Internal eliminated sequences interrupting the Oxytricha 81 locus: allelic divergence, conservation, conversions, and possible transposon origins

Internal eliminated sequences (IESs) often interrupt ciliate genes in the silent germline nucleus but are exactly excised and eliminated from the developing somatic nucleus from which genes are then expressed. Some long IESs are transposons, supporting the hypothesis that short IESs are ancient transposon relics. In light of that hypothesis and to explore the evolutionary history of a collection of IESs, we have compared various alleles of a particular locus (the 81 locus) of the ciliated protozoa Oxytricha trifallax and O. fallax. Three short IESs that interrupt two genes of the locus are found in alleles from both species, and thus must be relatively ancient, consistent with the hypothesis that short IESs are transposon relics. In contrast, TBE1 transposon interruptions of the locus are allele-specific and probably the results of recent transpositions. These IESs (and the TBE1s) are precisely excised from the DNA of the developing somatic macronucleus. Each IES interrupts a highly conserved sequence. A few nucleotides at the ends of each IES are also conserved, suggesting that they interact critically with IES excision machinery. However, most IES nucleotide positions have evolved at high rates, showing little or no selective constraint for function. Nonetheless, the length of each IES has been maintained (+/- 3 bp). While one IES is approximately 33 bp long, three other IESs have very similar sizes, approximately 70 bp long. Two IESs are surrounded by direct repeats of the sequence TTCTT. No other sequence similarities were found between any of the four IESs. However, the ends of one IES do match the inverted terminal repeat consensus sequence of the "TA" IESs of Paramecium. Three O. trifallax alleles appear to have been recipients in recent conversion events that could have been provoked by double-strand breaks associated with IES ends subsequent to IES transposition. Our findings support the hypothesis that short IESs evolved from ancient transposons that have lost most of their sequences, except those necessary for precise excision during macronuclear development.      

Biodiversity and ecology of epilithic diatoms in the Agnéby River,Ivory Coast

The ecology and taxonomy of the epilithic diatom flora of the Agnéby River, Ivory Coast were studied in 2012. Ten sites were investigated and diatoms were sampled on glass slides immersed for a period of 30 days during the wet and dry seasons. Physico-chemical parameters were measured at each site while sampling diatoms. Five taxa were largely dominant: Planothidium comperei CE Wetzel, N’Guessan and Tison-Rosebery, Eolimna minima (Grunow) Lange-Bertalot, Planothidium piaficum (JR Carter and Denny) CE Wetzel and Ector, Cocconeis schroederi Foged and Cocconeis scutellum var. parva (Grunow in Van Heurck) Cleve. Electrical conductivity, temperature, dissolved oxygen, nitrite and phosphorus were found to influence the distribution of taxa.     

Minnelide: A Novel Therapeutic That Promotes Apoptosis in Non-Small Cell Lung Carcinoma In Vivo

Background

Minnelide, a pro-drug of triptolide, has recently emerged as a potent anticancer agent. The precise mechanisms of its cytotoxic effects remain unclear.

Methods

Cell viability was studied using CCK8 assay. Cell proliferation was measured real-time on cultured cells using Electric Cell Substrate Impedence Sensing (ECIS). Apoptosis was assayed by Caspase activity on cultured lung cancer cells and TUNEL staining on tissue sections. Expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA, APAF-1) was estimated by qRTPCR. Effect of Minnelide on proliferative cells in the tissue was estimated by Ki-67 staining of animal tissue sections.

Results

In this study, we investigated in vitro and in vivo antitumor effects of triptolide/Minnelide in non-small cell lung carcinoma (NSCLC). Triptolide/Minnelide exhibited anti-proliferative effects and induced apoptosis in NSCLC cell lines and NSCLC mouse models. Triptolide/Minnelide significantly down-regulated the expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA) and up-regulated pro-apoptotic APAF-1 gene, in part, via attenuating the NF-κB signaling activity.

Conclusion

In conclusion, our results provide supporting mechanistic evidence for Minnelide as a potential in NSCLC.     

The relationship of nitrogen source and in vivo nitrate reductase actiity to root formation in Euphorbia esula cell suspension cultures

Root formation and in vivo nitrate reductase (NR) activity were determined in leafy spurge cell suspensions. Cells grown in B5 media with 1 mg L^–1 2,4-D were transferred to B5 media without 2,4-D, but containing either high (92:8) or low (15:85) ratios of nitrogen as NO ₃ ^– -N:NH ₄ ⁺ -N. In older cell lines root formation occurred only in the low NO ₃ ^– medium with =<30 roots per flask. In younger cell lines root numbers were greatest in the high NO ₃ ^– medium (1000 to 3000 per flask). Cells grown in low NO ₃ ^– medium were about one-third the final dry weight as those in high NO ₃ ^– medium. Root length was consistently greater for cell lines of all ages in the low NO ₃ ^– medium. Developmental profiles of NR activity were similar in cell lines of all ages, whether or not roots were formed. NR activity was lower, however, in cultures grown in low NO ₃ ^– medium compared to high NO ₃ ^– medium. There was no consistent relationship between NR activity and root initiation. Therefore, nitrate reductase does not appear to be a primary target for regulation of leafy spurge growth by chemical application.     

The effects of dissolved heavy metals on attached diatoms in the Uintah Basin of Utah,U.S.A.

The relationships of diatom species to dissolved heavy metals in the streams of the Uintah Basin of Utah were studied through four seasons of 1977–1978. Niche center gradient analysis, cluster analysis and correlation analysis were performed.Achnanthes minutissima, Cyclotella meneghiniana, Cymbella minuta, Gomphonema parvulum, Navicula secreta var.apiculata, Nitzschia frustulum, Nitzschia frustulum var.perminuta, Nitzschia frustulum var.perpusilla, Nitzschia palea, andSynedra ulna appear to be indicator species of high or low heavy metal concentrations. Several other species also showed meaningful relationships to high or low heavy metal concentrations.Dept. of Physics and Astronomy, Brigham Young UniversityDept. of Physics and Astronomy, Brigham Young University     

EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P‐bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P‐body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P‐body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P‐bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer‐relevant functions and suggest that modulation of P‐body activity may represent a new paradigm for cancer treatment.     

Enhancing Personal Wellness in Counselor Trainees Using Biofeedback: An Exploratory Study

The purpose of this study was to explore whether biofeedback-assisted relaxation training could reduce stress-related symptoms and enhance personal well-being in a group of counselor trainees enrolled in a basic counseling skills course. Treatment participants received ten sessions of weekly biofeedback-assisted relaxation training, whereas the control participants received no intervention. The treatment group showed, significant improvements in several symptom areas measured by the Symptom Checklist 90-Revised: physical complaints (Somatization), personal inadequacy (Interpersonal Sensitivity), confused thinking or alienation or both (Psychoticism), and the overall number and severity of symptoms (Global Severity Index and Positive Symptom Total). The control participants showed no significant improvements in any symptom areas. Biofeedback-assisted relaxation was shown to be an effective stress-reducing intervention for counselor trainees, which resulted in a greater sense of personal well-being.     

Nontarget arthropod abundance in areawide-managed corn habitats treated with semiochemical-based bait insecticide for corn rootworm (Coleoptera: Chrysomelidae) control

Impacts of semiochemical-based insecticidal bait applications on beneficial arthropod groups common to field corn, Zea mays L., habitats were assessed in areawide-managed field sites in South Dakota and Iowa during 1997 and 1998. Slam, a commercial bait formulation comprised of 87% cucurbitacin and 13% carbaryl insecticide, was used for management of adult rootworm, Diabrotica spp., and controls consisted of cornfield habitats without bait applications. Effects on beneficial organisms were variable, and negative impacts were infrequent. Coccinellidae, Staphylinidae, and Anthocoridae were usually more abundant in bait-treated plots than in controls that received at-plant soil insecticides, especially by 4 wk postapplication. Carabid beetle activity also had increased in bait-treated corn by proportionally greater rates than in control plots at 4 wk postapplication in two of the four site by year combinations in this study. Impacts of semiochemical-based adulticide applications on Formicidae were not consistently negative or positive. The relative lack of consistent negative impacts on nontarget arthropods suggests that other biotic and abiotic factors leading to natural population fluxes may have more influence on these groups of beneficial organisms than applications of semiochemical-based bait containing carbaryl. Overall, it seems that areawide applications of these baits for managing rootworm populations in corn are not likely to impose deleterious effects on the nontarget faunal groups we surveyed, especially in comparison with the at-plant applications of soil insecticides used as experimental controls in this study.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

Comparison of human and mouse sperm chromatin structure by flow cytometry

Human and mouse sperm nuclei obtained by sonication or mechanical agitation of freshly isolated sperm in the presence of anionic detergent were purified through a sucrose gradient and stained with acridine orange (AO); their fluorescence intensity was measured by flow cytometry. The green fluorescence, characteristic of AO binding to DNA by intercalation, was twice lower per unit of DNA for human sperm nuclei than for human peripheral blood lymphocytes. After extraction of basic proteins with 0.08 N HCl, AO binding to DNA increased 3.2-fold for lymphocytes and only 1.3-fold for sperm indicating that, in contrast to somatic cells, the proteins restricting AO binding to DNA are essentially non-extractable from sperm at that low pH. Treatment of human and mouse nuclei with dithiothreitol (DTT), a sulfhydryl reducing agent, and trypsin, removed constraints responsible for the restriction of AO binding. Specifically, as a result of DTT treatment alone there was up to a 20–30% increase of AO binding; upon subsequent addition of trypsin there was a further rapid rise in AO binding up to a final level of approximately 5 times the original AO binding to isolated sperm nuclei. Electron microscopy of DTT-treated human sperm nuclei showed that the reducing agent caused chromatin decondensation to a level whereby 20–30 Å diameter fibers interconnecting chromatin bodies about 30–75 nm in diameter were revealed. Trypsin digestion in the presence of DTT converted the chromatin bodies into a network of fibrous structures about 150 Å in diameter. Both electron microscopy and flow cytometry demonstrated an extremely large intercellular variation among human sperm nuclei in response to DTT and trypsin treatment indicating heterogeneity of chromatin structure. In contrast, AO staining of mouse sperm nuclei increased homogeneously in response to DTT and trypsin treatment.