Outcomes following Exchange and Upsizing of Malfunctioning Small-Caliber Double-J Ureteral Stents.

PURPOSE: To determine the effectiveness of exchange and upsizing of malfunctioning small-caliber double-J (JJ) ureteral stents. MATERIALS AND METHODS: Thirty-one patients with malfunctioning cystoscopically placed small-caliber (6 or 7 F) JJ stents underwent transurethral (n = 28) or transrenal (n = 3) exchange and upsizing to a large-caliber (10 F) JJ stent from 2013 to 2022. Ureteral obstruction was malignant in 20 patients (65%) and benign in 11 (35%). Fifteen patients (48%) presented with persistent hydroureteronephrosis and 16 patients (52%) with worsening hydronephrosis. Acute kidney injury (AKI) was present in 19 patients (61%) at the time of stent malfunction. Therapeutic success was defined as resolution of hydronephrosis and AKI, if present. RESULTS: JJ stent exchange and upsizing was technically successful in 31 patients (100%) with no immediate adverse events. Therapeutic success was achieved in 27 patients (87%). During follow-up (median, 97 days; IQR, 32-205 days), 2 patients who initially achieved therapeutic success had stent malfunction, requiring conversion to percutaneous nephrostomy drainage (2/27, 7%). CONCLUSIONS: Exchange and upsizing to large-caliber JJ stents can relieve urinary obstruction and resolve AKI in patients with malfunctioning small-caliber JJ stents. Large-caliber JJ stents should be considered as a salvage option for patients who wish to continue internal drainage and avoid percutaneous nephrostomy.

CRISPR-Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters.

Here we report an efficient CRISPR-Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities.

Characterization of the flavin monooxygenase involved in biosynthesis of the antimalarial FR-900098.

The latter steps in this biosynthetic pathway for the antimalarial phosphonic acid FR-900098 include the installation of a hydroxamate onto 3-aminopropylphosphonate, which is catalyzed by the consecutive actions of an acetyltransferase and an amine hydroxylase. Here, we present the 1.6 Å resolution co-crystal structure and accompanying biochemical characterization of FrbG, which catalyzes the hydroxylation of aminopropylphosphonate. We show that FrbG is a flavin-dependent N-hydroxylating monooxygenase (NMO), which shares a similar overall structure with flavin-containing monooxygenases (FMOs). Notably, we also show that the cytidine-5'-monophosphate moiety of the substrate is a critical determinant of specificity, distinguishing FrbG from other FMOs in that the nucleotide cofactor-binding domain also serves in conferring substrate recognition. In the FrbG-FAD+-NADPH co-crystal structure, the C4 of the NADPH nicotinamide is situated near the N5 of the FAD isoalloxazine, and is oriented with a distance and stereochemistry to facilitate hydride transfer.

CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973.

BACKGROUND: As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions. RESULTS: First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructs containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain. CONCLUSIONS: High expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.

Directed evolution as a powerful synthetic biology tool.

At the heart of synthetic biology lies the goal of rationally engineering a complete biological system to achieve a specific objective, such as bioremediation and synthesis of a valuable drug, chemical, or biofuel molecule. However, the inherent complexity of natural biological systems has heretofore precluded generalized application of this approach. Directed evolution, a process which mimics Darwinian selection on a laboratory scale, has allowed significant strides to be made in the field of synthetic biology by allowing rapid identification of desired properties from large libraries of variants. Improvement in biocatalyst activity and stability, engineering of biosynthetic pathways, tuning of functional regulatory systems and logic circuits, and development of desired complex phenotypes in industrial host organisms have all been achieved by way of directed evolution. Here, we review recent contributions of directed evolution to synthetic biology at the protein, pathway, network, and whole cell levels.

DNA assembly techniques for next-generation combinatorial biosynthesis of natural products.

Natural product scaffolds remain important leads for pharmaceutical development. However, transforming a natural product into a drug entity often requires derivatization to enhance the compound's therapeutic properties. A powerful method by which to perform this derivatization is combinatorial biosynthesis, the manipulation of the genes in the corresponding pathway to divert synthesis towards novel derivatives. While these manipulations have traditionally been carried out via restriction digestion/ligation-based cloning, the shortcomings of such techniques limit their throughput and thus the scope of corresponding combinatorial biosynthesis experiments. In the burgeoning field of synthetic biology, the demand for facile DNA assembly techniques has promoted the development of a host of novel DNA assembly strategies. Here we describe the advantages of these recently developed tools for rapid, efficient synthesis of large DNA constructs. We also discuss their potential to facilitate the simultaneous assembly of complete libraries of natural product biosynthetic pathways, ushering in the next generation of combinatorial biosynthesis.

Radiofrequency guidewire-facilitated recanalization of chronic thoracic central venous occlusions in hemodialysis patients.

PURPOSE: To assess the outcome and safety of radiofrequency (RF) wire recanalization in patients with end-stage renal disease (ESRD) and chronic central venous occlusions (CVO). MATERIALS AND METHODS: A retrospective review of ESRD patients who underwent RF-wire recanalization of symptomatic chronic thoracic CVO from January 2017 to August 2022 yielded 20 patients who underwent 21 procedures. All patients had undergone at least one prior unsuccessful attempt at central venous recanalization using conventional catheter-based techniques. Technical success was defined by the ability to cross the CVO using RF-wire recanalization enabling endovascular treatment. Access circuit patency was evaluated based on follow-up imaging and symptomatic improvement. RESULTS: Radiofrequency wire recanalization was successful in 17/21 procedures (81%) with all patients (100%) reporting resolution of arm ± facial swelling. Three major complications occurred (14%): two hemothoraces and one hemopericardium. Medial stent diameter was 13 mm (range, 9-14 mm). Mean duration of hospital stay was 2 days ± 3 days. Mean procedure time was 158 ± 46 min with a mean fluoroscopy time of 31.7 ± 16.3 min. Primary unassisted patency at 6 and 12 months was 94 ± 6% and 85 ± 10%, respectively. Additional interventions resulted in significantly increased stent graft patency (P = 0.006). CONCLUSION: Radiofrequency wire-enabled recanalization of CVO in symptomatic dialysis patients has a high rate of technical success with resolution of arm and facial swelling and resumed use of the ipsilateral dialysis access. Although a superior safety profile was seen than with needle-based techniques such as sharp recanalization, major complications were not infrequent indicating that this RF-wire procedure should be performed in centers equipped to manage central venous perforations.

Left Inferior Vena Cava With Infrahepatic Disruption, Azygos Continuation, and Retroaortic Left Renal Vein - A Complex, Symptomatic Caval Anomaly Managed With Endovascular Intervention.

Background: Complex congenital anomalies of the inferior vena cava (IVC) are rare sequelae of inappropriate persistence or regression of embryological precursor veins. These anomalies are typically asymptomatic and generally do not warrant intervention. Case Presentation: Here we present a case of severely symptomatic left IVC with infrahepatic disruption, azygos continuation, and retroaortic left renal vein causing symptoms of severe pelvic congestion and recurrent miscarriages (8 total) in a 41 year old female. The patient was treated with stenting of the compressed retroaortic portion of the IVC/left renal vein. Four months post-procedure, the stent remained patent and the patient reported considerable improvement in their venous congestion symptoms. Most notably, as of the writing of this report, the patient is 38 weeks pregnant. Conclusions: The case is notable for its severe symptomatology of pelvic venous disease including recurrent miscarriage. More importantly, it represents the first documented case of successful retroaortic endovascular management of such a venous anomaly, in which the entirety of the typical IVC drainage occurred via a compressed left-to-right retroaortic crossover.

New N-acetyltransferase fold in the structure and mechanism of the phosphonate biosynthetic enzyme FrbF.

The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 Å resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.

Gata4 and Gata5 cooperatively regulate cardiac myocyte proliferation in mice.

GATA5 is a member of the zinc finger transcription factor GATA family (GATA1-6) that plays a wide variety of roles in embryonic and adult development. Experiments in multiple model systems have emphasized the importance of the GATA family members 4-6 in the development of the endoderm and mesoderm. Yet despite overlapping expression patterns, there is little evidence of an important role for GATA5 in mammalian cardiac development. We have generated a new Gata5 mutant allele lacking exons 2 and 3 that encodes both zinc finger domains (Gata5(tm)(2)(Eem)), and we show that although Gata5(-/-) mice are viable, Gata4(+/-)5(-/-) mutants die at mid-gestation and exhibit profound cardiovascular defects, including abnormalities of cardiomyocyte proliferation and cardiac chamber maturation. These results demonstrate functional redundancy between Gata4 and Gata5 during cardiac development and implicate Gata5 as a candidate modifier gene for congenital heart disease.

Untreated Superior Vena Cava Aneurysm: Radiological Significance and Review of the Literature.

Superior vena cava (SVC) aneurysms are a rare entity. The majority of the literature is in the form of case reports. SVC aneurysms are often an incidental finding with iatrogenic, congenital, or idiopathic etiologies. Treatment goals focus on preventing theoretical rupture or thrombus formation. Management options include observation, conservative medical management, surgical excision, and thrombin injection. We present a 73-year-old female with an incidental SVC aneurysm discovered on computed tomography (CT) of the thorax. The patient was observed without intervention for greater than 6 years. No complications were attributable to the SVC aneurysm during follow-up or over the course of the patient's life.

Accelerated genome engineering through multiplexing.

Throughout the biological sciences, the past 15 years have seen a push toward the analysis and engineering of biological systems at the organism level. Given the complexity of even the simplest organisms, though, to elicit a phenotype of interest often requires genotypic manipulation of several loci. By traditional means, sequential editing of genomic targets requires a significant investment of time and labor, as the desired editing event typically occurs at a very low frequency against an overwhelming unedited background. In recent years, the development of a suite of new techniques has greatly increased editing efficiency, opening up the possibility for multiple editing events to occur in parallel. Termed as multiplexed genome engineering, this approach to genome editing has greatly expanded the scope of possible genome manipulations in diverse hosts, ranging from bacteria to human cells. The enabling technologies for multiplexed genome engineering include oligonucleotide-based and nuclease-based methodologies, and their application has led to the great breadth of successful examples described in this review. While many technical challenges remain, there also exists a multiplicity of opportunities in this rapidly expanding field.

High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system.

Actinobacteria, particularly those of genus Streptomyces, remain invaluable hosts for the discovery and engineering of natural products and their cognate biosynthetic pathways. However, genetic manipulation of these bacteria is often labor and time intensive. Here, we present an engineered CRISPR/Cas system for rapid multiplex genome editing of Streptomyces strains, demonstrating targeted chromosomal deletions in three different Streptomyces species and of various sizes (ranging from 20 bp to 30 kb) with efficiency ranging from 70 to 100%. The designed pCRISPomyces plasmids are amenable to assembly of spacers and editing templates via Golden Gate assembly and isothermal assembly (or traditional digestion/ligation), respectively, allowing rapid plasmid construction to target any genomic locus of interest. As such, the pCRISPomyces system represents a powerful new tool for genome editing in Streptomyces.

Structure-guided design and biosynthesis of a novel FR-900098 analogue as a potent Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) inhibitor.

We report here the enzymatic biosynthesis of FR-900098 analogues and establish an in vivo platform for the biosynthesis of an N-propionyl derivative FR-900098P. FR-900098P is found to be a significantly more potent inhibitor of Plasmodium falciparum 1-deoxy-D-xylulose 5-phosphate reductoisomerase (PfDxr) than the parent compound, and thus a more promising antimalarial drug candidate.

Recent advances in natural product discovery.

Natural products have been and continue to be the source and inspiration for a substantial fraction of human therapeutics. Although the pharmaceutical industry has largely turned its back on natural product discovery efforts, such efforts continue to flourish in academia with promising results. Natural products have traditionally been identified from a top-down perspective, but more recently genomics- and bioinformatics-guided bottom-up approaches have provided powerful alternative strategies. Here we review recent advances in natural product discovery from both angles, including diverse sampling and innovative culturing and screening approaches, as well as genomics-driven discovery and genetic manipulation techniques for both native and heterologous expression.

Directed Evolution: Past, Present and Future.

Directed evolution, the laboratory process by which biological entities with desired traits are created through iterative rounds of genetic diversification and library screening or selection, has become one of the most useful and widespread tools in basic and applied biology. From its roots in classical strain engineering and adaptive evolution, modern directed evolution came of age twenty years ago with the demonstration of repeated rounds of PCR-driven random mutagenesis and activity screening to improve protein properties. Since then, numerous techniques have been developed that have enabled the evolution of virtually any protein, pathway, network or entire organism of interest. Here we recount some of the major milestones in the history of directed evolution, highlight the most promising recent developments in the field, and discuss the future challenges and opportunities that lie ahead.

Activation and characterization of a cryptic polycyclic tetramate macrolactam biosynthetic gene cluster.

Polycyclic tetramate macrolactams (PTMs) are a widely distributed class of natural products with important biological activities. However, many of these PTMs have not been characterized. Here we apply a plug-and-play synthetic biology strategy to activate a cryptic PTM biosynthetic gene cluster SGR810-815 from Streptomyces griseus and discover three new PTMs. This gene cluster is highly conserved in phylogenetically diverse bacterial strains and contains an unusual hybrid polyketide synthase-nonribosomal peptide synthetase, which resembles iterative polyketide synthases known in fungi. To further characterize this gene cluster, we use the same synthetic biology approach to create a series of gene deletion constructs and elucidate the biosynthetic steps for the formation of the polycyclic system. The strategy we employ bypasses the traditional laborious processes to elicit gene cluster expression and should be generally applicable to many other silent or cryptic gene clusters for discovery and characterization of new natural products.

Directed evolution: an evolving and enabling synthetic biology tool.

Synthetic biology, with its goal of designing biological entities for wide-ranging purposes, remains a field of intensive research interest. However, the vast complexity of biological systems has heretofore rendered rational design prohibitively difficult. As a result, directed evolution remains a valuable tool for synthetic biology, enabling the identification of desired functionalities from large libraries of variants. This review highlights the most recent advances in the use of directed evolution in synthetic biology, focusing on new techniques and applications at the pathway and genome scale.