Inhibition of checkpoint kinase 1 sensitizes lung cancer brain metastases to radiotherapy

The most important therapeutic tool in brain metastasis is radiation therapy. However, resistance to radiation is a possible cause of recurrence or treatment failure. Recently, signal pathways about DNA damage checkpoints after irradiation have been noticed. We investigated the radiosensitivity can be enhanced with treatment of Chk1 inhibitor, AZD7762 in lung cancer cell lines and xenograft models of lung cancer brain metastasis. Clonogenic survival assays showed enhancement of radiosensitivity with AZD7762 after irradiation of various doses. AZD7762 increased ATR/ATM-mediated Chk1 phosphorylation and stabilized Cdc25A, suppressed cyclin A expression in lung cancer cell lines. In xenograft models of lung cancer (PC14PE6) brain metastasis, AZD7762 significantly prolonged the median survival time in response to radiation. Depletion of Chk1 using shRNA also showed an enhancement of sensitivity to radiation in PC14PE6 cells. The results of this study support that Chk1 can be a good target for enhancement of radiosensitivity.     

Photophysical properties of Prochlorococcus marinus SS120 divinyl chlorophylls and phycoerythrin in vitro and in vivo

Prochlorococcus marinus SS120 is an ecologically important and biochemically intriguing marine cyanobacterium. In addition to divinyl chlorophylls (DV-Chls) a and b it possesses a particular form of phycoerythrin (PE), but no other phycobilins and therefore no complete phycobilisomes. Here, a spectroscopic characterisation of these DV-Chls and PE is provided. Comparison of fluorescence quantum yields, excited state lifetimes and absorption characteristics indicate similar light-harvesting properties of the DV-Chls as their monovinyl counterparts. PE, which is present only in tiny amounts, was purified and considerably enriched. A phycourobilin to phycoerythrobilin ratio of 3:1 chromophores per (alphabeta) PE monomer is suggested. The in vitro fluorescence lifetime of PE is 1.74 ns. In vivo time-resolved fluorescence measurements with synchrotron radiation were used to investigate the possible role of PE in light-harvesting. The fluorescence decay time for PE is about 550 ps, indicating an unusually slow excitation energy transfer. The decay time slowed to 1 ns after addition of glycerol to cell cultures. The contribution of PE to total light-harvesting capacity was estimated to be about one (alphabeta) PE monomer per 330 DV-Chl b molecules. Thus, the capacity of PE to function primarily as a photosynthetic light-harvesting pigment in P. marinus SS120 is low.     

Transformation of mortal human fibroblasts and activation of a growth inhibitory pathway by the bovine papillomavirus E5 oncoprotein.

The 44-amino acid bovine papillomavirus E5 protein induces tumorigenic transformation of immortal rodent fibroblasts by binding to and activating the platelet-derived growth factor beta receptor (PDGFbetaR). Here E5 was expressed in mortal human diploid fibroblasts (HDFs), which lack the accumulated genetic changes that are present in immortal rodent cells. E5 induced focus formation and morphological transformation of HDFs without inducing anchorage independence or immortalization. Similar effects were observed with the v-sis and neu* oncogenes. E5-PDGFbetaR complexes were observed in the E5-expressing HDFs, as was constitutive PDGFbetaR activation, which was required for the transforming activity of E5. The E5 HDFs attained a higher saturation density than the control cells, expressing increased levels of hyperphosphorylated retinoblastoma protein at subconfluent densities. However, when these cells reached confluence, growth inhibition accompanied by dramatic down-regulation of the PDGFbetaR, and retinoblastoma protein was induced apparently by a factor secreted into the medium. This may represent a novel negative feedback mechanism controlling PDGFbetaR-induced proliferation and thereby protecting against complete transformation.     

宁夏中部干旱带潜在蒸散量变化及影响因素

潜在蒸散量(PE,Potential Evapotranspiration)的估算与分析对于研究气候变化、监测农业旱情、提高农业水资源利用率具有十分重要的意义。在利用FAO Penman-Monteith(FPM)公式计算研究区1975—2012年PE日值的基础上,采用去趋势预置白Mann-Kendall检验法及敏感性分析探讨了历年来PE的变化规律和影响因素,将Matlab与Arc GIS相结合,研究了PE及其时序趋势的空间特征。结果显示:研究区多年平均PE月值呈现倒U形的变化规律,最大值和最小值分别出现在7月和1月;多年来,同心县PE具有明显的增长趋势,盐池和海原县则表现为微弱的减少。就影响因素而言,年际尺度上,同心县PE的主导因素为温度和风速,海原县为风速和水汽压,盐池县则以风速为主;月际水平上,温度的变化幅度最大,特别是在植物的生长季节(5—9月份)明显高于其他因素。研究区内PE具有明显的空间变化规律,盐池县表现为沿经向递减,同心和海原县则表现为沿纬向递增;就PE变化趋势的空间特征而言,盐池县大部分区域的PE变化不明显,显著增加的区域仅占该县总面积的2.52%,同心县显著和极显著增加的区域占全县面积的61.98%,海原县PE则以微弱减少和微弱增加为主,显著增加的区域面积比例小于30.00%。     

Role of Transthoracic Lung Ultrasonography in the Diagnosis of Pulmonary Embolism: A Systematic Review and Meta-Analysis

Background

Pulmonary embolism (PE) is a potentially life-threatening condition. Although computed tomography pulmonary angiography (CTPA) is the reference standard for diagnosis, its early diagnosis remains a challenge, and the concerns about the radiation exposures further limit the general use of CTPA. The primary aim of this meta-analysis was to evaluate the overall diagnostic accuracy of transthoracic lung ultrasound (TLS) in the diagnosis of PE.

Methods

PubMed, Web of science, OvidSP, ProQuest, EBSCO, Cochrane Library and Clinicaltrial.gov were searched systematically. The quality of included studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. The sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and hierarchical summary receiver operating characteristic (HSROC) curves were used to examine the TS performance. The Bayes analysis was used to calculate the post-test probability of PE. Publication bias was assessed with Deeks funnel plot.

Results

The results indicated that the sensitivity, specificity, PLR and NLR were 0.85 (95% confidence interval (CI), 0.78 to 0.90), and 0.83 (95% CI, 0.73 to 0.90). And the DOR and HSROC were 28.82 (95% CI, 17.60 to 47.21), 0.91(95% CI, 0.88, 0.93).

Conclusions

The present meta-analysis suggested that transthoracic lung ultrasonography is helpful in diagnosing pulmonary embolism. Although the application of transthoracic lung ultrasound may change some patients’ diagnostic processes, it is inappropriate to generally use transthoracic ultrasonography in diagnosing pulmonary embolism currently.     

X-ray-induced mutants resistant to 8-azaguanine. II. Cell cycle dose response.

Synchronous Chinese hamster ovary cells were irradiated in G1 or S phase. Colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 mug/ml 8-azaguanine (AG). An expression period of over three generations (multiplicity of 20) was utilized, with expression times ranging from 58 to 114 h. Both G1 and S phase were practically identical in sensitivity to X-ray-induced mutations, with mutant frequency/viable cell/rad ranging from 1 X 10(-7) (75-100 rad) to 8 X 10(-7) (1000 rad). The spontaneous mutation rate, shown by Luria-Delbruck fluctuation analysis, was 5 X 10(-7) per generation. Thirty-three mutants, isolated at random and grown for over 30 generations in the absence of AG, were analyzed for plating efficiency (PE) in different concentrations of AG or in hypoxanthine-aminopterin-thymidine (HAT) medium. Of these, 64% were resistant (PE greater than 0.1) to 7.5 mug/ml AG, 85% to 5.0 mug/ml, and 91% to 3.5 mug/ml. Only 42% showed possible hypoxanthine-phosphoribosyltransferase (hprtase) deficiency as evidenced by HAT sensitivity (PE less than 0.1). Wild type controls exhibited PE's in 3.5 mug/ml AG of less than 0.001 and in HAT of greater than 0.5. Of ten mutants studied, all demonstrated survival response to radiation similar to wild type cells (D0 of approx. 120 rad). For radiation protection standards, the radiation dose required to induce mutations at a rate equal to that occurring spontaneously is called the doubling dose. The doubling dose observed for acute irradiation was about 3 rad and was estimated to be 10-60 rad for chronic irradiation, similar to that often reported for in vivo studies.     

UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells

Epidermal growth factor receptor (EGFR) is overexpressed in human pancreatic cancer and is one of the clinical targets in its treatment. In the present study we investigated the mechanism underlying ultraviolet C (UVC)-radiation-induced cell growth inhibition and downregulation of EGFR in human pancreatic cancer cells (Panc1 and KP3). The cell proliferation assay indicated that Panc1 and KP3 cells were more sensitive to UVC radiation, and their growth was significantly inhibited compared to cells of the normal human pancreatic epithelial cell line, PE. Although EGFR levels was extremely low in PE cells, EGFR were highly overexpressed in Panc1 and KP3 cells, and UVC radiation downregulated the expression of EGFR in a time-dependent manner and induced phosphorylation of EGFR at Ser1046/1047 (S1046/7) in Panc1 and KP3 cells. UVC radiation induced activation of p38 mitogen-activated protein kinase (MAPK), and EGFR phosphorylation at S1046/7 induced by UVC radiation was markedly attenuated by the inhibition of p38 MAPK. Moreover, fluorescence microscopy revealed that p38 MAPK activated by UVC radiation triggered EGFR internalization and that this was not correlated with c-Cbl, an ubiquitin ligase, which plays an important role in EGF-induced EGFR downregulation. Taken together, our results suggest that in pancreatic cancer cells UVC radiation induced desensitization of the cells to EGFR stimuli via phosphorylation of EGFR at S1046/7 by activation of p38 MAPK, independent of c-Cbl.     

The genetic basis of resistance to ionising radiation damage in cultured mammalian cells

To test the genetic similarity of independently-isolated hamster cell mutants sensitive to ionising radiation, these were fused in pairs and the hybrids exposed to X-rays. Some mutants (irs1, irs3, xrs-1, XR-1, BLM2) were found to complement all others tested for radiosensitivity in hybrids, and are therefore in separate genetic groups. The mutants irs2 and V-E5, both isolated from V79 cells, did not complement and therefore belong to the same group. Another pair, EM7 and irs1SF, formed hybrids with intermediate levels of survival between mutant and wild-type. However, the parental cells fused to irs1SF also showed intermediate sensitivity, suggesting a semi-dominant mutant phenotype rather than a lack of complementation. Crosses of some of these hamster mutants to the radiosensitive mouse mutant M10 showed clear complementation (irs1 x M10, irs2 x M10) but for others the complementation did not greatly exceed the sensitivity of one (irs3 x M10) or both mutants (XR-1 x M10). Taken with our previously-published data, these results show that there are at least 8 genetic groups determining resistance to ionising radiation damage in rodent cells.     

High resistance of cultured Mongolian gerbil cells to X-ray-induced killing and chromosome aberrations.

The Mongolian gerbil (Meriones unguiculatus) is known to be one of the most radioresistant animals. We have examined the X-ray sensitivity of normal diploid fibroblasts from Mongolian gerbil embryos compared with those of cultured embryo cells obtained from various laboratory animals and a normal human. There was a wide difference in X-ray sensitivity for cell killing among different mammalian species. The D0 values for Mongolian gerbil cells ranged from 2.08 to 2.28 Gy, values which are twice as high as those for human cells. The mean D0 value for human cells was 1.06 Gy. Mouse, rat, Chinese hamster, and Syrian/golden hamster cells showed similar D0 values ranging from 1.30 to 1.56 Gy. When cells were irradiated with X rays, ten times more chromosome aberrations were detected in human cells than in Mongolian gerbil cells. The frequencies of chromosome aberrations in other rodent cells were between the values for cells from humans and those from gerbils. These data indicate that the Mongolian gerbil cells are resistant to X-ray-induced cell killing and chromosome aberrations, and that the radiation sensitivity of mammalian cells in primary culture may be reflected by their radioresistance in vivo.     

Evaluation of the GreenScreen GADD45alpha-GFP indicator assay with non-proprietary and proprietary compounds

The GreenScreen GADD45alpha indicator assay has been assessed for its concordance with in vitro genotoxicity and rodent carcinogenicity bioassay data. To test robustness, sensitivity, and specificity of the assay, 91 compounds with known genotoxicity results were screened in a blinded manner. Fifty seven of the compounds were classified as in vitro genotoxic whereas 34 were non-genotoxic. Out of the 91 compounds, 50 had been tested in 2-year carcinogenicity assays, with 33 identified to be rodent carcinogens and 17 non-carcinogens. Gadd45alpha assay sensitivity and specificity for genotoxicity was 30% and 97%, respectively (17/57 and 33/34), whereas its sensitivity and specificity for rodent carcinogenicity was 30% and 88%, respectively (10/33 and 15/17). Gadd45alpha assay genotoxicity results from this validation study exhibited a high concordance with previously published results as well as for compound test results generated at two different sites (91%, 19/21), indicating that the assay is both robust and reproducible. In conclusion, results from this blinded and independent validation study indicate that the GreenScreen GADD45 indicator assay is reproducible and reliable with low sensitivity and high specificity for identifying genotoxic and carcinogenic compounds.     

Influence of waste water from the paper industry and UV-B radiation on the photosynthetic efficiency of Euglena gracilis

The green flagellate Euglena gracilis has been used as a model organism to elucidate the possible large-scale and short-term effects of waste substances from the pulp and paper industry on photosynthetic efficiency (PE). Different concentrations of waste substances before and after treatment in a cleaning system were studied. The uncleaned sample at concentrations up to 1:10 and the cleaned sample at concentrations up to 1:5 showed stimulating effects on the PE after 7 days of incubation compared to the control. The effects of waste substances on the PE of E. gracilis were also studied in combination with short-term studies (20 and 40 min) of ultraviolet-B radiation (UV-B, 280–320 nm). It was shown that increasing concentrations of the uncleaned sample had continuously stimulating effects on the PE and worked protectively against UV-B radiation. The cleaned sample exhibited no effects, or negative effects, on the PE of E. gracilis together with UV-B radiation compared to the experiments with only UV-B radiation. At the concentration 1:1 of the cleaned sample an increase in the PE was detected due to the high concentration of the coloured substances and a decrease in the UV-B penetration. PE revealed itself to be highly sensitive for detecting toxic effects on E. gracilis and is thus very promising for use in regular toxicity tests of waste water from pulp and paper industry. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phycoerythrin synthesis is induced by solar UV-B in the cyanobacterium Nostoc

Solar UV-B (280–315 nm) induces the synthesis of phycoerythrin (PE) in a Nostoc species isolated from the Andean high altitude lake Yanaqocha. The outdoor experiments were carried out in a small lake in Erlangen, Germany, using natural conditions. After 2- and 4-h exposure to solar radiation, the immunodetection signal using monoclonal antibodies anti-PE was lower in control cells (exposed to PAR + UV-A) than in cells exposed to total solar radiation (PAR + UV-A + UV-B). Cells exposed at depths in which no UV-B penetrated showed no differences from control cells regarding PE content. When exposed to monochromatic radiation of 280, 300 or 360 nm, purified PE was photodegraded in a wavelength dependent manner resulting in different polypeptide fragments carrying chromophore groups. Immunodetection revealed active synthesis of PE in parallel to photodamage by solar UV-B indicating that PE is important for photoadaptation to shorter wavelengths in the cyanobacterium Nostoc sp.     

Metabolic Injury in Frozen Bacteria

S ummary : Aerobacter aerogenes populations, partly killed by freezing and thawing, usually showed more viable bacteria when plated on a rich medium than on a poor one. This 'metabolic injury' was not due to osmotic sensitivity or to toxic materials in the agar. Freezing and thawing was not significantly mutagenic and mutation to nutritional exigence did not account for the phenomenon. The survivors of freezing and thawing showed long lag phases in the recovery media, during which lag periods further mortality occurred.     

Rodent phylogeny and a timescale for the evolution of Glires: evidence from an extensive taxon sampling using three nuclear genes

Rodentia is the largest order of placental mammals, with approximately 2,050 species divided into 28 families. It is also one of the most controversial with respect to its monophyly, relationships between families, and divergence dates. Here, we have analyzed and compared the performance of three nuclear genes (von Willebrand Factor, interphotoreceptor retinoid-binding protein, and Alpha 2B adrenergic receptor) for a large taxonomic sampling, covering the whole rodent and placental diversity. The phylogenetic results significantly support rodent monophyly, the association of Rodentia with Lagomorpha (the Glires clade), and a Glires + Euarchonta (Primates, Dermoptera, and Scandentia) clade. The resolution of relationships among rodents is also greatly improved. The currently recognized families are divided here into seven well-defined clades (Anomaluromorpha, Castoridae, Ctenohystrica, Geomyoidea, Gliridae, Myodonta, and Sciuroidea) that can be grouped into three major clades: Ctenohystrica, Gliridae + Sciuroidea, and a mouse-related clade (Anomaluromorpha, Castoridae + Geomyoidea, and Myodonta). Molecular datings based on these three genes suggest that the rodent radiation took place at the transition between Paleocene and Eocene. The divergence between rodents and lagomorphs is placed just at the K-T boundary and the first splits among placentals in the Late Cretaceous. Our results thus tend to reconcile molecular and morphological-paleontological insights.     

Effects of increasing doses of UV-B radiation on photosynthesis and motility inChlamydomonas reinhardtii

Effects of increasing doses of UV-B radiation on photosynthetic efficiency (PE) and motility inChlamydomonas reinhardtii were investigated. The exposure time ranged from 10 to 120 min at 0.73 W/m² UV-B radiation. A decrease in compensation points (CP) was followed by an increase in PE on extending the UV-B treatment up to 60 min. The subsequent increase in PE was accompanied by increasing CPs. Enhanced doses of UV-B radiation thus have a stimulatory effect on the PE inC. reinhardtii. No inhibitory effects could be detected. No statistically significant differences in motility could be observed due to an extremely high variation of values.     

Clinical considerations in rodent bioimaging

Imaging modalities such as micro-computed tomography (micro-CT), micro-positron emission tomography (micro-PET), high-resolution magnetic resonance imaging (MRI), optical imaging, and high-resolution ultrasound are rapidly becoming invaluable research tools. These advanced imaging technologies are now commonly used to investigate rodent biology, metabolism, pharmacokinetics, and disease in vivo. Choosing an appropriate anesthetic regimen as well as monitoring and supporting the animal's physiologic balance is key to obtaining images that truly represent the biologic process or disease state of interest. However, there are many challenges in rodent bioimaging such as limited animal access, small sample volumes, anesthetic complications, strain and gender variability, and the introduction of image artifacts. Because each imaging study presents unique challenges, a thorough understanding of the imaging modality used, the animal's health status, and the research data desired is required. This article addresses these issues along with other common laboratory animal clinical considerations such as biosecurity and radiation safety in in vivo rodent bioimaging.     

Bone and glucose metabolism: A two-way street

Evidence from rodent models indicates that undercarboxylated osteocalcin (ucOC), a product of osteoblasts, is a hormone affecting insulin production by the pancreas and insulin sensitivity in peripheral tissues, at least in part through enhanced secretion of adiponectin from adipocytes. Clinical research to test whether this relationship is found in humans is just beginning to emerge. Cross-sectional studies confirm associations between total osteocalcin (OC), ucOC and glucose metabolism but cannot distinguish causality. To date, longitudinal studies have not provided a consistent picture of the effects of ucOC or OC on fasting glucose and insulin sensitivity. Further exploration into the physiological and mechanistic effects of ucOC and OC, in rodent models and clinical studies, is necessary to determine to what extent the skeleton regulates energy metabolism in humans.     

In vivo bupivacaine reduces the in vitro sensitivity of the human vas deferens to phenylephrine

The isolated human vas deferens (HVD) has been the subject of a limited number of pharmacological studies. Furthermore, it has not been established whether the local or general anesthetics used in obtaining the HVD affect the responses of this isolated tissue. Therefore, the aim of this study was to determine if the local anesthetic, bupivacaine (BPV), alters the sensitivity of the HVD to agonists. It was shown that BPV, injected into subjects undergoing vasectomy, was present in the excised HVD and in sufficient concentration to attenuate the phenylephrine (PE)-induced contraction. Bupivacaine assays were done by gas chromatography - mass spectrometry. Following 3 h of equilibration with repetitive washing, 95.0 +/- 2.1% (SD) of the BPV could be eliminated from the tissue, which correlated well with an observed increase in sensitivity of the HVD to PE. The sensitivity to PE of HVD isolated from subjects under general anesthesia was less than the sensitivity of tissues obtained with bupivacaine and in this case the depressant effect was not reversed by washing. These results indicate that it is important to consider the anesthetic procedure used to acquire the HVD and also the equilibration procedure prior to pharmacological studies.     

Power of exclusion of 19 microsatellite markers for parentage testing in river buffalo (Bubalus bubalis)

In the present study, 19 microsatellite markers were assessed for their power of exclusion to test parentage in river buffalo. Microsatellite genotypes of 216 unrelated buffaloes belonging to five different breeds were utilized for the study. The probabilities of exclusion were calculated for three hypothetical situations viz. paternity testing (PE1), one parental genotype unavailable (PE2) and exclusion of both parents i.e. substituted offspring (PE3). The mean probability of exclusion across 19 investigated markers in buffalo was 0.578 (PE1), 0.405 (PE2) and 0.764 (PE3) respectively. The probability of exclusion for paternity (PE1) ranged between 0.297 and 0.814 across different markers. The exclusion probability for the cases one parent unavailable (PE2) and substituted offspring (PE3) varied from 0.143 to 0.688 and 0.465 to 0.946 respectively. Polymorphism information content and expected heterozygosity were found to have significantly high correlation with probability of exclusion of microsatellite markers. The cumulative PE1 of nine marker loci was estimated to be 0.9999 while in case of absence of one of the parental genotypes, a minimum of 11 markers were required to achieve a cumulative PE2 of 0.999. In conclusion, the present study proposes two multiplex sets with four and five markers respectively for routine parentage testing in buffalo and an additional set of four markers for doubtful cases of paternity.