Benjamin A. Pinsky

The Ipl1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores

The spindle checkpoint ensures accurate chromosome segregation by delaying cell-cycle progression until all sister kinetochores capture microtubules from opposite poles and come under tension (for reviews, see refs 1, 2). Although the checkpoint is activated by either the lack of kinetochore-microtubule attachments or defects in the tension exerted by microtubule-generated forces, it is not clear whether these signals are linked. We investigated the connection between tension and attachment by studying the conserved budding yeast Ipl1Aurora protein kinase that is required for checkpoint activation in the absence of tension but not attachment. Here, we show that spindle-checkpoint activation in kinetochore mutants that seem to have unattached kinetochores depends on Ipl1 activity. When Ipl1 function was impaired in these kinetochore mutants, the attachments were restored and the checkpoint was turned off. These data indicate that Ipl1 activates the checkpoint in response to tension defects by creating unattached kinetochores. Moreover, although the Dam1 kinetochore complex has been implicated as a key downstream target, we found the existence of unidentified Ipl1 sites on Dam1 or additional important substrates that regulate both microtuble detachment and the checkpoint.

Protein Phosphatase 1 Regulates Exit from the Spindle Checkpoint in Budding Yeast

Accurate chromosome segregation depends on sister kinetochores coming under tension when they make bioriented attachments to microtubules from opposite poles. The spindle checkpoint halts the cell cycle in response to defects in generating proper attachments or tension on kinetochores, although the precise signal that triggers the checkpoint is unclear because tension and attachment are coupled. The target of the checkpoint is the Cdc20 protein, which initiates the anaphase-promoting complex (APC)-dependent degradation of the anaphase inhibitor Pds1/securin. Although the molecular details of spindle checkpoint activation are still being elucidated, phosphorylation by at least four kinases is a crucial requirement. However, less is known about the mechanisms that silence the checkpoint after kinetochores biorient. Here, we show that the catalytic subunit of the budding yeast protein phosphatase 1 (PP1) homolog, Glc7, regulates exit from the checkpoint. Glc7 overexpression prevents spindle checkpoint activation in response to both tension and attachment defects. Although glc7 mutant cells are able to efficiently release from a non-checkpoint-mediated metaphase arrest, they are uniquely sensitive to transient spindle checkpoint activation as a result of a failure in spindle checkpoint exit. We therefore propose that PP1 activity silences the checkpoint by reversing key phosphorylation events.

Zika Virus: Diagnostics for an Emerging Pandemic Threat

ABSTRACT Zika virus (ZIKV) is an Aedes mosquito-borne flavivirus that emerged in Brazil in 2015 and then rapidly spread throughout the tropical and subtropical Americas. Based on clinical criteria alone, ZIKV cannot be reliably distinguished from infections with other pathogens that cause an undifferentiated systemic febrile illness, including infections with two common arboviruses, dengue virus and chikungunya virus. This minireview details the methods that are available to diagnose ZIKV infection.

An Mtw1 Complex Promotes Kinetochore Biorientation that Is Monitored by the Ipl1/Aurora Protein Kinase

Chromosome segregation depends on kinetochore biorientation so that sister kinetochores attach to microtubules from opposite poles and come under tension. The budding yeast Ipl1/Aurora protein kinase allows the absence of tension to activate the spindle checkpoint. We found that checkpoint activation in the mtw1-1 kinetochore mutant requires Ipl1p, suggesting that Mtw1p promotes tension. We isolated mtw1-1 dosage suppressors and identified Dsn1, a kinetochore protein that immunoprecipitates with the Mif2/CENP-C and Cse4/CENP-A proteins, as well as the Mtw1, Nnf1, and Nsl1 kinetochore proteins. mtw1 and dsn1 mutant strains exhibit similar phenotypes, suggesting that Mtw1p and Dsn1p act together. Although mtw1 mutant cells contained unattached chromosomes, attachment was restored by impairing Ipl1p function. These results suggest that mtw1 mutant kinetochores are competent to bind microtubules but Ipl1p generates unattached chromosomes. We therefore propose that an Mtw1 complex is required for kinetochore biorientation that is monitored by the Ipl1p kinase.

An International Multicenter Performance Analysis of Cytomegalovirus Load Tests

A new quantitative polymerase chain reaction assay, COBAS AmpliPrep/COBAS TaqMan CMV Test, was developed using the first World Health Organization cytomegalovirus standard. It demonstrated a high level of interlaboratory agreement and precision compared to quantitative results obtained with tests used by 5 different laboratories.

Multiplex Real-Time PCR Assay for Rapid Identification of Mycobacterium tuberculosis Complex Members to the Species Level

ABSTRACT The species identification of members of the Mycobacterium tuberculosis complex is critical to the timely initiation of both appropriate antibiotic therapy and proper public health control measures. However, the current commercially available molecular assays identify mycobacteria only to the complex level and are unable to differentiate M. tuberculosis from the closely related M. bovis and M. bovis BCG. We describe here a rapid and robust two-step, multiplex, real-time PCR assay based on genomic deletions to definitively identify M. tuberculosis, M. bovis, M. bovis BCG, and other members of the complex. When tested against a panel of well-characterized mycobacterial reference strains, the assay was both sensitive and specific, correctly identifying all strains. We applied this assay to 60 clinical isolates previously identified as M. tuberculosis complex and found 57 M. tuberculosis isolates and 3 M. bovis BCG isolates from patients who had received intravesical BCG. Furthermore, analysis of 15 clinical specimens previously identified as M. bovis by spoligotyping revealed an isolate of M. tuberculosis that had been misidentified. We propose that this assay will allow the routine identification of M. tuberculosis complex members in the clinical laboratory.

Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

Viremia and Clinical Presentation in Nicaraguan Patients Infected with Zika Virus, Chikungunya Virus, and Dengue Virus

Zika virus, chikungunya virus, and dengue virus result in similar clinical presentations, and coinfections may be relatively common. Accurate, multiplex diagnostics are necessary to detect and differentiate these arboviruses for patient care and epidemiologic surveillance.

An International Multicenter Performance Analysis of CMV Viral Load Tests

A new quantitative polymerase chain reaction assay, COBAS AmpliPrep/COBAS TaqMan CMV Test, was developed using the first World Health Organization cytomegalovirus standard. It demonstrated a high level of interlaboratory agreement and precision compared to quantitative results obtained with tests used by 5 different laboratories.

An International Collaboration to Harmonize the Quantitative Plasma Epstein-Barr Virus DNA Assay for Future Biomarker-Guided Trials in Nasopharyngeal Carcinoma

Purpose: Persistently elevated posttreatment plasma EBV DNA is a robust predictor of relapse in nasopharyngeal carcinoma (NPC). However, assay standardization is necessary for use in biomarker-driven trials. We conducted a study to harmonize the method between four centers with expertise in EBV DNA quantitation. Experimental Design: Plasma samples of 40 patients with NPC were distributed to four centers. DNA was extracted and EBV DNA copy number was determined by real-time quantitative PCR (BamHI-W primer/probe). Centers used the same protocol but generated their own calibrators. A harmonization study was then conducted using the same calibrators and PCR master mix and validated with ten pooled samples. Results: The initial intraclass correlations (ICC) for the first 40 samples between each center and the index center were 0.62 [95% confidence interval (CI): 0.39–0.78], 0.70 (0.50–0.83), and 0.59 (0.35–0.76). The largest variability was the use of different PCR master mixes and calibrators. Standardization improved ICC to 0.83 (0.5–0.95), 0.95 (0.83–0.99) and 0.96 (0.86–0.99), respectively, for ten archival frozen samples. For fresh plasma with spiked-in EBV DNA, correlations were more than 0.99 between the centers. At 5 EBV DNA copies per reaction or above, the coefficient of variance (CV) was less than 10% for the cycle threshold (Ct) among all centers, suggesting this concentration can be reliably used as a cutoff for defining the presence of detectable EBV DNA. Conclusions: Quantitative PCR assays, even when conducted in experienced clinical labs, can yield large variability in plasma EBV DNA copy numbers without harmonization. The use of common calibrators and PCR master mix can help to reduce variability. Clin Cancer Res; 19(8); 2208–15. ©2013 AACR.