naïve B cells. It thus may be a candidate antibody for the autoimmune disease therapy by modifying B cell functions.     

Down-regulated NOD2 by immunosuppressants in peripheral blood cells in patients with SLE reduces the muramyl dipeptide-induced IL-10 production

Background

Pattern recognition receptors (PRRs) such as Toll-like receptors are aberrantly expressed of peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients, for playing immunopathological roles.

Methodology/Principal Findings

We investigated the expression and function of the PRR nucleotide-binding oligomerization domain (NOD2) in SLE. NOD2 expression in T, B lymphocytes, monocytes, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) was assessed in SLE patients and healthy controls (HCs) using flow cytometric analysis. Ex vivo production of cytokines from PBMCs upon NOD2 agonist muramyl dipeptide (MDP) stimulation was assessed using Cytometric Bead Array. Over-expression of NOD2 in monocytes was observed in immunosuppressant naïve SLE patients, and was positively associated with longer disease duration. Immunosuppressive therapy was an independent explanatory variable for downregulating NOD2 expression in CD8+ T, monocytes, mDCs and pDCs. Ex vivo basal productions of cytokines (IL-6, IL-8 and IL-10) were significantly increased in immunosuppressant naïve patients and patients with active disease despite immunosuppressants compared with HCs. Upon MDP stimulaiton, relative induction (%) of cytokines (IL-1β) from PBMC was significantly increased in immunosuppressant naïve patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with HCs. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naïve patients and HCs.

Conclusions/Significance

Bacterial exposure may increase the NOD2 expression in monocytes in immunosuppressant naïve SLE patients which can subsequently lead to aberrant activation of PBMCs to produce proinflammatory cytokines, implicating the innate immune response for extracellular pathogens in the immunopathological mechanisms in SLE. Immunosuppressant therapy may downregulate NOD2 expression in CD8+ T lymphocytes, monocytes, and DCs in SLE patients which subsequently IL-10 reduction, contributing towards the regulation of immunopathological mechanisms of SLE, at the expense of increasing risk of bacterial infection.     

Interleukin-7 enhances proliferation responses to T-cell receptor stimulation in naïve CD4+ T cells from human immunodeficiency virus-infected persons

Proliferation responses of naïve CD4⁺ T cells to T-cell receptor and interleukin-7 (IL-7) stimulation were evaluated by using cells from human immunodeficiency virus-positive (HIV⁺) donors. IL-7 enhanced responses to T-cell receptor stimulation, and the magnitude of this enhancement was similar in cells from healthy controls and from HIV⁺ subjects. The overall response to T-cell receptor stimulation alone or in combination with IL-7, however, was diminished among viremic HIV⁺ donors and occurred independent of antigen-presenting cells. Frequencies of CD127⁺ cells were related to the magnitudes of proliferation enhancement that were mediated by IL-7. Thus, IL-7 enhances but does not fully restore the function of naïve CD4⁺ T cells from HIV-infected persons.Interleukin-7 (IL-7) plays an important role in T-cell homeostasis by modulating thymic output (1, 16, 22) and by enhancing the peripheral expansion and survival of both naïve and memory T-cell subsets (12, 18, 20, 25, 26, 31, 32). Under normal circumstances, the homeostatic maintenance of naïve CD4⁺ T cells is regulated by at least two types of signals that include T-cell receptor (TCR) engagement and IL-7 (10, 26, 30). In addition, IL-7 may play an important role in the conversion of effector T cells into long-term memory cells (12, 14).Homeostasis of T cells is dysregulated in human immunodeficiency virus (HIV) infection such that there is a marked depletion of CD4⁺ cells and a progressive loss of naïve CD4 and CD8⁺ T cells (24). Although the mechanisms for these deficiencies are not fully understood, it is possible that impairments in T-cell proliferation and responsiveness to immunomodulatory cytokines could play a role. In HIV disease, IL-7 is increased in plasma (2, 5, 11, 15, 19, 21, 23) and the alpha chain of the IL-7 receptor, CD127, is less frequently expressed among T lymphocytes (2, 5, 11, 21, 23). The ability of patient T cells to respond to IL-7 stimulation may be diminished in HIV disease but may not be related to the density of CD127 expression as it is in T cells from healthy controls (4). Moreover, the responsiveness of T cells, including naïve CD4⁺ lymphocytes, to TCR stimulation is diminished in HIV disease (27-29). Thus, defects in responsiveness to cytokines or TCR stimulation could contribute to the perturbations in T-cell proliferation and survival in HIV disease.In these studies, we examined the responsiveness of naïve CD4⁺ T cells from viremic HIV-positive (HIV⁺) donors (median plasma HIV RNA level, 25,200 copies/ml [range, 1,015 to 1,000,000 copies/ml]; median CD4 cell count, 429 cells/μl [range, 41 to 950 cells/μl]; median age, 38 years [range, 22 to 64 years]; n = 25) and aviremic, highly active antiretroviral therapy (HAART)-treated HIV⁺ donors (plasma HIV RNA level, <400 copies/ml; median CD4 cell count, 309 cells/μl [range, 74 to 918 cells/μl]; median age, 48 years [range, 37 to 55 years]; n = 12) to the combined stimulus of recombinant IL-7 (Cytheris) plus agonistic anti-CD3 antibody. Peripheral blood mononuclear cells (PBMC) were depleted of CD45RO⁺ cells by magnetic bead depletion (>90% purity) and were incubated in medium alone or were stimulated with anti-CD3 antibody, IL-7, or anti-CD3 antibody plus IL-7. CD4⁺CD45RO⁻CD28⁺CD27⁺ cells were assessed for the expression of Ki67 2 days poststimulation by flow cytometric analyses. The addition of IL-7 to anti-CD3 antibody enhanced the induction of Ki67 expression in cells from both HIV⁺ and HIV-negative (HIV⁻) donors (Fig. ​(Fig.11 and Fig. ​Fig.2).2). A diminished response to anti-CD3 antibody was observed among naïve CD4⁺ T cells from viremic HIV⁺ donors. In contrast, cells from aviremic HIV⁺ donors (all receiving antiretroviral therapy) had normal responses to anti-CD3 antibody compared to cells from healthy donors (Fig. ​(Fig.2).2). Importantly, the addition of IL-7 to the cultures significantly improved the responses to above those observed with anti-CD3 alone in HIV⁻ and HIV⁺ donors, regardless of viremia (Wilcoxon signed ranks test; for each comparison, P was <0.04), and the magnitude of that enhancement, although slightly diminished in cells from HIV⁺ donors, was not significantly different between groups of subjects when measured as either the enhancement (n-fold; not shown) or as the change in percent Ki67⁺ cells above the background observed for cells stimulated with anti-CD3 alone (Fig. ​(Fig.3).3). Although IL-7 enhanced responses to TCR stimulation in HIV⁻ subjects, the overall magnitude of the responses among cells from HIV viremic subjects did not reach the levels seen with cells from healthy donors, even in the presence of IL-7 (Fig. ​(Fig.2).2). It should be noted, however, that these functional readouts were not related to clinical indices of plasma HIV RNA level, CD4 cell count, or age when considered as continuous variables, suggesting that the functional perturbations in naïve CD4⁺ T cells are probably undermined by complexities extending beyond HIV replication (not shown). Together, these results suggest that TCR responsiveness is diminished in naïve CD4⁺ T cells from viremic HIV⁺ subjects, whereas responsiveness to IL-7 stimulation is relatively preserved.Open in a separate windowFIG. 1.IL-7 enhances the induction of Ki67 expression in naïve CD4⁺ T cells from healthy controls and HIV⁺ donors. CD45RO-depleted PBMC were incubated with anti-CD3 antibody (100 ng/ml), IL-7 (50 ng/ml), anti-CD3 antibody plus IL-7, or medium alone (RPMI with 10% fetal bovine serum). Cells were gated on CD4⁺CD27⁺CD28⁺ lymphocytes and examined for Ki67 expression by intracellular flow cytometry.Open in a separate windowFIG. 2.IL-7 responsiveness in cells from viremic and aviremic HIV⁺ donors. Plotted values represent the percentages of CD4⁺CD27⁺CD28⁺CD45RO⁻ T cells that expressed Ki67 after a 2-day incubation with anti-CD3 or with anti-CD3 plus IL-7. Percentages of Ki67⁺ cells in cultures without stimulation or with IL-7 only were subtracted from the values shown. Responses of cells from healthy controls (n = 9), HIV⁺ subjects with plasma HIV RNA levels of >400 copies/ml (n = 25), and HIV⁺ subjects on HAART with suppressed viral replication (<400 copies/ml; n = 12) are shown. Statistically significant differences between cells from controls and HIV⁺ donors are indicated. Analyses included Kruskal-Wallis test (P = 0.002) for multigroup comparisons and Mann-Whitney U test for comparison of two groups (*, P < 0.05).Open in a separate windowFIG. 3.IL-7 enhances responses to anti-CD3 antibody stimulation to a similar degree in cells from HIV⁺ and HIV⁻ donors. Naïve CD4⁺ T cells were incubated with IL-7, anti-CD3, anti-CD3 plus IL-7, or medium alone for 2 days. Background division (percent Ki67⁺ cells) in medium alone or IL-7 alone was first subtracted from the responses observed with cells stimulated with anti-CD3 alone or with anti-CD3 plus IL-7, respectively. The magnitude of IL-7 enhancement was then calculated by subtracting the percentage of naïve CD4⁺ cells that expressed Ki67⁺ after anti-CD3 antibody stimulation from the percentage of naïve CD4⁺ cells that expressed Ki67 after stimulation with anti-CD3 plus IL-7. n = 9, 25, and 12 for healthy controls, viremic subjects, and aviremic subjects, respectively.Previous studies indicate that the frequency of CD127⁺ T cells, particularly memory T-cell subsets, is reduced in patients with HIV disease (5, 11, 21, 23). This could, in part, result from the modulation of receptor expression through increased exposure to IL-7 in vivo and also may reflect accumulation of CD127⁻ effector memory cells (21). We assessed the expression of CD127 in naïve CD4⁺CD45RA⁺CD28⁺CD27⁺ and memory CD4⁺CD45RO⁺ T cells in a subset of patients and asked if the frequencies of CD127⁺ cells were related to the induction of Ki67 expression by anti-CD3 or by anti-CD3 plus IL-7 among naïve CD4⁺ T cells. We reasoned that the ability of IL-7 to enhance responses to TCR stimulation might be limited if CD127 expression was diminished among naïve CD4⁺ T cells from HIV⁺ donors. Alternatively, a defect in functional responses also could be related to increased exposure to IL-7 in vivo, as may be reflected by the absence of CD127 receptor expression on memory T-cell subsets.In agreement with previous studies, our results suggest that CD127 expression is relatively preserved in naïve CD4⁺ T cells from HIV⁺ donors (representative histograms in Fig. ​Fig.4)4) (mean percentage of CD127⁺ cells, 87 and 83 for HIV⁻ donors [n = 5] and HIV⁺ donors [n = 17], respectively; P = 0.96) but is diminished in memory CD4⁺ T cells from HIV⁺ donors (mean percentage of CD127⁺ cells, 83 and 59 for HIV⁻ and HIV⁺ donors, respectively; P = 0.01). The frequencies of CD127⁺ naïve T cells were directly related to the frequencies of CD127⁺ memory T cells (Spearman''s correlations; r = 0.711, P = 0.001; n = 18) in HIV⁺ subjects. This result suggests that a similar mechanism modulates the expression of CD127 in these T-cell subsets, even though the loss of CD127 expression is clearly greater among the memory T cells in HIV disease. Neither CD127 expression among naïve CD4⁺ T cells nor CD127 expression among memory CD4⁺ T cells was related to the functional response of naïve CD4⁺ T cells to anti-CD3 (r = 0.238 and P = 0.36 for naïve CD127 expression; r = 0.293 and P = 0.25 for memory CD127 expression) or to anti-CD3 plus IL-7 (r = 0.32 and P = 0.21 for naïve CD127 expression; r = 0.31 and P = 0.22 for memory CD127 expression). There was a relationship between the percentage of CD127⁺ naïve T cells and the delta Ki67 expression that resulted from the addition of IL-7 to anti-CD3-treated cultures (percentage of Ki67⁺ cells in cultures treated with anti-CD3 plus IL-7 minus the percentage of Ki67⁺ cells in cultures treated with anti-CD3 alone) (Fig. ​(Fig.4).4). This relationship was statistically significant by Pearson''s correlation (r = 0.5, P = 0.041), the use of which was justified based on the normal distribution of the data. Spearman''s analysis, which is independent of data distribution, indicated a similar trend that was not statistically significant (r = 0.41, P = 0.1). The mean fluorescence intensity of CD127 expression on CD4⁺CD45RA⁺CD27⁺CD28⁺ T cells was not significantly related to the delta Ki67 expression induced by IL-7 but also suggested a trend consistent with a direct relationship between these indices (r = 0.45 and P = 0.07 by Pearson''s correlation; r = 0.34 and P = 0.18 by Spearman''s correlation). Despite the relative preservation of IL-7 receptor in naïve CD4⁺ T cells from HIV⁺ donors, the association between the frequencies of CD127⁺ cells and CD4⁺ T-cell proliferation responses to TCR plus IL-7 suggests that subtle IL-7 receptor perturbations might contribute to functional defects of naïve CD4⁺ T cells in HIV-infected persons.Open in a separate windowFIG. 4.CD127 receptor expression is related to enhancement of proliferation by IL-7. (A) Whole blood from a healthy control and an HIV-infected person was examined by flow cytometry for expression of CD127 on CD4⁺CD45RA⁺CD27⁺CD28⁺ (naïve) T cells. The gating strategy for identifying naïve cells involved an initial gate for lymphocyte forward and side scatter (SSC) characteristics (not shown) and then sequential gates for CD4 positive, CD45RA positive and, finally, CD28⁺CD27⁺ double-positive cells. (B) Plotted values indicating the relationship between the delta Ki67 expression in naïve CD4⁺ T cells and the percentage of CD127⁺ naïve T cells that was determined by using freshly isolated whole blood. The delta Ki67 expression was calculated by subtracting the percentage of naïve CD4⁺ cells that expressed Ki67⁺ after anti-CD3 antibody stimulation from the percentage of naïve CD4⁺ cells that expressed Ki67 after stimulation with anti-CD3 plus IL-7.To consider the possibility that antigen-presenting cells could contribute to the diminished response of T-cells to stimulation with TCR plus IL-7, we next asked if defects in TCR-plus-IL-7 stimulation could be detected in purified naïve CD4⁺ T-cell populations. CD4⁺CD45RO⁻ cells were negatively selected by magnetic bead depletion, achieving a purity of >90% as determined by flow cytometric analyses. Purified naïve CD4⁺ T cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) tracking dye and incubated with IL-7, anti-CD3 antibody that was immobilized on a plate, anti-CD3 plus IL-7, or medium alone. The induction of proliferation was measured 7 days later by the dilution of CFSE tracking dye among CD4⁺CD27⁺ cells by calculating the division index (average number of cell divisions of all CD4⁺CD27⁺ cells) and the proliferation index (average number of divisions of CD4⁺CD27⁺ cells that had diluted tracking dye; Flow-Jo analysis software). These purified CD4⁺ T cells proliferated poorly in response to anti-CD3 antibody stimulation alone, providing functional evidence that the samples were free of antigen-presenting cell contamination (Fig. ​(Fig.5A).5A). The combined treatment of anti-CD3 and IL-7 induced cellular expansion, whereas alone, neither stimulus induced cellular proliferation during the 7-day period (Fig. ​(Fig.5A).5A). Responses of cells from HIV⁺ donors were reduced compared to those of cells from healthy donors, confirming that the defects in naïve CD4⁺ T-cell expansion are independent of antigen-presenting cells and not fully corrected by IL-7 (Fig. ​(Fig.5B5B).Open in a separate windowFIG. 5.Diminished responses to TCR plus IL-7 in purified naïve CD4⁺ T cells from HIV⁺ donors. CD4⁺CD45RO⁻ cells were purified from PBMC by negative selection. Cells from HIV⁺ donors (n = 7) and healthy controls (n = 7) were labeled with CSFE and incubated with anti-CD3 immobilized on a plate (5 μg/ml, overnight at 4°C) plus IL-7 (10 ng/ml). CFSE dye dilution was measured among the CD4⁺CD27⁺ cells. (A) Representative histograms showing the dilution of CFSE and CD27 expression among cells incubated with anti-CD3 antibody alone, IL-7 alone, or the combination of anti-CD3 plus IL-7. Placements of quadrant gates were based on an isotype control antibody stain (for CD27 expression) and on cells that had been incubated in medium alone (for CFSE dye dilution). (B) Division indices (average number of cell divisions among CD4⁺CD27⁺ cells) and proliferation indices (average number of cell divisions among CD4⁺CD27⁺ cells that had diluted tracking dye) are shown.IL-7 is a promising candidate for therapeutic and vaccine adjuvant applications in HIV disease. This cytokine may be especially beneficial in circumstances of immune reconstitution, since it normally plays an essential role in T-cell proliferation and survival. Here, we demonstrate that IL-7 efficiently enhances TCR-triggered naïve CD4⁺ T-cell expansion in cells from healthy individuals and from HIV⁺ donors. The mechanism of IL-7 activity is not discerned in these experiments but may involve effects on survival, such as the induction of Bcl-2 (9), or may involve the enhancement of IL-2 or IL-2 receptor expression (6, 8). In any case, our studies provide evidence that IL-7 should provide an effective therapy for the regulation of naïve CD4⁺ T-cell homeostasis and may be useful for vaccine adjuvant applications in HIV disease. The potential of this approach has been illustrated by recent human trials of IL-7 that demonstrated the expansion of naïve T cells in vivo after IL-7 administration to HIV-infected persons (13) and by animal studies, wherein IL-7 administration enhanced T-cell responses to immunization in mice (17).Notably, the depletion studies and purification methods employed here did not necessarily eliminate terminally differentiated effector memory CD4⁺ T cells from our cultures; however, studies of CMV-specific terminally differentiated cells suggested that these cells are primarily CD27⁻ (3), and the use of three markers to identify naïve CD4⁺ T cells, including the ones used here (CD27, CD28, and CD45RO) is estimated to provide 98% assurance that the cells being examined are truly naïve (7). Thus, it is likely that terminally differentiated cells were largely removed from our analyses.Our observations provide confirmation of a significant defect in the responses of naïve CD4⁺ T cells to TCR triggering in HIV disease, and this defect is not fully corrected by IL-7, as shown here, or by IL-2, as we demonstrated previously (27). These deficiencies are reproduced even among naïve CD4⁺ T cells that are purified from professional antigen-presenting cells, indicating that the defects are intrinsic to the T cells and not a consequence of dysfunctional antigen-presenting cells. We propose that functional defects in naïve CD4⁺ T cells from HIV⁺ donors stem primarily from deficiencies in TCR signaling. Further studies that define the nature of naïve CD4⁺ T-cell defects in HIV disease will be required to address the underlying mechanisms.     

Genome Reshuffling for Advanced Intercross Permutation (GRAIP): simulation and permutation for advanced intercross population analysis

Background

Advanced intercross lines (AIL) are segregating populations created using a multi-generation breeding protocol for fine mapping complex trait loci (QTL) in mice and other organisms. Applying QTL mapping methods for intercross and backcross populations, often followed by naïve permutation of individuals and phenotypes, does not account for the effect of AIL family structure in which final generations have been expanded and leads to inappropriately low significance thresholds. The critical problem with naïve mapping approaches in AIL populations is that the individual is not an exchangeable unit.

Methodology/Principal Findings

The effect of family structure has immediate implications for the optimal AIL creation (many crosses, few animals per cross, and population expansion before the final generation) and we discuss these and the utility of AIL populations for QTL fine mapping. We also describe Genome Reshuffling for Advanced Intercross Permutation, (GRAIP) a method for analyzing AIL data that accounts for family structure. GRAIP permutes a more interchangeable unit in the final generation crosses – the parental genome – and simulating regeneration of a permuted AIL population based on exchanged parental identities. GRAIP determines appropriate genome-wide significance thresholds and locus-specific P-values for AILs and other populations with similar family structures. We contrast GRAIP with naïve permutation using a large densely genotyped mouse AIL population (1333 individuals from 32 crosses). A naïve permutation using coat color as a model phenotype demonstrates high false-positive locus identification and uncertain significance levels, which are corrected using GRAIP. GRAIP also detects an established hippocampus weight locus and a new locus, Hipp9a.

Conclusions and Significance

GRAIP determines appropriate genome-wide significance thresholds and locus-specific P-values for AILs and other populations with similar family structures. The effect of family structure has immediate implications for the optimal AIL creation and we discuss these and the utility of AIL populations.     

A randomized controlled phase Ib trial of the malaria vaccine candidate GMZ2 in African children

Background

GMZ2 is a fusion protein of Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP) that mediates an immune response against the blood stage of the parasite. Two previous phase I clinical trials, one in naïve European adults and one in malaria-exposed Gabonese adults showed that GMZ2 was well tolerated and immunogenic. Here, we present data on safety and immunogenicity of GMZ2 in one to five year old Gabonese children, a target population for future malaria vaccine efficacy trials.

Methodology/Principal Findings

Thirty children one to five years of age were randomized to receive three doses of either 30 µg or 100 µg of GMZ2, or rabies vaccine. GMZ2, adjuvanted in aluminum hydroxide, was administered on Days 0, 28 and 56. All participants received a full course of their respective vaccination and were followed up for one year. Both 30 µg and 100 µg GMZ2 vaccine doses were well tolerated and induced antibodies and memory B-cells against GMZ2 as well as its antigenic constituents MSP3 and GLURP. After three doses of vaccine, the geometric mean concentration of antibodies to GMZ2 was 19-fold (95%CI: 11,34) higher in the 30 µg GMZ2 group than in the rabies vaccine controls, and 16-fold (7,36) higher in the 100 µg GMZ2 group than the rabies group. Geometric mean concentration of antibodies to MSP3 was 2.7-fold (1.6,4.6) higher in the 30 µg group than in the rabies group and 3.8-fold (1.5,9.6) higher in the 100 µg group. Memory B-cells against GMZ2 developed in both GMZ2 vaccinated groups.

Conclusions/Significance

Both 30 µg as well as 100 µg intramuscular GMZ2 are immunogenic, well tolerated, and safe in young, malaria-exposed Gabonese children. This result confirms previous findings in naïve and malaria-exposed adults and supports further clinical development of GMZ2.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00703066","term_id":"NCT00703066"}}NCT00703066     

Kraken: ultrafast metagenomic sequence classification using exact alignments

Kraken is an ultrafast and highly accurate program for assigning taxonomic labels to metagenomic DNA sequences. Previous programs designed for this task have been relatively slow and computationally expensive, forcing researchers to use faster abundance estimation programs, which only classify small subsets of metagenomic data. Using exact alignment of k-mers, Kraken achieves classification accuracy comparable to the fastest BLAST program. In its fastest mode, Kraken classifies 100 base pair reads at a rate of over 4.1 million reads per minute, 909 times faster than Megablast and 11 times faster than the abundance estimation program MetaPhlAn. Kraken is available at http://ccb.jhu.edu/software/kraken/.     

Census-based rapid and accurate metagenome taxonomic profiling

Background

Understanding the taxonomic composition of a sample, whether from patient, food or environment, is important to several types of studies including pathogen diagnostics, epidemiological studies, biodiversity analysis and food quality regulation. With the decreasing costs of sequencing, metagenomic data is quickly becoming the preferred typed of data for such analysis.

Results

Rapidly defining the taxonomic composition (both taxonomic profile and relative frequency) in a metagenomic sequence dataset is challenging because the task of mapping millions of sequence reads from a metagenomic study to a non-redundant nucleotide database such as the NCBI non-redundant nucleotide database (nt) is a computationally intensive task. We have developed a robust subsampling-based algorithm implemented in a tool called CensuScope meant to take a ‘sneak peak’ into the population distribution and estimate taxonomic composition as if a census was taken of the metagenomic landscape. CensuScope is a rapid and accurate metagenome taxonomic profiling tool that randomly extracts a small number of reads (based on user input) and maps them to NCBI’s nt database. This process is repeated multiple times to ascertain the taxonomic composition that is found in majority of the iterations, thereby providing a robust estimate of the population and measures of the accuracy for the results.

Conclusion

CensuScope can be run on a laptop or on a high-performance computer. Based on our analysis we are able to provide some recommendations in terms of the number of sequence reads to analyze and the number of iterations to use. For example, to quantify taxonomic groups present in the sample at a level of 1% or higher a subsampling size of 250 random reads with 50 iterations yields a statistical power of >99%. Windows and UNIX versions of CensuScope are available for download at https://hive.biochemistry.gwu.edu/dna.cgi?cmd=censuscope. CensuScope is also available through the High-performance Integrated Virtual Environment (HIVE) and can be used in conjunction with other HIVE analysis and visualization tools.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-918) contains supplementary material, which is available to authorized users.     

Feature selection for the prediction of translation initiation sites

Translation initiation sites （TISs） are important signals in cDNA sequences. In many previous attempts to predict TISs in cDNA sequences, three major factors affect the prediction performance： the nature of the cDNA sequence sets, the relevant features selected. and the classification methods used. In this paper, we examine different approaches to select and integrate relevant features for TIS prediction. The top selected significant features include the features from the position weight matrix and the propensity matrix, the number of nucleotide C in the sequence downstream ATG, the number of downstream stop codons. the number of upstream ATGs, and the number of some amino acids, such as amino acids A and D. With the numerical data generated from these features, different classification methods, including decision tree. naive Bayes, and support vector machine, were applied to three independent sequence sets. The identified significant features were found to be biologically meaningful. while the experiments showed promising results.