Implementation of electronic charting is not associated with significant change in physician productivity in an academic emergency department.

OBJECTIVES: To compare physician productivity and billing before and after implementation of electronic charting in an academic emergency department (ED). MATERIALS AND METHODS: This retrospective, blinded, observational study compared the 6 months pre-implementation (January to June 2012) with the 6 months post-implementation 1 year later (January to June 2013). Thirty-one ED physicians were recruited, with each physician acting as his/her own control in a before-after design. Productivity was measured via total number of encounters and "productivity index" defined as worked relative value units divided by the clinical full-time equivalent. Values for charges, encounters, and productivity index were determined during each study period and separately for procedures, observational stays, and critical care. RESULTS: No differences were found for total productivity index per month (758 [623-876] pre-group vs. 756 [673-886] post-group; P = 0.30). There was, however, a 9% decrease in total encounters per month (138 [101-163] pre-group vs. 125 [99-159] post-group; P = 0.01). Significant decreases were seen across all observation stay categories. Conversely, significant increases were seen across all critical care categories. There was no difference in total charges per month. DISCUSSION: This is one of few studies to demonstrate minimal disruption in physician productivity after transitioning to electronic documentation. The reasons for these findings are likely multi-factorial. CONCLUSION: In this study, implementation of electronic charting was not associated with decreases in productivity or billing for total ED care, but may be associated with increases for critical care and decreases for observational stays.

Crystal structure of NucB, a biofilm-degrading endonuclease.

Bacterial biofilms are a complex architecture of cells that grow on moist interfaces, and are held together by a molecular glue of extracellular proteins, sugars and nucleic acids. Biofilms are particularly problematic in human healthcare as they can coat medical implants and are thus a potential source of disease. The enzymatic dispersal of biofilms is increasingly being developed as a new strategy to treat this problem. Here, we have characterized NucB, a biofilm-dispersing nuclease from a marine strain of Bacillus licheniformis, and present its crystal structure together with the biochemistry and a mutational analysis required to confirm its active site. Taken together, these data support the categorization of NucB into a unique subfamily of the ßßα metal-dependent non-specific endonucleases. Understanding the structure and function of NucB will facilitate its future development into an anti-biofilm therapeutic agent.

Structure and function of a spectrin-like regulator of bacterial cytokinesis.

Bacterial cell division is facilitated by a molecular machine--the divisome--that assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.

A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression.

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

Control of the diadenylate cyclase CdaS in Bacillus subtilis: an autoinhibitory domain limits cyclic di-AMP production.

The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination.

Characterisation of a Tip60 specific inhibitor, NU9056, in prostate cancer.

Tip60 (KAT5) is a histone acetyltransferase (HAT enzyme) involved in multiple cellular processes including transcriptional regulation, DNA damage repair and cell signalling. In prostate cancer, aggressive cases over-express Tip60 which functions as an androgen receptor co-activator via direct acetylation of lysine residues within the KLKK motif of the receptor hinge region. The purpose of this study was to identify and characterise a Tip60 acetylase inhibitor. High-throughput screening revealed an isothiazole that inhibited both Tip60 and p300 HAT activity. This substance (initially identified as 4-methyl-5-bromoisothiazole) and other isothiazoles were synthesised and assayed against Tip60. Although an authentic sample of 4-methyl-5-bromoisothiazole was inactive against Tip60, in an in vitro HAT assay, 1,2-bis(isothiazol-5-yl)disulfane (NU9056) was identified as a relatively potent inhibitor (IC(50) 2 µM). Cellular activity was confirmed by analysis of acetylation of histone and non-histone proteins in a prostate cancer cell line model. NU9056 treatment inhibited cellular proliferation in a panel of prostate cancer cell lines (50% growth inhibition, 8-27 µM) and induced apoptosis via activation of caspase 3 and caspase 9 in a concentration- and time-dependent manner. Also, decreased androgen receptor, prostate specific antigen, p53 and p21 protein levels were demonstrated in response to treatment with NU9056. Furthermore, pre-treatment with NU9056 inhibited both ATM phosphorylation and Tip60 stabilization in response to ionising radiation. Based on the activity of NU9056 and the specificity of the compound towards Tip60 relative to other HAT enzymes, these chemical biology studies have identified Tip60 as a potential therapeutic target for the treatment of prostate cancer.

Dissection of the network of interactions that links RNA processing with glycolysis in the Bacillus subtilis degradosome.

The RNA degradosome is a multiprotein macromolecular complex that is involved in the degradation of messenger RNA in bacteria. The composition of this complex has been found to display a high degree of evolutionary divergence, which may reflect the adaptation of species to different environments. Recently, a degradosome-like complex identified in Bacillus subtilis was found to be distinct from those found in proteobacteria, the degradosomes of which are assembled around the unstructured C-terminus of ribonuclease E, a protein not present in B. subtilis. In this report, we have investigated in vitro the binary interactions between degradosome components and have characterized interactions between glycolytic enzymes, RNA-degrading enzymes, and those that appear to link these two cellular processes. The crystal structures of the glycolytic enzymes phosphofructokinase and enolase are presented and discussed in relation to their roles in the mediation of complex protein assemblies. Taken together, these data provide valuable insights into the structure and dynamics of the RNA degradosome, a fascinating and complex macromolecular assembly that links RNA degradation with central carbon metabolism.

Unusual, dual endo- and exonuclease activity in the degradosome explained by crystal structure analysis of RNase J1.

RNase J is an essential enzyme in Bacillus subtilis with unusual dual endonuclease and 5'-to-3' exonuclease activities that play an important role in the maturation and degradation of mRNA. RNase J is also a component of the recently identified "degradosome" of B. subtilis. We report the crystal structure of RNase J1 from B. subtilis to 3.0 Å resolution, analysis of which reveals it to be in an open conformation suitable for binding substrate RNA. RNase J is a member of the ß-CASP family of zinc-dependent metallo-ß-lactamases. We have exploited this similarity in constructing a model for an RNase J1:RNA complex. Analysis of this model reveals candidate-stacking interactions with conserved aromatic side chains, providing a molecular basis for the observed enzyme activity. Comparisons of the B. subtilis RNase J structure with related enzymes reveal key differences that provide insights into conformational changes during catalysis and the role of the C-terminal domain.

Functional dissection of a trigger enzyme: mutations of the bacillus subtilis glutamate dehydrogenase RocG that affect differentially its catalytic activity and regulatory properties.

Any signal transduction requires communication between a sensory component and an effector. Some enzymes engage in signal perception and transduction, as well as in catalysis, and these proteins are known as "trigger" enzymes. In this report, we detail the trigger properties of RocG, the glutamate dehydrogenase of Bacillus subtilis. RocG not only deaminates the key metabolite glutamate to form alpha-ketoglutarate but also interacts directly with GltC, a LysR-type transcription factor that regulates glutamate biosynthesis from alpha-ketoglutarate, thus linking the two metabolic pathways. We have isolated mutants of RocG that separate the two functions. Several mutations resulted in permanent inactivation of GltC as long as a source of glutamate was present. These RocG proteins have lost their ability to catabolize glutamate due to a strongly reduced affinity for glutamate. The second class of mutants is exemplified by the replacement of aspartate residue 122 by asparagine. This mutant protein has retained enzymatic activity but has lost the ability to control the activity of GltC. Crystal structures of glutamate dehydrogenases that permit a molecular explanation of the properties of the various mutants are presented. Specifically, we may propose that D122N replacement affects the surface of RocG. Our data provide evidence for a correlation between the enzymatic activity of RocG and its ability to inactivate GltC, and thus give insights into the mechanism that couples the enzymatic activity of a trigger enzyme to its regulatory function.

Screening and optimizing protein production in E. coli.

Significant improvements in the technologies used for protein production have been driven by impending genome-scale proteomics projects. These initiatives have favored Escherichia coli-based expression systems, which allow rapid cloning and expression of proteins at low cost. The range of commercially available molecular biology kits, vectors, affinity tags, and host cell lines have increased dramatically in recent years. For the structural biology community, where protein production is often a rate-limiting step, these developments have made the process of producing and purifying large amounts of protein for structural studies simpler and faster. The large-scale automated screening approaches for optimizing protein production employed by structural genomics initiatives can be adapted to a more practical targeted approach appropriate for individual structural biology groups. This chapter describes simple, rapid screening methods for testing optimal vector/host combinations using a 96-well format.