gebhart/patrepeval huggingface dataset:
Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme (2005)

Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.
Porphyrin Assemblies and Their Scaffolds (2013)

The chlorophyll and heme molecules of chloroplasts and mitochondria are brought to life by “the global fold of the protein scaffolds”. Proteins in hydrophobic cell regions touch the dye platelets from both sides, pushing and orienting them according to their life-spending activities in light and electron transfers. The conjugated π-electron systems or planarity of the porphyrin macrocycles are never disturbed. Most artificial porphyrin assemblies contain meso-tetraphenylporphyrins (TPPs), because the four phenyl groups rotate freely and carry their substituents above or below the macrocycle. A single porphyrin molecule can, for example, be attached to an anionic surface with ammonium groups on its 2,3-carbons, be located within a hydrophobic membrane with its alkyl chains on the 4-position, and then fixate a cationic polymer with 4,5-sulphonates. Charged TPPs also show unique spectroscopic changes at different pH values and a reversible loss of the macrocycle’s planarity. On smooth silicate, graphite, or gold scaffolds TPPs have been attached irreversibly as single molecules, as extended non-covalent H or J aggregates as well as acetylene or thiophene-linked polymers. Soft, mobile porphyrin ladders conduct excited electrons (“excitons”) better than rigid porphyrin wires (“polarons”).
A chimeric haemagglutinin-based influenza split virion vaccine adjuvanted with AS03 induces protective stalk-reactive antibodies in mice (2016)

Seasonal influenza virus vaccines are generally effective at preventing disease, but need to be well matched to circulating virus strains for maximum benefit. Influenza viruses constantly undergo antigenic changes because of their high mutation rate in the immunodominant haemagglutinin (HA) head domain, which necessitates annual re-formulation and re-vaccination for continuing protection. In case of pandemic influenza virus outbreaks, new vaccines need to be produced and quickly distributed. Novel influenza virus vaccines that redirect the immune response towards more conserved epitopes located in the HA stalk domain may remove the need for annual vaccine re-formulation and could also protect against emergent pandemic strains to which the human population is immunologically naive. One approach to create such universal influenza virus vaccines is the use of constructs expressing chimeric HAs. By sequential immunization with vaccine strains expressing the same conserved HA stalk domain and exotic HA heads to which the host is naive, antibodies against the stalk can be boosted to high titres. Here we tested a monovalent chimeric HA-based prototype universal influenza virus split virion vaccine candidate with and without AS03 adjuvant in primed mice. We found that the chimeric HA-based vaccination regimen induced higher stalk antibody titres than the seasonal vaccine. The stalk antibody responses were long lasting, cross-reactive to distantly related HAs and provided protection <i>in vivo</i> in a serum transfer challenge model. The results of this study are promising and support further development of a universal influenza vaccine candidate built on the chimeric HA technology platform.
Molecular Analysis of the Short Arm of Chromosome 3 in Small-Cell and Non-Small-Cell Carcinoma of the Lung (1987)

Previous studies have suggested that the loss of DNA sequences on the short arm of chromosome 3 (3p) is associated with small-cell lung carcinoma. We therefore looked for loss of 3p alleles in tumor tissue from 42 patients with either small-cell or non-small-cell lung carcinoma. All 13 patients with small-cell lung carcinoma who were heterozygous for one or more alleles at 3p in normal tissue had the loss of at least one codominant allele in the tumor tissue. Cell lines of small-cell lung carcinoma from an additional eight patients were homozygous for 3p alleles; this result was significantly different from the predicted frequency of homozygosity. The tumor tissue studied included cell lines of small-cell lung carcinoma obtained from biopsy specimens, an autopsy sample, and an excised lymph node containing tumor cells. Loss of alleles at 3p was observed in tumor samples obtained before and after chemotherapy. Four of 15 evaluable patients with non-small-cell carcinoma of the lung had loss of 3p alleles. We conclude that loss of alleles at 3p is a change found consistently in small-cell lung carcinoma and occasionally in non-small-cell lung carcinoma.
Sequence-specific binding and photocrosslinking of alpha and beta oligodeoxynucleotides to the major groove of DNA via triple-helix formation. (1988)

A photocrosslinking reagent (p-azidophenacyl) was covalently linked to an octathymidylate synthesized with either the natural (beta) anomer of thymidine or the synthetic (alpha) anomer. The oligothymidylate was further substituted by an acridine derivative to stabilize the hybrid formed with a complementary octadeoxyadenylate sequence via intercalation. A single-stranded 27-mer containing a (dA)8 sequence and a 27-mer duplex containing a (dA.dT)8 sequence were used as targets. Upon UV irradiation, photocrosslinking of the octathymidylate to its target sequence was observed, generating bands that migrated more slowly in denaturing gels. In the 27-mer duplex, both strands were photocrosslinked to the octathymidylate. Upon alkaline treatment of the irradiated samples, cleavage of the 27-mers was observed at specific sites. These reactions were analyzed at different salt concentrations. The location of the cleavage sites allowed us to demonstrate the following. (i) Both alpha and beta oligothymidylates can recognize a DNA double helix containing an oligo(dA).oligo(dT) sequence; the oligothymidylate binds to the major groove of DNA in a parallel orientation with respect to the adenine-containing strand of the DNA double helix. (ii) alpha oligothymidylates form helices with a complementary single-stranded oligodeoxyadenylate; the two strands have a parallel orientation independently of whether or not an intercalating agent is attached to the oligothymidylate. (iii) At low salt concentration, beta oligothymidylates form a double helix with an oligodeoxyadenylate in which, as expected, the two strands are antiparallel; at high salt concentration, a triple helix is formed in which the second oligothymidylate is oriented parallel to the adenine-containing strand. These results show that it is possible to recognize an oligopurine.oligopyrimidine sequence in a DNA double helix via local triple-helix formation and to target photochemical reactions to specific sequences in both double-stranded and single-stranded nucleic acids.
Synthesis of Bis(phosphane) Palladium and Rhodium Complexes on a Polyethylene Oxide Grafted Polystyrene Matrix (TentaGel) and the Catalytic Behavior of the Rhodium Complexes (1996)

A new polymer-attached rhodium catalyst on a polyethylene oxide-grafted styrene matrix (TentaGel) is described. The corresponding anchor molecules were prepared by starting from different Boc-protected chiral pyrrolidinebis(ph0sphane)Pd complexes. The crystal structure of [3R,4R)-N-(4-carboxybutanoyl) -3,4 -bis (diphenylphosphany1)pyrrolidine-P, P’] diiodopalladium ( lc ) was determined. The formation of an amide bond between TentaGel and anchor molecules resulted in polymer-bound Pd bisphosphane complexes. Decomplexation of the Pd with cyanide and reaction of the free polymeric ligand with [Rh(COD),]BF, led to the active hydrogenation catalyst. The influence of different solvents on the swelling volume and the catalytic behavior was tested. The polymer-bound rhodium complex [(3R,4R)-N-( 1,5-dioxo-5TentaGel-amino-pentyl) -3,4-bis (diphenylphosphany1)pyrrolidineP,P‘] (1,5-cyclooctadiene)rhodim tetrafluoroborate (7) has about the same catalytic activity and enantioselectivity as the homogeneous complex [(3R,4R)-N-(fert-butoxycarbonyl)-3,4-bis( diphenylphosphany1)pyrrolidine-P, P’] ( 1,5 -cyc1ooctadiene)rhodium tetrafluoroborate (9) in the same solvent mixture. It is possible to reuse 7 once, then it abruptly loses activity. This behavior remains to be clarified.
Fail-stop processors (1983)

A methodology that facilitates the design of fault-tolerant computing systems is presented. It is based on the notion of a failstop processor. Such a processor automatically halts in response to any internal failure and does so before the effects of that failure become visible. The problem of implementing processors that, with high probability, behave like fail-stop processors is addressed. Axiomatic program verification techniques are described for use in developing provably correct programs for failstop processors. The design of a process control system illustrates the use of our methodology. Categories and Subject Descriptors: C.2.4 [Computer-Communications Networks]: Distributed Systems--network operating systems; C.3 [Special-Purpose and Application-Based Systems]--real-time systems; D.4.5 [Operating Systems]: Reliability--verification; F.3.1 [Logics and Meaning of Programs]: Specifying and Verifying and Reasoning about Programs. General Terms: Reliability, Verification. ###...
Enzymic oligoribonucleotide synthesis with T4 RNA ligase (1978)

The substrate specificity of T4 RNA ligase has been examined to determine whether the intermolecular reaction is sufficiently general to realize its potential in the enzymatic synthesis of oligoribonucleotides of defined sequence. Reactions between a variety of acceptor molecules with 3'- and 5'-bydroxyl groups and donor molecules with 3'- and 5'-phosphates indicate that the minimal substrates are a trinucleoside diphosphate acceptor and a nucleoside 3',5'-bisphosphate donor. Increasing the chain length of either the acceptor or donnor has little effect on the rate or extent of reaction. Although the base composition of the donor has only a small effect on the reaction rate, the presence of uridine in the acceptor greatly reduces the amount of product formed. The presence of a phosphate on the 3' terminus of the donor molecule permits a unique intermolecular product with a 5'-hydroxyl and a 3'-phosphate. By enzymatically either adding a 5'-phosphate or removing the 3'-phosphate, a new donor or acceptor is prepared so synthesis of an oligomer chain can proceed in either direction. With the simplicity of this enzymatic pathway and the rather broad substrate specificity of T4 RNA ligase, a convenient method for the synthesis of oligoribonucleotides is established.
Oral administration of lipopolysaccharides for the prevention of various diseases: benefit and usefulness. (2011)

It is well known that intravenous administration of lipopolysaccharide (LPS) induces severe toxicity in mammals. The maximum tolerated dose of intravenous administration of LPS in humans is reported to be only 1 to 4 ng/kg body weight. However, oral administration of a high dose of LPS caused no toxicity or systemic inflammation in other mammals, birds, or fish. Two weeks of oral administration of a high dose of LPS (2 mg/kg) did not induce toxicity in a rat experiment. Moreover, several experiments have reported that oral administration of LPS had preventative and curative properties against various diseases, including allergic, and lifestyle-related diseases. These results demonstrate that mucosal administration of LPS acts via a different regulatory mechanism in biological responses from that of parenteral administration. Mucosal administration of LPS is thought to be quite promising for prevention of diseases, but LPS is rarely used. In order to expand the usage of oral administration of LPS for preventing lifestyle and allergic diseases, it will be necessary to clarify the mechanisms that arouse immune responses after oral administration of LPS. This short review presents a recent observation of the usefulness of orally administered LPS.
Garp: a MIPS processor with a reconfigurable coprocessor (2002)

Typical reconfigurable machines exhibit shortcomings that make them less than ideal for general-purpose computing. The Garp Architecture combines reconfigurable hardware with a standard MIPS processor on the same die to retain the better features of both. Novel aspects of the architecture are presented, as well as a prototype software environment and preliminary performance results. Compared to an UltraSPARC, a Garp of similar technology could achieve speedups ranging from a factor of 2 to as high as a factor of 24 for some useful applications.
Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions (1982)

The phosphorylation sites of simian virus 40 large T antigen were determined within the primary structure of the molecule. Exhaustive digestion of (32)P-labeled large T antigen with trypsin generated six major phosphopeptides which could be separated in a newly developed isobutyric acid-containing chromatography system. By partial tryptic digestion, large T antigen was cleaved into an amino-terminal fragment of 17,000 daltons and overlapping fragments from the carboxy-terminal region ranging in size between 71,000 and 13,000 daltons. The location of the phosphopeptides was then determined by fingerprint analyses of individual fragments. Their physical properties were analyzed by sizing on polyacrylamide gels and by sequential digestion and peptide mapping; their amino acid composition was determined by differential labeling with various amino acids. The amino-terminal 17,000-dalton fragment gave rise to only one phosphopeptide (phosphopeptide 3) that contained half of the phosphate label incorporated into large T antigen. It contained phosphoserine and phosphothreonine sites, all of which were clustered within a small segment between Cys(105) and Lys(127). This segment contained five serines and two threonines. Among these, Ser(106), Ser(123), and Thr(124) were identified as phosphorylated residues; in addition, either one or both of Ser(111) and Ser(112) were phosphorylated. The neighboring residues, Ser(123) and Thr(124), were found in three different phosphorylation states in that either Ser(123) or Thr(124) or both were phosphorylated. Phosphopeptides 1, 2, 4, 5, and 6 were all derived from a single fragment extending 26,000 daltons upstream from the carboxy terminus of large T antigen. Phosphopeptide 6 was identical with the previously determined phosphothreonine peptide phosphorylated at Thr(701). Phosphopeptides 1, 2, 4, and 5 contained only serine-bound phosphate. Phosphopeptides 1, 2, and 4 represented overlapping peptides, all of which were phosphorylated at Ser(639) located next to a cluster of six acidic residues. In phosphopeptide 5, a large peptide ranging from Asn(653) to Arg(691), at least two of seven serines were phosphorylated. Thus, large T antigen contains at least eight phosphorylation sites. Their clustering within two separate regions might correlate with structural and functional domains of this protein.
Design, synthesis and biological evaluation of novel nucleotide prodrugs as potential anti hepatitis C virus agents. (2007)

Hepatitis C virus (HCV) represents the leading cause of liver disease. Most of the patented potential anti-HCV agents are nucleosides. Usually, to be active, nucleosides need to be phosphorylated three times in order to generate the corresponding 5'-triphosphate inside cells. Our group has developed the phosphoramidate approach that bypasses the first phosphorylation step and releases the 5'-monophosphate intracellulary. Part of the presented work was focused on the synthesis and the biological evaluation of phosphoramidates of novel 4'-modifed nucleosides (AZC, AZU, AZA, dipentanoyl-AZC and ETU). Phosphoramidates with different natural (L-alanine, L-valine, glycine, L-leucine, L-isoleucine, L-methionine, L-proline, L-valine and L-phenylalanine) and unnatural (cyclopentylglycine, dimethylglycine, b-alanine, N-methylglycine, L-ethylaspartate and D-alanine) amino acid were synthesised. Octyl, dodecyl (never synthesised before), methyl, ethyl, isopropyl, benzyl, 2-butyl, butyl and terf-butyl are all the variation on the ester part of the phosphoramidate structures synthesised. a- and b-naphthyl, 8-quinoline and different substituents on the phenyl ring (para-chloro, 3,4-dichloro, para-methyl and para-methoxy) are the major examples of aryl moiety variations applied to these nucleosides. The protection of the 2'- and 3'-positions with cyclopentylidene and subsequent deprotection significantly increased the overall yield of these reactions. The optimisation of the synthesis of AZA was achieved and it represents the first example of a 4'-modified ribo-purine nucleoside active against HCV. The application of our technology to AZA and AZU converted an inactive nucleoside into a sub-micromolar active phosphoramidate. The separation of the two diastereoisomers was achieved for some of the AZC phosphoramidates synthesised. A series of novel phosphoramidates of 2'-methyl modified purine (adenosine and guanosine) were synthesised using the 2'-, 3'-protection previously optimised. The aryl moiety (a-naphthyl and phenyl) and ester (methyl, ethyl, /erf-butyl, benzyl) variations were explored using L-alanine as amino acid. A general enhancement of activity was observed applying the phosphoramidate technology to these nucleosides.
OX40 controls effector CD4+ T‐cell expansion, not follicular T helper cell generation in acute Listeria infection (2014)

To investigate the importance of OX40 signals for physiological CD4(+) T-cell responses, an endogenous antigen-specific population of CD4(+) T cells that recognise the 2W1S peptide was assessed and temporal control of OX40 signals was achieved using blocking or agonistic antibodies (Abs) in vivo. Following infection with Listeria monocytogenes expressing 2W1S peptide, OX40 was briefly expressed by the responding 2W1S-specific CD4(+) T cells, but only on a subset that co-expressed effector cell markers. This population was specifically expanded by Ab-ligation of OX40 during priming, which also caused skewing of the memory response towards effector memory cells. Strikingly, this greatly enhanced effector response was accompanied by the loss of T follicular helper (TFH) cells and germinal centres. Mice deficient in OX40 and CD30 showed normal generation of TFH cells but impaired numbers of 2W1S-specific effector cells. OX40 was not expressed by 2W1S-specific memory cells, although it was rapidly up-regulated upon challenge whereupon Ab-ligation of OX40 specifically affected the effector subset. In summary, these data indicate that for CD4(+) T cells, OX40 signals are important for generation of effector T cells rather than TFH cells in this response to acute bacterial infection.
Methylation of TFPI2 in Stool DNA: A Potential Novel Biomarker for the Detection of Colorectal Cancer (2009)

We have used a gene expression array-based strategy to identify the methylation of tissue factor pathway inhibitor 2 (TFPI2), a potential tumor suppressor gene, as a frequent event in human colorectal cancers (CRC). TFPI2 belongs to the recently described group of embryonic cell Polycomb group (PcG)-marked genes that may be predisposed to aberrant DNA methylation in early stages of colorectal carcinogenesis. Aberrant methylation of TFPI2 was detected in almost all CRC adenomas (97%, n = 56) and stages I to IV CRCs (99%, n = 115). We further explored the potential of TFPI2 as a biomarker for the early detection of CRC using stool DNA-based assays in patients with nonmetastatic CRC and average-risk noncancer controls who were candidates for screening. TFPI2 methylation was detected in stool DNA from stage I to III CRC patients with a sensitivity of 76% to 89% and a specificity of 79% to 93%. Detection of TFPI2 methylation in stool DNA may act as a useful adjunct to the noninvasive strategies for screening of CRCs in the future.
Solution phase synthesis of short oligoribonucleotides on a precipitative tetrapodal support (2014)

An effective method for the synthesis of short oligoribonucleotides in solution has been elaborated. Novel 2'-O-(2-cyanoethyl)-5'-O-(1-methoxy-1-methylethyl) protected ribonucleoside 3'-phosphoramidites have been prepared and their usefulness as building blocks in RNA synthesis on a soluble support has been demonstrated. As a proof of concept, a pentameric oligoribonucleotide, 3'-UUGCA-5', has been prepared on a precipitative tetrapodal tetrakis(4-azidomethylphenyl)pentaerythritol support. The 3'-terminal nucleoside was coupled to the support as a 3'-O-(4-pentynoyl) derivative by Cu(I) promoted 1,3-dipolar cycloaddition. Couplings were carried out with 1.5 equiv of the building block. In each coupling cycle, the small molecular reagents and byproducts were removed by two quantitative precipitations from MeOH, one after oxidation and the second after the 5'-deprotection. After completion of the chain assembly, treatment with triethylamine, ammonia and TBAF released the pentamer in high yields.
The Conversion of Vitamin K Epoxide to Vitamin K Quinone and Vitamin K Quinone to Vitamin K Hydroquinone Uses the Same Active Site Cysteines (2007)

Vitamin K epoxide (or oxido) reductase (VKOR) is the target of warfarin and provides vitamin K hydroquinone for the carboxylation of select glutamic acid residues of the vitamin K-dependent proteins which are important for coagulation, signaling, and bone metabolism. It has been known for at least 20 years that cysteines are required for VKOR function. To investigate their importance, we mutated each of the seven cysteines in VKOR. In addition, we made VKOR with both C43 and C51 mutated to alanine (C43A/C51A), as well as a VKOR with residues C43-C51 deleted. Each mutated enzyme was purified and characterized. We report here that C132 and C135 of the CXXC motif are essential for both the conversion of vitamin K epoxide to vitamin K and the conversion of vitamin K to vitamin K hydroquinone. Surprisingly, conserved cysteines, 43 and 51, appear not to be important for either reaction. For the in vitro reaction driven by dithiothreitol, the 43-51 deletion mutation retained 85% and C43A/C51A 112% of the wild-type activity. The facile purification of the nine different mutations reported here illustrates the ease and reproducibility of VKOR purification by the method reported in our recent publication [Chu, P.-H., Huang, T.-Y., Williams, J., and Stafford, D. W. (2006) Proc. Natl. Acad. Sci. U S A. 103, 19308-19313].