Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity.

The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein.

Finding the key to a better code: code team restructure to improve performance and outcomes.

Code teams respond to acute life threatening changes in a patient's status 24 hours a day, 7 days a week. If any variable, whether a medical skill or non-medical quality, is lacking, the effectiveness of a code team's resuscitation could be hindered. To improve the overall performance of our hospital's code team, we implemented an evidence-based quality improvement restructuring plan. The code team restructure, which occurred over a 3-month period, included a defined number of code team participants, clear identification of team members and their primary responsibilities and position relative to the patient, and initiation of team training events and surprise mock codes (simulations). Team member assessments of the restructured code team and its performance were collected through self-administered electronic questionnaires. Time-to-defibrillation, defined as the time the code was called until the start of defibrillation, was measured for each code using actual time recordings from code summary sheets. Significant improvements in team member confidence in the skills specific to their role and clarity in their role's position were identified. Smaller improvements were seen in team leadership and reduction in the amount of extra talking and noise during a code. The average time-to-defibrillation during real codes decreased each year since the code team restructure. This type of code team restructure resulted in improvements in several areas that impact the functioning of the team, as well as decreased the average time-to-defibrillation, making it beneficial to many, including the team members, medical institution, and patients.

Functional screening reveals HORMAD1-driven gene dependencies associated with translesion synthesis and replication stress tolerance.

HORMAD1 expression is usually restricted to germline cells, but it becomes mis-expressed in epithelial cells in ~60% of triple-negative breast cancers (TNBCs), where it is associated with elevated genomic instability (1). HORMAD1 expression in TNBC is bimodal with HORMAD1-positive TNBC representing a biologically distinct disease group. Identification of HORMAD1-driven genetic dependencies may uncover novel therapies for this disease group. To study HORMAD1-driven genetic dependencies, we generated a SUM159 cell line model with doxycycline-inducible HORMAD1 that replicated genomic instability phenotypes seen in HORMAD1-positive TNBC (1). Using small interfering RNA screens, we identified candidate genes whose depletion selectively inhibited the cellular growth of HORMAD1-expressing cells. We validated five genes (ATR, BRIP1, POLH, TDP1 and XRCC1), depletion of which led to reduced cellular growth or clonogenic survival in cells expressing HORMAD1. In addition to the translesion synthesis (TLS) polymerase POLH, we identified a HORMAD1-driven dependency upon additional TLS polymerases, namely POLK, REV1, REV3L and REV7. Our data confirms that out-of-context somatic expression of HORMAD1 can lead to genomic instability and reveals that HORMAD1 expression induces dependencies upon replication stress tolerance pathways, such as translesion synthesis. Our data also suggest that HORMAD1 expression could be a patient selection biomarker for agents targeting replication stress.

Integrated genomics and functional validation identifies malignant cell specific dependencies in triple negative breast cancer.

Triple negative breast cancers (TNBCs) lack recurrent targetable driver mutations but demonstrate frequent copy number aberrations (CNAs). Here, we describe an integrative genomic and RNAi-based approach that identifies and validates gene addictions in TNBCs. CNAs and gene expression alterations are integrated and genes scored for pre-specified target features revealing 130 candidate genes. We test functional dependence on each of these genes using RNAi in breast cancer and non-malignant cells, validating malignant cell selective dependence upon 37 of 130 genes. Further analysis reveals a cluster of 13 TNBC addiction genes frequently co-upregulated that includes genes regulating cell cycle checkpoints, DNA damage response, and malignant cell selective mitotic genes. We validate the mechanism of addiction to a potential drug target: the mitotic kinesin family member C1 (KIFC1/HSET), essential for successful bipolar division of centrosome-amplified malignant cells and develop a potential selection biomarker to identify patients with tumors exhibiting centrosome amplification.

Expression of the murine norovirus (MNV) ORF1 polyprotein is sufficient to induce apoptosis in a virus-free cell model.

Investigations into human norovirus infection, replication and pathogenesis, as well as the development of potential antiviral agents, have been restricted by the lack of a cell culture system for human norovirus. To date, the optimal cell culture surrogate virus model for studying human norovirus biology is the murine norovirus (MNV). In this report we generate a tetracycline-regulated, inducible eukaryotic cell system expressing the entire MNV ORF1 polyprotein. Once induced, the MNV ORF1 polyprotein was faithfully processed to the six mature non-structural proteins that predominately located to a discrete perinuclear region, as has been observed in active MNV infection. Furthermore, we found that expression of the ORF1 polyprotein alone was sufficient to induce apoptosis, characterised by caspase-9 activation and survivin down-regulation. This cell line provides a valuable new tool for studying MNV ORF1 non-structural protein function, screening for potential antiviral agents and acts as a proof-of-principle for such systems to be developed for human noroviruses.