Interfacial plasticity facilitates high reaction rate of E. coli FAS malonyl-CoA:ACP transacylase, FabD.

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.

Structural and dynamical rationale for fatty acid unsaturation in Escherichia coli.

Fatty acid biosynthesis in α- and γ-proteobacteria requires two functionally distinct dehydratases, FabA and FabZ. Here, mechanistic cross-linking facilitates the structural characterization of a stable hexameric complex of six Escherichia coli FabZ dehydratase subunits with six AcpP acyl carrier proteins. The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is preorganized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization.

Influence of water and enzyme SpnF on the dynamics and energetics of the ambimodal [6+4]/[4+2] cycloaddition.

SpnF is the first monofunctional Diels-Alder/[6+4]-ase that catalyzes a reaction leading to both Diels-Alder and [6+4] adducts through a single transition state. The environment-perturbed transition-state sampling method has been developed to calculate free energies, kinetic isotope effects, and quasi-classical reaction trajectories of enzyme-catalyzed reactions and the uncatalyzed reaction in water. Energetics calculated in this way reproduce the experiment and show that the normal Diels-Alder transition state is stabilized by H bonds with water molecules, while the ambimodal transition state is favored in the enzyme SpnF by both intramolecular hydrogen bonding and hydrophobic binding. Molecular dynamics simulations show that trajectories passing through the ambimodal transition state bifurcate to the [6+4] adduct and the Diels-Alder adduct with a ratio of 1:1 in the gas phase, 1:1.6 in water, and 1:11 in the enzyme. This example shows how an enzyme acts on a vibrational time scale to steer post-transition state trajectories toward the Diels-Alder adduct.

Telemedicine Versus Traditional for Follow-Up Evaluation of Enuresis.

Introduction: Telemedicine allows health care professionals to diagnose and treat patients remotely. Enuresis is one of the most common chronic problems in childhood and specialized care can be limited. Utilization of telemedicine in this setting has not been previously analyzed. Materials and Methods: This study's aim is to evaluate the feasibility and effectiveness of telemedicine follow-up treatment of enuresis compared with traditional follow-up at our institution. A retrospective review of patients treated for nocturnal enuresis with either telemedicine (Group 1) or traditional (Group2) follow-up care was conducted. Patients, aged 5-18 years, treated for enuresis between July 2016 and December 2017 were included. Patients with confounding disease were excluded. Resolution of enuresis was the primary outcome as categorized by the International Children's Continence Society standards. Results: Seventy-seven (n = 77) patients met inclusion criteria with 23 patients in Group 1 and 54 patients in Group 2. Two patients in each group were lost to follow-up and 61.9% in Group 1 and 48.1% in Group 2 responded to treatment. The average age for both groups was 9.2 years. Patients in Group 1 averaged four appointments per patient and patients in Group 2 averaged 3.04 appointments per patient. Telemedicine follow-up patients missed fewer appointments (0.14) than patients with traditional follow-up (0.5) (p-value = 0.016). Thirteen of 21 patients (61.9%) responded to treatment in Group 1 (7 partial and 6 complete responders) as compared with 25 of 52 patients (48.1%) responding to treatment in Group 2 (8 partial and 17 complete responders) (p = 0.22). Of patients in Group 1, 87% reported they would use telemedicine again. Conclusions: Telemedicine follow-up of patients with enuresis demonstrated comparable effectiveness. Most patient families demonstrate a favorable opinion of using telemedicine again for this problem. Further research to understand the efficacy and benefits of telemedicine in this setting is needed.

An Unexpected Ireland-Claisen Rearrangement Cascade During the Synthesis of the Tricyclic Core of Curcusone C: Mechanistic Elucidation by Trial-and-Error and Automatic Artificial Force-Induced Reaction (AFIR) Computations.

In the course of a total synthesis effort directed toward the natural product curcusone C, the Stoltz group discovered an unexpected thermal rearrangement of a divinylcyclopropane to the product of a formal Cope/1,3-sigmatropic shift sequence. Since the involvement of a thermally forbidden 1,3-shift seemed unlikely, theoretical studies involving two approaches, the "trial-and-error" testing of various conceivable mechanisms (Houk group) and an "automatic" approach using the Maeda-Morokuma AFIR method (Morokuma group) were applied to explore the mechanism. Eventually, both approaches converged on a cascade mechanism shown to have some partial literature precedent: Cope rearrangement/1,5-sigmatropic silyl shift/Claisen rearrangement/retro-Claisen rearrangement/1,5-sigmatropic silyl shift, comprising a quintet of five sequential thermally allowed pericyclic rearrangements.

Mechanistic Probes for the Epimerization Domain of Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases (NRPSs) are responsible for the synthesis of a variety of bioactive natural products with clinical and economic significance. Interestingly, these large multimodular enzyme machineries incorporate nonproteinogenic d-amino acids through the use of auxiliary epimerization domains, converting l-amino acids into d-amino acids that impart into the resulting natural products unique bioactivity and resistance to proteases. Due to the large and complex nature of NRPSs, several questions remain unanswered about the mechanism of the catalytic domain reactions. We have investigated the use of mechanism-based crosslinkers to probe the mechanism of an epimerization domain in gramicidin S biosynthesis. In addition, MD simulations were performed, showcasing the possible roles of catalytic residues within the epimerization domain.

2-octyl cyanoacrylate hypospadias repair dressing: a retrospective, controlled comparison.

INTRODUCTION: 2-octyl cyanoacrylate (OC) has been shown to be a viable option for usage following standard circumcision but data on its utilization following hypospadias repair is limited. Both OC and a standard waterproof transparent dressing (WD) are used following hypospadias repair at our children's hospital. Our hypothesis is that patients with distal hypospadias repair using OC for surgical dressing have similar outcomes as compared to patients with WD. MATERIALS AND METHODS: A retrospective study was performed evaluating all patients with distal hypospadias repair during a 2 year period. OC was primarily used by one of the three physicians in the practice with the other two primarily used WD for surgical dressing. The primary endpoints evaluated include hematoma requiring surgical drainage, infection, meatal stenosis, urethrocutaneous fistula, dehiscence, and diverticulum. Standard follow up after hypospadias repair includes a 1 week follow up for patients requiring urethral stent removal and reevaluation for all patients 3-4 months after surgery. REDCap was used in order to compile the database used in this study. RESULTS: A total of 280 patients underwent distal hypospadias repair during this interval. One hundred twenty-two patients had OC used with 3 (2.4%) having complications: 2 fistulas and 1 with both meatal stenosis and fistula. One hundred fifty-eight patients were dressed with WD with 5 (3.2%) complications: 4 fistulas and 1 meatal stenosis. No patients had hematoma, wound dehiscence, diverticulum, or infection. CONCLUSION: A low rate of complication was observed following distal hypospadias repair using both 2-octyl cyanoacrylate and a standard waterproof transparent dressing. 2-octyl cyanoacrylate is a safe option for surgical dressing following distal hypospadias repair but its utilization in this setting is surgeon dependent.

Modifying the Thioester Linkage Affects the Structure of the Acyl Carrier Protein.

At the center of many complex biosynthetic pathways, the acyl carrier protein (ACP) shuttles substrates to appropriate enzymatic partners to produce fatty acids and polyketides. Carrier proteins covalently tether their cargo via a thioester linkage to a phosphopantetheine cofactor. Due to the labile nature of this linkage, chemoenzymatic methods have been developed that involve replacement of the thioester with a more stable amide or ester bond. We explored the importance of the thioester bond to the structure of the carrier protein by using solution NMR spectroscopy and molecular dynamics simulations. Remarkably, the replacement of sulfur with other heteroatoms results in significant structural changes, thus suggesting more rigorous selections of isosteric substitutes is needed.

Manipulating Protein-Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins.

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein-protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities.

Mechanisms and Origins of Periselectivity of the Ambimodal [6 + 4] Cycloadditions of Tropone to Dimethylfulvene.

The mechanisms and selectivities of the cycloadditions of tropone to dimethylfulvene have been investigated with M06-2X and B3LYP-D3 density functional theory (DFT) calculations and quasi-classical direct molecular dynamics simulations. The originally proposed reaction mechanism (Houk) involves a highly peri-, regio-, and stereoselective [6F + 4T] cycloaddition of tropone [4π] to dimethylfulvene [6π], followed by a [1,5] hydrogen shift, and, finally, a second [6 + 4] cycloaddition of tropone [6π] to the cyclopentadiene moiety [4π]. Paddon-Row and Warrener proposed an alternative mechanism: the initial cycloaddition involves a different [6T + 4F] cycloaddition in which fulvene acts as the 4π component, and a subsequent Cope rearrangement produces the formal [6F + 4T] adduct. Computations now demonstrate that the initial cycloaddition proceeds via an ambimodal transition state that can lead to both of the proposed [6 + 4] adducts. These adducts can interconvert through a [3,3] sigmatropic shift (Cope rearrangement). Molecular dynamics simulations reveal the initial distribution of products and provide insights into the time-resolved mechanism of this ambimodal cycloaddition. Competing [4 + 2] cycloadditions and various sigmatropic shifts are also explored.

Effect of donor atom identity on metal-binding pharmacophore coordination.

The inhibition and binding of three metal-binding pharmacophores (MBPs), 2-hydroxycyclohepta-2,4,6-trien-1-one (tropolone), 2-mercaptopyridine-N-oxide (1,2-HOPTO), and 2-hydroxycyclohepta-2,4,6-triene-1-thione (thiotropolone) to human carbonic anhydrase II (hCAII) and a mutant protein hCAII L198G were investigated. These MBPs displayed bidentate coordination to the active site Zn(II) metal ion, but the MBPs respond to the mutation of L198G differently, as characterized by inhibition activity assays and X-ray crystallography. The L198G mutation increases the active site volume thereby decreasing the steric pressure exerted on MBPs upon binding, allowing changes in MBP coordination to be observed. When comparing the binding mode of tropolone to thiotropolone or 1,2-HOPTO (O,O versus O,S donor sets), structural modifications of the hCAII active site were shown to have a stronger effect on MBPs with an O,O versus O,S donor set. These findings were corroborated with density functional theory (DFT) calculations of model coordination complexes. These results suggest that the MBP binding geometry is a malleable interaction, particularly for certain ligands, and that the identity of the donor atoms influences the response of the ligand to changes in the protein active site environment. Understanding underlying interactions between a MBP and a metalloenzyme active site may aid in the design and development of potent metalloenzyme inhibitors.

Analysis of preoperative antibiotic prophylaxis in stented, distal hypospadias repair.

INTRODUCTION: Surgical site infection [SSI] is a risk for any surgical procedure, including hypospadias repair. Prophylactic antibiotic therapy for patients having surgery is often effective in preventing SSIs, but with increasing rates of antibiotic resistance, this practice has been questioned. The objectives of this study are 1) to assess the incidence of SSIs in patients following stented, distal hypospadias repair and 2) to observe for any potential difference in the incidence of SSIs for patients with and without preoperative antibiotic utilization in this setting. MATERIALS AND METHODS: We retrospectively reviewed consecutive patients treated with stented, distal hypospadias repair from 2011 to 2014 by three surgeons and compared two groups: patients who received preoperative antibiotics and patients who did not. Patients with a history of previous hypospadias repair were excluded from the study. RESULTS: Two hundred twenty-four subjects were identified. Group 1 (135) received preoperative antibiotic and Group 2 (89) did not receive preoperative antibiotics. There was no statistically significant difference in SSI prevalence with 0 patients in Group 1 and 1 patient in Group 2 having a SSI. CONCLUSION: Although prophylactic antibiotics prior to hypospadias repair are most often used by pediatric urologists, this study demonstrates further evidence that antibiotics prior to this procedure do not appear to lower the rate of SSI. This study is limited by its retrospective nature and disparate mean follow up in the two cohorts. Surgical site infection does not appear to be decreased by prophylactic antibiotic therapy before distal hypospadias repair.

Association of O-Antigen Serotype with the Magnitude of Initial Systemic Cytokine Responses and Persistence in the Urinary Tract.

UNLABELLED: Urinary tract infection (UTI) is one of the most common ailments requiring both short-term and prophylactic antibiotic therapies. Progression of infection from the bladder to the kidney is associated with more severe clinical symptoms (e.g., fever and vomiting) as well as with dangerous disease sequelae (e.g., renal scaring and sepsis). Host-pathogen interactions that promote bacterial ascent to the kidney are not completely understood. Prior studies indicate that the magnitude of proinflammatory cytokine elicitation in vitro by clinical isolates of uropathogenic Escherichia coli (UPEC) inversely correlates with the severity of clinical disease. Therefore, we hypothesize that the magnitude of initial proinflammatory responses during infection defines the course and severity of disease. Clinical UPEC isolates obtained from patients with a nonfebrile UTI elicited high systemic proinflammatory responses early during experimental UTI in a murine model and were attenuated in bladder and kidney persistence. Conversely, UPEC isolates obtained from patients with febrile UTI elicited low systemic proinflammatory responses early during experimental UTI and exhibited prolonged persistence in the bladder and kidney. Soluble factors in the supernatant from saturated cultures as well as the lipopolysaccharide (LPS) serotype correlated with the magnitude of proinflammatory responses in vitro. Our data suggest that the structure of the O-antigen sugar moiety of the LPS may determine the strength of cytokine induction by epithelial cells. Moreover, the course and severity of disease appear to be the consequence of the magnitude of initial cytokines produced by the bladder epithelium during infection. IMPORTANCE: The specific host-pathogen interactions that determine the extent and course of disease are not completely understood. Our studies demonstrate that modest changes in the magnitude of cytokine production observed using in vitro models of infection translate into significant ramifications for bacterial persistence and disease severity. While many studies have demonstrated that modifications of the LPS lipid A moiety modulate the extent of Toll-like receptor 4 (TLR4) activation, our studies implicate the O-antigen sugar moiety as another potential rheostat for the modulation of proinflammatory cytokine production.

Dynamically Complex [6+4] and [4+2] Cycloadditions in the Biosynthesis of Spinosyn A.

SpnF, an enzyme involved in the biosynthesis of spinosyn A, catalyzes a transannular Diels-Alder reaction. Quantum mechanical computations and dynamic simulations now show that this cycloaddition is not well described as either a concerted or stepwise process, and dynamical effects influence the identity and timing of bond formation. The transition state for the reaction is ambimodal and leads directly to both the observed Diels-Alder and an unobserved [6+4] cycloadduct. The potential energy surface bifurcates and the cycloadditions occur by dynamically stepwise modes featuring an "entropic intermediate". A rapid Cope rearrangement converts the [6+4] adduct into the observed [4+2] adduct. Control of nonstatistical dynamical effects may serve as another way by which enzymes control reactions.

P450-Mediated Coupling of Indole Fragments To Forge Communesin and Unnatural Isomers.

Dimeric indole alkaloids are structurally diverse natural products that have attracted significant attention from the synthetic and biosynthetic communities. Here, we describe the characterization of a P450 monooxygenase CnsC from Penicillium that catalyzes the heterodimeric coupling between two different indole moieties, tryptamine and aurantioclavine, to construct vicinal quaternary stereocenters and yield the heptacyclic communesin scaffold. We show, via biochemical characterization, substrate analogues, and computational methods that CnsC catalyzes the C3-C3' carbon-carbon bond formation and controls the regioselectivities of the pair of subsequent aminal bond formations to yield the communesin core. Use of ω-N-methyltryptamine and tryptophol in place of tryptamine led to the enzymatic synthesis of isocommunesin compounds, which have not been isolated to date.

A carbonate-forming Baeyer-Villiger monooxygenase.

Despite the remarkable versatility displayed by flavin-dependent monooxygenases (FMOs) in natural product biosynthesis, one notably missing activity is the oxidative generation of carbonate functional groups. We describe a multifunctional Baeyer-Villiger monooxygenase, CcsB, which catalyzes the formation of an in-line carbonate in the macrocyclic portion of cytochalasin E. This study expands the repertoire of activities of FMOs and provides a possible synthetic strategy for transformation of ketones into carbonates.

Shortened operative time for pediatric robotic versus laparoscopic dismembered pyeloplasty.

INTRODUCTION: Robotic-assisted laparoscopic pyeloplasty (RALP) is increasingly becoming the standard procedure for management of ureteropelvic junction obstruction (UPJO) in the pediatric population, but few studies have shown a clear advantage over the more technically demanding laparoscopic pyeloplasty (LP) in children. The objective was to study the patients treated with RALP or LP at our institution and the associated outcomes for each minimally invasive approach for the correction of UPJO. MATERIALS AND METHODS: Our laparoscopic and robotic database was queried to identify all patients with a history of primary robotic-assisted or laparoscopic dismembered pyeloplasty for the correction of UPJO performed at our institution from January 2010 through November 2013 and were retrospectively reviewed. We analyzed age, surgical time, blood loss, hospital stay length, postoperative complications, and success rate. RESULTS: Seventy-three total patients were identified as having RALP or LP during this time period with five patients excluded from the analysis. We identified 55 patients with RALP and 13 patients with LP. No differences in success rate or postoperative complications were found for the two cohorts. The length of procedure was significantly shorter for the RALP group compared to the LP group.

Ultrasound Guided In-Plane Penile Nerve Block for Circumcision: A New, Modified Technique Suggests Lower Anesthetic Volume and Narcotic Use.

Context: Circumcision is one of the most common surgical procedures in pediatric males. Anesthesia is often the classic dorsal penile nerve block (DPNB), which is based on landmark identification and tactile feel of tissue resistance during needle advancement. However, this technique is associated with technical failures and vascular complications. Objective: We used an ultrasound-guided in-plane technique to avoid injury of penile vascular and neural tissues during DPNB. The aims of this retrospective study were to compare the success rate and efficacy of these two penile block techniques. Methods: Male pediatric patients undergoing circumcision received general anesthesia before the penis and surrounding area were prepared with 0.5% chlorhexidine in 70% alcohol. Sixteen patients underwent classic DPNB, and 16 underwent the modified ultrasound-guided inplane technique. The ultrasound machine was adjusted to the musculoskeletal setting, and a linear ultrasound probe with a frequency range of 5 to 10 MHz was placed transversely along the base of the penis, which received gentle traction. Results: Though not statistically significant, patients who underwent the classic DPNB were approximately 1.8 times more likely to require rescue analgesia and approximately 2 times more likely to have a complication than those in the ultrasound-guided group. Results also showed lower volume requirements for local anesthetic and intraoperative narcotics, longer time until rescue analgesic, and lower incidence of vomiting in the ultrasound-guided group than in the landmark-guided group. Conclusions: The ultrasound-guided DPNB technique appears to offer advantages over classic DPNB and warrants a prospective controlled trial to confirm these findings.

Transannular [6 + 4] and Ambimodal Cycloaddition in the Biosynthesis of Heronamide A.

The transannular [6 + 4] cycloaddition proposed as a step in the biosynthesis of heronamide A has been modeled using density functional theory. The proposed cycloaddition is highly stereoselective, affording a single product. The reaction proceeds through an ambimodal transition state that directly leads to a [4 + 2] adduct in addition to the observed [6 + 4] adduct. Interconversion of these adducts is possible via a facile Cope rearrangement. The [6 + 4] adduct is thermodynamically more stable than the [4 + 2] adduct by 5.2 kcal mol(-1) due to a combination of the ring and steric strain in the [4 + 2] product. The results strongly support the plausibility of the proposed transannular [6 + 4] cycloaddition in the biogenesis of heronamide A and may provide insights to designing substrates that selectively undergo [6 + 4] cycloaddition to form unbridged 10-membered rings.

Involvement of Lipocalin-like CghA in Decalin-Forming Stereoselective Intramolecular [4+2] Cycloaddition.

Understanding enzymatic Diels-Alder (DA) reactions that can form complex natural product scaffolds is of considerable interest. Sch 210972 1, a potential anti-HIV fungal natural product, contains a decalin core that is proposed to form through a DA reaction. We identified the gene cluster responsible for the biosynthesis of 1 and heterologously reconstituted the biosynthetic pathway in Aspergillus nidulans to characterize the enzymes involved. Most notably, deletion of cghA resulted in a loss of stereoselective decalin core formation, yielding both an endo (1) and a diastereomeric exo adduct of the proposed DA reaction. Complementation with cghA restored the sole formation of 1. Density functional theory computation of the proposed DA reaction provided a plausible explanation of the observed pattern of product formation. Based on our study, we propose that lipocalin-like CghA is responsible for the stereoselective intramolecular [4+2] cycloaddition that forms the decalin core of 1.