The impact of low pressure pneumoperitoneum in robotic assisted radical prostatectomy II: a prospective, randomized, double blinded trial.

OBJECTIVE: To analyze postoperative ileus rates and postoperative complications between the different pneumoperitoneum settings. The secondary objective was to evaluate narcotic use and intraoperative blood loss between the different pneumoperitoneum settings. METHODS: A prospective, randomized, double blinded study was conducted at pneumoperitoneum pressures of either 12 mmHg or 15 mmHg for patients undergoing robotic assisted radical prostatectomy with bilateral pelvic lymph node dissection by a single high volume surgeon. RESULTS: The risk of ileus in the 12 mmHg group was 1.9% (2/105) compared to 3.2% (3/93) in the 15 mmHg group (OR 0.58, 95%CI 0.1-3.6). There was no difference in the risk of any complication with a complication rate of 4.8% (5/105) in the 12 mmHg arm compared to 4.3% (4/93) in the 15 mmHg arm (OR 1.1, 95% CI 0.3 - 4.3). CONCLUSION: Pneumoperitoneum pressure setting of 12 mmHg has no significant difference to 15 mmHg in the rate of postoperative complications, narcotic use, and intraoperative bleeding. Additional research is warranted to understand the optimal.

Visualizing the ai5γ group IIB intron.

It has become apparent that much of cellular metabolism is controlled by large well-folded noncoding RNA molecules. In addition to crystallographic approaches, computational methods are needed for visualizing the 3D structure of large RNAs. Here, we modeled the molecular structure of the ai5γ group IIB intron from yeast using the crystal structure of a bacterial group IIC homolog. This was accomplished by adapting strategies for homology and de novo modeling, and creating a new computational tool for RNA refinement. The resulting model was validated experimentally using a combination of structure-guided mutagenesis and RNA structure probing. The model provides major insights into the mechanism and regulation of splicing, such as the position of the branch-site before and after the second step of splicing, and the location of subdomains that control target specificity, underscoring the feasibility of modeling large functional RNA molecules.

Ensemble Detection of DNA Engineering Signatures.

Synthetic biology is creating genetically engineered organisms at an increasing rate for many potentially valuable applications, but this potential comes with the risk of misuse or accidental release. To begin to address this issue, we have developed a system called GUARDIAN that can automatically detect signatures of engineering in DNA sequencing data, and we have conducted a blinded test of this system using a curated Test and Evaluation (T&E) data set. GUARDIAN uses an ensemble approach based on the guiding principle that no single approach is likely to be able to detect engineering with perfect accuracy. Critically, ensembling enables GUARDIAN to detect sequence inserts in 13 target organisms with a high degree of specificity that requires no subject matter expert (SME) review.

A new way to see RNA.

Unlike proteins, the RNA backbone has numerous degrees of freedom (eight, if one counts the sugar pucker), making RNA modeling, structure building and prediction a multidimensional problem of exceptionally high complexity. And yet RNA tertiary structures are not infinite in their structural morphology; rather, they are built from a limited set of discrete units. In order to reduce the dimensionality of the RNA backbone in a physically reasonable way, a shorthand notation was created that reduced the RNA backbone torsion angles to two (η and θ, analogous to φ and ψ in proteins). When these torsion angles are calculated for nucleotides in a crystallographic database and plotted against one another, one obtains a plot analogous to a Ramachandran plot (the η/θ plot), with highly populated and unpopulated regions. Nucleotides that occupy proximal positions on the plot have identical structures and are found in the same units of tertiary structure. In this review, we describe the statistical validation of the η/θ formalism and the exploration of features within the η/θ plot. We also describe the application of the η/θ formalism in RNA motif discovery, structural comparison, RNA structure building and tertiary structure prediction. More than a tool, however, the η/θ formalism has provided new insights into RNA structure itself, revealing its fundamental components and the factors underlying RNA architectural form.

Solving nucleic acid structures by molecular replacement: examples from group II intron studies.

Structured RNA molecules are key players in ensuring cellular viability. It is now emerging that, like proteins, the functions of many nucleic acids are dictated by their tertiary folds. At the same time, the number of known crystal structures of nucleic acids is also increasing rapidly. In this context, molecular replacement will become an increasingly useful technique for phasing nucleic acid crystallographic data in the near future. Here, strategies to select, create and refine molecular-replacement search models for nucleic acids are discussed. Using examples taken primarily from research on group II introns, it is shown that nucleic acids are amenable to different and potentially more flexible and sophisticated molecular-replacement searches than proteins. These observations specifically aim to encourage future crystallographic studies on the newly discovered repertoire of noncoding transcripts.

Semiautomated model building for RNA crystallography using a directed rotameric approach.

Structured RNA molecules play essential roles in a variety of cellular processes; however, crystallographic studies of such RNA molecules present a large number of challenges. One notable complication arises from the low resolutions typical of RNA crystallography, which results in electron density maps that are imprecise and difficult to interpret. This problem is exacerbated by the lack of computational tools for RNA modeling, as many of the techniques commonly used in protein crystallography have no equivalents for RNA structure. This leads to difficulty and errors in the model building process, particularly in modeling of the RNA backbone, which is highly error prone due to the large number of variable torsion angles per nucleotide. To address this, we have developed a method for accurately building the RNA backbone into maps of intermediate or low resolution. This method is semiautomated, as it requires a crystallographer to first locate phosphates and bases in the electron density map. After this initial trace of the molecule, however, an accurate backbone structure can be built without further user intervention. To accomplish this, backbone conformers are first predicted using RNA pseudotorsions and the base-phosphate perpendicular distance. Detailed backbone coordinates are then calculated to conform both to the predicted conformer and to the previously located phosphates and bases. This technique is shown to produce accurate backbone structure even when starting from imprecise phosphate and base coordinates. A program implementing this methodology is currently available, and a plugin for the Coot model building program is under development.

Systematic Part Transfer by Extending a Modular Toolkit to Diverse Bacteria.

It is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively transferable, but there is little quantitative information defining transferability. Here, we systematically quantified the behavior of a parts set across multiple hosts. To do this, we developed a broad host range (BHR) plasmid system compatible with the large, modular CIDAR parts collection for E. coli, which we named openCIDAR. This enabled testing of a library of DNA constructs across the Pseudomonadota─Escherichia coli, Pseudomonas putida, Cupriavidus necator, and Komagataeibacter nataicola. Part performance was evaluated with a standardized characterization procedure that quantified expression in terms of molecules of equivalent fluorescein (MEFL), an objective unit of measure. The results showed that the CIDAR parts enable graded gene expression across all organisms─meaning that the same parts can be used to program E. coli, P. putida, C. necator, and K. nataicola. Most parts had a similar expression trend across hosts, although each organism had a different average gene expression level. The variability is enough that to achieve the same MEFL in a different organism, a lookup table is required to translate a design from one host to another. To identify truly divergent parts, we applied linear regression to a combinatorial set of promoters and ribosome binding sites, finding that the promoter J23100 behaves very differently in K. nataicola than in the other hosts. Thus, it is now possible to evaluate any CIDAR compatible part in three other hosts of interest, and the diversity of these hosts implies that the collection will also be compatible with many other Proteobacteria (Pseudomonadota). Furthermore, this work defines an approach to generalize modular synthetic biology parts sets beyond a single host, implying that only a few parts sets may be needed to span the tree of life. This will accelerate current efforts to engineer diverse species for environmental, biotechnological, and health applications.

Adolescent male with bilateral succinate dehydrogenase-deficient renal cell carcinoma in a horseshoe kidney managed successfully with staged bilateral robotic-assisted partial nephrectomies: A case report.

Succinate dehydrogenase (SDH) deficient renal cell carcinoma (RCC) is a rare subset of familial RCC with only 59 cases reported. SDH deficiency is associated with hereditary paraganglioma/pheochromocytoma syndrome. Most of the cases are solitary tumors with only two reported cases of bilateral tumor. The identification of SDH deficient RCC is often the sentinel event of patient's syndromic diagnosis. We present a case of an adolescent male with bilateral tumors in a horseshoe kidney who was treated with staged robotic-assisted partial nephrectomies without complication. Both tumors were SDH negative on immunohistochemical staining.

RCrane: semi-automated RNA model building.

RNA crystals typically diffract to much lower resolutions than protein crystals. This low-resolution diffraction results in unclear density maps, which cause considerable difficulties during the model-building process. These difficulties are exacerbated by the lack of computational tools for RNA modeling. Here, RCrane, a tool for the partially automated building of RNA into electron-density maps of low or intermediate resolution, is presented. This tool works within Coot, a common program for macromolecular model building. RCrane helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RCrane then allows the crystallographer to review the newly built structure and select alternative backbone conformations where desired. This tool can also be used to automatically correct the backbone structure of previously built nucleotides. These automated corrections can fix incorrect sugar puckers, steric clashes and other structural problems.

A structural analysis of the group II intron active site and implications for the spliceosome.

Group II introns are self-splicing, mobile genetic elements that have fundamentally influenced the organization of terrestrial genomes. These large ribozymes remain important for gene expression in almost all forms of bacteria and eukaryotes and they are believed to share a common ancestry with the eukaryotic spliceosome that is required for processing all nuclear pre-mRNAs. The three-dimensional structure of a group IIC intron was recently determined by X-ray crystallography, making it possible to visualize the active site and the elaborate network of tertiary interactions that stabilize the molecule. Here we describe the molecular features of the active site in detail and evaluate their correspondence with prior biochemical, genetic, and phylogenetic analyses on group II introns. In addition, we evaluate the structural significance of RNA motifs within the intron core, such as the major-groove triple helix and the domain 5 bulge. Having combined what is known about the group II intron core, we then compare it with known structural features of U6 snRNA in the eukaryotic spliceosome. This analysis leads to a set of predictions for the molecular structure of the spliceosomal active site.

Tertiary architecture of the Oceanobacillus iheyensis group II intron.

Group II introns are large ribozymes that act as self-splicing and retrotransposable RNA molecules. They are of great interest because of their potential evolutionary relationship to the eukaryotic spliceosome, their continued influence on the organization of many genomes in bacteria and eukaryotes, and their potential utility as tools for gene therapy and biotechnology. One of the most interesting features of group II introns is their relative lack of nucleobase conservation and covariation, which has long suggested that group II intron structures are stabilized by numerous unusual tertiary interactions and backbone-mediated contacts. Here, we provide a detailed description of the tertiary interaction networks within the Oceanobacillus iheyensis group IIC intron, for which a crystal structure was recently solved to 3.1 A resolution. The structure can be described as a set of several intricately constructed tertiary interaction nodes, each of which contains a core of extended stacking networks and elaborate motifs. Many of these nodes are surrounded by a web of ribose zippers, which appear to further stabilize local structure. As predicted from biochemical and genetic studies, the group II intron provides a wealth of new information on strategies for RNA folding and tertiary structural organization.

Discovering Content through Text Mining for a Synthetic Biology Knowledge System.

Scientific articles contain a wealth of information about experimental methods and results describing biological designs. Due to its unstructured nature and multiple sources of ambiguity and variability, extracting this information from text is a difficult task. In this paper, we describe the development of the synthetic biology knowledge system (SBKS) text processing pipeline. The pipeline uses natural language processing techniques to extract and correlate information from the literature for synthetic biology researchers. Specifically, we apply named entity recognition, relation extraction, concept grounding, and topic modeling to extract information from published literature to link articles to elements within our knowledge system. Our results show the efficacy of each of the components on synthetic biology literature and provide future directions for further advancement of the pipeline.

Are Postoperative Laboratory Studies Following Robotic Assisted Radical Prostatectomy Necessary?

INTRODUCTION: We evaluated the necessity of obtaining routine postoperative laboratory studies, such as complete blood count and basic metabolic panel, after robotic assisted radical prostatectomy. METHODS: This study is a retrospective review of 200 robotic assisted radical prostatectomy cases performed over a year and a half at our institution. The incidences of laboratory abnormalities were examined along with any clinical intervention. Patient demographics, tumor stage, Gleason score, operative time, estimated blood loss, length of hospital stay, presence of comorbidities and postoperative laboratory studies were extracted from the electronic medical record. The costs of laboratory studies were tabulated to further analyze potential savings to patients. RESULTS: Only 15 (7.5%) patients demonstrated laboratory abnormalities that required medical intervention. Of these 15 patients, all demonstrated hypokalemia that was treated with potassium supplementation. Patients with longer lengths of stay demonstrated higher percentages of medical intervention. The costs of these laboratory studies were calculated at $8,840. CONCLUSIONS: Lower blood loss and transfusion rates with the advent of robotic assisted radical prostatectomy along with the results described in this study provide greater evidence that postoperative laboratory studies may be futile. By eliminating these laboratory studies, substantial cost savings are realized if extrapolated across the United States. This study is limited in its evaluation of complications from different types of medical centers, higher risk patients, postoperative laboratory studies impact on symptomatic patients, and absence of emergency room visits or hospital readmissions.

Curation Principles Derived from the Analysis of the SBOL iGEM Data Set.

As an engineering endeavor, synthetic biology requires effective sharing of genetic design information that can be reused in the construction of new designs. While there are a number of large community repositories of design information, curation of this information has been limited. This in turn limits the ways in which design information can be put to use. The aim of this work was to improve this situation by creating a curated library of parts from the International Genetically Engineered Machines (iGEM) registry data set. To this end, an analysis of the Synthetic Biology Open Language (SBOL) version of the iGEM registry was carried out using four different approaches-simple statistics, SnapGene autoannotation, SYNBICT autoannotation, and expert analysis-the results of which are presented herein. Key challenges encountered include the use of free text, insufficient part provenance, part duplication, lack of part removal, and insufficient continuous curation. On the basis of these analyses, the focus has shifted from the creation of a curated iGEM part library to instead the extraction of a set of lessons, which are presented here. These lessons can be exploited to facilitate the creation and curation of other part libraries using a simpler and less labor intensive process.

Engineered yeast genomes accurately assembled from pure and mixed samples.

Yeast whole genome sequencing (WGS) lacks end-to-end workflows that identify genetic engineering. Here we present Prymetime, a tool that assembles yeast plasmids and chromosomes and annotates genetic engineering sequences. It is a hybrid workflow-it uses short and long reads as inputs to perform separate linear and circular assembly steps. This structure is necessary to accurately resolve genetic engineering sequences in plasmids and the genome. We show this by assembling diverse engineered yeasts, in some cases revealing unintended deletions and integrations. Furthermore, the resulting whole genomes are high quality, although the underlying assembly software does not consistently resolve highly repetitive genome features. Finally, we assemble plasmids and genome integrations from metagenomic sequencing, even with 1 engineered cell in 1000. This work is a blueprint for building WGS workflows and establishes WGS-based identification of yeast genetic engineering.

Synthetic Biology Knowledge System.

The Synthetic Biology Knowledge System (SBKS) is an instance of the SynBioHub repository that includes text and data information that has been mined from papers published in ACS Synthetic Biology. This paper describes the SBKS curation framework that is being developed to construct the knowledge stored in this repository. The text mining pipeline performs automatic annotation of the articles using natural language processing techniques to identify salient content such as key terms, relationships between terms, and main topics. The data mining pipeline performs automatic annotation of the sequences extracted from the supplemental documents with the genetic parts used in them. Together these two pipelines link genetic parts to papers describing the context in which they are used. Ultimately, SBKS will reduce the time necessary for synthetic biologists to find the information necessary to complete their designs.

RNA backbone: consensus all-angle conformers and modular string nomenclature (an RNA Ontology Consortium contribution).

A consensus classification and nomenclature are defined for RNA backbone structure using all of the backbone torsion angles. By a consensus of several independent analysis methods, 46 discrete conformers are identified as suitably clustered in a quality-filtered, multidimensional dihedral angle distribution. Most of these conformers represent identifiable features or roles within RNA structures. The conformers are given two-character names that reflect the seven-angle delta epsilon zeta alpha beta gamma delta combinations empirically found favorable for the sugar-to-sugar "suite" unit within which the angle correlations are strongest (e.g., 1a for A-form, 5z for the start of S-motifs). Since the half-nucleotides are specified by a number for delta epsilon zeta and a lowercase letter for alpha beta gamma delta, this modular system can also be parsed to describe traditional nucleotide units (e.g., a1) or the dinucleotides (e.g., a1a1) that are especially useful at the level of crystallographic map fitting. This nomenclature can also be written as a string with two-character suite names between the uppercase letters of the base sequence (N1aG1gN1aR1aA1cN1a for a GNRA tetraloop), facilitating bioinformatic comparisons. Cluster means, standard deviations, coordinates, and examples are made available, as well as the Suitename software that assigns suite conformer names and conformer match quality (suiteness) from atomic coordinates. The RNA Ontology Consortium will combine this new backbone system with others that define base pairs, base-stacking, and hydrogen-bond relationships to provide a full description of RNA structural motifs.

Robotic management of large stone disease: a case series.

The gold standard for urologic management of large stone disease traditionally has been percutaneous nephrolithotomy (PCNL). An alternative to PCNL is robotic pyelolithotomy (RP), which continues to gain traction. This study is a retrospective review of ten cases performed over a 2 year period presenting operative outcomes for large stone disease treated with RP. The mean and standard deviation were calculated for age, body mass index, stone volume, stone diameter, pre-operative creatinine, operative time, robot-docked time, length of stay, post-operative creatinine, and estimated blood loss. In addition, results were collected for post-operative complications and secondary procedure requirements. Complete stone clearance was successful in 9 of 10 cases. The average renal function remained stable from a pre-operative creatinine of 0.917 mg/dL to a post-operative creatinine level of 0.943 mg/dL. This case series demonstrates that robotic assisted surgery has practical application when managing large stone disease.