Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans▿

PubMed Central

Xie, Zhoujie; Okinaga, Toshinori; Qi, Fengxia; Zhang, Zhijun; Merritt, Justin

2011-01-01

Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches. PMID:21948849

Mapping Eight Male-Sterile, Female-Sterile Soybean Mutants

USDA-ARS?s Scientific Manuscript database

In soybean, mutations in genes involved in meiosis can lead to altered chromosome pairing and result in non-functional gametes.Mutability of the w4 flower color locus in soybean is due to an unstable allele designated w4-m (mutable). Several germinal revertant studies using the w4-m system resulted ...

Maintaining Genome Stability: The Role of Helicases and Deaminases

DTIC Science & Technology

2006-07-01

functional for replication. Then, we screened the surviving transformants for sensitivity to hydroxyurea . Our first screen was conducted under conditions...that are lethal for checkpoint mutants such as ∆rad3. We did not observe any hydroxyurea -sensitive MCM clones. However, following the old genetics...this kinase. In particular, we observe that an mcm4ts mutant blocked in hydroxyurea , and then released to restrictive temperature, is competent to

Maintaining Genome Stability: The Role of Helicases and Deaminases

DTIC Science & Technology

2007-07-01

transformants for sensitivity to hydroxyurea . Our first screen was conducted under conditions that are lethal for checkpoint mutants such as ∆rad3...We did not observe any hydroxyurea -sensitive MCM clones. However, following the old genetics adage “absence of evidence is not evidence of... hydroxyurea , and then released to restrictive temperature, is competent to complete replication and go on to divide. In contrast, this mutant shifted

Clonal cell populations unresponsive to radiosensitization induced by telomerase inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, Yeun-Jin; Shin, Hyun-Jin; Park, Jeong-Eun

Research highlights: {yields} In our present manuscript, we have clearly showed an interesting but problematic obstacle of a radiosensitization strategy based on telomerase inhibition by showing that: Clonal population unresponsive to this radiosensitization occasionally arise. {yields} The telomere length of unsensitized clones was reduced, as was that of most sensitized clones. {yields} The unsensitized clones did not show chromosome end fusion which was noted in all sensitized clones. {yields} P53 status is not associated with the occurrence of unsensitized clone. {yields} Telomere end capping in unsensitized clone is operative even under telomerase deficiency. -- Abstract: A combination of a radiotherapeuticmore » regimen with telomerase inhibition is valuable when tumor cells are to be sensitized to radiation. Here, we describe cell clones unresponsive to radiosensitization after telomere shortening. After extensive division of individual transformed clones of mTERC{sup -/-} cells, about 22% of clones were unresponsive to radiosensitization even though telomerase action was inhibited. The telomere lengths of unsensitized mTERC{sup -/-} clones were reduced, as were those of most sensitized clones. However, the unsensitized clones did not exhibit chromosomal end-to-end fusion to the extent noted in all sensitized clones. Thus, a defense mechanism preventing telomere erosion is operative even when telomeres become shorter under conditions of telomerase deficiency, and results in unresponsiveness to the radiosensitization generally mediated by telomere shortening.« less

Typed Regions

DTIC Science & Technology

2005-01-01

specifically, we show how to write a two-generations collector for a language with mutable ref cells. 1 Introduction As the technology of certifying...how to write a two-generations collector for a language with mutable ref cells. 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF...stated goals, we make the following contributions: – A language that can seamlessly combine the benefits of traditional intuitionistic references and

Why Information Matters: Examining the Consequences of Suggesting That Pedophilia Is Immutable.

PubMed

Tozdan, Safiye; Kalt, Anna; Dekker, Arne; Keller, Livia B; Thiel, Stephanie; Müller, Jürgen L; Briken, Peer

2018-04-01

In this study, the impact of suggesting that pedophilia is immutable on a man's specific self-efficacy for modifying his sexual interest in children was examined in 94 men with a sexual interest in children. The participants were selected from differing contexts and included non-forensic patients, forensic patients, and participants from the Internet. Randomly distributed to two conditions, the mutable condition group received the information that experts consider pedophilia to be modifiable, whereas the immutable condition group received the information that experts consider pedophilia to be stable. Afterward, the participants' levels of specific self-efficacy for modifying their sexual interest in children were assessed. Non-forensic participants in the mutable condition reported higher levels of specific self-efficacy than those in the immutable condition. No differences in specific self-efficacy were revealed for the forensic and Internet participants when comparing the mutable and immutable conditions. It would appear appropriate to avoid generalized and absolute statements about the (im)mutability of sexual interest in children, as scientific research on this topic remains insufficient. Furthermore, given the present results, such statements might have serious consequences for an individual's belief in being able to change his sexual interest in children.

A Search for a General Phenomenon of Adaptive Mutability

PubMed Central

Galitski, T.; Roth, J. R.

1996-01-01

The most prominent systems for the study of adaptive mutability depend on the specialized activities of genetic elements like bacteriophage Mu and the F plasmid. Searching for general adaptive mutability, we have investigated the behavior of Salmonella typhimurium strains with chromosomal lacZ mutations. We have studied 30 revertible nonsense, missense, frameshift, and insertion alleles. One-third of the mutants produced >=10 late revertant colonies (appearing three to seven days after plating on selective medium). For the prolific mutants, the number of late revertants showed rank correlation with the residual β-galactosidase activity; for the same mutants, revertant number showed no correlation with the nonselective reversion rate (from fluctuation tests). Leaky mutants, which grew slowly on selective medium, produced late revertants whereas tight nongrowing mutants generally did not produce late revertants. However, the number of late revertants was not proportional to residual growth. Using total residual growth and the nonselective reversion rate, the expected number of late revertants was calculated. For several leaky mutants, the observed revertant number exceeded the expected number. We suggest that excess late revertants from these mutants arise from general adaptive mutability available to any chromosomal gene. PMID:8725216

The microbiology of mutability.

PubMed

Sundin, George W; Weigand, Michael R

2007-12-01

Bacteria possessing elevated spontaneous mutation rates are prevalent in certain environments, which is a paradox because most mutations are deleterious. For example, cells with defects in the methyl-directed mismatch repair (MMR) system, termed mutators or hypermutators, are overrepresented in populations of bacterial pathogens, with the mutator trait hypothesized to be advantageous in the changing host enviroments faced during colonization and establishment of chronic infections. Error-prone DNA polymerases, such as polIV and polV, function in translesion DNA synthesis, a DNA damage response that ensures genome integrity with a cost of increased mutation. While the biochemical aspects of these mutability pathways are well understood, the biological impacts have received less attention. Here, an examination of bacterial mutability systems and specifically the ecological and evolutionary context resulting in the selection of these systems is carried out.

A Laboratory for Characterizing the Efficacy of Moving Target Defense

DTIC Science & Technology

2016-10-25

of William and Mary are developing a scalable, dynamic, adaptive security system that combines virtualization , emulation, and mutable network...goal with the resource constraints of a small number of servers, and making virtual nodes “real enough” from the view of attackers. Unfortunately, with...we at College of William and Mary are developing a scalable, dynamic, adaptive security system that combines virtualization , emulation, and mutable

Projecting potential adoption of genetically engineered freeze-tolerant Eucalyptus in the United States

Treesearch

David N. Wear; Ernest Dixon IV; Robert C. Abt; Navinder Singh

2015-01-01

Development of commercial Eucalyptus plantations has been limited in the United States because of the species’ sensitivity to freezing temperatures. Recently developed genetically engineered clones of a Eucalyptus hybrid, which confer freeze tolerance, could expand the range of commercial plantations. This study explores how...

Isolation of an essential Schizosaccharomyces pombe gene, prp31+, that links splicing and meiosis

PubMed Central

Bishop, Danielle T.; McDonald, W. Hayes; Gould, Kathleen L.; Forsburg, Susan L.

2000-01-01

We carried out a screen for mutants that arrest prior to premeiotic S phase. One of the strains we isolated contains a temperature-sensitive allele mutation in the fission yeast prp31+ gene. The prp31-E1 mutant is defective in vegetative cell growth and in meiotic progression. It is synthetically lethal with prp6 and displays a pre-mRNA splicing defect at the restrictive temperature. We cloned the wild-type gene by complementation of the temperature-sensitive mutant phenotype. Prp31p is closely related to human and budding yeast PRP31 homologs and is likely to function as a general splicing factor in both vegetative growth and sexual differentiation. PMID:10871341

Interfibrillar stiffening of echinoderm mutable collagenous tissue demonstrated at the nanoscale

PubMed Central

Mo, Jingyi; Blowes, Liisa M.; Egertová, Michaela; Terrill, Nicholas J.; Wang, Wen; Elphick, Maurice R.; Gupta, Himadri S.

2016-01-01

The mutable collagenous tissue (MCT) of echinoderms (e.g., sea cucumbers and starfish) is a remarkable example of a biological material that has the unique attribute, among collagenous tissues, of being able to rapidly change its stiffness and extensibility under neural control. However, the mechanisms of MCT have not been characterized at the nanoscale. Using synchrotron small-angle X-ray diffraction to probe time-dependent changes in fibrillar structure during in situ tensile testing of sea cucumber dermis, we investigate the ultrastructural mechanics of MCT by measuring fibril strain at different chemically induced mechanical states. By measuring a variable interfibrillar stiffness (EIF), the mechanism of mutability at the nanoscale can be demonstrated directly. A model of stiffness modulation via enhanced fibrillar recruitment is developed to explain the biophysical mechanisms of MCT. Understanding the mechanisms of MCT quantitatively may have applications in development of new types of mechanically tunable biomaterials. PMID:27708167

Interfibrillar stiffening of echinoderm mutable collagenous tissue demonstrated at the nanoscale.

PubMed

Mo, Jingyi; Prévost, Sylvain F; Blowes, Liisa M; Egertová, Michaela; Terrill, Nicholas J; Wang, Wen; Elphick, Maurice R; Gupta, Himadri S

2016-10-18

The mutable collagenous tissue (MCT) of echinoderms (e.g., sea cucumbers and starfish) is a remarkable example of a biological material that has the unique attribute, among collagenous tissues, of being able to rapidly change its stiffness and extensibility under neural control. However, the mechanisms of MCT have not been characterized at the nanoscale. Using synchrotron small-angle X-ray diffraction to probe time-dependent changes in fibrillar structure during in situ tensile testing of sea cucumber dermis, we investigate the ultrastructural mechanics of MCT by measuring fibril strain at different chemically induced mechanical states. By measuring a variable interfibrillar stiffness (E IF ), the mechanism of mutability at the nanoscale can be demonstrated directly. A model of stiffness modulation via enhanced fibrillar recruitment is developed to explain the biophysical mechanisms of MCT. Understanding the mechanisms of MCT quantitatively may have applications in development of new types of mechanically tunable biomaterials.

Experimental analysis of a TEM plane transmission line for DNA studies at 900 MHz EM fields

NASA Astrophysics Data System (ADS)

Belloni, F.; Doria, D.; Lorusso, A.; Nassisi, V.; Velardi, L.; Alifano, P.; Monaco, C.; Talà, A.; Tredici, M.; Rainò, A.

2006-07-01

A suitable plane transmission line was developed and its behaviour analysed at 900 MHz radiofrequency fields to study DNA mutability and the repair of micro-organisms. In this work, utilizing such a device, we investigated the behaviour of DNA mutability and repair of Escherichia coli strains. The transmission line was very simple and versatile in changing its characteristic resistance and field intensity by varying its sizes. In the absence of cell samples inside the transmission line, the relative modulation of the electric and/or magnetic field was ±31% with respect to the mean values, allowing the processing of more samples at different exposure fields in a single run. A slight decrease in spontaneous mutability to rifampicin-resistance of the E. coli JC411 strain was demonstrated in mismatch-repair proficient samples exposed to the radio-frequency fields during their growth on solid medium.

Cloning of the altered mRNA stability (ams) gene of Escherichia coli K-12.

PubMed Central

Claverie-Martin, F; Diaz-Torres, M R; Yancey, S D; Kushner, S R

1989-01-01

A temperature-sensitive mutation in the ams gene of Escherichia coli causes an increase in the chemical half-life of pulse-labeled RNA at the nonpermissive temperature. Using lambda clones containing DNA fragments from the 23- to 24-min region on the E. coli chromosome, we have isolated a 5.8-kilobase DNA fragment which, when present in a low-copy-number plasmid, complements the conditional lethality and increased mRNA stability associated with the ams-1 mutation. The approximate initiation site and the direction of transcription of the ams gene were determined from the size of truncated polypeptides produced by Tn1000 insertions and Bal 31 deletions. Overexpression of the ams locus by using a T7 RNA polymerase-promoter system permitted the identification of an ams-encoded polypeptide of 110 kilodaltons. Images PMID:2477358

Integrating content and structure aspects of the self: traits, values, and self-improvement.

PubMed

Roccas, Sonia; Sagiv, Lilach; Oppenheim, Shani; Elster, Andrey; Gal, Avigail

2014-04-01

Research on the structure of the self has mostly developed separately from research on its content. Taking an integrative approach, we studied two structural aspects of the self associated with self-improvement--self-discrepancies and perceived mutability--by focusing on two content areas, traits and values. In Studies 1A-C, 337 students (61% female) reported self-discrepancies in values and traits, with the finding that self-discrepancies in values are smaller than in traits. In Study 2 (80 students, 41% female), we experimentally induced either high or low mutability and measured perceived mutability of traits and values. We found that values are perceived as less mutable than traits. In Study 3, 99 high school students (60% female) reported their values, traits, and the extent to which they wish to change them. We found that values predict the wish to change traits, whereas traits do not predict the wish to change values. In Study 4, 172 students (47.7% female) were assigned to one of four experimental conditions in which they received feedback denoting either uniqueness or similarity to others, on either their values or their traits. The results indicated that feedback that one's values (but not traits) are unique affected self-esteem. Integrating between theories of content and structure of the self can contribute to the development of both. © 2013 Wiley Periodicals, Inc.

A Heat-Sensitive TRP Channel Expressed in Keratinocytes

NASA Astrophysics Data System (ADS)

Peier, Andrea M.; Reeve, Alison J.; Andersson, David A.; Moqrich, Aziz; Earley, Taryn J.; Hergarden, Anne C.; Story, Gina M.; Colley, Sian; Hogenesch, John B.; McIntyre, Peter; Bevan, Stuart; Patapoutian, Ardem

2002-06-01

Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. Here, we describe the cloning and characterization of a novel thermosensitive TRP channel. TRPV3 has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes; hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons.

Evidence that selected amplification of a bacterial lac frameshift allele stimulates Lac(+) reversion (adaptive mutation) with or without general hypermutability.

PubMed Central

Slechta, E Susan; Liu, Jing; Andersson, Dan I; Roth, John R

2002-01-01

In the genetic system of Cairns and Foster, a nongrowing population of an E. coli lac frameshift mutant appears to specifically accumulate Lac(+) revertants when starved on medium including lactose (adaptive mutation). This behavior has been attributed to stress-induced general mutagenesis in a subpopulation of starved cells (the hypermutable state model). We have suggested that, on the contrary, stress has no direct effect on mutability but favors only growth of cells that amplify their leaky mutant lac region (the amplification mutagenesis model). Selection enhances reversion primarily by increasing the mutant lac copy number within each developing clone on the selection plate. The observed general mutagenesis is attributed to a side effect of growth with an amplification-induction of SOS by DNA fragments released from a tandem array of lac copies. Here we show that the S. enterica version of the Cairns system shows SOS-dependent general mutagenesis and behaves in every way like the original E. coli system. In both systems, lac revertants are mutagenized during selection. Eliminating the 35-fold increase in mutation rate reduces revertant number only 2- to 4-fold. This discrepancy is due to continued growth of amplification cells until some clones manage to revert without mutagenesis solely by increasing their lac copy number. Reversion in the absence of mutagenesis is still dependent on RecA function, as expected if it depends on lac amplification (a recombination-dependent process). These observations support the amplification mutagenesis model. PMID:12136002

[Offspring quality and its related factors of different Brachionus calyciflorus clones].

PubMed

Dong, Lili; Xi, Yilong; Zhang, Lei

2006-12-01

This paper studied the neonate starvation-endurance duration of four Brachionus calyciflorus clones (Clone A, B, C and D) with different biochemical-genetic characteristics at 15 degrees C, 20 degrees C, 25 degrees C and 30 degrees C, and the relationships of this duration with the temperature and the body- and egg volumes of B. calyciflorus. The results showed that at 15 degrees C, the neonates of Clone B had the shortest starvation-endurance duration (45.67 h); at 20 degrees C and 25 degrees C, the neonates' starvation-endurance duration of Clone C was the longest, being 61.33 h and 72.01 h, respectively; while at 30 degrees C, this duration of Clone A was the longest (40.11 h). The neonates' starvation-endurance duration of Clone A was the longest at 15 degrees C, those of Clone B and C were the shortest at 30 degrees C, while that of Clone D decreased with raising temperature. The neonates' starvation-endurance duration of all the four clones was negatively correlated with temperature. There was a negative correlation between this duration of Clone A and its egg volume, and the reverse was true for Clone C. The neonates' starvation-endurance duration of Clone B and D was positively correlated with the body volume of rotifer mother.

Requirement of the yeast MSH3 and MSH6 genes for MSH2-dependent genomic stability.

PubMed

Johnson, R E; Kovvali, G K; Prakash, L; Prakash, S

1996-03-29

Defects in DNA mismatch repair result in instability of simple repetitive DNA sequences and elevated levels of spontaneous mutability. The human G/T mismatch binding protein, GTBP/p160, has been suggested to have a role in the repair of base-base and single nucleotide insertion-deletion mismatches. Here we examine the role of the yeast GTBP homolog, MSH6, in mismatch repair. We show that both MSH6 and MSH3 genes are essential for normal genomic stability. Interestingly, although mutations in either MSH3 or MSH6 do not cause the extreme microsatellite instability and spontaneous mutability observed in the msh2 mutant, yeast cells harboring null mutations in both the MSH3 and MSH6 genes exhibit microsatellite instability and mutability similar to that in the msh2 mutant. Results from epistasis analyses indicate that MSH2 functions in mismatch repair in conjunction with MSH3 or MSH6 and that MSH3 and MSH6 constitute alternate pathways of MSH2-dependent mismatch repair.

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. II. Attributes of virus cloned at different dog kidney passage levels.

PubMed

Halstead, S B; Marchette, N J; Diwan, A R; Palumbo, N E; Putvatana, R

1984-07-01

Uncloned dengue (DEN) 4 (H-241) which had been passaged 15, 30 and 50 times in primary dog kidney (PDK) cells were subjected to two successive terminal dilution procedures. In the first (3Cl), virus was diluted in 10-fold steps in 10 replicate tubes. An infected tube from a dilution row with three or fewer virus-infected tubes was selected for two further passages. In the second (TD3), virus was triple terminal diluted using 2-fold dilution steps and selecting one positive tube out of 10. Both procedures selected virus population which differed from antecedents. Plaque size of PDK 15 was medium, PDK 30, small and PDK 50, pin-point. PDK 19-3Cl were medium and 56-3Cl, 24-TD3, 35-TD3 and 61-TD3 were all small. All cloned virus replication was completely shut-off at 38.5 degrees C; PDK 15 and 30 continued to replicate at this temperature. Uncloned viruses showed a graduated decrease in monkey virulence with PDK passage; cloned viruses were either avirulent for monkeys (19-3Cl, 56-31Cl, 24-TD3 and 35-TD3) or produced revertant large plaque parental-type viremia (35-3Cl and 61-TD3). Those cloned viruses which exhibited temperature sensitivity, reduced monkey virulence and stability after monkey passage may be suitable as vaccine candidates for evaluation in human beings.

Interactive effects of a bacterial parasite and the insecticide carbaryl to life-history and physiology of two Daphnia magna clones differing in carbaryl sensitivity.

PubMed

De Coninck, Dieter I M; De Schamphelaere, Karel A C; Jansen, Mieke; De Meester, Luc; Janssen, Colin R

2013-04-15

Natural and chemical stressors occur simultaneously in the aquatic environment. Their combined effects on biota are usually difficult to predict from their individual effects due to interactions between the different stressors. Several recent studies have suggested that synergistic effects of multiple stressors on organisms may be more common at high compared to low overall levels of stress. In this study, we used a three-way full factorial design to investigate whether interactive effects between a natural stressor, the bacterial parasite Pasteuria ramosa, and a chemical stressor, the insecticide carbaryl, were different between two genetically distinct clones of Daphnia magna that strongly differ in their sensitivity to carbaryl. Interactive effects on various life-history and physiological endpoints were assessed as significant deviations from the reference Independent Action (IA) model, which was implemented by testing the significance of the two-way carbaryl×parasite interaction term in two-way ANOVA's on log-transformed observational data for each clone separately. Interactive effects (and thus significant deviations from IA) were detected in both the carbaryl-sensitive clone (on survival, early reproduction and growth) and in the non-sensitive clone (on growth, electron transport activity and prophenoloxidase activity). No interactions were found for maturation rate, filtration rate, and energy reserve fractions (carbohydrate, protein, lipid). Furthermore, only antagonistic interactions were detected in the non-sensitive clone, while only synergistic interactions were observed in the carbaryl sensitive clone. Our data clearly show that there are genetically determined differences in the interactive effects following combined exposure to carbaryl and Pasteuria in D. magna. Copyright © 2013 Elsevier B.V. All rights reserved.

Patterns of cross-sensitivity in the responses of clonal subpopulations isolated from the RIF-1 mouse sarcoma to selected nitrosoureas and nitrogen mustards.

PubMed Central

Reeve, J. G.; Wright, K. A.; Workman, P.

1984-01-01

The response of clonal subpopulations isolated from the RIF-1 mouse sarcoma to melphalan treatment is independent of cell ploidy, whereas a clear relationship exists between ploidy and cell sensitivity to CCNU treatment. In the present study RIF-1 clones have been exposed to nitrogen mustard, aniline mustard and chlorambucil, and to nitrosoureas BCNU, MeCCNU and chlorozotocin, in order to evaluate whether or not the different physiochemical and biological activities of these agents would affect the patterns of drug sensitivity obtained for melphalan and CCNU. Irrespective of the different lipophilicities, transport properties and chemical reactivities of the nitrogen mustards, RIF-1 clones showed the same pattern of sensitivity as previously observed for melphalan. Similarly, RIF-1 clones when exposed to nitrosoureas BCNU, MeCCNU and chlorozotocin, showed the same pattern of sensitivity as that obtained for CCNU exposure. These data suggest (a) that the variation in the sensitivity of RIF-1 clones to treatment by the nitrogen mustards is unlikely to reflect differences in either membrane permeability or in drug transport and (b) that the ploidy dependent nitrosourea responses shown by RIF-1 clones similarly do not reflect differences in drug uptake. PMID:6466534

Comparison of randomly cloned and whole genomic DNA probes for the detection of Porphyromonas gingivalis and Bacteroides forsythus

PubMed Central

Wong, M.; DiRienzo, J.M.; Lai, C.-H.; Listgarten, M. A.

2012-01-01

Whole genomic and randomly-cloned DNA probes for two fastidious periodontal pathogens, Porphyromonas gingivalis and Bacteroides forsythus were labeled with digoxigenin and detected by a colorimetric method. The specificity and sensitivity of the whole genomic and cloned probes were compared. The cloned probes were highly specific compared to the whole genomic probes. A significant degree of cross-reactivity with Bacteroides species. Capnocytophaga sp. and Prevotella sp. was observed with the whole genomic probes. The cloned probes were less sensitive than the whole genomic probes and required at least 106 target cells or a minimum of 10 ng of target DNA to be detected during hybridization. Although a ten-fold increase in sensitivity was obtained with the whole genomic probes, cross-hybridization to closely related species limits their reliability in identifying target bacteria in subgingival plaque samples. PMID:8636873

Temperature-sensitive albino gene TCD5, encoding a monooxygenase, affects chloroplast development at low temperatures.

PubMed

Wang, Yufeng; Zhang, Jianhui; Shi, Xiaoliang; Peng, Yu; Li, Ping; Lin, Dongzhi; Dong, Yanjun; Teng, Sheng

2016-09-01

Chloroplasts are essential for photosynthesis and play critical roles in plant development. In this study, we characterized the temperature-sensitive chlorophyll-deficient rice mutant tcd5, which develops albino leaves at low temperatures (20 °C) and normal green leaves at high temperatures (32 °C). The development of chloroplasts and etioplasts is impaired in tcd5 plants at 20 °C, and the temperature-sensitive period for the albino phenotype is the P4 stage of leaf development. The development of thylakoid membranes is arrested at the mid-P4 stage in tcd5 plants at 20 °C. We performed positional cloning of TCD5 and then complementation and knock-down experiments, and the results showed that the transcript LOC_Os05g34040.1 from the LOC_Os05g34040 gene corresponded to the tcd5 phenotype. TCD5 encodes a conserved plastid-targeted monooxygenase family protein which has not been previously reported associated with a temperature-sensitive albino phenotype in plants. TCD5 is abundantly expressed in young leaves and immature spikes, and low temperatures increased this expression. The transcription of some genes involved in plastid transcription/translation and photosynthesis varied in the tcd5 mutant. Although the phenotype and temperature dependence of the TCD5 orthologous mutant phenotype were different in rice and Arabidopsis, OsTCD5 could rescue the phenotype of the Arabidopsis mutant, suggesting that TCD5 function is conserved between monocots and dicots. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis

PubMed Central

1994-01-01

We have isolated and characterized temperature-sensitive endocytosis mutants in Dictyostelium discoideum. Dictyostelium is an attractive model for genetic studies of endocytosis because of its high rates of endocytosis, its reliance on endocytosis for nutrient uptake, and tractable molecular genetics. Endocytosis-defective mutants were isolated by a fluorescence-activated cell sorting (FACS) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indy1) was characterized in detail and found to exhibit a complete block in fluid phase endocytosis at the restrictive temperature, but normal rates of endocytosis at the permissive temperature. Likewise, a potential cell surface receptor that was rapidly internalized in wild-type cells and indy1 cells at the permissive temperature was poorly internalized in indy1 under restrictive conditions. Growth was also completely arrested at the restrictive temperature. The endocytosis block was rapidly induced upon shift to the restrictive temperature and reversed upon return to normal conditions. Inhibition of endocytosis was also specific, as other membrane-trafficking events such as phagocytosis, secretion of lysosomal enzymes, and contractile vacuole function were unaffected at the restrictive temperature. Because recycling and transport to late endocytic compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indy1 cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indy1 mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning. PMID:7929583

The effect of phenolic and polyphenolic compounds on the development of drug resistance.

PubMed

Birosová, Lucia; Mikulásová, Mária; Chromá, Magdaléna

2005-12-01

The effect of two phenolic compounds vanillin (4-hydroxy-3-methoxybenzaldehyde) and lignin on the development of drug/antibiotic resistance in Salmonella typhimurium was studied. Using the modified Ames test we have shown that vanillin alone has negligible effect on spontaneous mutability to ciprofloxacin and gentamicin resistance. At the tested concentrations vanillin reduces the toxicity of 4-nitroquinoline-N-oxide (4NQO) and reduces the ability of this compound to induce mutations leading to ciprofloxacin but not to gentamicin resistance. Lignin at higher concentrations increases mutagenicity to ciprofloxacin resistance and possess considerable inhibition effect on the spontaneous and 4NQO induced mutability to gentamicin resistance.

Design of stapled DNA-minor-groove-binding molecules with a mutable atom simulated annealing method

NASA Astrophysics Data System (ADS)

Walker, Wynn L.; Kopka, Mary L.; Dickerson, Richard E.; Goodsell, David S.

1997-11-01

We report the design of optimal linker geometries for the synthesis of stapledDNA-minor-groove-binding molecules. Netropsin, distamycin, and lexitropsinsbind side-by-side to mixed-sequence DNA and offer an opportunity for thedesign of sequence-reading molecules. Stapled molecules, with two moleculescovalently linked side-by-side, provide entropic gains and restrain theposition of one molecule relative to its neighbor. Using a free-atom simulatedannealing technique combined with a discrete mutable atom definition, optimallengths and atomic composition for covalent linkages are determined, and anovel hydrogen bond `zipper' is proposed to phase two molecules accuratelyside-by-side.

Better, Stronger, Faster: Self-Serving Judgment, Affect Regulation, and the Optimal Vigilance Hypothesis.

PubMed

Roese, Neal J; Olson, James M

2007-06-01

Self-serving judgments, in which the self is viewed more favorably than other people, are ubiquitous. Their dynamic variation within individuals may be explained in terms of the regulation of affect. Self-serving judgments produce positive emotions, and threat increases self-serving judgments (a compensatory pattern that restores affect to a set point or baseline). Perceived mutability is a key moderator of these judgments; low mutability (i.e., the circumstance is closed to modification) triggers a cognitive response aimed at affect regulation, whereas high mutability (i.e., the circumstance is open to further modification) activates direct behavioral remediation. Threats often require immediate response, whereas positive events do not. Because of this brief temporal window, an active mechanism is needed to restore negative (but not positive) affective shifts back to a set point. Without this active reset, an earlier threat would make the individual less vigilant toward a new threat. Thus, when people are sad, they aim to return their mood to baseline, often via self-serving judgments. We argue that asymmetric homeostasis enables optimal vigilance, which establishes a coherent theoretical account of the role of self-serving judgments in affect regulation. © 2007 Association for Psychological Science.

Better, Stronger, Faster Self-Serving Judgment, Affect Regulation, and the Optimal Vigilance Hypothesis

PubMed Central

Roese, Neal J.; Olson, James M.

2008-01-01

Self-serving judgments, in which the self is viewed more favorably than other people, are ubiquitous. Their dynamic variation within individuals may be explained in terms of the regulation of affect. Self-serving judgments produce positive emotions, and threat increases self-serving judgments (a compensatory pattern that restores affect to a set point or baseline). Perceived mutability is a key moderator of these judgments; low mutability (i.e., the circumstance is closed to modification) triggers a cognitive response aimed at affect regulation, whereas high mutability (i.e., the circumstance is open to further modification) activates direct behavioral remediation. Threats often require immediate response, whereas positive events do not. Because of this brief temporal window, an active mechanism is needed to restore negative (but not positive) affective shifts back to a set point. Without this active reset, an earlier threat would make the individual less vigilant toward a new threat. Thus, when people are sad, they aim to return their mood to baseline, often via self-serving judgments. We argue that asymmetric homeostasis enables optimal vigilance, which establishes a coherent theoretical account of the role of self-serving judgments in affect regulation. PMID:18552989

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

PubMed Central

Correia, Rodolfo Patussi; Bento, Laiz Cameirão; Bortolucci, Ana Carolina Apelle; Alexandre, Anderson Marega; Vaz, Andressa da Costa; Schimidell, Daniela; Pedro, Eduardo de Carvalho; Perin, Fabricio Simões; Nozawa, Sonia Tsukasa; Mendes, Cláudio Ernesto Albers; Barroso, Rodrigo de Souza; Bacal, Nydia Strachman

2016-01-01

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones. PMID:27759825

Competitive release of drug resistance following drug treatment of mixed Plasmodium chabaudi infections.

PubMed

de Roode, Jacobus C; Culleton, Richard; Bell, Andrew S; Read, Andrew F

2004-09-14

Malaria infections are often genetically diverse, potentially leading to competition between co-infecting strains. Such competition is of key importance in the spread of drug resistance. The effects of drug treatment on within-host competition were studied using the rodent malaria Plasmodium chabaudi. Mice were infected simultaneously with a drug-resistant and a drug-sensitive clone and were then either drug-treated or left untreated. Transmission was assessed by feeding mice to Anopheles stephensi mosquitoes. In the absence of drugs, the sensitive clone competitively suppressed the resistant clone; this resulted in lower asexual parasite densities and also reduced transmission to the mosquito vector. Drug treatment, however, allowed the resistant clone to fill the ecological space emptied by the removal of the sensitive clone, allowing it to transmit as well as it would have done in the absence of competition. These results show that under drug pressure, resistant strains can have two advantages: (1) they survive better than sensitive strains and (2) they can exploit the opportunities presented by the removal of their competitors. When mixed infections are common, such effects could increase the spread of drug resistance.

Effects of Tropospheric O3 on Trembling Aspen and Interaction with CO2: Results from an O3-Gradient and a FACE Experiment

Treesearch

D. F. Karnosky; B. Mankovska; K. Percy; R. E. Dickson; G. K. Podila; J. Sober; A. Noormets; G. Hendrey; M. D. Coleman; M. Kubiske; K. S. Pregitzer; J. G. Isebrands

1999-01-01

Over the years, a series of trembling aspen (Populus tremuloides Michx.) clones differing in O3 sensitivity have been identified from OTC studies. Three clones (216 and 271[O3 tolerant] and 259 [O3 sensitive]) have been characterized for O3 sensitivity by growth and biomass...

Temperature-sensitive mutants of influenza A virus. XIV. Production and evaluation of influenza A/Georgia/74-ts-1[E] recombinant viruses in human adults.

PubMed

Richman, D D; Murphy, B R; Belshe, R B; Rusten, H M; Chanock, R M; Blacklow, N R; Parrino, T A; Rose, F B; Levine, M M; Caplan, E

1977-08-01

The two temperature-sensitive (ts) lesions present in influenza A/Hong Kong/68-ts-1[E] (H3N2 68) virus were transferred via genetic reassortment to influenza A/Georgia/74 (H3N2 74) wild-type virus. A recombinant clone possessing both ts lesions and the shutoff temperature of 38 C of the Hong Kong/68 ts donor and the two surface antigens of the Georgia/74 wild-type virus was administered to 32 seronegative adult volunteers. Thirty-one volunteers were infected, of whom only five experienced mild afebrile upper respiratory tract illness. The wild-type recipient virus was a cloned population that induced illness in five of six infected volunteers. Therfore, the attenuation exhibited by the Georgia/74-ts-1[E] virus could reasonably be assumed to be due to the acquisition of the two ts-1[E] lesions by the Georgia/74 wild-type virus. The serum and nasal wash antibody responses of the ts-1[E] vaccinees were equivalent to those of the volunteers who received wild-type virus. The two ts lesions present in the Hong Kong/68-ts-1[E] virus have now been transferred three times to a wild-type virus bearing a new hemagglutinin, and in each instance the new ts recombination exhibited a similar, satisfactory level of attenuation and antigenicity for adults. It seems likely that the transfer of the ts-1[E] lesions to any new influenza virus will regularly result in attenuation of a recombinat virus possessing the new surface antigens.

A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80.

PubMed Central

Chen, M X; Bouquin, N; Norris, V; Casarégola, S; Séror, S J; Holland, I B

1991-01-01

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein. Images PMID:1915285

The novel extremely psychrophilic luciferase from Metridia longa: Properties of a high-purity protein produced in insect cells.

PubMed

Larionova, Marina D; Markova, Svetlana V; Vysotski, Eugene S

2017-01-29

The bright bioluminescence of copepod Metridia longa is conditioned by a small secreted coelenterazine-dependent luciferase (MLuc). To date, three isoforms of MLuc differing in length, sequences, and some properties were cloned and successfully applied as high sensitive bioluminescent reporters. In this work, we report cloning of a novel group of genes from M. longa encoding extremely psychrophilic isoforms of MLuc (MLuc2-type). The novel isoforms share only ∼54-64% of protein sequence identity with the previously cloned isoforms and, consequently, are the product of a separate group of paralogous genes. The MLuc2 isoform with consensus sequence was produced as a natively folded protein using baculovirus/insect cell expression system, purified, and characterized. The MLuc2 displays a very high bioluminescent activity and high thermostability similar to those of the previously characterized M. longa luciferase isoform MLuc7. However, in contrast to MLuc7 revealing the highest activity at 12-17 °C and 0.5 M NaCl, the bioluminescence optima of MLuc2 isoforms are at ∼5 °C and 1 M NaCl. The MLuc2 adaptation to cold is also accompanied by decrease of melting temperature and affinity to substrate suggesting a more conformational flexibility of a protein structure. The luciferase isoforms with different temperature optima may provide adaptability of the M. longa bioluminescence to the changes of water temperature during diurnal vertical migrations. Copyright © 2016 Elsevier Inc. All rights reserved.

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawata, Shigehisa; Suzuki, Jun; Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871

2006-11-10

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types ofmore » cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.« less

Intracellular population genetics: evidence for random drift of mitochondrial allele frequencies in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

PubMed

Thrailkill, K M; Birky, C W

1980-09-01

We report evidence for random drift of mitochondrial allele frequencies in zygote clones of Saccharomyces cerevisiae and Schizosaccharomyces pombe. Monofactorial and bifactorial crosses were done, using strains resistant or sensitive to erythromycin (alleles Er, Es), oligomycin (Or, Os), or diuron (Dr, Ds). The frequencies of resistant and sensitive cells (and thus the frequencies of the resistant and sensitive alleles) were determined for each of a number of clones of diploid cells arising from individual zygotes. Allele frequencies were extremely variable among these zygote clones; some clones were "uniparental," with mitochondrial alleles from only one parent present. These observations suggest random drift of the allele frequencies in the population of mitochondrial genes within an individual zygote and its diploid progeny. Drift would cease when all the cells in a clone become homoplasmic, due to segregation of the mitochondrial genomes during vegetative cell divisions. To test this, we delayed cell division (and hence segregation) for varying times by starving zygotes in order to give drift more time to operate. As predicted, delaying cell division resulted in an increase in the variance of allele frequencies among the zygote clones and an increase in the proportion of uniparental zygote clones. The changes in form of the allele frequency distributions resembled those seen during random drift in finite Mendelian populations. In bifactorial crosses, genotypes as well as individual alleles were fixed or lost in some zygote clones. However, the mean recombination frequency for a large number of clones did not increase when cell division was delayed. Several possible molecular mechanisms for intracellular random drift are discussed.

'Diffuse skin browning' in 1-MCP-treated apples: etiology and systems of control.

PubMed

Larrigaudière, Christian; Vilaplana, Rosa; Recasens, Inmaculada; Soria, Yolanda; Dupille, Eve

2010-11-01

'Diffuse skin browning' (DSB) is a physiological disorder that affects Golden Delicious apples treated with 1-methylcyclopropene (1-MCP). Although a very high incidence is found, very little is known about the etiology of this disorder. This study aims to provide an understanding of the causes of this disorder and prevent it. A very high incidence of DSB was found in 1-MCP-treated apples independent of the location of the orchard. Similar to superficial scald, harvest maturity determines the DSB incidence, with the more mature fruit being less sensitive. The 1-MCP dose (156 nL L(-1) or 625 nL L(-1)) and the temperature at which the 1-MCP treatment was applied (0.5 or 20 °C) did not affect the incidence of DSB. Diphenylamine (DPA) treatment did not prevent DSB, contrary to superficial scald. Additionally, controlled atmosphere storage only partially reduced the incidence of DSB, whereas progressive cooling strategies completely inhibited DSB occurrence. A direct correlation was found between the sensitivity of the Golden Delicious clone to russeting and its sensitivity to develop DSB during storage. Our results indicated that DSB and superficial scald are two different disorders involving different oxidative processes. DSB can be prevented by progressive cooling and selection of russeting-resistant clones. 2010 Society of Chemical Industry

Polarity-defective mutants of Aspergillus nidulans.

PubMed

Osherov, N; Mathew, J; May, G S

2000-12-01

We have identified two polarity-defective (pod) mutants in Aspergillus nidulans from a collection of heat-sensitive lethal mutants. At restrictive temperature, these mutants are capable of nuclear division but are unable to establish polar hyphal growth. We cloned the two pod genes by complementation of their heat-sensitive lethal phenotypes. The libraries used to clone the pod genes are under the control of the bidirectional niaD and niiA promoters. Complementation of the pod mutants is dependent on growth on inducing medium. We show that rescue of the heat-sensitive phenotype on inducing media is independent of the orientation of the gene relative to the niaD or niiA promoters, demonstrating that the intergenic region between the niaD and the niiA genes functions as an orientation-independent enhancer and repressor that is capable of functioning over long distances. The products of the podG and the podH genes were identified as homologues of the alpha subunit of yeast mitochondrial phenylalanyl--tRNA synthetase and transcription factor IIF interacting component of the CTD phosphatase. Neither of these gene products would have been predicted to produce a pod mutant phenotype based on studies of cellular polarity mutants in other organisms. The implications of these results are discussed. Copyright 2000 Academic Press.

The Mutable Nature of Risk and Acceptability: A Hybrid Risk Governance Framework.

PubMed

Wong, Catherine Mei Ling

2015-11-01

This article focuses on the fluid nature of risk problems and the challenges it presents to establishing acceptability in risk governance. It introduces an actor-network theory (ANT) perspective as a way to deal with the mutable nature of risk controversies and the configuration of stakeholders. To translate this into a practicable framework, the article proposes a hybrid risk governance framework that combines ANT with integrative risk governance, deliberative democracy, and responsive regulation. This addresses a number of the limitations in existing risk governance models, including: (1) the lack of more substantive public participation throughout the lifecycle of a project; (2) hijacking of deliberative forums by particular groups; and (3) the treatment of risk problems and their associated stakeholders as immutable entities. The framework constitutes a five-stage process of co-selection, co-design, co-planning, and co-regulation to facilitate the co-production of collective interests and knowledge, build capacities, and strengthen accountability in the process. The aims of this article are twofold: conceptually, it introduces a framework of risk governance that accounts for the mutable nature of risk problems and configuration of stakeholders. In practice, this article offers risk managers and practitioners of risk governance a set of procedures with which to operationalize this conceptual approach to risk and stakeholder engagement. © 2015 Society for Risk Analysis.

Room-Temperature Quantum Cloning Machine with Full Coherent Phase Control in Nanodiamond

PubMed Central

Chang, Yan-Chun; Liu, Gang-Qin; Liu, Dong-Qi; Fan, Heng; Pan, Xin-Yu

2013-01-01

In contrast to the classical world, an unknown quantum state cannot be cloned ideally, as stated by the no-cloning theorem. However, it is expected that approximate or probabilistic quantum cloning will be necessary for different applications, and thus various quantum cloning machines have been designed. Phase quantum cloning is of particular interest because it can be used to attack the Bennett-Brassard 1984 (BB84) states used in quantum key distribution for secure communications. Here, we report the first room-temperature implementation of quantum phase cloning with a controllable phase in a solid-state system: the nitrogen-vacancy centre of a nanodiamond. The phase cloner works well for all qubits located on the equator of the Bloch sphere. The phase is controlled and can be measured with high accuracy, and the experimental results are consistent with theoretical expectations. This experiment provides a basis for phase-controllable quantum information devices. PMID:23511233

Detecting Horizontal Gene Transfer between Closely Related Taxa

PubMed Central

Adato, Orit; Ninyo, Noga; Gophna, Uri; Snir, Sagi

2015-01-01

Horizontal gene transfer (HGT), the transfer of genetic material between organisms, is crucial for genetic innovation and the evolution of genome architecture. Existing HGT detection algorithms rely on a strong phylogenetic signal distinguishing the transferred sequence from ancestral (vertically derived) genes in its recipient genome. Detecting HGT between closely related species or strains is challenging, as the phylogenetic signal is usually weak and the nucleotide composition is normally nearly identical. Nevertheless, there is a great importance in detecting HGT between congeneric species or strains, especially in clinical microbiology, where understanding the emergence of new virulent and drug-resistant strains is crucial, and often time-sensitive. We developed a novel, self-contained technique named Near HGT, based on the synteny index, to measure the divergence of a gene from its native genomic environment and used it to identify candidate HGT events between closely related strains. The method confirms candidate transferred genes based on the constant relative mutability (CRM). Using CRM, the algorithm assigns a confidence score based on “unusual” sequence divergence. A gene exhibiting exceptional deviations according to both synteny and mutability criteria, is considered a validated HGT product. We first employed the technique to a set of three E. coli strains and detected several highly probable horizontally acquired genes. We then compared the method to existing HGT detection tools using a larger strain data set. When combined with additional approaches our new algorithm provides richer picture and brings us closer to the goal of detecting all newly acquired genes in a particular strain. PMID:26439115

Examination of food chain-derived Listeria monocytogenes strains of different serotypes reveals considerable diversity in inlA genotypes, mutability, and adaptation to cold temperatures.

PubMed

Kovacevic, Jovana; Arguedas-Villa, Carolina; Wozniak, Anna; Tasara, Taurai; Allen, Kevin J

2013-03-01

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.

Protective Properties of Radio-Chemoresistant Glioblastoma Stem Cell Clones Are Associated with Metabolic Adaptation to Reduced Glucose Dependence

PubMed Central

Yamada, Kazunari; Tso, Jonathan L.; Menjivar, Jimmy C.; Tian, Jane Y.; Yong, William H.; Schaue, Dörthe; Mischel, Paul S.; Cloughesy, Timothy F.; Nelson, Stanley F.; Liau, Linda M.; McBride, William; Tso, Cho-Lea

2013-01-01

Glioblastoma stem cells (GSC) are a significant cell model for explaining brain tumor recurrence. However, mechanisms underlying their radiochemoresistance remain obscure. Here we show that most clonogenic cells in GSC cultures are sensitive to radiation treatment (RT) with or without temozolomide (TMZ). Only a few single cells survive treatment and regain their self-repopulating capacity. Cells re-populated from treatment-resistant GSC clones contain more clonogenic cells compared to those grown from treatment-sensitive GSC clones, and repeated treatment cycles rapidly enriched clonogenic survival. When compared to sensitive clones, resistant clones exhibited slower tumor development in animals. Upregulated genes identified in resistant clones via comparative expression microarray analysis characterized cells under metabolic stress, including blocked glucose uptake, impaired insulin/Akt signaling, enhanced lipid catabolism and oxidative stress, and suppressed growth and inflammation. Moreover, many upregulated genes highlighted maintenance and repair activities, including detoxifying lipid peroxidation products, activating lysosomal autophagy/ubiquitin-proteasome pathways, and enhancing telomere maintenance and DNA repair, closely resembling the anti-aging effects of caloric/glucose restriction (CR/GR), a nutritional intervention that is known to increase lifespan and stress resistance in model organisms. Although treatment–introduced genetic mutations were detected in resistant clones, all resistant and sensitive clones were subclassified to either proneural (PN) or mesenchymal (MES) glioblastoma subtype based on their expression profiles. Functional assays demonstrated the association of treatment resistance with energy stress, including reduced glucose uptake, fatty acid oxidation (FAO)-dependent ATP maintenance, elevated reactive oxygen species (ROS) production and autophagic activity, and increased AMPK activity and NAD+ levels accompanied by upregulated mRNA levels of SIRT1/PGC-1α axis and DNA repair genes. These data support the view that treatment resistance may arise from quiescent GSC exhibiting a GR-like phenotype, and suggest that targeting stress response pathways of resistant GSC may provide a novel strategy in combination with standard treatment for glioblastoma. PMID:24260384

Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair.

PubMed

Chatterjee, Nimrat; Lin, Yunfu; Yotnda, Patricia; Wilson, John H

2016-07-31

Multiple pathways modulate the dynamic mutability of trinucleotide repeats (TNRs), which are implicated in neurodegenerative disease and evolution. Recently, we reported that environmental stresses induce TNR mutagenesis via stress responses and rereplication, with more than 50% of mutants carrying deletions or insertions-molecular signatures of DNA double-strand break repair. We now show that knockdown of alt-nonhomologous end joining (alt-NHEJ) components-XRCC1, LIG3, and PARP1-suppresses stress-induced TNR mutagenesis, in contrast to the components of homologous recombination and NHEJ, which have no effect. Thus, alt-NHEJ, which contributes to genetic mutability in cancer cells, also plays a novel role in environmental stress-induced TNR mutagenesis. Published by Elsevier Ltd.

Evidence of drug-response heterogeneity rapidly generated from a single cancer cell.

PubMed

Wang, Rong; Jin, Chengmeng; Hu, Xun

2017-06-20

One cancer cell line is believed to be composed of numerous clones with different drug sensitivity. We sought to investigate the difference of drug-response pattern in clones from a cell line or from a single cell. We showed that 22 clones derived from 4T1 cells were drastically different from each other with respect to drug-response pattern against 11 anticancer drugs and expression profile of 19 genes associated with drug resistance or sensitivity. Similar results were obtained using daughter clones derived from a single 4T1 cell. Each daughter clone showed distinct drug-response pattern and gene expression profile. Similar results were also obtained using Bcap37 cells. We conclude that a single cancer cell can rapidly produce a population of cells with high heterogeneity of drug response and the acquisition of drug-response heterogeneity is random.

Identification of a phorbol ester-repressible v-src-inducible gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simmons, D.L.; Levy, D.B.; Yannoni, Y.

1989-02-01

Chicken embryo fibroblasts (CEF) infected with a temperature-sensitive Rous sarcoma virus (RSV) mutant, tsNY72-4, express a set of pp60{sup v-src}-induced RNAs soon after shift to the permissive temperature. By subtractive and differential screening, the authors have cloned 12 of these sequences, 2 of which were c-fos and krox-24. Serum induced all the v-src-inducible genes tested, suggesting that these genes serve roles in normal cell division and are not specific to transformation per se. Significantly, however, v-src produced prolonged, and in some cases kinetically complex, patterns of induction compared to serum. For most of the clones, phorbol 12-tetradecanoate 13-acetate (TPA) inducedmore » mRNAs with kinetics similar to that of serum. However, one clone (CEF-4) was expressed in a biphasic manner. Another (CEF-10) was repressed by TPA at 1 hr, after which this mRNA was permanently induced. The pattern of repression-induction of CEF-10 mRNA is the inverse of protein kinase C (PKC) activity in the cell, suggesting that PKC actively represses this gene. In vivo expression of CEF-10 mRNA is restricted predominantly to the lung. A full-length CEF-10 cDNA encodes a 41-kDa protein that has an amino-terminal signal peptide for secretion, contains a markedly high number of cysteine residues, and shows no sequence similarity to known proteins.« less

Effects of Tropospheric O3 on Trembling Aspen and Interaction with CO2: Results From An O3-Gradient and a Face Experiment

Treesearch

D.F. Karnosky; B. Mankovska; K. Percy; R.E. Dickson; G.K. Podila; J. Sober; A. Noormets; G. Hendrey; Mark D. Coleman; M. Kubiske; K.S. Pregitzer; J.G. Isebrands

1999-01-01

Abstract. Over the years, a series of trembling aspen (Populus tremuloides Michx.) clones differing in O3 sensitivity have been identified from OTC studies. Three clones (216 and 271[(O3 tolerant] and 259 [O3 sensitive]) have been characterized for O3...

Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

PubMed

Lu, Yifei; Yan, Hongxiang; Deng, Jiezhong; Huang, Zhigang; Jin, Xurui; Yu, Yanlan; Hu, Qiwen; Hu, Fuquan; Wang, Jing

2017-09-18

Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

Differential Effects of Elevated Ozone on Two Hybrid Aspen Genotypes Predisposed to Chronic Ozone Fumigation. Role of Ethylene and Salicylic Acid1

PubMed Central

Vahala, Jorma; Keinänen, Markku; Schützendübel, Andres; Polle, Andrea; Kangasjärvi, Jaakko

2003-01-01

The role of ethylene (ET) signaling in the responses of two hybrid aspen (Populus tremula L. × P. tremuloides Michx.) clones to chronic ozone (O3; 75 nL L−1) was investigated. The hormonal responses differed between the clones; the O3-sensitive clone 51 had higher ET evolution than the tolerant clone 200 during the exposure, whereas the free salicylic acid concentration in clone 200 was higher than in clone 51. The cellular redox status, measured as glutathione redox balance, did not differ between the clones suggesting that the O3 lesions were not a result of deficient antioxidative capacity. The buildup of salicylic acid during chronic O3 exposure might have prevented the up-regulation of ET biosynthesis in clone 200. Blocking of ET perception with 1-methylcyclopropene protected both clones from the decrease in net photosynthesis during chronic exposure to O3. After a pretreatment with low O3 for 9 d, an acute 1.5-fold O3 elevation caused necrosis in the O3-sensitive clone 51, which increased substantially when ET perception was blocked. The results suggest that in hybrid aspen, ET signaling had a dual role depending on the severity of the stress. ET accelerated leaf senescence under low O3, but under acute O3 elevation, ET signaling seemed to be required for protection from necrotic cell death. PMID:12746525

Deletions in the tetracycline resistance determinant reduce the thermosensitivity of a trfA(Ts) derivative of plasmid RP1 in Pseudomonas aeruginosa.

PubMed

Rella, M; Watson, J M; Thomas, C M; Haas, D

1987-01-01

A derivative of the broad-host-range plasmid RP1, pME301, was temperature-sensitive (Ts) at 43 degrees C for maintenance in Pseudomonas aeruginosa, P. mendocina, Klebsiella aerogenes and Escherichia coli. In E. coli, the Ts defect of pME301 could be complemented in trans by the cloned trfA gene, which is known to be essential for RP1 replication in E. coli and P. aeruginosa. Because pME301 expressed a Ts phenotype in P. mendocina and K. aerogenes, we assume that the trfA function is also vital in these organisms. When plasmid-encoded carbenicillin resistance (on transposon Tn801) was selected at non-permissive temperatures in P. aeruginosa strain PAO carrying pME301, we obtained either Tn801 insertions into the chromosome or pME301 derivatives having a deletion (or point mutation) in their tet genes, which determine resistance to tetracycline and are not transposable. From cloning experiments, we infer that the tet gene product(s) destabilize the pME301 replicon in P. aeruginosa at 40-43 degrees C.

The DCL gene of tomato is required for chloroplast development and palisade cell morphogenesis in leaves.

PubMed

Keddie, J S; Carroll, B; Jones, J D; Gruissem, W

1996-08-15

The defective chloroplasts and leaves-mutable (dcl-m) mutation of tomato was identified in a Ds mutagenesis screen. This unstable mutation affects both chloroplast development and palisade cell morphogenesis in leaves. Mutant plants are clonally variegated as a result of somatic excision of Ds and have albino leaves with green sectors. Leaf midribs and stems are light green with sectors of dark green tissue but fruit and petals are wild-type in appearance. Within dark green sectors of dcl-m leaves, palisade cells are normal, whereas in albino areas of dcl-m leaves, palisade cells do not expand to become their characteristic columnar shape. The development of chloroplasts from proplastids in albino areas is apparently blocked at an early stage. DCL was cloned using Ds as a tag and encodes a novel protein of approximately 25 kDa, containing a chloroplast transit peptide and an acidic alpha-helical region. DCL protein was imported into chloroplasts in vitro and processed to a mature form. Because of the ubiquitous expression of DCL and the proplastid-like appearance of dcl-affected plastids, the DCL protein may regulate a basic and universal function of the plastid. The novel dcl-m phenotype suggests that chloroplast development is required for correct palisade cell morphogenesis during leaf development.

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups B and E to genome segments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gombold, J.L.; Estes, M.K.; Ramig, R.F.

1985-05-01

Recombinant (reassortant) viruses were selected from crosses between temperature-sensitive (ts) mutants of simian rotavirus SA11 and wild-type human rotavirus Wa. The double-stranded genome RNAs of the reassortants were examined by electrophoresis in Tris-glycine-buffered polyacrylamide gels and by dot hybridization with a cloned DNA probe for genome segment 2. Analysis of replacements of genome segments in the reassortants allowed construction of a map correlating genome segments providing functions interchangeable between SA11 and Wa. The reassortants revealed a functional correspondence in order of increasing electrophoretic mobility of genome segments. Analysis of the parental origin of genome segments in ts+ SA11/Wa reassortants derivedmore » from the crosses SA11 tsB(339) X Wa and SA11 tsE(1400) X Wa revealed that the group B lesion of tsB(339) was located on genome segment 3 and the group E lesion of tsE(1400) was on segment 8.« less

Cloning of organic solvent tolerance gene ostA that determines n-hexane tolerance level in Escherichia coli.

PubMed Central

Aono, R; Negishi, T; Nakajima, H

1994-01-01

A variety of genes are involved in determining the level of organic solvent tolerance of Escherichia coli K-12. Gene ostA is one of the genes contributing to the level of organic solvent tolerance. This gene was cloned from an n-hexane-tolerant strain of E. coli, JA300. A JA300-based n-hexane-sensitive strain, OST4251, was converted to the n-hexane-tolerant phenotype by transformation with DNA containing the ostA gene derived from JA300. Thus, the cloned ostA gene complemented the n-hexane-sensitive phenotype of OST4251. Images PMID:7811102

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors.

PubMed

Anaka, Matthew; Hudson, Christopher; Lo, Pu-Han; Do, Hongdo; Caballero, Otavia L; Davis, Ian D; Dobrovic, Alexander; Cebon, Jonathan; Behren, Andreas

2013-10-11

Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant.

PubMed

Singh, B N; Mudgil, Yashwanti; Sopory, S K; Reddy, M K

2003-07-01

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5' and 3' splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg(2+)- and ATP-dependent manner, and this activity was inhibited by 4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII(ts) yeast mutant.

Longitudinal analysis of large social networks: Estimating the effect of health traits on changes in friendship ties

PubMed Central

O'Malley, A James; Christakis, Nicholas A

2011-01-01

We develop novel mixed effects models to examine the role of health traits on the status of peoples' close friendship nominations in the Framingham Heart Study. The health traits considered are both mutable (body mass index (BMI), smoking, blood pressure, body proportion, muscularity, and depression) and, for comparison, basically immutable (height, birth order, personality type, only child, and handedness); and the traits have varying degrees of observability. We test the hypotheses that existing ties (i.e. close friendship nominations) are more likely to dissolve between people with dissimilar (mutable and observable) health traits whereas new ties are more likely to form between those with similar (mutable and observable) traits while controlling for persons' age, gender, geographic separation, and education. The mixed effects models contain random effects for both the nominator (ego) and nominated (alter) persons in a tie to account for the fact that people were involved in multiple relationships and contributed observations at multiple exams. Results for BMI support the hypotheses that people of similar BMI are less likely to dissolve existing ties and more likely to form ties, while smoker to non-smoker ties were the least likely to dissolve and smoker to smoker ties were the most likely to form. We also validated previously known findings regarding homophily on age and gender, and found evidence that homophily also depends upon geographic separation. Copyright © 2011 John Wiley & Sons, Ltd. PMID:21287589

Longitudinal analysis of large social networks: estimating the effect of health traits on changes in friendship ties.

PubMed

O'Malley, A James; Christakis, Nicholas A

2011-04-30

We develop novel mixed effects models to examine the role of health traits on the status of peoples' close friendship nominations in the Framingham Heart Study. The health traits considered are both mutable (body mass index (BMI), smoking, blood pressure, body proportion, muscularity, and depression) and, for comparison, basically immutable (height, birth order, personality type, only child, and handedness); and the traits have varying degrees of observability. We test the hypotheses that existing ties (i.e. close friendship nominations) are more likely to dissolve between people with dissimilar (mutable and observable) health traits whereas new ties are more likely to form between those with similar (mutable and observable) traits while controlling for persons' age, gender, geographic separation, and education. The mixed effects models contain random effects for both the nominator (ego) and nominated (alter) persons in a tie to account for the fact that people were involved in multiple relationships and contributed observations at multiple exams. Results for BMI support the hypotheses that people of similar BMI are less likely to dissolve existing ties and more likely to form ties, while smoker to non-smoker ties were the least likely to dissolve and smoker to smoker ties were the most likely to form. We also validated previously known findings regarding homophily on age and gender, and found evidence that homophily also depends upon geographic separation. Copyright © 2011 John Wiley & Sons, Ltd.

MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

PubMed Central

Puziss, J W; Hardy, T A; Johnson, R B; Roach, P J; Hieter, P

1994-01-01

The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3. Images PMID:8264650

Metabolic vulnerability of cisplatin-resistant cancers.

PubMed

Obrist, Florine; Michels, Judith; Durand, Sylvere; Chery, Alexis; Pol, Jonathan; Levesque, Sarah; Joseph, Adrien; Astesana, Valentina; Pietrocola, Federico; Wu, Gen Sheng; Castedo, Maria; Kroemer, Guido

2018-06-06

Cisplatin is the most widely used chemotherapeutic agent, and resistance of neoplastic cells against this cytoxicant poses a major problem in clinical oncology. Here, we explored potential metabolic vulnerabilities of cisplatin-resistant non-small human cell lung cancer and ovarian cancer cell lines. Cisplatin-resistant clones were more sensitive to killing by nutrient deprivation in vitro and in vivo than their parental cisplatin-sensitive controls. The susceptibility of cisplatin-resistant cells to starvation could be explained by a particularly strong dependence on glutamine. Glutamine depletion was sufficient to restore cisplatin responses of initially cisplatin-resistant clones, and glutamine supplementation rescued cisplatin-resistant clones from starvation-induced death. Mass spectrometric metabolomics and specific interventions on glutamine metabolism revealed that, in cisplatin-resistant cells, glutamine is mostly required for nucleotide biosynthesis rather than for anaplerotic, bioenergetic or redox reactions. As a result, cisplatin-resistant cancers became exquisitely sensitive to treatment with antimetabolites that target nucleoside metabolism. © 2018 The Authors.

The modest beginnings of one genome project.

PubMed

Kaback, David B

2013-06-01

One of the top things on a geneticist's wish list has to be a set of mutants for every gene in their particular organism. Such a set was produced for the yeast, Saccharomyces cerevisiae near the end of the 20th century by a consortium of yeast geneticists. However, the functional genomic analysis of one chromosome, its smallest, had already begun more than 25 years earlier as a project that was designed to define most or all of that chromosome's essential genes by temperature-sensitive lethal mutations. When far fewer than expected genes were uncovered, the relatively new field of molecular cloning enabled us and indeed, the entire community of yeast researchers to approach this problem more definitively. These studies ultimately led to cloning, genomic sequencing, and the production and phenotypic analysis of the entire set of knockout mutations for this model organism as well as a better concept of what defines an essential function, a wish fulfilled that enables this model eukaryote to continue at the forefront of research in modern biology.

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors

PubMed Central

2013-01-01

Background Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Methods Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. Results MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Conclusion Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma. PMID:24119551

An inter-laboratory comparison of PNH clone detection by high-sensitivity flow cytometry in a Russian cohort.

PubMed

Sipol, Alexandra A; Babenko, Elena V; Borisov, Vyacheslav I; Naumova, Elena V; Boyakova, Elena V; Yakunin, Dimitry I; Glazanova, Tatyana V; Chubukina, Zhanna V; Pronkina, Natalya V; Popov, Alexander M; Saveliev, Leonid I; Lugovskaya, Svetlana A; Lisukov, Igor A; Kulagin, Alexander D; Illingworth, Andrea J

2015-01-01

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by partial or absolute deficiency of glycophosphatidyl-inositol (GPI) anchor-linked surface proteins on blood cells. A lack of precise diagnostic standards for flow cytometry has hampered useful comparisons of data between laboratories. We report data from the first study evaluating the reproducibility of high-sensitivity flow cytometry for PNH in Russia. PNH clone sizes were determined at diagnosis in PNH patients at a central laboratory and compared with follow-up measurements in six laboratories across the country. Analyses in each laboratory were performed according to recommendations from the International Clinical Cytometry Society (ICCS) and the more recent 'practical guidelines'. Follow-up measurements were compared with each other and with the values determined at diagnosis. PNH clone size measurements were determined in seven diagnosed PNH patients (five females, two males: mean age 37 years); five had a history of aplastic anemia and three (one with and two without aplastic anemia) had severe hemolytic PNH and elevated plasma lactate dehydrogenase. PNH clone sizes at diagnosis were low in patients with less severe clinical symptoms (0.41-9.7% of granulocytes) and high in patients with severe symptoms (58-99%). There were only minimal differences in the follow-up clone size measurement for each patient between the six laboratories, particularly in those with high values at diagnosis. The ICCS-recommended high-sensitivity flow cytometry protocol was effective for detecting major and minor PNH clones in Russian PNH patients, and showed high reproducibility between laboratories.

Expression of Small Heat-Shock Proteins at Low Temperatures1

PubMed Central

Sabehat, Adnan; Lurie, Susan; Weiss, David

1998-01-01

We previously reported that short exposure of tomato (Lycopersicon esculentum L.) fruits to high temperature protects them from chilling injury. To study the involvement of heat-shock proteins (HSPs) in the acquisition of low-temperature tolerance, we cloned two heat-shock-induced genes that are also expressed at low temperatures. The cloned cDNAs belong to the small HSP group. Sequence analyses of the clones showed perfect homology to the tomato-ripening gene tom66 and to the tomato chloroplastic HSP21 gene tom111. The expression of both genes was induced by high temperature in fruits, flowers, leaves, and stems, but not by low or ambient temperatures or by other stresses such as drought and anaerobic conditions. When the heated fruits were transferred to low temperature, tom66 and tom111 mRNA levels first decreased but were then reinduced. Induction was not observed in nonheated fruits at low temperature. Immunodetection of tom111-encoded protein indicated that this protein is present at low temperatures in the heated fruits. The results of this study show that the expression of tom66 and tom111 is correlated with protection against some, but not all, symptoms of chilling injury. PMID:9625718

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning

PubMed Central

Meghdadi, Hossein; Khosravi, Azar D.; Ghadiri, Ata A.; Sina, Amir H.; Alami, Ameneh

2015-01-01

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39–31.27% for rpoB-PCR, 36.44–60.83% for IS6110- PCR, 75.29–92.93% for nested-rpoB PCR, and 87.98–99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100% specificity of each PCR method were calculated as 69.15–100%. Our results indicated that nested-rpoB PCR combined with TA cloning and sequencing is a preferred method for the detection of MTB DNA in EPTB samples with high sensitivity and specificity which confirm the histopathology results. PMID:26191059

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning.

PubMed

Meghdadi, Hossein; Khosravi, Azar D; Ghadiri, Ata A; Sina, Amir H; Alami, Ameneh

2015-01-01

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39-31.27% for rpoB-PCR, 36.44-60.83% for IS6110- PCR, 75.29-92.93% for nested-rpoB PCR, and 87.98-99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100% specificity of each PCR method were calculated as 69.15-100%. Our results indicated that nested-rpoB PCR combined with TA cloning and sequencing is a preferred method for the detection of MTB DNA in EPTB samples with high sensitivity and specificity which confirm the histopathology results.

A Microbial Community in Sediments Beneath the Western Antarctic Ice Sheet, Ice Stream C (Kamb)

NASA Astrophysics Data System (ADS)

Skidmore, M.; Han, S.; Foo, W.; Bui, D.; Lanoil, B.

2004-12-01

In 2000, an ice-drilling project focusing on the "sticky spot" of Ice Stream C recovered cores of sub-glacial sediments from beneath the Western Antarctic Ice Sheet. We have characterized several chemical and microbiological parameters of the sole intact sediment core. Pore waters extracted from these sediments were brackish and some were supersaturated with respect to calcite. Ion chromatography demonstrated the presence of several organic acids at low, but detectable, levels in the pore water. DAPI direct cell counts were approximately 107 cells g-1. Aerobic viable plate counts were much lower than direct cell counts; however, they were two orders of magnitude higher on plates incubated at low temperature (4 ° C; 3.63 x 105 CFU ml-1) than at higher temperatures (ca. 22° C; 1.5 x 103 CFU ml-1); no colonies were detected on plates incubated anaerobically at either temperature. 16S rDNA clone library analysis indicates extremely limited bacterial diversity in these samples: six phylogenetic clades were detected. The three dominant bacterial phylogenetic clades in the clone libraries (252 clones total) were most closely related to Thiobacillus thioparus (180 clones), Polaromonas vacuolata (34 clones), and Gallionella ferruginea (35 clones) and their relatives; one clone each represented the other three phylogenetic clades (most closely related to Ralstonia pickettii, Lysobacter antibioticus, and Xylella fastidiosa, respectively). These sequences match closely with sequences previously obtained from other subglacial environments in Alaska, Ellesmere Island, Canada and New Zealand. Implications of this microbial community to subglacial chemistry and microbial biogeography will be discussed.

Levels of HIV1 gp120 3D B-cell epitopes mutability and variability: searching for possible vaccine epitopes.

PubMed

Khrustalev, Vladislav Victorovich

2010-01-01

We used a DiscoTope 1.2 (http://www.cbs.dtu.dk/services/DiscoTope/), Epitopia (http://epitopia.tau.ac.il/) and EPCES (http://www.t38.physik.tu-muenchen.de/programs.htm) algorithms to map discontinuous B-cell epitopes in HIV1 gp120. The most mutable nucleotides in HIV genes are guanine (because of G to A hypermutagenesis) and cytosine (because of C to U and C to A mutations). The higher is the level of guanine and cytosine usage in third (neutral) codon positions and the lower is their level in first and second codon positions of the coding region, the more stable should be an epitope encoded by this region. We compared guanine and cytosine usage in regions coding for five predicted 3D B-cell epitopes of gp120. To make this comparison we used GenBank resource: 385 sequences of env gene obtained from ten HIV1-infected individuals were studied (http://www.barkovsky.hotmail.ru/Data/Seqgp120.htm). The most protected from nonsynonymous nucleotide mutations of guanine and cytosine 3D B-cell epitope is situated in the first conserved region of gp120 (it is mapped from 66th to 86th amino acid residue). We applied a test of variability to confirm this finding. Indeed, the less mutable predicted B-cell epitope is the less variable one. MEGA4 (standard PAM matrix) was used for the alignments and "VVK Consensus" algorithm (http://www.barkovsky.hotmail.ru) was used for the calculations.

Collagenous Extracellular Matrix Biomaterials for Tissue Engineering: Lessons from the Common Sea Urchin Tissue.

PubMed

Goh, Kheng Lim; Holmes, David F

2017-04-25

Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action-the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced composites to a wide variety of collagen reinforcing (non-mutable) connective tissue, has allowed us to draw general conclusions concerning the mechanical response of the MCT at specific mechanical states, namely the stiff and complaint states. The intent of this review is to provide the latest insights, as well as identify technical challenges and opportunities, that may be useful for developing methods for effective mechanical support when adapting decellularised connective tissues from the sea urchin for tissue engineering or for the design of a synthetic analogue.

Collagenous Extracellular Matrix Biomaterials for Tissue Engineering: Lessons from the Common Sea Urchin Tissue

PubMed Central

Goh, Kheng Lim; Holmes, David F.

2017-01-01

Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action—the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced composites to a wide variety of collagen reinforcing (non-mutable) connective tissue, has allowed us to draw general conclusions concerning the mechanical response of the MCT at specific mechanical states, namely the stiff and complaint states. The intent of this review is to provide the latest insights, as well as identify technical challenges and opportunities, that may be useful for developing methods for effective mechanical support when adapting decellularised connective tissues from the sea urchin for tissue engineering or for the design of a synthetic analogue. PMID:28441344

Polymer ligand–induced autonomous sorting and reversible phase separation in binary particle blends

DOE PAGES

Schmitt, Michael; Zhang, Jianan; Lee, Jaejun; ...

2016-12-23

The tethering of ligands to nanoparticles has emerged as an important strategy to control interactions and organization in particle assembly structures. Here, we demonstrate that ligand interactions in mixtures of polymer-tethered nanoparticles (which are modified with distinct types of polymer chains) can impart upper or lower critical solution temperature (UCST/LCST)–type phase behavior on binary particle mixtures in analogy to the phase behavior of the corresponding linear polymer blends. Therefore, cooling (or heating) of polymer-tethered particle blends with appropriate architecture to temperatures below (or above) the UCST (or LCST) results in the organization of the individual particle constituents into monotype microdomainmore » structures. The shape (bicontinuous or island-type) and lengthscale of particle microdomains can be tuned by variation of the composition and thermal process conditions. Thermal cycling of LCST particle brush blends through the critical temperature enables the reversible growth and dissolution of monoparticle domain structures. The ability to autonomously and reversibly organize multicomponent particle mixtures into monotype microdomain structures could enable transformative advances in the high-throughput fabrication of solid films with tailored and mutable structures and properties that play an important role in a range of nanoparticle-based material technologies.« less

Polymer ligand–induced autonomous sorting and reversible phase separation in binary particle blends

PubMed Central

Schmitt, Michael; Zhang, Jianan; Lee, Jaejun; Lee, Bongjoon; Ning, Xin; Zhang, Ren; Karim, Alamgir; Davis, Robert F.; Matyjaszewski, Krzysztof; Bockstaller, Michael R.

2016-01-01

The tethering of ligands to nanoparticles has emerged as an important strategy to control interactions and organization in particle assembly structures. We demonstrate that ligand interactions in mixtures of polymer-tethered nanoparticles (which are modified with distinct types of polymer chains) can impart upper or lower critical solution temperature (UCST/LCST)–type phase behavior on binary particle mixtures in analogy to the phase behavior of the corresponding linear polymer blends. Therefore, cooling (or heating) of polymer-tethered particle blends with appropriate architecture to temperatures below (or above) the UCST (or LCST) results in the organization of the individual particle constituents into monotype microdomain structures. The shape (bicontinuous or island-type) and lengthscale of particle microdomains can be tuned by variation of the composition and thermal process conditions. Thermal cycling of LCST particle brush blends through the critical temperature enables the reversible growth and dissolution of monoparticle domain structures. The ability to autonomously and reversibly organize multicomponent particle mixtures into monotype microdomain structures could enable transformative advances in the high-throughput fabrication of solid films with tailored and mutable structures and properties that play an important role in a range of nanoparticle-based material technologies. PMID:28028538

3D calcite heterostructures for dynamic and deformable mineralized matrices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi, Jaeseok; Wang, Yucai; Jiang, Yuanwen

Scales are rooted in soft tissues, and are regenerated by specialized cells. The realization of dynamic synthetic analogues with inorganic materials has been a significant challenge, because the abiological regeneration sites that could yield deterministic growth behavior are hard to form. Here we overcome this fundamental hurdle by constructing a mutable and deformable array of three-dimensional calcite heterostructures that are partially locked in silicone. Individual calcite crystals exhibit asymmetrical dumbbell shapes and are prepared by a parallel tectonic approach under ambient conditions. Furthermore, the silicone matrix immobilizes the epitaxial nucleation sites through self-templated cavities, which enables symmetry breaking in reactionmore » dynamics and scalable manipulation of the mineral ensembles. With this platform, we devise several mineral-enabled dynamic surfaces and interfaces. For example, we show that the induced growth of minerals yields localized inorganic adhesion for biological tissue and reversible focal encapsulation for sensitive components in flexible electronics.« less

3D calcite heterostructures for dynamic and deformable mineralized matrices

DOE PAGES

Yi, Jaeseok; Wang, Yucai; Jiang, Yuanwen; ...

2017-09-11

Scales are rooted in soft tissues, and are regenerated by specialized cells. The realization of dynamic synthetic analogues with inorganic materials has been a significant challenge, because the abiological regeneration sites that could yield deterministic growth behavior are hard to form. Here we overcome this fundamental hurdle by constructing a mutable and deformable array of three-dimensional calcite heterostructures that are partially locked in silicone. Individual calcite crystals exhibit asymmetrical dumbbell shapes and are prepared by a parallel tectonic approach under ambient conditions. Furthermore, the silicone matrix immobilizes the epitaxial nucleation sites through self-templated cavities, which enables symmetry breaking in reactionmore » dynamics and scalable manipulation of the mineral ensembles. With this platform, we devise several mineral-enabled dynamic surfaces and interfaces. For example, we show that the induced growth of minerals yields localized inorganic adhesion for biological tissue and reversible focal encapsulation for sensitive components in flexible electronics.« less

Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

PubMed Central

Lucero, David E.; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W.; Peña, Reynaldo; Morrissey, Leslie A.; Rizzo, Donna M.; Stevens, Lori

2014-01-01

Background In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. Methodology/Principal Findings We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). Conclusions/Significance We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors. PMID:25474154

Minimal Residual Disease Detection and Evolved IGH Clones Analysis in Acute B Lymphoblastic Leukemia Using IGH Deep Sequencing.

PubMed

Wu, Jinghua; Jia, Shan; Wang, Changxi; Zhang, Wei; Liu, Sixi; Zeng, Xiaojing; Mai, Huirong; Yuan, Xiuli; Du, Yuanping; Wang, Xiaodong; Hong, Xueyu; Li, Xuemei; Wen, Feiqiu; Xu, Xun; Pan, Jianhua; Li, Changgang; Liu, Xiao

2016-01-01

Acute B lymphoblastic leukemia (B-ALL) is one of the most common types of childhood cancer worldwide and chemotherapy is the main treatment approach. Despite good response rates to chemotherapy regiments, many patients eventually relapse and minimal residual disease (MRD) is the leading risk factor for relapse. The evolution of leukemic clones during disease development and treatment may have clinical significance. In this study, we performed immunoglobulin heavy chain ( IGH ) repertoire high throughput sequencing (HTS) on the diagnostic and post-treatment samples of 51 pediatric B-ALL patients. We identified leukemic IGH clones in 92.2% of the diagnostic samples and nearly half of the patients were polyclonal. About one-third of the leukemic clones have correct open reading frame in the complementarity determining region 3 (CDR3) of IGH , which demonstrates that the leukemic B cells were in the early developmental stage. We also demonstrated the higher sensitivity of HTS in MRD detection and investigated the clinical value of using peripheral blood in MRD detection and monitoring the clonal IGH evolution. In addition, we found leukemic clones were extensively undergoing continuous clonal IGH evolution by variable gene replacement. Dynamic frequency change and newly emerged evolved IGH clones were identified upon the pressure of chemotherapy. In summary, we confirmed the high sensitivity and universal applicability of HTS in MRD detection. We also reported the ubiquitous evolved IGH clones in B-ALL samples and their response to chemotherapy during treatment.

Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus.

PubMed

McGoldrick, A; Macadam, A J; Dunn, G; Rowe, A; Burlison, J; Minor, P D; Meredith, J; Evans, D J; Almond, J W

1995-12-01

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.

An Escherichia coli strain, PGB01, isolated from feral pigeon Faeces, thermally fit to survive in pigeon, shows high level resistance to trimethoprim.

PubMed

Kumar, Arvind; Tiwary, Bipransh Kumar; Kachhap, Sangita; Nanda, Ashis Kumar; Chakraborty, Ranadhir

2015-01-01

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon's gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr.

A role for chromosomal instability in the development of and selection for radioresistant cell variants

NASA Technical Reports Server (NTRS)

Limoli, C. L.; Corcoran, J. J.; Jordan, R.; Morgan, W. F.; Schwartz, J. L.

2001-01-01

Chromosome instability is a common occurrence in tumour cells. We examined the hypothesis that the elevated rate of mutation formation in unstable cells can lead to the development of clones of cells that are resistant to the cancer therapy. To test this hypothesis, we compared chromosome instability to radiation sensitivity in 30 independently isolated clones of GM10115 human-hamster hybrid cells. There was a broader distribution of radiosensitivity and a higher mean SF(2)in chromosomally unstable clones. Cytogenetic and DNA double-strand break rejoining assays suggest that sensitivity was a function of DNA repair efficiency. In the unstable population, the more radioresistant clones also had significantly lower plating efficiencies. These observations suggest that chromosome instability in GM10115 cells can lead to the development of cell variants that are more resistant to radiation. In addition, these results suggest that the process of chromosome breakage and recombination that accompanies chromosome instability might provide some selective pressure for more radioresistant variants. Copyright 2001 Cancer Research Campaign.

Memory-built-in quantum cloning in a hybrid solid-state spin register

NASA Astrophysics Data System (ADS)

Wang, W.-B.; Zu, C.; He, L.; Zhang, W.-G.; Duan, L.-M.

2015-07-01

As a way to circumvent the quantum no-cloning theorem, approximate quantum cloning protocols have received wide attention with remarkable applications. Copying of quantum states to memory qubits provides an important strategy for eavesdropping in quantum cryptography. We report an experiment that realizes cloning of quantum states from an electron spin to a nuclear spin in a hybrid solid-state spin register with near-optimal fidelity. The nuclear spin provides an ideal memory qubit at room temperature, which stores the cloned quantum states for a millisecond under ambient conditions, exceeding the lifetime of the original quantum state carried by the electron spin by orders of magnitude. The realization of a cloning machine with built-in quantum memory provides a key step for application of quantum cloning in quantum information science.

Memory-built-in quantum cloning in a hybrid solid-state spin register.

PubMed

Wang, W-B; Zu, C; He, L; Zhang, W-G; Duan, L-M

2015-07-16

As a way to circumvent the quantum no-cloning theorem, approximate quantum cloning protocols have received wide attention with remarkable applications. Copying of quantum states to memory qubits provides an important strategy for eavesdropping in quantum cryptography. We report an experiment that realizes cloning of quantum states from an electron spin to a nuclear spin in a hybrid solid-state spin register with near-optimal fidelity. The nuclear spin provides an ideal memory qubit at room temperature, which stores the cloned quantum states for a millisecond under ambient conditions, exceeding the lifetime of the original quantum state carried by the electron spin by orders of magnitude. The realization of a cloning machine with built-in quantum memory provides a key step for application of quantum cloning in quantum information science.

Quick and clean cloning.

PubMed

Thieme, Frank; Marillonnet, Sylvestre

2014-01-01

Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.

Profiling secondary metabolites of needles of ozone-fumigated white pine (Pinus strobus) clones by thermally assisted hydrolysis/methylation GC/MS.

PubMed

Shadkami, F; Helleur, R J; Cox, R M

2007-07-01

Plant secondary metabolites have an important role in defense responses against herbivores and pathogens, and as a chemical barrier to elevated levels of harmful air pollutants. This study involves the rapid chemical profiling of phenolic and diterpene resin acids in needles of two (ozone-tolerant and ozone-sensitive) white pine (Pinus strobus) clones, fumigated with different ozone levels (control, and daily events peaking at 80 and 200 ppb) for 40 days. The phenolic and resin acids were measured using thermally assisted hydrolysis and methylation (THM) gas chromatography/mass spectrometry. Short-term fumigation affected the levels of two phenolic acids, i.e., 3-hydroxybenzoic and 3,4-dihydroxybenzoic acids, in that both showed a substantial decrease in concentration with increased ozone dose. The decrease in concentration of these THM products may be caused by inhibition of the plant's shikimate biochemical pathway caused by ozone exposure. The combined occurrence of these two ozone-sensitive indicators has a role in biomonitoring of ozone levels and its impact on forest productivity. In addition, chromatographic profile differences in the major diterpene resin acid components were observed between ozone-tolerant and ozone-sensitive clones. The resin acids anticopalic, 3-oxoanticopalic, 3beta-hydroxyanticopalic, and 3,4-cycloanticopalic acids were present in the ozone-sensitive pine; however, only anticopalic acid was present in the ozone-tolerant clone. This phenotypic variation in resin acid composition may be useful in distinguishing populations that are differentially adapted to air pollutants.

Comprehensive analysis of an Antarctic bacterial community with the adaptability of growth at higher temperatures than those in Antarctica.

PubMed

Hosoi-Tanabe, Shoko; Zhang, Hongyan; Zhu, Daochen; Nagata, Shinichi; Ban, Syuhei; Imura, Satoshi

2010-06-01

To investigate the adaptability to higher temperatures of Antarctic microorganisms persisting in low temperature conditions for a long time, Antarctic lake samples were incubated in several selection media at 25 degrees C and 30 degrees C. The microorganisms did not grow at 30 degrees C; however, some of them grew at 25 degrees C, indicating that the bacteria in Antarctic have the ability to grow at a wide range of temperatures. Total DNA was extracted from these microorganisms and amplified using the bacteria-universal primers. The amplified fragments were cloned, and randomly selected 48 clones were sequenced. The sequenced clones showed high similarity to the alpha-subdivision of the Proteobacteria with specific affinity to the genus Agrobacterium, Caulobacter and Brevundimonas, the ss-subdivision of Proteobacteria with specific affinity to the genus Cupriavidus, and Bacillus of the phylum Firmicutes. These results showed the presence of universal genera, suggesting that the bacteria in the Antarctic lake were not specific to this environment.

DNA probe for lactobacillus delbrueckii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delley, M.; Mollet, B.; Hottinger, H.

1990-06-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

DNA Probe for Lactobacillus delbrueckii

PubMed Central

Delley, Michèle; Mollet, Beat; Hottinger, Herbert

1990-01-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-labeled DNA probe. Images PMID:16348233

Teaching Time-Space Compression

ERIC Educational Resources Information Center

Warf, Barney

2011-01-01

Time-space compression shows students that geographies are plastic, mutable and forever changing. This paper justifies the need to teach this topic, which is rarely found in undergraduate course syllabi. It addresses the impacts of transportation and communications technologies to explicate its dynamics. In summarizing various conceptual…

Photosynthetic productivity of aspen clones varying in sensitivity to tropospheric ozone

Treesearch

M.D. Coleman; J.G. Isebrands; R.E. Dickson; D.F. Karnosky

1995-01-01

Rooted cuttings from three aspen (Populus tremuloides Michx.) clones (216, 271 and 259, classified as high, intermediate and low in O3 tolerance, respectively) were exposed to either diumal O3 profiles simulating those of Michigan's Lower Peninsula (episodic treatments), or diurnal square-wave O

Memory-built-in quantum cloning in a hybrid solid-state spin register

PubMed Central

Wang, W.-B.; Zu, C.; He, L.; Zhang, W.-G.; Duan, L.-M.

2015-01-01

As a way to circumvent the quantum no-cloning theorem, approximate quantum cloning protocols have received wide attention with remarkable applications. Copying of quantum states to memory qubits provides an important strategy for eavesdropping in quantum cryptography. We report an experiment that realizes cloning of quantum states from an electron spin to a nuclear spin in a hybrid solid-state spin register with near-optimal fidelity. The nuclear spin provides an ideal memory qubit at room temperature, which stores the cloned quantum states for a millisecond under ambient conditions, exceeding the lifetime of the original quantum state carried by the electron spin by orders of magnitude. The realization of a cloning machine with built-in quantum memory provides a key step for application of quantum cloning in quantum information science. PMID:26178617

Ultrastructural and biochemical characterization of mechanically adaptable collagenous structures in the edible sea urchin Paracentrotus lividus.

PubMed

Barbaglio, Alice; Tricarico, Serena; Ribeiro, Ana R; Di Benedetto, Cristiano; Barbato, Marta; Dessì, Desirèe; Fugnanesi, Valeria; Magni, Stefano; Mosca, Fabio; Sugni, Michela; Bonasoro, Francesco; Barbosa, Mario A; Wilkie, Iain C; Candia Carnevali, M Daniela

2015-06-01

The viscoelastic properties of vertebrate connective tissues rarely undergo significant changes within physiological timescales, the only major exception being the reversible destiffening of the mammalian uterine cervix at the end of pregnancy. In contrast to this, the connective tissues of echinoderms (sea urchins, starfish, sea cucumbers, etc.) can switch reversibly between stiff and compliant conditions in timescales of around a second to minutes. Elucidation of the molecular mechanism underlying such mutability has implications for the zoological, ecological and evolutionary field. Important information could also arise for veterinary and biomedical sciences, particularly regarding the pathological plasticization or stiffening of connective tissue structures. In the present investigation we analyzed aspects of the ultrastructure and biochemistry in two representative models, the compass depressor ligament and the peristomial membrane of the edible sea urchin Paracentrotus lividus, compared in three different mechanical states. The results provide further evidence that the mechanical adaptability of echinoderm connective tissues does not necessarily imply changes in the collagen fibrils themselves. The higher glycosaminoglycan (GAG) content registered in the peristomial membrane with respect to the compass depressor ligament suggests a diverse role of these molecules in the two mutable collagenous tissues. The possible involvement of GAG in the mutability phenomenon will need further clarification. During the shift from a compliant to a standard condition, significant changes in GAG content were detected only in the compass depressor ligament. Similarities in terms of ultrastructure (collagen fibrillar assembling) and biochemistry (two alpha chains) were found between the two models and mammalian collagen. Nevertheless, differences in collagen immunoreactivity, alpha chain migration on SDS-PAGE and BLAST alignment highlighted the uniqueness of sea urchin collagen with respect to mammalian collagen. Copyright © 2015 Elsevier GmbH. All rights reserved.

Quantifying Wheat Sensitivities to Environmental Constraints to Dissect Genotype × Environment Interactions in the Field1[OPEN

PubMed Central

Maphosa, Lance; Kovalchuk, Alex

2017-01-01

Yield is subject to strong genotype-by-environment (G × E) interactions in the field, especially under abiotic constraints such as soil water deficit (drought [D]) and high temperature (heat [H]). Since environmental conditions show strong fluctuations during the whole crop cycle, geneticists usually do not consider environmental measures as quantitative variables but rather as factors in multienvironment analyses. Based on 11 experiments in a field platform with contrasting temperature and soil water deficit, we determined the periods of sensitivity to drought and heat constraints in wheat (Triticum aestivum) and determined the average sensitivities for major yield components. G × E interactions were separated into their underlying components, constitutive genotypic effect (G), G × D, G × H, and G × H × D, and were analyzed for two genotypes, highlighting contrasting responses to heat and drought constraints. We then tested the constitutive and responsive behaviors of two strong quantitative trait loci (QTLs) associated previously with yield components. This analysis confirmed the constitutive effect of the chromosome 1B QTL and explained the G × E interaction of the chromosome 3B QTL by a benefit of one allele when temperature rises. In addition to the method itself, which can be applied to other data sets and populations, this study will support the cloning of a major yield QTL on chromosome 3B that is highly dependent on environmental conditions and for which the climatic interaction is now quantified. PMID:28546436

A brief history of trp: commentary and personal perspective.

PubMed

Hardie, Roger C

2011-05-01

The history of the discovery of the transient receptor potential (TRP) cation channel superfamily began in 1969 with Cosens and Manning's isolation of the Drosophila transient receptor potential mutant, in which the photoreceptor response decays during continuous illumination. Early studies from Minke found that the elementary light response was unaffected in trp mutants, and he attributed the defect to an intermediate stage of phototransduction. Montell and Rubin cloned the trp gene in 1989: they recognised it as a transmembrane protein, but also concluded that it did not encode the light-sensitive channels. In 1991, Minke and Selinger proposed that TRP represented a Ca2+ transporter required for refilling intracellular InsP3-sensitive Ca2+ stores, in turn required for activation of the light-sensitive channels. Also in 1991, after developing a photoreceptor patch clamp preparation, I showed that the light-sensitive channels themselves were highly permeable to Ca2+, questioning the need for such a dedicated Ca2+ transporter. In 1992, in collaboration with Minke, I resolved this paradox by showing there were two classes of light-sensitive channels, one highly Ca2+ permeable and eliminated in trp mutants. This represented the first and compelling evidence that TRP represented a light-sensitive channel and was supported by the cloning of the second light-sensitive channel, TRPL, by Kelly's lab. Three years later, in 1995, the labs of Montell and Birnbaumer independently cloned TRPC1, the first of 29 vertebrate TRP isoforms distributed amongst seven subfamilies.

Improved Soluble ScFv ELISA Screening Approach for Antibody Discovery Using Phage Display Technology.

PubMed

Tohidkia, Mohammad R; Sepehri, Maryam; Khajeh, Shirin; Barar, Jaleh; Omidi, Yadollah

2017-09-01

Phage display technology (PDT) is a powerful tool for the isolation of recombinant antibody (Ab) fragments. Using PDT, target molecule-specific phage-Ab clones are enriched through the "biopanning" process. The individual specific binders are screened by the monoclonal scFv enzyme-linked immunosorbent assay (ELISA) that may associate with inevitable false-negative results. Thus, in this study, three strategies were investigated for optimization of the scFvs screening using Tomlinson I and J libraries, including (1) optimizing the expression of functional scFvs, (2) improving the sensitivity of ELISA, and (3) preparing different samples containing scFvs. The expression of all scFv Abs was significantly enhanced when scFv clones were cultivated in the terrific broth (TB) medium at the optimum temperature of 30 °C. The protein A-conjugated with horseradish peroxidase (HRP) was found to be a well-suited reagent for the detection of Ag-bound scFvs in comparison with either anti-c-myc Ab or the mixing procedure. Based on our findings, it seems there is no universal media supplement for an improved expression of all scFvs derived from both Tomlinson I and J libraries. We thus propose that expression of scFv fragments in a microplate scale is largely dependent on a variety of parameters, in particular the scFv clones and relevant sequences.

Host plant development, water level and water parameters shape Phragmites australis-associated oomycete communities and determine reed pathogen dynamics in a large lake.

PubMed

Wielgoss, Anna; Nechwatal, Jan; Bogs, Carolin; Mendgen, Kurt

2009-08-01

In a 3-year-study, we analysed the population dynamics of the reed pathogen Pythium phragmitis and other reed-associated oomycetes colonizing fresh and dried reed leaves in the littoral zone of a large lake. Oomycete communities derived from internal transcribed spacer clone libraries were clearly differentiated according to substrate and seasonal influences. In fresh leaves, diverse communities consisting of P. phragmitis and other reed-associated pathogens were generally dominant. Pythium phragmitis populations peaked in spring with the emergence of young reed shoots, and in autumn after extreme flooding events. In summer it decreased with falling water levels, changing water chemistry and rising temperatures. Another Pythium species was also highly abundant in fresh leaves throughout the year and might represent a new, as-yet uncultured reed pathogen. In dried leaves, reed pathogens were rarely detected, whereas saprophytic species occurred abundantly during all seasons. Saprophyte communities were less diverse, less temperature sensitive and independent of reed development. In general, our results provide evidence for the occurrence of highly specialized sets of reed-associated oomycetes in a natural reed ecosystem. Quantitative analyses (clone abundances and quantitative real-time PCR) revealed that the reed pathogen P. phragmitis is particularly affected by changing water levels, water chemistry and the stage of reed development.

Sterility and hypermutability in the P-M system of hybrid dysgenesis in Drosophila melanogaster.

PubMed

Kocur, G J; Drier, E A; Simmons, M J

1986-12-01

Inbred wild strains of Drosophila melanogaster derived from the central and eastern United States were used to make dysgenic hybrids in the P-M system. These strains possessed P elements and the P cytotype, the condition that represses P element transposition. Their hybrids were studied for the mutability of the P element insertion mutation, snw, and for the incidence of gonadal dysgenesis (GD) sterility. All the strains tested were able to induce hybrid dysgenesis by one or both of these assays; however, high levels of dysgenesis were rare. Sets of X chromosomes and autosomes from the inbred wild strains were more effective at inducing GD sterility than were sets of Y chromosomes and autosomes. In two separate analyses, GD sterility was positively correlated with snw mutability, suggesting a linear relationship. However, one strain appeared to induce too much GD sterility for its level of snw destabilization, indicating an uncoupling of these two manifestations of hybrid dysgenesis.

Sterility and Hypermutability in the P-M System of Hybrid Dysgenesis in DROSOPHILA MELANOGASTER

PubMed Central

Kocur, Gordon J.; Drier, Eric A.; Simmons, Michael J.

1986-01-01

Inbred wild strains of Drosophila melanogaster derived from the central and eastern United States were used to make dysgenic hybrids in the P-M system. These strains possessed P elements and the P cytotype, the condition that represses P element transposition. Their hybrids were studied for the mutability of the P element insertion mutation, snw, and for the incidence of gonadal dysgenesis (GD) sterility. All the strains tested were able to induce hybrid dysgenesis by one or both of these assays; however, high levels of dysgenesis were rare. Sets of X chromosomes and autosomes from the inbred wild strains were more effective at inducing GD sterility than were sets of Y chromosomes and autosomes. In two separate analyses, GD sterility was positively correlated with snw mutability, suggesting a linear relationship. However, one strain appeared to induce too much GD sterility for its level of snw destabilization, indicating an uncoupling of these two manifestations of hybrid dysgenesis. PMID:3100389

How to hit HIV where it hurts

NASA Astrophysics Data System (ADS)

Chakraborty, Arup

No medical procedure has saved more lives than vaccination. But, today, some pathogens have evolved which have defied successful vaccination using the empirical paradigms pioneered by Pasteur and Jenner. One characteristic of many pathogens for which successful vaccines do not exist is that they present themselves in various guises. HIV is an extreme example because of its high mutability. This highly mutable virus can evade natural or vaccine induced immune responses, often by mutating at multiple sites linked by compensatory interactions. I will describe first how by bringing to bear ideas from statistical physics (e.g., maximum entropy models, Hopfield models, Feynman variational theory) together with in vitro experiments and clinical data, the fitness landscape of HIV is beginning to be defined with explicit account for collective mutational pathways. I will describe how this knowledge can be harnessed for vaccine design. Finally, I will describe how ideas at the intersection of evolutionary biology, immunology, and statistical physics can help guide the design of strategies that may be able to induce broadly neutralizing antibodies.

Characterizing Phase Transitions in a Model of Neutral Evolutionary Dynamics

NASA Astrophysics Data System (ADS)

Scott, Adam; King, Dawn; Bahar, Sonya

2013-03-01

An evolutionary model was recently introduced for sympatric, phenotypic evolution over a variable fitness landscape with assortative mating (Dees & Bahar 2010). Organisms in the model are described by coordinates in a two-dimensional phenotype space, born at random coordinates with limited variation from their parents as determined by a mutation parameter, mutability. The model has been extended to include both neutral evolution and asexual reproduction in Scott et al (submitted). It has been demonstrated that a second order, non-equilibrium phase transition occurs for the temporal dynamics as the mutability is varied, for both the original model and for neutral conditions. This transition likely belongs to the directed percolation universality class. In contrast, the spatial dynamics of the model shows characteristics of an ordinary percolation phase transition. Here, we characterize the phase transitions exhibited by this model by determining critical exponents for the relaxation times, characteristic lengths, and cluster (species) mass distributions. Missouri Research Board; J.S. McDonnell Foundation

Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds.

PubMed

Troncoso-Ponce, M A; Rivoal, J; Venegas-Calerón, M; Dorion, S; Sánchez, R; Cejudo, F J; Garcés, R; Martínez-Force, E

2012-07-01

Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues. Copyright © 2012 Elsevier Ltd. All rights reserved.

Adaptation to fluctuations in temperature by nine species of bacteria.

PubMed

Saarinen, Kati; Laakso, Jouni; Lindström, Leena; Ketola, Tarmo

2018-03-01

Rapid environmental fluctuations are ubiquitous in the wild, yet majority of experimental studies mostly consider effects of slow fluctuations on organism. To test the evolutionary consequences of fast fluctuations, we conducted nine independent experimental evolution experiments with bacteria. Experimental conditions were same for all species, and we allowed them to evolve either in fluctuating temperature alternating rapidly between 20°C and 40°C or at constant 30°C temperature. After experimental evolution, we tested the performance of the clones in both rapid fluctuation and in constant environments (20°C, 30°C and 40°C). Results from experiments on these nine species were combined meta-analytically. We found that overall the clones evolved in the fluctuating environment had evolved better efficiency in tolerating fluctuations (i.e., they had higher yield in fluctuating conditions) than the clones evolved in the constant environment. However, we did not find any evidence that fluctuation-adapted clones would have evolved better tolerance to any measured constant environments (20°C, 30°C, and 40°C). Our results back up recent empirical findings reporting that it is hard to predict adaptations to fast fluctuations using tolerance curves.

Carbon allocation and partitioning in aspen clones varying in sensitivity to tropospheric ozone

Treesearch

M.D. Coleman; R.E. Dickson; J.G. Isebrands; D.F. Karnosky

1995-01-01

Clones of aspen (Populus tremuloides Michx.) were identified that differ in biomass production in response to O3exposure. 14Carbon tracer studies were used to determine if the differences in biomass response were linked to shifts in carbon allocation and carbon partitioning patterns. Rooted cuttings from...

Tissue Gene Expression Analysis Using Arrayed Normalized cDNA Libraries

PubMed Central

Eickhoff, Holger; Schuchhardt, Johannes; Ivanov, Igor; Meier-Ewert, Sebastian; O'Brien, John; Malik, Arif; Tandon, Neeraj; Wolski, Eryk-Witold; Rohlfs, Elke; Nyarsik, Lajos; Reinhardt, Richard; Nietfeld, Wilfried; Lehrach, Hans

2000-01-01

We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AL360374–AL36537.] PMID:10958641

Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

PubMed

Kurata, Morito; Rathe, Susan K; Bailey, Natashay J; Aumann, Natalie K; Jones, Justine M; Veldhuijzen, G Willemijn; Moriarity, Branden S; Largaespada, David A

2016-11-03

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

Diagnostic Markers of Ovarian Cancer by High-Throughput Antigen Cloning and Detection on Arrays

PubMed Central

Chatterjee, Madhumita; Mohapatra, Saroj; Ionan, Alexei; Bawa, Gagandeep; Ali-Fehmi, Rouba; Wang, Xiaoju; Nowak, James; Ye, Bin; Nahhas, Fatimah A.; Lu, Karen; Witkin, Steven S.; Fishman, David; Munkarah, Adnan; Morris, Robert; Levin, Nancy K.; Shirley, Natalie N.; Tromp, Gerard; Abrams, Judith; Draghici, Sorin; Tainsky, Michael A.

2008-01-01

A noninvasive screening test would significantly facilitate early detection of epithelial ovarian cancer. This study used a combination of high-throughput selection and array-based serologic detection of many antigens indicative of the presence of cancer, thereby using the immune system as a biosensor. This high-throughput selection involved biopanning of an ovarian cancer phage display library using serum immunoglobulins from an ovarian cancer patient as bait. Protein macroarrays containing 480 of these selected antigen clones revealed 65 clones that interacted with immunoglobulins in sera from 32 ovarian cancer patients but not with sera from 25 healthy women or 14 patients having other benign or malignant gynecologic diseases. Sequence analysis data of these 65 clones revealed 62 different antigens. Among the markers, we identified some known antigens, including RCAS1, signal recognition protein-19, AHNAK-related sequence, nuclear autoantogenic sperm protein, Nijmegen breakage syndrome 1 (Nibrin), ribosomal protein L4, Homo sapiens KIAA0419 gene product, eukaryotic initiation factor 5A, and casein kinase II, as well as many previously uncharacterized antigenic gene products. Using these 65 antigens on protein microarrays, we trained neural networks on two-color fluorescent detection of serum IgG binding and found an average sensitivity and specificity of 55% and 98%, respectively. In addition, the top 6 of the most specific clones resulted in an average sensitivity and specificity of 32% and 94%, respectively. This global approach to antigenic profiling, epitomics, has applications to cancer and autoimmune diseases for diagnostic and therapeutic studies. Further work with larger panels of antigens should provide a comprehensive set of markers with sufficient sensitivity and specificity suitable for clinical testing in high-risk populations. PMID:16424057

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landry, L.G.; Pell, E.J.

Plants exposed to ozone (O{sub 3}) exhibited symptoms of premature senescence, including early decline in quantity of rubisco. O{sub 3}-induced oxidation may cause changes in protein conformation of rubisco, resulting in enhanced proteolysis. To test this hypothesis, rubisco was purified from two hybrid clones of Populus maximowizii x trichocarpa, clones 388 and 245, and treated in vitro with O{sub 3} or air. Rubisco was then challenged with bromelain, papain, chymotrypsin, carboxypeptidase A, or endoproteinase Glu-C and percent degradation measured by SDS-PAGE and densitometric scanning of the gels. Degree of rubisco sensitivity to oxidation may be related to available sulfhydryl (SH)more » groups on the protein. The number of SH groups in native and denatured rubisco was measured for purified rubisco of both clones by DTNB titration method. The relationship between sensitivity to proteolysis and number and availability of SH groups is discussed.« less

Islamic perspective on human cloning and stem cell research.

PubMed

Larijani, B; Zahedi, F

2004-12-01

Recent advances in the field of cloning and stem cell research have introduced new hope for treatment of serious diseases. But this promise has been accompanied by enormous questions. Currently, cloning is a matter of public discussion. It is rare that a field of science causes debate and challenge not only among scientists but also among ethicists, religious scholars, governments, and politicians. One important concern is religious arguments. Various religions have different attitudes toward the morality of these subjects; even within a particular religious tradition there is a diversity of opinions. The following article briefly reviews Islamic perspectives about reproductive/therapeutic cloning and stem cell research. The majority of Muslim jurists distinguish between reproductive and therapeutic cloning. The moral status of the human embryo, the most sensitive and disputed point in this debate, is also discussed according to Holy Quran teachings.

Cloning of heat shock protein genes (hsp70, hsc70 and hsp90) and their expression in response to larval diapause and thermal stress in the wheat blossom midge, Sitodiplosis mosellana.

PubMed

Cheng, Weining; Li, Dan; Wang, Yue; Liu, Yang; Zhu-Salzman, Keyan

2016-12-01

Sitodiplosis mosellana Géhin, one of the most important pests of wheat, undergoes obligatory diapause as a larva to survive unfavorable temperature extremes during hot summers and cold winters. To explore the potential roles of heat shock proteins (hsp) in this process, we cloned full-length cDNAs of hsp70, hsc70 and hsp90 from S. mosellana larvae, and examined their expression in response to diapause and short-term temperature stresses. Three hsps included all signature sequences of corresponding protein family and EEVD motifs. They showed high homology to their counterparts in other species, and the phylogenetic analysis of hsp90 was consistent with the known classification of insects. Expression of hsp70 and hsp90 were highly induced by diapause, particularly pronounced during summer and winter. Interestingly, hsp70 was more strongly expressed in summer than in winter whereas hsp90 displayed the opposite pattern. Abundance of hsc70 mRNA was comparable prior to and during diapauses and was highly up-regulated when insects began to enter the stage of post-diapause quiescence. Heat-stressed over-summering larvae (⩾30°C) or cold-stressed over-wintering larvae (⩽0°C) could further elevate expression of these three genes, but temperature extremes i.e. as high as 45°C or as low as -15°C failed to trigger such expression patterns. Notably, hsp70 was most sensitive to heat stress and hsp90 was most sensitive to cold stress. These results suggested that hsp70 and hsp90 play key roles in diapause maintenance and thermal stress; the former may be more prominent contributor to heat tolerance and the latter for cold tolerance. In contrast, hsc70 most likely is involved in developmental transition from diapause to post-diapause quiescence, and thus may serve as a molecular marker to predict diapause termination. Copyright © 2016 Elsevier Ltd. All rights reserved.

An Escherichia coli Strain, PGB01, Isolated from Feral Pigeon Faeces, Thermally Fit to Survive in Pigeon, Shows High Level Resistance to Trimethoprim

PubMed Central

Kachhap, Sangita; Nanda, Ashis Kumar; Chakraborty, Ranadhir

2015-01-01

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon’s gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr. PMID:25750990

Stabilization of biothreat diagnostic samples through vitrification matrices.

PubMed

Minogue, Timothy Devin; Kalina, Warren Vincent; Coyne, Susan Rajnik

2014-06-01

Diagnostics for biothreat agents require sample shipment to reference labs for diagnosis of disease; however high/fluctuating temperatures during sample transport negatively affect sample quality and results. Vitrification additives preserve sample integrity for molecular-based assay diagnostics in the absence of refrigeration by imparting whole molecule stability to a plethora of environmental insults. Therefore, we have evaluated commercially available vitrification matrices' (Biomatrica's CloneStable® and RNAStable®) ability to stabilize samples of Yersinia pestis and Venezuelan Equine Encephalitis Virus. When heated to 95°C in RNAStable®, Y. pestis had a 13-fold improvement in detection via real-time PCR compared to heated samples in buffer. VEEV, in RNAStable® at 55°C, had a ~10-fold improved detection versus heated samples in buffer. CloneStable® also preserved Y. pestis antigens for 7days after exposure to cycling temperatures. Overall, RNAStable® and CloneStable® respectively offered superior stabilization to nucleic acids and proteins in response to temperature fluctuations. Copyright © 2014. Published by Elsevier B.V.

Up-regulation of antioxidant enzymes and coenzyme Q(10) in a human oral cancer cell line with acquired bleomycin resistance.

PubMed

Yen, Hsiu-Chuan; Li, Sin-Hua; Majima, Hideyuki J; Huang, Yu-Hsiang; Chen, Chiu-Ping; Liu, Chia-Chi; Tu, Ya-Chi; Chen, Chih-Wei

2011-06-01

Bleomycin (BLM) is an anti-cancer drug that can induce formation of reactive oxygen species (ROS). To investigate the association between up-regulation of antioxidant enzymes and coenzyme Q(10) (CoQ(10)) in acquired BLM resistance, one BLM-resistant clone, SBLM24 clone, was selected from a human oral cancer cell line, SCC61 clone. The BLM resistance of SBLM24 clone relative to a sub-clone of SCC61b cells was confirmed by analysis of clonogenic ability and cell cycle arrest. CoQ(10) levels and levels of Mn superoxide dismutase, glutathione peroxidase 1, catalase and thioredoxin reductase 1 were augmented in SBLM24 clone although there was also a mild increase in the expression of BLM hydrolase. Suppression of CoQ(10) levels by 4-aminobenzoate sensitized BLM-induced cytotoxicity. The results of suppression on enhanced ROS production by BLM and the cross-resistance to hydrogen peroxide in SBLM24 clone further demonstrated the development of adaptation to oxidative stress during the formation of acquired BLM resistance.

Expression of CdDHN4, a Novel YSK2-Type Dehydrin Gene from Bermudagrass, Responses to Drought Stress through the ABA-Dependent Signal Pathway

PubMed Central

Lv, Aimin; Fan, Nana; Xie, Jianping; Yuan, Shili; An, Yuan; Zhou, Peng

2017-01-01

Dehydrin improves plant resistance to many abiotic stresses. In this study, the expression profiles of a dehydrin gene, CdDHN4, were estimated under various stresses and abscisic acid (ABA) treatments in two bermudagrasses (Cynodon dactylon L.): Tifway (drought-tolerant) and C299 (drought-sensitive). The expression of CdDHN4 was up-regulated by high temperatures, low temperatures, drought, salt and ABA. The sensitivity of CdDHN4 to ABA and the expression of CdDHN4 under drought conditions were higher in Tifway than in C299. A 1239-bp fragment, CdDHN4-P, the partial upstream sequence of the CdDHN4 gene, was cloned by genomic walking from Tifway. Bioinformatic analysis showed that the CdDHN4-P sequence possessed features typical of a plant promoter and contained many typical cis elements, including a transcription initiation site, a TATA-box, an ABRE, an MBS, a MYC, an LTRE, a TATC-box and a GT1-motif. Transient expression in tobacco leaves demonstrated that the promoter CdDHN4-P can be activated by ABA, drought and cold. These results indicate that CdDHN4 is regulated by an ABA-dependent signal pathway and that the high sensitivity of CdDHN4 to ABA might be an important mechanism enhancing the drought tolerance of bermudagrass. PMID:28559903

Expression of CdDHN4, a Novel YSK2-Type Dehydrin Gene from Bermudagrass, Responses to Drought Stress through the ABA-Dependent Signal Pathway.

PubMed

Lv, Aimin; Fan, Nana; Xie, Jianping; Yuan, Shili; An, Yuan; Zhou, Peng

2017-01-01

Dehydrin improves plant resistance to many abiotic stresses. In this study, the expression profiles of a dehydrin gene, CdDHN4 , were estimated under various stresses and abscisic acid (ABA) treatments in two bermudagrasses ( Cynodon dactylon L.): Tifway (drought-tolerant) and C299 (drought-sensitive). The expression of CdDHN4 was up-regulated by high temperatures, low temperatures, drought, salt and ABA. The sensitivity of CdDHN4 to ABA and the expression of CdDHN4 under drought conditions were higher in Tifway than in C299. A 1239-bp fragment, CdDHN4-P, the partial upstream sequence of the CdDHN4 gene, was cloned by genomic walking from Tifway. Bioinformatic analysis showed that the CdDHN4-P sequence possessed features typical of a plant promoter and contained many typical cis elements, including a transcription initiation site, a TATA-box, an ABRE, an MBS, a MYC, an LTRE, a TATC-box and a GT1-motif. Transient expression in tobacco leaves demonstrated that the promoter CdDHN4-P can be activated by ABA, drought and cold. These results indicate that CdDHN4 is regulated by an ABA-dependent signal pathway and that the high sensitivity of CdDHN4 to ABA might be an important mechanism enhancing the drought tolerance of bermudagrass.

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: a solvent gate for the active site of pH-sensitive luciferases.

PubMed

Viviani, Vadim R; Silva Neto, Antonio J; Arnoldi, Frederico G C; Barbosa, João A R G; Ohmiya, Yoshihiro

2008-01-01

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors.

Potential contributions of heat shock proteins to temperature-dependent sex determination in the American alligator.

PubMed

Kohno, S; Katsu, Y; Urushitani, H; Ohta, Y; Iguchi, T; Guillette, L J

2010-01-01

Sex determination in the American alligator depends on the incubation temperature experienced during a thermo-sensitive period (TSP), although sex determination can be 'reversed' by embryonic exposure to an estrogenic compound. Thus, temperature and estrogenic signals play essential roles during temperature-dependent sex determination (TSD). The genetic basis for TSD is poorly understood, although previous studies observed that many of the genes associated with genetic sex determination (GSD) are expressed in species with TSD. Heat shock proteins (HSPs), good candidates because of their temperature-sensitive expression, have not been examined in regard to TSD but HSPs have the ability to modify steroid receptor function. A number of HSP cDNAs (HSP27, DNAJ, HSP40, HSP47, HSP60, HSP70A, HSP70B, HSP70C, HSP75, HSP90alpha, HSP90beta, and HSP108) as well as cold-inducible RNA binding protein (CIRBP) and HSP-binding protein (HSPBP) were cloned, and expression of their mRNA in the gonadal-adrenal-mesonephros complex (GAM) was investigated. Embryonic and neonatal GAMs exhibited mRNA for all of the HSPs examined during and after the TSP. One-month-old GAMs were separated into 3 portions (gonad, adrenal gland, and mesonephros), and sexual dimorphism in the mRNA expression of gonadal HSP27 (male > female), gonadal HSP70A (male < female), and adrenal HSP90 alpha (male > female) was observed. These findings provide new insights on TSD and suggest that further studies examining the role of HSPs during gonadal development are needed. (c) 2009 S. Karger AG, Basel.

Quantifying Wheat Sensitivities to Environmental Constraints to Dissect Genotype × Environment Interactions in the Field.

PubMed

Parent, Boris; Bonneau, Julien; Maphosa, Lance; Kovalchuk, Alex; Langridge, Peter; Fleury, Delphine

2017-07-01

Yield is subject to strong genotype-by-environment (G × E) interactions in the field, especially under abiotic constraints such as soil water deficit (drought [D]) and high temperature (heat [H]). Since environmental conditions show strong fluctuations during the whole crop cycle, geneticists usually do not consider environmental measures as quantitative variables but rather as factors in multienvironment analyses. Based on 11 experiments in a field platform with contrasting temperature and soil water deficit, we determined the periods of sensitivity to drought and heat constraints in wheat ( Triticum aestivum ) and determined the average sensitivities for major yield components. G × E interactions were separated into their underlying components, constitutive genotypic effect (G), G × D, G × H, and G × H × D, and were analyzed for two genotypes, highlighting contrasting responses to heat and drought constraints. We then tested the constitutive and responsive behaviors of two strong quantitative trait loci (QTLs) associated previously with yield components. This analysis confirmed the constitutive effect of the chromosome 1B QTL and explained the G × E interaction of the chromosome 3B QTL by a benefit of one allele when temperature rises. In addition to the method itself, which can be applied to other data sets and populations, this study will support the cloning of a major yield QTL on chromosome 3B that is highly dependent on environmental conditions and for which the climatic interaction is now quantified. © 2017 American Society of Plant Biologists. All Rights Reserved.

A cell clone strain from Mythimna separata (Lepidoptera: Noctuidae) highly susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata NPV (MsNPV).

PubMed

Meng, Xiang-Qian; Zheng, Gui-Ling; Zhao, Chuan-De; Wan, Fang-Hao; Li, Chang-You

2017-08-01

In this study, we describe a cell line, Ms-10C, cloned from the line QAU-Ms-E-10 (simplified Ms-10), an embryonic line from Mythimna separata. The cloned cell line was significantly more sensitive to nucleopolyhedrovirus (NPV). Ms-10C cells were mainly spherical with a diameter of 14.42 ± 2.23 μm. DNA amplification fingerprinting (DAF) confirmed the profile of PCR-amplified bands of the cloned cell line was consistent with those of the parental cell line, Ms-10. The sequencing result of the mitochondrial cytochrome c oxidase I (mtCO I) fragment confirmed that the amplified 636-bps mtCOI fragment was 100% identical to that of M. separata. Its chromosomes exhibited the typical characters of lepidopteran cell lines. Its population doubling time was 42.2 h at 27°C. Ms-10C was more sensitive than Ms-10 to both Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata nucleopolyhedrovirus (MsNPV). At 4 d post infection, the infection rates of two viruses reached 94.2 and 92.3%, respectively. The availability of this cell clone strain will provide a useful tool for the basic research on nucleopolyhedrovirus and for potential application in expression of recombinant proteins with baculovirus expression vector system.

Ozone sensitivity in hybrid poplar correlates with insensitivity to both salicylic acid and jasmonic acid. The role of programmed cell death in lesion formation.

PubMed

Koch, J R; Creelman, R A; Eshita, S M; Seskar, M; Mullet, J E; Davis, K R

2000-06-01

Our earlier studies demonstrated that the ozone-sensitive hybrid poplar clone NE-388 displays an attenuated level of ozone-, wound-, and phytopathogen-induced defense gene expression. To determine if this reduced gene activation involves signal transduction pathways dependent on salicylic acid (SA) and/or jasmonic acid (JA), we compared the responses of NE-388 and an ozone-tolerant clone, NE-245, to these signal molecules. JA levels increased in both clones in response to ozone, but only minimal increases in SA levels were measured for either clone. Treatment with SA and methyl jasmonate induced defense gene expression only in NE-245, indicating that NE-388 is insensitive to these signal molecules. DNA fragmentation, an indicator of programmed cell death (PCD), was detected in NE-245 treated with either ozone or an avirulent phytopathogen, but was not detected in NE-388. We conclude that these clones undergo two distinct mechanisms of ozone-induced lesion formation. In NE-388, lesions appear to be due to toxic cell death resulting from a limited ability to perceive and subsequently activate SA- and/or JA-mediated antioxidant defense responses. In NE-245, SA-dependent PCD precedes lesion formation via a process related to the PCD pathway activated by phytopathogenic bacteria. These results support the hypothesis that ozone triggers a hypersensitive response.

Ozone Sensitivity in Hybrid Poplar Correlates with Insensitivity to Both Salicylic Acid and Jasmonic Acid. The Role of Programmed Cell Death in Lesion Formation1

PubMed Central

Koch, Jennifer Riehl; Creelman, Robert A.; Eshita, Steven M.; Seskar, Mirjana; Mullet, John E.; Davis, Keith R.

2000-01-01

Our earlier studies demonstrated that the ozone-sensitive hybrid poplar clone NE-388 displays an attenuated level of ozone-, wound-, and phytopathogen-induced defense gene expression. To determine if this reduced gene activation involves signal transduction pathways dependent on salicylic acid (SA) and/or jasmonic acid (JA), we compared the responses of NE-388 and an ozone-tolerant clone, NE-245, to these signal molecules. JA levels increased in both clones in response to ozone, but only minimal increases in SA levels were measured for either clone. Treatment with SA and methyl jasmonate induced defense gene expression only in NE-245, indicating that NE-388 is insensitive to these signal molecules. DNA fragmentation, an indicator of programmed cell death (PCD), was detected in NE-245 treated with either ozone or an avirulent phytopathogen, but was not detected in NE-388. We conclude that these clones undergo two distinct mechanisms of ozone-induced lesion formation. In NE-388, lesions appear to be due to toxic cell death resulting from a limited ability to perceive and subsequently activate SA- and/or JA-mediated antioxidant defense responses. In NE-245, SA-dependent PCD precedes lesion formation via a process related to the PCD pathway activated by phytopathogenic bacteria. These results support the hypothesis that ozone triggers a hypersensitive response. PMID:10859179

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

Comparison of the X-radiation, drug and ultraviolet-radiation responses of clones isolated from a human colorectal tumor cell line.

PubMed

Qutob, Sami S; Multani, Asha S; Pathak, S; Feng, Y; Kendal, Wayne S; Ng, Cheng E

2004-03-01

We isolated several clones with a wide range of responses to X radiation from an unirradiated human colorectal (HCT 116) tumor cell line. The responses of one of these clones (HCT116-Clone10) and nine other clones to either fractionated or acute (i.e. single, nonfractionated doses) X irradiation in vitro was similar to that of the parental cell line. By contrast, after the same types of treatment, another clone (HCT116-Clone2) manifested a significantly increased survival whereas a third clone (HCT116-CloneK) manifested a significantly decreased survival relative to the parental cell line. This suggested that they were, respectively, a radioresistant and a radiosensitive clone. All three clones (clones 2, 10, K) retained their tumorigenic phenotype and formed tumors in nude mice. G-banding studies demonstrated that they were of human origin and were derived from the same parental cell line. The metaphases of HCT116-Clone2 demonstrated features commonly associated with genomic instability (i.e. mitotic catastrophe including chromosome and chromatid breaks, dicentrics and additional nonclonal markers). Data obtained by quantitative fluorescence in situ hybridization (Q- FISH) analysis failed to demonstrate any apparent correlation between the radiosensitivity and the relative telomere content of these three clones. Interestingly, HCT116-CloneK was the most resistant to several chemotherapeutic drugs (topotecan, camptothecin, etoposide and cisplatin) with diverse mechanisms of action. Also, there were no significant differences in the survivals of the three clones after treatment with UV radiation. Because of the lack of overlap among the relative sensitivities of these clones to X radiation, chemotherapeutic drugs and UV radiation, these clones may be useful models for evaluating the genetic basis of the response of human tumor cells to these treatment agents both in vitro and in vivo.

Temporal and intraclonal variation of flowering and pseudovivipary in Poa bulbosa

PubMed Central

Ofir, Micha; Kigel, Jaime

2014-01-01

Background and Aims Versatility in the reproductive development of pseudoviviparous grasses in response to growth conditions is an intriguing reproduction strategy. To better understand this strategy, this study examined variation in flowering and pseudovivipary among populations, co-occurring clones within populations, and among tillers in individual clones of Poa bulbosa, a summer-dormant geophytic grass that reproduces sexually by seed, and asexually by basal tiller bulbs and bulbils formed in proliferated panicles. Methods Clones were collected from 17 populations across a rainfall gradient. Patterns of reproduction were monitored for 11 years in a common garden experiment and related to interannual differences in climatic conditions. Intraclonal variation in flowering and pseudovivipary was studied in a phytotron, under daylengths marginal for flowering induction. Key Results Clones showed large temporal variability in their reproductive behaviour. They flowered in some years but not in others, produced normal or proliferated panicles in different years, or became dormant without flowering. Proliferating clones did not show a distinct time sequence of flowering and proliferation across years. Populations differed in incidence of flowering and proliferation. The proportion of flowering clones increased with decreasing rainfall at the site of population origin, but no consistent relationship was found between flowering and precipitation in the common garden experiment across years. In contrast, flowering decreased at higher temperatures during early growth stages after bulb sprouting. Pulses of soil fertilization greatly increased the proportion of flowering clones and panicle production. High intraclonal tiller heterogeneity was observed, as shown by the divergent developmental fates of daughter plants arising from bulbs from the same parent clone and grown under similar conditions. Panicle proliferation was enhanced by non-inductive 8 h short days, while marginally inductive 12 h days promoted normal panicles. Conclusions Interannual variation in flowering and proliferation in P. bulbosa clones was attributed to differences in the onset of the rainy season, resulting in different daylength and temperature conditions during the early stages of growth, during which induction of flowering and dormancy occurs. PMID:24685715

Overexpression of Three Heat Shock Proteins Protects Monochamus alternatus (Coleoptera: Cerambycidae) From Thermal Stress

PubMed Central

Cai, Ziling; Chen, Jingxiang; Cheng, Jie

2017-01-01

Abstract Ambient temperature is an important factor limiting the abundance and distribution of insects, and heat shock protein (Hsp) gene expression is sensitive to extremes of cold and heat. In order to explore the role of Hsps during thermal stress and development in Monochamus alternatus Hope (Coleoptera: Cerambycidae), we cloned and characterized full-length Hsp genes, including MaHsp60, MaHsp70, and MaHsp90. M. alternatus were exposed to different temperatures (−15, −5, 5, 15, 25, 35, and 40℃) for 1 h and was allowed to recover at 25℃ for 1 h. Following the treatments, we investigated the expression of the Hsps by quantitative real-time polymerase chain reaction. In third instar larvae, MaHsp60, MaHsp70, and MaHsp90 expression was upregulated in response to cold and heat, but the three Hsps were especially sensitive to heat, specifically at 35℃ and 40℃. After heating M. alternatus to 35℃, the expression of MaHsp60, MaHsp70, and MaHsp90 was higher than at 5℃ and 25℃ in nearly all developmental stages. MaHsp60, MaHsp70, and MaHsp90 expression was highest in later pupal, early adult, and early adult stages, respectively. These results suggest that compared with normal ambient temperatures, thermal stress could induce high expression of the three Hsps.

Sensitive and substrate-specific detection of metabolically active microorganisms in natural microbial consortia using community isotope arrays.

PubMed

Tourlousse, Dieter M; Kurisu, Futoshi; Tobino, Tomohiro; Furumai, Hiroaki

2013-05-01

The goal of this study was to develop and validate a novel fosmid-clone-based metagenome isotope array approach - termed the community isotope array (CIArray) - for sensitive detection and identification of microorganisms assimilating a radiolabeled substrate within complex microbial communities. More specifically, a sample-specific CIArray was used to identify anoxic phenol-degrading microorganisms in activated sludge treating synthetic coke-oven wastewater in a single-sludge predenitrification-nitrification process. Hybridization of the CIArray with DNA from the (14) C-phenol-amended sample indicated that bacteria assimilating (14) C-atoms, presumably directly from phenol, under nitrate-reducing conditions were abundant in the reactor, and taxonomic assignment of the fosmid clone end sequences suggested that they belonged to the Gammaproteobacteria. The specificity of the CIArray was validated by quantification of fosmid-clone-specific DNA in density-resolved DNA fractions from samples incubated with (13) C-phenol, which verified that all CIArray-positive probes stemmed from microorganisms that assimilated isotopically labeled carbon. This also demonstrated that the CIArray was more sensitive than DNA-SIP, as the former enabled positive detection at a phenol concentration that failed to yield a 'heavy' DNA fraction. Finally, two operational taxonomic units distantly related to marine Gammaproteobacteria were identified to account for more than half of 16S rRNA gene clones in the 'heavy' DNA library, corroborating the CIArray-based identification. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy.

PubMed

Gritsun, T S; Gould, E A

1998-12-01

In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 microl of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3' antisense primer concentration was essential to achieve PCR synthesis of an 11 kb cDNA copy of RNA from infectious virus. A novel system utilising two antisense primers, a 14-mer for reverse transcription and a 35-mer for long PCR, produced high yields of genomic length cDNA. Use of low copy number Able K cells and an incubation temperature of 28 degrees C increased the genetic stability of cloned cDNA. Clones containing 11 kb cDNA inserts produced colonies of reduced size, thus providing a positive selection system for full length clones. Sequencing of the infectious clone emphasised the improved fidelity of the method compared with conventional PCR and cloning methods. A simple and rapid strategy for genetic manipulation of the infectious clone is also described. These developments represent a significant advance in recombinant technology and should be applicable to positive stranded RNA viruses which cannot easily be purified or genetically manipulated.

Paroxysmal nocturnal hemoglobinuria clones in severe aplastic anemia patients treated with horse anti-thymocyte globulin plus cyclosporine

PubMed Central

Scheinberg, Phillip; Marte, Michael; Nunez, Olga; Young, Neal S.

2010-01-01

Background Clones of glycosylphosphatidylinositol-anchor protein-deficient cells are characteristic in paroxysmal nocturnal hemoglobinuria and are present in about 40–50% of patients with severe aplastic anemia. Flow cytometry has allowed for sensitive and precise measurement of glycosylphosphatidylinositol-anchor protein-deficient red blood cells and neutrophils in severe aplastic anemia. Design and Methods We conducted a retrospective analysis of paroxysmal nocturnal hemoglobinuria clones measured by flow cytometry in 207 consecutive severe aplastic anemia patients who received immunosuppressive therapy with a horse anti-thymocyte globulin plus cyclosporine regimen from 2000 to 2008. Results The presence of a glycosylphosphatidylinositol-anchor protein-deficient clone was detected in 83 (40%) patients pre-treatment, and the median clone size was 9.7% (interquartile range 3.5–29). In patients without a detectable clone pre-treatment, the appearance of a clone after immunosuppressive therapy was infrequent, and in most with a clone pre-treatment, clone size often decreased after immunosuppressive therapy. However, in 30 patients, an increase in clone size was observed after immunosuppressive therapy. The majority of patients with a paroxysmal nocturnal hemoglobinuria clone detected after immunosuppressive therapy did not have an elevated lactate dehydrogenase, nor did they experience hemolysis or thrombosis, and they did not require specific interventions with anticoagulation and/or eculizumab. Of the 7 patients who did require therapy for clinical paroxysmal nocturnal hemoglobinuria symptoms and signs, all had an elevated lactate dehydrogenase and a clone size greater than 50%. In all, 18 (8.6%) patients had a clone greater than 50% at any given time of sampling. Conclusions The presence of a paroxysmal nocturnal hemoglobinuria clone in severe aplastic anemia is associated with low morbidity and mortality, and specific measures to address clinical paroxysmal nocturnal hemoglobinuria are seldom required. PMID:20595102

Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response.

PubMed

Reichstetter, S; Ettinger, R A; Liu, A W; Gebe, J A; Nepom, G T; Kwok, W W

2000-12-15

The polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16(369-379)-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16(369-379) peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16(369-379) tetramer at either 23 or 37 degrees C. Weak staining was also observed at 4 degrees C. Clone 5 showed weaker staining compared with clone 48 at 37 degrees C, and no staining was observed at 23 degrees C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16(369-379) complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.

Why Izzie Didn't Go to College: Choosing Work over College as Latina Feminism

ERIC Educational Resources Information Center

Harklau, Linda

2013-01-01

Background/Context: Explanations for the relatively low numbers of Latinas pursuing higher education have tended to focus on socialization into traditional gender roles. However, recent scholarship has challenged this view, suggesting that gender roles--particularly among recent immigrants--are mutable and subject to constant renegotiation.…

Digital Photography and Its Impact on Instruction.

ERIC Educational Resources Information Center

Lantz, Chris

Today the chemical processing of film is being replaced by a virtual digital darkroom. Digital image storage makes new levels of consistency possible because its nature is less volatile and more mutable than traditional photography. The potential of digital imaging is great, but issues of disk storage, computer speed, camera sensor resolution,…

Social Class as Flow and Mutability: The Barbados Case

ERIC Educational Resources Information Center

Greenhalgh-Spencer, Heather; Castro, Michelle; Bulut, Ergin; Goel, Koeli; Lin, Chunfeng; McCarthy, Cameron

2015-01-01

This article draws on ethnographic research that examines the contemporary articulation of class identity in the postcolonial elite school setting of Old College high school in Barbados. From the qualitative data derived from this study, we argue that social class is better conceived as a series of flows, mutations, performances and performatives.…

Mutable Mobiles: Online Journals and the Evolving Genre Ecosystem of Science

ERIC Educational Resources Information Center

Casper, Christian Fredrick

2009-01-01

This dissertation addresses the related questions of how online communication technologies affect communication in science and, more broadly, how new ways of interaction in online spaces affect how texts enact genres. Genres have been usefully thought of as typified discursive responses to recurrent social exigences, and much recent work has shown…

Optimal immunization cocktails can promote induction of broadly neutralizing Abs against highly mutable pathogens.

PubMed

Shaffer, J Scott; Moore, Penny L; Kardar, Mehran; Chakraborty, Arup K

2016-10-24

Strategies to elicit Abs that can neutralize diverse strains of a highly mutable pathogen are likely to result in a potent vaccine. Broadly neutralizing Abs (bnAbs) against HIV have been isolated from patients, proving that the human immune system can evolve them. Using computer simulations and theory, we study immunization with diverse mixtures of variant antigens (Ags). Our results show that particular choices for the number of variant Ags and the mutational distances separating them maximize the probability of inducing bnAbs. The variant Ags represent potentially conflicting selection forces that can frustrate the Darwinian evolutionary process of affinity maturation. An intermediate level of frustration maximizes the chance of evolving bnAbs. A simple model makes vivid the origin of this principle of optimal frustration. Our results, combined with past studies, suggest that an appropriately chosen permutation of immunization with an optimally designed mixture (using the principles that we describe) and sequential immunization with variant Ags that are separated by relatively large mutational distances may best promote the evolution of bnAbs.

Diversity and Community Can Coexist.

PubMed

Stivala, Alex; Robins, Garry; Kashima, Yoshihisa; Kirley, Michael

2016-03-01

We examine the (in)compatibility of diversity and sense of community by means of agent-based models based on the well-known Schelling model of residential segregation and Axelrod model of cultural dissemination. We find that diversity and highly clustered social networks, on the assumptions of social tie formation based on spatial proximity and homophily, are incompatible when agent features are immutable, and this holds even for multiple independent features. We include both mutable and immutable features into a model that integrates Schelling and Axelrod models, and we find that even for multiple independent features, diversity and highly clustered social networks can be incompatible on the assumptions of social tie formation based on spatial proximity and homophily. However, this incompatibility breaks down when cultural diversity can be sufficiently large, at which point diversity and clustering need not be negatively correlated. This implies that segregation based on immutable characteristics such as race can possibly be overcome by sufficient similarity on mutable characteristics based on culture, which are subject to a process of social influence, provided a sufficiently large "scope of cultural possibilities" exists. © Society for Community Research and Action 2016.

Long-term effects of inducible mutagenic DNA repair on relative fitness and phenotypic diversification in Pseudomonas cichorii 302959.

PubMed

Weigand, Michael R; Sundin, George W

2009-01-01

Mutagenic DNA repair (MDR) employs low-fidelity DNA polymerases capable of replicating past DNA lesions resulting from exposure to high-energy ultraviolet radiation (UVR). MDR confers UVR tolerance and activation initiates a transient mutator phenotype that may provide opportunities for adaptation. To investigate the potential role of MDR in adaptation, we have propagated parallel lineages of the highly mutable epiphytic plant pathogen Pseudomonas cichorii 302959 with daily UVR activation (UVR lineages) for approximately 500 generations. Here we examine those lineages through the measurement of relative fitness and observation of distinct colony morphotypes that emerged. Isolates and population samples from UVR lineages displayed gains in fitness relative to the ancestor despite increased rates of inducible mutation to rifampicin resistance. Regular activation of MDR resulted in the maintenance of genetic diversity within UVR lineages, including the reproducible diversification and coexistence of "round" and "fuzzy" colony morphotypes. These results suggest that inducible mutability may present a reasonable strategy for adaptive evolution in stressful environments by contributing to gains in relative fitness and diversification.

Optimal immunization cocktails can promote induction of broadly neutralizing Abs against highly mutable pathogens

PubMed Central

Shaffer, J. Scott; Moore, Penny L.; Kardar, Mehran; Chakraborty, Arup K.

2016-01-01

Strategies to elicit Abs that can neutralize diverse strains of a highly mutable pathogen are likely to result in a potent vaccine. Broadly neutralizing Abs (bnAbs) against HIV have been isolated from patients, proving that the human immune system can evolve them. Using computer simulations and theory, we study immunization with diverse mixtures of variant antigens (Ags). Our results show that particular choices for the number of variant Ags and the mutational distances separating them maximize the probability of inducing bnAbs. The variant Ags represent potentially conflicting selection forces that can frustrate the Darwinian evolutionary process of affinity maturation. An intermediate level of frustration maximizes the chance of evolving bnAbs. A simple model makes vivid the origin of this principle of optimal frustration. Our results, combined with past studies, suggest that an appropriately chosen permutation of immunization with an optimally designed mixture (using the principles that we describe) and sequential immunization with variant Ags that are separated by relatively large mutational distances may best promote the evolution of bnAbs. PMID:27791170

An Objectivist Critique of Relativism in Mathematics Education

NASA Astrophysics Data System (ADS)

Rowlands, Stuart; Graham, Ted; Berry, John

Many constructivists tag as `absolutist' references to mathematics as an abstract body of knowledge, and stake-out the moral high-ground with the argument that mathematics is not only utilised oppressively but that mathematics is, in-itself, oppressive. With much reference to Ernest's (1991) Philosophy of Mathematics Education this tag has been justified on the grounds that if mathematics is a social-cultural creation that is mutable and fallible then it must be social acceptance that confers the objectivity of mathematics. This paper argues that mathematics, albeit a social-cultural creation that is mutable and fallible, is a body of knowledge the objectivity of which is independent of origin or social acceptance. Recently, Ernest (1998) has attempted to express social constructivism as a philosophy of mathematics and has included the category of logical necessity in his elaboration of the objectivity of mathematics. We argue that this inclusion of logical necessity not only represents a U-turn, but that the way in which Ernest has included this category is an attempt to maintain his earlier position that it is social acceptance that confers the objectivity of mathematics.

News from the protein mutability landscape.

PubMed

Hecht, Maximilian; Bromberg, Yana; Rost, Burkhard

2013-11-01

Some mutations of protein residues matter more than others, and these are often conserved evolutionarily. The explosion of deep sequencing and genotyping increasingly requires the distinction between effect and neutral variants. The simplest approach predicts all mutations of conserved residues to have an effect; however, this works poorly, at best. Many computational tools that are optimized to predict the impact of point mutations provide more detail. Here, we expand the perspective from the view of single variants to the level of sketching the entire mutability landscape. This landscape is defined by the impact of substituting every residue at each position in a protein by each of the 19 non-native amino acids. We review some of the powerful conclusions about protein function, stability and their robustness to mutation that can be drawn from such an analysis. Large-scale experimental and computational mutagenesis experiments are increasingly furthering our understanding of protein function and of the genotype-phenotype associations. We also discuss how these can be used to improve predictions of protein function and pathogenicity of missense variants. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Adaptive phenotypic plasticity and local adaptation for temperature tolerance in freshwater zooplankton

PubMed Central

Yampolsky, Lev Y.; Schaer, Tobias M. M.; Ebert, Dieter

2014-01-01

Many organisms have geographical distributions extending from the tropics to near polar regions or can experience up to 30°C temperature variation within the lifespan of an individual. Two forms of evolutionary adaptation to such wide ranges in ambient temperatures are frequently discussed: local adaptation and phenotypic plasticity. The freshwater planktonic crustacean Daphnia magna, whose range extends from South Africa to near arctic sites, shows strong phenotypic and genotypic variation in response to temperature. In this study, we use D. magna clones from 22 populations (one clone per population) ranging from latitude 0° (Kenya) to 66° North (White Sea) to explore the contributions of phenotypic plasticity and local adaptation to high temperature tolerance. Temperature tolerance was studied as knockout time (time until immobilization, Timm) at 37°C in clones acclimatized to either 20°C or 28°C. Acclimatization to 28°C strongly increased Timm, testifying to adaptive phenotypic plasticity. At the same time, Timm significantly correlated with average high temperature at the clones’ sites of origin, suggesting local adaptation. As earlier studies have found that haemoglobin expression contributes to temperature tolerance, we also quantified haemoglobin concentration in experimental animals and found that both acclimatization temperature (AccT) and temperature at the site of origin are positively correlated with haemoglobin concentration. Furthermore, Daphnia from warmer climates upregulate haemoglobin much more strongly in response to AccT, suggesting local adaptation for plasticity in haemoglobin expression. Our results show that both local adaptation and phenotypic plasticity contribute to temperature tolerance, and elucidate a possible role of haemoglobin in mediating these effects that differs along a cold–warm gradient. PMID:24352948

Microsatellite Interruptions Stabilize Primate Genomes and Exist as Population-Specific Single Nucleotide Polymorphisms within Individual Human Genomes

PubMed Central

Ananda, Guruprasad; Hile, Suzanne E.; Breski, Amanda; Wang, Yanli; Kelkar, Yogeshwar; Makova, Kateryna D.; Eckert, Kristin A.

2014-01-01

Interruptions of microsatellite sequences impact genome evolution and can alter disease manifestation. However, human polymorphism levels at interrupted microsatellites (iMSs) are not known at a genome-wide scale, and the pathways for gaining interruptions are poorly understood. Using the 1000 Genomes Phase-1 variant call set, we interrogated mono-, di-, tri-, and tetranucleotide repeats up to 10 units in length. We detected ∼26,000–40,000 iMSs within each of four human population groups (African, European, East Asian, and American). We identified population-specific iMSs within exonic regions, and discovered that known disease-associated iMSs contain alleles present at differing frequencies among the populations. By analyzing longer microsatellites in primate genomes, we demonstrate that single interruptions result in a genome-wide average two- to six-fold reduction in microsatellite mutability, as compared with perfect microsatellites. Centrally located interruptions lowered mutability dramatically, by two to three orders of magnitude. Using a biochemical approach, we tested directly whether the mutability of a specific iMS is lower because of decreased DNA polymerase strand slippage errors. Modeling the adenomatous polyposis coli tumor suppressor gene sequence, we observed that a single base substitution interruption reduced strand slippage error rates five- to 50-fold, relative to a perfect repeat, during synthesis by DNA polymerases α, β, or η. Computationally, we demonstrate that iMSs arise primarily by base substitution mutations within individual human genomes. Our biochemical survey of human DNA polymerase α, β, δ, κ, and η error rates within certain microsatellites suggests that interruptions are created most frequently by low fidelity polymerases. Our combined computational and biochemical results demonstrate that iMSs are abundant in human genomes and are sources of population-specific genetic variation that may affect genome stability. The genome-wide identification of iMSs in human populations presented here has important implications for current models describing the impact of microsatellite polymorphisms on gene expression. PMID:25033203

The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein.

PubMed

Lim, Hyoun-Sub; Nam, Jiryun; Seo, Eun-Young; Nam, Moon; Vaira, Anna Maria; Bae, Hanhong; Jang, Chan-Yong; Lee, Cheol Ho; Kim, Hong Gi; Roh, Mark; Hammond, John

2014-03-01

Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CPSP) with that from AltMV-Po (CP(Po)) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP(Po) [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CPSP but not CP(Po) interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CPSP than CP(Po) in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN. Published by Elsevier Inc.

[Cloning of cDNA for RNA polymerase subunit from the fission yeast Schizosaccharomyces pombe by heterospecific complementation in Saccharomyces cerevisiae].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N; Thuriaux, P

1997-02-01

The rpb10 cDNA of the fission yeast Schizosaccharomyces pombe, encoding one of the five small subunits common to all three nuclear DNA-dependent RNA polymerases, was isolated from an expression cDNA library by two independent approaches: PCR-based screening and direct suppression by means of heterospecific complementation of a temperature-sensitive mutant defective in the corresponding gene of Saccharomyces cerevisiae. The cloned Sz. pombe cDNA encodes a protein Rpb10 of 71 amino acids with an M of 8,275 Da, sharing 51 amino acids (71% identity) with the subunit ABC10 beta of RNA polymerases I-III from S. cerevisiae. All eukaryotic members of this protein family have the same general organization featuring two highly conserved motifs (RCFT/SCGK and RYCCRRM) around an atypical zinc finger and an additional invariant HVDLIEK motif toward the C-terminal end. The last motif is only characteristics for homologs from eukaryotes. In keeping with this remarkable structural conservation, the Sz. pombe cDNA also fully complemented a S. cerevisiae deletion mutant lacking subunit ABC10 beta (null allele rpb10-delta 1::HIS3).

Defense Against Chip Cloning Attacks Based on Fractional Hopfield Neural Networks.

PubMed

Pu, Yi-Fei; Yi, Zhang; Zhou, Ji-Liu

2017-06-01

This paper presents a state-of-the-art application of fractional hopfield neural networks (FHNNs) to defend against chip cloning attacks, and provides insight into the reason that the proposed method is superior to physically unclonable functions (PUFs). In the past decade, PUFs have been evolving as one of the best types of hardware security. However, the development of the PUFs has been somewhat limited by its implementation cost, its temperature variation effect, its electromagnetic interference effect, the amount of entropy in it, etc. Therefore, it is imperative to discover, through promising mathematical methods and physical modules, some novel mechanisms to overcome the aforementioned weaknesses of the PUFs. Motivated by this need, in this paper, we propose applying the FHNNs to defend against chip cloning attacks. At first, we implement the arbitrary-order fractor of a FHNN. Secondly, we describe the implementation cost of the FHNNs. Thirdly, we propose the achievement of the constant-order performance of a FHNN when ambient temperature varies. Fourthly, we analyze the electrical performance stability of the FHNNs under electromagnetic disturbance conditions. Fifthly, we study the amount of entropy of the FHNNs. Lastly, we perform experiments to analyze the pass-band width of the fractor of an arbitrary-order FHNN and the defense against chip cloning attacks capability of the FHNNs. In particular, the capabilities of defense against chip cloning attacks, anti-electromagnetic interference, and anti-temperature variation of a FHNN are illustrated experimentally in detail. Some significant advantages of the FHNNs are that their implementation cost is considerably lower than that of the PUFs, their electrical performance is much more stable than that of the PUFs under different temperature conditions, their electrical performance stability of the FHNNs under electromagnetic disturbance conditions is much more robust than that of the PUFs, and their amount of entropy is significantly higher than that of the PUFs with the same rank circuit scale.

A Diverse Panel of Hepatitis C Virus Glycoproteins for Use in Vaccine Research Reveals Extremes of Monoclonal Antibody Neutralization Resistance.

PubMed

Urbanowicz, Richard A; McClure, C Patrick; Brown, Richard J P; Tsoleridis, Theocharis; Persson, Mats A A; Krey, Thomas; Irving, William L; Ball, Jonathan K; Tarr, Alexander W

2015-12-23

Despite significant advances in the treatment of hepatitis C virus (HCV) infection, the need to develop preventative vaccines remains. Identification of the best vaccine candidates and evaluation of their performance in preclinical and clinical development will require appropriate neutralization assays utilizing diverse HCV isolates. We aimed to generate and characterize a panel of HCV E1E2 glycoproteins suitable for subsequent use in vaccine and therapeutic antibody testing. Full-length E1E2 clones were PCR amplified from patient-derived serum samples, cloned into an expression vector, and used to generate viral pseudoparticles (HCVpp). In addition, some of these clones were used to generate cell culture infectious (HCVcc) clones. The infectivity and neutralization sensitivity of these viruses were then determined. Bioinformatic and HCVpp infectivity screening of approximately 900 E1E2 clones resulted in the assembly of a panel of 78 functional E1E2 proteins representing distinct HCV genotypes and different stages of infection. These HCV glycoproteins differed markedly in their sensitivity to neutralizing antibodies. We used this panel to predict antibody efficacy against circulating HCV strains, highlighting the likely reason why some monoclonal antibodies failed in previous clinical trials. This study provides the first objective categorization of cross-genotype patient-derived HCV E1E2 clones according to their sensitivity to antibody neutralization. It has shown that HCV isolates have clearly distinguishable neutralization-sensitive, -resistant, or -intermediate phenotypes, which are independent of genotype. The panel provides a systematic means for characterization of the neutralizing response elicited by candidate vaccines and for defining the therapeutic potential of monoclonal antibodies. Hepatitis C virus (HCV) has a global burden of more than 170 million people, many of whom cannot attain the new, expensive, direct-acting antiviral therapies. A safe and effective vaccine that generates both T cell responses and neutralizing antibodies is required to eradicate the disease. Regions within the HCV surface glycoproteins E1 and E2 are essential for virus entry and are targets for neutralizing antibodies. Screening of vaccine candidates requires suitable panels of glycoproteins that represent the breadth of neutralization resistance. Use of a standard reference panel for vaccine studies will ensure comparability of data sets, as has become routine for HIV-1. Here, we describe a large panel of patient-derived HCV glycoproteins with an assessment of their neutralization sensitivity to defined monoclonal antibodies, which has enabled us to predict their likely efficacy in the wider HCV-infected population. The panel could also be important for future selection of additional therapeutic antibodies and for vaccine design. Copyright © 2016 Urbanowicz et al.

Cloning and sequencing of Staphylococcus aureus murC, a gene essential for cell wall biosynthesis.

PubMed

Lowe, A M; Deresiewicz, R L

1999-01-01

Staphylococcus aureus is a major human pathogen that is increasingly resistant to clinically useful antimicrobial agents. While screening for S. aureus genes expressed during mammalian infection, we isolated murC. This gene encodes UDP-N-acetylmuramoyl-L-alanine synthetase, an enzyme essential for cell wall biosynthesis in a number of bacteria. S. aureus MurC has a predicted mass 49,182 Da and complements the temperature-sensitive murC mutation of E. coli ST222. Sequence data on the DNA flanking staphylococcal murC suggests that the local gene organization there parallels that found in B. subtilis, but differs from that found in gram-negative bacterial pathogens. MurC proteins represent promising targets for broad spectrum antimicrobial drug development.

Genetic aftereffects of increased temperature in Larix

Treesearch

Michael S. Greenwood; Keith W. Hutchinson

1996-01-01

We tested the hypothesis that temperature during gametogenesis and embryogenesis can affect progeny genotype and phenotype. Identical crosses were made among cloned parents of Larix spp. inside and outside a greenhouse, where the temperature inside averaged 4?C above the outside temperature. Significant growth differences as a function of crossing...

Isolation of uv-sensitive variants of human FL cells by a viral suicide method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiomi, T.; Sato, K.

A new method (viral suicide method) for the isolation of uv-sensitive mutants is described. Colonies of mutagenized human FL cells were infected with uv-irradiated Herpes simplex viruses and surviving ones which seemed to be deficient in host cell reactivation (HCR) were examined for their uv sensitivity. Nineteen of 238 clones examined were sensitive to uv irradiation at the time of the isolation. After recloning, four of these clones have been studied and two (UVS-1 and UVS-2) of them are stable in their uv sensitivity for 4 months in culture. uv sensitivity of UVS-1, UVS-2, and the parental FL cells aremore » as follows: the extrapolation numbers (n) are 2.2, 2.1, and 1.8 and mean lethal doses (DO) are 2.9, 3.7, and 7.8 J/m/sup 2/ for UVS-1, UVS-2, and the parental FL cells, respectively. They are no more sensitive than FL cells to x-irradiation. The ability of HCR in UVS-2 cells is apparently lower than that in FL cells, whereas UVS-1 cells are the same as FL cells in the ability.« less

Further insights into the components of resistance to Ophiostoma novo-ulmi in Ulmus minor: hydraulic conductance, stomatal sensitivity and bark dehydration.

PubMed

Pita, Pilar; Rodríguez-Calcerrada, Jesús; Medel, David; Gil, Luis

2018-02-01

Dutch elm disease (DED) is a vascular disease that has killed over 1 billion elm trees. The pathogen spreads throughout the xylem network triggering vessel blockage, which results in water stress, tissue dehydration and extensive leaf wilting in susceptible genotypes. We investigated the differences between four Ulmus minor Mill. clones of contrasting susceptibility to Ophiostoma novo-ulmi Brasier regarding morphological, anatomical and physiological traits affecting water transport, in order to gain a better understanding of the mechanisms underlying DED susceptibility. We analyzed the differential response to water shortage and increased air vapor pressure deficit (VPD) to investigate whether resistance to water stress might be related to DED tolerance. Sixteen plants per clone, aged 2 years, were grown inside a greenhouse under differential watering. Stomatal conductance was measured under ambient and increased VPD. Growth, bark water content and stem hydraulic and anatomical parameters were measured 22 days after starting differential watering. Vessel lumen area, lumen fraction and hydraulic conductance were highest in susceptible clones. Stomatal conductance was lowest under low VPD and decreased faster under increased VPD in resistant clones. We found a negative relationship between the decrease in stomatal conductance at increased VPD and specific hydraulic conductance, revealing a narrower hydraulic margin for sustaining transpiration in resistant clones. The effect of water shortage was greater on radial stem growth than on leaf area, which could be explained through an extensive use of capacitance water to buffer xylem water potential. Water shortage reduced stomatal conductance and vessel lumen area. Bark water content under conditions of water shortage only decreased in susceptible clones. Higher hydraulic constraints to sap flow in resistant clones may determine higher stomatal sensitivity to VPD and so contribute to DED resistance by limiting pathogen expansion and reducing water loss and metabolic impairment in cells involved in fighting against infection. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Long-term shifts in the cyclicity of outbreaks of a forest-defoliating insect

Treesearch

Andrew J. ​Allstadt; Kyle J. Haynes; Andrew M. Liebhold; Derek M. Johnson

2013-01-01

Recent collapses of population cycles in several species highlight the mutable nature of population behavior as well as the potential role of human-induced environmental change in causing population dynamics to shift. We investigate changes in the cyclicity of gypsy moth (Lymantria dispar) outbreaks by applying wavelet analysis to an 86-year time...

Using Sexually Explicit Material in a Therapeutic Context

ERIC Educational Resources Information Center

Darnell, Cyndi

2015-01-01

For most of us, sex is a subjective, lived experience that is as unique as our genetic make-up, our upbringing, our thoughts, values, feelings, beliefs and ideas. It is through our erotic interactions, or the absence thereof, that we form aspects of our fluid and mutable erotic paths and identities. Despite the proliferation of sexual imagery…

Mutant maize variety containing the glt1-1 allele

DOEpatents

Nelson, Oliver E.; Pan, David

1994-01-01

A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindquist, N.; Hay, M.E.

Marine larvae are consumed by a wide variety of generalist fishes and particle-feeding invertebrates, but larvae of any particular species probably constitute a small and variable portion of the diet for these consumers. Because virtually all consumers can ingest small quantities of noxious compounds with minimal detrimental effects, it is uncertain that toxic chemicals in larvae could be consumed in quantities sufficient to select for predator recognition and avoidance. Despite this, chemically defended larvae do occur. We show that, at low doses, secondary metabolites (the didemnins) from adults and larvae of the Caribbean tunicate Trididenmum solidum induced vomiting in fish,more » resulting in rapid learned aversion to the dedemnin-defended food. The particle-feeding anemone Aiptasia pallida did not learn to avoid the chemically defended food. When anemones ingested the chemical equivalent of 15 larvae/d, representing <2% of the mass of their total daily diet, the didemnins in the {open_quotes}larvae{close_quotes} significantly reduced: (1) growth of adults by 82%, (2) combined growth of adults and daughter clones by 76%, (3) production of daughter clones by 44%, and (4) average mass of individual daughter clones by 41%. At higher water temperatures, anemones cloned more rapidly. Significant differences in the number of daughter clones produced between treatment and control anemones occurred after only 4 d at seawater temperatures of 27{degrees}-29{degrees}C vs. 32 d at seawater temperatures of 18{degrees}-21{degrees}C. Consumption of even very small quantities of secondary metabolites can decrease consumer fitness substantially and select for predators that recognize and avoid chemically defended larvae, as do many consumers that co-occur with Trididemnum solidum larvae. This is the first rigorous demonstration that consumption of marine secondary metabolites can decrease consumer fitness when ingested at ecologically realistic doses. 59 refs., 5 figs., 3 tabs.« less

Genetic Determinism of Sensitivity to Corynespora cassiicola Exudates in Rubber Tree (Hevea brasiliensis).

PubMed

Tran, Dinh Minh; Clément-Demange, André; Déon, Marine; Garcia, Dominique; Le Guen, Vincent; Clément-Vidal, Anne; Soumahoro, Mouman; Masson, Aurélien; Label, Philippe; Le, Mau Tuy; Pujade-Renaud, Valérie

2016-01-01

An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated.

Genetic Determinism of Sensitivity to Corynespora cassiicola Exudates in Rubber Tree (Hevea brasiliensis)

PubMed Central

Tran, Dinh Minh; Clément-Demange, André; Déon, Marine; Garcia, Dominique; Le Guen, Vincent; Clément-Vidal, Anne; Soumahoro, Mouman; Masson, Aurélien; Label, Philippe; Le, Mau Tuy; Pujade-Renaud, Valérie

2016-01-01

An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated. PMID:27736862

cDNA Clones with Rare and Recurrent Mutations Found in Cancers | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at UT- MD Anderson Cancer Center has developed High-Throughput Mutagenesis and Molecular Barcoding (HiTMMoB)1,2 pipeline to construct mutant alleles open reading frame expression clones that are either recurrent or rare in cancers. These barcoded genes can be used for context-specific functional validation, detection of novel biomarkers (pathway activation) and targets (drug sensitivity).

A nuclear mutation defective in mitochondrial recombination in yeast.

PubMed

Ling, F; Makishima, F; Morishima, N; Shibata, T

1995-08-15

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.

[Presence of the double pfmdr1 mutation 86Tyr and 1246 Tyr in clones of a chloroquine-resistant west African isolate of Plasmodium falciparum].

PubMed

Pinheiro, L; Franco, S; Adagu, I S; Rosa, R; Rosário, V E; Warhurst, D C

2003-01-01

Isolates of Plasmodium falciparum from three areas of West Africa were recovered from cryopreservation and their chloroquine-sensitivity were determined in vitro. Of the 90 samples studied, 60 were from Guinea-Bissau (30Resistant/30Sensitive), 15 were from S. Tomé and Príncipe (11Resistant/4Sensitive) and 15 were from Angola (11Resistant/4Sensitive). All the isolates were sensitive to mefloquine. Using the polymerase chain reaction/restriction fragment length polymorphism technique (PCR/RFLP) it was possible to detect two mutations in the pfmdr1 gene, often associated with chloroquine-resistance. 66% of the samples from Guiné-Bissau showed a correlation with chloroquine-resistance while 73% of the samples from São Tomé and Angola altogether had the 86Tyr mutation. The present study on West African isolates and clones showed, for the first time, the presence of a double point mutation in the pfmdr1 gene one being found, up to now, only in South America isolates of Plasmodium falciparum.

Evaluation of microbial community in hydrothermal field by direct DNA sequencing

NASA Astrophysics Data System (ADS)

Kawarabayasi, Y.; Maruyama, A.

2002-12-01

Many extremophiles have been discovered from terrestrial and marine hydrothermal fields. Some thermophiles can grow beyond 90°C in culture, while direct microscopic analysis occasionally indicates that microbes may survive in much hotter hydrothermal fluids. However, it is very difficult to isolate and cultivate such microbes from the environments, i.e., over 99% of total microbes remains undiscovered. Based on experiences of entire microbial genome analysis (Y.K.) and microbial community analysis (A.M.), we started to find out unique microbes/genes in hydrothermal fields through direct sequencing of environmental DNA fragments. At first, shotgun plasmid libraries were directly constructed with the DNA molecules prepared from mixed microbes collected by an in situ filtration system from low-temperature fluids at RM24 in the Southern East Pacific Rise (S-EPR). A gene amplification (PCR) technique was not used for preventing mutation in the process. The nucleotide sequences of 285 clones indicated that no sequence had identical data in public databases. Among 27 clones determined entire sequences, no ORF was identified on 14 clones like intron in Eukaryote. On four clones, tetra-nucleotide-long multiple tandem repetitive sequences were identified. This type of sequence was identified in some familiar disease in human. The result indicates that living/dead materials with eukaryotic features may exist in this low temperature field. Secondly, shotgun plasmid libraries were constructed from the environmental DNA prepared from Beppu hot springs. In randomly-selected 143 clones used for sequencing, no known sequence was identified. Unlike the clones in S-EPR library, clear ORFs were identified on all nine clones determined the entire sequence. It was found that one clone, H4052, contained the complete Aspartyl-tRNA synthetase. Phylogenetic analysis using amino acid sequences of this gene indicated that this gene was separated from other Euryarchaea before the differentiation of species. Thus, some novel archaeal species are expected to be in this field. The present direct cloning and sequencing technique is now opening a window to the new world in hydrothermal microbial community analysis.

Memory-built-in quantum cloning in a hybrid solid-state spin register

NASA Astrophysics Data System (ADS)

Wang, Weibin; Zu, Chong; He, Li; Zhang, Wengang; Duan, Luming

2015-05-01

As a way to circumvent the quantum no-cloning theorem, approximate quantum cloning protocols have received wide attention with remarkable applications. Copying of quantum states to memory qubits provides an important strategy for eavesdropping in quantum cryptography. We report an experiment that realizes cloning of quantum states from an electron spin to a nuclear spin in a hybrid solid-state spin register with near-optimal fidelity. The nuclear spin provides an ideal memory qubit at room temperature, which stores the cloned quantum states for a millisecond under ambient conditions, exceeding the lifetime of the original quantum state carried by the electron spin by orders of magnitude, and making it an ideal memory qubit. Our experiment is based on control of an individual nitrogen vacancy (NV) center in the diamond, which is a diamond defect that attracts strong interest in recent years with great potential for implementation of quantum information protocols.

Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase.

PubMed

Fu, Changlin; Donovan, William P; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H

2014-01-01

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17-30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50 °C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90-95%.

Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

PubMed Central

Fu, Changlin; Donovan, William P.; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H.

2014-01-01

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17–30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50°C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90–95%. PMID:25551825

High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides.

PubMed

Bahniuk, Markian S; Alshememry, Abdullah K; Unsworth, Larry D

2016-12-01

The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250-660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides.

Testing a growth efficiency hypothesis with continental-scale phenological variations of common and cloned plants.

PubMed

Liang, Liang; Schwartz, Mark D

2014-10-01

Variation in the timing of plant phenology caused by phenotypic plasticity is a sensitive measure of how organisms respond to weather and climate variability. Although continental-scale gradients in climate and consequential patterns in plant phenology are well recognized, the contribution of underlying genotypic difference to the geography of phenology is less well understood. We hypothesize that different temperate plant genotypes require varying amount of heat energy for resuming annual growth and reproduction as a result of adaptation and other ecological and evolutionary processes along climatic gradients. In particular, at least for some species, the growing degree days (GDD) needed to trigger the same spring phenology events (e.g., budburst and flower bloom) may be less for individuals originated from colder climates than those from warmer climates. This variable intrinsic heat energy requirement in plants can be characterized by the term growth efficiency and is quantitatively reflected in the timing of phenophases-earlier timing indicates higher efficiency (i.e., less heat energy needed to trigger phenophase transitions) and vice versa compared to a standard reference (i.e., either a uniform climate or a uniform genotype). In this study, we tested our hypothesis by comparing variations of budburst and bloom timing of two widely documented plants from the USA National Phenology Network (i.e., red maple-Acer rubrum and forsythia-Forsythia spp.) with cloned indicator plants (lilac-Syringa x chinensis 'Red Rothomagensis') at multiple eastern US sites. Our results indicate that across the accumulated temperature gradient, the two non-clonal plants showed significantly more gradual changes than the cloned plants, manifested by earlier phenology in colder climates and later phenology in warmer climates relative to the baseline clone phenological response. This finding provides initial evidence supporting the growth efficiency hypothesis, and suggests more work is warranted. More studies investigating genotype-determined phenological variations will be useful for better understanding and prediction of the continental-scale patterns of biospheric responses to climate change.

Assessment of mitochondrial functions in Daphnia pulex clones using high-resolution respirometry.

PubMed

Kake-Guena, Sandrine A; Touisse, Kamal; Vergilino, Roland; Dufresne, France; Blier, Pierre U; Lemieux, Hélène

2015-06-01

The objectives of our study were to adapt a method to measure mitochondrial function in intact mitochondria from the small crustacean Daphnia pulex and to validate if this method was sensitive enough to characterize mitochondrial metabolism in clones of the pulex complex differing in ploidy levels, mitochondrial DNA haplotypes, and geographic origins. Daphnia clones belonging to the Daphnia pulex complex represent a powerful model to delineate the link between mitochondrial DNA evolution and mitochondrial phenotypes, as single genotypes with divergent mtDNA can be grown under various experimental conditions. Our study included two diploid clones from temperate environments and two triploid clones from subarctic environments. The whole animal permeabilization and measurement of respiration with high-resolution respirometry enabled the measurement of the functional capacity of specific mitochondrial complexes in four clones. When expressing the activity as ratios, our method detected significant interclonal variations. In the triploid subarctic clone from Kuujjurapik, a higher proportion of the maximal physiological oxidative phosphorylation (OXPHOS) capacity of mitochondria was supported by complex II, and a lower proportion by complex I. The triploid subarctic clone from Churchill (Manitoba) showed the lowest proportion of the maximal OXPHOS supported by complex II. Additional studies are required to determine if these differences in mitochondrial functions are related to differences in mitochondrial haplotypes or ploidy level and if they might be associated with fitness divergences and therefore selective value. © 2015 Wiley Periodicals, Inc.

Immunohistochemical application of a highly sensitive and specific murine monoclonal antibody recognising the extracellular domain of the human hepatocyte growth factor receptor (MET).

PubMed

Gruver, Aaron M; Liu, Ling; Vaillancourt, Peter; Yan, Sau-Chi B; Cook, Joel D; Roseberry Baker, Jessica A; Felke, Erin M; Lacy, Megan E; Marchal, Christophe C; Szpurka, Hadrian; Holzer, Timothy R; Rhoads, Emily K; Zeng, Wei; Wortinger, Mark A; Lu, Jirong; Chow, Chi-kin; Denning, Irene J; Beuerlein, Gregory; Davies, Julian; Hanson, Jeff C; Credille, Kelly M; Wijayawardana, Sameera R; Schade, Andrew E

2014-12-01

Development of novel targeted therapies directed against hepatocyte growth factor (HGF) or its receptor (MET) necessitates the availability of quality diagnostics to facilitate their safe and effective use. Limitations of some commercially available anti-MET antibodies have prompted development of the highly sensitive and specific clone A2H2-3. Here we report its analytical properties when applied by an automated immunohistochemistry method. Excellent antibody specificity was demonstrated by immunoblot, ELISA, and IHC evaluation of characterised cell lines including NIH3T3 overexpressing the related kinase MST1R (RON). Sensitivity was confirmed by measurements of MET in cell lines or characterised tissues. IHC correlated well with FISH and quantitative RT-PCR assessments of MET (P < 0.001). Good total agreement (89%) was observed with the anti-MET antibody clone SP44 using whole-tissue sections, but poor positive agreement (21-47%) was seen in tissue microarray cores. Multiple lots displayed appropriate reproducibility (R(2)  > 0.9). Prevalence of MET positivity by IHC was higher in non-squamous cell NSCLC, MET or EGFR amplified cases, and in tumours harbouring abnormalities in EGFR exon 19 or 21. The anti-MET antibody clone A2H2-3 displays excellent specificity and sensitivity. These properties make it suitable for clinical trial investigations and development as a potential companion diagnostic. © 2014 The Authors. Histopathology Published by John Wiley & Sons Ltd.

Stable MSAP markers for the distinction of Vitis vinifera cv Pinot noir clones.

PubMed

Ocaña, Juan; Walter, Bernard; Schellenbaum, Paul

2013-11-01

Grapevine is one of the most economically important fruit crops. Molecular markers have been used to study grapevine diversity. For instance, simple sequence repeats are a powerful tool for identification of grapevine cultivars, while amplified fragment length polymorphisms have shown their usefulness in intra-varietal diversity studies. Other techniques such as sequence-specific amplified polymorphism are based on the presence of mobile elements in the genome, but their detection lies upon their activity. Relevant attention has been drawn toward epigenetic sources of variation. In this study, a set of Vitis vinifera cv Pinot noir clones were analyzed using the methylation-sensitive amplified polymorphism technique with isoschizomers MspI and HpaII. Nine out of fourteen selective primer combinations were informative and generated two types of polymorphic fragments which were categorized as "stable" and "unstable." In total, 23 stable fragments were detected and they discriminated 92.5 % of the studied clones. Detected stable polymorphisms were either common to several clones, restricted to a few clones or unique to a single clone. The identification of these stable epigenetic markers will be useful in clonal diversity studies. We highlight the relevance of stable epigenetic variation in V. vinifera clones and analyze at which level these markers could be applicable for the development of forthright techniques for clonal distinction.

Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development.

PubMed

Smith, Craig A; Shoemaker, Christina M; Roeszler, Kelly N; Queen, Joanna; Crews, David; Sinclair, Andrew H

2008-07-24

R-Spondin1 (Rspo1) is a novel regulator of the Wnt/beta-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway. Gonadal RSPO1 gene expression is female up-regulated in the embryonic gonads in each species at the onset of sexual differentiation. In the mouse gonad, Rspo1 mRNA is expressed in the somatic cell lineage at the time of ovarian differentiation (E12.5-E15.5), with little expression in germ cells. However, the protein is localised in the cytoplasm and at the cell surface of both somatic (pre-follicular) and germ cells. In the chicken embryo, RSPO1 expression becomes elevated in females at the time of ovarian differentiation, coinciding with female-specific activation of the FOXL2 gene and estrogen synthesis. RSPO1 protein in chicken is localised in the outer cortical zone of the developing ovary, the site of primordial follicle formation and germ cell differentiation. Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen. In the red-eared slider turtle, which exhibits temperature-dependent sex determination, up-regulation of RSPO1 occurs during the temperature-sensitive period, when gonadal development is responsive to temperature. Accordingly, RSPO1 expression is temperature-responsive, and is down-regulated in embryos shifted from female- to male-producing incubation temperatures. These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms. In all instances, RSPO1 is expressed in the incipient ovary. These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway.

Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development

PubMed Central

Smith, Craig A; Shoemaker, Christina M; Roeszler, Kelly N; Queen, Joanna; Crews, David; Sinclair, Andrew H

2008-01-01

Background R-Spondin1 (Rspo1) is a novel regulator of the Wnt/β-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway. Results Gonadal RSPO1 gene expression is female up-regulated in the embryonic gonads in each species at the onset of sexual differentiation. In the mouse gonad, Rspo1 mRNA is expressed in the somatic cell lineage at the time of ovarian differentiation (E12.5–E15.5), with little expression in germ cells. However, the protein is localised in the cytoplasm and at the cell surface of both somatic (pre-follicular) and germ cells. In the chicken embryo, RSPO1 expression becomes elevated in females at the time of ovarian differentiation, coinciding with female-specific activation of the FOXL2 gene and estrogen synthesis. RSPO1 protein in chicken is localised in the outer cortical zone of the developing ovary, the site of primordial follicle formation and germ cell differentiation. Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen. In the red-eared slider turtle, which exhibits temperature-dependent sex determination, up-regulation of RSPO1 occurs during the temperature-sensitive period, when gonadal development is responsive to temperature. Accordingly, RSPO1 expression is temperature-responsive, and is down-regulated in embryos shifted from female- to male-producing incubation temperatures. Conclusion These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms. In all instances, RSPO1 is expressed in the incipient ovary. These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway. PMID:18651984

The groESL Chaperone Operon of Lactobacillus johnsonii†

PubMed Central

Walker, D. Carey; Girgis, Hany S.; Klaenhammer, Todd R.

1999-01-01

The Lactobacillus johnsonii VPI 11088 groESL operon was localized on the chromosome near the insertion element IS1223. The operon was initially cloned as a series of three overlapping PCR fragments, which were sequenced and used to design primers to amplify the entire operon. The amplified fragment was used as a probe to recover the chromosomal copy of the groESL operon from a partial library of L. johnsonii VPI 11088 (NCK88) DNA, cloned in the shuttle vector pTRKH2. The 2,253-bp groESL fragment contained three putative open reading frames, two of which encoded the ubiquitous GroES and GroEL chaperone proteins. Analysis of the groESL promoter region revealed three transcription initiation sites, as well as three sets of inverted repeats (IR) positioned between the transcription and translation start sites. Two of the three IR sets bore significant homology to the CIRCE elements, implicated in negative regulation of the heat shock response in many bacteria. Northern analysis and primer extension revealed that multiple temperature-sensitive promoters preceded the groESL chaperone operon, suggesting that stress protein production in L. johnsonii is strongly regulated. Maximum groESL transcription activity was observed following a shift to 55°C, and a 15 to 30-min exposure of log-phase cells to this temperature increased the recovery of freeze-thawed L. johnsonii VPI 11088. These results suggest that a brief, preconditioning heat shock can be used to trigger increased chaperone production and provide significant cross-protection from the stresses imposed during the production of frozen culture concentrates. PMID:10388700

Mutant maize variety containing the glt1-1 allele

DOEpatents

Nelson, O.E.; Pan, D.

1994-07-19

A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating. 2 figs.

Identification of an active endogenous transposon from the W4 locus in soybean

USDA-ARS?s Scientific Manuscript database

In soybean [Glycine max (L.) Merr.], W4 is one of the loci that control anthocyanin biosynthesis in flowers and hypocotyls. A putative transposable element was suggested to reside within or adjacent to this locus in the mutable T322 line resulting in the w4-m allele. We have shown that the W4 locu...

Identification and characterization of the first active endogenous transposable element in soybean

USDA-ARS?s Scientific Manuscript database

In soybean [Glycine max (L.) Merr.], W4 is one of the loci that control anthocyanin biosynthesis in flowers and hypocotyls. A putative transposable element was suggested to reside within or adjacent to this locus in the mutable T322 line resulting in the w4-m allele. We have shown that the W4 locu...

National or Global: The Mutable Concepts of Identity and Home for International School Students

ERIC Educational Resources Information Center

Bagnall, Nigel

2012-01-01

This article examines a selection of responses about identity and belonging among students in an international school in Rio de Janeiro, Brazil, who must often move from one continent to another because of the nature of their parents' work. A review of the literature highlights some of the issues these students face within an international school…

An inter-residue network model to identify mutational-constrained regions on the Ebola coat glycoprotein

PubMed Central

Quinlan, Devin S.; Raman, Rahul; Tharakaraman, Kannan; Subramanian, Vidya; del Hierro, Gabriella; Sasisekharan, Ram

2017-01-01

Recently, progress has been made in the development of vaccines and monoclonal antibody cocktails that target the Ebola coat glycoprotein (GP). Based on the mutation rates for Ebola virus given its natural sequence evolution, these treatment strategies are likely to impose additional selection pressure to drive acquisition of mutations in GP that escape neutralization. Given the high degree of sequence conservation among GP of Ebola viruses, it would be challenging to determine the propensity of acquiring mutations in response to vaccine or treatment with one or a cocktail of monoclonal antibodies. In this study, we analyzed the mutability of each residue using an approach that captures the structural constraints on mutability based on the extent of its inter-residue interaction network within the three-dimensional structure of the trimeric GP. This analysis showed two distinct clusters of highly networked residues along the GP1-GP2 interface, part of which overlapped with epitope surfaces of known neutralizing antibodies. This network approach also permitted us to identify additional residues in the network of the known hotspot residues of different anti-Ebola antibodies that would impact antibody-epitope interactions. PMID:28397835

Ram locus is a key regulator to trigger multidrug resistance in Enterobacter aerogenes.

PubMed

Molitor, Alexander; James, Chloë E; Fanning, Séamus; Pagès, Jean-Marie; Davin-Regli, Anne

2018-02-01

Several genetic regulators belonging to AraC family are involved in the emergence of MDR isolates of E. aerogenes due to alterations in membrane permeability. Compared with the genetic regulator Mar, RamA may be more relevant towards the emergence of antibiotic resistance. Focusing on the global regulators, Mar and Ram, we compared the amino acid sequences of the Ram repressor in 59 clinical isolates and laboratory strains of E. aerogenes. Sequence types were associated with their corresponding multi-drug resistance phenotypes and membrane protein expression profiles using MIC and immunoblot assays. Quantitative gene expression analysis of the different regulators and their targets (porins and efflux pump components) were performed. In the majority of the MDR isolates tested, ramR and a region upstream of ramA were mutated but marR or marA were unchanged. Expression and cloning experiments highlighted the involvement of the ram locus in the modification of membrane permeability. Overexpression of RamA lead to decreased porin production and increased expression of efflux pump components, whereas overexpression of RamR had the opposite effects. Mutations or deletions in ramR, leading to the overexpression of RamA predominated in clinical MDR E. aerogenes isolates and were associated with a higher-level of expression of efflux pump components. It was hypothesised that mutations in ramR, and the self-regulating region proximal to ramA, probably altered the binding properties of the RamR repressor; thereby producing the MDR phenotype. Consequently, mutability of RamR may play a key role in predisposing E. aerogenes towards the emergence of a MDR phenotype.

Status of vaccine research and development of vaccines for HIV-1.

PubMed

Safrit, Jeffrey T; Fast, Patricia E; Gieber, Lisa; Kuipers, Hester; Dean, Hansi J; Koff, Wayne C

2016-06-03

Human immunodeficiency virus (HIV) is the cause of one of the most lethal pandemics in human history, although in recent years access to highly effective anti-retroviral therapy has provided new hope worldwide. Transmission of HIV by sexual contact, childbirth and injection drug use has been reduced, but 2 million are newly infected each year, and much of the transmission is from people who do not know their status. In addition to known methods, a preventive vaccine is needed to end the pandemic. The extraordinary mutability and genetic diversity of HIV is an enormous challenge, but vaccines are being designed for broad coverage. Computer-aided design of mosaic immunogens, incorporating many epitopes from the entire genome or from conserved regions aim to induce CD8+ T cells to kill virus-infected cells or inhibit virus replication, while trimeric envelope proteins or synthetic mimics aim to induce broadly reactive neutralizing antibodies similar to those cloned from some infected patients. Induction of more potent and durable responses may require new adjuvants or replicating chimeric vectors chimeras that bear HIV genes. Passive or genetic delivery of broadly neutralizing antibodies may provide broad protection and/or lead to insights for vaccine designers. Proof-of-concept trials in non-human primates and in one human efficacy trial have provided scientific clues for a vaccine that could provide broad and durable protection against HIV. The use of vaccines to destroy HIV reservoirs as part of therapy or cure is now also being explored. Copyright © 2016 World Health Organization. Published by Elsevier Ltd.. All rights reserved.

A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pH-sensitivity in firefly luciferases.

PubMed

Viviani, Vadim R; Oehlmeyer, T L; Arnoldi, F G C; Brochetto-Braga, M R

2005-01-01

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases.

Expression of dog1 in low-grade fibromyxoid sarcoma: A study of 19 cases and review of the literature.

PubMed

Vallejo-Benítez, Ana; Rodríguez-Zarco, Enrique; Carrasco, Sara Pabón; Pereira-Gallardo, Sofia; Brugal Molina, Javier; García-Escudero, Antonio; Robles Frías, Antonio; Marcilla, David; González-Cámpora, Ricardo

2017-10-01

DOG1 is a highly-sensitive marker often included in the immunohistochemical panel for the diagnosis of gastrointestinal stromal tumors (GISTs). Recent research has shown that DOG1 may also be expressed by low-grade fibromyxoid sarcomas (LGFMSs); this may give rise to diagnostic error when the sarcoma is located in the abdominal cavity. This paper reports on immnohistochemical expression of DOG1 in 19 LGFMSs using two different monoclonal antibodies: K9 (Leica, Novocastra Laboratories, Newcastle upon Tyne, UK) and SP31 (Thermo Scientific, Freemont, USA). All LGFMSs displayed the standard histological pattern of alternating myxoid and fibrous areas, low cellularity and bland spindle-cell morphology. Positive staining for MUC4 was observed in 18/19 cases (94.7%), while there was rearrangement of the FUS gene in 14/19 (73.7%) cases and of the EWR1 gene in 2/19 (10.5%). The sarcoma staining negative for MUC4 displayed FUS gene rearrangement. Whole-section immunohistochemistry revealed positive staining for DOG1 in 8/19 cases (42.1%), though only with clone K9. Cytoplasmic as well as membrane staining was observed in all cases; staining was focal (10-30%) and of varying intensity (1+ to 2+). In conclusion, DOG1 clone K9 exhibited low sensitivity (42.1%) for the diagnosis of LGFMS, although higher than clone SP31. Since the two clones display similar sensitivity and specificity for GIST diagnosis, SP31 would appear to be more specific for this purpose, since no reaction was observed here with LGFMS, a GIST-mimicking lesion. Copyright © 2017 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guta, Madalin; Matsumoto, Keiji; Quantum Computation and Information Project, JST, Hongo 5-28-3, Bunkyo-ku, Tokyo 113-0033

We construct the optimal one to two cloning transformation for the family of displaced thermal equilibrium states of a harmonic oscillator, with a fixed and known temperature. The transformation is Gaussian and it is optimal with respect to the figure of merit based on the joint output state and norm distance. The proof of the result is based on the equivalence between the optimal cloning problem and that of optimal amplification of Gaussian states which is then reduced to an optimization problem for diagonal states of a quantum oscillator. A key concept in finding the optimum is that of stochasticmore » ordering which plays a similar role in the purely classical problem of Gaussian cloning. The result is then extended to the case of n to m cloning of mixed Gaussian states.« less

T-cell stimuli independently sum to regulate an inherited clonal division fate

PubMed Central

Marchingo, J. M.; Prevedello, G.; Kan, A.; Heinzel, S.; Hodgkin, P. D.; Duffy, K. R.

2016-01-01

In the presence of antigen and costimulation, T cells undergo a characteristic response of expansion, cessation and contraction. Previous studies have revealed that population-level reproducibility is a consequence of multiple clones exhibiting considerable disparity in burst size, highlighting the requirement for single-cell information in understanding T-cell fate regulation. Here we show that individual T-cell clones resulting from controlled stimulation in vitro are strongly lineage imprinted with highly correlated expansion fates. Progeny from clonal families cease dividing in the same or adjacent generations, with inter-clonal variation producing burst-size diversity. The effects of costimulatory signals on individual clones sum together with stochastic independence; therefore, the net effect across multiple clones produces consistent, but heterogeneous population responses. These data demonstrate that substantial clonal heterogeneity arises through differences in experience of clonal progenitors, either through stochastic antigen interaction or by differences in initial receptor sensitivities. PMID:27869196

Participation of the arcRACME protein in self-activation of the arc operon located in the arginine catabolism mobile element in pandemic clone USA300.

PubMed

Rozo, Zayda Lorena Corredor; Márquez-Ortiz, Ricaurte Alejandro; Castro, Betsy Esperanza; Gómez, Natasha Vanegas; Escobar-Pérez, Javier

2017-07-01

Staphylococcus aureus pandemic clone USA300 has, in addition to its constitutive arginine catabolism (arc) gene cluster, an arginine catabolism mobile element (ACME) carrying another such cluster, which gives this clone advantages in colonisation and infection. Gene arcR, which encodes an oxygen-sensitive transcriptional regulator, is inside ACME and downstream of the constitutive arc gene cluster, and this situation may have an impact on its activation. Different relative expression behaviours are proven here for arcRACME and the arcACME operon compared to the constitutive ones. We also show that the artificially expressed recombinant ArcRACME protein binds to the promoter region of the arcACME operon; this mechanism can be related to a positive feedback model, which may be responsible for increased anaerobic survival of the USA300 clone during infection-related processes.

Molecular cloning and expression analysis of KIN10 and cold-acclimation related genes in wild banana 'Huanxi' (Musa itinerans).

PubMed

Liu, Weihua; Cheng, Chunzhen; Lai, Gongti; Lin, Yuling; Lai, Zhongxiong

2015-01-01

Banana cultivars may experience chilling or freezing injury in some of their cultivated regions, where wild banana can still grow very well. The clarification of the cold-resistant mechanism of wild banana is vital for cold-resistant banana breeding. In this study, the central stress integrator gene KIN10 and some cold-acclimation related genes (HOS1 and ICE1s) from the cold-resistant wild banana 'Huanxi' (Musa itinerans) were cloned and their expression patterns under different temperature treatments were analyzed. Thirteen full-length cDNA transcripts including 6 KIN10s, 1 HOS1 and 6 ICE1s were successfully cloned. Quantitative real-time PCR (qRT-PCR) results showed that all these genes had the highest expression levels at the critical temperature of banana (13 °C). Under chilling temperature (4 °C), the expression level of KIN10 reduced significantly but the expression of HOS1 was still higher than that at the optimal temperature (28 °C, control). Both KIN10 and HOS1 showed the lowest expression levels at 0 °C, the expression level of ICE1, however, was higher than control. As sucrose plays role in plant cold-acclimation and in regulation of KIN10 and HOS1 bioactivities, the sucrose contents of wild banana under different temperatures were detected. Results showed that the sucrose content increased as temperature lowered. Our result suggested that KIN10 may participate in cold stress response via regulating sucrose biosynthesis, which is helpful in regulating cold acclimation pathway in wild banana.

Hd6, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2

PubMed Central

Takahashi, Yuji; Shomura, Ayahiko; Sasaki, Takuji; Yano, Masahiro

2001-01-01

Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the α subunit of protein kinase CK2 (CK2α) in this region. The Nipponbare allele of CK2α contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2α increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype. PMID:11416158

Detection of anticentromere antibodies using cloned autoantigen CENP-B.

PubMed

Rothfield, N; Whitaker, D; Bordwell, B; Weiner, E; Senecal, J L; Earnshaw, W

1987-12-01

A solid-phase enzyme-linked immunosorbent assay has been established using a cloned fusion protein, CtermCENP-B [beta-gal], as antigen. The fusion protein carries the major epitope of CENP-B, the major centromeric autoantigen. The enzyme-linked immunosorbent assay was more sensitive than immunofluorescence techniques in detecting anticentromere antibodies in patients with scleroderma or Raynaud's disease, and was weakly positive in 3% of normal controls and in 3% of 70 patients with other connective tissue diseases.

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites.

PubMed

Hunt, Paul; Afonso, Ana; Creasey, Alison; Culleton, Richard; Sidhu, Amar Bir Singh; Logan, John; Valderramos, Stephanie G; McNae, Iain; Cheesman, Sandra; do Rosario, Virgilio; Carter, Richard; Fidock, David A; Cravo, Pedro

2007-07-01

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites§

PubMed Central

Hunt, Paul; Afonso, Ana; Creasey, Alison; Culleton, Richard; Sidhu, Amar Bir Singh; Logan, John; Valderramos, Stephanie G; McNae, Iain; Cheesman, Sandra; do Rosario, Virgilio; Carter, Richard; Fidock, David A; Cravo, Pedro

2007-01-01

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed. PMID:17581118

Activation of an ATP-dependent K(+) conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules.

PubMed

Brochiero, E; Coady, M J; Klein, H; Laprade, R; Lapointe, J Y

2001-02-09

In rabbit proximal convoluted tubules, an ATP-sensitive K(+) (K(ATP)) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na(+) transport and basolateral K(+) conductance. This K(+) conductance is inhibited by taurine. We sought to isolate this K(+) channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K(+) conductance, largely inhibited by extracellular Ba(2+) and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K(+) current which was Ba(2+)-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K(+) channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K(+) current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K(+) current. The possible involvement of AK in the K(ATP) channel's regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations.

Targeting the T-Lak cell originated protein kinase by OTS964 shrinks the size of power-law coded heterogeneous glioma stem cell populations

PubMed Central

Sugimori, Michiya; Hayakawa, Yumiko; Koh, Masaki; Hayashi, Tomohide; Tamura, Ryoi; Kuroda, Satoshi

2018-01-01

Glioblastoma resists chemoradiotherapy, then, recurs to be a fatal space-occupying lesion. The recurrence is caused by re-growing cell populations such as glioma stem cells (GSCs), suggesting that GSC populations should be targeted. This study addressed whether a novel anti-cancer drug, OTS964, an inhibitor for T-LAK cell originated protein kinase (TOPK), is effective in reducing the size of the heterogeneous GSC populations, a power-law coded heterogeneous GSC populations consisting of glioma sphere (GS) clones, by detailing quantitative growth properties. We found that OTS964 killed GS clones while suppressing the growth of surviving GS clones, thus identifying clone-eliminating and growth-disturbing efficacies of OTS964. The efficacies led to a significant size reduction in GS populations in a dose-dependent manner. The surviving GS clones reconstructed GS populations in the following generations; the recovery of GS populations fits a recurrence after the chemotherapy. The recovering GS clones resisted the clone-eliminating effect of OTS964 in sequential exposure during the growth recovery. However, surprisingly, the resistant properties of the recovered-GS clones had been plastically canceled during self-renewal, and then the GS clones had become re-sensitive to OTS964. Thus, OTS964 targets GSCs to eliminate them or suppress their growth, resulting in shrinkage of the power-law coded GSC populations. We propose a therapy focusing on long-term control in recurrence of glioblastoma via reducing the size of the GSC populations by OTS964. PMID:29423027

Targeting the T-Lak cell originated protein kinase by OTS964 shrinks the size of power-law coded heterogeneous glioma stem cell populations.

PubMed

Sugimori, Michiya; Hayakawa, Yumiko; Koh, Masaki; Hayashi, Tomohide; Tamura, Ryoi; Kuroda, Satoshi

2018-01-09

Glioblastoma resists chemoradiotherapy, then, recurs to be a fatal space-occupying lesion. The recurrence is caused by re-growing cell populations such as glioma stem cells (GSCs), suggesting that GSC populations should be targeted. This study addressed whether a novel anti-cancer drug, OTS964, an inhibitor for T-LAK cell originated protein kinase (TOPK), is effective in reducing the size of the heterogeneous GSC populations, a power-law coded heterogeneous GSC populations consisting of glioma sphere (GS) clones, by detailing quantitative growth properties. We found that OTS964 killed GS clones while suppressing the growth of surviving GS clones, thus identifying clone-eliminating and growth-disturbing efficacies of OTS964. The efficacies led to a significant size reduction in GS populations in a dose-dependent manner. The surviving GS clones reconstructed GS populations in the following generations; the recovery of GS populations fits a recurrence after the chemotherapy. The recovering GS clones resisted the clone-eliminating effect of OTS964 in sequential exposure during the growth recovery. However, surprisingly, the resistant properties of the recovered-GS clones had been plastically canceled during self-renewal, and then the GS clones had become re-sensitive to OTS964. Thus, OTS964 targets GSCs to eliminate them or suppress their growth, resulting in shrinkage of the power-law coded GSC populations. We propose a therapy focusing on long-term control in recurrence of glioblastoma via reducing the size of the GSC populations by OTS964.

Analysis of proviral integration in human mammary epithelial cell lines immortalized by retroviral infection with a temperature-sensitive SV40 T-antigen construct.

PubMed

Stamps, A C; Davies, S C; Burman, J; O'Hare, M J

1994-06-15

A panel of eight conditionally immortal lines derived by infection of human breast epithelial cells with an amphotropic retrovirus transducing a ts mutant of SV40 large T-antigen was analyzed with respect to individual retroviral integration patterns. Each line contained multiple integration sites which were clonal and stable over extended passage. Similar integration patterns were observed between individual lines arising separately from the same stock of pre-immortal cells, suggesting a common progenitor. Retroviral integration analysis of pre-immortal cells at different stages of pre-crisis growth showed changes indicative of a progressive transition from polyclonality to clonality as the cells approached crisis. Each of the immortal lines contained a sub-set of the integration sites of their pre-immortal progenitors, with individual combinations and copy numbers of sites. Since all the cell lines appeared to originate from single foci in separate flasks, it is likely that each set arose from a common clone of pre-immortal cells as the result of separate genetic events. There was no evidence from this analysis to suggest that specific integration sites played any part either in the selection of pre-crisis clones or in the subsequent establishment of immortal lines.

Genetic code mutations: the breaking of a three billion year invariance.

PubMed

Mat, Wai-Kin; Xue, Hong; Wong, J Tze-Fei

2010-08-20

The genetic code has been unchanging for some three billion years in its canonical ensemble of encoded amino acids, as indicated by the universal adoption of this ensemble by all known organisms. Code mutations beginning with the encoding of 4-fluoro-Trp by Bacillus subtilis, initially replacing and eventually displacing Trp from the ensemble, first revealed the intrinsic mutability of the code. This has since been confirmed by a spectrum of other experimental code alterations in both prokaryotes and eukaryotes. To shed light on the experimental conversion of a rigidly invariant code to a mutating code, the present study examined code mutations determining the propagation of Bacillus subtilis on Trp and 4-, 5- and 6-fluoro-tryptophans. The results obtained with the mutants with respect to cross-inhibitions between the different indole amino acids, and the growth effects of individual nutrient withdrawals rendering essential their biosynthetic pathways, suggested that oligogenic barriers comprising sensitive proteins which malfunction with amino acid analogues provide effective mechanisms for preserving the invariance of the code through immemorial time, and mutations of these barriers open up the code to continuous change.

Nutlin‐3a selects for cells harbouring TP 53 mutations

PubMed Central

Hollstein, Monica; Arlt, Volker M.; Phillips, David H.

2016-01-01

TP53 mutations occur in half of all human tumours. Mutagen‐induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock‐in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen‐treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2–5 months) and much effort is expended maintaining TP53‐WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin‐3a, an MDM2 inhibitor that leads to stabilisation and activation of wild‐type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin‐3a to examine the effect on cell growth and p53 activation. Nutlin‐3a induced the p53 pathway in TP53‐WT HUFs and inhibited cell growth, whereas most TP53‐mutated HUFs were resistant to Nutlin‐3a. We then assessed whether Nutlin‐3a treatment could discriminate between TP53‐WT and TP53‐mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin‐3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin‐3a‐resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin‐3a‐sensitive clones were TP53‐WT. These data suggest that including a Nutlin‐3a counter‐screen significantly improves the specificity and efficiency of the HIMA, whereby TP53‐mutated clones are selected prior to sequencing and TP53‐WT clones can be discarded. PMID:27813088

Induction and cultivation of cloned filaments of Polysiphonia urceolata (Rhodomelaceae, Rhodophyta)

NASA Astrophysics Data System (ADS)

Wang, Jinxia; Shao, Kuishuang; Cheng, Bin; Lu, Qinqin; Zhou, Baicheng

2011-11-01

A filamentous clone of Polysiphonia urceolata was regenerated from segments cut from the fronds of gametophytes. Unlike wild thalli with short virgate branchlets, the clone was filamentous with few branches. Many transparent trichoblasts arose from pericentral cells during the induction culture, but these were seldom observed during normal growth. The trichoblasts were uniseriate, often colorless, and formed lobed rhizoids rapidly when they came into contact with solid substrates. In addition to morphological characteristics, the photosynthetic properties and growth conditions of the clone differed from those of the mother plant. Cross-gradient light and temperature culture experiments revealed that the most favorable conditions for culture of the filamentous clone were 22°C and 95-120 μE/(m2·s) light intensity. The photosynthetic light saturation value for filaments was approx. 100 μE/(m2·s), which is far lower than that of wild thalli. These results could be used to develop techniques for mass cultures of P. urceolata in photobioreactors for production of seed stock or bioactive products.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-05-15

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

PubMed

Alban, Silvana M; de Moura, Juliana Ferreira; Minozzo, João Carlos; Mira, Marcelo Távora; Soccol, Vanete Thomaz

2013-01-25

An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

Genomics approach to the environmental community of microorganisms

NASA Astrophysics Data System (ADS)

Kawarabayasi, Y.; Maruyama, A.

2004-12-01

It was indicated by microscopic observation or comparison of 16S rDNA sequence that many extremophiles were surviving in many hydrothermal environments. But it is generally said that over 99% of total microbes are now uncultivable. Thus, we planned to identify uncultivable microbes through direct sequencing of environmental DNA. At first, shotgun plasmid libraries were directly constructed with the DNA molecules prepared from mixed microbes collected from low-temperature hydrothermal water at RM24 in the Southern East Pacific Rise (S-EPR). It was shown that the sequences of some number of clones indicated the similar feature to the intron in eukaryote or tandem repetitive sequence identified in some human familiar diseases. The results indicated that many microorganisms with eukaryotic feature were dominant in low temperature water of S-EPR. Secondly, shotgun plasmid libraries were constructed from the environmental DNA prepared from Beppu hot springs. The ORFs were easily identified all clones determined entire sequence. Thus it can be said that hot springs is good resources for searching novel genes. At last, the mixed microbes isolated from Suiyo seamount were used for construction of shotgun library. The clones in this library contained the ORFs. From some clones in hot spring and Suiyo sample, aminoacyl-tRNA synthatase, which is generally present in all organisms, was isolated by similarity. The phylogenetic analysis of aminoacyl-tRNA synthetase identified indicated that novel and unidentified microorganisms should be present in hot spring or Suiyo seamount. The novel genes identified from Suiyo seamount were also utilized for expression in E. coli. Some gene products were successfully obtained from the E. coli cells as soluble proteins. Some protein indicated the thermostability up to 70_E#8249;C, meaning that the original host cell of this gene should be stable up to the same temperature. Our work indicates that environmental genomics, including the direct cloning, sequencing of environmental DNA and expression of gene identified, is powerful approach to collect novel uncultivable microbes or novel active genes.

Variations in phenology and growth of European white birch (Betula pendula) clones.

PubMed

Rousi, Matti; Pusenius, Jyrki

2005-02-01

Phenology can have a profound effect on growth and climatic adaptability of northern tree species. Although the large interannual variations in dates of bud burst and growth termination have been widely discussed, little is known about the genotypic and spatial variations in phenology and how these sources of variation are related to temporal variation. We measured bud burst of eight white birch (Betula pendula Roth) clones in two field experiments daily over 6 years, and determined the termination of growth for the same clones over 2 years. We also measured yearly height growth. We found considerable genetic variation in phenological characteristics among the birch clones. There was large interannual variation in the date of bud burst and especially in the termination of growth, indicating that, in addition to genetic effects, environmental factors have a strong influence on both bud burst and growth termination. Height growth was correlated with timing of growth termination, length of growth period and bud burst, but the relationships were weak and varied among years. We accurately predicted the date of bud burst from the temperature accumulation after January 1, and base temperatures between +2 and -1 degrees C. There was large clonal variation in the duration of bud burst. Interannual variation in bud burst may have important consequences for insect herbivory of birches.

Bacterial and archaeal diversity in two hot spring microbial mats from the geothermal region of Tengchong, China.

PubMed

Pagaling, Eulyn; Grant, William D; Cowan, Don A; Jones, Brian E; Ma, Yanhe; Ventosa, Antonio; Heaphy, Shaun

2012-07-01

We investigated the bacterial and archaeal diversity in two hot spring microbial mats from the geothermal region of Tengchong in the Yunnan Province, China, using direct molecular analyses. The Langpu (LP) laminated mat was found by the side of a boiling pool with temperature of 60-65 °C and a pH of 8.5, while the Tengchong (TC) streamer mat consisted of white streamers in a slightly acidic (pH 6.5) hot pool outflow with a temperature of 72 °C. Four 16S rRNA gene clone libraries were constructed and restriction enzyme analysis of the inserts was used to identify unique sequences and clone frequencies. From almost 200 clones screened, 55 unique sequences were retrieved. Phylogenetic analysis showed that the LP mat consisted of a diverse bacterial population [Cyanobacteria, Chloroflexi, Chlorobia, Nitrospirae, 'Deinococcus-Thermus', Proteobacteria (alpha, beta and delta subdivisions), Firmicutes, Bacteroidetes and Actinobacteria], while the archaeal population was dominated by methanogenic Euryarchaeota and Crenarchaeota. In contrast, the TC streamer mat consisted of a bacterial population dominated by Aquificae, while the archaeal population also contained Korarchaeota as well as Crenarchaeota and methanogenic Euryarchaeota. These mats harboured clone sequences affiliated to unidentified lineages, suggesting that they are a potential source for discovering novel bacteria and archaea.

[Cloning, expression and characterization of a novel esterase from marine sediment microbial metagenomic library].

PubMed

Xu, Shiqing; Hu, Yongfei; Yuan, Aihua; Zhu, Baoli

2010-07-01

To clone, express and characterize a novel esterase from marine sediment microbial metagenomic library. Using esterase segregation agar containing tributyrin, we obtained esterase positive fosmid clone FL10 from marine sediment microbial metagenomic library. This fosmid was partially digested with Sau3A I to construct the sublibrary, from which the esterase positive subclone pFLS10 was obtained. The full length of the esterase gene was amplified and cloned into the expressing vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 cells. We analyse the enzyme activity and study the characterization of the esterase after its expression and purification. An ORF (Open Reading Frame) of 924 bp was identified from the subclone pFLS10. Sequence analysis indicated that it showed 71% amino acid identity to esterase (ADA70030) from a marine sediment metagenomic library. The esterase is a novel low-temperature-active esterase and had highest lipolytic activity to the substrate of 4-nitrophenyl butyrate (C4). The optimum temperature of the esterase was 20 degrees C, the optimum pH was 7.5. The esterase in this study had good thermostability at 20 degrees C and good pH stability at pH8 -10. Significant increase in lipolytic activity was observed with addition of K+ and Mg2+, while decrease with Mn2+ etc. We obtained the novel esterase gene fls10 from the marine sediment microbial metagenomic library. The esterase had good thermostability and high lipolytic activity at low temperature and under basic conditions, which laid a basis for industrial application.

Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay.

PubMed

Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

2017-01-01

In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

Change in algal symbiont communities after bleaching, not prior heat exposure, increases heat tolerance of reef corals.

PubMed

Silverstein, Rachel N; Cunning, Ross; Baker, Andrew C

2015-01-01

Mutualistic organisms can be particularly susceptible to climate change stress, as their survivorship is often limited by the most vulnerable partner. However, symbiotic plasticity can also help organisms in changing environments by expanding their realized niche space. Coral-algal (Symbiodinium spp.) symbiosis exemplifies this dichotomy: the partnership is highly susceptible to 'bleaching' (stress-induced symbiosis breakdown), but stress-tolerant symbionts can also sometimes mitigate bleaching. Here, we investigate the role of diverse and mutable symbiotic partnerships in increasing corals' ability to thrive in high temperature conditions. We conducted repeat bleaching and recovery experiments on the coral Montastraea cavernosa, and used quantitative PCR and chlorophyll fluorometry to assess the structure and function of Symbiodinium communities within coral hosts. During an initial heat exposure (32 °C for 10 days), corals hosting only stress-sensitive symbionts (Symbiodinium C3) bleached, but recovered (at either 24 °C or 29 °C) with predominantly (>90%) stress-tolerant symbionts (Symbiodinium D1a), which were not detected before bleaching (either due to absence or extreme low abundance). When a second heat stress (also 32 °C for 10 days) was applied 3 months later, corals that previously bleached and were now dominated by D1a Symbiodinium experienced less photodamage and symbiont loss compared to control corals that had not been previously bleached, and were therefore still dominated by Symbiodinium C3. Additional corals that were initially bleached without heat by a herbicide (DCMU, at 24 °C) also recovered predominantly with D1a symbionts, and similarly lost fewer symbionts during subsequent thermal stress. Increased thermotolerance was also not observed in C3-dominated corals that were acclimated for 3 months to warmer temperatures (29 °C) before heat stress. These findings indicate that increased thermotolerance post-bleaching resulted from symbiont community composition changes, not prior heat exposure. Moreover, initially undetectable D1a symbionts became dominant only after bleaching, and were critical to corals' resilience after stress and resistance to future stress. © 2014 John Wiley & Sons Ltd.

Multiparameter FLAER-based flow cytometry for screening of paroxysmal nocturnal hemoglobinuria enhances detection rates in patients with aplastic anemia.

PubMed

Sachdeva, Man Updesh Singh; Varma, Neelam; Chandra, Dinesh; Bose, Parveen; Malhotra, Pankaj; Varma, Subhash

2015-05-01

Flow cytometry is the gold standard methodology for screening of paroxysmal nocturnal hemoglobinuria. In the last few years, proaerolysin conjugated with fluorescein (FLAER) has become an important component of antibody panel used for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clone. This study aimed to compare PNH clone detection by flow cytometry in the pre-FLAER era versus the FLAER era. This was a retrospective analysis of 4 years and included 1004 individuals screened for PNH clone, either presenting as hemolytic anemia or as aplastic anemia. In the pre-FLAER time period, the RBCs and neutrophils were screened with antibodies against CD55 and CD59. With the introduction of FLAER, neutrophils were screened with FLAER/CD24/CD15 and monocytes with FLAER/CD14/CD33 combination. A comparative analysis was done for detection of PNH clone in aplastic anemia patients versus non-aplastic anemia patients, as well as between pre-FLAER and FLAER era. Out of a total of 1004 individuals, 59 (5.8%) were detected to have PNH clone positivity. The frequency of PNH clone detected in aplastic anemia and non-aplastic anemia groups was 12.02 and 3.36%, respectively. The detection rate of PNH clone increased from 4.5% (32/711) in the pre-FLAER era to 9.2% (27/293) with the introduction of FLAER. However, this increase could be attributed to increased detection of PNH clone in the aplastic anemia group, which showed a significant increase from 8.3 to 18.2% after use of FLAER. In the non-aplastic group, PNH clone was detected with similar frequencies before and after use of FLAER (3.2 versus 3.8%, respectively). Mean PNH clone size was lower in the aplastic anemia group when compared with the non-aplastic group. RBCs always showed a lower clone size than neutrophils. PNH clone on neutrophils and monocytes was however similar. Inclusion of FLAER increases the sensitivity of the test which is especially useful in picking up small PNH clones in patients of aplastic anemia.

Spring leaf flush in aspen (Populus tremuloides) clones is altered by long-term growth at elevated carbon dioxide and elevated ozone concentration.

PubMed

McGrath, Justin M; Karnosky, David F; Ainsworth, Elizabeth A

2010-04-01

Early spring leaf out is important to the success of deciduous trees competing for light and space in dense forest plantation canopies. In this study, we investigated spring leaf flush and how long-term growth at elevated carbon dioxide concentration ([CO(2)]) and elevated ozone concentration ([O(3)]) altered leaf area index development in a closed Populus tremuloides (aspen) canopy. This work was done at the Aspen FACE experiment where aspen clones have been grown since 1997 in conditions simulating the [CO(2)] and [O(3)] predicted for approximately 2050. The responses of two clones were compared during the first month of spring leaf out when CO(2) fumigation had begun, but O(3) fumigation had not. Trees in elevated [CO(2)] plots showed a stimulation of leaf area index (36%), while trees in elevated [O(3)] plots had lower leaf area index (-20%). While individual leaf area was not significantly affected by elevated [CO(2)], the photosynthetic operating efficiency of aspen leaves was significantly improved (51%). There were no significant differences in the way that the two aspen clones responded to elevated [CO(2)]; however, the two clones responded differently to long-term growth at elevated [O(3)]. The O(3)-sensitive clone, 42E, had reduced individual leaf area when grown at elevated [O(3)] (-32%), while the tolerant clone, 216, had larger mature leaf area at elevated [O(3)] (46%). These results indicate a clear difference between the two clones in their long-term response to elevated [O(3)], which could affect competition between the clones, and result in altered genotypic composition in future atmospheric conditions. Published by Elsevier Ltd.

Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens.

PubMed Central

Gurney, E G; Harrison, R O; Fenno, J

1980-01-01

We have isolated three clones of hybrid cells which synthesize antibodies specific for determinants on simian virus 40 (SV40) T antigens. Mouse myeloma NS1 cells were fused with spleen cells from mice that had been immunized with SV40-transformed mouse cells. Hybrid cells were selected in HAT medium and cloned in soft agar. We used an enzyme-linked immunosorbent assay for detection and quantification of mouse antibodies against SV40 T antigens. Monoclonal antibodies from 3 of the 24 clones that scored as positive in the enzyme-linked immunosorbent assay were verified by immunoprecipitation to be specific for SV40 T antigens. Two clones (7 and 412) produced antibodies that recognized denaturation-sensitive antigenic determinants unique to large T antigen. Antibodies from clone 7 appeared to have a low affinity for large T antigen. Antibodies from clone 412 had a higher affinity for large T antigen but did not recognize a subclass of large T antigen that was recognized by tumor serum. Antibodies of the third clone, clone 122, recognized a denaturation-stable antigenic determinant of the 53,000-dalton mouse nonviral T antigen in SV40-transformed cells. Antibodies from clone 122 also recognized similar (51,000- to 56,000-dalton) nonviral T antigens in SV40-transormed or lytically infected cells from five mammalian species and in four uninfected mouse lines. From these observations, we have concluded that (i) the 94,000-dalton SV40 large T antigen may exist as immunologically distinguishable subclasses, and (ii) the nonviral T antigens of five mammalian species share at least one antigenic determinant. Images PMID:6155477

Soil temperature and precipitation affect the rooting ability of dormant hardwood cuttings of Populus

Treesearch

R.S., Jr. Zalesny; R.B. Hall; E.O. Bauer; D.E. Riemenschneider

2005-01-01

In addition to genetic control, responses to environmental stimuli affect the success of rooting. Our objectives were to: 1) assess the variation in rooting ability among 21 Populus clones grown under varying soil temperatures and amounts of precipitation and 2) identify combinations of soil temperature and precipitation that promote rooting. The...

Cytotoxic T cell clones isolated from ovarian tumor-infiltrating lymphocytes recognize multiple antigenic epitopes on autologous tumor cells.

PubMed

Ioannides, C G; Freedman, R S; Platsoucas, C D; Rashed, S; Kim, Y P

1991-03-01

CTL clones were developed from tumor infiltrating lymphocytes (TIL) from the ascites of a patient with ovarian carcinoma by coculture of TIL with autologous tumor cells and subsequent cloning in the presence of autologous tumor cells. These CTL clones expressed preferential cytolytic activity against autologous tumor cells but not against allogeneic ovarian tumor cells and the NK-sensitive cell line K562. The cytolytic activity of these CTL against autologous tumors was inhibited by anti-TCR (WT31 mAb), anti-HLA class I, and anti-CD3 mAb but not by the NK function antibody Leu 11b. Cloning of the autologous tumor cells in vitro revealed that the CTL clones of the ovarian TIL expressed differential abilities to lyse autologous tumor cell clones. The specificity analysis of these autologous tumor specific CTL suggested that they recognize several antigenic determinants present on the ovarian tumor cells. Our results indicate the presence of at least three antigenic epitopes on the tumor cells (designated OVA-1A, OVA-1B, and OVA-1C), one of which (OVA-1C) is unstable. These determinants are present either simultaneously or separately, and six types of ovarian clones can be distinguished on the basis of their expression. These results indicate that CTL of the TIL detect intratumor antigenic heterogeneity. The novel heterogeneity identified within the ovarian tumor cells in this report may be of significance for understanding cellular immunity in ovarian cancer and developing adoptive specific immunotherapeutic approaches in ovarian cancer.

A strategy for clone selection under different production conditions.

PubMed

Legmann, Rachel; Benoit, Brian; Fedechko, Ronald W; Deppeler, Cynthia L; Srinivasan, Sriram; Robins, Russell H; McCormick, Ellen L; Ferrick, David A; Rodgers, Seth T; Russo, A Peter

2011-01-01

Top performing clones have failed at the manufacturing scale while the true best performer may have been rejected early in the screening process. Therefore, the ability to screen multiple clones in complex fed-batch processes using multiple process variations can be used to assess robustness and to identify critical factors. This dynamic ranking of clones' strategy requires the execution of many parallel experiments than traditional approaches. Therefore, this approach is best suited for micro-bioreactor models which can perform hundreds of experiments quickly and efficiently. In this study, a fully monitored and controlled small scale platform was used to screen eight CHO clones producing a recombinant monoclonal antibody across several process variations, including different feeding strategies, temperature shifts and pH control profiles. The first screen utilized 240 micro-bioreactors were run for two weeks for this assessment of the scale-down model as a high-throughput tool for clone evaluation. The richness of the outcome data enable to clearly identify the best and worst clone as well as process in term of maximum monoclonal antibody titer. The follow-up comparison study utilized 180 micro-bioreactors in a full factorial design and a subset of 12 clone/process combinations was selected to be run parallel in duplicate shake flasks. Good correlation between the micro-bioreactor predictions and those made in shake flasks with a Pearson correlation value of 0.94. The results also demonstrate that this micro-scale system can perform clone screening and process optimization for gaining significant titer improvements simultaneously. This dynamic ranking strategy can support better choices of production clones. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

Longevity of crapemyrtle pollen stored at different temperatures

USDA-ARS?s Scientific Manuscript database

Temperatures for storage of crapemyrtle (Lagerstroemia app.) pollen over time were studied using clones of two interspecific hybrids (L. 'Cheyenne' and L. 'Wichita') and five species (L. indica 'Catawba', L. subcostata (NA 40181), L. limii, L. speciosa, and L. fauriei 'Kiowa'). Pollen samples were s...

Congressional Control of Navy Budget Execution: Acquisition of the A-6F Aircraft

DTIC Science & Technology

1988-06-01

understandable to Congress, and explaining the rationale may employ tricks of storytelling . Promotional efforts serve the purpose of 64 0 winning...Circumstances That Gain Congressional Empathy The second sub-category of financial manipulation is a tactic that gains understanding for the mutability...defeat at the polls. Similarly, the bid for empathy in coping with uncertainty creates an atmosphere of friendliness, trust and benevolence in which

The point mutation process in proteins

NASA Technical Reports Server (NTRS)

Schwartz, R. M.; Dayhoff, M. O.

1978-01-01

An optimized scoring matrix for residue-by-residue comparisons of distantly related protein sequences has been developed. The scoring matrix is based on observed exchanges and mutabilities of amino acids in 1572 closely related sequences derived from a cross-section of protein groups. Very few superimposed or parallel mutations are included in the data. The scoring matrix is most useful for demonstrating the relatedness of proteins between 65 and 85% different.

Synergistic Behavior of Tubes, Junctions, and Sheets Imparts Mechano-Mutable Functionality in 3D Porous Boron Nitride Nanostructures

PubMed Central

2015-01-01

One-dimensional (1D) boron nitride nanotube (BNNT) and 2D hexagonal BN (h-BN) are attractive for demonstrating fundamental physics and promising applications in nano-/microscale devices. However, there is a high anisotropy associated with these BN allotropes as their excellent properties are either along the tube axis or in-plane directions, posing an obstacle in their widespread use in technological and industrial applications. Herein, we report a series of 3D BN prototypes, namely, pillared boron nitride (PBN), by fusing single-wall BNNT and monolayer h-BN aimed at filling this gap. We use density functional theory and molecular dynamics simulations to probe the diverse mechano-mutable properties of PBN prototypes. Our results demonstrate that the synergistic effect of the tubes, junctions, and sheets imparts cooperative deformation mechanisms, which overcome the intrinsic limitations of the PBN constituents and provide a number of superior characteristics including 3D balance of strength and toughness, emergence of negative Poisson’s ratio, and elimination of strain softening along the armchair orientation. These features, combined with the ultrahigh surface area and lightweight structure, render PBN as a 3D multifunctional template for applications in graphene-based nanoelectronics, optoelectronics, gas storage, and functional composites with fascinating in-plane and out-of-plane tailorable properties. PMID:25289114

Genomic and Phenotypic Characterization of Yeast Biosensor for Deep-space Radiation

NASA Technical Reports Server (NTRS)

Marina, Diana B.; Santa Maria, Sergio; Bhattacharya, Sharmila

2016-01-01

The BioSentinel mission was selected to launch as a secondary payload onboard NASA Exploration Mission 1 (EM-1) in 2018. In BioSentinel, the budding yeast Saccharomyces cerevisiae will be used as a biosensor to measure the long-term impact of deep-space radiation to living organisms. In the 4U-payload, desiccated yeast cells from different strains will be stored inside microfluidic cards equipped with 3-color LED optical detection system to monitor cell growth and metabolic activity. At different times throughout the 12-month mission, these cards will be filled with liquid yeast growth media to rehydrate and grow the desiccated cells. The growth and metabolic rates of wild-type and radiation-sensitive strains in deep-space radiation environment will be compared to the rates measured in the ground- and microgravity-control units. These rates will also be correlated with measurements obtained from onboard physical dosimeters. In our preliminary long-term desiccation study, we found that air-drying yeast cells in 10% trehalose is the best method of cell preservation in order to survive the entire 18-month mission duration (6-month pre-launch plus 12-month full-mission periods). However, our study also revealed that desiccated yeast cells have decreasing viability over time when stored in payload-like environment. This suggests that the yeast biosensor will have different population of cells at different time points during the long-term mission. In this study, we are characterizing genomic and phenotypic changes in our yeast biosensor due to long-term storage and desiccation. For each yeast strain that will be part of the biosensor, several clones were reisolated after long-term storage by desiccation. These clones were compared to their respective original isolate in terms of genomic composition, desiccation tolerance and radiation sensitivity. Interestingly, clones from a radiation-sensitive mutant have better desiccation tolerance compared to their original isolate without losing radiation sensitivity. We employed Next-Generation Sequencing technology to better understand this phenotypic variation. Current effort is focusing on the analysis of high-throughput sequencing data to look for genomic changes in these reisolated clones compared to their original isolate.

PRP5: a helicase-like protein required for mRNA splicing in yeast.

PubMed Central

Dalbadie-McFarland, G; Abelson, J

1990-01-01

A 96-kDa protein predicted by the DNA sequence of the Saccharomyces cerevisiae PRP5 gene contains a domain that bears a striking resemblance to a family of RNA helicases characterized by the conserved amino acid sequence Asp-Glu-Ala-Asp (D-E-A-D). Previous work indicated that the product of the PRP5 gene is required for splicing and that spliceosome assembly does not occur in its absence. However, its precise role in splicing and the nature of its biochemical activity remained unknown. To examine the role of PRP5 in splicing, we cloned the gene by complementation of a temperature-sensitive mutation and determined its DNA sequence. We discuss here the possible roles for an RNA helicase in splicing and for the activity of the PRP5 protein. Images PMID:2349233

Architecture of a Species: Phylogenomics of Staphylococcus aureus.

PubMed

Planet, Paul J; Narechania, Apurva; Chen, Liang; Mathema, Barun; Boundy, Sam; Archer, Gordon; Kreiswirth, Barry

2017-02-01

A deluge of whole-genome sequencing has begun to give insights into the patterns and processes of microbial evolution, but genome sequences have accrued in a haphazard manner, with biased sampling of natural variation that is driven largely by medical and epidemiological priorities. For instance, there is a strong bias for sequencing epidemic lineages of methicillin-resistant Staphylococcus aureus (MRSA) over sensitive isolates (methicillin-sensitive S. aureus: MSSA). As more diverse genomes are sequenced the emerging picture is of a highly subdivided species with a handful of relatively clonal groups (complexes) that, at any given moment, dominate in particular geographical regions. The establishment of hegemony of particular clones appears to be a dynamic process of successive waves of replacement of the previously dominant clone. Here we review the phylogenomic structure of a diverse range of S. aureus, including both MRSA and MSSA. We consider the utility of the concept of the 'core' genome and the impact of recombination and horizontal transfer. We argue that whole-genome surveillance of S. aureus populations could lead to better forecasting of antibiotic resistance and virulence of emerging clones, and a better understanding of the elusive biological factors that determine repeated strain replacement. Copyright © 2016. Published by Elsevier Ltd.

Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast.

PubMed

Chan, C S; Botstein, D

1993-11-01

We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37 degrees. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37 degrees. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth.

Reactor performance and microbial community of an EGSB reactor operated at 20 and 15 degrees C.

PubMed

Xing, W; Zuo, J-E; Dai, N; Cheng, J; Li, J

2009-09-01

To investigate the effects of low temperatures on the performance and microbial community of anaerobic wastewater treatment. An expanded granular sludge bed (EGSB) reactor was employed to treat synthetic brewery wastewater at 20 and 15 degrees C. Reactor performance was represented by chemical oxygen demand (COD) removal efficiency, while the microbial community was analysed using denaturing gradient gel electrophoresis (DGGE) and clone technology. When the hydraulic retention time (HRT) was maintained at 18 h, COD removal efficiencies above 85% were obtained at both 20 and 15 degrees C, with influent COD concentrations up to 7300 and 4100 mg l(-1), respectively. At 15 degrees C, the COD removal efficiency was more easily manipulated by increasing the influent COD concentration. DGGE and clone results for both temperatures revealed that Methanosaeta and Methanobacterium were two dominant methanogens, and that the majority of the eubacterial clones were represented by Firmicutes. When the temperature decreased from 20 to 15 degrees C, both archaeal and eubacterial communities had higher diversity, and the proportion of Methanosaeta (acetate-utilizing methanogens) decreased markedly from 60.0% to 49.3%, together with an increase in proportions of hydrogen-utilizing methanogens (especially Methanospirillum). The feasibility of psychrophilic anaerobic treatment of low and medium strength organic wastewaters was demonstrated, although lower temperature could significantly affect both reactor performance and the anaerobic microbial community. The findings enrich the theory involving the microbial community and the application of anaerobic treatment in a psychrophilic environment.

Cloning, sequencing, and expression of cDNA for human. beta. -glucuronidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oshima, A.; Kyle, J.W.; Miller, R.D.

1987-02-01

The authors report here the cDNA sequence for human placental ..beta..-glucuronidase (..beta..-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH/sub 2/-terminal amino acid sequence determined for human spleen ..beta..-glucuronidase agreed with that inferred from the DNAmore » sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human ..beta..-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human ..beta..-glucuronidase, demonstrate the existence of two populations of mRNA for ..beta..-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.« less

Diagnostic algorithm for detection of targetable driver mutations in lung adenocarcinomas: Comprehensive analyses of 205 cases with immunohistochemistry, real-time PCR and fluorescence in situ hybridization methods.

PubMed

Kao, Hua-Lin; Yeh, Yi-Chen; Lin, Chin-Hsuan; Hsu, Wei-Fang; Hsieh, Wen-Yu; Ho, Hsiang-Ling; Chou, Teh-Ying

2016-11-01

Analysis of the targetable driver mutations is now recommended in all patients with advanced lung adenocarcinoma. Molecular-based methods are usually adopted, however, along with the implementation of highly sensitive and/or mutation-specific antibodies, immunohistochemistry (IHC) has been considered an alternative method for identifying driver mutations in lung adenocarcinomas. A total of 205 lung adenocarcinomas were examined for EGFR mutations and ALK and ROS1 rearrangements using real-time PCR, fluorescence in situ hybridization (FISH) and IHC in parallel. The performance of different commercially available IHC antibody clones toward targetable driver mutations was evaluated. The association between these driver mutations and clinicopathological characteristics was also analyzed. In 205 cases we studied, 58.5% were found to harbor EGFR mutations, 6.3% ALK rearrangements and 1.0% ROS1 rearrangements. Compared to molecular-based methods, IHC of EGFR mutations showed an excellent specificity but the sensitivity is suboptimal, while IHC of ALK and ROS1 rearrangements demonstrated high sensitivity and specificity. No significant difference regarding the performance of different antibody clones toward these driver mutations was observed, except that clone SP125 showed a higher sensitivity than 43B2 in the detection of p.L858R of EGFR. In circumstances such as poor quality of nucleic acids or low content of tumor cells, IHC of EGFR mutation-specific antibodies could be used as an alternative method. Patients negative for EGFR mutations are subjected to further analysis on ALK and ROS1 rearrangements using IHC methods. Herein, we proposed a lung adenocarcinoma testing algorithm for the application of IHC in therapeutic diagnosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

[Condition optimization for bio-oxidation of high-S and high-As gold concentrate].

PubMed

Yang, Caiyun; Dong, Bowen; Wang, Meijun; Ye, Zhiyong; Zheng, Tianling; Huang, Huaiguo

2015-12-04

To study the effects of temperature and lixivium return on the concentrate bio-oxidation and rate of gold cyanide leaching. The bioleaching of a high-sulphur (S) and high-arsenic (As) refractory gold concentrate was conducted, and we studied the effects of different temperature (40 ° and 45 °C) and lixivium return (0 and 600 mL) on the bio-oxidation efficiency. The bacterial community structure also was investigated by 16S rRNA gene clone library. The results showed that both the temperature and lixivium return significantly influenced the oxidation system. The temperature rising elevated the oxidation level, while the addition of lixivium depressed the oxidation. Dissimilarity and DCA (detrended correspondence analysis) indicated the effect of temperature on oxidation system was much greater than lixivium. The bacterial community was comprised by Acidithiocacillus caldu (71%) Leptospirillum ferriphilum (23%) and Sulfobacillus thermosulfidooxidans (6%) indicated by the clone library, and the OTU coverage based on 97% sequence similarity was as high as 93.67%. Temperature rising to 45 T would improve the oxidation efficiency while lixivium return would decrease it. This study is helpful to provide an important guiding value for the industry cost optimization of mesophile bacterial oxidation and reduction process.

Derivation and Characterization of Pathogenic Transmitted/Founder Molecular Clones from Simian Immunodeficiency Virus SIVsmE660 and SIVmac251 following Mucosal Infection

PubMed Central

Lopker, Michael J.; Del Prete, Gregory Q.; Estes, Jacob D.; Li, Hui; Reid, Carolyn; Newman, Laura; Lipkey, Leslie; Camus, Celine; Easlick, Juliet L.; Wang, Shuyi; Decker, Julie M.; Bar, Katharine J.; Learn, Gerald; Pal, Ranajit; Weiss, Deborah E.; Hahn, Beatrice H.; Lifson, Jeffrey D.; Shaw, George M.

2016-01-01

ABSTRACT Currently available simian immunodeficiency virus (SIV) infectious molecular clones (IMCs) and isolates used in nonhuman primate (NHP) models of AIDS were originally derived from infected macaques during chronic infection or end stage disease and may not authentically recapitulate features of transmitted/founder (T/F) genomes that are of particular interest in transmission, pathogenesis, prevention, and treatment studies. We therefore generated and characterized T/F IMCs from genetically and biologically heterogeneous challenge stocks of SIVmac251 and SIVsmE660. Single-genome amplification (SGA) was used to identify full-length T/F genomes present in plasma during acute infection resulting from atraumatic rectal inoculation of Indian rhesus macaques with low doses of SIVmac251 or SIVsmE660. All 8 T/F clones yielded viruses that were infectious and replication competent in vitro, with replication kinetics similar to those of the widely used chronic-infection-derived IMCs SIVmac239 and SIVsmE543. Phenotypically, the new T/F virus strains exhibited a range of neutralization sensitivity profiles. Four T/F virus strains were inoculated into rhesus macaques, and each exhibited typical SIV replication kinetics. The SIVsm T/F viruses were sensitive to TRIM5α restriction. All T/F viruses were pathogenic in rhesus macaques, resulting in progressive CD4+ T cell loss in gastrointestinal tissues, peripheral blood, and lymphatic tissues. The animals developed pathological immune activation; lymphoid tissue damage, including fibrosis; and clinically significant immunodeficiency leading to AIDS-defining clinical endpoints. These T/F clones represent a new molecular platform for the analysis of virus transmission and immunopathogenesis and for the generation of novel “bar-coded” challenge viruses and next-generation simian-human immunodeficiency viruses that may advance the HIV/AIDS vaccine agenda. IMPORTANCE Nonhuman primate research has relied on only a few infectious molecular clones for a myriad of diverse research projects, including pathogenesis, preclinical vaccine evaluations, transmission, and host-versus-pathogen interactions. With new data suggesting a selected phenotype of the virus that causes infection (i.e., the transmitted/founder virus), we sought to generate and characterize infectious molecular clones from two widely used simian immunodeficiency virus lineages (SIVmac251 and SIVsmE660). Although the exact requirements necessary to be a T/F virus are not yet fully understood, we generated cloned viruses with all the necessary characteristic of a successful T/F virus. The cloned viruses revealed typical acute and set point viral-load dynamics with pathological immune activation, lymphoid tissue damage progressing to significant immunodeficiency, and AIDS-defining clinical endpoints in some animals. These T/F clones represent a new molecular platform for studies requiring authentic T/F viruses. PMID:27412591

Molecular Mechanism of MART-1+/A*0201+ Human Melanoma Resistance to Specific CTL-Killing Despite Functional Tumor-CTL Interaction

PubMed Central

Jazirehi, Ali R.; Baritaki, Stavroula; Koya, Richard C.; Bonavida, Benjamin; Economou, James S.

2014-01-01

Durable responses in metastatic melanoma patients remain generally difficult to achieve. Adoptive cell therapy with ex vivo engineered lymphocytes expressing high affinity T cell receptors TCRα/β for the melanoma antigen MART-127-35/HLA A*0201 (recognized by F5 cytotoxic T lymphocytes [F5 CTLs]) has been found to benefit certain patients. However, many other patients are inherently unresponsive and/or relapse for unknown reasons. To analyze the basis for the acquired-resistance and strategies to reverse it, we established F5 CTLresistant (R) human melanoma clones from relatively sensitive parental lines under selective F5 CTL pressure. Surface MART-127-35/HLA-A*0201 in these clones was unaltered and F5 CTLs recognized and interacted with them similarly to the parental lines. Nevertheless, the R clones were resistant to F5 CTL killing, exhibited hyperactivation of the NF-κB survival pathway, and overexpression of the anti-apoptotic genes Bcl-2, Bcl-xL and Mcl-1. Sensitivity to F5 CTL-killing could be increased by pharmacological inhibition of the NF-κB pathway, Bcl-2 family members, or the proteasome, the latter of which reduced NF-κB activity and diminished anti-apoptotic gene expression. Specific gene-silencing (by siRNA) confirmed the protective role of anti-apoptotic factors by reversing R clone resistance. Together, our findings suggest that long-term immunotherapy may impose a selection for the development of resistant cells that are unresponsive to highly avid and specific melanoma-reactive CTLs, despite maintaining expression of functional peptide:MHC complexes, due to activation of anti-apoptotic signaling pathways. Though unresponsive to CTL, our results argue that resistant cells can be re-sensitized to immunotherapy with co-administration of targeted inhibitors to anti-apoptotic survival pathways. PMID:21159666

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed Central

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-01-01

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

Transposon tagging of a male-sterility, female-sterility gene, St8, revealed that the meiotic MER3 DNA helicase activity is essential for fertility in soybean

USDA-ARS?s Scientific Manuscript database

The W4 locus in soybean encodes a dihydroflavonol-4-reductase (DFR2) that regulates pigmentation patterns in flowers and hypocotyl. The mutable w4-m allele that governs variegated flowers has arisen through insertion of a CACTA-type transposable element, Tgm9, in DFR2. In the w4-m line, reversion fr...

Multiplex Real-Time PCR for Monitoring Heterobasidion annosum Colonization in Norway Spruce Clones That Differ in Disease Resistance

PubMed Central

Hietala, Ari M.; Eikenes, Morten; Kvaalen, Harald; Solheim, Halvor; Fossdal, Carl G.

2003-01-01

A multiplex real-time PCR assay was developed to monitor the dynamics of the Picea abies-Heterobasidion annosum pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host. The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation (product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host. In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen. Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, whereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance. PMID:12902224

Isolation and characterization of dexamethasone-resistant mutants from human lymphoid cell line CEM-C7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harmon, J.M.; Thompson, E.B.

1981-06-01

Fifty-four independent dexamethasone-resistant clones were isolated from the clonal, glucocorticoid-sensitive human leukemic T-cell line CEM-C7. Resistance to 1 ..mu..M dexamethasone was acquired spontaneously at a rate of 2.6 x 10/sup -5/ per cell per generation as determined by fluctuation analysis. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the phenotypic expression time for dexamethasone resistance was determined to be 3 days. The mutagens ICR 191 and MNNG were effective in increasing the dexamethasone-resistant fraction of cells in mutagenized cultures; ICR 191 produced a 35.6-fold increase, and MNNG produced an 8.5-fold increase. All the spontaneous dexamethasone-resistant clones contained glucocorticoid receptors, usually less than halfmore » of the amount found in the parental clone. They are therefore strikingly different from dexamethasone-resistant clones derived from the mouse cell lines S49 and W7. Dexamethasone-resistant clones isolated after mutagenesis of CEM-C7 contained, on the average, lower concentrations of receptor than did those isolated spontaneously, and one clone contained no detectable receptor. These results are consistent with a mutational origin for dexamethasone resistance in these human cells at a haploid or functionally hemizygous locus. They also suggest that this is a useful system for mutation assay.« less

The Multi-Resistant Reaction of Drought-Tolerant Coffee 'Conilon Clone 14' to Meloidogyne spp. and Late Hypersensitive-Like Response in Coffea canephora.

PubMed

Lima, Edriana A; Furlanetto, Cleber; Nicole, Michel; Gomes, Ana C M M; Almeida, Maria R A; Jorge-Júnior, Aldemiro; Correa, Valdir R; Salgado, Sônia Maria; Ferrão, Maria A G; Carneiro, Regina M D G

2015-06-01

Root-knot nematodes (RKN), Meloidogyne spp., have major economic impact on coffee production in Central and South America. Genetic control of RKN constitutes an essential part for integrated pest management strategy. The objective of this study was to evaluate the resistance of Coffea canephora genotypes (clones) to Meloidogyne spp. Sensitive and drought-tolerant coffee genotypes were used to infer their resistance using nematode reproduction factor and histopathology. Eight clonal genotypes were highly resistant to M. paranaensis. 'Clone 14' (drought-tolerant) and 'ESN2010-04' were the only genotypes highly resistant and moderately resistant, respectively, to both M. incognita races 3 and 1. Several clones were highly resistant to both avirulent and virulent M. exigua. Clone 14 and ESN2010-04 showed multiple resistance to major RKNs tested. Roots of 'clone 14' (resistant) and 'clone 22' (susceptible) were histologically studied against infection by M. incognita race 3 and M. paranaensis. Reduction of juvenile (J2) penetration in clone 14 was first seen at 2 to 6 days after inoculation (DAI). Apparent early hypersensitive reaction (HR) was seen in root cortex between 4 and 6 DAI, which led to cell death and prevention of some nematode development. At 12 to 20 DAI, giant cells formed in the vascular cylinder, besides normal development into J3/J4. From 32 to 45 DAI, giant cells were completely degenerated. Late, intense HR and cell death were frequently observed around young females and giant cells reported for the first time in coffee pathosystem. These results provide rational bases for future studies, including prospection, characterization, and expression profiling of genomic loci involved in both drought tolerance and resistance to multiple RKN species.

Cloning, expression, and crystallization of Cpn60 proteins from Thermococcus litoralis.

PubMed

Osipiuk, J; Sriram, M; Mai, X; Adams, M W; Joachimiak, A

2000-01-01

Two genes of the extreme thermophilic archaeon Thermococcus litoralis homologous to those that code for Cpn60 chaperonins were cloned and expressed in Escherichia coli. Each of the Cpn60 subunits as well as the entire Cpn60 complex crystallize in a variety of morphological forms. The best crystals diffract to 3.6 A resolution at room temperature and belong to the space group 1422 with unit cell parameters a = b = 193.5 A, c = 204.2 A.

Genomic amplification of the human DHFR/MSH3 locus remodels mismatch recognition and repair activities.

PubMed

Drummond, J T

1999-01-01

Mismatch recognition in human cells is mediated by two heterodimers, MutS alpha and MutS beta. MutS alpha appears to shoulder primary responsibility for mismatch correction during replication, based on its relative abundance and ability to recognize a broad spectrum of base-base and base-insertion mismatches. Because MutS alpha and MutS beta share a common component, MSH2, conditions that influence the expression or degradation of MSH3 or MSH6 can redistribute the profile of mismatch recognition and repair. MSH3 is linked by a shared promoter with DHFR, connecting two pathways with key roles in DNA metabolism. In a classic example of gene amplification, the DHFR (and MSH3) locus can become amplified to several hundred copies in the presence of methotrexate. Under these conditions, MutS beta forms at the expense of MutS alpha, and the mutation rate in these tumor cells rises more than 100-fold. The implications for cancer chemotherapy include a potential increase in mutability when tumors are treated with methotrexate, which could increase the frequency of subsequent mutations that influence the tumor's drug sensitivity or aggressiveness. Because processing certain types of DNA damage by the mismatch repair pathway has also been implicated in tumor sensitivity to agents such as cisplatin, changes in expression at the DHFR/MSH3 locus may have further relevance to the outcome of multi-drug treatment regimens.

Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo.

PubMed

Bol, S A; van Steeg, H; van Oostrom, C T; Tates, A D; Vrieling, H; de Groot, A J; Mullenders, L H; van Zeeland, A A; Jansen, J G

1999-05-01

The butylating agent N-n-butyl-N-nitrosourea (BNU) was employed to study the role of nucleotide excision repair (NER) in protecting mammalian cells against the genotoxic effects of monofunctional alkylating agents. The direct acting agent BNU was found to be mutagenic in normal and XPA mouse splenocytes after a single i.p. treatment in vivo. After 25 and 35 mg/kg BNU, but not after 75 mg/ kg, 2- to 3-fold more hprt mutants were detected in splenocytes from XPA mice than from normal mice. Using O6-alkylguanine-DNA alkyltransferase (AGT)-deficient hamster cells, it was found that NER-deficient CHO UV5 cells carrying a mutation in the ERCC-2 gene were 40% more mutable towards lesions induced by BNU when compared with parental NER-proficient CHO AA8 cells. UV5 cells were 1.4-fold more sensitive to the cytotoxic effects of BNU compared with AA8 cells. To investigate whether this increased sensitivity of NER-deficient cells is modulated by AGT activity, cell survival studies were performed in human and mouse primary fibroblasts as well. BNU was 2.7-fold more toxic for mouse XPA fibroblasts compared with normal mouse fibroblasts. Comparable results were found for human fibroblasts. Taken together these data indicate that the role of NER in protecting rodent cells against the mutagenic and cytotoxic effects of the alkylating agent BNU depends on AGT.

Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meining, Winfried, E-mail: wim@csb.ki.se; Scheuring, Johannes; Fischer, Markus

2006-06-01

SecA ATPase from E. faecalis has been cloned, overexpressed, purified and crystallized. Crystals belong to space group C2 and diffract to 2.4 Å resolution. The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 Å resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 Å,more » α = γ = 90.0, β = 119.1°. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.« less

A high-throughput screen for single gene activities: isolation of apoptosis inducers.

PubMed

Albayrak, Timur; Grimm, Stefan

2003-05-16

We describe a novel genetic screen that is performed by transfecting every individual clone of an expression library into a separate population of cells in a high-throughput mode. The screen allows one to achieve a hitherto unattained sensitivity in expression cloning which was exploited in a first read-out to clone apoptosis-inducing genes. This led to the isolation of several genes whose proteins induce distinct phenotypes of apoptosis in 293T cells. One of the isolated genes is the tumor suppressor cytochrome b(L) (cybL), a component of the respiratory chain complex II, that diminishes the activity of this complex for apoptosis induction. This gene is more efficient and specific for causing cell death than a drug with the same activity. These results suggest further applications, both of the isolated genes and the screen.

Genomics and Ethics: The Case of Cloned and/or Transgenic Animals

PubMed Central

2003-01-01

The point of the present study is to illustrate and, if possible, promote the existing link between genomics and ethics, taking the example of cloned and transgenic animals. These ‘new animals’ raise theoretical and practical problems that concern applied ethics. We will explore more particularly an original strategy showing that it is possible, starting from philosophical questioning about the nature of identity, to use a genomic approach, based on amplification fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) detection, to provide useful tools to define more rigorously what cloned animals are, by testing their genetic and epigenetic identity. We expect from the future results of this combined approach to stimulate the creativity of the philosophical and ethical reflection about the impact of biotechnology on animals, and to increase scientific involvement in such issues. PMID:18629111

Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes.

PubMed Central

Santangelo, J D; Jones, D T; Woods, D R

1991-01-01

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum. Images PMID:1991710

Elucidation of cross-species proteomic effects in human and hominin bone proteome identification through a bioinformatics experiment.

PubMed

Welker, F

2018-02-20

The study of ancient protein sequences is increasingly focused on the analysis of older samples, including those of ancient hominins. The analysis of such ancient proteomes thereby potentially suffers from "cross-species proteomic effects": the loss of peptide and protein identifications at increased evolutionary distances due to a larger number of protein sequence differences between the database sequence and the analyzed organism. Error-tolerant proteomic search algorithms should theoretically overcome this problem at both the peptide and protein level; however, this has not been demonstrated. If error-tolerant searches do not overcome the cross-species proteomic issue then there might be inherent biases in the identified proteomes. Here, a bioinformatics experiment is performed to test this using a set of modern human bone proteomes and three independent searches against sequence databases at increasing evolutionary distances: the human (0 Ma), chimpanzee (6-8 Ma) and orangutan (16-17 Ma) reference proteomes, respectively. Incorrectly suggested amino acid substitutions are absent when employing adequate filtering criteria for mutable Peptide Spectrum Matches (PSMs), but roughly half of the mutable PSMs were not recovered. As a result, peptide and protein identification rates are higher in error-tolerant mode compared to non-error-tolerant searches but did not recover protein identifications completely. Data indicates that peptide length and the number of mutations between the target and database sequences are the main factors influencing mutable PSM identification. The error-tolerant results suggest that the cross-species proteomics problem is not overcome at increasing evolutionary distances, even at the protein level. Peptide and protein loss has the potential to significantly impact divergence dating and proteome comparisons when using ancient samples as there is a bias towards the identification of conserved sequences and proteins. Effects are minimized between moderately divergent proteomes, as indicated by almost complete recovery of informative positions in the search against the chimpanzee proteome (≈90%, 6-8 Ma). This provides a bioinformatic background to future phylogenetic and proteomic analysis of ancient hominin proteomes, including the future description of novel hominin amino acid sequences, but also has negative implications for the study of fast-evolving proteins in hominins, non-hominin animals, and ancient bacterial proteins in evolutionary contexts.

Seasonal Changes in Carbohydrates and Ascorbic Acid and White Pine and Possible Relation to Tipburn Sensitivity

Treesearch

Robert L Barnes; Charles R. Berry

1969-01-01

Changes in amounts of total soluble carbohydrates and ascorbic acid were related to needle length of eastern white pine during June and July 1967 at Bent Creek Experimental Forest. Sugar values remained low through the early growing season, and several instances of injury to clones sensitive to tipburn occurred as late as mid-July. Sugar levels fluctuated somewhat,...

Spread of carbapenem-resistant international clones of Acinetobacter baumannii in Turkey and Azerbaijan: a collaborative study.

PubMed

Ahmed, S S; Alp, E; Ulu-Kilic, A; Dinc, G; Aktas, Z; Ada, B; Bagirova, F; Baran, I; Ersoy, Y; Esen, S; Guven, T G; Hopman, J; Hosoglu, S; Koksal, F; Parlak, E; Yalcin, A N; Yilmaz, G; Voss, A; Melchers, W

2016-09-01

Epidemic clones of Acinetobacter baumannii, described as European clones I, II, and III, are associated with hospital epidemics throughout the world. We aimed to determine the molecular characteristics and genetic diversity between European clones I, II, and III from Turkey and Azerbaijan. In this study, a total of 112 bloodstream isolates of carbapenem-resistant Acinetobacter spp. were collected from 11 hospitals across Turkey and Azerbaijan. The identification of Acinetobacter spp. using conventional and sensitivity tests was performed by standard criteria. Multiplex polymerase chain reaction (PCR) was used to detect OXA carbapenemase-encoding genes (bla OXA-23-like, bla OXA-24-like, bla OXA-51-like, and bla OXA-58-like). Pulsed-field gel electrophoresis (PFGE) typing was used to investigate genetic diversity. The bla OXA-51-like gene was present in all 112 isolates, 75 (67 %) carried bla OXA-23-like, 7 (6.2 %) carried bla OXA-58-like genes, and 5 (4.5 %) carried bla OXA-24-like genes. With a 90 % similarity cut-off value, 15 clones and eight unique isolates were identified. The largest clone was cluster D, with six subtypes. Isolates from clusters D and I were widely spread in seven different geographical regions throughout Turkey. However, F cluster was found in the northern and eastern regions of Turkey. EU clone I was grouped within J cluster with three isolates found in Antalya, Istanbul, and Erzurum. EU clone II was grouped in the U cluster with 15 isolates and found in Kayseri and Diyarbakır. The bla OXA-24-like gene in carbapenemases was identified rarely in Turkey and has been reported for the first time from Azerbaijan. Furthermore, this is the first multicenter study in Turkey and Azerbaijan to identify several major clusters belonging to European clones I and II of A. baumannii.

Evaluation of Interferon Resistance in Newly Established Genotype 1b Hepatitis C Virus Cell Culture System

PubMed Central

Taniguchi, Miki; Tasaka-Fujita, Megumi; Nakagawa, Mina; Watanabe, Takako; Kawai-Kitahata, Fukiko; Otani, Satoshi; Goto, Fumio; Nagata, Hiroko; Kaneko, Shun; Nitta, Sayuri; Murakawa, Miyako; Nishimura-Sakurai, Yuki; Azuma, Seishin; Itsui, Yasuhiro; Mori, Kenichi; Yagi, Shintaro; Kakinuma, Sei; Asahina, Yasuhiro; Watanabe, Mamoru

2016-01-01

Background and Aims: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. Methods: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. Results were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. Results: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). Conclusions: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment. PMID:27047766

Aldo-keto reductase and alcohol dehydrogenase contribute to benznidazole natural resistance in Trypanosoma cruzi.

PubMed

González, Laura; García-Huertas, Paola; Triana-Chávez, Omar; García, Gabriela Andrea; Murta, Silvane Maria Fonseca; Mejía-Jaramillo, Ana M

2017-12-01

The improvement of Chagas disease treatment is focused not only on the development of new drugs but also in understanding mechanisms of action and resistance to drugs conventionally used. Thus, some strategies aim to detect specific changes in proteins between sensitive and resistant parasites and to evaluate the role played in these processes by functional genomics. In this work, we used a natural Trypanosoma cruzi population resistant to benznidazole, which has clones with different susceptibilities to this drug without alterations in the NTR I gene. Using 2DE-gel electrophoresis, the aldo-keto reductase and the alcohol dehydrogenase proteins were found up regulated in the natural resistant clone and therefore their possible role in the resistance to benznidazole and glyoxal was investigated. Both genes were overexpressed in a drug sensitive T. cruzi clone and the biological changes in response to these compounds were evaluated. The results showed that the overexpression of these proteins enhances resistance to benznidazole and glyoxal in T. cruzi. Moreover, a decrease in mitochondrial and cell membrane damage was observed, accompanied by a drop in the intracellular concentration of reactive oxygen species after treatment. Our results suggest that these proteins are involved in the mechanism of action of benznidazole. © 2017 John Wiley & Sons Ltd.

Identification of a STOP1-like protein in Eucalyptus that regulates transcription of Al tolerance genes.

PubMed

Sawaki, Yoshiharu; Kobayashi, Yuriko; Kihara-Doi, Tomonori; Nishikubo, Nobuyuki; Kawazu, Tetsu; Kobayashi, Masatomo; Kobayashi, Yasufumi; Iuchi, Satoshi; Koyama, Hiroyuki; Sato, Shigeru

2014-06-01

Tolerance to soil acidity is an important trait for eucalyptus clones that are introduced to commercial forestry plantations in pacific Asian countries, where acidic soil is dominant in many locations. A conserved transcription factor regulating aluminum (Al) and proton (H⁺) tolerance in land-plant species, STOP1 (SENSITIVE TOPROTON RHIZOTOXICITY 1)-like protein, was isolated by polymerase chain reaction-based cloning, and then suppressed by RNA interference in hairy roots produced by Agrobacterium rhizogenes-mediated transformation. Eucalyptus STOP1-like protein complemented proton tolerance in an Arabidopsis thaliana stop1-mutant, and localized to the nucleus in a transient assay of a green fluorescent protein fusion protein expressed in tobacco leaves by Agrobacterium tumefaciens-mediated transformation. Genes encoding a citrate transporting MULTIDRUGS AND TOXIC COMPOUND EXTRUSION protein and an orthologue of ALUMINUM SENSITIVE 3 were suppressed in transgenic hairy roots in which the STOP1 orthologue was knocked down. In summary, we identified a series of genes for Al-tolerance in eucalyptus, including a gene for STOP1-like protein and the Al-tolerance genes it regulates. These genes may be useful for molecular breeding and genomic selection of elite clones to introduce into acid soil regions. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Hierarchical Structures in Biology as a Guide for New Materials Technology

DTIC Science & Technology

1994-01-01

properties--to changes in performance requirements. For example, while the spines of the sea urchin are moving, the attached ligaments are soft and...by studying how sea urchins control the material properties of their bodies as a function of the local environment. Sea stars, sea urchins , and sea ...Materials Research Society Bulletin 16(7):23-28. Trotter, J. A., and T. J. Koob. 1989. Collagen and proteoglycan in a sea urchin ligament with mutable

In Vitro Determination of Bicarbonate Dosage to Alkalinize Local Anesthetics to Physiologic pH

DTIC Science & Technology

1997-05-01

as "an unpleasant sensory and emotional experience associated with actual damage or injury to a body tissue" (Mersky, 1986, p.SI). However, this...definition does not adequately express the unique, mutable and subjective experience that those who suffer pain encounter. Milton alludes to the...emotional experience of pain in Paradise Lost when he writes "Pain is perfect miserie, the worst of evils, and excessive, overturns all patience" (Milton

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method.

PubMed

Sharma, Nandita; Tanksale, Himgouri; Kapley, Atya; Purohit, Hemant J

2012-12-01

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.

Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy

PubMed Central

Levin, Mattias; King, Jasmine J.; Glanville, Jacob; Jackson, Katherine J. L.; Looney, Timothy J.; Hoh, Ramona A.; Mari, Adriano; Andersson, Morgan; Greiff, Lennart; Fire, Andrew Z.; Boyd, Scott D.; Ohlin, Mats

2016-01-01

Background Specific immunotherapy (SIT) is the only treatment with proven long-term curative potential in allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B cell repertoires are not well understood. Objective To characterize the IgE sequences expressed by allergen-specific B cells, and track the fate of these B cell clones during SIT. Methods We have used high-throughput antibody gene sequencing and identification of allergen-specific IgE using combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and nasal mucosa of aeroallergen-sensitized individuals before and during the first year of subcutaneous SIT. Results Of 52 distinct allergen-specific IgE heavy chains from eight allergic donors, 37 were also detected by high-throughput antibody gene sequencing of blood, nasal mucosa, or both sample types. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships. Conclusion Combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies may in the future aid assessment of SIT mechanisms and efficacy. PMID:26559321

CD34 expression in human hair follicles and tricholemmoma: a comprehensive study.

PubMed

Misago, Noriyuki; Toda, Shuji; Narisawa, Yutaka

2011-08-01

There has recently been controversy regarding whether clone My10 is superior to clone QBEND-10 for labeling cells of tricholemmal lineage. Moreover, there have been no previous reports on the CD34 expression in human vellus hair follicles. We performed a comprehensive study of the CD34 expression in human terminal and vellus hair follicles and in 10 tricholemmomas using both the QBEND-10 and the My10 clones. We also performed two different procedures of immunostaining, which included the using of the standard avidin-biotin-peroxidase (ABC) complex system and the Envision system. The most sensitive marker of CD34 for normal human hair follicles and tricholemmomas is QBEND-10 using the ABC system. The degree and strength of the CD34 positive staining mainly depended on the method being used (whether it was the ABC system or the Envision system) rather than the clone. CD34 staining was rarely (20-30%) seen in the anagen and catagen vellus hair follicles, and could only be seen by the QBEND-10 clone using the ABC system. CD34 expression in the tricholemmomas represented either a diffuse or peripheral pattern. CD34 may not be a tricholemmal lineage-specific antigen, but may be related to certain functions of the cells. Copyright © 2011 John Wiley & Sons A/S.

Preparation and characterization of specific and high-affinity monoclonal antibodies against morphine.

PubMed

Rahbarizadeh, F; Rasaee, M J; Madani, R; Rahbarizadeh, M H; Omidfar, K

2000-10-01

A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. A C3-hemisuccinate derivative of morphine was prepared and conjugated to enzyme penicillinase used as a tracer molecule. A novel enzyme-linked immunoadsorbent assay was developed using this conjugate to screen and characterize the monoclonal antibody produced in these experiments. After two successive limiting dilutions, antibodies produced by 5 clones with good affinities ranging from 10(8) to 10(12) M(-1) and less cross-reaction (least for codeine and other structurally related molecules) were selected. These clones were found to be of IgG class with kappa light chain. Subclass determination showed that two of the clones produced IgG2b and three of them produced IgG1 type of antibody. Affinity purifications were performed for the selected clone (MOR-I). Purified antibody was coated onto the wells of microtiter plate. The standard curve was constructed with a sensitivity of 100 pg/mL covering up to 10 ng/mL in buffer and urine. The slope of the standard curve for selected clone in buffer and urine was calculated to be -0.7 and -0.64, respectively.

Genomic analysis of genetic heterogeneity and evolution in high-grade serous ovarian carcinoma

PubMed Central

Cooke, Susanna L; Ng, Charlotte KY; Melnyk, Nataliya; Garcia, Maria J; Hardcastle, Tom; Temple, Jillian; Langdon, Simon; Huntsman, David; Brenton, James D

2010-01-01

Resistance to chemotherapy in ovarian cancer is poorly understood. Evolutionary models of cancer predict that, following treatment, resistance emerges either due to outgrowth of an intrinsically resistant sub-clone, or evolves in residual disease under the selective pressure of treatment. To investigate genetic evolution in high-grade serous (HGS) ovarian cancers we first analysed cell line series derived from three cases of HGS carcinoma before and after platinum resistance had developed (PEO1, PEO4 and PEO6, PEA1 and PEA2, and PEO14 and PEO23). Analysis with 24-colour fluorescence in situ hybridisation and SNP array comparative genomic hybridisation (CGH) showed mutually exclusive endoreduplication and loss of heterozygosity events in clones present at different timepoints in the same individual. This implies that platinum sensitive and resistant disease was not linearly related but shared a common ancestor at an early stage of tumour development. Array CGH analysis of six paired pre- and post-neoadjuvant treatment HGS samples from the CTCR-OV01 clinical study did not show extensive copy number differences, suggesting that one clone was strongly dominant at presentation. These data show that cisplatin resistance in HGS carcinoma develops from pre-existing minor clones but that enrichment for these clones is not apparent during short-term chemotherapy treatment. PMID:20581869

Lineage-tracking of stem cell differentiation: a neutral model of hematopoiesis in rhesus macaque

NASA Astrophysics Data System (ADS)

Chou, Tom

How a potentially diverse population of hematopoietic stem cells (HSCs) differentiates and proliferates to supply more than 1011 mature blood cells every day in humans remains a key biological question. We investigated this process by quantitatively analyzing the clonal structure of peripheral blood that is generated by a population of transplanted lentivirus-marked HSCs in myeloablated rhesus macaques. Each transplanted HSC generates a clonal lineage of cells in the peripheral blood that is then detected and quantified through deep sequencing of the viral vector integration sites (VIS) common within each lineage. This approach allowed us to observe, over a period of 4-12 years, hundreds of distinct clonal lineages. Surprisingly, while the distinct clone sizes varied by three orders of magnitude, we found that collectively, they form a steady-state clone size-distribution with a distinctive shape. Our concise model shows that slow HSC differentiation followed by fast progenitor growth is responsible for the observed broad clone size-distribution. Although all cells are assumed to be statistically identical, analogous to a neutral theory for the different clone lineages, our mathematical approach captures the intrinsic variability in the times to HSC differentiation after transplantation. Steady-state solutions of our model show that the predicted clone size-distribution is sensitive to only two combinations of parameters. By fitting the measured clone size-distributions to our mechanistic model, we estimate both the effective HSC differentiation rate and the number of active HSCs. NSF and NIH.

Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes.

PubMed

Owor, Betty E; Shepherd, Dionne N; Taylor, Nigel J; Edema, Richard; Monjane, Adérito L; Thomson, Jennifer A; Martin, Darren P; Varsani, Arvind

2007-03-01

Leaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers' fields in 23 districts in Uganda in May/June 2005 by leaf-pressing infected samples onto FTA Classic Cards. Viral DNA was successfully extracted from cards stored at room temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage phi29 DNA polymerase using the TempliPhi system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced. Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned and that no errors were introduced by the phi29 polymerase. This is the first successful large-scale application of FTA card technology to the field, and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as diversity analysis and cloning of potentially new virus genomes.

Revival of extinct species using nuclear transfer: hope for the mammoth, true for the Pyrenean ibex, but is it time for "conservation cloning"?

PubMed

Piña-Aguilar, Raul E; Lopez-Saucedo, Janet; Sheffield, Richard; Ruiz-Galaz, Lilia I; Barroso-Padilla, Jose de J; Gutiérrez-Gutiérrez, Antonio

2009-09-01

Recent accomplishments in the fields of nuclear transfer and genomics, such as the cloned offspring production from frozen mouse cells, cryopreserved at not too low temperatures without cryoprotectors; or the sequencing of wooly mammoth genome, have opened the opportunity for the revival of extinct species. As expected, they are receiving a lot of publicity in the media and also scientific attention. Furthermore, it was recently published the "revival" of the first extinct subspecie: the Pyrenean ibex (Capra pyrenaica pyrenaica), a wild goat extinct in 2000. This strengthens the field of cloning as it had been tarnished by induced pluripotent stem cells (iPS) and other methods of reprogramming. However, for biological conservation purposes, cloning is not generally accepted as an alternative for animal conservation, and there is an ongoing debate between reproductive scientists and conservation specialists. Although we believe that nuclear transfer technologies have an opportunity in conservation efforts for some species that are on the brink of extinction and that population status, geographical isolation, reproductive characteristics, and human pressure create a situation that is almost unsustainable. In this article we discuss the barriers in cloning mammoths and cloning controversies in conservation from a zoological perspective, citing the species that might benefit from nuclear transfer techniques in the arduous journey so as not to disappear forever from this, our world.

Novel Prunus rootstock somaclonal variants with divergent ability to tolerate waterlogging.

PubMed

Pistelli, Laura; Iacona, Calogero; Miano, Dario; Cirilli, Marco; Colao, Maria Chiara; Mensuali-Sodi, Anna; Muleo, Rosario

2012-03-01

Plants require access to free water for nutrient uptake, but excess water surrounding the roots can be injurious or even lethal because it blocks the transfer of free oxygen between the soil and the atmosphere. Genetic improvement efforts in this study were focused on the increased tolerance in roots to waterlogging. Among a pool of clones generated in vitro from leaf explants of rootstock Mr.S.2/5 of Prunus cerasifera L., the S.4 clone was flood tolerant whereas the S.1 clone was sensitive. The S.4 clone formed adventitious roots on exposure to flooding. Moreover, the chlorophyll content and mitochondrial activity in the leaf and root, soluble sugar content, alcohol dehydrogenase activity and ethylene content were different between the clones. The sorbitol transporter gene (SOT1) was up-regulated during hypoxia, the alcohol dehydrogenase genes (ADH1 and ADH3) were up-regulated in the leaves and down-regulated in the roots of the S.4 clone during hypoxia, and the 1-aminocyclopropane-1-oxidase gene (ACO1) was up-regulated in the leaves and roots of the S.4 clone during hypoxia and down-regulated in the wild-type roots. In addition, in the S.4 root, hypoxia induced significant down-regulation of a glycosyltransferase-like gene (GTL), which has a yet-undefined role. Although the relevant variation in the S.4 genome has yet to be determined, genetic alteration clearly conferred a flooding-tolerant phenotype. The isolation of novel somaclonals with the same genomic background but with divergent tolerance to flooding may offer new insights in the elucidation of the genetic machinery of resistance to flooding and aid in the selection of new Prunus rootstocks to be used in various adverse environments.

Elevated ozone affects C, N and P ecological stoichiometry and nutrient resorption of two poplar clones.

PubMed

Shang, Bo; Feng, Zhaozhong; Li, Pin; Calatayud, Vicent

2018-03-01

The effects of elevated ozone on C (carbon), N (nitrogen) and P (phosphorus) ecological stoichiometry and nutrient resorption in different organs including leaves, stems and roots were investigated in poplar clones 546 (P. deltoides cv. '55/56' × P. deltoides cv. 'Imperial') and 107 (P. euramericana cv. '74/76') with a different sensitivity to ozone. Plants were exposed to two ozone treatments, NF (non-filtered ambient air) and NF60 (NF with targeted ozone addition of 60 ppb), for 96 days in open top chambers (OTCs). Significant ozone effects on most variables of C, N and P ecological stoichiometry were found except for the C concentration and the N/P in different organs. Elevated ozone increased both N and P concentrations of individual organs while for C/N and C/P ratios a reduction was observed. On these variables, ozone had a greater effect for clone 546 than for clone 107. N concentrations of different leaf positions ranked in the order upper > middle > lower, showing that N was transferred from the lower senescent leaves to the upper ones. This was also indicative of N resorption processes, which increased under elevated ozone. N resorption of clone 546 was 4 times larger than that of clone 107 under ambient air (NF). However, elevated ozone (NF60) had no significant effect on P resorption for both poplar clones, suggesting that their growth was only limited by N, while available P in the soil was enough to sustain growth. Understanding ecological stoichiometric responses under ozone stress is crucial to predict future effects on ecological processes and biogeochemical cycles. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii▿ †

PubMed Central

Brede, Dag Anders; Lothe, Sheba; Salehian, Zhian; Faye, Therese; Nes, Ingolf F.

2007-01-01

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 107 transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive PpampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ∼91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains. PMID:17933941

Identification of the propionicin F bacteriocin immunity gene (pcfI) and development of a food-grade cloning system for Propionibacterium freudenreichii.

PubMed

Brede, Dag Anders; Lothe, Sheba; Salehian, Zhian; Faye, Therese; Nes, Ingolf F

2007-12-01

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 10(7) transformants/microg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive P(pampS) promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by approximately 91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody-Ligand Interaction Using Chain Shuffling.

PubMed

Rangnoi, Kuntalee; Choowongkomon, Kiattawee; O'Kennedy, Richard; Rüker, Florian; Yamabhai, Montarop

2018-06-06

A human antiaflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a naı̈ve human phage-displayed scFv library, was used as a template for improving and analysis of antibody-ligand interactions using the chain-shuffling technique. The variable-heavy and variable-light (VH/VL)-shuffled library was constructed from the VH of 25 preselected clones recombined with the VL of yAFB1-c3 and vice versa. Affinity selection from these libraries demonstrated that the VH domain played an important role in the binding of scFv to free AFB1. Therefore, in the next step, VH-shuffled scFv library was constructed from variable-heavy (VH) chain repertoires, amplified from the naı̈ve library, recombined with the variable-light (VL) chain of the clone yAFB1-c3. This library was then used to select a specific scFv antibody against soluble AFB1 by a standard biopanning method. Three clones that showed improved binding properties were isolated. Amino acid sequence analysis indicated that the improved clones have amino acid mutations in framework 1 (FR1) and the complementarity determining region (CDR1) of the VH chain. One clone, designated sAFH-3e3, showed 7.5-fold improvement in sensitivity over the original scFv clone and was selected for molecular binding studies with AFB1. Homology modeling and molecular docking were used to compare the binding of this and the original clones. The results confirmed that VH is more important than VL for AFB1 binding.

Competitive release and facilitation of drug-resistant parasites after therapeutic chemotherapy in a rodent malaria model

USGS Publications Warehouse

Wargo, A.R.; Huijben, S.; De Roode, J. C.; Shepherd, J.; Read, A.F.

2007-01-01

Malaria infections frequently consist of mixtures of drug-resistant and drug-sensitive parasites. If crowding occurs, where clonal population densities are suppressed by the presence of coinfecting clones, removal of susceptible clones by drug treatment could allow resistant clones to expand into the newly vacated niche space within a host. Theoretical models show that, if such competitive release occurs, it can be a potent contributor to the strength of selection, greatly accelerating the rate at which resistance spreads in a population. A variety of correlational field data suggest that competitive release could occur in human malaria populations, but direct evidence cannot be ethically obtained from human infections. Here we show competitive release after pyrimethamine curative chemotherapy of acute infections of the rodent malaria Plasmodium chabaudi in laboratory mice. The expansion of resistant parasite numbers after treatment resulted in enhanced transmission-stage densities. After the elimination or near-elimination of sensitive parasites, the number of resistant parasites increased beyond that achieved when a competitor had never been present. Thus, a substantial competitive release occurred, markedly elevating the fitness advantages of drug resistance above those arising from survival alone. This finding may explain the rapid spread of drug resistance and the subsequently brief useful lifespans of some antimalarial drugs. In a second experiment, where subcurative chemotherapy was administered, the resistant clone was only partly released from competitive suppression and experienced a restriction in the size of its expansion after treatment. This finding raises the prospect of harnessing in-host ecology to slow the spread of drug resistance. ?? 2007 by The National Academy of Sciences of the USA.

Changes in the level of perforin and its transcript during effector and target cell interactions.

PubMed

Kim, K K; Blakely, A; Zhou, Z; Davis, J; Clark, W; Kwon, B S

1993-05-01

Perforin is a cytoplasmic granule protein expressed in cytotoxic lymphocytes, and is capable of lysing target cells. This protein is induced as cytotoxic T cells are activated, and the mRNA expression is modulated by various stimulators. These observations suggest possible changes in the level of perforin transcripts and protein when killer lymphocytes meet specific target cells leading to target cell death. To address this question, we examined three murine T-cell clones and primary human NK cells in perforin expression. When the cytotoxic lymphocytes were exposed to sensitive targets, perforin mRNA disappeared within 5 to 30 min and appeared within an hour thereafter. Among the murine T cell clones, L3 and OE4 showed two phases of mRNA decrease while human NK cells and the third murine T cell clone, AB.1, showed only one phase of mRNA loss during a 240 min period. The data indicate that when cytotoxic lymphocytes receive signals from a sensitive target, the cells rapidly degrade previously accumulated perforin mRNA and synthesize new transcripts. Interestingly, heat shock protein 70 mRNA was induced as the perforin mRNA levels recovered, while P55 Il-2 receptor mRNA was downregulated within 5 min after exposure to targets. The perforin protein level also rapidly decreased immediately after the interaction with the target, followed by a recovery, and then another decrease as seen in primary human NK cells, OE4 and L3 cells. However, in the AB.1 clone, no change in perforin content was detectable, despite the loss of perforin mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

[Antimutagenic activity of Armoracia rusticana, Zea mays and Ficus carica plant extracts and their mixture].

PubMed

Agabeĭli, R A; Kasimova, T E

2005-01-01

Antimutagenic action of plant extracts of Armoracia rusticana, Ficus carica, Zea mays and their mixture on environmental xenobiotics has been investigated. The plant extracts and their mixture decreased the level of mutations induced by N-metil-N'-nitro-N-nitrozoguanidin (MNNG) in Vicia faba cells, chlorophyll mutations in Arabidopsis thaliana and NaF induced mutability in rat marrow cells. The studied plant extracts and their mixture demonstrate the ability to decrease the genotoxicity of environmental mutagens.

Host Immunity via Mutable Virtualized Large-Scale Network Containers

DTIC Science & Technology

2016-07-25

and constrain the distributed persistent inside crawlers that have va.lid credentials to access the web services. The main idea is to add a marker...to each web page URL and use the URL path and user inforn1ation contained in the marker to help accurately detect crawlers at its earliest stage...more than half of all website traffic, and malicious bots contributes almost one third of the traffic. As one type of bots, web crawlers have been

Energy: Between Physics and Metaphysics

NASA Astrophysics Data System (ADS)

Bunge, Mario

The general concept of energy is somewhat unclear as long as it is confined to physics, since every chapter of it defines its own particular concept of energy. The general concept can be elucidated in terms of the hypergeneral (philosophical) concepts of concrete thing and changeability. In this way one succeeds in crafting a minitheory that identifies energy with mutability, and that regards it, as well as its conservation, as a universal property of concrete things. The moral is that physicists and philosophers can learn from one another.

Isolation and Characterization of Chromosome-Gain and Increase-in-Ploidy Mutants in Yeast

PubMed Central

Chan, CSM.; Botstein, D.

1993-01-01

We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37°. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37°. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth. PMID:8293973

The L1-type cell adhesion molecule neuroglian influences the stability of neural ankyrin in the Drosophila embryo but not its axonal localization.

PubMed

Bouley, M; Tian, M Z; Paisley, K; Shen, Y C; Malhotra, J D; Hortsch, M

2000-06-15

Ankyrins are linker proteins, which connect various membrane proteins, including members of the L1 family of neural cell adhesion molecules, with the submembranous actin-spectrin skeleton. Here we report the cloning and characterization of a second, novel Drosophila ankyrin gene (Dank2) that appears to be the result of a gene duplication event during arthropod evolution. The Drosophila L1-type protein neuroglian interacts with products from both Drosophila ankyrin genes. Whereas the previously described ankyrin gene is ubiquitously expressed during embryogenesis, the expression of Dank2 is restricted to the nervous system in the Drosophila embryo. The absence of neuroglian protein in a neuroglian null mutant line causes decreased levels of Dank2 protein in most neuronal cells. This suggests that neuroglian is important for the stability of Dank2 protein. However, neuroglian is not required for Dank2 axonal localization. In temperature-sensitive neuroglian mutants in which neuroglian protein is mislocated at the restrictive temperature to an intracellular location in the neuronal soma, Dank2 protein can still be detected along embryonic nerve tracts.

Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs.

PubMed

Orphan, V J; Taylor, L T; Hafenbradl, D; Delong, E F

2000-02-01

Recent investigations of oil reservoirs in a variety of locales have indicated that these habitats may harbor active thermophilic prokaryotic assemblages. In this study, we used both molecular and culture-based methods to characterize prokaryotic consortia associated with high-temperature, sulfur-rich oil reservoirs in California. Enrichment cultures designed for anaerobic thermophiles, both autotrophic and heterotrophic, were successful at temperatures ranging from 60 to 90 degrees C. Heterotrophic enrichments from all sites yielded sheathed rods (Thermotogales), pleomorphic rods resembling Thermoanaerobacter, and Thermococcus-like isolates. The predominant autotrophic microorganisms recovered from inorganic enrichments using H(2), acetate, and CO(2) as energy and carbon sources were methanogens, including isolates closely related to Methanobacterium, Methanococcus, and Methanoculleus species. Two 16S rRNA gene (rDNA) libraries were generated from total community DNA collected from production wellheads, using either archaeal or universal oligonucleotide primer sets. Sequence analysis of the universal library indicated that a large percentage of clones were highly similar to known bacterial and archaeal isolates recovered from similar habitats. Represented genera in rDNA clone libraries included Thermoanaerobacter, Thermococcus, Desulfothiovibrio, Aminobacterium, Acidaminococcus, Pseudomonas, Halomonas, Acinetobacter, Sphingomonas, Methylobacterium, and Desulfomicrobium. The archaeal library was dominated by methanogen-like rDNAs, with a lower percentage of clones belonging to the Thermococcales. Our results strongly support the hypothesis that sulfur-utilizing and methane-producing thermophilic microorganisms have a widespread distribution in oil reservoirs and the potential to actively participate in the biogeochemical transformation of carbon, hydrogen, and sulfur in situ.

Cloning, characterization and functional analysis of a 1-FEH cDNA from Vernonia herbacea (Vell.) Rusby.

PubMed

Asega, Amanda Francine; do Nascimento, João Roberto O; Schroeven, Lindsey; Van den Ende, Wim; Carvalho, Maria Angela M

2008-08-01

Variations in the inulin contents have been detected in rhizophores of Vernonia herbacea during the phenological cycle. These variations indicate the occurrence of active inulin synthesis and depolymerization throughout the cycle and a role for this carbohydrate as a reserve compound. 1-Fructan exohydrolase (1-FEH) is the enzyme responsible for inulin depolymerization, and its activity has been detected in rhizophores of sprouting plants. Defoliation and low temperature are enhancer conditions of this 1-FEH activity. The aim of the present work was the cloning of this enzyme. Rhizophores were collected from plants induced to sprout, followed by storage at 5 degrees C. A full length 1-FEH cDNA sequence was obtained by PCR and inverse PCR techniques, and expressed in Pichia pastoris. Cold storage enhances FEH gene expression. Vh1-FEH was shown to be a functional 1-FEH, hydrolyzing predominantly beta-2,1 linkages, sharing high identity with chicory FEH sequences, and its activity was inhibited by 81% in the presence of 10 mM sucrose. In V. herbacea, low temperature and sucrose play a role in the control of fructan degradation. This is the first study concerning the cloning and functional analysis of a 1-FEH cDNA of a native species from the Brazilian Cerrado. Results will contribute to understanding the role of fructans in the establishment of a very successful fructan flora of the Brazilian Cerrado, subjected to water limitation and low temperature during winter.

Cloning and Characterization of the Lipooligosaccharide Galactosyltransferase II Gene of Haemophilus ducreyi

PubMed Central

Sun, Shuhua; Schilling, Birgit; Tarantino, Laurie; Tullius, Michael V.; Gibson, Bradford W.; Munson, Robert S.

2000-01-01

Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the N-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of Haemophilus influenzae, Haemophilus somnus, Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77. PMID:10735874

Characterization of IDH1 p.R132H Mutant Clones Using Mutation-specific Antibody in Myeloid Neoplasms.

PubMed

Kurt, Habibe; Bueso-Ramos, Carlos E; Khoury, Joseph D; Routbort, Mark J; Kanagal-Shamanna, Rashmi; Patel, Umang V; Jorgensen, Jeffrey L; Wang, Sa A; Ravandi, Farhad; DiNardo, Courtney; Luthra, Rajyalakshmi; Medeiros, L Jeffrey; Patel, Keyur P

2018-05-01

Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations occur in a variety of myeloid neoplasms. Immunohistochemistry (IHC)-based direct visualization of mutant clones of hematopoietic cells can be useful for rapid diagnostic screening and for monitoring treatment response. In this study, we first evaluated the sensitivity and specificity of the IDH1 p.R132H mutation-specific antibody by IHC. All IDH1 wild type cases (n=11) and IDH1 mutant cases with a non-p.R132H mutation (n=30) were negative by IHC, demonstrating 100% antibody specificity. All the initial diagnostic specimens with IDH1 p.R132H mutation including acute myeloid leukemia (n=30), myelodysplastic syndromes (MDS) (n=10), MDS/myeloproliferative neoplasms (MPN) (n=4), and MPN (n=5) were positive by IHC, demonstrating 100% antibody sensitivity. Both immature and mature myeloid cells showed immunoreactivity. Erythroid precursors, lymphoid cells, endothelial cells, and osteoblasts were consistently negative by IHC. We then evaluated the follow-up specimens with a known IDH1 mutation status including acute myeloid leukemia (n=23), MDS (n=2), MDS/MPN (n=2), and MPN (n=2). Thirty-three IDH1 p.R132H mutant cases were positive by IHC and 12 IDH1 mutation negative cases were negative by IHC. However, IHC reactivity in up to 25% of bone marrow cells was noted in 8 of 20 polymerase chain reaction-negative cases, all from patients with a known history of IDH1 p.R132H mutation indicating sampling error or a sensitivity issue with molecular tests. These data indicate that IHC is a highly specific and sensitive tool to detect IDH1 p.R132H mutation in bone marrow involved by myeloid neoplasms. In addition, IDH1 p.R132H IHC also allows localization and assessment of the maturation stage of the clones carrying the mutation.

Avian influenza virus RNA extraction

USDA-ARS?s Scientific Manuscript database

The efficient extraction and purification of viral RNA is critical for down-stream molecular applications whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of avian influenza (AI) virus by molecular methods from expe...

Acidophiles of saline water at thermal vents of Vulcano, Italy.

PubMed

Simmons, Susan; Norris, R

2002-06-01

DNA was extracted from samples taken from close to acidic hydrothermal vents on shore of the Aeolian Island of Vulcano (Italy). RNA gene sequences were amplified by PCR, cloned, and sequenced. A sequence with an origin in samples at 35 degrees and 45 degrees C corresponded to that of a novel Acidithiobacillus species that was isolated from water close to the vents. Novel, iron-oxidizing mesophilic acidophiles were isolated through enrichment cultures with ferrous iron but were not represented in the clone banks of environmental rDNA. These acidophiles were related to Thiobacillus prosperus, which was isolated previously from Vulcano. The archaeal sequences that comprised a clone bank representing a high-temperature sample (75 degrees C) corresponded to those of Acidianus brierleyi and of thermophiles previously isolated from Vulcano, Thermoplasma volcanium and Acidianus infernus.

i-rDNA: alignment-free algorithm for rapid in silico detection of ribosomal gene fragments from metagenomic sequence data sets.

PubMed

Mohammed, Monzoorul Haque; Ghosh, Tarini Shankar; Chadaram, Sudha; Mande, Sharmila S

2011-11-30

Obtaining accurate estimates of microbial diversity using rDNA profiling is the first step in most metagenomics projects. Consequently, most metagenomic projects spend considerable amounts of time, money and manpower for experimentally cloning, amplifying and sequencing the rDNA content in a metagenomic sample. In the second step, the entire genomic content of the metagenome is extracted, sequenced and analyzed. Since DNA sequences obtained in this second step also contain rDNA fragments, rapid in silico identification of these rDNA fragments would drastically reduce the cost, time and effort of current metagenomic projects by entirely bypassing the experimental steps of primer based rDNA amplification, cloning and sequencing. In this study, we present an algorithm called i-rDNA that can facilitate the rapid detection of 16S rDNA fragments from amongst millions of sequences in metagenomic data sets with high detection sensitivity. Performance evaluation with data sets/database variants simulating typical metagenomic scenarios indicates the significantly high detection sensitivity of i-rDNA. Moreover, i-rDNA can process a million sequences in less than an hour on a simple desktop with modest hardware specifications. In addition to the speed of execution, high sensitivity and low false positive rate, the utility of the algorithmic approach discussed in this paper is immense given that it would help in bypassing the entire experimental step of primer-based rDNA amplification, cloning and sequencing. Application of this algorithmic approach would thus drastically reduce the cost, time and human efforts invested in all metagenomic projects. A web-server for the i-rDNA algorithm is available at http://metagenomics.atc.tcs.com/i-rDNA/

Identification of Novel Desiccation-Tolerant S. cerevisiae Strains for Deep Space Biosensors

NASA Technical Reports Server (NTRS)

Tieze, Sofia Massaro; Santa Maria, Sergio R.; Liddell, Lauren C.; Bhattacharya, Sharmila

2017-01-01

NASA's BioSentinel mission, a secondary payload that will fly on the Space Launch System's first Exploration Mission (EM-1), utilizes the budding yeast S. cerevisiae to study the biological response to the deep space radiation environment. Yeast samples are desiccated prior to launch to suspend growth and metabolism while the spacecraft travels to its target heliocentric orbit beyond Low Earth Orbit. Each sample is then rehydrated at the desired time points to reactivate the cells. A major risk in this mission is the loss of cell viability that occurs in the recovery period following the desiccation and rehydration process. Cell survival is essential for the detection of the biological response to features in the deep space environment, including ionizing radiation. The aim of this study is to mitigate viable cell loss in future biosensors by identifying mutations and genes that confer tolerance to desiccation stress in rad51, a radiation-sensitive yeast strain. We initiated a screen for desiccation-tolerance after rehydrating cells that were desiccated for three years, and selected various clones exhibiting robust growth. To verify retention of radiation sensitivity in the isolated clones - a crucial feature for a successful biosensor - we exposed them to ionizing radiation. Finally, to elucidate the genetic and molecular bases for observed desiccation-tolerance, we will perform whole-genome sequencing of those rad51 clones that exhibit both robust growth and radiation sensitivity following desiccation. The identification and characterization of desiccation-tolerant strains will allow us to engineer a biological model that will be resilient in face of the challenges of the deep space environment, and will thus ensure the experimental success of future biosensor missions.

Cloning, Codon Optimization, and Expression of Yersinia intermedia Phytase Gene in E. coli.

PubMed

Mirzaei, Maryam; Saffar, Behnaz; Shareghi, Behzad

2016-06-01

Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals' foods to hydrolyze phytate and increase absorption of phosphorus. Y. intermedia phytase is a new phytase with special characteristics such as high specific activity, pH stability, and thermostability. Our aim was to clone, express, and characterizea codon optimized Y. intermedia phytase gene in E. coli . The Y. intermedia phytase gene was optimized according to the codon usage in E. coli . The sequence was synthesized and sub-cloned in pET-22b (+) vector and transformed into E. coli Bl21 (DE3). The protein was expressed in the presence of IPTG at a final concentration of 1 mM at 30°C. The purification of recombinant protein was performed by Ni 2+ affinity chromatography. Phytase activity and stability were determined in various pH and temperatures. The codon optimized Y. intermedia phytase gene was sub-cloned successfully.The expression was confirmed by SDS-PAGE and Western blot analysis. The recombinant enzyme (approximately 45 kDa) was purified. Specific activity of enzyme was 3849 (U.mg -1 ) with optimal pH 5 and optimal temperature of 55°C. Thermostability (80°C for 15 min) and pH stability (3-6) of the enzyme were 56 and more than 80%, respectively. The results of the expression and enzyme characterization revealed that the optimized Y. intermedia phytase gene has a good potential to be produced commercially andto be applied in animals' foodsindustry.

Cloning of the cocaine-sensitive bovine dopamine transporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Usdin, T.B.; Chen, C.; Brownstein, M.J.

1991-12-15

A cDNA encoding the dopamine transporter from bovine brain substantia nigra was identified on the basis of its structural homology to other, recently cloned, neurotransmitter transporters. The sequence of the 693-amino acid protein is quite similar to those of the rat {gamma}-aminobutyric acid, human norepinephrine, and rat serotonin transporters. Dopamine transporter mRNA was detected by in situ hybridization in the substantia nigra but not in the locus coeruleus, raphe, caudate, or other brain areas. ({sup 3}H)Dopamine accumulation in tissue culture cells transfected with the cDNA was inhibited by amphetamine, cocaine, and specific inhibitors of dopamine transports, including GBR12909.

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

PubMed

Dron, M; Modjtahedi, N; Brison, O; Tovey, M G

1986-05-01

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.

Modeling forest ecosystem responses to elevated carbon dioxide and ozone using artificial neural networks.

PubMed

Larsen, Peter E; Cseke, Leland J; Miller, R Michael; Collart, Frank R

2014-10-21

Rising atmospheric levels of carbon dioxide and ozone will impact productivity and carbon sequestration in forest ecosystems. The scale of this process and the potential economic consequences provide an incentive for the development of models to predict the types and rates of ecosystem responses and feedbacks that result from and influence of climate change. In this paper, we use phenotypic and molecular data derived from the Aspen Free Air CO2 Enrichment site (Aspen-FACE) to evaluate modeling approaches for ecosystem responses to changing conditions. At FACE, it was observed that different aspen clones exhibit clone-specific responses to elevated atmospheric levels of carbon dioxide and ozone. To identify the molecular basis for these observations, we used artificial neural networks (ANN) to examine above and below-ground community phenotype responses to elevated carbon dioxide, elevated ozone and gene expression profiles. The aspen community models generated using this approach identified specific genes and subnetworks of genes associated with variable sensitivities for aspen clones. The ANN model also predicts specific co-regulated gene clusters associated with differential sensitivity to elevated carbon dioxide and ozone in aspen species. The results suggest ANN is an effective approach to predict relevant gene expression changes resulting from environmental perturbation and provides useful information for the rational design of future biological experiments. Copyright © 2014 Elsevier Ltd. All rights reserved.

Herpesvirus Saimiri Transforms Human T-Cell Clones to Stable Growth without Inducing Resistance to Apoptosis

PubMed Central

Kraft, Michael S.; Henning, Golo; Fickenscher, Helmut; Lengenfelder, Doris; Tschopp, Jürg; Fleckenstein, Bernhard; Meinl, Edgar

1998-01-01

Herpesvirus saimiri (HVS) transforms human T cells to stable growth in vitro. Since HVS codes for two different antiapoptotic proteins, growth transformation by HVS might be expected to confer resistance to apoptosis. We found that the expression of both viral antiapoptotic genes was restricted to cultures with viral replication and absent in growth-transformed human T cells. A comparative examination of HVS-transformed T-cell clones and their native parental clones revealed that the expression of Bcl-2, Bcl-XL, Bax, and members of the tumor necrosis factor receptor (TNF-R) superfamily with a death domain, namely, TNF-RI, CD95, and TRAMP, were not modulated by HVS. Expression of CD30 was induced in HVS-transformed T cells, and these cells also expressed the CD30 ligand. Uninfected and transformed T cells were sensitive to CD95 ligation but resistant to apoptosis mediated by TRAIL or soluble TNF-α. CD95 ligand was constitutively expressed on transformed but not uninfected parental T cells. Both cell types showed similar sensitivity to cell death induction or inhibition of T-cell activation mediated by irradiation, oxygen radicals, dexamethasone, cyclosporine, and prostaglandin E2. Altogether, this study strongly suggests that growth transformation by HVS is based not on resistance to apoptosis but, rather, on utilization of normal cellular activation pathways. PMID:9525639

Complementation of DNA repair defect in xeroderma pigmentosum cells of group C by the transfer of human chromosome 5

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaur, G.P.; Athwal, R.S.

1993-01-01

Complementation of DNA excision repair defect in xeroderma pigmentosum cells of group C (XP-C) has been achieved by the transfer of human chromosome 5. Individual human chromosomes tagged with a selectable marker were transferred to XP-C cells by microcell fusion from mouse-human hybrid cell lines each bearing a single different human chromosome. Analysis of the chromosome transfer clones revealed that introduction of chromosome 5 into XP-C cells corrected the DNA repair defect as well as UV-sensitive phenotypes, while chromosomes 2, 6, 7, 9, 13, 15, 17, and 21 failed to complement. The introduced chromosome 5 in complemented UV[sup r] clonesmore » was distinguished from the parental XP-C chromosomes by polymorphism for dinucleotide (CA)[sub n] repeats at two loci, D5S117 and D5S209. In addition, an intact marked chromosome 5 was rescued into mouse cells from a complemented UV[sup r] clone by microcell fusion. Five subclones of a complemented clone that had lost the marked chromosome 5 exhibited UV-sensitive and repair-deficient phenotypes identical to parental XP-C cells. Concordant loss of the transferred chromosome and reappearance of XP-C phenotype further confirmed the presence of a DNA repair gene on human chromosome 5. 38 refs., 7 figs., 1 tab.« less

A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes

PubMed Central

Meng, Jia; Kanzaki, Gregory; Meas, Diane; Lam, Christopher K.; Crummer, Heather; Tain, Justina; Xu, H. Howard

2013-01-01

Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published reports describing the application of asRNA gene silencing for comprehensive analyses of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250,000 library transformants for conditional growth-inhibitory recombinant clones from two shot-gun genomic libraries of E. coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer-sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes while 18 originated from non-essential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12 fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E. coli using regulated asRNA expression. PMID:22268863

Distinct signatures for mutator sensitivity of lacZ reversions and for the spectrum of lacI/lacO forward mutations on the chromosome of nondividing Escherichia coli.

PubMed Central

Bharatan, Shanti M; Reddy, Manjula; Gowrishankar, J

2004-01-01

A conditional lethal galE(Ts)-based strategy was employed in Escherichia coli, first to eliminate all growth-associated chromosomal reversions in lacZ or forward mutations in lacI/lacO by incubation at the restrictive temperature and subsequently to recover (as papillae) spontaneous mutations that had arisen in the population of nondividing cells after shift to the permissive temperature. Data from lacZ reversion studies in mutator strains indicated that the products of all genes for mismatch repair (mutHLS, dam, uvrD), of some for oxidative damage repair (mutMT), and of that for polymerase proofreading (dnaQ) are required in dividing cells; some others for oxidative damage repair (mutY, nth nei) are required in both dividing and nondividing cells; and those for alkylation damage repair (ada ogt) are required in nondividing cells. The spectrum of lacI/lacO mutations in nondividing cells was distinguished both by lower frequencies of deletions and IS1 insertions and by the unique occurrence of GC-to-AT transitions at lacO +5. In the second approach to study mutations that had occurred in nondividing cells, lacI/lacO mutants were selected as late-arising papillae from the lawn of a galE+ strain; once again, transitions at lacO +5 were detected among the mutants that had been obtained from populations initially grown on poor carbon sources such as acetate, palmitate, or succinate. Our results indicate that the lacO +5 site is mutable only in nondividing cells, one possible mechanism for which might be that random endogenous alkylation (or oxidative) damage to DNA in these cells is efficiently corrected by the Ada Ogt (or Nth Nei) repair enzymes at most sites but not at lacO +5. Furthermore, the late-arising papillae from the second approach were composed almost exclusively of dominant lacI/lacO mutants. This finding lends support to "instantaneous gratification" models in which a spontaneous lesion, occurring at a random site in DNA of a nondividing cell, is most likely to be fixed as a mutation if it allows the cell to immediately exit the nondividing state. PMID:15020459

DNA book.

PubMed

Kawai, Jun; Hayashizaki, Yoshihide

2003-06-01

We propose herein a new method of DNA distribution, whereby DNA clones or PCR products are printed directly onto the pages of books and delivered to users along with relevant scientific information. DNA sheets, comprising water-soluble paper onto which DNA is spotted, can be bound into books. Readers can easily extract the DNA fragments from DNA sheets and amplify them using PCR. We show that DNA sheets can withstand various conditions that may be experienced during bookbinding and delivery, such as high temperatures and humidity. Almost all genes (95%-100% of randomly selected RIKEN mouse cDNA clones) were recovered successfully by use of PCR. Readers can start their experiments after a 2-h PCR amplification without waiting for the delivery of DNA clones. The DNA Book thus provides a novel method for delivering DNA in a timely and cost-effective manner. A sample DNA sheet (carrying RIKEN mouse cDNA clones encoding genes of enzymes for the TCA cycle) is included in this issue for field-testing. We would greatly appreciate it if readers could attempt to extract DNA and report the results and whether the DNA sheet was shipped to readers in good condition.

Microbial community analysis of a coastal hot spring in Kagoshima, Japan, using molecular- and culture-based approaches.

PubMed

Nishiyama, Minako; Yamamoto, Shuichi; Kurosawa, Norio

2013-08-01

Ibusuki hot spring is located on the coastline of Kagoshima Bay, Japan. The hot spring water is characterized by high salinity, high temperature, and neutral pH. The hot spring is covered by the sea during high tide, which leads to severe fluctuations in several environmental variables. A combination of molecular- and culture-based techniques was used to determine the bacterial and archaeal diversity of the hot spring. A total of 48 thermophilic bacterial strains were isolated from two sites (Site 1: 55.6°C; Site 2: 83.1°C) and they were categorized into six groups based on their 16S rRNA gene sequence similarity. Two groups (including 32 isolates) demonstrated low sequence similarity with published species, suggesting that they might represent novel taxa. The 148 clones from the Site 1 bacterial library included 76 operational taxonomy units (OTUs; 97% threshold), while 132 clones from the Site 2 bacterial library included 31 OTUs. Proteobacteria, Bacteroidetes, and Firmicutes were frequently detected in both clone libraries. The clones were related to thermophilic, mesophilic and psychrophilic bacteria. Approximately half of the sequences in bacterial clone libraries shared <92% sequence similarity with their closest sequences in a public database, suggesting that the Ibusuki hot spring may harbor a unique and novel bacterial community. By contrast, 77 clones from the Site 2 archaeal library contained only three OTUs, most of which were affiliated with Thaumarchaeota.

An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

PubMed Central

Sterling, Catherine H.; Veksler-Lublinsky, Isana; Ambros, Victor

2015-01-01

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids. PMID:25056322

Amino acid changes in disease-associated variants differ radically from variants observed in the 1000 genomes project dataset.

PubMed

de Beer, Tjaart A P; Laskowski, Roman A; Parks, Sarah L; Sipos, Botond; Goldman, Nick; Thornton, Janet M

2013-01-01

The 1000 Genomes Project data provides a natural background dataset for amino acid germline mutations in humans. Since the direction of mutation is known, the amino acid exchange matrix generated from the observed nucleotide variants is asymmetric and the mutabilities of the different amino acids are very different. These differences predominantly reflect preferences for nucleotide mutations in the DNA (especially the high mutation rate of the CpG dinucleotide, which makes arginine mutability very much higher than other amino acids) rather than selection imposed by protein structure constraints, although there is evidence for the latter as well. The variants occur predominantly on the surface of proteins (82%), with a slight preference for sites which are more exposed and less well conserved than random. Mutations to functional residues occur about half as often as expected by chance. The disease-associated amino acid variant distributions in OMIM are radically different from those expected on the basis of the 1000 Genomes dataset. The disease-associated variants preferentially occur in more conserved sites, compared to 1000 Genomes mutations. Many of the amino acid exchange profiles appear to exhibit an anti-correlation, with common exchanges in one dataset being rare in the other. Disease-associated variants exhibit more extreme differences in amino acid size and hydrophobicity. More modelling of the mutational processes at the nucleotide level is needed, but these observations should contribute to an improved prediction of the effects of specific variants in humans.

Automatic page composition with nested sub-layouts

NASA Astrophysics Data System (ADS)

Hunter, Andrew

2013-03-01

This paper provides an overview of a system for the automatic composition of publications. The system first composes nested hierarchies of contents, then applies layout engines at branch points in the hierarchies to explore layout options, and finally selects the best overall options for the finished publications. Although the system has been developed as a general platform for automated publishing, this paper describes its application to the composition and layout of a magazine-like publication for social content from Facebook. The composition process works by assembling design fragments that have been populated with text and images from the Facebook social network. The fragments constitute a design language for a publication. Each design fragment is a nested mutable sub-layout that has no specific size or shape until after it has been laid-out. The layout process balances the space requirements of the fragment's internal contents with its external context in the publication. The mutability of sub-layouts requires that their layout options must be kept open until all the other contents that share the same space have been considered. Coping with large numbers of options is one of the greatest challenges in layout automation. Most existing layout methods work by rapidly elimination design options rather than by keeping options open. A further goal of this publishing system is to confirm that a custom publication can be generated quickly by the described methods. In general, the faster that publications can be created, the greater the opportunities for the technology.

Ozone and carbon dioxide effects on spider mites in white clover and peanut

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heagle, A.S.; Brandenburg, R.L.; Burns, J.C.

1994-11-01

Effects of O{sub 3} and/or elevated CO{sub 2} on two-spotted spider mites (Tetranychus urticae Koch) grown on an O{sub 3}-sensitive and an O{sub 3}-resistant clone of white clover (Trifolium repens L.) were measured in greenhouse and field experiments. Peanut (Arachis hypogeae L.) {open_quote}NC-9{close_quote} was used in one greenhouse study with O{sub 3}. In field studies, O{sub 3} treatments were charcoal filtered air (CF), nonfiltered air (NF), and two NF treatments with O{sub 3} added for 12 h d{sup {minus}1} at proportions of {approx} 1.25 and 1.50 times the ambient O{sub 3} concentration. In greenhouse studies, constant amounts of O{sub 3}more » were added to CF for 6 h d{sup {minus}1} to achieve mean concentrations ranging from 5 to 100 nL L{sup {minus}1}. For the greenhouse O{sub 3} x CO{sub 2} experiment, CO{sub 2} concentrations were ambient and approximately twice-ambient for 24 h d{sup {minus}1}. Plants were exposed to O{sub 3} and/or CO{sub 2} for {approx} 7 d before infestation with mites; daily exposures continued for 14 to 28 d to allow reproduction for at least two generations. Leaves were sampled to count eggs, larvae, nymphs, and adults. Ozone caused more chlorosis and necrosis on the O{sub 3}-sensitive clover clone (NC-S) than on the O{sub 3}-resistant clone (NC-R). Carbon dioxide enrichment increased shoot growth of both clones by {approx}33%. Statistical analyses indicated significant O{sub 3} effects in some experiments and nonsignificant O{sub 3} effects in others. A trend toward increased mite populations with increased O{sub 3} occurred, however, on NC-S in all trials. No consistent trends occurred with NC-R. With peanut, a significant linear increase in mite population occurred with increased O{sub 3}. Carbon dioxide enrichment increased the rate of population increase on both clover clones, but more so on NC-R. 47 refs., 2 figs., 7 tabs.« less

Comparing four different ALK antibodies with manual immunohistochemistry (IHC) to screen for ALK-rearranged non-small cell lung cancer (NSCLC).

PubMed

Shen, Qin; Wang, Xuan; Yu, Bo; Shi, Shanshan; Liu, Biao; Wang, Yanfen; Xia, Qiuyuan; Rao, Qiu; Zhou, Xiaojun

2015-12-01

Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) screening is essential to its treatment such as crizotinib. Different assays have been developed to detect ALK rearrangements, such as fluorescence in situ hybridization (FISH), reverse transcriptase-PCR (RT-PCR), and immunohistochemistry (IHC). However, ALK detection has not been applied widely in all hospitals. Moreover, IHC has been proposed to be a pre-screening tool because of its wide application in clinics. Since the low expression of ALK protein, the sensitivity and specificity of ALK antibody are the keys to the success of IHC screening. Therefore, we compared different antibodies to find the best one for IHC detection. We evaluated ALK expression by four different ALK antibodies: clone D5F3 (Ventana), clone D5F3 (CST), clone 1A4/1H7 (OriGene Tech.), and clone 5A4 (Abcam) based on manual IHC in a cohort of 60 NSCLCs. The results were compared with those from automated IHC (clone D5F3, Ventana). All cases were evaluated independently by ALK FISH. 32 ALK-positive and 28 ALK-negative NSCLCs were identified by automated IHC (D5F3, Ventana) and FISH analysis. Based on conventional manual IHC, the sensitivity of four antibodies-D5F3 (Ventana), D5F3 (CST), 1A4/1H7 (OriGene Tech.), and 5A4 (Abcam)-was 93.8%, 84.4%, 93.8%, and 56.3%, respectively. Their specificities and positive predictive values were 100%. The percentage of strong-moderate staining was 65.6%, 62.5%, 68.8%, and 21.9%, respectively. Compared with automated IHC (D5F3, Ventana), each staining concordance was 96.7%, 91.7%, 96.7%, and 76.7%, respectively, and each presented staining heterogeneity (weak-moderate-strong intensity). These data indicated that manual IHC with a more reliable ALK antibody might provide an effective strategy for screening ALK gene rearrangements in all NSCLC patients, followed by confirmatory FISH analysis in IHC-positive cases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Mesophilic and hyperthermophilic adenylate kinases differ in their tolerance to random fragmentation.

PubMed

Segall-Shapiro, Thomas H; Nguyen, Peter Q; Dos Santos, Edgardo D; Subedi, Saurav; Judd, Justin; Suh, Junghae; Silberg, Jonathan J

2011-02-11

The extent to which thermostability influences the location of protein fragmentation sites that allow retention of function is not known. To evaluate this, we used a novel transposase-based approach to create libraries of vectors that express structurally-related fragments of Bacillus subtilis adenylate kinase (BsAK) and Thermotoga neapolitana adenylate kinase (TnAK) with identical modifications at their termini, and we selected for variants in each library that complement the growth of Escherichia coli with a temperature-sensitive adenylate kinase (AK). Mutants created using the hyperthermophilic TnAK were found to support growth with a higher frequency (44%) than those generated from the mesophilic BsAK (6%), and selected TnAK mutants complemented E. coli growth more strongly than homologous BsAK variants. Sequencing of functional clones from each library also identified a greater dispersion of fragmentation sites within TnAK. Nondisruptive fission sites were observed within the AMP binding and core domains of both AK homologs. However, only TnAK contained sites within the lid domain, which undergoes dynamic fluctuations that are critical for catalysis. These findings implicate the flexible lid domain as having an increased sensitivity to fission events at physiological temperatures. In addition, they provide evidence that comparisons of nondisruptive fission sites in homologous proteins could be useful for finding dynamic regions whose conformational fluctuations are important for function, and they show that the discovery of protein fragments that cooperatively function in mesophiles can be aided by the use of thermophilic enzymes as starting points for protein design. Copyright © 2010 Elsevier Ltd. All rights reserved.

Identification and characterisation of DfCHS, a chalcone synthase gene regulated by temperature and ultraviolet in Dryopteris fragrans.

PubMed

Sun, L L; Li, Y; Li, S S; Wu, X J; Hu, B Z; Chang, Y

2014-12-30

Chalcone synthase (CHS) is an enzyme that catalyzes the first committed step in flavonoid biosynthesis, and its transcription level is regulated by light conditions. By using homology cloning and rapid amplification of cDNA ends, we cloned a chalcone synthase gene (DfCHS) from Dryopteris fragrans (L.) Schott. The full-length cDNA of DfCHS is 1,737 bp, with an open reading frame (ORF) of 1,122 bp (deposited in GenBank under Accession Number KF530802) encoding a predicted protein of 373 amino acids. The calculated molecular mass of DfCHS is 41.3 kDa. We studied the expression of DfCHS and total flavonoid contents in tissue culture seedlings cultured under the low temperature at 4ºC, high temperature at 35ºC and UV conditions, respectively. The results show that the expression of DfCHS are not the same, but all present rising trends, then flavonoid contents were increased. Overall, our results imply that the expression of DfCHS gene provide a certain theory basis in the status of evolution among ferns.

Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV).

PubMed

Wirth, Jennifer Fulton; Snyder, Jamie C; Hochstein, Rebecca A; Ortmann, Alice C; Willits, Deborah A; Douglas, Trevor; Young, Mark J

2011-06-20

Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82°C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication. Copyright © 2011. Published by Elsevier Inc.

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Eachmore » group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

Development and Application of Diagnostics in the National Schistosomiasis Control Programme in The People's Republic of China.

PubMed

Zhang, J-F; Xu, J; Bergquist, R; Yu, L-L; Yan, X-L; Zhu, H-Q; Wen, L-Y

2016-01-01

Schistosomiasis, caused by Schistosoma japonicum infection to human, has a documented history of more than 2100years in The People's Republic of China. In spite of great progress in controlling the disease, it is still one of the most serious parasitic diseases in the country. The study and use of diagnostic techniques play an important role in the targeting of chemotherapy that has been continuously applied in the national schistosomiasis control programme for several decades. This paper reviews the development and application of parasitological, immunodiagnostic and molecular diagnostic technology for S. japonicum in The People's Republic of China with a brief mention of diagnostic imagery, such as ultrasound and radiology. When analysing the efficacy and performance characteristics of the main diagnostic techniques in current use, it becomes apparent that approaches that worked well in the past are less suitable now as successful control has shifted the endemic situation towards control and interruption of transmission. The conclusion is that a mutable approach must be adopted choosing the most appropriate diagnostic technique for each control stage (and area), thus modifying the methodology according to the prevailing diagnostic needs in terms of sensitivity and specificity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

DOE PAGES

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.; ...

2014-01-01

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Eachmore » group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

PubMed

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-08-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula.

Preparation and characterization of monoclonal antibody against melatonin.

PubMed

Soukhtanloo, Mohammad; Ansari, Mohammad; Paknejad, Maliheh; Parizadeh, Mohammad Reza; Rasaee, Mohammad Javad

2008-06-01

Anti-melatonin monoclonal antibodies (MAb) were prepared following coupling melatonin to bovine serum albumin (BSA) by Mannich reaction. Balb/c mice were immunized via injection of the melatonin-BSA intraperitonally. The spleen cells producing high titer of antibody were fused with myeloma cells of SP2/0 origin. After two limiting dilutions, two stable clones (AS-H10 and AS-D26) exhibiting best properties were selected for further studies. The class and subclass of two MAbs were found to be IgG(1) and IgG(2a) with lambda and kappa light chains, respectively. Antibodies secreted by these two clones showed high affinity of about 10(9)M(1). Study of the specificity criteria showed that these clones had no cross reactivity with indolic, aromatic, and imidazole ring-containing compounds, and had high specificity towards melatonin. The calibration curve was constructed with a sensitivity range of 10 ng/mL to 10 microg/mL. In conclusion, these MAbs may be useful for immunoassay of melatonin.

Identification of a Highly Transmissible Animal-Independent Staphylococcus aureus ST398 Clone with Distinct Genomic and Cell Adhesion Properties

PubMed Central

Uhlemann, Anne-Catrin; Porcella, Stephen F.; Trivedi, Sheetal; Sullivan, Sean B.; Hafer, Cory; Kennedy, Adam D.; Barbian, Kent D.; McCarthy, Alex J.; Street, Craig; Hirschberg, David L.; Lipkin, W. Ian; Lindsay, Jodi A.; DeLeo, Frank R.; Lowy, Franklin D.

2012-01-01

ABSTRACT A methicillin-resistant Staphylococcus aureus (MRSA) clone known as ST398 has emerged as a major cause of acute infections in individuals who have close contact with livestock. More recently, the emergence of an animal-independent ST398 methicillin-sensitive S. aureus (MSSA) clone has been documented in several countries. However, the limited surveillance of MSSA has precluded an accurate assessment of the global spread of ST398 and its clinical relevance. Here we provide evidence that ST398 is a frequent source of MSSA infections in northern Manhattan and is readily transmitted between individuals in households. This contrasts with the limited transmissibility of livestock-associated ST398 (LA-ST398) MRSA strains between humans. Our whole-genome sequence analysis revealed that the chromosome of the human-associated ST398 MSSA clone is smaller than that of the LA-ST398 MRSA reference strain S0385, due mainly to fewer mobile genetic elements (MGEs). In contrast, human ST398 MSSA isolates harbored the prophage φ3 and the human-specific immune evasion cluster (IEC) genes chp and scn. While most of the core genome was conserved between the human ST398 MSSA clone and S0385, these strains differed substantially in their repertoire and composition of intact adhesion genes. These genetic changes were associated with significantly enhanced adhesion of human ST398 MSSA isolates to human skin keratinocytes and keratin. We propose that the human ST398 MSSA clone can spread independent of animal contact using an optimized repertoire of MGEs and adhesion molecules adapted to transmission among humans. PMID:22375071

Clonal success of piliated penicillin nonsusceptible pneumococci

PubMed Central

Sjöström, K.; Blomberg, C.; Fernebro, J.; Dagerhamn, J.; Morfeldt, E.; Barocchi, M. A.; Browall, S.; Moschioni, M.; Andersson, M.; Henriques, F.; Albiger, B.; Rappuoli, Rino; Normark, S.; Henriques-Normark, B.

2007-01-01

Antibiotic resistance in pneumococci is due to the spread of strains belonging to a limited number of clones. The Spain9V-3 clone of sequence type (ST)156 is one of the most successful clones with reduced susceptibility to penicillin [pneumococci nonsusceptible to penicillin (PNSP)]. In Sweden during 2000–2003, a dramatic increase in the number of PNSP isolates was observed. Molecular characterization of these isolates showed that a single clone of sequence type ST156 increased from 40% to 80% of all serotype 14, thus causing the serotype expansion. Additionally, during the same time period, we examined the clonal composition of two serotypes 9V and 19F: all 9V and 20% of 19F isolates belonged to the clonal cluster of ST156, and overall ≈50% of all PNSP belonged to the ST156 clonal cluster. Moreover, microarray and PCR analysis showed that all ST156 isolates, irrespective of capsular type, carried the rlrA pilus islet. This islet was also found to be present in the penicillin-sensitive ST162 clone, which is believed to be the drug-susceptible ancestor of ST156. Competitive experiments between related ST156 serotype 19F strains confirmed that those containing the rlrA pilus islet were more successful in an animal model of carriage. We conclude that the pilus island is an important biological factor common to ST156 isolates and other successful PNSP clones. In Sweden, a country where the low antibiotic usage does not explain the spread of resistant strains, at least 70% of all PNSP isolates collected during year 2003 carried the pilus islet. PMID:17644611

Effect of antibiotic pretreatment on survival, mutability, and electrophoretic spectrum of soluble proteins in a gamma-irradiated chlorella population

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ovsyannikova, M.N.; Osetrova, A.Ya.

1977-01-01

A study was made of the protective action of several antibiotics in a ..gamma..-irradiated chlorella population. It was shown that not all of the antibiotics tested had a protective effect with regard to survival. At the same time, all of the tested antibiotics, with the exception of nistatin, diminished the effectiveness of subsequent ..gamma..-irradiation with respect to induction of mutations. It is assumed that electrophoresis of readily soluble proteins can be used for biochemical identification of mutations in the population.

Mutants in Arabidopsis thaliana with altered shoot gravitropism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bullen, B.L.; Poff, K.L.

1987-04-01

A procedure has been developed and used to screen 40,000 m-2 seedlings of Arabidopsis thaliana for strains with altered shoot gravitropism. Several strains have been identified for which shoot gravitropism is considerably more random than that of their wild-type parent (based on frequency distribution histograms of the gravitropic response to a 1 g stimulus). One such strain exhibits normal hypocotyl phototropism and normal root gravitropism. Thus, the gravitropism pathway in the shoot contains at least one mutable element which is not required for root gravitropism.

T-cell clonality analysis in biopsy specimens from two different skin sites shows high specificity in the diagnosis of patients with suggested mycosis fungoides.

PubMed

Thurber, Stacy E; Zhang, Bing; Kim, Youn H; Schrijver, Iris; Zehnder, James; Kohler, Sabine

2007-11-01

The diagnosis of mycosis fungoides (MF) is often difficult because of significant clinical and histopathologic overlap with inflammatory dermatoses. T-cell receptor (TCR)gamma chain rearrangement by polymerase chain reaction (PCR) (TCR-PCR) is a helpful adjuvant tool in this setting, but several of the inflammatory dermatoses in the differential diagnosis of MF may contain a clonal T-cell proliferation. We examined whether analysis for T-cell clonality and comparison of the clones with the standardized BIOMED-2 PCR multiplex primers for the TCRgamma chain from two anatomically distinct skin sites improves diagnostic accuracy. We examined two biopsy specimens each from 10 patients with unequivocal MF, from 18 patients with inflammatory dermatoses, and from 18 patients who could initially not be definitively given a diagnosis based on clinical and histopathologic criteria. Eight of 10 patients with unequivocal MF had an identical clone in both biopsy specimens. Two of 18 patients with inflammatory dermatoses were found to have a clone in one of the biopsy specimens. On further follow-up of the 18 patients with morphologically nondiagnostic biopsy specimens, 13 of 18 were later confirmed to have MF and 5 of 18 had inflammatory dermatoses. Eleven of 13 patients with MF had an identical clone in both biopsy specimens; two of 13 had a polyclonal amplification pattern in both biopsy specimens. Four of 5 patients with inflammatory dermatoses had no clone in either biopsy specimen. One patient with an inflammatory dermatosis had an identical clone in both specimens. The sensitivity of TCR-PCR analysis to evaluate for an identical clone at different anatomic skin sites (dual TCR-PCR) is 82.6% and the specificity is 95.7%. The number of patients in the study group was limited. These data suggest that dual TCR-PCR is a very promising technique with high specificity in distinguishing MF from inflammatory dermatoses.

Steady-state nutrition of soil grown trembling aspen clones and the potential for gaseous pollutant experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coleman, M.D.; Dickson, R.E.; Isebrands, J.G.

To assess the interaction of gaseous pollutants and plant nutrition it is desirable to grow plants at a constant growth rate while maintaining constant nutrient status. Once constant, or steady-state, conditions are established relationships between growth, nutrition, physiology and stress responses are simplified. Relative nutrient additions are an effective way to maintain such constant conditions in solution culture; however, few experiments have applied such treatments to soil grown plants. This experiment evaluates the response of two aspen clones (259 and 271) to various relative nutrient addition rates (1,2,3,4,5 % per day) applied to the peat:sand:vermiculite growing media. Although the initialmore » lag phase (adjustment period) lasted up to 50 days, subsequent relative growth rates were uniform and related to treatment. Growth responses among treatments were distinct with final biomass in the higher addition rates (3,4,5% per day) as much as twice that of the next lower treatment. Clone 271 (ozone tolerant) produced only 61% of the biomass that clone 259 (ozone sensitive) produced in the 5% per day treatment. Final leaf nitrogen was 1.5, 2.1, 3.4, 3.8, 4.3% dry weight for 1 to 5% per day addition rate treatments respectively. Concentrations between clones were equal. Results demonstrate the effectiveness of steady-state nutrition in controlling growth and nutrient status of soil grown aspen, enabling more critical control of stress experiments.« less

Sphagnum palustre clone vs native Pseudoscleropodium purum: A first trial in the field to validate the future of the moss bag technique.

PubMed

Capozzi, F; Adamo, P; Di Palma, A; Aboal, J R; Bargagli, R; Fernandez, J A; Lopez Mahia, P; Reski, R; Tretiach, M; Spagnuolo, V; Giordano, S

2017-06-01

Although a large body of literature exists on the use of transplanted mosses for biomonitoring of air pollution, no article has addressed so far the use and the accumulation performance of a cloned moss for this purpose. In this work, a direct comparison of metal accumulation between bags filled with a Sphagnum palustre L. clone or with native Pseudoscleropodium purum Hedw., one of the most used moss species in biomonitoring surveys, was investigated. The test was performed in sites with different atmospheric contamination levels selected in urban, industrial, agricultural and background areas of Italy and Spain. Among the eighteen elements investigated, S. palustre was significantly enriched in 10 elements (Al, Ba, Cr, Cu, Fe, Hg, Pb, Sr, V and Zn), while P. purum was enriched only in 6 elements (Al, Ba, Cu, Hg, Pb and Sr), and had a consistently lower uptake capacity than S. palustre. The clone proved to be more sensitive in terms of metal uptake and showed a better performance as a bioaccumulator, providing a higher accumulation signal and allowing a finer distinction among the different land uses and levels of pollution. The excellent uptake performance of the S. palustre clone compared to the native P. purum and its low and stable baseline elemental content, evidenced in this work, are key features for the improvement of the moss bag approach and its large scale application. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past.

PubMed

Ribera, Judit; Estupiñán, Mónica; Fuentes, Alba; Fillat, Amanda; Martínez, Josefina; Diaz, Pilar

2017-01-01

A search for extremophile enzymes from ancient volcanic soils in El Hierro Island (Canary Islands, Spain) allowed isolation of a microbial sporulated strain collection from which several enzymatic activities were tested. Isolates were obtained after sample cultivation under several conditions of nutrient contents and temperature. Among the bacterial isolates, supernatants from the strain designated JR3 displayed high esterase activity at temperatures ranging from 30 to 100°C, suggesting the presence of at least a hyper-thermophilic extracellular lipase. Sequence alignment of known thermophilic lipases allowed design of degenerated consensus primers for amplification and cloning of the corresponding lipase, named LipJ. However, the cloned enzyme displayed maximum activity at 30°C and pH 7, showing a different profile from that observed in supernatants of the parental strain. Sequence analysis of the cloned protein showed a pentapeptide motif -GHSMG- distinct from that of thermophilic lipases, and much closer to that of esterases. Nevertheless, the 3D structural model of LipJ displayed the same folding as that of thermophilic lipases, suggesting a common evolutionary origin. A phylogenetic study confirmed this possibility, positioning LipJ as a new member of the thermophilic family of bacterial lipases I.5. However, LipJ clusters in a clade close but separated from that of Geobacillus sp. thermophilic lipases. Comprehensive analysis of the cloned enzyme suggests a common origin of LipJ and other bacterial thermophilic lipases, and highlights the most probable divergent evolutionary pathway followed by LipJ, which during the harsh past times would have probably been a thermophilic enzyme, having lost these properties when the environment changed to more benign conditions.

Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past

PubMed Central

Ribera, Judit; Estupiñán, Mónica; Fuentes, Alba; Fillat, Amanda; Martínez, Josefina

2017-01-01

A search for extremophile enzymes from ancient volcanic soils in El Hierro Island (Canary Islands, Spain) allowed isolation of a microbial sporulated strain collection from which several enzymatic activities were tested. Isolates were obtained after sample cultivation under several conditions of nutrient contents and temperature. Among the bacterial isolates, supernatants from the strain designated JR3 displayed high esterase activity at temperatures ranging from 30 to 100°C, suggesting the presence of at least a hyper-thermophilic extracellular lipase. Sequence alignment of known thermophilic lipases allowed design of degenerated consensus primers for amplification and cloning of the corresponding lipase, named LipJ. However, the cloned enzyme displayed maximum activity at 30°C and pH 7, showing a different profile from that observed in supernatants of the parental strain. Sequence analysis of the cloned protein showed a pentapeptide motif -GHSMG- distinct from that of thermophilic lipases, and much closer to that of esterases. Nevertheless, the 3D structural model of LipJ displayed the same folding as that of thermophilic lipases, suggesting a common evolutionary origin. A phylogenetic study confirmed this possibility, positioning LipJ as a new member of the thermophilic family of bacterial lipases I.5. However, LipJ clusters in a clade close but separated from that of Geobacillus sp. thermophilic lipases. Comprehensive analysis of the cloned enzyme suggests a common origin of LipJ and other bacterial thermophilic lipases, and highlights the most probable divergent evolutionary pathway followed by LipJ, which during the harsh past times would have probably been a thermophilic enzyme, having lost these properties when the environment changed to more benign conditions. PMID:28742841

Survival and growth of epidemically successful and nonsuccessful Salmonella enterica clones after freezing and dehydration.

PubMed

Müller, Karoline; Aabo, Søren; Birk, Tina; Mordhorst, Hanne; Bjarnadóttir, Björg; Agersø, Yvonne

2012-03-01

The spread of epidemically successful nontyphoidal Salmonella clones has been suggested as the most important cause of salmonellosis in industrialized countries. Factors leading to the emergence of success clones are largely unknown, but their ability to survive and grow after physical stress may contribute. During epidemiological studies, a mathematical model was developed that allowed estimation of a factor (q) accounting for the relative ability of Salmonella serovars with different antimicrobial resistances to survive in the food chain and cause human disease. Based on this q-factor, 26 Salmonella isolates were characterized as successful or nonsuccessful. We studied the survival and growth of stationary- and exponential-phase cells of these isolates after freezing for up to 336 days in minced meat. We also investigated survival and growth after dehydration at 10°C and 82% relative humidity (RH) and 25°C and 49% RH for 112 days. Stationary-phase cells were reduced by less than 1 log unit during 1 year of freezing, and growth was initiated with an average lag phase of 1.7 h. Survival was lower in exponentialphase cells, but lag phases tended to be shorter. High humidity and low temperature were less harmful to Salmonella than were low humidity and high temperature. Tolerance to adverse conditions was highest for Salmonella Infantis and one Salmonella Typhimurium U292 isolate and lowest for Salmonella Derby and one Salmonella Typhimurium DT170 isolate. Dehydration, in contrast to freezing, was differently tolerated by the Salmonella strains in this study, but tolerance to freezing and dehydration does not appear to contribute to the emergence of successful Salmonella clones.

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

NASA Technical Reports Server (NTRS)

Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

1996-01-01

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

Genetics of trehalose biosynthesis in desert-derived Aureobasidium melanogenum and role of trehalose in the adaptation of the yeast to extreme environments.

PubMed

Jiang, Hong; Liu, Guang-Lei; Chi, Zhe; Hu, Zhong; Chi, Zhen-Ming

2018-04-01

Melanin plays an important role in the stress adaptation of Aureobasidium melanogenum XJ5-1 isolated from the Taklimakan desert. A trehalose-6-phosphate synthase gene (TPS1 gene) was cloned from K5, characterized, and then deleted to determine the role of trehalose in the stress adaptation of the albino mutant K5. No stress response element and heat shock element were found in the promoter of the TPS1 gene. Deletion of the TPS1 gene in the albino mutant rendered a strain DT43 unable to synthesize any trehalose, but DT43 still could grow in glucose, suggesting that its hexokinase was insensitive to inhibition by trehalose-6-phosphate. Overexpression of the TPS1 gene enhanced trehalose biosynthesis in strain ET6. DT43 could not grow at 33 °C, whereas K5, ET6, and XJ5-1 could grow well at this temperature. Compared with K5 and ET6, DT43 was highly sensitive to heat shock treatment, high oxidation, and high desiccation, but all the three strains demonstrated the same sensitivity to UV light and high NaCl concentration. Therefore, trehalose played an important role in the adaptation of K5 to heat shock treatment, high oxidation, and high desiccation.

Genomic stability and physiological assessments of live offspring sired by a bull clone, Starbuck II.

PubMed

Ortegon, H; Betts, D H; Lin, L; Coppola, G; Perrault, S D; Blondin, P; King, W A

2007-01-01

It appears that overt phenotypic abnormalities observed in some domestic animal clones are not transmitted to their progeny. The current study monitored Holstein heifers sired by a bull clone, Starbuck II, from weaning to puberty. Genomic stability was assessed by telomere length status and chromosomal analysis. Growth parameters, blood profiles, physical exams and reproductive parameters were assessed for 12 months (and compared to age-matched control heifers). Progeny sired by the clone bull did not differ (P>0.05) in weight, length and height compared to controls. However, progeny had lower heart rates (HR) (P=0.009), respiratory rates (RR) (P=0.007) and body temperature (P=0.03). Hematological profiles were within normal ranges and did not differ (P>0.05) between both groups. External and internal genitalia were normal and both groups reached puberty at expected ages. Progeny had two or three ovarian follicular waves per estrous cycle and serum progesterone concentrations were similar (P=0.99) to controls. Telomere lengths of sperm and blood cells from Starbuck II were not different (P>0.05) than those of non-cloned cattle; telomere lengths of progeny were not different (P>0.05) from age-matched controls. In addition, progeny had normal karyotypes in peripheral blood leukocytes compared to controls (89.1% versus 86.3% diploid, respectively). In summary, heifers sired by a bull clone had normal chromosomal stability, growth, physical, hematological and reproductive parameters, compared to normal heifers. Furthermore, they had moderate stress responses to routine handling and restraint.

Microbial Diversity of Groundwater from Deep Subsurface Environment

NASA Astrophysics Data System (ADS)

Lin, L.; Onstott, T. C.; Hall, J.

2002-12-01

The subsurface environment harbors one of the most abundant reservoirs of biomass on Earth. The distribution of microbial ecosystems and the diversity of microbial metabolisms there remained poorly understood due to lack of detailed sampling over three-dimensional space with extremely heterogeneous characteristics. South African Au mines, however, provide the best access in the world to various types of groundwater and rocks at depths up to 4 km below surface. In this study, we present our recent analyses of microbial community structure of groundwater (with residence time of several million years) collected from depths between 850 to 1500 mbsl of Beatrix Au mine, South Africa. Five groundwater samples were collected anaerobically from freshly drilling boreholes with flow rates of 1 to 38 L/min. Cells were concentrated through filtration and total DNA were extracted from filters and PCR-amplified with primers targeting 16S rDNA gene. The amplicons were cloned and digested with restriction enzymes to identify the unique clone type. Sequences were obtained through direct sequencing of representative clones and compared with the closest matching sequences deposited in the gene bank for the construction of phylogenetic tree. The archaeal signatures were only found in one sample and close to the lineage of methanosarcina. The most predominant ribotype was similar to the environmental clone found in the same mine under the species level while the rest of ribotypes were either close to those capable of methanogenesis from long-chain alkanes or found in rice field or were distant from other environmental clones reported in previous study (Takai et al., 2001). The bacterial community exhibited a wide range of diversity among samples. Most samples were dominated by sequences close to alpha proteobacteria with various proportions of beta, gamma proteobacteria and environmental clones. A significant proportion of sequences close to thermophilic delta proteobacteria and clostridia were observed from one of the deepest samples. Since the in-situ water or rock temperature at sampling location is below the temperature range for thermophilic bacteria, this might indicate that the microorganisms once colonizing in the deeper and hotter portion of crust were transported upward with hydrothermal fluid and preserved in the sealed water pocket for a time scale of millions of years.

Characterization of Sarcoptes scabiei Tropomyosin and Paramyosin: Immunoreactive Allergens in Scabies.

PubMed

Naz, Shumaila; Desclozeaux, Marion; Mounsey, Kate E; Chaudhry, Farhana Riaz; Walton, Shelley F

2017-09-01

Scabies is a human skin disease due to the burrowing ectoparasite Sarcoptes scabiei var. hominis resulting in intense itching and inflammation and manifesting as a skin allergy. Because of insufficient mite material and lack of in vitro propagation system for antigen preparation, scabies is a challenging disease to develop serological diagnostics. For allergen characterization, full-length S. scabiei tropomyosin (Sar s 10) was cloned, expressed in pET-15b, and assessed for reactivity with IgE antibodies from human sera. IgE binding was observed to Sar s 10 with sera collected from subjects with ordinary scabies, house dust mite (HDM)-positive and naive subjects and a diagnostic sensitivity of < 30% was observed. S. scabiei paramyosin (Sar s 11) was cloned, and expressed in pET-28a in three overlapping fragments designated Sspara1, Sspara2, and Sspara3. IgE and IgG binding was observed to Sspara2 and Sspara3 antigens with sera collected from ordinary scabies, and HDM-positive subjects, but no binding was observed with sera collected from naive subjects. Sspara2 displayed excellent diagnostic potential with 98% sensitivity and 90% specificity observed for IgE binding and 70% sensitivity for IgG. In contrast, the diagnostic sensitivity of Sspara3 was 84% for IgE binding and 40% for IgG binding. In combination, Sspara2 and Sspara3 provided an IgE sensitivity of 94%. This study shows that IgE binding to Sspara2 and Sspara3 is a highly sensitive method for diagnosis of scabies infestation in clinical practice. The developed enzyme-linked immunosorbent assay helps direct future development of a specific diagnostic tool for scabies.

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

PubMed Central

Dron, M; Modjtahedi, N; Brison, O; Tovey, M G

1986-01-01

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells. Images PMID:3785169

Cloning, sequencing, and expression of interferon-γ from elk in North America

USGS Publications Warehouse

Sweeney, Steven J.; Emerson, Carlene; Eriks, Inge S.

2001-01-01

Eradication of Mycobacterium bovis relies on accurate detection of infected animals, including potential domestic and wildlife reservoirs. Available diagnostic tests lack the sensitivity and specificity necessary for accurate detection, particularly in infected wildlife populations. Recently, an in vitro diagnostic test for cattle which measures plasma interferon-gamma (IFN-γ) levels in blood following in vitro incubation with M. bovis purified protein derivative has been enveloped. This test appears to have increased sensitivity over traditional testing. Unfortunately, it does not detect IFN-γ from Cervidae. To begin to address this problem, the IFN-γ gene from elk (Cervus elaphus) was cloned, sequenced, expressed, and characterized. cDNA was cloned from mitogen stimulated peripheral blood mononuclear cells. The predicted amino acid (aa) sequence was compared to known sequences from cattle, sheep, goats, red deer (Cervus elaphus), humans, and mice. Biological activity of the recombinant elk IFN-γ (rElkIFN-γ) was confirmed in a vesicular stomatitis virus cytopathic effect reduction assay. Production of monoclonal antibodies to IFN-γ epitopes conserved between ruminant species could provide an important tool for the development of reliable, practical diagnostic assays for detection of a delayed type hypersensitivity response to a variety of persistent infectious agents in ruminants, including M. bovis and Brucella abortus. Moreover, development of these reagents will aid investigators in studies to explore immunological responses of elk that are associated with resistance to infectious diseases.

A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes.

PubMed

Meng, Jia; Kanzaki, Gregory; Meas, Diane; Lam, Christopher K; Crummer, Heather; Tain, Justina; Xu, H Howard

2012-04-01

Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published reports describing the application of asRNA gene silencing for comprehensive analyses of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250 000 library transformants for conditional growth inhibitory recombinant clones from two shotgun genomic libraries of E. coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes, while 18 originated from nonessential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12-fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E. coli using regulated asRNA expression. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

The Leaderless Bacteriocin Enterocin K1 Is Highly Potent against Enterococcus faecium: A Study on Structure, Target Spectrum and Receptor.

PubMed

Ovchinnikov, Kirill V; Kristiansen, Per Eugen; Straume, Daniel; Jensen, Marianne S; Aleksandrzak-Piekarczyk, Tamara; Nes, Ingolf F; Diep, Dzung B

2017-01-01

Enterocin K1 (EntK1), enterocin EJ97 (EntEJ97), and LsbB are three sequence related leaderless bacteriocins. Yet LsbB kills only lactococci while EntK1 and EntEJ97 target wider spectra with EntK1 being particularly active against Enterococcus faecium , including nosocomial multidrug resistant isolates. NMR study of EntK1 showed that it had a structure very similar to LsbB - both having an amphiphilic N-terminal α-helix and an unstructured C-terminus. The α-helix in EntK1 is, however, about 3-4 residues longer than that of LsbB. Enterococcal mutants highly resistant to EntEJ97 and EntK1 were found to have mutations within rseP , a gene encoding a stress response membrane-bound Zn-dependent protease. Heterologous expression of the enterococcal rseP rendered resistant cells of Streptococcus pneumoniae sensitive to EntK1 and EntEJ97, suggesting that RseP likely serves as the receptor for EntK1 and EntEJ97. It was also shown that the conserved proteolytic active site in E. faecalis RseP is partly required for EntK1 and EntEJ97 activity, since alanine substitutions of its conserved residues (HExxH) reduced the sensitivity of the clones to the bacteriocins. RseP is known to be involved in bacterial stress response. As expected, the growth of resistant mutants with mutations within rseP was severely affected when they were exposed to higher (stressing) growth temperatures, e.g., at 45°C, at which wild type cells still grew well. These findings allow us to design a hurdle strategy with a combination of the bacteriocin(s) and higher temperature that effectively kills bacteriocin sensitive bacteria and prevents the development of resistant cells.

Caffeine-enhanced survival of radiation-sensitive, repair-deficient Chinese hamster cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Utsumi, H.; Elkind, M.M.

1983-11-01

A clone of V79 Chinese hamster cells (V79-AL162/S-10) with unique properties has been isolated after a challenge of parental cells (V79-AL162) with 1 mM ouabain. Compared with parental cells, or with other clones isolated after the ouabain challenge, these cells form smaller colonies, are more sensitive to both x rays and fission-spectrum neutrons, and respond atypically to a postirradiation treatment with caffeine. Their enhanced response to x rays results mainly from a large reduction in the shoulder of their survival curve, probably because in late S phase, the most resistant phase in the cell cycle, the survival curve of thesemore » cells has a reduced shoulder width. Caffeine, and to a lesser extent theophylline, added to the colony-forming medium immediately after exposure appreciably increases the width of the shoulder of these sensitive cells, whereas caffeine has the opposite effect on the response of normal V79 cells. Thus the unique response of the V79-AL162/S-10 cells to a radiation posttreatment with caffeine (increased survival) results from a net increase in their ability to repair damage that is otherwise lethal; caffeine treatment ordinarly prevents normal V79 cells from repairing damage that is only potentially lethal.« less

Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

NASA Technical Reports Server (NTRS)

Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

1999-01-01

Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins.

PubMed

Higel, Fabian; Seidl, Andreas; Demelbauer, Uwe; Sörgel, Fritz; Frieß, Wolfgang

2014-01-01

N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins

PubMed Central

Higel, Fabian; Seidl, Andreas; Demelbauer, Uwe; Sörgel, Fritz; Frieß, Wolfgang

2014-01-01

N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented. PMID:24848368

DNA Book

PubMed Central

Kawai, Jun; Hayashizaki, Yoshihide

2003-01-01

We propose herein a new method of DNA distribution, whereby DNA clones or PCR products are printed directly onto the pages of books and delivered to users along with relevant scientific information. DNA sheets, comprising water-soluble paper onto which DNA is spotted, can be bound into books. Readers can easily extract the DNA fragments from DNA sheets and amplify them using PCR. We show that DNA sheets can withstand various conditions that may be experienced during bookbinding and delivery, such as high temperatures and humidity. Almost all genes (95%–100% of randomly selected RIKEN mouse cDNA clones) were recovered successfully by use of PCR. Readers can start their experiments after a 2-h PCR amplification without waiting for the delivery of DNA clones. The DNA Book thus provides a novel method for delivering DNA in a timely and cost-effective manner. A sample DNA sheet (carrying RIKEN mouse cDNA clones encoding genes of enzymes for the TCA cycle) is included in this issue for field-testing. We would greatly appreciate it if readers could attempt to extract DNA and report the results and whether the DNA sheet was shipped to readers in good condition. PMID:12819147

Clonal evolution and devolution after chemotherapy in adult acute myelogenous leukemia

PubMed Central

Parkin, Brian; Ouillette, Peter; Li, Yifeng; Keller, Jennifer; Lam, Cindy; Roulston, Diane; Li, Cheng; Shedden, Kerby

2013-01-01

The frequent occurrence of persistent or relapsed disease after induction chemotherapy in AML necessitates a better understanding of the clonal relationship of AML in various disease phases. In this study, we used SNP 6.0 array-based genomic profiling of acquired copy number aberrations (aCNA) and copy neutral LOH (cnLOH) together with sequence analysis of recurrently mutated genes to characterize paired AML genomes. We analyzed 28 AML sample pairs from patients who achieved complete remission with chemotherapy and subsequently relapsed and 11 sample pairs from patients with persistent disease after induction chemotherapy. Through review of aCNA/cnLOH and gene mutation profiles in informative cases, we demonstrate that relapsed AML invariably represents re-emergence or evolution of a founder clone. Furthermore, all individual aCNA or cnLOH detected at presentation persisted at relapse indicating that this lesion type is proximally involved in AML evolution. Analysis of informative paired persistent AML disease samples uncovered cases with 2 coexisting dominant clones of which at least one was chemotherapy sensitive and one resistant, respectively. These data support the conclusion that incomplete eradication of AML founder clones rather than stochastic emergence of fully unrelated novel clones underlies AML relapse and persistence with direct implications for clinical AML research. PMID:23175688

Novel Cell Culture-Adapted Genotype 2a Hepatitis C Virus Infectious Clone

PubMed Central

Date, Tomoko; Kato, Takanobu; Kato, Junko; Takahashi, Hitoshi; Morikawa, Kenichi; Akazawa, Daisuke; Murayama, Asako; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi

2012-01-01

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones. PMID:22787209

Highly complex neutralization determinants on a monophyletic lineage of newly transmitted subtype C HIV-1 Env clones from India.

PubMed

Kulkarni, Smita S; Lapedes, Alan; Tang, Haili; Gnanakaran, S; Daniels, Marcus G; Zhang, Ming; Bhattacharya, Tanmoy; Li, Ming; Polonis, Victoria R; McCutchan, Francine E; Morris, Lynn; Ellenberger, Dennis; Butera, Salvatore T; Bollinger, Robert C; Korber, Bette T; Paranjape, Ramesh S; Montefiori, David C

2009-03-15

Little is known about the neutralization properties of HIV-1 in India to optimally design and test vaccines. For this reason, a functional Env clone was obtained from each of ten newly acquired, heterosexually transmitted HIV-1 infections in Pune, Maharashtra. These clones formed a phylogenetically distinct genetic lineage within subtype C. As Env-pseudotyped viruses the clones were mostly resistant to IgG1b12, 2G12 and 2F5 but all were sensitive to 4E10. When compared to a large multi-subtype panel of Env-pseudotyped viruses (subtypes B, C and CRF02_AG) in neutralization assays with a multi-subtype panel of HIV-1-positive plasma samples, the Indian Envs were remarkably complex. With the exception of the Indian Envs, results of a hierarchical clustering analysis showed a strong subtype association with the patterns of neutralization susceptibility. From these patterns we were able to identify 19 neutralization cluster-associated amino acid signatures in gp120 and 14 signatures in the ectodomain and cytoplasmic tail of gp41. We conclude that newly transmitted Indian Envs are antigenically complex in spite of close genetic similarity. Delineation of neutralization-associated amino acid signatures provides a deeper understanding of the antigenic structure of HIV-1 Env.

Fab MAbs specific to HA of influenza virus with H5N1 neutralizing activity selected from immunized chicken phage library.

PubMed

Pitaksajjakul, Pannamthip; Lekcharoensuk, Porntippa; Upragarin, Narin; Barbas, Carlos F; Ibrahim, Madiha Salah; Ikuta, Kazuyoshi; Ramasoota, Pongrama

2010-05-14

Hemagglutinin protein (HA) was considered to be the primary target for monoclonal antibody production. This protein not only plays an important role in viral infections, but can also be used to differentiate H5N1 virus from other influenza A viruses. Hence, for diagnostic and therapeutic applications, it is important to develop anti-HA monoclonal antibody (MAb) with high sensitivity, specificity, stability, and productivity. Nine unique Fab MAbs were generated from chimeric chicken/human Fab phage display library constructed from cDNA derived from chickens immunized with recombinant hemagglutinin protein constructed from H5N1 avian influenza virus (A/Vietnam/1203/04). The obtained Fab MAbs showed several characteristics for further optimization and development-three clones were highly specific to only H5N1 virus. This finding can be applied to the development of H5N1 diagnostic testing. Another clone showed neutralization activity that inhibited H5N1 influenza virus infection in Madin-Darby canine kidney (MDCK) cells. In addition, one clone showed strong reactivity with several of the influenza A virus subtypes tested. The conversion of this clone to whole IgG is a promising study for a cross-neutralization activity test. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli.

PubMed

Stephenson, Stacy Ann-Marie; Brown, Paul D

2016-01-01

Urinary tract infections (UTI) are among the most frequently encountered infections in clinical practice globally. Predominantly a burden among female adults and infants, UTIs primarily caused by uropathogenic Escherichia coli (UPEC) results in high morbidity and fiscal health strains. During pathogenesis, colonization of the urinary tract via fimbrial adhesion to mucosal cells is the most critical point in infection and has been linked to DNA methylation. Furthermore, with continuous exposure to antibiotics as the standard therapeutic strategy, UPEC has evolved to become highly adaptable in circumventing the effect of antimicrobial agents and host defenses. Hence, the need for alternative treatment strategies arises. Since differential DNA methylation is observed as a critical precursor to virulence in various pathogenic bacteria, this body of work sought to assess the influence of the DNA adenine methylase (dam) gene on gene expression and cellular adhesion in UPEC and its potential as a therapeutic target. To monitor the influence of dam on attachment and FQ resistance, selected UPEC dam mutants created via one-step allelic exchange were transformed with cloned qnrA and dam complement plasmid for comparative analysis of growth rate, antimicrobial susceptibility, biofilm formation, gene expression, and mammalian cell attachment. The absence of DNA methylation among dam mutants was apparent. Varying deficiencies in cell growth, antimicrobial resistance and biofilm formation, alongside low-level increases in gene expression (recA and papI), and adherence to HEK-293 and HTB-9 mammalian cells were also detected as a factor of SOS induction to result in increased mutability. Phenotypic characteristics of parental strains were restored in dam complement strains. Dam's vital role in DNA methylation and gene expression in local UPEC isolates was confirmed. Similarly to dam-deficient Enterohemorrhagic E. coli (EHEC), these findings suggest unsuccessful therapeutic use of Dam inhibitors against UPEC or dam-deficient UPEC strains as attenuated live vaccines. However, further investigations are necessary to determine the post-transcriptional influence of dam on the regulatory network of virulence genes central to pathogenesis.

Molecular cloning and functional characterization of eleven subtypes of interferon-α in Amur tigers (Panthera tigris altaica).

PubMed

Zhao, Hongjing; Ma, Jian; Wang, Yu; Liu, Juanjuan; Shao, Yizhi; Li, Jinglun; Jiang, Guangshun; Xing, Mingwei

2017-12-01

Interferon has a broad-spectrum of antiviral effects and represents an ideal choice for the development of antiviral drugs. Nonetheless, information about alpha interferon (IFN-α) is vacant in Amur tiger (Panthera tigris altaica), an endangered species and indigenous to northeast Asia. Herein, 11 PtIFN-αs genes, which encoded proteins of 164-165 amino acids, were amplified. Afterwards, expression and purification were conducted in Escherichia coli. In physicochemical analysis, PtIFN-αs were shown to be highly sensitive to trypsin and remained stable despite changes in pH and temperature. In feline kidney cells (F81)/vesicular stomatitis virus (VSV)/canine distemper virus (CDV)/avian influenza virus (AIV) systems, PtIFN-αs were demonstrated to have distinct antiviral activities, some of them (PtIFN-α and PtIFN-α9) inhibited viral transcription levels more effectively than the other subtypes including Felis catus IFN-α, an effective therapeutic agent used for viral infections clinically. Additionally, PtIFN-α and PtIFN-α9 can up-regulate the transcription and expression of p53, a tumor suppressor factor, which could promote apoptosis of virus-infected cells. In conclusion, we cloned and expressed 11 subtypes of PtIFN-α for the first time. Furthermore, PtIFN-α and PtIFN-α9 were likely to be more efficient against both chronic viral infections and neoplastic diseases that affect the Amur tiger population. It will be of significant importance for further studies to protect this endangered species. Copyright © 2017. Published by Elsevier Ltd.

Optimization of the simultaneous saccharification and fermentation process using thermotolerant yeasts.

PubMed

Ballesteros, I; Oliva, J M; Ballesteros, M; Carrasco, J

1993-01-01

Different treatments to improve the thermotolerance of fermenting yeasts for simultaneous ethanol saccharification and fermentation process of cellulosic materials have been examined. Yeasts of the genera Saccharomyces and Kluyveromyces were tested for growth and fermentation at progressively higher temperatures in the range of 42-47 degrees C. The best results were obtained with K. marxianus LG, which was then submitted to different treatments in order to achieve thermotolerant clones. A total of 35 new clones were obtained that dramatically improved the SSF of 10% Solka-floc substrate at 45 degrees C when compared to the original strain, some with ethanol concentrations as high as 33 g/L.

Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

NASA Astrophysics Data System (ADS)

Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

2015-07-01

The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future.

Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

PubMed Central

Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

2015-01-01

The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future. PMID:26198671

China Report Science and Technology No. 196

DTIC Science & Technology

1983-05-02

Liang Shiyuan ; HANGKONG ZHISHI, Mar 83) 8 Briefs High Temperature Microscopic Television 10 LIFE SCIENCES Briefs Hepatitis B Virus Clone...Mar 83 pp 18-19 [Article by Liang Shiyuan [2733 1102 0337]: Salvaged Helicopter Rotor Blades and Windpower Stations"] [Excerpt] China is

Temperature Sensitivity as a Microbial Trait Using Parameters from Macromolecular Rate Theory

PubMed Central

Alster, Charlotte J.; Baas, Peter; Wallenstein, Matthew D.; Johnson, Nels G.; von Fischer, Joseph C.

2016-01-01

The activity of soil microbial extracellular enzymes is strongly controlled by temperature, yet the degree to which temperature sensitivity varies by microbe and enzyme type is unclear. Such information would allow soil microbial enzymes to be incorporated in a traits-based framework to improve prediction of ecosystem response to global change. If temperature sensitivity varies for specific soil enzymes, then determining the underlying causes of variation in temperature sensitivity of these enzymes will provide fundamental insights for predicting nutrient dynamics belowground. In this study, we characterized how both microbial taxonomic variation as well as substrate type affects temperature sensitivity. We measured β-glucosidase, leucine aminopeptidase, and phosphatase activities at six temperatures: 4, 11, 25, 35, 45, and 60°C, for seven different soil microbial isolates. To calculate temperature sensitivity, we employed two models, Arrhenius, which predicts an exponential increase in reaction rate with temperature, and Macromolecular Rate Theory (MMRT), which predicts rate to peak and then decline as temperature increases. We found MMRT provided a more accurate fit and allowed for more nuanced interpretation of temperature sensitivity in all of the enzyme × isolate combinations tested. Our results revealed that both the enzyme type and soil isolate type explain variation in parameters associated with temperature sensitivity. Because we found temperature sensitivity to be an inherent and variable property of an enzyme, we argue that it can be incorporated as a microbial functional trait, but only when using the MMRT definition of temperature sensitivity. We show that the Arrhenius metrics of temperature sensitivity are overly sensitive to test conditions, with activation energy changing depending on the temperature range it was calculated within. Thus, we propose the use of the MMRT definition of temperature sensitivity for accurate interpretation of temperature sensitivity of soil microbial enzymes. PMID:27909429

Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2.

PubMed

Leow, Chiuan Herng; Jones, Martina; Cheng, Qin; Mahler, Stephen; McCarthy, James

2014-07-18

Early and accurate diagnosis of Plasmodium falciparum infection is important for providing appropriate treatment to patients with malaria. However, technical limitations of currently available diagnostic tests limit their use in control programs. One possible explanation for the vulnerability of current antibodies used in RDTs is their propensity to degrade at high ambient temperatures. Isolation of new antibodies with better thermal stability represents an appealing approach to improve the performance of RDTs. In this study, phage display technology was deployed to isolate novel binders by screening a human naïve scFv antibody library against recombinant Plasmodium falciparum histidine rich protein 2 (rPfHRP2). The isolated scFv clones were reformatted to whole IgG and the recombinant mAbs were produced in a mammalian CHO cell expression system. To verify the biological activity of these purified recombinant mAbs, range of functional assays were characterized. Two unique clones (D2 and F9) were isolated after five rounds of biopanning. The reformatted and expressed antibodies demonstrated high binding specificity to malaria recombinant PfHRP2 and native proteins. When 5 μg/mL of mAbs applied, mAb C1-13 had the highest sensitivity, with an OD value of 1, the detection achieved 5 ng/mL of rPfHRP2, followed by mAbs D2 and F9 at 10 ng/mL and 100 ng/mL of rPfHRP2, respectively. Although the sensitivity of mAbs D2 and F9 was lower than the control, these recombinant human mAbs have shown better stability compared to mouse mAb C1-13 at various temperatures in DSC and blot assays. In view of epitope mapping, the predominant motif of rPfHRP2 recognized by mAb D2 was AHHAADAHHA, whereas mAb F9 was one amino acid shorter, resulting in AHHAADAHH. mAb F9 had the strongest binding affinity to rPfHRP2 protein, with a KD value of 4.27 × 10(-11) M, followed by control mAb C1-13 at 1.03 × 10(-10) M and mAb D2 at 3.05 × 10(-10) M. Overall, the performance of these mAbs showed comparability to currently available PfHRP2-specific mouse mAb C1-13. The stability of these novel binders indicate that they merit further work to evaluate their utility in the development of new generation point of care diagnosis of malaria.

Cloning, Expression Analysis and Enzyme Activity Assays of the α-Carbonic Anhydrase Gene from Chlamydomonas sp. ICE-L.

PubMed

Qu, Changfeng; He, Yingying; Zheng, Zhou; An, Meiling; Li, Lulu; Wang, Xixi; He, Xiaodong; Wang, Yibin; Liu, Fangming; Miao, Jinlai

2018-01-01

The α-carbonic anhydrase (α-CA) is a zinc ion-containing enzyme that catalyzes the hydration of carbon dioxide. In this paper, a full-length α-CA gene was cloned from Chlamydomonas sp. ICE-L using RT-PCR and RACE-PCR for bioinformatic analysis. The α-CA open reading frame obtained by PCR was cloned into a vector and transformed into Escherichia coli to generate α-CA-producing bacteria. The α-CA was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.8 mM. A single band with a molecular weight of approximate 40 kDa expressed in the recombinant E. coli strain harboring the α-CA vector was observed in SDS-PAGE analysis. The carbon dioxide hydration activity and esterase activity of α-CA expressed by the recombinant strain were 0.404 U/mg and 0.319 U, respectively. In addition, three conditions, temperature, salinity and UVB radiation exposure, were selected to analyze α-CA transcription levels by qRT-PCR. The results suggested UVB exposure increased the expression of relative mRNA; meanwhile, the α-CA mRNA expression was rapidly induced by temperature and salinity stress, indicating that Chlamydomonas sp. ICE-L might modulate the α-CA mRNA expression to adapt to the extreme environments.

Gene cloning, expression, and characterization of a new carboxylesterase from Serratia sp. SES-01: comparison with Escherichia coli BioHe enzyme.

PubMed

Kwon, Min-A; Kim, Hyun Suk; Oh, Joon Young; Song, Bong Keun; Song, Jae Kwang

2009-02-01

The carboxylesterase-encoding gene (bioHs) of a newly isolated strain, Serratia sp. SES-01, was cloned from the genomic DNA library by detecting formation of transparent halo around the colony on LB-tributyrin agar plates. The amino acid sequence of BioHs was highly similar to the members of the BioH enzyme family involved in the biotin biosynthetic pathway; it showed the highest similarity (91%) with that of Serratia proteamaculans. To compare BioHs with other BioH enzymes, the relatively well-known bioHe gene of E. coli was cloned with PCR. After we achieved high-level expression of soluble BioHs and BioHe through the exploration of different culture conditions, the purified BioHs and BioHe enzymes were characterized in terms of specificity, activity, and stability. BioHe was generally more robust to a change in temperature and pH and an addition of organic solvents than BioHs. The two enzymes exhibited a strong preference for carboxylesterase rather than for thioesterase and were optimal at relatively low temperatures (20-40 degrees ) and alkaline pHs (7.5-9.0). The results in this study strongly suggested that both the BioHs and BioHe enzymes would be potential candidates for use as a carboxylesterase in many industrial applications.

Quantitative Effects of P Elements on Hybrid Dysgenesis in Drosophila Melanogaster

PubMed Central

Rasmusson, K. E.; Simmons, M. J.; Raymond, J. D.; McLarnon, C. F.

1990-01-01

Genetic analyses involving chromosomes from seven inbred lines derived from a single M' strain were used to study the quantitative relationships between the incidence and severity of P-M hybrid dysgenesis and the number of genomic P elements. In four separate analyses, the mutability of sn(w), a P element-insertion mutation of the X-linked singed locus, was found to be inversely related to the number of autosomal P elements. Since sn(w) mutability is caused by the action of the P transposase, this finding supports the hypothesis that genomic P elements titrate the transposase present within a cell. Other analyses demonstrated that autosomal transmission ratios were distorted by P element action. In these analyses, the amount of distortion against an autosome increased more or less linearly with the number of P elements carried by the autosome. Additional analyses showed that the magnitude of this distortion was reduced when a second P element-containing autosome was present in the genome. This reduction could adequately be explained by transposase titration; there was no evidence that it was due to repressor molecules binding to P elements and inhibiting their movement. The influence of genomic P elements on the incidence of gonadal dysgenesis was also investigated. Although no simple relationship between the number of P elements and the incidence of the trait could be discerned, it was clear that even a small number of elements could increase the incidence markedly. The failure to find a quantitative relationship between P element number and the incidence of gonadal dysgenesis probably reflects the complex etiology of this trait. PMID:2155853

Structural insight into the binding interactions of modeled structure of Arabidopsis thaliana urease with urea: an in silico study.

PubMed

Yata, Vinod Kumar; Thapa, Arun; Mattaparthi, Venkata Satish Kumar

2015-01-01

Urease (EC 3.5.1.5., urea amidohydrolase) catalyzes the hydrolysis of urea to ammonia and carbon dioxide. Urease is present to a greater abundance in plants and plays significant role related to nitrogen recycling from urea. But little is known about the structure and function of the urease derived from the Arabidopsis thaliana, the model system of choice for research in plant biology. In this study, a three-dimensional structural model of A. thaliana urease was constructed using computer-aided molecular modeling technique. The characteristic structural features of the modeled structure were then studied using atomistic molecular dynamics simulation. It was observed that the modeled structure was stable and regions between residues index (50-80, 500-700) to be significantly flexible. From the docking studies, we detected the possible binding interactions of modeled urease with urea. Ala399, Ile675, Thr398, and Thr679 residues of A. thaliana urease were observed to be significantly involved in binding with the substrate urea. We also compared the docking studies of ureases from other sources such as Canavalia ensiformis, Helicobacter pylori, and Bacillus pasteurii. In addition, we carried out mutation analysis to find the highly mutable amino acid residues of modeled A. thaliana urease. In this particular study, we observed Met485, Tyr510, Ser786, Val426, and Lys765 to be highly mutable amino acids. These results are significant for the mutagenesis analysis. As a whole, this study expounds the salient structural features as well the binding interactions of the modeled structure of A. thaliana urease.

Extensive de novo mutation rate variation between individuals and across the genome of Chlamydomonas reinhardtii

PubMed Central

Ness, Rob W.; Morgan, Andrew D.; Vasanthakrishnan, Radhakrishnan B.; Colegrave, Nick; Keightley, Peter D.

2015-01-01

Describing the process of spontaneous mutation is fundamental for understanding the genetic basis of disease, the threat posed by declining population size in conservation biology, and much of evolutionary biology. Directly studying spontaneous mutation has been difficult, however, because new mutations are rare. Mutation accumulation (MA) experiments overcome this by allowing mutations to build up over many generations in the near absence of natural selection. Here, we sequenced the genomes of 85 MA lines derived from six genetically diverse strains of the green alga Chlamydomonas reinhardtii. We identified 6843 new mutations, more than any other study of spontaneous mutation. We observed sevenfold variation in the mutation rate among strains and that mutator genotypes arose, increasing the mutation rate approximately eightfold in some replicates. We also found evidence for fine-scale heterogeneity in the mutation rate, with certain sequence motifs mutating at much higher rates, and clusters of multiple mutations occurring at closely linked sites. There was little evidence, however, for mutation rate heterogeneity between chromosomes or over large genomic regions of 200 kbp. We generated a predictive model of the mutability of sites based on their genomic properties, including local GC content, gene expression level, and local sequence context. Our model accurately predicted the average mutation rate and natural levels of genetic diversity of sites across the genome. Notably, trinucleotides vary 17-fold in rate between the most and least mutable sites. Our results uncover a rich heterogeneity in the process of spontaneous mutation both among individuals and across the genome. PMID:26260971

Coherent Somatic Mutation in Autoimmune Disease

PubMed Central

Ross, Kenneth Andrew

2014-01-01

Background Many aspects of autoimmune disease are not well understood, including the specificities of autoimmune targets, and patterns of co-morbidity and cross-heritability across diseases. Prior work has provided evidence that somatic mutation caused by gene conversion and deletion at segmentally duplicated loci is relevant to several diseases. Simple tandem repeat (STR) sequence is highly mutable, both somatically and in the germ-line, and somatic STR mutations are observed under inflammation. Results Protein-coding genes spanning STRs having markers of mutability, including germ-line variability, high total length, repeat count and/or repeat similarity, are evaluated in the context of autoimmunity. For the initiation of autoimmune disease, antigens whose autoantibodies are the first observed in a disease, termed primary autoantigens, are informative. Three primary autoantigens, thyroid peroxidase (TPO), phogrin (PTPRN2) and filaggrin (FLG), include STRs that are among the eleven longest STRs spanned by protein-coding genes. This association of primary autoantigens with long STR sequence is highly significant (). Long STRs occur within twenty genes that are associated with sixteen common autoimmune diseases and atherosclerosis. The repeat within the TTC34 gene is an outlier in terms of length and a link with systemic lupus erythematosus is proposed. Conclusions The results support the hypothesis that many autoimmune diseases are triggered by immune responses to proteins whose DNA sequence mutates somatically in a coherent, consistent fashion. Other autoimmune diseases may be caused by coherent somatic mutations in immune cells. The coherent somatic mutation hypothesis has the potential to be a comprehensive explanation for the initiation of many autoimmune diseases. PMID:24988487

Simple, Inexpensive, and Rapid Way to Produce Bacillus subtilis Spores for the Guthrie Bioassay

PubMed Central

Franklin, Martha L.; Clark, William A.

1981-01-01

Esculin agar has been found to be a simple, inexpensive, rapid, and reliable means to promote production of spores of inhibitor-sensitive clones of Bacillus subtilis strains ATCC 6051 and 6633 for use in the Guthrie bioassay screening tests for genetic metabolic disorders. Images PMID:6790564

Validation of genome-wide association studies as a tool to identify virulence factors in Parastagonospora nodorum

USDA-ARS?s Scientific Manuscript database

Parastagonospora nodorum is a necrotrophic fungal pathogen causing Septoria nodorum blotch (SNB) on wheat. We have identified nine necrotrophic effector-host dominant sensitivity gene interactions, and we have cloned three of the necrotrophic effector (NE) genes, including SnToxA, SnTox1 and SnTox3...

Medicinal chemistry discoveries among 1,3,5-triazines: recent advances (2000-2013) as antimicrobial, anti-TB, anti-HIV and antimalarials.

PubMed

Patel, Rahul V; Keum, Young-Soo; Park, Se Won

2014-01-01

The chemistry and an extensive spectrum of biological activities of s-triazines have been examined since several decades and this heterocyclic core has received emerging consensus. This article aims to summarize recent advances (2000-2013) made towards the discovery of antimicrobial, antituberculosis, anti-HIV and antimalarial agents holding 1,3,5-triazine ring as a nucleus with the substitution of several types of nucleophiles. Molecular patterns associated with particular potency have been identified targeting several Gram-positive and Gram-negative bacteria and some fungal species, mycobacterium tuberculosis H37Rv, HIV type I and HIV type II, particularly, HIV-1I IIB and HIV- 1ROD strains as well as a variety of P. falciparum malarial strains as chloroquine-resistant K1, chloroquine-susceptible NF54, chloroquine-sensitive 3D7, P. falciparum (D6 clone), P. falciparum (W2 clone), cycloguanil-resistant FCR-3, chloroquine sensitive RKL2. The report will be of considerable interest to gain useful information for the furtherance of drug discovery with extended 1,3,5-triazine designs.

Abundance and composition dynamics of soil ammonia-oxidizing archaea in an alpine fir forest on the eastern Tibetan Plateau of China.

PubMed

Wang, Ao; Wu, Fu-Zhong; Yang, Wan-Qin; Wu, Zhi-Chao; Wang, Xu-Xi; Tan, Bo

2012-05-01

Real-time qPCR and clone library sequencing targeting amoA genes were used to investigate the seasonal dynamics of an ammonia-oxidizing archaea (AOA) community in an alpine fir forest in western China. AOA were detected at all sampling dates, and there were significant variations in archaeal amoA gene copy numbers (7.63 × 10(5) to 8.35 × 10(8) per gram of dry soil) throughout the nongrowing season. Compared with ammonia-oxidizing bacteria (AOB), the AOA displayed a higher abundance on the majority of sampling dates during the freeze-thaw period. All of the AOA sequences fell within soil and sediment lineages and were affiliated with 7 clusters. Compared with the other clusters, cluster 1 was more sensitive to low temperature and was the dominant group in August. In contrast, cluster 3 dominated the AOA community in winter and probably represents a group of cold-adapted archaea. Redundancy analysis (RDA) revealed that the seasonality of the AOA community was mainly attributed to changes in soil temperature and nutrient availability (e.g., dissolved organic nitrogen and carbon). Our results indicate that AOA exist in frozen soils in the alpine coniferous forest ecosystem of the eastern Tibetan Plateau. Moreover, soil temperature may directly and (or) indirectly affect AOA abundance and composition and may further influence the soil N cycle during the winter.

Cold-induced ependymin expression in zebrafish and carp brain: implications for cold acclimation.

PubMed

Tang, S J; Sun, K H; Sun, G H; Lin, G; Lin, W W; Chuang, M J

1999-10-01

Cold acclimation has been suggested to be mediated by alternations in the gene expression pattern in the cold-adapted fish. To investigate the mechanism of cold acclimation in fish brain at the molecular level, relevant subsets of differentially expressed genes of interest were identified and cloned by the PCR-based subtraction suppression hybridization. Characterization of the selected cold-induced cDNA clones revealed one encoding ependymin. This gene was shown to be brain-specific. The expression of ependymin was induced by a temperature shift from 25 degrees C to 6 degrees C in Cyprinus carpio or 12 degrees C in Danio rerio. Activation of ependymin was detected 2 h after cold exposure and peaked at more than 10-fold at 12 h. This peak level remains unchanged until the temperature returns to 25 degrees C. Although the amount of soluble ependymin protein in brain was not changed by cold treatment, its level in the fibrous insoluble polymers increased 2-fold after exposure to low temperature. These findings indicate that the increase in ependymin expression is an early event that may play an important role in the cold acclimation of fish.

Cloning and expression of cyclodextrin glycosyltransferase gene from Paenibacillus sp. T16 isolated from hot spring soil in northern Thailand.

PubMed

Charoensakdi, Ratiya; Murakami, Shuichiro; Aoki, Kenji; Rimphanitchayakit, Vichien; Limpaseni, Tipaporn

2007-05-31

Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower K(m) for coupling reaction using cellobiose and cyclodextrins as substrates.

Environmental Controls Over Actinobacteria Communities in Ecological Sensitive Yanshan Mountains Zone

PubMed Central

Tang, Hui; Shi, Xunxun; Wang, Xiaofei; Hao, Huanhuan; Zhang, Xiu-Min; Zhang, Li-Ping

2016-01-01

The Yanshan Mountains are one of the oldest mountain ranges in the world. They are located in an ecologically sensitive zone in northern China near the Hu Huanyong Line. In this metagenomic study, we investigated the diversity of Actinobacteria in soils at 10 sites (YS1–YS10) on the Yanshan Mountains. First, we assessed the effect of different soil prtreatment on Actinobacteria recovery. With the soil pretreatment method: air drying of the soil sample, followed by exposure to 120°C for 1 h, we observed the higher Actinobacteria diversity in a relatively small number of clone libraries. No significant differences were observed in the Actinobacterial diversity of soils from sites YS2, YS3, YS4, YS6, YS8, YS9, or YS10 (P > 0.1). However, there were differences (P < 0.05) from the YS7 site and other sites, especially in response to environmental change. And we observed highly significant differences (P < 0.001) in Actinobacterial diversity of the soil from YS7 and that from YS4 and YS8 sites. The climatic characteristics of mean active accumulated temperature, annual mean precipitation, and annual mean temperature, and biogeochemical data of total phosphorus contributed to the diversity of Actinobacterial communities in soils at YS1, YS3, YS4, and YS5 sites. Compared to the climatic factors, the biogeochemical factors mostly contributed in shaping the Actinobacterial community. This work provides evidence that the diversity of Actinobacterial communities in soils from the Yashan Mountains show regional biogeographic patterns and that community membership change along the north-south distribution of the Hu Huanyong Line. PMID:27047461

[Fungal community structure in phase II composting of Volvariella volvacea].

PubMed

Chen, Changqing; Li, Tong; Jiang, Yun; Li, Yu

2014-12-04

To understand the fungal community succession during the phase II of Volvariella volvacea compost and clarify the predominant fungi in different fermentation stages, to monitor the dynamic compost at the molecular level accurately and quickly, and reveal the mechanism. The 18S rDNA-denaturing gradient gel electrophoresis (DGGE) and sequencing methods were used to analyze the fungal community structure during the course of compost. The DGGE profile shows that there were differences in the diversity of fungal community with the fermentation progress. The diversity was higher in the stages of high temperature. And the dynamic changes of predominant community and relative intensity was observed. Among the 20 predominant clone strains, 9 were unknown eukaryote and fungi, the others were Eurotiales, Aspergillus sp., Melanocarpus albomyces, Colletotrichum sp., Rhizomucor sp., Verticillium sp., Penicillium commune, Microascus trigonosporus and Trichosporon lactis. The 14 clone strains were detected in the stages of high and durative temperature. The fungal community structure and predominant community have taken dynamic succession during the phase II of Volvariella volvacea compost.

The recA gene from the thermophile Thermus aquaticus YT-1: cloning, expression, and characterization.

PubMed Central

Angov, E; Camerini-Otero, R D

1994-01-01

We have cloned, expressed, and purified the RecA analog from the thermophilic eubacterium Thermus aquaticus YT-1. Analysis of the deduced amino acid sequence indicates that the T. aquaticus RecA is structurally similar to the Escherichia coli RecA and suggests that RecA-like function has been conserved in thermophilic organisms. Preliminary biochemical analysis indicates that the protein has an ATP-dependent single-stranded DNA binding activity and can pair and carry out strand exchange to form a heteroduplex DNA under reaction conditions previously described for E. coli RecA, but at 55 to 65 degrees C. Further characterization of a thermophilically derived RecA protein should yield important information concerning DNA-protein interactions at high temperatures. In addition, a thermostable RecA protein may have some general applicability in stabilizing DNA-protein interactions in reactions which occur at high temperatures by increasing the specificity (stringency) of annealing reactions. Images PMID:8113181

Expression of the cloned ColE1 kil gene in normal and Kilr Escherichia coli.

PubMed Central

Altieri, M; Suit, J L; Fan, M L; Luria, S E

1986-01-01

The kil gene of the ColE1 plasmid was cloned under control of the lac promoter. Its expression under this promoter gave rise to the same pattern of bacterial cell damage and lethality as that which accompanies induction of the kil gene in the colicin operon by mitomycin C. This confirms that cell damage after induction is solely due to expression of kil and is independent of the cea or imm gene products. Escherichia coli derivatives resistant to the lethal effects of kil gene expression under either the normal or the lac promoter were isolated and found to fall into several classes, some of which were altered in sensitivity to agents that affect the bacterial envelope. PMID:2946661

YAC cloning Mus musculus telomeric DNA: physical, genetic, in situ and STS markers for the distal telomere of chromosome 10.

PubMed

Kipling, D; Wilson, H E; Thomson, E J; Cooke, H J

1995-06-01

Three Mus musculus DBA/2 YAC libraries were constructed using a half-YAC telomere cloning vector. This functional complementation approach yields libraries which include terminal restriction fragments of the mouse genome. Screening all three libraries led to the isolation of 32 independent clones which carry linear YACs containing the mouse terminal repeat sequence, (TTAGGG)n. These YACs provide a resource to isolate regions of the mouse genome close to chromosome termini and excluded from existing conventional YAC libraries. To demonstrate their utility, a hybridization probe was isolated from Mtel-1, the first (TTAGGG)n-containing YAC isolated. This probe detects a approximately 70 kb Kpnl fragment in the mouse genome which is sensitive to pretreatment with BAL31 exonuclease. A PCR-based genetic marker generated from the sequence of this probe maps 4.4 cM from the most distal anchor locus on chromosome 10 in the EUCIB interspecific backcross. STS primers for this locus, D10Hgu1, were used to isolate YAC 110F4 from a commercially available mouse YAC library. Fluorescence in situ hybridization demonstrates that YAC 110F4 hybridizes to the distal telomere of chromosome 10. Clones in this collection of telomere YACs therefore partially overlap clones in conventional YAC libraries, and thus the previously unavailable terminal regions of the mouse genome can now be linked with the developing mouse STS YAC contig. Genetic markers such as D10Hgu1 allow the ends of the mouse genetic map to be defined, thus closing the map.

A fosmid cloning strategy for detecting the widest possible spectrum of microbes from the international space station drinking water system.

PubMed

Choi, Sangdun; Chang, Mi Sook; Stuecker, Tara; Chung, Christine; Newcombe, David A; Venkateswaran, Kasthuri

2012-12-01

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular-weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.

Computational smart polymer design based on elastin protein mutability.

PubMed

Tarakanova, Anna; Huang, Wenwen; Weiss, Anthony S; Kaplan, David L; Buehler, Markus J

2017-05-01

Soluble elastin-like peptides (ELPs) can be engineered into a range of physical forms, from hydrogels and scaffolds to fibers and artificial tissues, finding numerous applications in medicine and engineering as "smart polymers". Elastin-like peptides are attractive candidates as a platform for novel biomaterial design because they exhibit a highly tunable response spectrum, with reversible phase transition capabilities. Here, we report the design of the first virtual library of elastin-like protein models using methods for enhanced sampling to study the effect of peptide chemistry, chain length, and salt concentration on the structural transitions of ELPs, exposing associated molecular mechanisms. We describe the behavior of the local molecular structure under increasing temperatures and the effect of peptide interactions with nearest hydration shell water molecules on peptide mobility and propensity to exhibit structural transitions. Shifts in the magnitude of structural transitions at the single-molecule scale are explained from the perspective of peptide-ion-water interactions in a library of four unique elastin-like peptide systems. Predictions of structural transitions are subsequently validated in experiment. This library is a valuable resource for recombinant protein design and synthesis as it elucidates mechanisms at the single-molecule level, paving a feedback path between simulation and experiment for smart material designs, with applications in biomedicine and diagnostic devices. Copyright © 2017. Published by Elsevier Ltd.

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir.

PubMed Central

Stibitz, S; Weiss, A A; Falkow, S

1988-01-01

The vir locus of Bordetella pertussis apparently encodes a trans-acting positive regulator that is required for the coordinate expression of genes associated with virulence: pertussis toxin, filamentous hemagglutinin (FHA), hemolysin, and adenylate cyclase toxin. DNA clones of vir and of genes required for the synthesis of some of the factors under vir control were obtained with DNA probes from the chromosomal DNA surrounding sites of Tn5 insertion mutations that inactivated those genes. Two vir clones were found which also contained genes required for the proper expression of FHA in B. pertussis. The plasmids which contained both the fha and vir genes expressed immunologically reactive FHA in Escherichia coli, as detected by colony blots, whereas plasmids which contained only fha or vir were negative in this assay. The regulation of FHA production in E. coli, as in B. pertussis, was temperature dependent and inhibited by high concentrations of either magnesium ions or nicotinic acid, indicating that the sequences cloned in E. coli contained the information required to preserve the physiological responses seen in B. pertussis. Further characterization of the vir-fha clones by Tn5 mutagenesis in E. coli and by the return of cloned sequences to B. pertussis in trans and to the B. pertussis chromosome led to the localization of the vir locus, the structural gene for FHA, and genes that are possibly required for the synthesis and export of FHA. Images PMID:2898470

Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production

PubMed Central

Tajabadi, Naser; Baradaran, Ali; Ebrahimpour, Afshin; Rahim, Raha A; Bakar, Fatimah A; Manap, Mohd Yazid A; Mohammed, Abdulkarim S; Saari, Nazamid

2015-01-01

Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products. PMID:25757029

Stem respiration of Populus species in the third year of free-air CO2 enrichment.

PubMed

Gielen, Birgit; Scarascia-Mugnozza, Giuseppe; Ceulemans, Reinhart

2003-04-01

Carbon cycling in ecosystems, and especially in forests, is intensively studied to predict the effects of global climate change, and the role which forests may play in 'changing climate change'. One of the questions is whether the carbon balance of forests will be affected by increasing atmospheric CO2 concentrations. Regarding this question, effects of elevated [CO2] on woody-tissue respiration have frequently been neglected. Stem respiration of three Populus species (P. alba L. (Clone 2AS-11), P. nigra L. (Clone Jean Pourtet), and P. x euramericana (Clone I-214)) was measured in a managed, high-density forest plantation exposed to free-air CO2 enrichment (POPFACE). During the period of measurements, in May of the third year, stem respiration rates were not affected by the FACE treatment. Moreover, FACE did not influence the relationships between respiration rate and both stem temperature and relative growth rate. The results were supported by the reported absence of a FACE-effect on growth and stem wood density.

PrecisePrimer: an easy-to-use web server for designing PCR primers for DNA library cloning and DNA shuffling.

PubMed

Pauthenier, Cyrille; Faulon, Jean-Loup

2014-07-01

PrecisePrimer is a web-based primer design software made to assist experimentalists in any repetitive primer design task such as preparing, cloning and shuffling DNA libraries. Unlike other popular primer design tools, it is conceived to generate primer libraries with popular PCR polymerase buffers proposed as pre-set options. PrecisePrimer is also meant to design primers in batches, such as for DNA libraries creation of DNA shuffling experiments and to have the simplest interface possible. It integrates the most up-to-date melting temperature algorithms validated with experimental data, and cross validated with other computational tools. We generated a library of primers for the extraction and cloning of 61 genes from yeast DNA genomic extract using default parameters. All primer pairs efficiently amplified their target without any optimization of the PCR conditions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Tobacco class I cytosolic small heat shock proteins are under transcriptional and translational regulations in expression and heterocomplex prevails under the high-temperature stress condition in vitro.

PubMed

Park, Soo Min; Kim, Keun Pill; Joe, Myung Kuk; Lee, Mi Ok; Koo, Hyun Jo; Hong, Choo Bong

2015-04-01

Seven genomic clones of tobacco (Nicotiana tabacum W38) cytosolic class I small heat shock proteins (sHSPs), probably representing all members in the class, were isolated and found to have 66 to 92% homology between their nucleotide sequences. Even though all seven sHSP genes showed heat shock-responsive accumulation of their transcripts and proteins, each member showed discrepancies in abundance and timing of expression upon high-temperature stress. This was mainly the result of transcriptional regulation during mild stress conditions and transcriptional and translational regulation during strong stress conditions. Open reading frames (ORFs) of these genomic clones were expressed in Escherichia coli and the sHSPs were purified from E. coli. The purified tobacco sHSPs rendered citrate synthase and luciferase soluble under high temperatures. At room temperature, non-denaturing pore exclusion polyacrylamide gel electrophoresis on three sHSPs demonstrated that the sHSPs spontaneously formed homo-oligomeric complexes of 200 ∼ 240 kDa. However, under elevated temperatures, hetero-oligomeric complexes between the sHSPs gradually prevailed. Atomic force microscopy showed that the hetero-oligomer of NtHSP18.2/NtHSP18.3 formed a stable oligomeric particle similar to that of the NtHSP18.2 homo-oligomer. These hetero-oligomers positively influenced the revival of thermally inactivated luciferase. Amino acid residues mainly in the N-terminus are suggested for the exchange of the component sHSPs and the formation of dominant hetero-oligomers under high temperatures. © 2014 John Wiley & Sons Ltd.

An experimental test of the effects of gender constancy on sex typing.

PubMed

Arthur, Andrea E; Bigler, Rebecca S; Ruble, Diane N

2009-12-01

This study provides an experimental test of the hypothesis that level of gender constancy understanding affects children's sex typing. Preschool-age children (N=62, mean age=47 months) were randomly assigned to experimental lessons that taught that biological traits (including gender) are either fixed (pro-constancy condition) or mutable (anti-constancy condition). Posttests revealed that the lessons were effective; children in the pro-constancy condition showed higher gender constancy and appearance-reality distinction scores than did children in the anti-constancy condition. Sex typing did not, however, differ between treatment conditions at immediate and 3-month posttesting.

Allocating Virtual and Physical Flows for Multiagent Teams in Mutable, Networked Environments

DTIC Science & Technology

2012-08-01

dividing between z flow among the first l − 1 children and reserving the remaining y − z flow for the lth child , for some z ∈ [0..y]. In lines 13–21 we...use them when considering the parent of v, which must consider all possible ways to divide its flow between its children (i.e., v and v’s siblings ... studies the solution quality and runtime results for LP 2 for k = 1 and for k between 10 to 50 in increments of 10, as shown in Table 4.2. Note that

Christians and Jews in the Twelfth-Century Werewolf Renaissance.

PubMed

Shyovitz, David I

2014-10-01

In the late twelfth century, northern European Jewish mystics engaged in a sustained, unprecedented effort to explore the theological meaning of werewolves. This article seeks to anchor this surprising preoccupation in contemporary European religious culture, arguing that medieval Jews and Christians found werewolves "good to think with" in exploring the spiritual status of the (mutable, unstable) human body. Discourses of monstrosity were used as polemical ammunition in Jewish-Christian debates, but monstrous creatures were simultaneously held to be theologically resonant by both communities-a fact that sheds light upon the broader intellectual and cultural setting in which they were joint participants.

Generation of genetic constructs that simultaneously express several shRNAs.

PubMed

Kretova, Olga V; Alembekov, Ildar R; Tchurikov, Nickolai A

2012-05-01

RNAi has potential as an antiviral gene therapy strategy. Cassette constructs simultaneously expressing several siRNAs could prove to be the most efficient technique in developing gene therapy approaches for highly mutable viruses such as HIV-1. Here we describe a rapid and cost-saving protocol to generate cassettes that simultaneously express three siRNAs for repression of HIV-1 and CCR5 transcripts. siRNA biological activity was tested in a non-viral system, and exhibited both efficiency and specificity. Our results suggest this protocol can be used to rapidly generate cassette constructs for antiviral gene therapy applications.

Mutation of a single residue in the S2-S3 loop of CNG channels alters the gating properties and sensitivity to inhibitors.

PubMed

Crary, J I; Dean, D M; Maroof, F; Zimmerman, A L

2000-12-01

We previously found that native cyclic nucleotide-gated (CNG) cation channels from amphibian rod cells are directly and reversibly inhibited by analogues of diacylglycerol (DAG), but little is known about the mechanism of this inhibition. We recently determined that, at saturating cGMP concentrations, DAG completely inhibits cloned bovine rod (Brod) CNG channels while only partially inhibiting cloned rat olfactory (Rolf) channels (Crary, J.I., D.M. Dean, W. Nguitragool, P.T. Kurshan, and A.L. Zimmerman. 2000. J. Gen. Phys. 116:755-768; in this issue). Here, we report that a point mutation at position 204 in the S2-S3 loop of Rolf and a mouse CNG channel (Molf) found in olfactory epithelium and heart, increased DAG sensitivity to that of the Brod channel. Mutation of this residue from the wild-type glycine to a glutamate (Molf G204E) or aspartate (Molf G204D) gave dramatic increases in DAG sensitivity without changing the apparent cGMP or cAMP affinities or efficacies. However, unlike the wild-type olfactory channels, these mutants demonstrated voltage-dependent gating with obvious activation and deactivation kinetics. Interestingly, the mutants were also more sensitive to inhibition by the local anesthetic, tetracaine. Replacement of the position 204 glycine with a tryptophan residue (Rolf G204W) not only gave voltage-dependent gating and an increased sensitivity to DAG and tetracaine, but also showed reduced apparent agonist affinity and cAMP efficacy. Sequence comparisons show that the glycine at position 204 in the S2-S3 loop is highly conserved, and our findings indicate that its alteration can have critical consequences for channel gating and inhibition.

Molecular basis and drug sensitivity of the delayed rectifier (IKr) in the fish heart.

PubMed

Hassinen, Minna; Haverinen, Jaakko; Vornanen, Matti

2015-01-01

Fishes are increasingly used as models for human cardiac diseases, creating a need for a better understanding of the molecular basis of fish cardiac ion currents. To this end we cloned KCNH6 channel of the crucian carp (Carassius carassius) that produces the rapid component of the delayed rectifier K(+) current (IKr), the main repolarising current of the fish heart. KCNH6 (ccErg2) was the main isoform of the Kv11 potassium channel family with relative transcript levels of 98.9% and 99.6% in crucian carp atrium and ventricle, respectively. KCNH2 (ccErg1), an orthologue to human cardiac Erg (Herg) channel, was only slightly expressed in the crucian carp heart. The native atrial IKr and the cloned ccErg2 were inhibited by similar concentrations of verapamil, terfenadine and KB-R7943 (P>0.05), while the atrial IKr was about an order of magnitude more sensitive to E-4031 than ccErg2 (P<0.05) suggesting that some accessory β-subunits may be involved. Sensitivity of the crucian carp atrial IKr to E-4031, terfenadine and KB-R7943 was similar to what has been reported for the Herg channel. In contrast, the sensitivity of the crucian carp IKr to verapamil was approximately 30 times higher than the previously reported values for the Herg current. In conclusion, the cardiac IKr is produced by non-orthologous gene products in fish (Erg2) and mammalian hearts (Erg1) and some marked differences exist in drug sensitivity between fish and mammalian Erg1/2 which need to be taken into account when using fish heart as a model for human heart. Copyright © 2015 Elsevier Inc. All rights reserved.

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

PubMed Central

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-01-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula. PMID:3498590

[Molecular cloning and expression of the severe acute respiratory syndrome-associated coronavirus nucleocapsid protein and its clinical application].

PubMed

Lu, Jian; Zhou, Bai-ping; Zhou, Yu-sen; Jiang, Xiao-ling; Wen, Li-xia; Le, Xiao-hua; Li, Bing; Xu, Liu-mei; Li, Li-xiong

2005-03-01

To clone and express nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus, and to evaluate its antigenicity and application value in the development of serological diagnostic test for SARS. SARS-associated coronavirus N protein gene was amplified from its genomic RNA by reverse transcript nested polymerase chain reaction (RT-nested-PCR) and cloned into pBAD/Thio-TOPO prokaryotic expression vector. The recombinant N fusion protein was expressed and purified, and its antigenicity and specificity was analyzed by Western Blot, to establish the recombinant N protein-based ELISA for detection of IgG antibodies to SARS-associated coronavirus, and SARS-associated coronavirus lysates-based ELISA was compared parallelly. The recombinant expression vector produced high level of the N fusion protein after induction, and that protein was purified successfully by affinity chromatography and displayed higher antigenicity and specificity as compared with whole virus lysates. The recombinant SARS-associated coronavirus N protein possessed better antigenicity and specificity and could be employed to establish a new, sensitive, and specific ELISA for SARS diagnosis.

The M sub 1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mei, Lin.

1989-01-01

The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated ({sup 3}H)IP{sub 1} accumulation in the SH-SY5Y cells was decreased in the presence of 1{mu}g/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M{sub 1} mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m{sub 1} gene. The transfected B82 cells (cTB10) showed specific ({supmore » 3}H)(-)QNB binding activity. The mAChRs in these cells are of the M{sub 1} type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M{sub 1} mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M{sub 1} mAChR densities in these cells characterized by ({sup 3}H)(-)MQNB binding ranged from 12 fmol/10{sup 6} cells in LK3-1 cells to 260 fmol/10{sup 6} cells in the LK3-8 cells.« less

Growth and crown architecture of two aspen genotypes exposed to interacting ozone and carbon dioxide

Treesearch

Richard E. Dickson; M.D. Coleman; Priit Pechter; David Karnosky

2001-01-01

To study the impact of ozone (O3) and O3 plus CO2 on aspen growth, we planted two trembling aspen clones, differing in sensitivity to O3, in the ground in open-top chambers and exposed them to different concentrations of O3 and O3 plus CO2...

Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

PubMed

Privé, Florence; Newbold, C Jamie; Kaderbhai, Naheed N; Girdwood, Susan G; Golyshina, Olga V; Golyshin, Peter N; Scollan, Nigel D; Huws, Sharon A

2015-07-01

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

PubMed

Christiaens, H; Leer, R J; Pouwels, P H; Verstraete, W

1992-12-01

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

PubMed

Su, L N; Little, J B

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

Molecular and immunological characterization of subtilisin like serine protease, a major allergen of Curvularia lunata.

PubMed

Tripathi, Prabhanshu; Nair, Smitha; Singh, B P; Arora, Naveen

2011-03-01

Serine protease from numerous sources have been identified and characterized as major allergens. The present study aimed to clone, express and characterize a serine protease from Curvularia lunata. cDNA library screening identified partial protease clones. A clone showed significant homology to subtilisin like serine proteases from Aspergillus and Penicillium species. Full length sequence was generated by RACE PCR, subcloned in pET vector, protein expressed in Escherichia coli and purified from inclusion bodies yielding 0.5 mg/L of culture. Bioinformatic analysis identified serine protease motifs of subtilase family, catalytic triad and N-glycosylation sites on the primary sequence. The protein resolved at 54-kDa on SDS-PAGE and was recognized as a major allergen on immunoblot with 13/16 C. lunata sensitive patients' sera in ELISA and immunoblot. Recombinant protein reacted with rabbit polyclonal antibodies against alkaline serine proteases from C. lunata. Recombinant protein required 50-56 ng of same protein for 50% inhibition of IgE binding in competitive ELISA. In addition, 13 of 16 patients' samples showed significant basophil histamine release upon stimulation with purified recombinant protein. In conclusion, a 54 kDa major allergen of C. lunata was cloned, expressed, characterized and showed biological activity. It has potential to be used in molecule based approach for allergy diagnosis and therapy. Copyright © 2010 Elsevier GmbH. All rights reserved.

Design and Validation of Real-Time PCR: Quantitative Diagnosis of Common Leishmania Species in Iran.

PubMed

Fekri Soofi Abadi, Maryam; Dabiri, Shahriar; Fotouhi Ardakani, Reza; Fani Malaki, Lina; Amirpoor Rostami, Sahar; Ziasistani, Mahsa; Dabiri, Donya

2016-07-01

Design and validation of Real-time PCR on the protected gene region ITS2 to quantify the parasite load in common leishmania (L) species. Probe and primer were designed from the ITS2 region between the rRNA genes with minimum gene variation in three common leishmania species followed by a Real-time PCR using the Taq man probe method in the form of absolute quantification. A series of different concentrations of leishmania were analyzed. After the purified PCR product was successfully placed in a PTG19-T plasmid vector, specialized ITS2 region was cloned in this plasmid. In the last phase, the cloned gene was transferred to the Ecoli.Top10F bacteria. The standard plasmid was provided in 10(7) to 10(1) copies/rxn concentrations. The specification and clinical sensitivity of the data was analyzed using inter and intra scales. The probe and primer were designed using three species, including L. infantum, L. major, and L.tropica. Seven concentrations of purified parasite in culture media showed that the selected region for quantifying the parasite is suitable. Clinical and analytical specificity and sensitivity were both 100%, respectively. The Taq man method for the ITS2 region in leishmania is one the most sensitive diagnostic test for identifying the parasite load and is suggested as a tool for fast identification and quantification of species.

Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

PubMed Central

Osório, Nádia; Pereira, Carla; Simões, Sara; Delgadillo, Ivonne

2018-01-01

The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells. PMID:29518018

Partial complementation of the UV sensitivity of E. coli and yeast excision repair mutants by the cloned denV gene of bacteriophage T4.

PubMed

Chenevert, J M; Naumovski, L; Schultz, R A; Friedberg, E C

1986-04-01

The denV gene of bacteriophage T4 was reconstituted from two overlapping DNA fragments cloned in M13 vectors. The coding region of the intact gene was tailored into a series of plasmid vectors containing different promoters suitable for expression of the gene in E. coli and in yeast. Induction of the TAC promoter with IPTG resulted in overexpression of the gene, which was lethal to E. coli. Expression of the TACdenV gene in the absence of IPTG, or the use of the yeast GAL1 or ADH promoters resulted in partial complementation of the UV sensitivity of uvrA, uvrB, uvrC and recA mutants of E. coli and rad1, rad2, rad3, rad4 and rad10 mutants of S. cerevisiae. The extent of denV-mediated reactivation of excision-defective mutants was approximately equal to that of photoreactivation of such strains. Excision proficient E. coli cells transformed with a plasmid containing the denV gene were slightly more resistant to ultraviolet (UV) radiation than control cells without the denV gene. On the other hand, excision proficient yeast cells were slightly more sensitive to killing by UV radiation following transformation with a plasmid containing the denV gene. This effect was more pronounced in yeast mutants of the RAD52 epistasis group.

Population bottlenecks during the infectious cycle of the Lyme disease spirochete Borrelia burgdorferi.

PubMed

Rego, Ryan O M; Bestor, Aaron; Stefka, Jan; Rosa, Patricia A

2014-01-01

Borrelia burgdorferi is a zoonotic pathogen whose maintenance in nature depends upon an infectious cycle that alternates between a tick vector and mammalian hosts. Lyme disease in humans results from transmission of B. burgdorferi by the bite of an infected tick. The population dynamics of B. burgdorferi throughout its natural infectious cycle are not well understood. We addressed this topic by assessing the colonization, dissemination and persistence of B. burgdorferi within and between the disparate mammalian and tick environments. To follow bacterial populations during infection, we generated seven isogenic but distinguishable B. burgdorferi clones, each with a unique sequence tag. These tags resulted in no phenotypic changes relative to wild type organisms, yet permitted highly sensitive and specific detection of individual clones by PCR. We followed the composition of the spirochete population throughout an experimental infectious cycle that was initiated with a mixed inoculum of all clones. We observed heterogeneity in the spirochete population disseminating within mice at very early time points, but all clones displayed the ability to colonize most mouse tissues by 3 weeks of infection. The complexity of clones subsequently declined as murine infection persisted. Larval ticks typically acquired a reduced and variable number of clones relative to what was present in infected mice at the time of tick feeding, and maintained the same spirochete population through the molt to nymphs. However, only a random subset of infectious spirochetes was transmitted to naïve mice when these ticks next fed. Our results clearly demonstrate that the spirochete population experiences stochastic bottlenecks during both acquisition and transmission by the tick vector, as well as during persistent infection of its murine host. The experimental system that we have developed can be used to further explore the forces that shape the population of this vector-borne bacterial pathogen throughout its infectious cycle.

Chemokine (C-C motif) receptor 5-using envelopes predominate in dual/mixed-tropic HIV from the plasma of drug-naive individuals.

PubMed

Irlbeck, David M; Amrine-Madsen, Heather; Kitrinos, Kathryn M; Labranche, Celia C; Demarest, James F

2008-07-31

HIV-1 utilizes CD4 and either chemokine (C-C motif) receptor 5 (CCR5) or chemokine (C-X-C motif) receptor 4 (CXCR4) to gain entry into host cells. Small molecule CCR5 antagonists are currently being developed for the treatment of HIV-1 infection. Because HIV-1 may also use CXCR4 for entry, the use of CCR5 entry inhibitors is controversial for patients harboring CCR5-using and CXCR4-using (dual/mixed-tropic) viruses. The goal of the present study was to determine the proportion of CCR5-tropic and CXCR4-tropic viruses in dual/mixed-tropic virus isolates from drug-naïve patients and the phenotypic and genotypic relationships of viruses that use CCR5 or CXCR4 or both. Fourteen antiretroviral-naive HIV-1-infected patients were identified as having population coreceptor tropism readout of dual/mixed-tropic viruses. Intrapatient comparisons of coreceptor tropism and genotype of env clones were conducted on plasma virus from each patient. Population HIV-1 envelope tropism and susceptibility to the CCR5 entry inhibitor, aplaviroc, were performed using the Monogram Biosciences Trofile Assay. Twelve env clones from each patient were analyzed for coreceptor tropism, aplaviroc sensitivity, genotype, and intrapatient phylogenetic relationships. Viral populations from antiretroviral-naive patients with dual/mixed-tropic virus are composed primarily of CCR5-tropic env clones mixed with those that use both coreceptors (R5X4-tropic) and, occasionally, CXCR4-tropic env clones. Interestingly, the efficiency of CXCR4 use by R5X4-tropic env clones varied with their genetic relationships to CCR5-tropic env clones from the same patient. These data show that the majority of viruses in these dual/mixed-tropic populations use CCR5 and suggest that antiretroviral-naive patients may benefit from combination therapy that includes CCR5 entry inhibitors.

Enhanced Tumor Growth and Invasiveness in Vivo by a Carboxyl-Terminal Fragment of α1-Proteinase Inhibitor Generated by Matrix Metalloproteinases

PubMed Central

Kataoka, Hiroaki; Uchino, Hirofumi; Iwamura, Takeshi; Seiki, Motoharu; Nabeshima, Kazuki; Koono, Masashi

1999-01-01

Matrix metalloproteinases (MMPs) are believed to contribute to the complex process of cancer progression. They also exhibit an α1-proteinase inhibitor (αPI)-degrading activity generating a carboxyl-terminal fragment of ∼5 kd (αPI-C). This study reports that overexpression of αPI-C in S2–020, a cloned subline derived from the human pancreas adenocarcinoma cell line SUIT-2, potentiates the growth capability of the cells in nude mice. After stable transfection of a vector containing a chimeric cDNA encoding a signal peptide sequence of tissue inhibitor of metalloproteinase-1 followed by cDNA for αPI-C into S2–020 cells, three clones that stably secrete αPI-C were obtained. The ectopic expression of αPI-C did not alter in vitro cellular growth. However, subcutaneous injection of the αPI-C-secreting clones resulted in tumors that were 1.5 to 3-fold larger than those of control clones with an increased tendency to invasiveness and lymph node metastasis. These effects could be a result of modulation of natural killer (NK) cell-mediated control of tumor growth in nude mice, as the growth advantage of αPI-C-secreting clones was not observed in NK-depleted mice, and αPI-C-secreting clones showed decreased NK sensitivity in vitro. In addition, production of αPI and generation of the cleaved form of αPI by MMP were observed in various human tumor cell lines and in a highly metastatic subline of SUIT-2 in vitro. These results provide experimental evidence that the αPI-degrading activity of MMPs may play a role in tumor progression not only via the inactivation of αPI but also via the generation of αPI-C. PMID:10027404

Ebbie: automated analysis and storage of small RNA cloning data using a dynamic web server

PubMed Central

Ebhardt, H Alexander; Wiese, Kay C; Unrau, Peter J

2006-01-01

Background DNA sequencing is used ubiquitously: from deciphering genomes[1] to determining the primary sequence of small RNAs (smRNAs) [2-5]. The cloning of smRNAs is currently the most conventional method to determine the actual sequence of these important regulators of gene expression. Typical smRNA cloning projects involve the sequencing of hundreds to thousands of smRNA clones that are delimited at their 5' and 3' ends by fixed sequence regions. These primers result from the biochemical protocol used to isolate and convert the smRNA into clonable PCR products. Recently we completed a smRNA cloning project involving tobacco plants, where analysis was required for ~700 smRNA sequences[6]. Finding no easily accessible research tool to enter and analyze smRNA sequences we developed Ebbie to assist us with our study. Results Ebbie is a semi-automated smRNA cloning data processing algorithm, which initially searches for any substring within a DNA sequencing text file, which is flanked by two constant strings. The substring, also termed smRNA or insert, is stored in a MySQL and BlastN database. These inserts are then compared using BlastN to locally installed databases allowing the rapid comparison of the insert to both the growing smRNA database and to other static sequence databases. Our laboratory used Ebbie to analyze scores of DNA sequencing data originating from an smRNA cloning project[6]. Through its built-in instant analysis of all inserts using BlastN, we were able to quickly identify 33 groups of smRNAs from ~700 database entries. This clustering allowed the easy identification of novel and highly expressed clusters of smRNAs. Ebbie is available under GNU GPL and currently implemented on Conclusion Ebbie was designed for medium sized smRNA cloning projects with about 1,000 database entries [6-8].Ebbie can be used for any type of sequence analysis where two constant primer regions flank a sequence of interest. The reliable storage of inserts, and their annotation in a MySQL database, BlastN[9] comparison of new inserts to dynamic and static databases make it a powerful new tool in any laboratory using DNA sequencing. Ebbie also prevents manual mistakes during the excision process and speeds up annotation and data-entry. Once the server is installed locally, its access can be restricted to protect sensitive new DNA sequencing data. Ebbie was primarily designed for smRNA cloning projects, but can be applied to a variety of RNA and DNA cloning projects[2,3,10,11]. PMID:16584563

Extrachromosomal inheritance in Schizosaccharomyces pombe. I. Evidence for an extrakaryotically inherited mutation conferring resistance to antimycin.

PubMed

Wolf, K; Burger, G; Lang, B; Kaudewitz, F

1976-02-27

In crosses of [ANTr8] with auxotrophic strains, resistance to antimycin segregates almost 50:50 in random spore analysis with a slight preponderance for the sensitivity allele. Tetrad analysis, however, shows all possible types of tetrads (2:2; 3:1; 1:3; 4:0; 0:4 resistant versus sensitive) with an excess of 2:2 segregations and sectoring of colonies on antimycin medium indicating an extrachromosomal mode of inheritance. The overall ratio of resistant versus sensitive spores is the same as compared with random spore data. Using a mutant blocked in meiosis (mei 1) mitotic segregation of stable diploids is achieved, leading to a ratio of 20% resistant to 80% sensitive clones. Possible reasons for the bias in transmission of the resistance determinant is discussed.

Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase from Geobacillus sp. CHB1.

PubMed

Jia, Xianbo; Chen, Jichen; Lin, Chenqiang; Lin, Xinjian

2016-01-01

Catalases are widely used in many scientific areas. A catalase gene (Kat) from Geobacillus sp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed in Escherichia coli (E. coli), which was the first time to clone and express this type of catalase of genus Geobacillus strains as far as we know. This Kat gene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studied Bacillus sp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble in E. coli and made up 30% of the total E. coli protein. Fermentation broth of the recombinant E. coli showed a high catalase activity level up to 35,831 U/mL which was only lower than recombinant Bacillus sp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg and K m of 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications.

Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase from Geobacillus sp. CHB1

PubMed Central

2016-01-01

Catalases are widely used in many scientific areas. A catalase gene (Kat) from Geobacillus sp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed in Escherichia coli (E. coli), which was the first time to clone and express this type of catalase of genus Geobacillus strains as far as we know. This Kat gene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studied Bacillus sp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble in E. coli and made up 30% of the total E. coli protein. Fermentation broth of the recombinant E. coli showed a high catalase activity level up to 35,831 U/mL which was only lower than recombinant Bacillus sp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg and K m of 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications. PMID:27579320

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation.

PubMed

Vandorpe, D H; Shmukler, B E; Jiang, L; Lim, B; Maylie, J; Adelman, J P; de Franceschi, L; Cappellini, M D; Brugnara, C; Alper, S L

1998-08-21

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.

Treponema pallidum 3-Phosphoglycerate Mutase Is a Heat-Labile Enzyme That May Limit the Maximum Growth Temperature for the Spirochete

PubMed Central

Benoit, Stéphane; Posey, James E.; Chenoweth, Matthew R.; Gherardini, Frank C.

2001-01-01

In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP in T. pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this, T. pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective T. pallidum. Enzyme assays indicated that Gpm did not require Mn2+ while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25°C, retained only 50% activity after incubation for 20 min at 34°C or 10 min at 37°C, and was completely inactive after 10 min at 42°C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented E. coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of E. coli PL225 harboring pSLB2 from 34 to 42°C resulted in a 7- to 11-h period in which no growth occurred (compared to wild-type E. coli). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T. pallidum. PMID:11466272

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

PubMed

Nallapareddy, Sreedhar R; Singh, Kavindra V; Murray, Barbara E

2006-01-01

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Temperature induced variation in gene expression of thyroid hormone receptors and deiodinases of European eel (Anguilla anguilla) larvae.

PubMed

Politis, S N; Servili, A; Mazurais, D; Zambonino-Infante, J-L; Miest, J J; Tomkiewicz, J; Butts, I A E

2018-04-01

Thyroid hormones (THs) are key regulators of growth, development, and metabolism in vertebrates and influence early life development of fish. TH is produced in the thyroid gland (or thyroid follicles) mainly as T4 (thyroxine), which is metabolized to T3 (3,5,3'-triiodothyronine) and T2 (3,5-diiodothyronine) by deiodinase (DIO) enzymes in peripheral tissues. The action of these hormones is mostly exerted by binding to a specific nuclear thyroid hormone receptor (THR). In this study, we i) cloned and characterized thr sequences, ii) investigated the expression pattern of the different subtypes of thrs and dios, and iii) studied how temperature affects the expression of those genes in artificially produced early life history stages of European eel (Anguilla anguilla), reared in different thermal regimes (16, 18, 20 and 22 °C) from hatch until first-feeding. We identified 2 subtypes of thr (thrα and thrβ) with 2 isoforms each (thrαA, thrαB, thrβA, thrβB) and 3 subtypes of deiodinases (dio1, dio2, dio3). All thr genes identified showed high similarity to the closely related Japanese eel (Anguilla japonica). We found that all genes investigated in this study were affected by larval age (in real time or at specific developmental stages), temperature, and/or their interaction. More specifically, the warmer the temperature the earlier the expression response of a specific target gene. In real time, the expression profiles appeared very similar and only shifted with temperature. In developmental time, gene expression of all genes differed across selected developmental stages, such as at hatch, during teeth formation or at first-feeding. Thus, we demonstrate that thrs and dios show sensitivity to temperature and are involved in and during early life development of European eel. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimal use of novel agents in chronic lymphocytic leukemia.

PubMed

Smith, Mitchell R; Weiss, Robert F

2018-05-07

Novel agents are changing therapy for patients with CLL, but their optimal use remains unclear. We model the clinical situation in which CLL responds to therapy, but resistant clones, generally carrying del17p, progress and lead to relapse. Sub-clones of varying growth rates and treatment sensitivity affect predicted therapy outcomes. We explore effects of different approaches to starting novel agent in relation to bendamustine-rituximab induction therapy: at initiation of therapy, at the end of chemo-immunotherapy, at molecular relapse, or at clinical detection of relapse. The outcomes differ depending on the underlying clonal architecture, raising the concept that personalized approaches based on clinical evaluation of each patient's clonal architecture might optimize outcomes while minimizing toxicity and cost. Copyright © 2018 Elsevier Ltd. All rights reserved.

Heat stress affects carbohydrate metabolism during cold-induced sweetening of potato (Solanum tuberosum L.).

PubMed

Herman, Derek J; Knowles, Lisa O; Knowles, N Richard

2017-03-01

Tolerance to heat stress for retention of low-temperature sweetening-resistant phenotype in potato is conferred by insensitivity of acid invertase activity to cold induction. Heat stress exacerbated cold sweetening (buildup of reducing sugars) of the LTS (low-temperature sweetening)-susceptible potato (Solanum tuberosum L.) cultivars, Ranger Russet and Russet Burbank, and completely abolished the resistance to cold sweetening in the LTS-resistant cultivars/clones, Sage Russet, GemStar Russet, POR06V12-3 and A02138-2. Payette Russet and EGA09702-2, however, demonstrated considerable tolerance to heat stress for retention of their LTS-resistant phenotype. Heat-primed Payette Russet and EGA09702-2 tubers accumulated fourfold more sucrose when subsequently stored at 4 °C, while reducing sugar concentrations also increased marginally but remained low relative to the non-heat-tolerant LTS-resistant clones, resulting in light-colored fries. By contrast, sucrose concentrations in heat-primed tubers of the non-heat-tolerant clones remained unchanged during LTS, but reducing sugars increased fivefold, resulting in darkening of processed fries. Acid invertase activity increased in the LTS-susceptible and non-heat-tolerant LTS-resistant cultivars/clones during cold storage. However, Payette Russet tubers maintained very low invertase activity regardless of heat stress and cold storage treatments, as was the case for Innate ® Russet Burbank (W8) tubers, where silenced invertase conferred robust tolerance to heat stress for retention of LTS-resistant phenotype. Importantly, heat-stressed tubers of Payette Russet, EGA09702-2 and Innate ® Russet Burbank (W8) demonstrated similar low reducing sugar and high sucrose-accumulating phenotypes when stored at 4 °C. Tolerance to heat stress for retention of LTS-resistant phenotype in Payette Russet and likely its maternal parent, EGA09702-2, is, therefore, conferred by the ability to maintain low invertase activity during cold storage of heat-stressed tubers.

Binding of Amphipathic Cell Penetrating Peptide p28 to Wild Type and Mutated p53 as studied by Raman, Atomic Force and Surface Plasmon Resonance spectroscopies.

PubMed

Signorelli, Sara; Santini, Simona; Yamada, Tohru; Bizzarri, Anna Rita; Beattie, Craig W; Cannistraro, Salvatore

2017-04-01

Mutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in >50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding. Molecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28. We show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the β-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge. These results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function. Raman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53. Copyright © 2017 Elsevier B.V. All rights reserved.

ASC ATDM Level 2 Milestone #5325: Asynchronous Many-Task Runtime System Analysis and Assessment for Next Generation Platforms.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Gavin Matthew; Bettencourt, Matthew Tyler; Bova, Steven W.

2015-09-01

This report provides in-depth information and analysis to help create a technical road map for developing next- generation Orogramming mocleN and runtime systemsl that support Advanced Simulation and Computing (ASC) work- load requirements. The focus herein is on 4synchronous many-task (AMT) model and runtime systems, which are of great interest in the context of "Oriascale7 computing, as they hold the promise to address key issues associated with future extreme-scale computer architectures. This report includes a thorough qualitative and quantitative examination of three best-of-class AIM] runtime systemsHCharm-HE, Legion, and Uintah, all of which are in use as part of the Centers.more » The studies focus on each of the runtimes' programmability, performance, and mutability. Through the experiments and analysis presented, several overarching Predictive Science Academic Alliance Program II (PSAAP-II) Ascl findings emerge. From a performance perspective, AIVT11runtimes show tremendous potential for addressing extreme- scale challenges. Empirical studies show an AM11 runtime can mitigate performance heterogeneity inherent to the machine itself and that Message Passing Interface (MP1) and AM11runtimes perform comparably under balanced con- ditions. From a programmability and mutability perspective however, none of the runtimes in this study are currently ready for use in developing production-ready Sandia ASCIapplications. The report concludes by recommending a co- design path forward, wherein application, programming model, and runtime system developers work together to define requirements and solutions. Such a requirements-driven co-design approach benefits the community as a whole, with widespread community engagement mitigating risk for both application developers developers. and high-performance computing inntime systein« less

Natural selection promotes antigenic evolvability.

PubMed

Graves, Christopher J; Ros, Vera I D; Stevenson, Brian; Sniegowski, Paul D; Brisson, Dustin

2013-01-01

The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed 'cassettes' that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections.

Natural Selection Promotes Antigenic Evolvability

PubMed Central

Graves, Christopher J.; Ros, Vera I. D.; Stevenson, Brian; Sniegowski, Paul D.; Brisson, Dustin

2013-01-01

The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed ‘cassettes’ that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections. PMID:24244173

Natural and Chemotherapy-Induced Clonal Evolution of Tumors.

PubMed

Ibragimova, M K; Tsyganov, M M; Litviakov, N V

2017-04-01

Evolution and natural selection of tumoral clones in the process of transformation and the following carcinogenesis can be called natural clonal evolution. Its main driving factors are internal: genetic instability initiated by driver mutations and microenvironment, which enables selective pressure while forming the environment for cell transformation and their survival. We present our overview of contemporary research dealing with mechanisms of carcinogenesis in different localizations from precancerous pathologies to metastasis and relapse. It shows that natural clonal evolution establishes intratumoral heterogeneity and enables tumor progression. Tumors of monoclonal origin are of low-level intratumoral heterogeneity in the initial stages, and this increases with the size of the tumor. Tumors of polyclonal origin are of extremely high-level intratumoral heterogeneity in the initial stages and become more homogeneous when larger due to clonal expansion. In cases of chemotherapy-induced clonal evolution of a tumor, chemotherapy becomes the leading factor in treatment. The latest research shows that the impact of chemotherapy can radically increase the speed of clonal evolution and lead to new malignant and resistant clones that cause tumor metastasis. Another option of chemotherapy-induced clonal evolution is formation of a new dominant clone from a clone that was minor in the initial tumor and obtained free space due to elimination of sensitive clones by chemotherapy. As a result, in ~20% of cases, chemotherapy can stimulate metastasis and relapse of tumors due to clonal evolution. The conclusion of the overview formulates approaches to tumor treatment based on clonal evolution: in particular, precision therapy, prediction of metastasis stimulation in patients treated with chemotherapy, methods of genetic evaluation of chemotherapy efficiency and clonal-oriented treatment, and approaches to manipulating the clonal evolution of tumors are presented.

A Fosmid Cloning Strategy for Detecting the Widest Possible Spectrum of Microbes from the International Space Station Drinking Water System

PubMed Central

Choi, Sangdun; Chang, Mi Sook; Stuecker, Tara; Chung, Christine; Newcombe, David A.; Venkateswaran, Kasthuri

2012-01-01

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular-weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing. PMID:23346038

Carb-3 is the superior anti-CD15 monoclonal antibody for immunohistochemistry.

PubMed

Røge, Rasmus; Nielsen, Søren; Vyberg, Mogens

2014-07-01

Immunohistochemical detection of CD15 is important in the diagnosis of Hodgkin lymphoma and may play a role in the classification of renal cell tumors (RCTs). In the NordiQC external quality assessment scheme, 4 CD15 tests, each with 71 to 121 participating laboratories, showed that 24% to 50% of the stains were insufficient. This was mainly because of very low primary antibody (Ab) concentration and insufficient heat-induced epitope retrieval, whereas the Ab clone performance seemed of little importance. The purpose of this study was to evaluate the performance of the most commonly used CD15 Abs on the basis of vendor-recommended and in-house optimized protocols. Multitissue blocks with 199 specimens including various malignant lymphomas, RCTs, and normal tissues were stained with 3 different concentrated (conc) CD15 Ab clones Carb-3, MMA, and BY87 according to predetermined in-house optimized protocols on 2 automated immunostaining platforms. Carb-3 and MMA were also applied in ready-to-use (RTU) formats utilized according to vendor protocols. Extension and intensity of stains was determined using the H-score method. Clone Carb-3-conc gave with an in-house optimized protocol the highest H-scores in Hodgkin lymphoma, RCTs, and normal kidney tissue. Clones Carb-3-RTU and MMA-conc gave slightly lower scores, whereas clones MMA-RTU and BY87-conc gave the lowest scores and a large proportion of false-negative reactions. For all concentrated Abs, in-house optimized protocols resulted in increased sensitivity and improved overall staining results compared with vendor-recommended protocols. The importance of Ab selection and protocol optimization in immunohistochemical laboratories is emphasized.

Variation in drought resistance, drought acclimation and water conservation in four willow cultivars used for biomass production.

PubMed

Wikberg, Jenny; Ogren, Erling

2007-09-01

Growth and water-use parameters of four willow (Salix spp.) clones grown in a moderate drought regime or with ample water supply were determined to characterize their water-use efficiency, drought resistance and capacity for drought acclimation. At the end of the 10-week, outdoor pot experiment, clonal differences were observed in: (1) water-use efficiency of aboveground biomass production (WUE); (2) resistance to xylem cavitation; and (3) stomatal conductance to leaf-specific, whole-plant hydraulic conductance ratio (g(st)/K(P); an indicator of water balance). Across clones and regimes, WUE was positively correlated with the assimilation rate to stomatal conductance ratio (A/g(st)), a measure of instantaneous water-use efficiency. Both of these water-use efficiency indicators were generally higher in drought-treated trees compared with well-watered trees. However, the between-treatment differences in (shoot-based) WUE were smaller than expected, considering the differences in A/g(st) for two of the clones, possibly because plants reallocated dry mass from shoots to roots when subject to drought. Higher root hydraulic conductance to shoot hydraulic conductance ratios (K(R)/K(S)) during drought supports this hypothesis. The same clones were also the most sensitive to xylem cavitation and, accordingly, showed the strongest reduction in g(st)/K(P) in response to drought. Drought acclimation was manifested in decreased g(st), g(st)/K(P), osmotic potential and leaf area to vessel internal cross-sectional area ratio, and increased K(R), K(P) and WUE. Increased resistance to stem xylem cavitation in response to drought was observed in only one clone. It is concluded that WUE and drought resistance traits are inter-linked and that both may be enhanced by selection and breeding.

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH*

PubMed Central

Daidoji, Tomo; Watanabe, Yohei; Ibrahim, Madiha S.; Yasugi, Mayo; Maruyama, Hisataka; Masuda, Taisuke; Arai, Fumihito; Ohba, Tomoyuki; Honda, Ayae; Ikuta, Kazuyoshi; Nakaya, Takaaki

2015-01-01

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells. PMID:25673693

The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Hyoun-Sub, E-mail: hyounlim@cnu.ac.kr; Nam, Jiryun, E-mail: jilyoon@naver.com; Seo, Eun-Young, E-mail: sey22@cnu.ac.kr

Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CP{sub SP}) with that from AltMV-Po (CP{sub Po}) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution ofmore » only two residues from CP{sub Po} [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CP{sub SP} but not CP{sub Po} interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CP{sub SP} than CP{sub Po} in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN. - Highlights: • Alternanthera mosaic virus CP is an elicitor of systemic necrosis in N. benthamiana. • Virus-induced systemic necrosis is enhanced at 15 °C compared to 25 °C. • Induction of systemic necrosis is dependent on as few as two CP amino acid residues. • These residues are at subunit interfaces within the same turn of the virion helix. • Inducer/non-inducer CPs interact differentially with a boron transporter protein.« less

Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

PubMed

Gray, Sean A; Weigel, Kris M; Ali, Ibne K M; Lakey, Annie A; Capalungan, Jeremy; Domingo, Gonzalo J; Cangelosi, Gerard A

2012-01-01

The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

Biotechnology Opens New Routes to High-Performance Materials for Improved Photovoltaics, Batteries, Uncooled IR Detectors, Ferroelectrics and Optical Applications

DTIC Science & Technology

2006-11-01

for High Power-Density, Safe Batteries and Solar Energy applications Cloning reveals: Protein template is an enzyme catalyst: γ- Ga2O3 Enzyme that...catalyzes & templates synthesis of silica at low temperature also makes semiconductors from molecular precursors: TiO2 , Ga2O3 , ZnO...CoO, RuOx (311) γ- Ga2O3 Low-temperature catalysis & templating of semiconductor synthesis The catalyst IS the template! Catalytic & Structure

Multi-Enzyme Complexes in the Thermophilic Archaea: The Effects of Temperature on Stability, Catalysis and Enzyme Interactions in a Multi-Component System

DTIC Science & Technology

2012-01-01

COVERED (From - To) 4. TITLE AND SUBTITLE Multi- enzyme complexes in the thermophilic archaea: The effects of temperature on stability, catalysis and... enzyme interactions in a multi- component system 5a. CONTRACT NUMBER 5b. GRANT NUMBER FA9550-07-1-0058 5c. PROGRAM ELEMENT NUMBER 61102F 6...involves cloning of the genes for the relevant lipoylation enzymes , and characterisation of the protein products 15. SUBJECT TERMS 16. SECURITY

Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.

PubMed

Tajabadi, Naser; Baradaran, Ali; Ebrahimpour, Afshin; Rahim, Raha A; Bakar, Fatimah A; Manap, Mohd Yazid A; Mohammed, Abdulkarim S; Saari, Nazamid

2015-07-01

Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Role of P-450 aromatase in sex determination of the diamondback terrapin, Malaclemys terrapin.

PubMed

Jeyasuria, P; Roosenburg, W M; Place, A R

1994-09-15

Sex determination in the diamondback terrapin, Malaclemys terrapin, is temperature-dependent. Eggs incubated at 31 degrees C, and above, hatch in approximately 45 days as females. Eggs incubated below 27 degrees C hatch in about 60 days as males. Sex is not reversible after hatching. Nest temperatures in the wild can be as low as 20 degrees C and as high as 37 degrees C with as much as a 10 degrees C diel cycle. The shortest incubation time measured in nature was 56 days and the longest approaching 120 days. Nests in our study site produced predominantly (> 95%) male hatchlings. Treatment of developing embryos with estrogen produces females at male producing temperatures while treatment with fadrozole (a nonsteroidal aromatase inhibitor) induces partial male-like gonads. Treatment with a steroidal aromatase inhibitor (4-hydroxyandrostenedione, 4-OHA) had no effect on sex determination. Both fadrozole and 4-OHA are potent competitive inhibitors (Ki approximately 40-50 nM) for terrapin in vitro aromatase activity. These findings are consistent with aromatase expression being a key step in sex determination of terrapins. We have cloned a partial single copy P-450 aromatase from the terrapin using a cDNA library constructed from ovarian mRNA. This partial clone is highly homologous to other vertebrate aromatases.

Cloning and expression of γ-glutamyl transpeptidase and its relationship to greening in crushed garlic (Allium sativum) cloves.

PubMed

Cho, Jungeun; Park, Minkyu; Choi, Doil; Lee, Seung Koo

2012-01-30

Garlic greening occurs when garlic cloves are stored at low temperature, increasing 1-propenyl cysteine sulfoxide, which is induced by γ-glutamyl transpeptidase (GGT) activity. Although the metabolism of the γ-glutamyl peptide is important for the biosynthesis of green pigments in crushed garlic cloves, garlic GGT is poorly characterised. For the analysis of GGT at the gene level, the garlic GGT sequence was partially cloned using an onion GGT sequence. The relationship between garlic greening and related gene expressions, depending on storage condition, was investigated using reverse transcription polymerase chain reaction for garlic GGT and alliinase. Three storage conditions were set: A, storage at a constant temperature of 20 °C; B, storage at 20 °C for 3 months and then transfer to 0 °C for an additional 3 months; C, storage at 0 °C for 3 months and then transfer to 20 °C for an additional 3 months. GGT expression increased under storage condition B and decreased under storage condition C. However, alliinase expression was not affected by storage condition. Greening in crushed garlic cloves increases with increasing GGT expression at low temperature, while alliinase expression is not affected. Copyright © 2011 Society of Chemical Industry.

A new indicator cell line established to monitor bovine foamy virus infection.

PubMed

Guo, Hong-Yan; Liang, Zhi-Bin; Li, Yue; Tan, Juan; Chen, Qi-Min; Qiao, Wen-Tao

2011-10-01

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.

Evolution of the Drosophila melanogaster-sigma virus system in a natural population from Tübingen.

PubMed

Fleuriet, A; Sperlich, D

1992-11-01

In natural populations of D. melanogaster, usually, a minority of individuals are infected by a Rhabdovirus called sigma. This virus is not contagious but is vertically transmitted through the gametes. In D. melanogaster, a polymorphism for two alleles (O, permissive and P, restrictive) of a gene responsible for resistance to the virus is regularly observed in the wild. On the virus side two types are found, which differ in their sensitivity to the P allele: Type I is very sensitive, and Type II more resistant. Previous findings had led to the hypothesis that an invasion of Type II clones, starting from central France, might be spreading over European populations. This replacement of viral Type I by viral Type II in natural populations could be observed in Languedoc (southern France), where it led to a dramatic increase in the frequency of infected flies. The invasion hypothesis is confirmed by the data from samples collected at Tübingen, where the frequency of Type II clones increased from 0.27 to 0.93 over a 6-year period (1985-1991). However, over the same period, no increase in the frequency of infected flies was observed. The evolution of other viral characteristics is discussed.

Characterization and cloning of tripeptidyl peptidase II from the fruit fly, Drosophila melanogaster.

PubMed

Renn, S C; Tomkinson, B; Taghert, P H

1998-07-24

We describe the characterization, cloning, and genetic analysis of tripeptidyl peptidase II (TPP II) from Drosophila melanogaster. Mammalian TPP II removes N-terminal tripeptides, has wide distribution, and has been identified as the cholecystokinin-degrading peptidase in rat brain. Size exclusion and ion exchange chromatography produced a 70-fold purification of dTPP II activity from Drosophila tissue extracts. The substrate specificity and the inhibitor sensitivity of dTPP II is comparable to that of the human enzyme. In particular, dTPP II is sensitive to butabindide, a specific inhibitor of the rat cholecystokinin-inactivating activity. We isolated a 4309-base pair dTPP II cDNA which predicts a 1354-amino acid protein. The deduced human and Drosophila TPP II proteins display 38% overall identity. The catalytic triad, its spacing, and the sequences that surround it are highly conserved; the C-terminal end of dTPP II contains a 100-amino acid insert not found in the mammalian proteins. Recombinant dTPP II displays the predicted activity following expression in HEK cells. TPP II maps to cytological position 49F4-7; animals deficient for this interval show reduced TPP II activity.

Using Quantum Confinement to Uniquely Identify Devices

PubMed Central

Roberts, J.; Bagci, I. E.; Zawawi, M. A. M.; Sexton, J.; Hulbert, N.; Noori, Y. J.; Young, M. P.; Woodhead, C. S.; Missous, M.; Migliorato, M. A.; Roedig, U.; Young, R. J.

2015-01-01

Modern technology unintentionally provides resources that enable the trust of everyday interactions to be undermined. Some authentication schemes address this issue using devices that give a unique output in response to a challenge. These signatures are generated by hard-to-predict physical responses derived from structural characteristics, which lend themselves to two different architectures, known as unique objects (UNOs) and physically unclonable functions (PUFs). The classical design of UNOs and PUFs limits their size and, in some cases, their security. Here we show that quantum confinement lends itself to the provision of unique identities at the nanoscale, by using fluctuations in tunnelling measurements through quantum wells in resonant tunnelling diodes (RTDs). This provides an uncomplicated measurement of identity without conventional resource limitations whilst providing robust security. The confined energy levels are highly sensitive to the specific nanostructure within each RTD, resulting in a distinct tunnelling spectrum for every device, as they contain a unique and unpredictable structure that is presently impossible to clone. This new class of authentication device operates with minimal resources in simple electronic structures above room temperature. PMID:26553435

Functional characterizations of malonyl-CoA:acyl carrier protein transacylase (MCAT) in Eimeria tenella.

PubMed

Sun, Mingfei; Zhu, Guan; Qin, Zonghua; Wu, Caiyan; Lv, Minna; Liao, Shenquan; Qi, Nanshan; Xie, Mingquan; Cai, Jianping

2012-07-01

Eimeria tenella, an apicomplexan parasite in chickens, possesses an apicoplast and its associated metabolic pathways including the Type II fatty acid synthesis (FAS II). Malonyl-CoA:acyl-carry protein transacylase (MCAT) encoded by the fabD gene is one of the essential enzymes in the FAS II system. In the present study, the entire E. tenella MCAT gene (EtfabD) was cloned and sequenced. Immunolabeling located this protein in the apicoplast organelle in coccidial sporozoites. Functional replacement of the fabD gene with amber mutation of E. coli temperature-sensitive LA2-89 strain by E. tenella EtMCAT demonstrated that EcFabD and EtMCAT perform the same biochemical function. The recombinant EtMCAT protein was expressed and its general biochemical features were also determined. An alkaloid natural product corytuberine (CAS: 517-56-6) could specifically inhibit the EtMCAT activity (IC(50)=16.47μM), but the inhibition of parasite growth in vitro by corytuberine was very weak (the predicted MIC(50)=0.65mM). Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization and function of Mycobacterium tuberculosis H37Rv Lipase Rv1076 (LipU).

PubMed

Li, Chunyan; Li, Qiming; Zhang, Yuan; Gong, Zhen; Ren, Sai; Li, Ping; Xie, Jianping

2017-03-01

Lipids and lipases/esterases are essential for Mycobacterium tuberculosis (Mtb) survival and persistence, even virulence. Mycobacterium tuberculosis H37Rv Rv1076 (LipU), a member of lipase family, is homologous to the human Hormone Sensitive Lipase (HSL) based on the presence of conserved motif 'GXSXG'. To define the enzymatic characteristics of rv1076, the gene was cloned, and expressed in Escherichia coli. The protein was purified for enzymatic characterization. LipU showed high specific activity for the hydrolysis of short carbon chain substrates with optimal activity at 40°C/pH 8.0 and stability at low temperature and near-neutral pH. The specific activity, Km and Vmax of LipU was calculated to 176.7U/mg, 1.73μM and 62.24μM/min respectively. Ionic detergents can inhibit its activity. The active-site residues of LipU were determined to be Ser140, Asp244 and His269 by site-directed mutagenesis. The upregulation of Mycobacterium tuberculosis rv1076 under nutritive stress implicates a role in starvation. Copyright © 2016 Elsevier GmbH. All rights reserved.

The rice nuclear gene, VIRESCENT 2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria.

PubMed

Sugimoto, Hiroki; Kusumi, Kensuke; Noguchi, Ko; Yano, Masahiro; Yoshimura, Atsushi; Iba, Koh

2007-11-01

Guanylate kinase (GK) is a critical enzyme in guanine nucleotide metabolism pathways, catalyzing the phosphorylation of (d)GMP to (d)GDP. Here we show that a novel gene, VIRESCENT 2 (V2), encodes a new type of GK (designated pt/mtGK) that is localized in plastids and mitochondria. We initially identified the V2 gene by positional cloning of the rice v2 mutant. The v2 mutant is temperature-sensitive and develops chlorotic leaves at restrictive temperatures. The v2 mutation causes inhibition of chloroplast differentiation; in particular, it disrupts the chloroplast translation machinery during early leaf development [Sugimoto et al. (2004)Plant Cell Physiol. 45, 985]. In the bacterial and animal species studied to date, GK is localized in the cytoplasm and participates in maintenance of the guanine nucleotide pools required for many fundamental cellular processes. Phenotypic analysis of rice seedlings with RNAi knockdown of cytosolic GK (designated cGK) showed that cGK is indispensable for the growth and development of plants, but not for chloroplast development. Thus, rice has two types of GK, as does Arabidopsis, suggesting that higher plants have two types of GK. Our results suggest that, of the two types of GK, only pt/mtGK is essential for chloroplast differentiation.

cDNA cloning and characterization of an aryl hydrocarbon receptor from the harbor seal (Phoca vitulina): a biomarker of dioxin susceptibility?

PubMed

Kim, Eun-Young; Hahn, Mark E

2002-07-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are found at high concentrations in some marine mammals. Species differences in sensitivity to TCDD and PHAHs are a major limitation in assessing the ecological risk to these animals. Harbor seals accumulate high levels of PHAHs and are thought to be highly sensitive to the toxic effects of these compounds. To investigate the mechanistic basis for PHAH toxicity in harbor seals (Phoca vitulina), we sought to characterize the aryl hydrocarbon receptor (AHR), an intracellular protein that is responsible for PHAH effects. Here we report the cDNA cloning and characterization of a harbor seal AHR. The harbor seal AHR cDNA has an open reading frame of 2529 nucleotides that encodes a protein of 843 amino acids with a predicted molecular mass of 94.6 kDa. The harbor seal AHR protein possesses basic helix-loop-helix (bHLH) and Per-ARNT-Sim (PAS) domains. It is most closely related to the beluga AHR (82%) and human AHR (79%) in overall amino acid identity, indicating a high degree of conservation of AHR structure between terrestrial and some marine mammals. The ligand binding properties of the harbor seal AHR were determined using protein synthesized by in vitro transcription and translation from the cloned cDNA. Velocity sedimentation analysis on sucrose gradients showed that the harbor seal AHR exhibits specific binding of [(3)H]TCDD. The [(3)H]TCDD-binding affinity of the harbor seal AHR was compared with that of the AHR from a dioxin-sensitive mouse strain (C57BL/6) using a hydroxylapatite assay. The equilibrium dissociation constants of seal and mouse AHRs were 0.93+/-0.19 and 1.70+/-0.26 nM, respectively. Thus, the harbor seal AHR bound TCDD with an affinity that was at least as high as that of the mouse AHR, suggesting that this seal species may be sensitive to PHAH effects. The characteristics of the AHR potentially can be used as a biomarker of susceptibility to dioxin-like compounds, contributing to the assessment of the risk of these compounds to marine mammals and other protected animals.

Use of Fatty Acid Methyl Ester Profiles to Compare Copper-Tolerant and Copper-Sensitive Strains of Pantoea ananatis.

PubMed

Nischwitz, C; Gitaitis, R; Sanders, H; Langston, D; Mullinix, B; Torrance, R; Boyhan, G; Zolobowska, L

2007-10-01

ABSTRACT A survey was conducted to evaluate differences in fatty acid methyl ester (FAME) profiles among strains of Pantoea ananatis, causal agent of center rot of onion (Allium cepa), isolated from 15 different onion cultivars in three different sites in Georgia. Differences in FAME composition were determined by plotting principal components (PCs) in two-dimensional plots. Euclidean distance squared (ED(2)) values indicated a high degree of similarity among strains. Plotting of PCs calculated from P. ananatis strains capable of growing on media amended with copper sulfate pentahydrate (200 mug/ml) indicated that copper-tolerant strains grouped into tight clusters separate from clusters formed by wild-type strains. However, unlike copper-sensitive strains, the copper-tolerant strains tended to cluster by location. A total of 80, 60, and 73% of the strains from Tift1, Tift2, and Tattnall, respectively, exhibited either confluent growth or partial growth on copper-amended medium. However, all strains were sensitive to a mixture of copper sulfate pentahydrate (200 mug/ml) and maneb (40 mug/ml). When copper-tolerant clones were analyzed and compared with their wild-type parents, in all cases the plotting of PCs developed from copper-tolerant clones formed tight clusters separate from clusters formed by the parents. Eigenvalues generated from these tests indicated that two components provided a good summary of the data, accounting for 98, 98, and 96% of the standardized variance for strains Pna 1-15B, Pna 1-12B, and Pna 2-5A, respectively. Furthermore, feature 4 (cis-9-hexadecenoic acid/2-hydroxy-13-methyltetradecanoic acid) and feature 7 (cis-9/trans-12/cis-7-octadecenoic acid) were the highest or second highest absolute values for PC1 in all three strains of the parents versus copper-tolerant clones, and hexadecanoic acid was the highest absolute value for PC2 in all three strains. Along with those fatty acids, dodecanoic acid and feature 3 (3-hydroxytetradecanoic acid/14-methylpentadecenoic acid) also had an impact on the differences observed between copper-sensitive parents and copper-resistant mutants. Finding these changes in bacterial fatty acid composition could lead to the development of a laboratory assay to identify copper-tolerant strains using gas chromatography as well as providing clues to further elucidate the mode of action of copper tolerance.

Comparison of global brain gene expression profiles between inbred long-sleep and inbred short-sleep mice by high-density gene array hybridization.

PubMed

Xu, Y; Ehringer, M; Yang, F; Sikela, J M

2001-06-01

Inbred long-sleep (ILS) and short-sleep (ISS) mice show significant central nervous system-mediated differences in sleep time for sedative dose of ethanol and are frequently used as a rodent model for ethanol sensitivity. In this study, we have used complementary DNA (cDNA) array hybridization methodology to identify genes that are differentially expressed between the brains of ILS and ISS mice. To carry out this analysis, we used both the gene discovery array (GDA) and the Mouse GEM 1 Microarray. GDA consists of 18,378 nonredundant mouse cDNA clones on a single nylon filter. Complex probes were prepared from total brain mRNA of ILS or ISS mice by using reverse transcription and 33P labeling. The labeled probes were hybridized in parallel to the gene array filters. Data from GDA experiments were analyzed with SQL-Plus and Oracle 8. The GEM microarray includes 8,730 sequence-verified clones on a glass chip. Two fluorescently labeled probes were used to hybridize a microarray simultaneously. Data from GEM experiments were analyzed by using the GEMTools software package (Incyte). Differentially expressed genes identified from each method were confirmed by relative quantitative reverse transcription-polymerase chain reaction (RT-PCR). A total of 41 genes or expressed sequence tags (ESTs) display significant expression level differences between brains of ILS and ISS mice after GDA, GEM1 hybridization, and quantitative RT-PCR confirmation. Among them, 18 clones were expressed higher in ILS mice, and 23 clones were expressed higher in ISS mice. The individual gene or EST's function and mapping information have been analyzed. This study identified 41 genes that are differentially expressed between brains of ILS and ISS mice. Some of them may have biological relevance in mediation of phenotypic variation between ILS and ISS mice for ethanol sensitivity. This study also demonstrates that parallel gene expression comparison with high-density cDNA arrays is a rapid and efficient way to discover potential genes and pathways involved in alcoholism and alcohol-related physiologic processes.

Changes in coral-associated microbial communities during a bleaching event.

PubMed

Bourne, David; Iida, Yuki; Uthicke, Sven; Smith-Keune, Carolyn

2008-04-01

Environmental stressors such as increased sea surface temperatures are well-known for contributing to coral bleaching; however, the effect of increased temperatures and subsequent bleaching on coral-associated microbial communities is poorly understood. Colonies of the hard coral Acropora millepora were tagged on a reef flat off Magnetic Island (Great Barrier Reef) and surveyed over 2.5 years, which included a severe bleaching event in January/February 2002. Daily average water temperatures exceeded the previous 10-year average by more than 1 degrees C for extended periods with field-based visual surveys recording all tagged colonies displaying signs of bleaching. During the bleaching period, direct counts of coral zooxanthellae densities decreased by approximately 64%, before recovery to pre-bleaching levels after the thermal stress event. A subset of three tagged coral colonies were sampled through the bleaching event and changes in the microbial community elucidated. Denaturing gradient gel electrophoresis (DGGE) analysis demonstrated conserved bacterial banding profiles between the three coral colonies, confirming previous studies highlighting specific microbial associations. As coral colonies bleached, the microbial community shifted and redundancy analysis (RDA) of DGGE banding patterns revealed a correlation of increasing temperature with the appearance of Vibrio-affiliated sequences. Interestingly, this shift to a Vibrio-dominated community commenced prior to visual signs of bleaching. Clone libraries hybridized with Vibrio-specific oligonucleotide probes confirmed an increase in the fraction of Vibrio-affiliated clones during the bleaching period. Post bleaching, the coral microbial associations again shifted, returning to a profile similar to the fingerprints prior to bleaching. This provided further evidence for corals selecting and shaping their microbial partners. For non-bleached samples, a close association with Spongiobacter-related sequences were revealed by both clone libraries and DGGE profiling. Despite Vibrio species being previously implicated in bleaching of specific coral species, it is unsure if the relative increase in retrieved Vibrio sequences is due to bacterial infection or an opportunistic response to compromised health and changing environmental parameters of the coral host. This study provides the first molecular-based study demonstrating changes in coral-associated bacterial assemblages during a bleaching event on a natural reef system.

Brain aromatase (Cyp19A2) and estrogen receptors, in larvae and adult pejerrey fish Odontesthes bonariensis: Neuroanatomical and functional relations

USGS Publications Warehouse

Strobl-Mazzulla, P. H.; Lethimonier, C.; Gueguen, M.M.; Karube, M.; Fernandino, J.I.; Yoshizaki, G.; Patino, R.; Strussmann, C.A.; Kah, O.; Somoza, G.M.

2008-01-01

Although estrogens exert many functions on vertebrate brains, there is little information on the relationship between brain aromatase and estrogen receptors. Here, we report the cloning and characterization of two estrogen receptors, ?? and ??, in pejerrey. Both receptors' mRNAs largely overlap and were predominantly expressed in the brain, pituitary, liver, and gonads. Also brain aromatase and estrogen receptors were up-regulated in the brain of estradiol-treated males. In situ hybridization was performed to study in more detail, the distribution of the two receptors in comparison with brain aromatase mRNA in the brain of adult pejerrey. The estrogen receptors' mRNAs exhibited distinct but partially overlapping patterns of expression in the preoptic area and the mediobasal hypothalamus, as well as in the pituitary gland. Moreover, the estrogen receptor ??, but not ??, were found to be expressed in cells lining the preoptic recess, similarly as observed for brain aromatase. Finally, it was shown that the onset expression of brain aromatase and both estrogen receptors in the head of larvae preceded the morphological differentiation of the gonads. Because pejerrey sex differentiation is strongly influenced by temperature, brain aromatase expression was measured during the temperature-sensitive window and was found to be significantly higher at male-promoting temperature. Taken together these results suggest close neuroanatomical and functional relationships between brain aromatase and estrogen receptors, probably involved in the sexual differentiation of the brain and raising interesting questions on the origin (central or peripheral) of the brain aromatase substrate. ?? 2008 Elsevier Inc.

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

PubMed

Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

2017-12-01

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

Differences in antigen presentation to MHC class I-and class II- restricted influenza virus-specific cytolytic T lymphocyte clones

PubMed Central

1986-01-01

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA- specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II- restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets. PMID:3485173

Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors.

PubMed

Taniguchi, Hirokazu; Takeuchi, Shinji; Fukuda, Koji; Nakagawa, Takayuki; Arai, Sachiko; Nanjo, Shigeki; Yamada, Tadaaki; Yamaguchi, Hiroyuki; Mukae, Hiroshi; Yano, Seiji

2017-01-01

Crizotinib, a first-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, is known to be effective against echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive non-small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib-resistant cells (A925LPE3-CR) via long-term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4-ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib-resistant cells, and these cells were cross-resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3-CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine-kinase inhibitor (erlotinib) or an anti-EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4-ALK lung cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Detection of Epidemic USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus Strains by Use of a Single Allele-Specific PCR Assay Targeting a Novel Polymorphism of Staphylococcus aureus pbp3

PubMed Central

Chadwick, Sean G.; Prasad, Aditya; Smith, W. Lamar; Mordechai, Eli; Adelson, Martin E.

2013-01-01

In recent years, the dramatic increase in community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections has become a significant health care challenge. Early detection of CA-MRSA is important because of its increased virulence associated with the arginine catabolic mobile element (ACME), Panton-Valentine leukocidin (PVL), and other toxins that may contribute to disease severity. In particular, the USA300 epidemic clone has emerged and now represents the cause of as much as 98% of CA-MRSA skin and soft tissue infections in the United States. Current diagnostic assays used to identify CA-MRSA strains are based on complex multiplex PCRs targeting the staphylococcal cassette chromosome mec (SCCmec) DNA junction, a multitude of genes, and noncoding DNA fragments or on a number of lengthy sequence-typing methods. Here, two nucleotide polymorphisms, G88A and G2047A, that were found to be in strict linkage disequilibrium in the S. aureus penicillin-binding protein 3 (pbp3) gene were also found to be highly associated with the USA300 clone of CA-MRSA. Clinical isolates that contained this pbp3 allele were also positive for the presence of SCCmec type IV, the ACME, and the PVL toxin gene and matched the t008 or t121 molecular spa types, which are associated specifically with the USA300 CA-MRSA clone. A single allele-specific PCR targeting the G88A polymorphism was developed and was found to be 100% sensitive and specific for the detection of USA300 CA-MRSA and 91.5% sensitive and 100% specific for the detection of all CA-MRSA isolates in this study. PMID:23698534

Clonal variation in physiological responses of Daphnia magna to the strobilurin fungicide azoxystrobin.

PubMed

Warming, Trine Perlt; Mulderij, Gabi; Christoffersen, Kirsten Seestern

2009-02-01

Because of its high grazing potential, Daphnia magna is an ecologically important species in aquatic food webs. This is especially true in small, shallow ponds lacking fish, where grazing by D. magna may have a relatively higher impact on water clarity as compared to larger lakes. Thus, a reduction in daphnid abundance may have dramatic ecological consequences for shallow ponds. At the same time, shallow ponds in close proximity to agricultural areas likely experience higher concentrations of pesticides because of runoff, spray drift, and drain flow. In the present study, the acute and chronic physiological effects of the strobilurin fungicide azoxystrobin on three clones of D. magna originating from different Danish lakes were evaluated. Significant clonal variation in the sensitivity of D. magna toward azoxystrobin was demonstrated. One clone had a 48-h median lethal concentration (LC50) of 0.277 mg/L (95% confidence limits [CL], 0.145 and 0.427 mg/L), which is comparable to the value widely used in risk assessments (0.259 mg/L). The two remaining clones were far more sensitive, however, and had LC50s of 0.071 mg/L (95% CL, 0.034 and 0.126 mg/L) and 0.098 mg/L (95% CL, 0.066 and 0.139 mg/L), respectively. Furthermore, through respiration measurements and life-table experiments, sublethal stress was shown to exist at exposure to an ecologically relevant concentration (0.026 microg/L). Based on these results, we may expect changes in daphnid populations at azoxystrobin concentrations much lower than previously thought. Thus, ponds in the agricultural areas may experience changes in food-web structure even at very low concentrations of azoxystrobin.

Diversity and community structure of fungi through a permafrost core profile from the Qinghai-Tibet Plateau of China.

PubMed

Hu, Weigang; Zhang, Qi; Li, Dingyao; Cheng, Gang; Mu, Jing; Wu, Qingbai; Niu, Fujun; An, Lizhe; Feng, Huyuan

2014-12-01

While a vast number of studies have addressed the prokaryotic diversity in permafrost, characterized by subzero temperatures, low water activity, and extremely low rates of nutrient and metabolite transfer, fungal patterns have received surprisingly limited attention. Here, the fungal diversity and community structure were investigated by culture-dependent technique combined with cloning-restriction fragment length polymorphism (RFLP) analysis of sediments in a 10-m-long permafrost core from the Qinghai-Tibet Plateau of China. A total of 62 fungal phylotypes related to 10 distinct classes representing three phyla were recovered from 5031 clones generated in 13 environmental gene libraries. A large proportion of the phylotypes (25/62) that were distantly related to described fungal species appeared to be novel diversity. Ascomycota was the predominant group of fungi, with respect to both clone and phylotype number. Our results suggested there was the existence of cosmopolitan psychrophilic or psychrotolerant fungi in permafrost sediments, the community composition of fungi varied with increasing depth, while these communities largely distributed according to core layers. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Singular Universe and the Reality of Time

NASA Astrophysics Data System (ADS)

Mangabeira Unger, Roberto; Smolin, Lee

2015-01-01

Introduction; Part I. Roberto Mangabeira Unger: 1. The science of the one universe in time; 2. The context and consequences of the argument; 3. The singular existence of the universe; 4. The inclusive reality of time; 5. The mutability of the laws of nature; 6. The selective realism of mathematics; Part II. Lee Smolin: 1. Cosmology in crisis; 2. Principles for a cosmological theory; 3. The setting: the puzzles of contemporary cosmology; 4. Hypotheses for a new cosmology; 5. Mathematics; 6. Approaches to solving the metalaw dilemma; 7. Implications of temporal naturalism for philosophy of mind; 8. An agenda for science; 9. Concluding remarks; A note concerning disagreements between our views.

The augmentation algorithm and molecular phylogenetic trees

NASA Technical Reports Server (NTRS)

Holmquist, R.

1978-01-01

Moore's (1977) augmentation procedure is discussed, and it is concluded that the procedure is valid for obtaining estimates of the total number of fixed nucleotide substitutions both theoretically and in practice, for both simulated and real data, and in agreement, for experimentally dense data sets, with stochastic estimates of the divergence, provided the restrictions on codon mutability resulting from natural selection are explicitly allowed for. Tateno and Nei's (1978) critique that the augmentation procedure has a systematic bias toward overestimation of the total number of nucleotide replacements is disputed, and a data analysis suggests that ancestral sequences inferred by the method of parsimony contain a large number of incorrectly assigned nucleotides.

Effect of dynamic factors of space flights on the green alga Chlorella vulgaris.

PubMed

Moskvitin, E V; Vaulina, E N

1974-01-01

The biological effects of vibrational and linear acceleration on the alga Chlorella vulgaris were studied. Periodic vibration in the frequency range of 4-4000 Hz with vibrational acceleration up to 16 g did not affect the survival and mutability of Chlorella cells and did not modify the effects of acute gamma-radiation. However, random vibration similar to that occurring during launch of spaceships, combined with linear acceleration increased the radiation damage to algae produced by acute gamma-radiation at a dose of 10000 r. This effect is seen only in cells at the beginning of the G1 stage, which precedes DNA synthesis.

Meditations on the ubiquity and mutability of nano-sized materials in the environment.

PubMed

Wiesner, Mark R; Lowry, Gregory V; Casman, Elizabeth; Bertsch, Paul M; Matson, Cole W; Di Giulio, Richard T; Liu, Jie; Hochella, Michael F

2011-11-22

A wide variety of nanomaterials can be found naturally occurring in the environment, although finding and characterizing these materials remains a challenge due to their size. Recent studies in the field have shown that natural nanomaterials are common in many geochemical systems. In this issue of ACS Nano, Hutchison and co-workers make us realize that manmade nanomaterials can often be practically identical to those that spontaneously form in the environment. This Perspective discusses the prevalence of nanomaterials in nature, including anthropogenic and naturally occurring nanomaterials, and the dynamic behavior of these materials in the environment. © 2011 American Chemical Society

Challenging empowerment: AIDS-affected South African children and the need for a multi-level relational approach.

PubMed

Ansell, Nicola

2014-01-01

Critics of empowerment have highlighted the concept's mutability, focus on individual transformation, one-dimensionality and challenges of operationalisation. Relating these critiques to children's empowerment raises new challenges. Drawing on scholarship on children's subjecthood and exercise of power, alongside empirical research with children affected by AIDS, I argue that empowerment envisaged as individual self-transformation and increased capacity to act independently offers little basis for progressive change. Rather it is essential to adopt a relational approach that recognises the need to transform power relationships at multiple levels. This analysis has implications for our wider understanding of empowerment in the 21st century.

'More than skin-deep': biological essentialism in response to a distinctiveness threat in a stigmatized fan community.

PubMed

Plante, Courtney N; Roberts, Sharon E; Snider, Jamie S; Schroy, Catherine; Reysen, Stephen; Gerbasi, Kathleen

2015-06-01

We investigated how group distinctiveness threats affect essentialist beliefs about group membership in a stigmatized fan community. An experiment conducted on 817 members of the fan community revealed that highly identified fans who perceived significant stigmatization were the most likely to endorse essentialist beliefs about group membership when exposed to a distinctiveness threat via comparison to a highly similar (vs. dissimilar) outgroup. These results bridge essentialism research and research on distinctiveness threat by demonstrating the mutability of group essentialism beliefs as a defensive response to distinctiveness threats. Implications for future research are discussed. © 2014 The British Psychological Society.

Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system.

PubMed

Tashakkori, Maryam Mohammadi; Tebianian, Majid; Tabatabaei, Mohammad; Mosavari, Nader

2016-12-01

Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium Tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2. In this study, we cloned, expressed, and purified MPT-64 as a potent M. bovis antigen in a prokaryotic system for use in future diagnostic studies. The antigenic region of the Mpt64 gene was investigated by bioinformatics methods, cloned into the PQE-30 plasmid, and expressed in Escherichia coli M15 cells, followed by isopropyl β-d-1-thiogalactopyranoside induction. The expressed protein was analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified using a nickel-affinity column. Biological activity was confirmed by western blot using specific antibodies. Our data verified the successful cloning of the Mpt64 gene (687-bp segment) via the expression vector and purification of recombinant MPT-64 as a 24-kDa protein. These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB. Copyright © 2016.

Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

PubMed Central

Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

2013-01-01

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy. PMID:23469246

Comparative assessment of genetic and epigenetic variation among regenerants of potato (Solanum tuberosum) derived from long-term nodal tissue-culture and cell selection.

PubMed

Dann, Alison L; Wilson, Calum R

2011-04-01

Three long-term nodal tissued cultured Russet Burbank potato clones and nine thaxtomin A-treated regenerant lines, derived from the nodal lines, were assessed for genetic and epigenetic (in the form of DNA methylation) differences by AFLP and MSAP. The treated regenerant lines were originally selected for superior resistance to common scab disease and acceptable tuber yield in pot and field trials. The long-term, tissue culture clone lines exhibited genetic (8.75-15.63% polymorphisms) and epigenetic (12.56-26.13% polymorphisms) differences between them and may represent a stress response induced by normal plant growth disruption. The thaxtomin A-treated regenerant lines exhibited much higher significant (p < 0.05) genetic (2-29.38%) and epigenetic (45.22-51.76%) polymorphisms than the nodal cultured parent clones. Methylation-sensitive mutations accumulated within the regenerant lines are significantly correlated (p < 0.05) to disease resistance. However, linking phenotypic differences that could be of benefit to potato growers, to single gene sequence polymorphisms in a tetraploid plant such as the potato would be extremely difficult since it is assumed many desirable traits are under polygenic control.

Disappearance of Ph1 chromosome with intensive chemotherapy and detection of minimal residual disease by polymerase chain reaction in a patient with blast crisis of chronic myelogenous leukemia.

PubMed

Honda, H; Miyagawa, K; Endo, M; Takaku, F; Yazaki, Y; Hirai, H

1993-06-01

We diagnosed a patient with chronic myelogenous leukemia (CML) in chronic phase (CP) on the basis of clinical findings, Ph1 chromosome detected by cytogenetic analysis, and bcr-abl fusion mRNA detected by reverse transcriptase-dependent polymerase chain reaction (RT-PCR). One month after diagnosis, the patient developed extramedullary blast crisis in the lymph nodes, and then medullary blast crisis in the bone marrow, in which different surface markers were shown. Combination chemotherapy with BH-AC, VP16, and mitoxantrone was administered; this resulted in rapid disappearance of the lymphadenopathy, restoration of normal hematopoiesis, and no Ph1 chromosome being detected by cytogenetic analysis. RT-PCR performed to detect the residual Ph1 clone revealed that although the Ph1 clone was preferentially suppressed, it was still residual. The intensive chemotherapy regimen preferentially suppressed the Ph1-positive clone and led to both clinical and cytogenetic remission in this patient with BC of CML; we suggest that RT-PCR is a sensitive and useful method for detecting minimal residual disease during the clinical course of this disease.

Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells.

PubMed

Andréasson, Claes; Schick, Anna J; Pfeiffer, Susanne M; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A

2013-01-01

Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.

Highly sensitive long-period fiber-grating strain sensor with low temperature sensitivity

NASA Astrophysics Data System (ADS)

Wang, Yi-Ping; Xiao, Limin; Wang, D. N.; Jin, Wei

2006-12-01

A long-period fiber-grating sensor with a high strain sensitivity of -7.6 pm/μɛ and a low temperature sensitivity of 3.91 pm/°C is fabricated by use of focused CO2 laser beam to carve periodic grooves on a large- mode-area photonic crystal fiber. Such a strain sensor can effectively reduce the cross-sensitivity between strain and temperature, and the temperature-induced strain error obtained is only 0.5 μɛ/°C without using temperature compensation.

Growth associated protein (GAP-43): cloning and the development of a sensitive ELISA for neurological disorders.

PubMed

Gnanapavan, Sharmilee; Yousaf, Nasim; Heywood, Wendy; Grant, Donna; Mills, Kevin; Chernajovsky, Yuti; Keir, Geoff; Giovannoni, Gavin

2014-11-15

GAP-43 has been studied in the rodent and mammalian brain and shown to be present specifically in areas undergoing axonal elongation and synapse formation. GAP-43 was cloned using the baculovirus expression system and purified. A sandwich ELISA was developed using the recombinant GAP-43 as standard and validated. CSF GAP-43 levels were analysed in benign intracranial hypertension, movement disorders, multiple sclerosis, neuropathy, CNS infections, motor neuron disease, and headache (neurological controls). GAP-43 levels were low in all disorders analysed (in particular motor neuron disease; p=0.001, and movement disorders and multiple sclerosis; p<0.0001) compared to controls, aside from CNS infections. GAP-43 is preferentially reduced in the CSF of neurological disorders associated with neurodegeneration. Copyright © 2014. Published by Elsevier B.V.

Sensitivity of Small RNA-Based Detection of Plant Viruses.

PubMed

Santala, Johanna; Valkonen, Jari P T

2018-01-01

Plants recognize unrelated viruses by the antiviral defense system called RNA interference (RNAi). RNAi processes double-stranded viral RNA into small RNAs (sRNAs) of 21-24 nucleotides, the reassembly of which into longer strands in silico allows virus identification by comparison with the sequences available in databases. The aim of this study was to compare the virus detection sensitivity of sRNA-based virus diagnosis with the established virus species-specific polymerase chain reaction (PCR) approach. Viruses propagated in tobacco plants included three engineered, infectious clones of Potato virus A (PVA), each carrying a different marker gene, and an infectious clone of Potato virus Y (PVY). Total RNA (containing sRNA) was isolated and subjected to reverse-transcription real-time PCR (RT-RT-PCR) and sRNA deep-sequencing at different concentrations. RNA extracted from various crop plants was included in the reactions to normalize RNA concentrations. Targeted detection of selected viruses showed a similar threshold for the sRNA and reverse-transcription quantitative PCR (RT-qPCR) analyses. The detection limit for PVY and PVA by RT-qPCR in this study was 3 and 1.5 fg of viral RNA, respectively, in 50 ng of total RNA per PCR reaction. When knowledge was available about the viruses likely present in the samples, sRNA-based virus detection was 10 times more sensitive than RT-RT-PCR. The advantage of sRNA analysis is the detection of all tested viruses without the need for virus-specific primers or probes.

Sensitivity enhanced strain and temperature measurements based on FBG and frequency chirp magnification.

PubMed

Du, Jiangbing; He, Zuyuan

2013-11-04

In this work, highly sensitive measurements of strain and temperature have been demonstrated using a fiber Bragg grating (FBG) sensor with significantly enhance sensitivity by all-optical signal processing. The sensitivity enhancement is achieved by degenerated Four Wave Mixing (FWM) for frequency chirp magnification (FCM), which can be used for magnifying the wavelength drift of the FBG sensor induced by strain and temperature change. Highly sensitive measurements of static strain and temperature have been experimentally demonstrated with strain sensitivity of 5.36 pm/με and temperature sensitivity of 54.09 pm/°C. The sensitivity has been enhanced by a factor of five based on a 4-order FWM in a highly nonlinear fiber (HNLF).

The link between CD8⁺ T-cell antigen-sensitivity and HIV-suppressive capacity depends on HLA restriction, target epitope and viral isolate.

PubMed

Lissina, Anna; Fastenackels, Solène; Inglesias, Maria C; Ladell, Kristin; McLaren, James E; Briceño, Olivia; Gostick, Emma; Papagno, Laura; Autran, Brigitte; Sauce, Delphine; Price, David A; Saez-Cirion, Asier; Appay, Victor

2014-02-20

Although it is established that CD8 T-cell immunity is critical for the control of HIV replication in vivo, the key factors that determine antiviral efficacy are yet to be fully elucidated. Antigen-sensitivity and T-cell receptor (TCR) avidity have been identified as potential determinants of CD8⁺ T-cell efficacy. However, there is no general consensus in this regard because the relationship between these parameters and the control of HIV infection has been established primarily in the context of immunodominant CD8⁺ T-cell responses against the Gag₂₆₃₋₂₇₂ KK10 epitope restricted by human leukocyte antigen (HLA)-B27. To investigate the relationship between antigen-sensitivity, TCR avidity and HIV-suppressive capacity in vitro across epitope specificities and HLA class I restriction elements, we used a variety of techniques to study CD8⁺ T-cell clones specific for Nef₇₃₋₈₂ QK10 and Gag₂₀₋₂₉ RY10, both restricted by HLA-A3, alongside CD8⁺ T-cell clones specific for Gag₂₆₃₋₂₇₂ KK10. For each targeted epitope, the linked parameters of antigen-sensitivity and TCR avidity correlated directly with antiviral efficacy. However, marked differences in HIV-suppressive capacity were observed between epitope specificities, HLA class I restriction elements and viral isolates. Collectively, these data emphasize the central role of the TCR as a determinant of CD8⁺ T-cell efficacy and demonstrate that the complexities of antigen recognition across epitope and HLA class I boundaries can confound simple relationships between TCR engagement and HIV suppression.

Selective sensitization to DNA-damaging agents in a human rhabdomyosarcoma cell line with inducible wild-type p53 overexpression.

PubMed

Gibson, A A; Harwood, F G; Tillman, D M; Houghton, J A

1998-01-01

Drug-induced cytotoxicity or apoptosis may be influenced by the expression of the p53 tumor suppressor gene and by the specific oncogene expressed, which may dictate the threshold at which a cytotoxic response may by induced. The objective of the study was to elucidate how DNA-damaging agents with different mechanisms of action were sensitized in the context of expression of the Pax3/FKHR fusion protein, a transformation event unique to alveolar rhabdomyosarcomas (ARMSs), and wild-type p53 (wtp53). A wtp53 cDNA was subcloned into the pGRE5-2/EBV vector with dexamethasone-inducible overexpression and transfected into Rh30 ARMS cells that express Pax3/FKHR and a mutant p53 phenotype. Following dexamethasone induction of wtp53 overexpression in a derived clone (Cl.#27), growth was slowed, and cells accumulated in G1. Functional wtp53 activity was demonstrated by selective transactivation of p50-2, a wtp53 chloramphenicol acetyltransferase reporter construct, and by up-regulated expression of endogenous p21Waf1. Data demonstrated p53-dependent sensitization (> or = 4-fold) to bleomycin, actinomycin D, and 5-fluorouracil and considerably less p53-dependence (< or = 2-fold) for doxorubicin, topotecan, etoposide, and cisplatin in Cl.#27 compared to an equivalent clone containing the pGRE5-EBV vector alone (VC#3). Data demonstrate that ARMS cells show a selective sensitization to DNA-damaging agents when wtp53 is overexpressed. The cytotoxic activity of agents that are not potentiated substantially must, therefore, depend upon p53-independent factors that relate to the mechanism of drug action.

Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells.

PubMed

Ireno, Ivanildce C; Baumann, Cindy; Stöber, Regina; Hengstler, Jan G; Wiesmüller, Lisa

2014-05-01

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens ("true positives"), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect ("true negatives") and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays ("false positives"). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities <60 % with a sensitivity of >85 %, specificity of ≥90 % and detection of false-positive compounds <17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system.

Moth Sex Pheromone Receptors and Deceitful Parapheromones

PubMed Central

Xu, Pingxi; Garczynski, Stephen F.; Atungulu, Elizabeth; Syed, Zainulabeuddin; Choo, Young-Moo; Vidal, Diogo M.; Zitelli, Caio H. L.; Leal, Walter S.

2012-01-01

The insect's olfactory system is so selective that male moths, for example, can discriminate female-produced sex pheromones from compounds with minimal structural modifications. Yet, there is an exception for this “lock-and-key” tight selectivity. Formate analogs can be used as replacement for less chemically stable, long-chain aldehyde pheromones, because male moths respond physiologically and behaviorally to these parapheromones. However, it remained hitherto unknown how formate analogs interact with aldehyde-sensitive odorant receptors (ORs). Neuronal responses to semiochemicals were investigated with single sensillum recordings. Odorant receptors (ORs) were cloned using degenerate primers, and tested with the Xenopus oocyte expression system. Quality, relative quantity, and purity of samples were evaluated by gas chromatography and gas chromatography-mass spectrometry. We identified olfactory receptor neurons (ORNs) housed in trichoid sensilla on the antennae of male navel orangeworm that responded equally to the main constituent of the sex pheromone, (11Z,13Z)-hexadecadienal (Z11Z13-16Ald), and its formate analog, (9Z,11Z)-tetradecen-1-yl formate (Z9Z11-14OFor). We cloned an odorant receptor co-receptor (Orco) and aldehyde-sensitive ORs from the navel orangeworm, one of which (AtraOR1) was expressed specifically in male antennae. AtraOR1•AtraOrco-expressing oocytes responded mainly to Z11Z13-16Ald, with moderate sensitivity to another component of the sex pheromone, (11Z,13Z)-hexadecadien-1-ol. Surprisingly, this receptor was more sensitive to the related formate than to the natural sex pheromone. A pheromone receptor from Heliothis virescens, HR13 ( = HvirOR13) showed a similar profile, with stronger responses elicited by a formate analog than to the natural sex pheromone, (11Z)-hexadecenal thus suggesting this might be a common feature of moth pheromone receptors. PMID:22911835

Molecular cloning and in-silico characterization of high temperature stress responsive pAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765.

PubMed

Padaria, Jasdeep Chatrath; Vishwakarma, Harinder; Biswas, Koushik; Jasrotia, Rahul Singh; Singh, Gyanendra Pratap

2014-10-10

Heat stress leads to accelerated production of reactive oxygen species (ROS) which causes a huge amount of oxidative damage to the cellular components of plants. A large number of heat stress related genes as HSPs, catalases, peroxidases are overexpressed at the time of stress. A potent stress responsive gene peroxisomal ascorbate peroxidase (TapAPX) obtained from heat stress (42 °C) responsive subtractive cDNA library from a thermo tolerant wheat cv. Raj3765 at anthesis stage was cloned, characterized and its role was validated under heat stress by proteomics and in-silico studies. In the present study we report the characterization at molecular and in-silico level of peroxisomal TapAPX gene isolated from heat tolerant wheat cultivar of India. qPCR studies of TapAPX gene displayed up to 203 fold level of expression at 42 °C heat stress exposure. A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling. TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting. TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.

Genetic Selection for Improved Abzymes in E. Coli

DTIC Science & Technology

1994-08-03

immunoassay to scemen antibody phage libraries directly for catalysis of a bimolecular Diels - Alder reaction and have identified several active clones...sensitive growth selection assay in F coli for catalysts with chorismate mutase activity; and (3) we identified new abzymes for a Diels - Alder ...generated combinatorial antibody libraries from mRNA isolated from the spleens of mice hyperimmunized with transition state analogs for Diels - Alder and

Acute Chagas outbreaks: molecular and biological features of Trypanosoma cruzi isolates, and clinical aspects of acute cases in Santander, Colombia.

PubMed

Díaz, Martha Lucía; Leal, Sandra; Mantilla, Julio César; Molina-Berríos, Alfredo; López-Muñoz, Rodrigo; Solari, Aldo; Escobar, Patricia; González Rugeles, Clara Isabel

2015-11-26

Outbreaks of acute Chagas disease associated with oral transmission are easily detected nowadays with trained health personnel in areas of low endemicity, or in which the vector transmission has been interrupted. Given the biological and genetic diversity of Trypanosoma cruzi, the high morbidity, mortality, and the observed therapeutic failure, new characteristics of these outbreaks need to be addressed at different levels, both in Trypanosoma cruzi as in patient response. The aim of this work was to evaluate the patient's features involved in six outbreaks of acute Chagas disease which occurred in Santander, Colombia, and the characteristics of Trypanosoma cruzi clones isolated from these patients, to establish the potential relationship between the etiologic agent features with host behavior. The clinical, pathological and epidemiological aspects of outbreaks were analyzed. In addition, Trypanosoma cruzi clones were biologically characterized both in vitro and in vivo, and the susceptibility to the classical trypanocidal drugs nifurtimox and benznidazole was evaluated. Trypanosoma cruzi clones were genotyped by means of mini-exon intergenic spacer and cytochrome b genes sequencing. All clones were DTU I, and based on the mini-exon intergenic spacer, belong to two genotypes: G2 related with sub-urban, and G11 with rural outbreaks. Girón outbreak clones with higher susceptibility to drugs presented G2 genotype and C/T transition in Cyt b. The outbreaks affected mainly young population (±25.9 years), and the mortality rate was 10 %. The cardiac tissue showed intense inflammatory infiltrate, myocardial necrosis and abundant amastigote nests. However, although the gastrointestinal tissue was congestive, no inflammation or parasites were observed. Although all clones belong to DTU I, two intra-DTU genotypes were found with the sequencing of the mini-exon intergenic spacer, however there is no strict correlation between genetic groups, the cycles of the parasite or the clinical forms of the disease. Trypanosoma cruzi clones from Girón with higher sensitivity to nifurtimox presented a particular G2 genotype and C/T transition in Cyt b. When the diagnosis was early, the patients responded well to antichagasic treatment, which highlights the importance of diagnosis and treatment early to prevent fatal outcomes associated with these acute episodes.

Development of a gene cloning system in a fast-growing and moderately thermophilic Streptomyces species and heterologous expression of Streptomyces antibiotic biosynthetic gene clusters

PubMed Central

2011-01-01

Background Streptomyces species are a major source of antibiotics. They usually grow slowly at their optimal temperature and fermentation of industrial strains in a large scale often takes a long time, consuming more energy and materials than some other bacterial industrial strains (e.g., E. coli and Bacillus). Most thermophilic Streptomyces species grow fast, but no gene cloning systems have been developed in such strains. Results We report here the isolation of 41 fast-growing (about twice the rate of S. coelicolor), moderately thermophilic (growing at both 30°C and 50°C) Streptomyces strains, detection of one linear and three circular plasmids in them, and sequencing of a 6996-bp plasmid, pTSC1, from one of them. pTSC1-derived pCWH1 could replicate in both thermophilic and mesophilic Streptomyces strains. On the other hand, several Streptomyces replicons function in thermophilic Streptomyces species. By examining ten well-sporulating strains, we found two promising cloning hosts, 2C and 4F. A gene cloning system was established by using the two strains. The actinorhodin and anthramycin biosynthetic gene clusters from mesophilic S. coelicolor A3(2) and thermophilic S. refuineus were heterologously expressed in one of the hosts. Conclusions We have developed a gene cloning and expression system in a fast-growing and moderately thermophilic Streptomyces species. Although just a few plasmids and one antibiotic biosynthetic gene cluster from mesophilic Streptomyces were successfully expressed in thermophilic Streptomyces species, we expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed. PMID:22032628

Temperature sensitive surfaces and methods of making same

DOEpatents

Liang, Liang [Richland, WA; Rieke, Peter C [Pasco, WA; Alford, Kentin L [Pasco, WA

2002-09-10

Poly-n-isopropylacrylamide surface coatings demonstrate the useful property of being able to switch charateristics depending upon temperature. More specifically, these coatings switch from being hydrophilic at low temperature to hydrophobic at high temperature. Research has been conducted for many years to better characterize and control the properties of temperature sensitive coatings. The present invention provides novel temperature sensitive coatings on articles and novel methods of making temperature sensitive coatings that are disposed on the surfaces of various articles. These novel coatings contain the reaction products of n-isopropylacrylamide and are characterized by their properties such as advancing contact angles. Numerous other characteristics such as coating thickness, surface roughness, and hydrophilic-to-hydrophobic transition temperatures are also described. The present invention includes articles having temperature-sensitve coatings with improved properties as well as improved methods for forming temperature sensitive coatings.

GmFT2a, a soybean homolog of FLOWERING LOCUS T, is involved in flowering transition and maintenance.

PubMed

Sun, Hongbo; Jia, Zhen; Cao, Dong; Jiang, Bingjun; Wu, Cunxiang; Hou, Wensheng; Liu, Yike; Fei, Zhihong; Zhao, Dazhong; Han, Tianfu

2011-01-01

Flowering reversion can be induced in soybean (Glycine max L. Merr.), a typical short-day (SD) dicot, by switching from SD to long-day (LD) photoperiods. This process may involve florigen, putatively encoded by FLOWERING LOCUS T (FT) in Arabidopsis thaliana. However, little is known about the potential function of soybean FT homologs in flowering reversion. A photoperiod-responsive FT homologue GmFT (renamed as GmFT2a hereafter) was cloned from the photoperiod-sensitive cultivar Zigongdongdou. GmFT2a gene expression under different photoperiods was analyzed by real-time quantitative PCR. In situ hybridization showed direct evidence for its expression during flowering-related processes. GmFT2a was shown to promote flowering using transgenic studies in Arabidopsis and soybean. The effects of photoperiod and temperature on GmFT2a expression were also analyzed in two cultivars with different photoperiod-sensitivities. GmFT2a expression is regulated by photoperiod. Analyses of GmFT2a transcripts revealed a strong correlation between GmFT2a expression and flowering maintenance. GmFT2a transcripts were observed continuously within the vascular tissue up to the shoot apex during flowering. By contrast, transcripts decreased to undetectable levels during flowering reversion. In grafting experiments, the early-flowering, photoperiod-insensitive stock Heihe27 promotes the appearance of GmFT2a transcripts in the shoot apex of scion Zigongdongdou under noninductive LD conditions. The photothermal effects of GmFT2a expression diversity in cultivars with different photoperiod-sensitivities and a hypothesis is proposed. GmFT2a expression is associated with flowering induction and maintenance. Therefore, GmFT2a is a potential target gene for soybean breeding, with the aim of increasing geographic adaptation of this crop.

Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

PubMed

Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

2014-01-01

The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

Three-dimensional solutions for the thermal buckling and sensitivity derivatives of temperature-sensitive multilayered angle-ply plates

NASA Technical Reports Server (NTRS)

Noor, A. K.; Burton, W. S.

1992-01-01

Analytic three-dimensional thermoelasticity solutions are presented for the thermal buckling of multilayered angle-ply composite plates with temperature-dependent thermoelastic properties. Both the critical temperatures and the sensitivity derivatives are computed. The sensitivity derivatives measure the sensitivity of the buckling response to variations in the different lamination and material parameters of the plate. The plates are assumed to have rectangular geometry and an antisymmetric lamination with respect to the middle plane. The temperature is assumed to be independent of the surface coordinates, but has an arbitrary symmetric variation through the thickness of the plate. The prebuckling deformations are accounted for. Numerical results are presented, for plates subjected to uniform temperature increase, showing the effects of temperature-dependent material properties on the prebuckling stresses, critical temperatures, and their sensitivity derivatives.

Densified waste form and method for forming

DOEpatents

Garino, Terry J.; Nenoff, Tina M.; Sava Gallis, Dorina Florentina

2015-08-25

Materials and methods of making densified waste forms for temperature sensitive waste material, such as nuclear waste, formed with low temperature processing using metallic powder that forms the matrix that encapsulates the temperature sensitive waste material. The densified waste form includes a temperature sensitive waste material in a physically densified matrix, the matrix is a compacted metallic powder. The method for forming the densified waste form includes mixing a metallic powder and a temperature sensitive waste material to form a waste form precursor. The waste form precursor is compacted with sufficient pressure to densify the waste precursor and encapsulate the temperature sensitive waste material in a physically densified matrix.

Survival of Salmonella enterica serovar infantis on and within stored table eggs.

PubMed

Lublin, Avishai; Maler, Ilana; Mechani, Sara; Pinto, Riky; Sela-Saldinger, Shlomo

2015-02-01

Contaminated table eggs are considered a primary source of foodborne salmonellosis globally. Recently, a single clone of Salmonella enterica serovar Infantis emerged in Israel and became the predominant serovar isolated in poultry. This clone is currently the most prevalent strain in poultry and is the leading cause of salmonellosis in humans. Because little is known regarding the potential transmission of this strain from contaminated eggs to humans, the objective of this study was to evaluate the ability of Salmonella Infantis to survive on the eggshell or within the egg during cold storage or at room temperature. Salmonella cells (5.7 log CFU per egg) were inoculated on the surface of 120 intact eggs or injected into the egg yolk (3.7 log CFU per egg) of another 120 eggs. Half of the eggs were stored at 5.5 ± 0.3°C and half at room temperature (25.5 ± 0.1°C) for up to 10 weeks. At both temperatures, the number of Salmonella cells on the shell declined by 2 log up to 4 weeks and remained constant thereafter. Yolk-inoculated Salmonella counts at cold storage declined by 1 log up to 4 weeks and remained constant, while room-temperature storage supported the growth of the pathogen to a level of 8 log CFU/ml of total egg content, as early as 4 weeks postinoculation. Examination of egg content following surface inoculation revealed the presence of Salmonella in a portion of the eggs at both temperatures up to 10 weeks, suggesting that this strain can also penetrate through the shell and survive within the egg. These findings imply that Salmonella enterica serovar Infantis is capable of survival both on the exterior and interior of table eggs and even multiply inside the egg at room temperature. Our findings support the need for prompt refrigeration to prevent Salmonella multiplication during storage of eggs at room temperature.

Molecular Cloning and Functional Characterization of Xenopus tropicalis Frog Transient Receptor Potential Vanilloid 1 Reveal Its Functional Evolution for Heat, Acid, and Capsaicin Sensitivities in Terrestrial Vertebrates*

PubMed Central

Ohkita, Masashi; Saito, Shigeru; Imagawa, Toshiaki; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

2012-01-01

The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca2+]i). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ∼60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca2+]i increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function. PMID:22130664

Development of a Serological Test for Haemophilus ducreyi for Seroprevalence Studies

PubMed Central

Elkins, Christopher; Yi, Kyungcheol; Olsen, Bonnie; Thomas, Christopher; Thomas, Kevin; Morse, Stephen

2000-01-01

We developed a new enzyme immunoassay (rpEIA) for use in determining the seroprevalence of chancroid. Three highly conserved outer membrane proteins from Haemophilus ducreyi strain 35000 were cloned, overexpressed, and purified from Escherichia coli for use as antigens in the rpEIA. Serum specimens from patients with and without chancroid were assayed to determine optimum sensitivity and specificity and to establish cutoff values. On the basis of these data, rpEIA was found to be both sensitive and specific when used to test a variety of serum specimens from patients with genital ulcers and urethritis and from healthy blood donors. PMID:10747137

Cloning, expression and characterization of a lipase gene from marine bacterium Pseudoalteromonas lipolytica SCSIO 04301

NASA Astrophysics Data System (ADS)

Su, Hongfei; Mai, Zhimao; Zhang, Si

2016-12-01

A lipase gene, lip1233, isolated from Pseudoalteromonas lipolytica SCSIO 04301, was cloned and expressed in E. coli. The enzyme comprised 810 amino acid residues with a deduced molecular weight of 80 kDa. Lip1233 was grouped into the lipase family X because it contained a highly conserved motif GHSLG. The recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimal temperature and pH value of Lip1233 were 45°C and 8.0, respectively. It retained more than 70% of original activity after being incubated in pH ranging from 6.0 to 9.5 for 30 min. It was stable when the temperature was below 45°C, but was unstable when the temperature was above 55°C. Most metal ions tested had no significant effect on the activity of Lip1233. Lip1233 remained more than original activity in some organic solvents at the concentration of 30% (v/v). It retained more than 30% activity after incubated in pure organic solvents for 12 h, while in hexane the activity was nearly 100%. Additionally, Lip1233 exhibited typical halotolerant characteristic as it was active under 4M NaCl. Lip1233 powder could catalyze efficiently the synthesis of fructose esters in hexane at 40°C. These characteristics demonstrated that Lip1233 is applicable to elaborate food processing and organic synthesis.

Cloning and high-level expression of β-xylosidase from Selenomonas ruminantium in Pichia pastoris by optimizing of pH, methanol concentration and temperature conditions.

PubMed

Dehnavi, Ehsan; Ranaei Siadat, Seyed Omid; Fathi Roudsari, Mehrnoosh; Khajeh, Khosro

2016-08-01

β-xylosidase and several other glycoside hydrolase family members, including xylanase, cooperate together to degrade hemicelluloses, a commonly found xylan polymer of plant-cell wall. β-d-xylosidase/α-l-arabinofuranosidase from the ruminal anaerobic bacterium Selenomonas ruminantium (SXA) has potential utility in industrial processes such as production of fuel ethanol and other bioproducts. The optimized synthetic SXA gene was overexpressed in methylotrophic Pichia pastoris under the control of alcohol oxidase I (AOX1) promoter and secreted into the medium. Recombinant protein showed an optimum pH 4.8 and optimum temperature 50 °C. Furthermore, optimization of growth and induction conditions in shake flask was carried out. Using the optimum expression condition (pH 6, temperature 20 °C and 1% methanol induction), protein production was increased by about three times in comparison to the control. The recombinant SXA we have expressed here showed higher turnover frequency using ρ-nitrophenyl β-xylopyranoside (PNPX) substrate, in contrast to most xylosidase experiments reported previously. This is the first report on the cloning and expression of a β-xylosidase gene from glycoside hydrolase (GH) family 43 in Pichia pastoris. Our results confirm that P. pastoris is an appropriate host for high level expression and production of SXA for industrial applications. Copyright © 2016. Published by Elsevier Inc.

Molecular cloning and expression analysis of major intrinsic protein gene in Chlamydomonas sp. ICE-L from Antarctica.

PubMed

Li, Lulu; An, Meiling; Qu, Changfeng; Zheng, Zhou; Wang, Yibin; Liu, Fangming; He, Yingying; He, Xiaodong; Miao, Jinlai

2017-07-01

Major intrinsic proteins (MIPs) form channels facilitating the passive transport of water and other small polar molecules across membranes. In this study, the complete open reading frame (ORF) of CiMIP1 (GenBank ID KY316061) encoding one kind of MIPs in the Antarctic ice microalga Chlamydomonas sp. ICE-L is successfully cloned using RACE. In addition, the expression patterns of CiMIP1 gene under different conditions of temperature and salinity are determined by qRT-PCR. The ORF of CiMIP1 gene encodes 308 amino acids, and the deduced amino acid sequence shows 74% homology with Chlamydomonas reinhardtii CrMIP1 (GenBank number 159471952). Phylogenetic analysis reveals that algal MIPs are divided into seven groups, and it is speculated that CiMIP1 most likely belongs to the MIPD subfamily. In addition, we are surprised to find that a third NPA motif exists at the carboxy terminus of the target protein except for two highly conserved ones. Expression analysis shows that the transcriptional levels of CiMIP1 gene are upregulated under either lower temperature or higher temperature and high salinity. In summary, the results together have provide new insights into the newly discovered gene in green algae and lay the foundation for further studies on the adaptation mechanism of Chlamydomonas sp. ICE-L to abiotic stresses.

Sexual Plasticity and Self-Fertilization in the Sea Anemone Aiptasia diaphana

PubMed Central

Schlesinger, Ami; Kramarsky-Winter, Esti; Rosenfeld, Hanna; Armoza-Zvoloni, Rachel; Loya, Yossi

2010-01-01

Traits that influence reproductive success and contribute to reproductive isolation in animal and plant populations are a central focus of evolutionary biology. In the present study we used an experimental approach to demonstrate the occurrence of environmental effects on sexual and asexual reproduction, and provide evidence for sexual plasticity and inter-clonal fertilization in laboratory-cultured lines of the sea anemone Aiptasia diaphana. We showed that in A. diaphana, both asexual reproduction by pedal laceration, and sexual reproduction have seasonal components. The rate of pedal laceration was ten-fold higher under summer photoperiod and water temperature conditions than under winter conditions. The onset of gametogenesis coincided with the rising water temperatures occurring in spring, and spawning occurred under parameters that emulated summer photoperiod and temperature conditions. In addition, we showed that under laboratory conditions, asexually produced clones derived from a single founder individual exhibit sexual plasticity, resulting in the development of both male and female individuals. Moreover, a single female founder produced not only males and females but also hermaphrodite individuals. We further demonstrated that A. diaphana can fertilize within and between clone lines, producing swimming planula larvae. These diverse reproductive strategies may explain the species success as invader of artificial marine substrates. We suggest that these diverse reproductive strategies, together with their unique evolutionary position, make Aiptasia diaphana an excellent model for studying the evolution of sex. PMID:20686700

Cloning and expression of two 9-cis-epoxycarotenoid dioxygenase genes during fruit development and under stress conditions from Malus.

PubMed

Xia, Hui; Wu, Shan; Ma, Fengwang

2014-10-01

There is now biochemical and genetic evidence that oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. To understand the expression characteristics of NCED during ABA biosynthesis in apple (Malus), two NCED genes cDNA sequence were cloned from Malus prunifolia using RT-PCR techniques, named MpNCED1 and MpNCED2. The two cDNA sequences have full-length open reading frame, encoding a polypeptide of 607 and 614 amino acids, respectively. Sequences analysis showed that the deduced two apple NCED proteins were highly homologous to other NCED proteins from different plant species. Real-time PCR analysis revealed MpNCED2 were expressed continuously during the whole period of apple fruit development with the pattern of "higher-low-highest", while the expression of MpNCED1 clearly declined to a steady low level in the mid-later period of fruit development. Expression of the MpNCED2 increased under the drought stress, high temperature and low temperature strongly and rapidly, whereas expression of the MpNCED1 was detected in response to temperature stress, but did not detected under drought stress. These results revealed that MpNCED1 and MpNCED2 may play different roles in regulation of the ABA biosynthesis in fruit development and various stresses response.

Method for rapid isolation of sensitive mutants

DOEpatents

Freyer, James P.

1997-01-01

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

Method for rapid isolation of sensitive mutants

DOEpatents

Freyer, J.P.

1997-07-29

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

What is Cloning?

MedlinePlus

Donate Home Cloning What is Cloning What is Cloning Clones are organisms that are exact genetic copies. ... clones made through modern cloning technologies. How Is Cloning Done? Many people first heard of cloning when ...

Molecular Analysis of Endolithic Microbial Communities in Volcanic Glasses

NASA Astrophysics Data System (ADS)

di Meo, C. A.; Giovannoni, S.; Fisk, M.

2002-12-01

Terrestrial and marine volcanic glasses become mineralogically and chemically altered, and in many cases this alteration has been attributed to microbial activity. We have used molecular techniques to study the resident microbial communities from three different volcanic environments that may be responsible for this crustal alteration. Total microbial DNA was extracted from rhyolite glass of the 7 million year old Rattlesnake Tuff in eastern Oregon. The DNA was amplified using the polymerase chain reaction (PCR) with bacterial primers targeting the 16S rRNA gene. This 16S rDNA was cloned and screened with restriction fragment length polymorphism (RFLP). Out of 89 total clones screened, 46 belonged to 13 different clone families containing two or more members, while 43 clones were unique. Sequences of eight clones representing the most dominant clone families in the library were 92 to 97% similar to soil bacterial species. In a separate study, young pillow basalts (<20 yrs old) from six different sites along the ridge axis at 9°N, East Pacific Rise were examined for microbial life. Total DNA was extracted from the basalt glass and screened for the presence of both bacteria and archaea using the PCR. Repeated attempts with different primer sets yielded no bacterial genes, whereas archaeal genes were quite abundant. A genetic fingerprinting technique, terminal restriction fragment length polymorphism (T-RFLP), was used to compare the archaeal community compositions among the six different basalts. Filtered deep-sea water samples (~15 L) were examined in parallel to identify any overlap between rock- and seawater-associated archaea. The six rock community profiles were quite similar to each other, and the background water communities were also similar, respectively. Both the rock and water communities shared the same dominant peak. To identify the T-RFLP peaks corresponding to the individual members of the rock and seawater communities, clone libraries of the archaeal 16S rDNA for one basalt sample (Dive 3718) and its corresponding background water sample were constructed. The most abundant archaeal genes were closely related to uncultured Group I marine Crenarchaeota that have been previously identified from similar deep-sea habitats. These archaeal genes collectively correspond to the dominant T-RFLP peak present in both the rock and water samples. In a third study, we investigated the microbial community residing in a Hawaiian Scientific Drilling Program core collected near Hilo, Hawaii. Total microbial DNA was extracted from a depth of 1351 m in the drill core (ambient temperature in the drill hole ~16°C), where petrographic evidence suggested the presence of microbial alteration. Archaeal 16S rRNA genes were amplified, cloned, and twelve clones representing the most abundant groups were sequenced. Eleven out of the twelve clones were 97 to 99% similar to Group I marine Crenarchaeota, while the remaining clone was 95% similar to Euryarchaeota, based on BLAST searches of the GenBank database. Our community-level approach to studying microbes living in volcanic glasses has provided a greater understanding of the microbial communities that potentially alter these materials.

POPCORN functions in the auxin pathway to regulate embryonic body plan and meristem organization in Arabidopsis.

PubMed

Xiang, Daoquan; Yang, Hui; Venglat, Prakash; Cao, Yongguo; Wen, Rui; Ren, Maozhi; Stone, Sandra; Wang, Edwin; Wang, Hong; Xiao, Wei; Weijers, Dolf; Berleth, Thomas; Laux, Thomas; Selvaraj, Gopalan; Datla, Raju

2011-12-01

The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are temperature sensitive, showing defective embryo development when grown at 22°C that is rescued when grown at 29°C. In pcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link PCN to auxin signaling and meristem function: ectopic expression of DR5(rev):green fluorescent protein (GFP), pBDL:BDL-GFP, and pMP:MP-β-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn, suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM.

POPCORN Functions in the Auxin Pathway to Regulate Embryonic Body Plan and Meristem Organization in Arabidopsis[W][OA

PubMed Central

Xiang, Daoquan; Yang, Hui; Venglat, Prakash; Cao, Yongguo; Wen, Rui; Ren, Maozhi; Stone, Sandra; Wang, Edwin; Wang, Hong; Xiao, Wei; Weijers, Dolf; Berleth, Thomas; Laux, Thomas; Selvaraj, Gopalan; Datla, Raju

2011-01-01

The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are temperature sensitive, showing defective embryo development when grown at 22°C that is rescued when grown at 29°C. In pcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link PCN to auxin signaling and meristem function: ectopic expression of DR5rev:green fluorescent protein (GFP), pBDL:BDL-GFP, and pMP:MP-β-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn, suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM. PMID:22158464

Catalase characterization and implication in bleaching of a symbiotic sea anemone.

PubMed

Merle, Pierre-Laurent; Sabourault, Cécile; Richier, Sophie; Allemand, Denis; Furla, Paola

2007-01-15

Symbiotic cnidarians are marine invertebrates harboring photosynthesizing microalgae (named zooxanthellae), which produce great amounts of oxygen and free radicals upon illumination. Studying antioxidative balance is then crucial to understanding how symbiotic cnidarians cope with ROS production. In particular, it is suspected that oxidative stress triggers cnidarian bleaching, i.e., the expulsion of zooxanthellae from the animal host, responsible for symbiotic cnidarian mass mortality worldwide. This study therefore investigates catalase antioxidant enzymes and their role in bleaching of the temperate symbiotic sea anemone Anemonia viridis. Using specific separation of animal tissues (ectoderm and endoderm) from the symbionts (zooxanthellae), spectrophotometric assays and native PAGE revealed both tissue-specific and activity pattern distribution of two catalase electrophoretypes, E1 and E2. E1, expressed in all three tissues, presents high sensitivity to the catalase inhibitor aminotriazole (ATZ) and elevated temperatures. The ectodermal E1 form is responsible for 67% of total catalase activity. The E2 form, expressed only within zooxanthellae and their host endodermal cells, displays low sensitivity to ATZ and relative thermostability. We further cloned an ectodermal catalase, which shares 68% identity with mammalian monofunctional catalases. Last, 6 days of exposure of whole sea anemones to ATZ (0.5 mM) led to effective catalase inhibition and initiated symbiont expulsion. This demonstrates the crucial role of this enzyme in cnidarian bleaching, a phenomenon responsible for worldwide climate-change-induced mass mortalities, with catastrophic consequences for marine biodiversity.

High refractive index and temperature sensitivity LPGs for high temperature operation

NASA Astrophysics Data System (ADS)

Nascimento, I. M.; Gouveia, C.; Jana, Surnimal; Bera, Susanta; Baptista, J. M.; Moreira, Paulo; Biwas, Palas; Bandyopadhyay, Somnath; Jorge, Pedro A. S.

2013-11-01

A fiber optic sensor for high sensitivity refractive index and temperature measurement able to withstand temperature up to 450 °C is reported. Two identical LPG gratings were fabricated, whereas one was coated with a high refractive index (~1.78) sol-gel thin film in order to increase its sensitivity to the external refractive index. The two sensors were characterized and compared in refractive index and temperature. Sensitivities of 1063 nm/RIU (1.338 - 1.348) and 260 pm/°C were achieved for refractive index and temperature, respectively.

Spatial patterns of stream temperatures and electric conductivity in a mesoscale catchment

NASA Astrophysics Data System (ADS)

Lieder, Ernestine; Weiler, Markus; Blume, Theresa

2017-04-01

Stream temperature and electric conductivity (EC) are both relatively easily measured and can provide valuable information on runoff generation processes and catchment storage.This study investigates the spatial variability of stream temperature and EC in a mesoscale basin. We focus on the mesoscale (sub-catchments and reach scale), and long term (seasonal / annual) stream temperature and EC patterns. Our study basin is the Attert catchment in Luxembourg (288km2), which contains multiple sub-catchments of different geology, topography and land use patterns. We installed 90 stream temperature and EC sensors at sites across the basin in summer 2015. The collected data is complemented by land use and discharge data and an extensive climate data set. Thermal sensitivity was calculated as the slope of daily air temperature-water-temperature regression line and describes the sensitivity of stream temperature to long term environmental change. Amplitude sensitivity was calculated as slope of the daily air and water temperature amplitude regression and describes the short term warming capacity of the stream. We found that groups with similar long term thermal and EC patterns are strongly related to different geological units. The sandstone reaches show the coldest temperatures and lowest annual thermal sensitivity to air temperature. The slate reaches are characterized by comparably low EC and high daily temperature amplitudes and amplitude sensitivity. Furthermore, mean annual temperatures and thermal sensitivities increase exponentially with drainage area, which can be attributed to the accumulation of heat throughout the system. On the reach scale, daily stream temperature fluctuations or sensitivities were strongly influenced by land cover distribution, stream shading and runoff volume. Daily thermal sensitivities were low for headwater streams; peaked for intermediate reaches in the middle of the catchment and then decreased again further downstream with increasing drainage area. Combining spatially distributed time series of stream temperatures and EC with information about geology, landscape and climate provides insight into the underlying hydrological processes and allows for the identification of thermally sensitive regions and reaches.

In Vitro Studies of Neurotoxic Substances

DTIC Science & Technology

1985-12-31

crotonamide (m-.-m) on the neuron specific enolase activity of differentiated N1E - 115 neuroblastoma cells (I S.E.). ’p.-.•,- -42- 1000 1C*1 802 "" 2...40 3. The Effect of Acrylamide, N-Methylacrylamide, and Crotonamide on Acetylcholinesterase Activity of Differentiated NIE- 115 Neurcblastoma Cells...NTE and OP sensitivity in differentiated and undifferentiated cultures of NIE- 115 . Clone NIE- 115 can be induced to differentiate morphologically

Plants as indicators of urban air pollution (ozone and trace elements) in Pisa, Italy.

PubMed

Nali, Cristina; Crocicchi, Lara; Lorenzini, Giacomo

2004-07-01

A biennial integrated survey, based on the use of vascular plants for the bioindication of the effects of tropospheric ozone, was performed in the area of Pisa (Tuscany, Central Italy). It also investigated the distribution of selected trace elements in plants and the data were compared with those obtained from the use of passive samplers, automatic analysers of ozone and lichen biodiversity. Photochemically produced ozone proved to be present during the warm season, with maximum hourly means surpassing 100 ppb: the use of supersensitive tobacco Bel-W3 confirmed the value of detailed, cost-effective, monitoring surveys. Trials with clover clones demonstrate that sensitive plants undergo severe biomass reduction in the current ozone regime. The mean NC-S (clover clone sensitive to ozone):NC-R (resistant) biomass ratio ranged from 0.7 (in 1999) to 0.5 (in 2000). The economic impact of these reductions deserves attention. The data obtained using passive ozone samplers exceeded those obtained using an automatic analyser. The mapping of epiphytic lichen biodiversity was not related to the geographical ozone distribution as can be seen from the tobacco's response. Lettuce plants grown under standardized conditions were used positively as bioaccumulators of trace elements: Pb was abundantly recovered, but a large portion of this element was removed by washing.

The smallest natural high-active luciferase: cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa.

PubMed

Markova, Svetlana V; Larionova, Marina D; Burakova, Ludmila P; Vysotski, Eugene S

2015-01-30

Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 SS bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of ∼3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. Copyright © 2014 Elsevier Inc. All rights reserved.

H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

PubMed

Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-08-01

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

Insertional Mutagenesis for Genes involved in Otic/Vestibular Development and Function in Xenopus Tropicalis

NASA Technical Reports Server (NTRS)

Torrejon, Marcela; Li, Erica; Nguyen, Minh; Winfree, Seth; Wang, Esther; Reinsch, Sigrid; Dalton, Bonnie (Technical Monitor)

2002-01-01

Sensitivity to gravity is essential for spatial orientation. Consequently, the gravity receptor system is one of the phylogenetically oldest sensory systems, and the special adaptations that enhance sensitivity to gravity are highly conserved. The main goal of this project is to use Xenopus (frog) to identify genes expressed during vestibular and auditory development. These studies will lead a better understanding of the molecular mechanisms involved in vestibular and auditory development and function. We are using a gene-trap approach in Xenopus tropicalis with the green fluorescent protein (GFP) gene as the transgene reporter. GFP expression occurs only when the GFP gene is correctly integrated in actively transcribed genes. Using the GFP as a tag we can easily identify and clone the mutated gene. In addition, we can study the function of the mutated gene by analyzing the defects generated by insertion of the GFP transgene. To date we have tissue specific GFP expression in X. tropicalis including expression in ear, neural tube, kidney, muscle, eyes and nose. Our transgenic animals will soon reach maturity so that we can outcross them and analyze their progeny. Our next goal is to isolate RNA from our transgenics and clone the tagged genes using RACE-PCR. Currently we are optimizing the RACE-PCR method using transgenics with crystallin GFP expression.

Densified waste form and method for forming

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garino, Terry J.; Nenoff, Tina M.; Sava Gallis, Dorina Florentina

Materials and methods of making densified waste forms for temperature sensitive waste material, such as nuclear waste, formed with low temperature processing using metallic powder that forms the matrix that encapsulates the temperature sensitive waste material. The densified waste form includes a temperature sensitive waste material in a physically densified matrix, the matrix is a compacted metallic powder. The method for forming the densified waste form includes mixing a metallic powder and a temperature sensitive waste material to form a waste form precursor. The waste form precursor is compacted with sufficient pressure to densify the waste precursor and encapsulate themore » temperature sensitive waste material in a physically densified matrix.« less

Reconstitution of wild type viral DNA in simian cells transfected with early and late SV40 defective genomes.

PubMed

O'Neill, F J; Gao, Y; Xu, X

1993-11-01

The DNAs of polyomaviruses ordinarily exist as a single circular molecule of approximately 5000 base pairs. Variants of SV40, BKV and JCV have been described which contain two complementing defective DNA molecules. These defectives, which form a bipartite genome structure, contain either the viral early region or the late region. The defectives have the unique property of being able to tolerate variable sized reiterations of regulatory and terminus region sequences, and portions of the coding region. They can also exchange coding region sequences with other polyomaviruses. It has been suggested that the bipartite genome structure might be a stage in the evolution of polyomaviruses which can uniquely sustain genome and sequence diversity. However, it is not known if the regulatory and terminus region sequences are highly mutable. Also, it is not known if the bipartite genome structure is reversible and what the conditions might be which would favor restoration of the monomolecular genome structure. We addressed the first question by sequencing the reiterated regulatory and terminus regions of E- and L-SV40 DNAs. This revealed a large number of mutations in the regulatory regions of the defective genomes, including deletions, insertions, rearrangements and base substitutions. We also detected insertions and base substitutions in the T-antigen gene. We addressed the second question by introducing into permissive simian cells, E- and L-SV40 genomes which had been engineered to contain only a single regulatory region. Analysis of viral DNA from transfected cells demonstrated recombined genomes containing a wild type monomolecular DNA structure. However, the complete defectives, containing reiterated regulatory regions, could often compete away the wild type genomes. The recombinant monomolecular genomes were isolated, cloned and found to be infectious. All of the DNA alterations identified in one of the regulatory regions of E-SV40 DNA were present in the recombinant monomolecular genomes. These and other findings indicate that the bipartite genome state can sustain many mutations which wtSV40 cannot directly sustain. However, the mutations can later be introduced into the wild type genomes when the E- and L-SV40 DNAs recombine to generate a new monomolecular genome structure.

The Number of Overlapping AID Hotspots in Germline IGHV Genes Is Inversely Correlated with Mutation Frequency in Chronic Lymphocytic Leukemia.

PubMed

Yuan, Chaohui; Chu, Charles C; Yan, Xiao-Jie; Bagnara, Davide; Chiorazzi, Nicholas; MacCarthy, Thomas

2017-01-01

The targeting of mutations by Activation-Induced Deaminase (AID) is a key step in generating antibody diversity at the Immunoglobulin (Ig) loci but is also implicated in B-cell malignancies such as chronic lymphocytic leukemia (CLL). AID has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) hotspots. WGCW sites, which contain an overlapping WRC hotspot on both DNA strands, mutate at much higher frequency than single hotspots. Human Ig heavy chain (IGHV) genes differ in terms of WGCW numbers, ranging from 4 for IGHV3-48*03 to as many as 12 in IGHV1-69*01. An absence of V-region mutations in CLL patients ("IGHV unmutated", or U-CLL) is associated with a poorer prognosis compared to "IGHV mutated" (M-CLL) patients. The reasons for this difference are still unclear, but it has been noted that particular IGHV genes associate with U-CLL vs M-CLL. For example, patients with IGHV1-69 clones tend to be U-CLL with a poor prognosis, whereas patients with IGHV3-30 tend to be M-CLL and have a better prognosis. Another distinctive feature of CLL is that ~30% of (mostly poor prognosis) patients can be classified into "stereotyped" subsets, each defined by HCDR3 similarity, suggesting selection, possibly for a self-antigen. We analyzed >1000 IGHV genes from CLL patients and found a highly significant statistical relationship between the number of WGCW hotspots in the germline V-region and the observed mutation frequency in patients. However, paradoxically, this correlation was inverse, with V-regions with more WGCW hotspots being less likely to be mutated, i.e., more likely to be U-CLL. The number of WGCW hotspots in particular, are more strongly correlated with mutation frequency than either non-overlapping (WRC) hotspots or more general models of mutability derived from somatic hypermutation data. Furthermore, this correlation is not observed in sequences from the B cell repertoires of normal individuals and those with autoimmune diseases.

The Number of Overlapping AID Hotspots in Germline IGHV Genes Is Inversely Correlated with Mutation Frequency in Chronic Lymphocytic Leukemia

PubMed Central

Yuan, Chaohui; Chu, Charles C.; Yan, Xiao-Jie; Bagnara, Davide; Chiorazzi, Nicholas

2017-01-01

The targeting of mutations by Activation-Induced Deaminase (AID) is a key step in generating antibody diversity at the Immunoglobulin (Ig) loci but is also implicated in B-cell malignancies such as chronic lymphocytic leukemia (CLL). AID has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) hotspots. WGCW sites, which contain an overlapping WRC hotspot on both DNA strands, mutate at much higher frequency than single hotspots. Human Ig heavy chain (IGHV) genes differ in terms of WGCW numbers, ranging from 4 for IGHV3-48*03 to as many as 12 in IGHV1-69*01. An absence of V-region mutations in CLL patients (“IGHV unmutated”, or U-CLL) is associated with a poorer prognosis compared to “IGHV mutated” (M-CLL) patients. The reasons for this difference are still unclear, but it has been noted that particular IGHV genes associate with U-CLL vs M-CLL. For example, patients with IGHV1-69 clones tend to be U-CLL with a poor prognosis, whereas patients with IGHV3-30 tend to be M-CLL and have a better prognosis. Another distinctive feature of CLL is that ~30% of (mostly poor prognosis) patients can be classified into “stereotyped” subsets, each defined by HCDR3 similarity, suggesting selection, possibly for a self-antigen. We analyzed >1000 IGHV genes from CLL patients and found a highly significant statistical relationship between the number of WGCW hotspots in the germline V-region and the observed mutation frequency in patients. However, paradoxically, this correlation was inverse, with V-regions with more WGCW hotspots being less likely to be mutated, i.e., more likely to be U-CLL. The number of WGCW hotspots in particular, are more strongly correlated with mutation frequency than either non-overlapping (WRC) hotspots or more general models of mutability derived from somatic hypermutation data. Furthermore, this correlation is not observed in sequences from the B cell repertoires of normal individuals and those with autoimmune diseases. PMID:28125682

Molecular and Microscopical Investigation of the Microflora Inhabiting a Deteriorated Italian Manuscript Dated from the Thirteenth Century

PubMed Central

Michaelsen, Astrid; Piñar, Guadalupe

2010-01-01

This case study shows the application of nontraditional diagnostic methods to investigate the microbial consortia inhabiting an ancient manuscript. The manuscript was suspected to be biologically deteriorated and SEM observations showed the presence of fungal spores attached to fibers, but classic culturing methods did not succeed in isolating microbial contaminants. Therefore, molecular methods, including PCR, denaturing gradient gel electrophoresis (DGGE), and clone libraries, were used as a sensitive alternative to conventional cultivation techniques. DGGE fingerprints revealed a high biodiversity of both bacteria and fungi inhabiting the manuscript. DNA sequence analysis confirmed the existence of fungi and bacteria in manuscript samples. A number of fungal clones identified on the manuscript showed similarity to fungal species inhabiting dry or saline environments, suggesting that the manuscript environment selects for osmophilic or xerophilic fungal species. Most of the bacterial sequences retrieved from the manuscript belong to phylotypes with cellulolytic activities. PMID:20449583

Cloning, sequence, and disruption of the Saccharomyces diastaticus DAR1 gene encoding a glycerol-3-phosphate dehydrogenase.

PubMed Central

Wang, H T; Rahaim, P; Robbins, P; Yocum, R R

1994-01-01

The Saccharomyces diastaticus DAR1 gene was cloned by complementation in an Escherichia coli strain auxogrophic for glycerol-3-phosphate. DAR1 encodes an NADH-dependent dihydroxyacetone phosphate reductase (sn-glycerol-3-phosphate dehydrogenase [G3PDase; EC 1.1.1.8]) homologous to several other eukaryotic G3PDases. DAR1 is distinct from GUT2, which encodes a glucose-repressed mitochondrial G3PDase, but is identical to GPD1 from S. cerevisiae, a close relative of S. diastaticus. The level of DAR1-encoded G3PDase was increased about threefold in a medium of high osmolarity. Disruption of DAR1 in a haploid S. cerevisiae was not lethal but led to a decrease in cytoplasmic NADH-dependent G3PDase activity, an increase in osmotic sensitivity, and a 25% reduction in glycerol secretion from cells grown anaerobically on glucose. PMID:7961476

Cloning, sequence, and disruption of the Saccharomyces diastaticus DAR1 gene encoding a glycerol-3-phosphate dehydrogenase.

PubMed

Wang, H T; Rahaim, P; Robbins, P; Yocum, R R

1994-11-01

The Saccharomyces diastaticus DAR1 gene was cloned by complementation in an Escherichia coli strain auxogrophic for glycerol-3-phosphate. DAR1 encodes an NADH-dependent dihydroxyacetone phosphate reductase (sn-glycerol-3-phosphate dehydrogenase [G3PDase; EC 1.1.1.8]) homologous to several other eukaryotic G3PDases. DAR1 is distinct from GUT2, which encodes a glucose-repressed mitochondrial G3PDase, but is identical to GPD1 from S. cerevisiae, a close relative of S. diastaticus. The level of DAR1-encoded G3PDase was increased about threefold in a medium of high osmolarity. Disruption of DAR1 in a haploid S. cerevisiae was not lethal but led to a decrease in cytoplasmic NADH-dependent G3PDase activity, an increase in osmotic sensitivity, and a 25% reduction in glycerol secretion from cells grown anaerobically on glucose.

Analysis of methylated patterns and quality-related genes in tobacco (Nicotiana tabacum) cultivars.

PubMed

Jiao, Junna; Jia, Yanlong; Lv, Zhuangwei; Sun, Chuanfei; Gao, Lijie; Yan, Xiaoxiao; Cui, Liusu; Tang, Zongxiang; Yan, Benju

2014-08-01

Methylation-sensitive amplified polymorphism was used in this study to investigate epigenetic information of four tobacco cultivars: Yunyan 85, NC89, K326, and Yunyan 87. The DNA fragments with methylated information were cloned by reamplified PCR and sequenced. The results of Blast alignments showed that the genes with methylation information included chitinase, nitrate reductase, chloroplast DNA, mitochondrial DNA, ornithine decarboxylase, ribulose carboxylase, and promoter sequences. Homologous comparison in three cloned gene sequences (nitrate reductase, ornithine decarboxylase, and ribulose decarboxylase) indicated that geographic factors had significant influence on the whole genome methylation. Introns also contained different information in different tobacco cultivars. These findings suggest that synthetic mechanisms for tobacco aromatic components could be affected by different environmental factors leading to variation of noncoding regions in the genome, which finally results in different fragrance and taste in different tobacco cultivars.

Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

NASA Astrophysics Data System (ADS)

Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

1995-07-01

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods▿ †

PubMed Central

La Duc, Myron T.; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J. A.; Venkateswaran, Kasthuri

2009-01-01

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments. PMID:19700540

Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora

PubMed Central

Marraccini, Pierre; Vinecky, Felipe; Alves, Gabriel S.C.; Ramos, Humberto J.O.; Elbelt, Sonia; Vieira, Natalia G.; Carneiro, Fernanda A.; Sujii, Patricia S.; Alekcevetch, Jean C.; Silva, Vânia A.; DaMatta, Fábio M.; Ferrão, Maria A.G.; Leroy, Thierry; Pot, David; Vieira, Luiz G.E.; da Silva, Felipe R.; Andrade, Alan C.

2012-01-01

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora. PMID:22511801

Screening and identification of RhD antigen mimic epitopes from a phage display random peptide library for the serodiagnosis of haemolytic disease of the foetus and newborn.

PubMed

Wang, Jiao; Song, Jingjing; Zhou, Shuimei; Fu, Yourong; Bailey, Jeffrey A; Shen, Changxin

2018-01-16

Identification of RhD antigen epitopes is a key component in understanding the pathogenesis of haemolytic disease of the foetus and newborn. Research has indicated that phage display libraries are useful tools for identifying novel mimic epitopes (mimotopes) which may help to determine antigen specificity. We selected the mimotopes of blood group RhD antigen by affinity panning a phage display library using monoclonal anti-D. After three rounds of biopanning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and then sent for sequencing and peptides synthesis. Next, competitive ELISA and erythrocyte haemagglutination inhibition tests were carried out to confirm the inhibitory activity of the synthetic peptide. To evaluate the diagnostic performance of the synthetic peptide, a diagnostic ELISA was examined. Fourteen of 35 phage clones that were chosen randomly from the titering plate were considered to be positive. Following DNA sequencing and translation, 11 phage clones were found to represent the same peptide - RMKMLMMLMRRK (P4) - whereas each of the other three clones represented a unique peptide. Through the competitive ELISA and erythrocyte haemagglutination inhibition tests, the peptide (P4) was verified to have the ability to mimic the RhD antigen. The diagnostic ELISA for P4 proved to be sensitive (82.61%) and specific (88.57%). This study reveals that the P4 peptide can mimic RhD antigen and paves the way for the development of promising targeted diagnostic and therapeutic platforms for haemolytic disease of the foetus and newborn.

Clonality, outer-membrane proteins profile and efflux pump in KPC- producing Enterobacter sp. in Brazil.

PubMed

Rosa, Juliana Ferraz; Rizek, Camila; Marchi, Ana Paula; Guimaraes, Thais; Miranda, Lourdes; Carrilho, Claudia; Levin, Anna S; Costa, Silvia F

2017-03-17

Carbapenems resistance in Enterobacter spp. has increased in the last decade, few studies, however, described the mechanisms of resistance in this bacterium. This study evaluated clonality and mechanisms of carbapenems resistance in clinical isolates of Enterobacter spp. identified in three hospitals in Brazil (Hospital A, B and C) over 7-year. Antibiotics sensitivity, pulsed-field gel electrophoresis (PFGE), PCR for carbapenemase and efflux pump genes were performed for all carbapenems-resistant isolates. Outer-membrane protein (OMP) was evaluated based on PFGE profile. A total of 130 isolates of Enterobacter spp were analyzed, 44/105 (41, 9%) E. aerogenes and 8/25 (32,0%) E. cloacae were resistant to carbapenems. All isolates were susceptible to fosfomycin, polymyxin B and tigecycline. KPC was present in 88.6% of E. aerogenes and in all E. cloacae resistant to carbapenems. The carbapenems-resistant E. aerogenes identified in hospital A belonged to six clones, however, a predominant clone was identified in this hospital over the study period. There is a predominant clone in Hospital B and Hospital C as well. The mechanisms of resistance to carbapenems differ among subtypes. Most of the isolates co-harbored blaKPC, blaTEM and /or blaCTX associated with decreased or lost of 35-36KDa and or 39 KDa OMP. The efflux pump AcrAB-TolC gene was only identified in carbapenems-resistant E. cloacae. There was a predominant clone in each hospital suggesting that cross-transmission of carbapenems-resistant Enterobacter spp. was frequent. The isolates presented multiple mechanisms of resistance to carbapenems including OMP alteration.

Comprehensive census of bacteria in clean rooms by using DNA microarray and cloning methods.

PubMed

La Duc, Myron T; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J A; Venkateswaran, Kasthuri

2009-10-01

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.