Deficient innate immunity,thymopoiesis, and gene expression response to radiation in survivors of childhood acute lymphoblastic leukemia

Background: Survivors of childhood acute lymphoblastic leukemia (ALL) are at an increased risk of developing secondary malignant neoplasms. Radiation and chemotherapy can cause mutations and cytogenetic abnormalities and induce genomic instability. Host immunity and appropriate DNA damage responses are critical inhibitors of carcinogenesis. Therefore, we sought to determine the long-term effects of ALL treatment on immune function and response to DNA damage. Methods: Comparative studies on 14 survivors in first complete remission and 16 siblings were conducted. Results: In comparison to siblings on the cells that were involved in adaptive immunity, the patients had either higher numbers (CD19+ B cells and CD4+CD25+ T regulatory cells) or similar numbers (αβT cells and CD45RO+/RA? memory T cells) in the blood. In contrast, patients had lower numbers of all lymphocyte subsets involved in innate immunity (γδT cells and all NK subsets, including KIR2DL1+ cells, KIR2DL2/L3+ cells, and CD16+ cells), and lower natural cytotoxicity against K562 leukemia cells. Thymopoiesis was lower in patients, as demonstrated by less CD45RO?/RA+ naïve T cell and less SjTREC levels in the blood, whereas the Vβ spectratype complexity score was similar. Array of gene expression response to low-dose radiation showed that about 70% of the probesets had a reduced response in patients. One of these genes, SCHIP-1, was also among the top-ranked single nucleotide polymorphisms (SNPs) during the whole-genome scanning by SNP microarray analysis. Conclusion: ALL survivors were deficient in innate immunity, thymopoiesis, and DNA damage responses to radiation. These defects may contribute to their increased likelihood of second malignancy.     

RNAi-based discovery and validation of new drug targets in filarial nematodes

Human filarial nematodes cause river blindness and lymphatic filariasis, both of which are diseases that produce considerable morbidity. Control of these diseases relies on drug treatments that are ineffective against macrofilariae and are threatened by the development of resistance. New validated drug targets are required to allow development of new classes of antifilarial drugs. To identify and validate potential new drug targets, we propose a collaborative research strategy utilizing bioinformatic filters and assessment of gene function by RNA interference in Caenorhabditis elegans and Brugia malayi.     

VHA-19 is essential in Caenorhabditis elegans oocytes for embryogenesis and is involved in trafficking in oocytes

There is an urgent need to develop new drugs against parasitic nematodes, which are a significant burden on human health and agriculture. Information about the function of essential nematode-specific genes provides insight to key nematode-specific processes that could be targeted with drugs. We have characterized the function of a novel, nematode-specific Caenorhabditis elegans protein, VHA-19, and show that VHA-19 is essential in the germline and, specifically, the oocytes, for the completion of embryogenesis. VHA-19 is also involved in trafficking the oocyte receptor RME-2 to the oocyte plasma membrane and is essential for osmoregulation in the embryo, probably because VHA-19 is required for proper eggshell formation via exocytosis of cortical granules or other essential components of the eggshell. VHA-19 may also have a role in cytokinesis, either directly or as an indirect effect of its role in osmoregulation. Critically, VHA-19 is expressed in the excretory cell in both larvae and adults, suggesting that it may have a role in osmoregulation in C. elegans more generally, probably in trafficking or secretion pathways. This is the first time a role for VHA-19 has been described.     

Hobbes: optimized gram-based methods for efficient read alignment

Recent advances in sequencing technology have enabled the rapid generation of billions of bases at relatively low cost. A crucial first step in many sequencing applications is to map those reads to a reference genome. However, when the reference genome is large, finding accurate mappings poses a significant computational challenge due to the sheer amount of reads, and because many reads map to the reference sequence approximately but not exactly. We introduce Hobbes, a new gram-based program for aligning short reads, supporting Hamming and edit distance. Hobbes implements two novel techniques, which yield substantial performance improvements: an optimized gram-selection procedure for reads, and a cache-efficient filter for pruning candidate mappings. We systematically tested the performance of Hobbes on both real and simulated data with read lengths varying from 35 to 100 bp, and compared its performance with several state-of-the-art read-mapping programs, including Bowtie, BWA, mrsFast and RazerS. Hobbes is faster than all other read mapping programs we have tested while maintaining high mapping quality. Hobbes is about five times faster than Bowtie and about 2–10 times faster than BWA, depending on read length and error rate, when asked to find all mapping locations of a read in the human genome within a given Hamming or edit distance, respectively. Hobbes supports the SAM output format and is publicly available at http://hobbes.ics.uci.edu.     

Tuning BRCA1 and BARD1 activity to investigate RING ubiquitin ligase mechanisms

The tumor‐suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N‐terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain‐containing proteins. RING domains bind and activate E2 ubiquitin‐conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer‐associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1.     

Shigella flexneri Infection in Caenorhabditis elegans: Cytopathological Examination and Identification of Host Responses

The Gram-negative bacterium Shigella flexneri is the causative agent of shigellosis, a diarrhoeal disease also known as bacillary dysentery. S. flexneri infects the colonic and rectal epithelia of its primate host and induces a cascade of inflammatory responses that culminates in the destruction of the host intestinal lining. Molecular characterization of host-pathogen interactions in this infection has been challenging due to the host specificity of S. flexneri strains, as it strictly infects humans and non-human primates. Recent studies have shown that S. flexneri infects the soil dwelling nematode Caenorhabditis elegans, however, the interactions between S. flexneri and C. elegans at the cellular level and the cause of nematode death are unknown. Here we attempt to gain insight into the complex host-pathogen interactions between S. flexneri and C. elegans. Using transmission electron microscopy, we show that live S. flexneri cells accumulate in the nematode intestinal lumen, produce outer membrane vesicles and invade nematode intestinal cells. Using two-dimensional differential in-gel electrophoresis we identified host proteins that are differentially expressed in response to S. flexneri infection. Four of the identified genes, aco-1, cct-2, daf-19 and hsp-60, were knocked down using RNAi and ACO-1, CCT-2 and DAF-19, which were identified as up-regulated in response to S. flexneri infection, were found to be involved in the infection process. aco-1 RNAi worms were more resistant to S. flexneri infection, suggesting S. flexneri-mediated disruption of host iron homeostasis. cct-2 and daf-19 RNAi worms were more susceptible to infection, suggesting that these genes are induced as a protective mechanism by C. elegans. These observations further our understanding of the processes involved in S. flexneri infection of C. elegans, which is immensely beneficial to the routine use of this new in vivo model to study S. flexneri pathogenesis.     

Aminomethylpiperazines as selective urotensin antagonists

Aminomethylpiperazines, reported previously as being kappa-opioid receptor agonists, were identified as lead compounds in the development of selective urotensin receptor antagonists. Optimized substitution of the piperazine moiety has provided high affinity urotensin receptor antagonists with greater than 100-fold selectivity over the kappa-opioid receptor. Select compounds were found to inhibit urotensin-induced vasoconstriction in isolated rat aortic rings consistent with the hypothesis that an urotensin antagonist may be useful for the treatment of hypertension.     

The Effect of Snow on Plant Chemistry and Invertebrate Herbivory: Experimental Manipulations Along a Natural Snow Gradient

Changing snow conditions have strong effects on northern ecosystems, but these effects are rarely incorporated into ecosystem models and our perception of how the ecosystems will respond to a warmer climate. We investigated the relationships between snow cover, plant phenology, level of invertebrate herbivory and leaf chemical traits in Betula nana in four different habitats located along a natural snow cover gradient. To separate the effect of snow per se from other differences, we manipulated the snow cover with snow fences in three habitats. The experimentally prolonged snow cover delayed plant phenology, but not as much as expected based on the pattern along the natural gradient. The positive effect of the snow treatment on plant nitrogen concentration was also weaker than expected, because plant nitrogen concentration closely followed plant phenology. The level of herbivory by leaf-chewing invertebrates increased in response to an increased snow cover, at least at the end of the growing season. The concentration of phenolic substances varied among habitats, treatments and sampling occasions, indicating that B. nana shrubs were able to retain a mosaic of secondary chemical quality despite altered snow conditions. This study shows that the effect of the snow cover period on leaf nitrogen concentration and level of herbivory can be predicted based on differences between habitats, whereas the effect of a changed plant phenology on plant nitrogen concentration is better explained by temporal trends within habitats. These results have important implications for how northern ecosystems should respond to future climate changes.     

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Development of the nitrogen fixing apparatus in the legumes,Centrosema pubescens Benth., and Vigna unguiculata L. Walp.

The sequence of events leading up to the establishment of symbiotic nitrogen-fixation were studied in two tropical legumes, Centrosema pubescens Benth, and Vigna unguiculata L. Walp. Parameters measured included fresh and dry weights, chlorophyll and leghaemoglobin contents, as well as the activities of NADH-nitrate reductase (EC 1.6.6.1), and nitrogenase (nitric-oxide reductase-EC 1.7.99.2) in plants that were inoculated with suitable rhizobia or which were watered with potassium nitrate. Dry weight and photosynthetic activity of both species followed the sigmoidal pattern which is characteristic of most plants. Growth was little different in either a qualitative or quantitative sense whether nitrogen was supplied as nitrate or through dinitrogen fixation. Although the biochemical sequence of events was dependent on the limiting sensitivities of the individual assays used, the data suggest that nitrate reductase is the first measurable enzymatic activity in the nodules (and roots), followed by acetylene reduction and leghaemoglobin in that order. It is possible therefore, that low levels of symbiotic nitrogen fixation occur in the nodules in the absence of leghaemoglobin. Nitrate reductase activity in C. pubescens nodules was negatively exponentially correlated with nitrogenase activity of the same nodules, suggesting a changing metabolism in old nodules. These data are discussed in terms of environmental and physical factors known to control nitrogen fixation.