Growth hormone mitigates against lethal irradiation and enhances hematologic and immune recovery in mice and nonhuman primates.

Medications that can mitigate against radiation injury are limited. In this study, we investigated the ability of recombinant human growth hormone (rhGH) to mitigate against radiation injury in mice and nonhuman primates. BALB/c mice were irradiated with 7.5 Gy and treated post-irradiation with rhGH intravenously at a once daily dose of 20 microg/dose for 35 days. rhGH protected 17 out of 28 mice (60.7%) from lethal irradiation while only 3 out of 28 mice (10.7%) survived in the saline control group. A shorter course of 5 days of rhGH post-irradiation produced similar results. Compared with the saline control group, treatment with rhGH on irradiated BALB/c mice significantly accelerated overall hematopoietic recovery. Specifically, the recovery of total white cells, CD4 and CD8 T cell subsets, B cells, NK cells and especially platelets post radiation exposure were significantly accelerated in the rhGH-treated mice. Moreover, treatment with rhGH increased the frequency of hematopoietic stem/progenitor cells as measured by flow cytometry and colony forming unit assays in bone marrow harvested at day 14 after irradiation, suggesting the effects of rhGH are at the hematopoietic stem/progenitor level. rhGH mediated the hematopoietic effects primarily through their niches. Similar data with rhGH were also observed following 2 Gy sublethal irradiation of nonhuman primates. Our data demonstrate that rhGH promotes hematopoietic engraftment and immune recovery post the exposure of ionizing radiation and mitigates against the mortality from lethal irradiation even when administered after exposure.

Expression in aneuploid Drosophila S2 cells.

Extensive departures from balanced gene dose in aneuploids are highly deleterious. However, we know very little about the relationship between gene copy number and expression in aneuploid cells. We determined copy number and transcript abundance (expression) genome-wide in Drosophila S2 cells by DNA-Seq and RNA-Seq. We found that S2 cells are aneuploid for >43 Mb of the genome, primarily in the range of one to five copies, and show a male genotype ( approximately two X chromosomes and four sets of autosomes, or 2X;4A). Both X chromosomes and autosomes showed expression dosage compensation. X chromosome expression was elevated in a fixed-fold manner regardless of actual gene dose. In engineering terms, the system "anticipates" the perturbation caused by X dose, rather than responding to an error caused by the perturbation. This feed-forward regulation resulted in precise dosage compensation only when X dose was half of the autosome dose. Insufficient compensation occurred at lower X chromosome dose and excessive expression occurred at higher doses. RNAi knockdown of the Male Specific Lethal complex abolished feed-forward regulation. Both autosome and X chromosome genes show Male Specific Lethal-independent compensation that fits a first order dose-response curve. Our data indicate that expression dosage compensation dampens the effect of altered DNA copy number genome-wide. For the X chromosome, compensation includes fixed and dose-dependent components.

Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.

BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.

Myosin VIIA, important for human auditory function, is necessary for Drosophila auditory organ development.

BACKGROUND: Myosin VIIA (MyoVIIA) is an unconventional myosin necessary for vertebrate audition [1]-[5]. Human auditory transduction occurs in sensory hair cells with a staircase-like arrangement of apical protrusions called stereocilia. In these hair cells, MyoVIIA maintains stereocilia organization [6]. Severe mutations in the Drosophila MyoVIIA orthologue, crinkled (ck), are semi-lethal [7] and lead to deafness by disrupting antennal auditory organ (Johnston's Organ, JO) organization [8]. ck/MyoVIIA mutations result in apical detachment of auditory transduction units (scolopidia) from the cuticle that transmits antennal vibrations as mechanical stimuli to JO. PRINCIPAL FINDINGS: Using flies expressing GFP-tagged NompA, a protein required for auditory organ organization in Drosophila, we examined the role of ck/MyoVIIA in JO development and maintenance through confocal microscopy and extracellular electrophysiology. Here we show that ck/MyoVIIA is necessary early in the developing antenna for initial apical attachment of the scolopidia to the articulating joint. ck/MyoVIIA is also necessary to maintain scolopidial attachment throughout adulthood. Moreover, in the adult JO, ck/MyoVIIA genetically interacts with the non-muscle myosin II (through its regulatory light chain protein and the myosin binding subunit of myosin II phosphatase). Such genetic interactions have not previously been observed in scolopidia. These factors are therefore candidates for modulating MyoVIIA activity in vertebrates. CONCLUSIONS: Our findings indicate that MyoVIIA plays evolutionarily conserved roles in auditory organ development and maintenance in invertebrates and vertebrates, enhancing our understanding of auditory organ development and function, as well as providing significant clues for future research.

c-Myc is required for maintenance of glioma cancer stem cells.

BACKGROUND: Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.

Risk of ovarian cancer and inherited variants in relapse-associated genes.

BACKGROUND: We previously identified a panel of genes associated with outcome of ovarian cancer. The purpose of the current study was to assess whether variants in these genes correlated with ovarian cancer risk. METHODS AND FINDINGS: Women with and without invasive ovarian cancer (749 cases, 1,041 controls) were genotyped at 136 single nucleotide polymorphisms (SNPs) within 13 candidate genes. Risk was estimated for each SNP and for overall variation within each gene. At the gene-level, variation within MSL1 (male-specific lethal-1 homolog) was associated with risk of serous cancer (p = 0.03); haplotypes within PRPF31 (PRP31 pre-mRNA processing factor 31 homolog) were associated with risk of invasive disease (p = 0.03). MSL1 rs7211770 was associated with decreased risk of serous disease (OR 0.81, 95% CI 0.66-0.98; p = 0.03). SNPs in MFSD7, BTN3A3, ZNF200, PTPRS, and CCND1A were inversely associated with risk (p<0.05), and there was increased risk at HEXIM1 rs1053578 (p = 0.04, OR 1.40, 95% CI 1.02-1.91). CONCLUSIONS: Tumor studies can reveal novel genes worthy of follow-up for cancer susceptibility. Here, we found that inherited markers in the gene encoding MSL1, part of a complex that modifies the histone H4, may decrease risk of invasive serous ovarian cancer.

Consumption of cyanogenic bamboo by a newly discovered species of bamboo lemur

Three species of bamboo‐eating lemurs were found to be sympatric in the southeastern rain forests of Madagascar. Sympatric species generally differ in habitat utilization or diet, but these three closely related bamboo lemurs lived in the same habitat and all ate bamboo. Behavioral observation revealed that they did select different parts of the bamboo, and chemical analyses confirmed that there was a difference in the secondary compound content present in those selections. The growing tips of Cephalostachyum ef uiguieri selected by the golden bamboo lemur (Hapalemuraureus) contained 15 mg of cyanide per 100 g fresh weight bamboo while the leaves of C. perrieri selected by the gentle bamboo lemur (H. griseus)and the mature culms of C. cf uiguieri selected by the greater bamboolemur (H. simus) did not contain cyanide. Since each individual golden bamboo lemur ate about 500 g of bamboo per day, they daily ingestedabout 12 times the lethal dose of cyanide. The mechanism by which this small primate avoids the acute and chronic symptoms of cyanide poisioning is unknown. Copyright © 1989 Wiley‐Liss, Inc., A Wiley Company

Human dimensions of bycatch reduction technology: Current assumptions and directions for future research

Bycatch reduction technology (BRT) modifies fishing gear to increase selectivity and avoid capture of non-target species, or to facilitate their non-lethal release. As a solution to fisheries-related mortality of non-target species, BRT is an attractive option; effectively implemented, BRT presents a technical 'fix' that can reduce pressure for politically contentious and economically detrimental interventions, such as fisheries closures. While a number of factors might contribute to effective implementation, our review of BRT literature finds that research has focused on technical design and experimental performance of individual technologies. In contrast, and with a few notable exceptions, research on the human and institutional context of BRT, and more specifically on how fishers respond to BRT, is limited. This is not to say that fisher attitudes are ignored or overlooked, but that incentives for fisher uptake of BRT are usually assumed rather than assessed or demonstrated. Three assumptions about fisher incentives dominate: (1) economic incentives will generate acceptance of BRT; (2) enforcement will generate compliance with BRT; and (3) 'participation' by fishers will increase acceptance and compliance, and overall support for BRT. In this paper, we explore evidence for and against these assumptions and situate our analysis in the wider social science literature on fisheries. Our goal is to highlight the need and suggest focal areas for further research. © Inter-Research 2008.

Copper signaling axis as a target for prostate cancer therapeutics.

Previously published reports indicate that serum copper levels are elevated in patients with prostate cancer and that increased copper uptake can be used as a means to image prostate tumors. It is unclear, however, to what extent copper is required for prostate cancer cell function as we observed only modest effects of chelation strategies on the growth of these cells in vitro. With the goal of exploiting prostate cancer cell proclivity for copper uptake, we developed a "conditional lethal" screen to identify compounds whose cytotoxic actions were manifested in a copper-dependent manner. Emerging from this screen was a series of dithiocarbamates, which, when complexed with copper, induced reactive oxygen species-dependent apoptosis of malignant, but not normal, prostate cells. One of the dithiocarbamates identified, disulfiram (DSF), is an FDA-approved drug that has previously yielded disappointing results in clinical trials in patients with recurrent prostate cancer. Similarly, in our studies, DSF alone had a minimal effect on the growth of prostate cancer tumors when propagated as xenografts. However, when DSF was coadministered with copper, a very dramatic inhibition of tumor growth in models of hormone-sensitive and of castrate-resistant disease was observed. Furthermore, we determined that prostate cancer cells express high levels of CTR1, the primary copper transporter, and additional chaperones that are required to maintain intracellular copper homeostasis. The expression levels of most of these proteins are increased further upon treatment of androgen receptor (AR)-positive prostate cancer cell lines with androgens. Not surprisingly, robust CTR1-dependent uptake of copper into prostate cancer cells was observed, an activity that was accentuated by activation of AR. Given these data linking AR to intracellular copper uptake, we believe that dithiocarbamate/copper complexes are likely to be effective for the treatment of patients with prostate cancer whose disease is resistant to classical androgen ablation therapies.

Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis.

Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, includingChlamydia trachomatis Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogenC. trachomatislacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against aC. trachomatisgenomic library using a conditional-lethallpxHmutantEscherichia colistrain, we have identified an open reading frame (Ct461, renamedlpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function inE. coliand catalyzes the conversion of UDP-DAGn to lipid Xin vitro LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity ofC. trachomatis, validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools forC. trachomatisand should be broadly applicable for the functional characterization of other essentialC. trachomatisgenes.IMPORTANCEChlamydia trachomatisis a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of manyChlamydiagenes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against theC. trachomatisgenomic library using a conditional-lethal mutant ofE. coliand identified the missing essential gene in the lipid A biosynthetic pathway, which we designatedlpxG We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activityin vitro Overexpression of LpxG inC. trachomatisleads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating thein vivofunction of LpxG and highlighting the importance of regulated lipid A biosynthesis inC. trachomatis.