Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast.

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.

Phosphorylation controls RNA binding and transcription by the influenza virus polymerase.

The influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. Genome replication is controlled in part by phosphorylation of NP that regulates assembly of the replication machinery. However, it remains unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle.

Dual metabolomic profiling uncovers Toxoplasma manipulation of the host metabolome and the discovery of a novel parasite metabolic capability.

The obligate intracellular parasite Toxoplasma gondii is auxotrophic for several key metabolites and must scavenge these from the host. It is unclear how T. gondii manipulates host metabolism to support its overall growth rate and non-essential metabolites. To investigate this question, we measured changes in the joint host-parasite metabolome over a time course of infection. Host and parasite transcriptomes were simultaneously generated to determine potential changes in expression of metabolic enzymes. T. gondii infection changed metabolite abundance in multiple metabolic pathways, including the tricarboxylic acid cycle, the pentose phosphate pathway, glycolysis, amino acid synthesis, and nucleotide metabolism. Our analysis indicated that changes in some pathways, such as the tricarboxylic acid cycle, were mirrored by changes in parasite transcription, while changes in others, like the pentose phosphate pathway, were paired with changes in both the host and parasite transcriptomes. Further experiments led to the discovery of a T. gondii enzyme, sedoheptulose bisphosphatase, which funnels carbon from glycolysis into the pentose phosphate pathway through an energetically driven dephosphorylation reaction. This additional route for ribose synthesis appears to resolve the conflict between the T. gondii tricarboxylic acid cycle and pentose phosphate pathway, which are both NADP+ dependent. Sedoheptulose bisphosphatase represents a novel step in T. gondii central carbon metabolism that allows T. gondii to energetically-drive ribose synthesis without using NADP+.

Group 2 ITI Consensus Report: Prosthodontics and implant dentistry.

OBJECTIVES: Working Group 2 was convened to address topics relevant to prosthodontics and dental implants. Systematic reviews were developed according to focused questions addressing (a) the number of implants required to support fixed full-arch restorations, (b) the influence of intentionally tilted implants compared to axial positioned implants when supporting fixed dental prostheses (FDPs), (c) implant placement and loading protocols, (d) zirconia dental implants, (e) zirconia and metal ceramic implant supported single crowns and (f) zirconia and metal ceramic implant supported FDPs. MATERIALS AND METHODS: Group 2 considered and discussed information gathered in six systematic reviews. Group participants discussed statements developed by the authors and developed consensus. The group developed and found consensus for clinical recommendations based on both the statements and the experience of the group. The consensus statements and clinical recommendations were presented to the plenary (gathering of all conference attendees) and discussed. Final versions were developed after consensus was reached. RESULTS: A total of 27 consensus statements were developed from the systematic reviews. Additionally, the group developed 24 clinical recommendations based on the combined expertise of the participants and the developed consensus statements. CONCLUSIONS: The literature supports the use of various implant numbers to support full-arch fixed prostheses. The use of intentionally tilted dental implants is indicated when appropriate conditions exist. Implant placement and loading protocols should be considered together when planning and treating patients. One-piece zirconia dental implants can be recommended when appropriate clinical conditions exist although two-piece zirconia implants should be used with caution as a result of insufficient data. Clinical performance of zirconia and metal ceramic single implant supported crowns is similar and each demonstrates significant, though different, complications. Zirconia ceramic FDPs are less reliable than metal ceramic. Implant supported monolithic zirconia prostheses may be a future option with more supporting evidence.

Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes.

Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5' to 3' exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs.

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase.

The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes, but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine.IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis.

Phosphorylation at the homotypic interface regulates nucleoprotein oligomerization and assembly of the influenza virus replication machinery.

Negative-sense RNA viruses assemble large ribonucleoprotein (RNP) complexes that direct replication and transcription of the viral genome. Influenza virus RNPs contain the polymerase, genomic RNA and multiple copies of nucleoprotein (NP). During RNP assembly, monomeric NP oligomerizes along the length of the genomic RNA. Regulated assembly of the RNP is essential for virus replication, but how NP is maintained as a monomer that subsequently oligomerizes to form RNPs is poorly understood. Here we elucidate a mechanism whereby NP phosphorylation regulates oligomerization. We identified new evolutionarily conserved phosphorylation sites on NP and demonstrated that phosphorylation of NP decreased formation of higher-order complexes. Two phosphorylation sites were located on opposite sides of the NP:NP interface. In both influenza A and B virus, mutating or mimicking phosphorylation at these residues blocked homotypic interactions and drove NP towards a monomeric form. Highlighting the central role of this process during infection, these mutations impaired RNP formation, polymerase activity and virus replication. Thus, dynamic phosphorylation of NP regulates RNP assembly and modulates progression through the viral life cycle.

Understanding how junction resistances impact the conduction mechanism in nano-networks.

Networks of nanowires, nanotubes, and nanosheets are important for many applications in printed electronics. However, the network conductivity and mobility are usually limited by the resistance between the particles, often referred to as the junction resistance. Minimising the junction resistance has proven to be challenging, partly because it is difficult to measure. Here, we develop a simple model for electrical conduction in networks of 1D or 2D nanomaterials that allows us to extract junction and nanoparticle resistances from particle-size-dependent DC network resistivity data. We find junction resistances in porous networks to scale with nanoparticle resistivity and vary from 5 Ω for silver nanosheets to 24 GΩ for WS2 nanosheets. Moreover, our model allows junction and nanoparticle resistances to be obtained simultaneously from AC impedance spectra of semiconducting nanosheet networks. Through our model, we use the impedance data to directly link the high mobility of aligned networks of electrochemically exfoliated MoS2 nanosheets (≈ 7 cm2 V-1 s-1) to low junction resistances of ∼2.3 MΩ. Temperature-dependent impedance measurements also allow us to comprehensively investigate transport mechanisms within the network and quantitatively differentiate intra-nanosheet phonon-limited bandlike transport from inter-nanosheet hopping.

A Practical Guide to Performance Improvement: Change Acceleration Process and Techniques to Maintain Improvements.

Performance improvement (PI) team members and leaders can use a variety of tools to help accelerate change and sustain performance for lasting success. Key factors for PI team members to consider for change management are the ability to work together to problem solve and the involvement of the project's champion or sponsor for the duration of the project. The PI team can use the Change Acceleration Process model, which includes the Threats & Opportunities Matrix; backward imaging; and the goals, roles, processes, and interpersonal relationships checklist to accelerate and maintain their improvement efforts. After reviewing this article, the reader should have a better understanding of how to use the Change Acceleration Process model and tools to promote teamwork, recognize external factors that may hinder improvement, and stay focused on the project goals for ultimate PI project success. This is the sixth article of a six-part series about performance improvement.

Post-Translation Regulation of Influenza Virus Replication.

Influenza virus exploits cellular factors to complete each step of viral replication. Yet, multiple host proteins actively block replication. Consequently, infection success depends on the relative speed and efficacy at which both the virus and host use their respective effectors. Post-translational modifications (PTMs) afford both the virus and the host means to readily adapt protein function without the need for new protein production. Here we use influenza virus to address concepts common to all viruses, reviewing how PTMs facilitate and thwart each step of the replication cycle. We also discuss advancements in proteomic methods that better characterize PTMs. Although some effectors and PTMs have clear pro- or antiviral functions, PTMs generally play regulatory roles to tune protein functions, levels, and localization. Synthesis of our current understanding reveals complex regulatory schemes where the effects of PTMs are time and context dependent as the virus and host battle to control infection.

Engaging the third estate: the Transplant Growth and Management Collaborative.

The Organ Donation and Transplantation Collaboratives that occurred within the United States from 2004 to 2008 helped contribute to a significant increase in organ donors and transplants across the country. Centers were needed to accommodate and maintain this increase in capacity to perform successful transplantations for candidates on the waiting list. The Transplant Growth and Management Collaborative was created to help fulfill this new performance level expectation. In 2007 the US Department of Health and Human Services, Health Resources and Services Administration published a best-practice report based on high-performing centers that experienced a significant increase in volume while maintaining expected, or higher than expected, outcomes. The report produced a change package that outlined common strategies, key change concepts, and actions used at the best-practice centers that could be adapted by other transplant programs by using Plan-Do-Study-Act cycles to test the impact of the changes. This change package and use of the Plan-Do-Study-Act cycles formed the foundation of the Collaborative that occurred from October 2007 through October 2008 to spread best practices to transplant programs willing to commit to making changes that could result in a 20% increase in transplant volume. More than 120 transplant centers participated at some point in the Collaborative. Although preliminary results of the Collaborative show that only a few participating programs achieved the 20% volume increase goal, many participating centers reported putting successful models in place for each of the strategies identified in the best-practice change package.

A Practical Guide to Performance Improvement: Implementation of Systematic Methodologies.

This article discusses performance improvement (PI) and the various methods that PI teams can use to provide a framework for improvement. Teams that complete successful PI projects use a systematic methodology that guides them through the process in a step-by-step manner, with each step building upon the previous one using data collection methods and analytics. Personnel involved with PI projects may need to use various methodologies to achieve improvement. These tools range from Plan-Do-Study-Act cycles to more complex methods such as Six Sigma, which uses a define, measure, analyze, improve, and control process as its foundation. This article includes examples of how PI project team members can implement various methodologies and analysis tools to improve processes across the PI project continuum. After reviewing this article, the reader should have a better understanding of the systematic methodologies supporting the PI process. This is the third article of a six-part series about performance improvement.

A Practical Guide to Performance Improvement: Beginning the Process.

This article discusses the history and background of performance improvement (PI) processes and describes the creation of the foundational document for a PI project: the project charter. It is important for PI teams to create a complete charter before the project begins to give structure to the project. Teams involved in PI should use the charter as a roadmap for project completion because it helps to keep the team focused on issues within the scope of the project. This article reviews the steps to begin creating the charter and provides examples of the various sections that should be included. By reviewing this article, the reader should have a better understanding of this part of the PI process.

A Practical Guide to Performance Improvement: Data Collection and Analysis.

This article discusses performance improvement (PI) data collection processes and the various tools that can be used to analyze and display data through the duration of a PI project. It describes the importance of data, how to determine the required amount of data, how to collect and analyze data, and in what format data should be presented. Personnel involved with PI projects may need to use various data collection methods and tools to ensure an effective project with a successful outcome. This article includes examples of how PI project team members can implement various data collection and analysis tools. After reviewing this article, the reader should have a better understanding of this part of the PI process.

A Practical Guide to Performance Improvement: Tools and Tips to Sustain and Control Project Improvements.

This article discusses some of the tools and tips to sustain performance improvement (PI) and explores some of the reasons why PI projects may fail. Although some PI projects seem simple and easy to implement, PI team members sometimes find it difficult to keep momentum going, maintain gains, and sustain the changes when the project is nearing completion and afterward. Team member and leader discipline, including use of multidisciplinary communication and standard work processes, can facilitate ongoing improvement. Communication may include visual displays of information and routine huddles. After reviewing this article, the reader should have a better understanding of sustaining change and promoting continuous PI. This is the fifth article of a six-part series about PI.

A Practical Guide to Performance Improvement: Failure Mode and Effects Analysis.

This article discusses the process of Failure Mode and Effects Analysis (FMEA) and how it relates to performance improvement (PI) and the development of high reliability organizations. As a proactive process, PI team members can use FMEA to identify and prioritize risk before errors occur in health care environments. This tool comprises steps to assess the failure risk of a process, system, or function before the failure occurs. Performance improvement team members can use FMEA as an additional tool to guide them when working to create a culture of safe patient care and improve patient outcomes. After reviewing this article, the reader should have a better understanding of FMEA, how to implement it, and how it supports PI processes and high reliability organizations. This is the fourth article of a six-part series about performance improvement.

A Genome-Wide Screen Reveals a Role for the HIR Histone Chaperone Complex in Preventing Mislocalization of Budding Yeast CENP-A.

Centromeric localization of the evolutionarily conserved centromere-specific histone H3 variant CENP-A (Cse4 in yeast) is essential for faithful chromosome segregation. Overexpression and mislocalization of CENP-A lead to chromosome segregation defects in yeast, flies, and human cells. Overexpression of CENP-A has been observed in human cancers; however, the molecular mechanisms preventing CENP-A mislocalization are not fully understood. Here, we used a genome-wide synthetic genetic array (SGA) to identify gene deletions that exhibit synthetic dosage lethality (SDL) when Cse4 is overexpressed. Deletion for genes encoding the replication-independent histone chaperone HIR complex (HIR1, HIR2, HIR3, HPC2) and a Cse4-specific E3 ubiquitin ligase, PSH1, showed highest SDL. We defined a role for Hir2 in proteolysis of Cse4 that prevents mislocalization of Cse4 to noncentromeric regions for genome stability. Hir2 interacts with Cse4 in vivo, and hir2∆ strains exhibit defects in Cse4 proteolysis and stabilization of chromatin-bound Cse4 Mislocalization of Cse4 to noncentromeric regions with a preferential enrichment at promoter regions was observed in hir2∆ strains. We determined that Hir2 facilitates the interaction of Cse4 with Psh1, and that defects in Psh1-mediated proteolysis contribute to increased Cse4 stability and mislocalization of Cse4 in the hir2∆ strain. In summary, our genome-wide screen provides insights into pathways that regulate proteolysis of Cse4 and defines a novel role for the HIR complex in preventing mislocalization of Cse4 by facilitating proteolysis of Cse4, thereby promoting genome stability.

Influenza virus recruits host protein kinase C to control assembly and activity of its replication machinery.

Influenza virus expresses transcripts early in infection and transitions towards genome replication at later time points. This process requires de novo assembly of the viral replication machinery, large ribonucleoprotein complexes (RNPs) composed of the viral polymerase, genomic RNA and oligomeric nucleoprotein (NP). Despite the central role of RNPs during infection, the factors dictating where and when they assemble are poorly understood. Here we demonstrate that human protein kinase C (PKC) family members regulate RNP assembly. Activated PKCδ interacts with the polymerase subunit PB2 and phospho-regulates NP oligomerization and RNP assembly during infection. Consistent with its role in regulating RNP assembly, knockout of PKCδ impairs virus infection by selectively disrupting genome replication. However, primary transcription from pre-formed RNPs deposited by infecting particles is unaffected. Thus, influenza virus exploits host PKCs to regulate RNP assembly, a step required for the transition from primary transcription to genome replication during the infectious cycle.

Endodontics versus implantology: to extirpate or integrate?

An increasingly frequent dilemma in restorative dentistry is the question of whether to retain a badly damaged tooth and restore it, or to remove and possibly replace it. Given the ethical difficulties inherent in testing this question, it is unlikely that we will ever see a properly designed clinical trial that will provide an answer. One of the most strongly debated components of this question is whether endodontic treatment and restoration can compete with a dental implant-borne prosthesis in terms of longevity and success. In the current paper, the authors will attempt to: (i) answer this question based on our current understanding of the dental literature; and (ii) provide practitioners with guidelines that might be applied to their daily practices.