Distinct blood transcriptomic signature of treatment in latent tuberculosis infected individuals at risk of developing active disease.

Although only a small fraction will ever develop the active form of tuberculosis (ATB) disease, chemoprophylaxis treatment in latent TB infected (LTBI) individuals is an effective strategy to control pathogen transmission. Characterizing immune responses in LTBI upon chemoprophylactic treatment is important to facilitate treatment monitoring, and thus improve TB control strategies. Here, we studied changes in the blood transcriptome in a cohort of 42 LTBI and 8 ATB participants who received anti-TB therapy. Based on the expression of previously published gene signatures of progression to ATB, we stratified the LTBI cohort in two groups and examined if individuals deemed to be at elevated risk of developing ATB before treatment (LTBI-Risk) differed from others (LTBI-Other). We found that LTBI-Risk and LTBI-Other groups were associated with two distinct transcriptomic treatment signatures, with the LTBI-Risk signature resembling that of treated ATB patients. Notably, overlapping genes between LTBI-Risk and ATB treatment signatures were associated with risk of progression to ATB and interferon (IFN) signaling, and were selectively downregulated upon treatment in the LTBI-Risk but not the LTBI-Other group. Our results suggest that transcriptomic reprogramming following treatment of LTBI is heterogeneous and can be used to distinguish LTBI-Risk individuals from the LTBI cohort at large.

Author Correction: The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.

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Development of an objective gene expression panel as an alternative to self-reported symptom scores in human influenza challenge trials.

BACKGROUND: Influenza challenge trials are important for vaccine efficacy testing. Currently, disease severity is determined by self-reported scores to a list of symptoms which can be highly subjective. A more objective measure would allow for improved data analysis. METHODS: Twenty-one volunteers participated in an influenza challenge trial. We calculated the daily sum of scores (DSS) for a list of 16 influenza symptoms. Whole blood collected at baseline and 24, 48, 72 and 96 h post challenge was profiled on Illumina HT12v4 microarrays. Changes in gene expression most strongly correlated with DSS were selected to train a Random Forest model and tested on two independent test sets consisting of 41 individuals profiled on a different microarray platform and 33 volunteers assayed by qRT-PCR. RESULTS: 1456 probes are significantly associated with DSS at 1% false discovery rate. We selected 19 genes with the largest fold change to train a random forest model. We observed good concordance between predicted and actual scores in the first test set (r = 0.57; RMSE = -16.1%) with the greatest agreement achieved on samples collected approximately 72 h post challenge. Therefore, we assayed samples collected at baseline and 72 h post challenge in the second test set by qRT-PCR and observed good concordance (r = 0.81; RMSE = -36.1%). CONCLUSIONS: We developed a 19-gene qRT-PCR panel to predict DSS, validated on two independent datasets. A transcriptomics based panel could provide a more objective measure of symptom scoring in future influenza challenge studies. Trial registration Samples were obtained from a clinical trial with the ClinicalTrials.gov Identifier: NCT02014870, first registered on December 5, 2013.

The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.

The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays.

Structural basis for duplex RNA recognition and cleavage by Archaeoglobus fulgidus C3PO.

Oligomeric complexes of Trax and Translin proteins, known as C3POs, participate in several eukaryotic nucleic acid metabolism pathways, including RNA interference and tRNA processing. In RNA interference in humans and Drosophila, C3PO activates the RNA-induced silencing complex (RISC) by removing the passenger strand of the small interfering RNA precursor duplex, using nuclease activity present in Trax. How C3POs engage with nucleic acid substrates is unknown. Here we identify a single protein from Archaeoglobus fulgidus that assembles into an octamer highly similar to human C3PO. The structure in complex with duplex RNA reveals that the octamer entirely encapsulates a single 13-base-pair RNA duplex inside a large inner cavity. Trax-like-subunit catalytic sites target opposite strands of the duplex for cleavage separated by 7 base pairs. The structure provides insight into the mechanism of RNA recognition and cleavage by an archaeal C3PO-like complex.

LOST MERISTEMS genes regulate cell differentiation of central zone descendants in Arabidopsis shoot meristems.

Meristems of seed plants continuously produce new cells for incorporation into maturing tissues. A tightly controlled balance between cell proliferation in the center and cell differentiation at the periphery of the shoot meristem maintains its integrity. Here, we describe the role of three GRAS genes, named LOST MERISTEMS genes, in shoot apical meristem maintenance and axillary meristem formation. Under short photoperiods, the lom1 lom2 and lom1 lom2 lom3 mutants have arrested meristems characterized by an over-proliferation of meristematic cells and loss of polar organization. They also show early arrest of axillary meristem development and formation of ectopic meristematic cell clusters within the stem. LOM1 and LOM2 transcripts accumulate in the peripheral and basal zones of the SAM and in vascular strands. We show that LOM1 and LOM2 promote cell differentiation at the periphery of shoot meristems and help to maintain their polar organization.

Enhancement of the seed-target recognition step in RNA silencing by a PIWI/MID domain protein.

Target recognition in RNA silencing is governed by the "seed sequence" of a guide RNA strand associated with the PIWI/MID domain of an Argonaute protein in RISC. Using a reconstituted in vitro target recognition system, we show that a model PIWI/MID domain protein confers position-dependent tightening and loosening of guide-strand-target interactions. Over the seed sequence, the interaction affinity is enhanced up to approximately 300-fold. Enhancement is achieved through a reduced entropy penalty for the interaction. In contrast, interactions 3' of the seed are inhibited. We quantified mismatched target recognition inside and outside the seed, revealing amplified discrimination at the third position in the seed mediated by the PIWI/MID domain. Thus, association of the guide strand with the PIWI/MID domain generates an enhanced affinity anchor site over the seed that can promote fidelity in target recognition and stabilize and guide the assembly of the active silencing complex.

Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicer-like 2, and is compromised by viral-encoded suppressor proteins.

In plants, worms, and fungi, RNA-dependent RNA polymerases (RDRs) amplify the production of short-interfering RNAs (siRNAs) that mediate RNA silencing. In Arabidopsis, RDR6 is thought to copy endogenous and exogenous RNA templates into double-stranded RNAs (dsRNAs), which are subsequently processed into siRNAs by one or several of the four Dicer-like enzymes (DCL1-->4). This reaction produces secondary siRNAs corresponding to sequences outside the primary targeted regions of a transcript, a phenomenon called transitivity. One recognized role of RDR6 is to strengthen the RNA silencing response mounted by plants against viruses. Accordingly, suppressor proteins deployed by viruses inhibit this defense. However, interactions between silencing suppressors and RDR6 have not yet been documented. Additionally, the mechanism underlying transitivity remains poorly understood. Here, we report how several viral silencing suppressors inhibit the RDR6-dependent amplification of virus-induced and transgene-induced gene silencing. Viral suppression of primary siRNA accumulation shows that transitivity can be initiated with minute amounts of DCL4-dependent 21-nucleotide (nt)-long siRNAs, whereas DCL3-dependent 24-nt siRNAs appear dispensable for this process. We further show that unidirectional (3-->5') transitivity requires the hierarchical and redundant functions of DCL4 and DCL2 acting downstream from RDR6 to produce 21- and 22-nt-long siRNAs, respectively. The 3-->5' transitive reaction is likely to be processive over >750 nt, with secondary siRNA production progressively decreasing as the reaction proceeds toward the 5'-proximal region of target transcripts. Finally, we show that target cleavage by a primary small RNA and 3-->5' transitivity can be genetically uncoupled, and we provide in vivo evidence supporting a key role for priming in this specific reaction.

In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA.

We show, with miR171, that plant miRNA genes are modular independent transcription units in which the fold-back pre-miRNA is sufficient for miRNA processing, and that the upstream region contains highly specific promoter elements. Processing depends on flanking sequences within the miRNA stem-loop precursor rather than the miRNA sequence itself, and mutations affecting target pairing at the center and 5' but not 3' region of the miRNA compromise its function in vivo. Inactivation of the SDE1 RNA-dependent-RNA-polymerase was mandatory for accurate representation of miRNA activity by sensor constructs in Arabidopsis. Work in sde1 background revealed a near-perfect spatial overlap between the patterns of miR171 transcription and activity, supporting the idea that plant miRNAs enable cell differentiation.

Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing.

In plants, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effectors of RNA silencing, a process involved in defense through RNA interference (RNAi) and in development. Plant viruses are natural targets of RNA silencing, and as a counterdefensive strategy, they have evolved highly diverse silencing suppressor proteins. Although viral suppressors are usually thought to act at distinct steps of the silencing machinery, there had been no consensus system so far that allowed a strict side-by-side analysis of those factors. We have set up such a system in Arabidopsis thaliana and used it to compare the effects of five unrelated viral silencing suppressors on the siRNA and miRNA pathways. Although all the suppressors inhibited RNAi, only three of them induced developmental defects, indicating that the two pathways are only partially overlapping. These developmental defects were remarkably similar, and their penetrance correlated with inhibition of miRNA-guided cleavage of endogenous transcripts and not with altered miRNA accumulation per se. Among the suppressors investigated, the tombusviral P19 protein coimmunoprecipitated with siRNA duplexes and miRNA duplexes corresponding to the primary cleavage products of miRNA precursors. Thus, it is likely that P19 prevents RNA silencing by sequestering both classes of small RNAs. Moreover, the finding here that P19 binds siRNAs and suppresses RNAi in Hela cells also suggests that this factor may be useful to dissect the RNA silencing pathways in animals. Finally, the differential effects of the silencing suppressors tested here upon other types of Arabidopsis silencing-related small RNAs revealed a surprising variety of biosynthetic and, presumably, functional pathways for those molecules. Therefore, silencing suppressors are valuable probes of the complexity of RNA silencing.