Vertebrate cells genetically deficient for Cdc14A or Cdc14B retain DNA damage checkpoint proficiency but are impaired in DNA repair

Cdc14A and Cdc14B knockout cells with double-strand breaks still arrest in G2, but they fail to repair the damage

Dengue Reporter Virus Particles for Measuring Neutralizing Antibodies against Each of the Four Dengue Serotypes

The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination

Probenecid Inhibits the Human Bitter Taste Receptor TAS2R16 and Suppresses Bitter Perception of Salicin

Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to β-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance

Functional Dissection of Mitotic Regulators Through Gene Targeting in Human Somatic Cells

International audienc

Strict pairing of var promoters and introns is required for var gene silencing in the malaria parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum, maintains a persistent infection altering the proteins expressed on the surface of the infected red blood cells, thus avoiding the host’s immune response. The primary surface antigen, a protein called PfEMP1, is encoded by a multicopy gene family called var. Each individual parasite only expresses a single var gene at a time, maintaining all other members of the family in a transcriptionally silent state. Previous work using reporter genes in transiently transfected plasmid constructs implicated a conserved intron found in all var genes in the silencing process. Here we utilize episomal recombination within stably transformed parasites to generate different var promoter and intron arrangements and show that loss of the intron results in var promotor activation. Further, in multicopy plasmid concatamers, each intron could only silence a single promoter, suggesting a one to one pairing requirement for silencing. Transcriptionally active, “unpaired” promoters remained active after integration into a chromosome, however they were not recognized by the pathway that maintains mutually exclusive var gene expression. The data indicate that intron/promoter pairing is responsible for silencing each individual var gene, and that disruption of silencing of one gene does not affect the transcriptional activity of neighboring var promoters. This suggests that silencing is regulated at the level of individual genes rather than assembly of silent chromatin throughout a chromosomal region, thus providing a possible explanation of how a var gene can be maintained in a silent state while the immediately adjacent var gene is transcriptionally active

Managing communications activities for a multi-drug, multi-indication oncology portfolio

Objective: To describe an effective approach that addresses the challenges and needs associated with managing the communications activities for a multi-drug, multi-indication oncology portfolio. • Challenge/problem: Oncology agents hold a distinct position in the pantheon of therapeutics, as many of these agents can treat >1 type of cancer. Correspondingly, communication strategies surrounding these agents present a unique challenge in that information must simultaneously maintain a consistent scientific narrative for the drug itself while meeting the specific needs for the differing tumor types. This problem is further complicated when a franchise of agents is considered. • Solution: In collaboration with a medical communications consultancy partner, a multi-pronged strategy for aligning scientific messaging among key oncology brands was developed. Key components of this strategy included: (1) development of and achieving consensus on a core clinical narrative for each agent; (2) alignment of the scientific platforms to the consensus narratives; (3) development of medical objectives based on the communications needs; and (4) creation of a unified communication strategy that capitalizes on synergies within and between agents. Novel strategies for developing consensus and tracking metrics will be discussed. • Outcome: Consensus was reached quickly, informed by novel live and virtual interactions in 2016-2017. Communication plans were subsequently developed and actioned, and metrics were monitored over time. Plan revisions were minimal and consistent with natural expansions of brand strategy. • Benefit: A collaborative unified approach to a portfolio strategy for communicating key scientific messages for oncology assets proved to be an effective and efficient means of disseminating pertinent information