The RNA-Binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria.

PIWI-interacting RNAs (piRNAs) silence transposons in Drosophila ovaries, ensuring female fertility. Two coupled pathways generate germline piRNAs: the ping-pong cycle, in which the PIWI proteins Aubergine and Ago3 increase the abundance of pre-existing piRNAs, and the phased piRNA pathway, which generates strings of tail-to-head piRNAs, one after another. Proteins acting in the ping-pong cycle localize to nuage, whereas phased piRNA production requires Zucchini, an endonuclease on the mitochondrial surface. Here, we report that Armitage (Armi), an RNA-binding ATPase localized to both nuage and mitochondria, links the ping-pong cycle to the phased piRNA pathway. Mutations that block phased piRNA production deplete Armi from nuage. Armi ATPase mutants cannot support phased piRNA production and inappropriately bind mRNA instead of piRNA precursors. We propose that Armi shuttles between nuage and mitochondria, feeding precursor piRNAs generated by Ago3 cleavage into the Zucchini-dependent production of Aubergine- and Piwi-bound piRNAs on the mitochondrial surface.

Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Coconversion.

Adoption of a streamlined version of the bacterial clustered regular interspersed short palindromic repeat (CRISPR)/Cas9 defense system has accelerated targeted genome engineering. The Streptococcus pyogenes Cas9 protein, directed by a simplified, CRISPR-like single-guide RNA, catalyzes a double-stranded DNA break at a specific genomic site; subsequent repair by end joining can introduce mutagenic insertions or deletions, while repair by homologous recombination using an exogenous DNA template can incorporate new sequences at the target locus. However, the efficiency of Cas9-directed mutagenesis is low in Drosophila melanogaster Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end-joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. When single injected G0 flies produce individual G1 broods, flies carrying edits at a gene-of-interest were readily found in broods in which all G1 offspring carried white mutations. Thus, visual assessment of eye color substitutes for wholesale PCR screening of large numbers of G1 offspring. We find that end-joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy.

Slicing and Binding by Ago3 or Aub Trigger Piwi-Bound piRNA Production by Distinct Mechanisms.

In Drosophila ovarian germ cells, PIWI-interacting RNAs (piRNAs) direct Aubergine and Argonaute3 to cleave transposon transcripts and instruct Piwi to repress transposon transcription, thereby safeguarding the germline genome. Here, we report that RNA cleavage by Argonaute3 initiates production of most Piwi-bound piRNAs. We find that the cardinal function of Argonaute3, whose piRNA guides predominantly correspond to sense transposon sequences, is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather than to make piRNAs that guide post-transcriptional silencing by Aubergine. We also find that the Tudor domain protein Qin prevents Aubergine's cleavage products from becoming Piwi-bound piRNAs, ensuring that antisense piRNAs guide Piwi. Although Argonaute3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. This alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs.

Small RNA-directed silencing: the fly finds its inner fission yeast?

Several recent studies demonstrate that piRNAs guide Piwi protein to repress transposon transcription in fly ovaries, much as fission yeast use siRNAs to silence repeat sequences. Still mysterious though is how Piwi targets euchromatic transposons for silencing, but not the specialized heterochromatic loci that produce piRNA precursors.

Salidroside protects human erythrocytes against hydrogen peroxide-induced apoptosis.

Rhodiola rosea is a commonly used folk medicine for the treatment of high altitude sickness, mountain malhypoxia, and anoxia. Its active ingredient, salidroside [2-(4-hydroxyphenyl)ethyl ß-D-glucopyranoside (1)], has been reported to have a broad spectrum of biological effects. However, the protective role of 1 in human erythrocytes remains unclear. This study therefore has investigated the effects of 1 on oxidative stress-induced apoptosis in human erythrocytes (also known as eryptosis or erythroptosis). Compound 1 increased cell survival significantly and prevented human erythrocytes from undergoing eryptosis/erythroptosis mediated by H(2)O(2), as confirmed by the decreased expression of phosphatidylserine on the cell surface and reduced leakage of calcein through the damaged membrane. Mechanistically, 1 was found to exert its protective effects through its antioxidative activity and the inhibition of caspase-3 activation and stress-induced intracellular Ca(2+) rise in a dose-dependent manner. Compound 1 is a protective agent in human erythrocytes against oxidative stress and may be a good adaptogen to enhance the body's resistance to stress and fatigue.

Salidroside promotes erythropoiesis and protects erythroblasts against oxidative stress by up-regulating glutathione peroxidase and thioredoxin.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola rosea is commonly used in China and Tibet folk medicine for the treatment of high altitude sickness, anoxia and mountain malhypoxia. AIM OF STUDY: Salidroside (SDS) is an active ingredient of Rhodiola rosea. This study attempted to examine the potential erythropoiesis-stimulating and anti-oxidative effect of SDS in TF-1 erythroblasts. MATERIALS AND METHODS: The erythropoiesis-promoting effect was determined by treating human TF-1 cells, one of the popular in vitro models for studying erythropoiesis, with SDS in the presence and absence of erythropoietin (EPO) through the measurement of the expression of a series of erythroid markers such as glycophorin A (GPA), transferrin receptor (CD71) and hemoglobin (Hb). The potential protective effect of SDS against H(2)O(2)-induced apoptosis and its underlying mechanism in TF-1 erythroblasts were examined by flow cytometry and Western blot analysis. RESULTS: SDS promotes erythropoiesis in the EPO-treated cells and it also reduces the number of apoptotic cells in TF-1 erythroblasts after H(2)O(2) treatment probably through the up-regulation of protective proteins thioredoxin-1 (Trx1) and glutathione peroxidase-1 (GPx1). CONCLUSION: Our study provides evidence to explain the ethnopharmacological role of SDS and Rhodiola rosea in Chinese medicine. Our findings also support the use of SDS as an erythropoiesis-adjuvant agent to correct anemia and malhypoxia.

Disturbance of cellular glucose transport by two prevalently used fluoroquinolone antibiotics ciprofloxacin and levofloxacin involves glucose transporter type 1.

Dysglycemia and central nervous system (CNS) complications are the known adverse effects of fluoroquinolone antibiotics. Ciprofloxacin and levofloxacin are among the most prescribed antibiotics. In this study we demonstrate that ciprofloxacin and levofloxacin disturb glucose transport into HepG2 cells and such inhibition is associated with inhibited glucose transporter type 1 (GLUT1) function. When exposed to ciprofloxacin or levofloxacin at maximum plasma concentrations (C(max)) and 5x of C(max) concentrations, GLUT1 mRNA expression, cell surface GLUT1 protein expression and glucose uptake were significantly reduced. These findings imply that disturbed cellular glucose transport and GLUT1 function may underlie the dysglycemic and CNS effects of ciprofloxacin and levofloxacin.