Evaluation of high-dose rifampin in patients with new, smear-positive tuberculosis (HIRIF): study protocol for a randomized controlled trial.

BACKGROUND: Evidence has existed for decades that higher doses of rifampin may be more effective, but potentially more toxic, than standard doses used in tuberculosis treatment. Whether increased doses of rifampin could safely shorten treatment remains an open question. METHODS/DESIGN: The HIRIF study is a phase II randomized trial comparing rifampin doses of 20 and 15 mg/kg/day to the standard 10 mg/kg/day for the first 2 months of tuberculosis treatment. All participants receive standard doses of companion drugs and a standard continuation-phase treatment (4 months, 2 drugs). They are followed for 6 months post treatment. Study participants are adults with newly diagnosed, previously untreated, smear positive (≥2+) pulmonary tuberculosis. The primary outcome is rifampin area under the plasma concentration-time curve (AUC0-24) after at least 14 days of study treatment/minimum inhibitory concentration. 180 randomized participants affords 90 % statistical power to detect a difference of at least 14 mcg/mL*hr between the 20 mg/kg group and the 10 mg/kg group, assuming a loss to follow-up of up to 17 %. DISCUSSION: Extant evidence suggests the potential for increased doses of rifampin to shorten tuberculosis treatment duration. Early studies that explored this potential using intermittent, higher dosing were derailed by toxicity. Given the continued large, global burden of tuberculosis with nearly 10 million new cases annually, shortened regimens with existing drugs would offer an important advantage to patients and health systems. TRIAL REGISTRATION: This trial was registered with clinicaltrials.gov (registration number: NCT01408914 ) on 2 August 2011

Increased Maternal Genome Dosage Bypasses the Requirement of the FIS Polycomb Repressive Complex 2 in Arabidopsis Seed Development

<div><p>Seed development in flowering plants is initiated after a double fertilization event with two sperm cells fertilizing two female gametes, the egg cell and the central cell, leading to the formation of embryo and endosperm, respectively. In most species the endosperm is a polyploid tissue inheriting two maternal genomes and one paternal genome. As a consequence of this particular genomic configuration the endosperm is a dosage sensitive tissue, and changes in the ratio of maternal to paternal contributions strongly impact on endosperm development. The FERTILIZATION INDEPENDENT SEED (FIS) Polycomb Repressive Complex 2 (PRC2) is essential for endosperm development; however, the underlying forces that led to the evolution of the FIS-PRC2 remained unknown. Here, we show that the functional requirement of the FIS-PRC2 can be bypassed by increasing the ratio of maternal to paternal genomes in the endosperm, suggesting that the main functional requirement of the FIS-PRC2 is to balance parental genome contributions and to reduce genetic conflict. We furthermore reveal that the AGAMOUS LIKE (AGL) gene <em>AGL62</em> acts as a dosage-sensitive seed size regulator and that reduced expression of <em>AGL62</em> might be responsible for reduced size of seeds with increased maternal genome dosage.</p> </div

FIS-PRC2 Target Genes Are Deregulated in Response to Maternal Excess Interploidy Hybridizations.

<p>Venn diagrams showing overlap of genes being deregulated in seeds derived from <i>osd1</i>×2n crosses with genes deregulated in seeds of 4n×2n crosses <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003163#pgen.1003163-Tiwari1" target="_blank">[24]</a> (upper panel); overlap of deregulated genes in seeds derived from <i>osd1</i>×2n crosses with H3K27me3 target genes in the endosperm <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003163#pgen.1003163-Weinhofer1" target="_blank">[49]</a> (middle panel), and genes that are upregulated in <i>fis2</i> mutant seeds <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003163#pgen.1003163-Erilova1" target="_blank">[9]</a> (lower panel). In the upper panel only deregulated genes that are present on the ATH1 array have been used to calculate the overlap.</p

Endosperm Cellularization Is Restored in Triploid <i>mea</i> Seeds.

<p>Sections of seeds from 2n×2n (left panels), <i>mea</i>/<i>MEA</i>×2n (middle panels) and <i>mea</i>/<i>MEA</i>; <i>osd1</i>/<i>osd1</i>×2n (right panels) crosses at 6–12 days after pollination (DAP). Bar = 100 µm. Images of mature seeds are shown in bottom panels. Bar = 0.5 mm.</p

Maternal Loss of NRPD1a Does Not Restore Wild-Type Levels of <i>AGL</i> Expression and Wild-Type Seed Size.

<p>(A) Quantitative RT-PCR analysis of <i>PHE1</i>, <i>AGL62</i>, <i>AGL90</i>, <i>AGL40</i>, <i>AGL28</i> and <i>AGL36</i> in seeds derived from crosses 2n×2n, <i>nrpd1a/nrpd1a</i>×2n, <i>osd1/osd1</i>×2n, and <i>nrpd1a/nrpd1a; osd1/osd1</i>×2n at 3 DAP. Error bars indicate s.e.m. (B) Distribution of seed sizes from different crosses. A minimum of 250 seeds was analyzed for each cross.</p

Expression of <i>AGL</i> MADS Box Genes Is Normalized in Triploid <i>mea</i> and <i>fis2</i> Seeds.

<p>Quantitative RT-PCR analysis of <i>AGL62</i>, <i>PHE1</i>, <i>AGL90</i>, <i>AGL40</i>, <i>AGL28</i> and <i>AGL36</i> at 8 DAP in <i>mea</i> and <i>fis2</i> seeds (upper and lower panel, respectively). Enlarged seeds with a <i>fis</i> phenotype were selected as 3n <i>mea</i> and <i>fis2</i> seeds from <i>mea</i>/<i>MEA</i>; <i>osd1</i>/<i>osd1</i>×2n and <i>fis2</i>/<i>FIS2</i>; <i>osd1</i>/<i>osd1</i>×2n crosses, respectively. Diploid <i>mea</i> seeds are a mixture of wild-type and <i>mea</i> seeds. Error bars indicate s.e.m.</p

Expression Level of <i>FIS2</i> but Not of <i>MEA</i> Is Increased in <i>osd1</i>×2n Crosses.

<p>Quantitative RT-PCR analysis of <i>MEA</i> and <i>FIS2</i> expression in seeds derived from <i>osd1</i>×2n crosses at 1–4 days after pollination (DAP). Error bars indicate s.e.m.</p

Expression of <i>AGL</i> MADS Box Genes Is Decreased in <i>osd1</i>×2n Crosses.

<p>Quantitative RT-PCR analysis of <i>AGL62</i>, <i>PHE1</i>, <i>AGL90</i>, <i>AGL36</i>, <i>AGL40</i> and <i>AGL28</i>. Error bars indicate s.e.m.</p

Maternal Excess Triploid <i>mea</i> and <i>fis2</i> Seeds Are Strongly Enlarged.

<p>(A) Distribution of seed sizes from different crosses. A minimum of 250 seeds was analyzed for each cross. Inset shows percentage of seeds larger than 0.14 mm². (B) Percentages of normal, enlarged and collapsed seeds from different crosses. Numbers in parenthesis correspond to numbers of analyzed seeds.</p

Seeds Derived from Crosses <i>osd1</i>×2n Mimic the Effect of Maternal Excess Interploidy Hybridizations.

<p>(A) Seeds from crosses of Col 2n×2n (upper panel) and <i>osd1</i>×2n (diploid and triploid seeds, middle and lower panel, respectively. Ploidy refers to ploidy of the embryo). Bar = 0.5 mm. (B) Average seed size (upper panel) and seed weight (lower panel) of different crosses. Numbers on top of bars correspond to number of analyzed seeds. For seed weight the average of 100 seeds was calculated in triplicates. Error bars indicate SD. The reference line corresponds to wild-type seed size and weight. (C) Number of endosperm nuclei in Col 2n×2n and <i>osd1</i>×2n crosses. n = 5. Error bars indicate SD. (D) Sections of seeds from Col 2n×2n and <i>osd1</i>×2n crosses. Bar = 100 µm.</p