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.

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.

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.

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.

Robotic-assisted nephrectomy with level II IVC thrombectomy using Rummel Tourniquets

ABSTRACT Background: Inferior vena cava (IVC) invasion from renal cell carcinoma (RCC) occurs at a rate of 4-10% (1). IVC thrombectomy (IVC-TE) can be an open procedure because of the need for handling of the IVC (2). The first reported series of robotic management of IVC-TE started in 2011 for the management of Level I - II thrombi with subsequent case reports in recent years (2-5). Materials and Methods: The following is a patient in his 50's with no significant medical history. Magnetic resonance imaging and IR venogram were performed preoperatively. The tumor was clinical stage T3b with a 4.3cm inferior vena cava thrombus. The patient underwent robotic assisted nephrectomy and IVC-TE. Rummel tourniquets were used for the contralateral kidney and the IVC. The tourniquets were created using vessel loops, a 24 French foley catheter and hem-o-lock clips. Results: The patient tolerated the surgical procedure well with no intraoperative complications. Total surgical time was 274 min with 200 minutes of console time and 22 minutes of IVC occlusion. Total blood loss in the surgery was 850cc. The patient was discharged from the hospital on post-operative day 3 without any complications. The final pathology of the specimen was pT3b clear cell renal cell carcinoma Fuhrman grade 2. The patient followed up post-operatively at both four months and six months without disease recurrence. The patient continues annual follow-up with no recurrence. Conclusions: Surgeon experience is a key factor in radical nephrectomy with thrombectomy as patients have a reported 50-65% survival rate after IVC-TE (4).

How we do it: robotic-assisted distal ureterectomy with ureteral reimplantation

ABSTRACT Background: High risk upper tract urothelial carcinoma (UTUC) is typically managed with radical nephroureterectomy, however, renal preservation can be attempted when UTUC is localized to the distal ureter in the presence of chronic kidney disease (1-3). Distal ureterectomy is typically managed with a ureteral reimplantation and psoas hitch in order to maintain urothelial continuity, to avoid comprising the contralateral ureter, and reducing risk of chronic urinary tract infections and electrolyte abnormalities (4). We present our case of distal ureteral UTUC managed robotically with a distal ureterectomy with ureteral reimplantation. Technique and Follow-Up: Initially, an Orandi needle on a resectoscope circumscribed the left ureteral orifice. Next, robotically, the retroperitoneum was exposed and a left sided pelvic lymphadenectomy was completed. The left ureter was mobilized and the diseased ureteral segment was transected. The mobilized bladder was sutured to psoas fascia. After a cystotomy, the ureter was re-anastomosed to the bladder. The patient was discharged on postoperative day three and re-evaluated one week later with a cystogram. Final pathology was downgraded to non-invasive low-grade papillary urothelial carcinoma with negative lymph nodes and margins. Conclusion: High risk UTUC localized to the distal ureter in the setting of chronic kidney disease can be managed with a distal ureterectomy (3). Robotic distal ureterectomy with ureteral reimplantation can be assisted by an Orandi needle to achieve negative margins. Utilizing a robotic technique can offer challenges with the ureteral spatulation and reanastomosis (5-7). By fixating the ureter to the bladder prior to reanastomosis, our technique offers a solution for these difficulties.

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.

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.

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.

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.

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.

Intra-Testicular Papaverine Injection for Testicular Salvage after Blunt Testicular Trauma: A Case Report.

Blunt trauma is the most common mechanism of injury to the scrotum and testicle. Surgical exploration with primary repair, hematoma evacuation, and de-torsion are common surgical interventions. A 20-year-old male with no previous medical history presented after a high-speed motor vehicle collision. Ultrasonography demonstrated heterogeneous changes of the tunica albuginea and decreased arterial flow to bilateral testicles. He was subsequently taken to the operating room for surgical exploration, which revealed bilateral mottled testes with questionable viability. Papaverine was injected into each testicle, which resulted in visibly increased perfusion and subsequent preservation of the testicles. Conclusion: Current evidence on the use of papaverine is isolated to testicular torsion. Additional research should be conducted on the use of papaverine in blunt testicular trauma. Papaverine injection may be a valuable treatment option when inadequate perfusion is observed intra-operatively.

Identification of a GUAAY Pentaloop Sequence Involved in a Novel RNA Loop-Helix Interaction.

Large RNAs often utilize GNRA tetraloops as structural elements to stabilize the overall tertiary fold. These tetraloop-receptor (TR) interactions have a conserved geometry in which the tetraloop docks into the receptor at an angle of ~15° from the helix containing the receptor. Here, we show that the conserved GUAAY pentaloop found in domain III of group IIB1 introns participates in a novel class of RNA tertiary interaction with a geometry and mode of binding that are significantly different from that found in GNRA TR interactions. This pentaloop is highly conserved within the IIB1 class and interacts with the minor groove of the catalytic domain V. The base planes of the loop and receptor nucleotides are not coplanar and greatly deviate from standard A-minor motifs. The helical axis of the GUAAY stem loop diverges ~70° from the angle of insertion found in a typical GNRA TR interaction. Therefore, the loop architecture and insertion orientation are distinctive, with in vitro splicing data indicating that a GNRA tetraloop is incompatible at this position. The GUAAY pentaloop-receptor motif is also found in the structure of the eukaryotic thiamine pyrophosphate riboswitch in the context of a hexanucleotide loop sequence. We therefore propose, based on phylogenetic, structural, and biochemical data, that the GUAAY pentaloop-receptor interaction represents a novel structural motif that is present in multiple structured RNAs.

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.

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.

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 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.

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.