gebhart/patrepeval huggingface dataset:
Characterization of an Autonomous Subgenomic Pestivirus RNA Replicon (1998)

As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of the Flaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5' and 3' untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3' end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5' third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5' end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.
Genetic diversity of the attachment protein of subgroup B respiratory syncytial viruses (1991)

Respiratory syncytial (RS) virus causes repeated infections throughout life. Between the two main antigenic subgroups of RS virus, there is antigenic variation in the attachment protein G. The antigenic differences between the subgroups appear to play a role in allowing repeated infections to occur. Antigenic differences also occur within subgroups; however, neither the extent of these differences nor their contributions to repeat infections are known. We report a molecular analysis of the extent of diversity within the subgroup B RS virus attachment protein genes of viruses isolated from children over a 30-year period. Amino acid sequence differences as high as 12% were observed in the ectodomains of the G proteins among the isolates, whereas the cytoplasmic and transmembrane domains were highly conserved. The changes in the G-protein ectodomain were localized to two areas on either side of a highly conserved region surrounding four cysteine residues. Strikingly, single-amino-acid coding changes generated by substitution mutations were not the only means by which change occurred. Changes also occurred by (i) substitutions that changed the available termination codons, resulting in proteins of various lengths, and (ii) a mutation introduced by a single nucleotide deletion and subsequent nucleotide insertion, which caused a shift in the open reading frame of the protein in comparison to the other G genes analyzed. Fifty-one percent of the G-gene nucleotide changes observed among the isolates resulted in amino acid coding changes in the G protein, indicating a selective pressure for change. Maximum-parsimony analysis demonstrated that distinct evolutionary lineages existed. These data show that sequence diversity exists among the G proteins within the subgroup B RS viruses, and this diversity may be important in the immunobiology of the RS viruses.
Recombinant human activated protein C attenuates the inflammatory response in endothelium and monocytes by modulating nuclear factor-κB (2002)

Drotrecogin alfa (activated) acts as a modulator of nuclear factor-kappaB to aid in the host immune response in endothelium and monocytes. Extrapolation of gene array findings to explain apoptosis in endothelium and monocytes, coupled with emerging preclinical reports, provides evidence to support the role of drotrecogin alfa (activated) in modulating nuclear factor-kappaB.
Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates (2000)

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.
COMPASS, a Histone H3 (Lysine 4) Methyltransferase Required for Telomeric Silencing of Gene Expression (2002)

The trithorax (Trx) family of proteins is required for maintaining a specific pattern of gene expression in some organisms. Recently we reported the isolation and characterization of COMPASS, a multiprotein complex that includes the Trx-related protein Set1 of the yeast Saccharomyces cerevisiae. Here we report that COMPASS catalyzes methylation of the fourth lysine of histone H3 in vitro. Set1 and several other components of COMPASS are also required for histone H3 methylation in vivo and for transcriptional silencing of a gene located near a chromosome telomere.
Synthesis of 3-(1-aryl-2-mercapto-imidazolyl)-2H-1-benzopyran-2-one derivatives (2002)

Reaction of 3-(2-bromoacetyl) coumarins with various substituted aromatic primary amines resulted in the formation of 3-(2-anilino acetyl) coumarins 1 which on subsequent reaction with potassium thiocyanate furnishes corresponding 3-(1-aryl-2-mercapto-imidazolyl)-2H-1-benzopyran-2-ones 2. The latter on treatment with alkyl halides/phenacyl halideslacyl halides in the presence of anhydrous ethanol and DMF gave the corresponding thioethers and thioesters 3.
Main Approaches to Target Discovery and Validation (2006)

The identification and validation of disease-causing target genes is an essential first step in drug discovery and development. Genomics and proteomics technologies have already begun to uncover novel functional pathways and therapeutic targets in several human diseases such as cancers and autoimmunity. Also, bioinformatics approaches have highlighted several key targets and functional networks. In contrast to gene-profiling approaches, phenotype-oriented target identification allows direct link between the genetic alterations and a disease phenotype. Therefore, identified genes are more likely to be a cause rather than a consequence of the disease. Once a gene target or a mechanistic pathway is identified, the next step is to demonstrate that it does play a critical role in disease initiation, perpetuation, or both. A range of strategies exists for modulating gene expression in vitro and in vivo. These strategies include the use of antibodies, negative dominant controls, antisense oligonucleotides, ribozymes, and small-interfering RNAs. In contrast to in vitro assays, mouse reverse genetics such as knockout phenotypes has become a powerful approach for deciphering gene function and target validation in the context of mammalian physiology. In addition to disease-causing genes, the identification of antigens that stimulate both arms of the immune system is the major goal for effective vaccine development. The hope is that target discovery and validation processes will concurrently identify and validate therapeutic targets for drug intervention in human diseases.
Design of Escherichia coli host strains for improved recombinant protein purification: An approach that bridges the upstream and downstream realms of bioprocessing (2011)

Escherichia coli is a favored host for rapid, scalable expression of recombinant proteins for academic, commercial or therapeutic use. To maximize its economic advantages, however, it must be coupled with robust downstream processes. These are typically composed of three or fewer chromatography steps that remove the majority of the host proteins to achieve a reasonable degree of purification. Removal of remaining impurities is often very difficult and costly due similarity among physicochemical properties with the target. This work implements a novel approach that overcomes such limitations by bridging upstream and downstream realms, that is, by subjecting lysed cell material to a variety of purification procedures, identifying individual impurities and mitigating their removal by genetic modification. Successful knockout of three prominent contaminants of immobilized metal affinity chromatography (IMAC) without detriment to cell growth or recombinant protein expression was demonstrated. Elution of the recombinant target was strategically manipulated with peptide tags that allowed purification to virtual homogeneity. Additional IMAC studies focused on the most problematic host proteins, those that retained binding affinity under stringent conditions. Two-dimensional difference gel electrophoresis discerned variation in the soluble extract pools loaded in IMAC and the subsequent impurities, with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein were shown to be the most persistent contaminants and had greater prevalence at low target protein expression. Since genetic removal of the transcription activator would negatively impact cellular function, I substituted specific residues to eliminate its IMAC affinity with minimal impact on its activity. I applied this integrative strategy seeking to improve performance of cheaper, non-affinity based processes. Phosphoenolpyruvate carboxykinase and peptidase D were significant contaminants during serial purification of a target by hydrophobic interaction and anion exchange chromatography. Ribosomal protein L25 dominated non-target binding of a polyarginine tagged recombinant on cation exchange resin. With the development of comprehensive genomic manipulation in higher order species, such integrative approaches will be conventional in the development of coupled expression systems for the production of complex biologics.
Transduction of cellular neo mRNA by retrovirus-mediated recombination (1990)

Transduction of cellular oncogenes by retroviruses is thought to be a multistep process, involving transcriptional activation of a cellular gene by upstream proviral integration and joining of cellular DNA to retroviral transcriptional signals, followed by copackaging and recombination with a helper virus genome during reverse transcription. To examine the molecular mechanism of the reverse transcriptase-mediated recombination, we introduced into mouse fibroblast cells a variety of constructs in which the neo selectable marker was joined to flanking retroviruslike or cell-like sequences. After superinfection and copackaging with a replication-competent Mo-MuLVsupF virus, the formation of recombinant neo transducing viruses was assessed in a second round of virus infection by the ability to confer G418 resistance to infected cells. Our results showed that recombinant neo proviruses were generated from neo RNA containing either a 5' or 3' retroviral end, implying that one recombination event with helper virus RNA was sufficient to incorporate the neo gene into proviral DNA. Recombination occurred with an apparent frequency of 10(-4) to 10(-5) per replication cycle in the absence of homology between the two recombining partners. This frequency, however, increased at least 100-fold if homology was provided at the site of recombination. Our results support the hypothesis that neo-transducing viruses arise via reverse transcriptase-mediated recombination of RNA rather than by recombination proceeding through DNA intermediates. Unexpectedly, removal of the retroviral packaging site psi reduced the number of neo recombinants only slightly. Our data indicated that although RNAs lacking the psi site are poorly packaged into virions, those RNAs that are included in the virions undergo frequent recombination, even if there is no selection for recombination. Many of the neo recombinants formed with the psi- constructs had undergone additional recombinations and often incorporated the psi site from the helper RNA.
Camptothecins in clinical development (2004)

Following the realisation that DNA topoisomerase I is a useful therapeutic target to be exploited for the design of potential inhibitors, topoisomerase I inhibitors now represent an established class of effective agents. In spite of intense efforts in the field, only camptothecins have a clinical relevance. Several options in chemical manipulation of natural camptothecin have been explored to overcome the major drawbacks of the drug, which include water insolubility, lactone instability, reversibility of the drug–target interaction and drug resistance. Several analogues are currently in clinical development, including water soluble camptothecins, lipophilic camptothecins and polymer-bound camptothecins. The therapeutic advantages of novel camptothecins over the two analogues (topotecan and irinotecan) approved for clinical use remain to be defined. This article is an overview of the relevant features of the analogues that are undergoing clinical development.
Mutation of the IFNAR-1 Receptor Binding Site of Human IFN-α2 Generates Type I IFN Competitive Antagonists (2008)

Type I interferons (IFNs) are multifunctional cytokines that activate cellular responses by binding a common receptor consisting of two subunits, IFNAR-1 and IFNAR-2. Although the binding of IFNs to IFNAR-2 is well characterized, the binding to the lower affinity IFNAR-1 remains less well understood. Previous reports identified a region of human IFN-alpha2 on the B and C helices ("site 1A": N65, L80, Y85, Y89) that plays a key role in binding IFNAR-1 and contributes strongly to differential activation by various type I IFNs. The current studies demonstrate that residues on the D helix are also involved in IFNAR-1 binding. In particular, residue 120 (Arg in IFN-alpha2; Lys in IFN-alpha2/alpha1) appears to be a "hot-spot" residue: substitution by alanine significantly decreased biological activity, and the charge-reversal mutation of residue 120 to Glu caused drastic loss of antiviral and antiproliferative activity for both IFN-alpha2 and IFN-alpha2/alpha1. Mutations in residues of helix D maintained their affinity for IFNAR-2 but had decreased affinity for IFNAR-1. Single-site or multiple-site mutants in the IFNAR-1 binding site that had little or no detectable in vitro biological activity were capable of blocking in vitro antiviral and antiproliferative activity of native IFN-alpha2; i.e., they are type I IFN antagonists. These prototype IFN antagonists can be developed further for possible therapeutic use in systemic lupus erythematosus, and analogous molecules can be designed for use in animal models.
TRIBUTE - A phase III trial of erlotinib HCl (OSI-774) combined with carboplatin and paclitaxel (CP) chemotherapy in advanced non-small cell lung cancer (NSCLC) (2004)

7011 Background: Tarceva™ (erlotinib) is a potent reversible HER1/EGFR tyrosine kinase inhibitor with single-agent antitumor activity. TRIBUTE was a prospective, placebo-controlled study randomizing patients (pts) with previously untreated advanced (Stage IIIB / IV) NSCLC to receive erlotinib at 150 mg/d or placebo (PBO) with a course of 6 cycles of CP followed by maintenance monotherapy. Methods: Pts with performance status (PS) of 0 or 1 were eligible. Randomization was stratified by stage, >5% weight loss prior 6 months, measurable disease, and study site. The primary endpoint was overall survival (OS). Secondary endpoints included time to progression (TTP), objective response (OR), duration of response and time to symptomatic progression (TTSP; as determined by the Lung Cancer Symptom Scale). Sample size of 1050 was based on 80% power to detect a 25% benefit in OS, α=0.05. Results: 1059 pts were randomized / treated (526 erlotinib; 533 PBO). As shown in the table below, there was no difference in median OS for patients treated with erlotinib with CP over CP alone, and no difference in OR or in median TTP. TTSP results to be presented. PBO and erlotinib were equivalent in terms of overall adverse (99.5% erlotinib, 99.5% PBO) and serious adverse events (excepting rash and diarrhea; 47.7% erlotinib, 43.2% PBO). There was an imbalance in the SAEs attributed by the reporting investigator as leading to death (53 erlotinib; 27 PBO). Only 5 of the 80 fatal SAEs were partially related to study drug (i.e., causality was also related to concurrent therapy or underlying condition) by the reporting investigators. Conclusions: Erlotinib combined with carboplatin and paclitaxel chemotherapy did not confer a survival advantage over carboplatin and paclitaxel alone in pts with previously untreated advanced NSCLC. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Genentech Genentech Genentech AstraZeneca; Genentech Genentech
Non-uniform recursive subdivision surfaces (1998)

Doo-Sabin and Catmull-Clark subdivision surfaces are based on the notion of repeated knot insertion of uniform tensor product B-spline surfaces. This paper develops rules for non-uniform Doo-Sabin and Catmull-Clark surfaces that generalize non-uniform tensor product Bspline surfaces to arbitrary topologies. This added flexibility allows, among other things, the natural introduction of features such as cusps, creases, and darts, while elsewhere maintaining the same order of continuity as their uniform counterparts.
Parallel syntheses of disulfide inhibitors of the thioredoxin redox system as potential antitumor agents. (1999)

We have reported previously that unsymmetrical disulfide inhibitors of the human thioredoxin/thioredoxin reductase redox system (hTrx/TR) possess antitumor activity. We have broadened the search for more potent inhibitors and evaluated a large range of mono- and bis-disulfide compounds, prepared using parallel syntheses. Reaction of isothioisourea-HCI salts (R') or bis-salts (R) with aromatic or aryl thiols (R") in wells of 96-well plates produced >450 derivatives with the structures R"SSR' and R"SSRSSR". The excellent yield and purity of the disulfides provided sufficient material for evaluations of enzyme inhibition and cytotoxicity. Selection criteria based on the IC50 values for hTrx/TR inhibition and for cytotoxicities of the disulfides identified agents for subsequent scale-up syntheses and in vivo evaluations of antitumor activity. These scale-up studies confirmed the original activities of agents synthesized in the plates and validated the parallel synthetic approach. Structure-activity information derived from the hTrx/TR IC50 data allow for a number of generalizations. The most potent inhibitors of the Trx system contained two heteroatoms ortho to the disulfide moiety in an aromatic functionality. The thioalkylating moieties had greatest activity with one branch point alpha to the disulfide. In the absence of branching, more potent inhibition was observed with the electron withdrawing functionalities. Bis-disulfides showed patterns of activity which depended on chain length, with optimum activity observed when the disulfide units were separated by 3.9 A, a similar distance to that separating the thioredoxin active site cysteine residues. From the agents selected for scale-up syntheses, three disulfide compounds were studied for their antitumor activity in vivo against human tumor xenografts in scid mice. One of the analogues discovered through the combinatorial syntheses/screening for Trx inhibition, 1-phenylethyl 2-imidazolyl disulfide, N1 (ProlX agent PX-C5), has demonstrated excellent in vivo activity against the MCF-7 human breast cancer and the HL-60 human leukemia, thus validating this approach for novel drug discovery.
Stable Capillary Coating with Successive Multiple Ionic Polymer Layers (1998)

A stable modification of the inner wall of a fused silica capillary was established by a simple coating procedure, successive multiple ionic-polymer layer (SMIL) coating. An anionic polymer was tightly fixed to the capillary wall by the SMIL coating, in which a cationic polymer was sandwiched between the anionic polymer and the uncoated fused silica capillary by noncovalent bonding. The SMIL-coated capillary showed a long lifetime. The endurance of the SMIL-coated capillary was more than 100 runs, and it was also tolerant to organic solvents, 1 M NaOH, and a surfactant. The coating efficiency did not depend on capillary sources, and the relative standard deviation of capillary-to-capillary reproducibility was less than 1%. In this study, dextran sulfate (DS) was used as the anionic polymer, and Polybrene was used as the cationic polymer for SMIL modification. The DS-modified capillary (SMIL-DS capillary) exhibited a pH-independent electroosmotic flow (EOF) from anode to cathode in the pH range of 2-11. The SMIL-DS capillary showed good performance for acidic protein analyses under physiological conditions (pH 7.4). Also, the presence of EOF under acidic conditions permitted new applications. Simultaneous separations of cationic, anionic, and neutral amino acids were achieved by capillary zone electrophoresis, and separations of cresol isomers were achieved by micellar electrokinetic chromatography under the acidic conditions. The SMIL-DS capillary was also useful for fast and precise determination of the pKa of acidic functional groups.