Characterisation of Modular Polyketide Synthases Designed to Make Pentaene Analogues of Amphotericin B.

Glycosylated polyene macrolides are important antifungal agents that are produced by many actinomycete species. Development of new polyenes may deliver improved antibiotics. Here, Streptomyces nodosus was genetically re-programmed to synthesise pentaene analogues of the heptaene amphotericin B. These pentaenes are of interest as surrogate substrates for enzymes catalysing unusual, late-stage biosynthetic modifications. The previous deletion of amphotericin polyketide synthase modules 5 and 6 generated S. nodosus M57, which produces an inactive pentaene. Here, the chain-terminating thioesterase was fused to module 16 to generate strain M57-16TE, in which cycles 5, 6, 17 and 18 are eliminated from the biosynthetic pathway. Another variant of M57 was obtained by replacing modules 15, 16 and 17 with a single 15-17 hybrid module. This gave strain M57-1517, in which cycles 5, 6, 15 and 16 are deleted. M57-16TE and M57-1517 gave reduced pentaene yields. Only M57-1517 delivered its predicted full-length pentaene macrolactone in low amounts. For both mutants, the major pentaenes were intermediates released from modules 10, 11 and 12. Longer pentaene chains were unstable. The novel pentaenes were not glycosylated and were not active against Candida albicans. However, random mutagenesis and screening may yet deliver new antifungal producers from the M57-16TE and M57-1517 strains.

Biosynthesis of a new skyllamycin in Streptomyces nodosus: a cytochrome P450 forms an epoxide in the cinnamoyl chain.

Activation of a silent gene cluster in Streptomyces nodosus leads to synthesis of a cinnamoyl-containing non-ribosomal peptide (CCNP) that is related to skyllamycins. This novel CCNP was isolated and its structure was interrogated using mass spectrometry and nuclear magnetic resonance spectroscopy. The isolated compound is an oxidised skyllamycin A in which an additional oxygen atom is incorporated in the cinnamoyl side-chain in the form of an epoxide. The gene for the epoxide-forming cytochrome P450 was identified by targeted disruption. The enzyme was overproduced in Escherichia coli and a 1.43 Å high-resolution crystal structure was determined. This is the first crystal structure for a P450 that forms an epoxide in a substituted cinnamoyl chain of a lipopeptide. These results confirm the proposed functions of P450s encoded by biosynthetic gene clusters for other epoxidized CCNPs and will assist investigation of how epoxide stereochemistry is determined in these natural products.

Long-term safety and functional outcomes of delandistrogene moxeparvovec gene therapy in patients with Duchenne muscular dystrophy: A phase 1/2a nonrandomized trial.

INTRODUCTION/AIMS: Delandistrogene moxeparvovec is indicated in the United States for the treatment of ambulatory pediatric patients aged 4 through 5 years with Duchenne muscular dystrophy (DMD) with a confirmed mutation in the DMD gene. Long-term delandistrogene moxeparvovec microdystrophin protein (a shortened dystrophin that retains key functional domains of the wild-type protein) expression may positively alter disease progression in patients with DMD. We evaluated long-term safety and functional outcomes of delandistrogene moxeparvovec in patients with DMD. METHODS: An open-label, phase 1/2a, nonrandomized controlled trial (Study 101; NCT03375164) enrolled ambulatory males, ≥4 to <8 years old, with DMD. Patients received a single intravenous infusion (2.0 × 1014 vg/kg by supercoiled quantitative polymerase chain reaction) of delandistrogene moxeparvovec and prednisone (1 mg/kg/day) 1 day before to 30 days after treatment. The primary endpoint was safety. Functional outcomes were change from baseline in North Star Ambulatory Assessment (NSAA) and timed function tests. RESULTS: Four patients (mean age, 5.1 years) were enrolled. There were 18 treatment-related adverse events; all occurred within 70 days posttreatment and resolved. Mean NSAA total score increased from 20.5 to 27.5, baseline to year 4, with a mean (standard deviation) change of +7.0 (2.9). Post hoc analysis demonstrated a statistically significant and clinically meaningful 9-point difference in NSAA score, relative to a propensity-score-weighted external control cohort (least-squares mean [standard error] = 9.4 [3.4]; P = .0125). DISCUSSION: Gene transfer therapy with delandistrogene moxeparvovec treatment is well tolerated, with a favorable safety profile. Functional improvements are sustained through 4 years, suggesting delandistrogene moxeparvovec may positively alter disease progression.

Generation of ultrahigh-brightness pre-bunched beams from a plasma cathode for X-ray free-electron lasers.

The longitudinal coherence of X-ray free-electron lasers (XFELs) in the self-amplified spontaneous emission regime could be substantially improved if the high brightness electron beam could be pre-bunched on the radiated wavelength-scale. Here, we show that it is indeed possible to realize such current modulated electron beam at angstrom scale by exciting a nonlinear wake across a periodically modulated plasma-density downramp/plasma cathode. The density modulation turns on and off the injection of electrons in the wake while downramp provides a unique longitudinal mapping between the electrons' initial injection positions and their final trapped positions inside the wake. The combined use of a downramp and periodic modulation of micrometers is shown to be able to produces a train of high peak current (17 kA) electron bunches with a modulation wavelength of 10's of angstroms - orders of magnitude shorter than the plasma density modulation. The peak brightness of the nano-bunched beam can be O(1021A/m2/rad2) orders of magnitude higher than current XFEL beams. Such prebunched, high brightness electron beams hold the promise for compact and lower cost XEFLs that can produce nanometer radiation with hundreds of GW power in a 10s of centimeter long undulator.

New Glycosylated Polyene Macrolides: Refining the Ore from Genome Mining.

Glycosylated polyene macrolides include effective antifungal agents, such as pimaricin, nystatin, candicidin, and amphotericin B. For the treatment of systemic mycoses, amphotericin B has been described as a gold-standard antibiotic because of its potent activity against a broad spectrum of fungal pathogens, which do not readily become resistant. However, amphotericin B has severe toxic side effects, and the development of safer alternatives remains an important objective. One approach towards obtaining such compounds is to discover new related natural products. Advances in next-generation sequencing have delivered a wealth of microbial genome sequences containing polyene biosynthetic gene clusters. These typically encode a modular polyketide synthase that catalyzes the assembly of the aglycone core, a cytochrome P450 that oxidizes a methyl branch to a carboxyl group, and additional enzymes for synthesis and attachment of a single mycosamine sugar residue. In some cases, further P450s catalyze epoxide formation or hydroxylation within the macrolactone. Bioinformatic analyses have identified over 250 of these clusters. Some are predicted to encode potentially valuable new polyenes that have not been uncovered by traditional screening methods. Recent experimental studies have characterized polyenes with new polyketide backbones, previously unknown late oxygenations, and additional sugar residues that increase water-solubility and reduce hemolytic activity. Here we review these studies and assess how this new knowledge can help to prioritize silent polyene clusters for further investigation. This approach should improve the chances of discovering better antifungal antibiotics.

Generation of Terawatt Attosecond Pulses from Relativistic Transition Radiation.

When a femtosecond duration and hundreds of kiloampere peak current electron beam traverses the vacuum and high-density plasma interface, a new process, that we call relativistic transition radiation (RTR), generates an intense ∼100 as pulse containing ∼1 terawatt power of coherent vacuum ultraviolet (VUV) radiation accompanied by several smaller femtosecond duration satellite pulses. This pulse inherits the radial polarization of the incident beam field and has a ring intensity distribution. This RTR is emitted when the beam density is comparable to the plasma density and the spot size much larger than the plasma skin depth. Physically, it arises from the return current or backward relativistic motion of electrons starting just inside the plasma that Doppler up shifts the emitted photons. The number of RTR pulses is determined by the number of groups of plasma electrons that originate at different depths within the first plasma wake period and emit coherently before phase mixing.

Extremely Dense Gamma-Ray Pulses in Electron Beam-Multifoil Collisions.

Sources of high-energy photons have important applications in almost all areas of research. However, the photon flux and intensity of existing sources is strongly limited for photon energies above a few hundred keV. Here we show that a high-current ultrarelativistic electron beam interacting with multiple submicrometer-thick conducting foils can undergo strong self-focusing accompanied by efficient emission of gamma-ray synchrotron photons. Physically, self-focusing and high-energy photon emission originate from the beam interaction with the near-field transition radiation accompanying the beam-foil collision. This near field radiation is of amplitude comparable with the beam self-field, and can be strong enough that a single emitted photon can carry away a significant fraction of the emitting electron energy. After beam collision with multiple foils, femtosecond collimated electron and photon beams with number density exceeding that of a solid are obtained. The relative simplicity, unique properties, and high efficiency of this gamma-ray source open up new opportunities for both applied and fundamental research including laserless investigations of strong-field QED processes with a single electron beam.

Assessment of Systemic Delivery of rAAVrh74.MHCK7.micro-dystrophin in Children With Duchenne Muscular Dystrophy: A Nonrandomized Controlled Trial.

Importance: Micro-dystrophin gene transfer shows promise for treating patients with Duchenne muscular dystrophy (DMD) using recombinant adeno-associated virus serotype rh74 (rAAVrh74) and codon-optimized human micro-dystrophin driven by a skeletal and cardiac muscle-specific promoter with enhanced cardiac expression (MHCK7). Objective: To identify the 1-year safety and tolerability of intravenous rAAVrh74.MHCK7.micro-dystrophin in patients with DMD. Design, Setting, and Participants: This open-label, phase 1/2a nonrandomized controlled trial was conducted at the Nationwide Children's Hospital in Columbus, Ohio. It began on November 2, 2017, with a planned duration of follow-up of 3 years, ending in March 2021. The first 4 patients who met eligibility criteria were enrolled, consisting of ambulatory male children with DMD without preexisting AAVrh74 antibodies and a stable corticosteroid dose (≥12 weeks). Interventions: A single dose of 2.0 × 1014 vg/kg rAAVrh74.MHCK7.micro-dystrophin was infused through a peripheral limb vein. Daily prednisolone, 1 mg/kg, started 1 day before gene delivery (30-day taper after infusion). Main Outcomes and Measures: Safety was the primary outcome. Secondary outcomes included micro-dystrophin expression by Western blot and immunohistochemistry. Functional outcomes measured by North Star Ambulatory Assessment (NSAA) and serum creatine kinase were exploratory outcomes. Results: Four patients were included (mean [SD] age at enrollment, 4.8 [1.0] years). All adverse events (n = 53) were considered mild (33 [62%]) or moderate (20 [38%]), and no serious adverse events occurred. Eighteen adverse events were considered treatment related, the most common of which was vomiting (9 of 18 events [50%]). Three patients had transiently elevated γ-glutamyltransferase, which resolved with corticosteroids. At 12 weeks, immunohistochemistry of gastrocnemius muscle biopsy specimens revealed robust transgene expression in all patients, with a mean of 81.2% of muscle fibers expressing micro-dystrophin with a mean intensity of 96% at the sarcolemma. Western blot showed a mean expression of 74.3% without fat or fibrosis adjustment and 95.8% with adjustment. All patients had confirmed vector transduction and showed functional improvement of NSAA scores and reduced creatine kinase levels (posttreatment vs baseline) that were maintained for 1 year. Conclusions and Relevance: This trial showed rAAVrh74.MHCK7.micro-dystrophin to be well tolerated and have minimal adverse events; the safe delivery of micro-dystrophin transgene; the robust expression and correct localization of micro-dystrophin protein; and improvements in creatine kinase levels and NSAA scores. These findings suggest that rAAVrh74.MHCK7.micro-dystrophin can provide functional improvement that is greater than that observed under standard of care. Trial Registration: ClinicalTrials.gov Identifier: NCT03375164.

Suppression of Deflecting Forces in Planar-Symmetric Dielectric Wakefield Accelerating Structures with Elliptical Bunches.

Wakefield based accelerators capable of accelerating gradients 2 orders of magnitude higher than present accelerators offer a path to compact high energy physics instruments and light sources. However, for high gradient accelerators, beam instabilities driven by commensurately high transverse wakefields limit beam quality. Previously, it has been theoretically shown that transverse wakefields can be reduced by elliptically shaping the transverse sizes of beams in dielectric structures with planar symmetry. Here, we report experimental measurements that demonstrate reduced transverse wakefields for elliptical beams in planar symmetric structures which are consistent with theoretical models. These results may enable the design of gigavolt-per-meter gradient wakefield based accelerators that produce and stably accelerate high quality beams.

The use of rotational fluoroscopy and 3-D reconstruction in the diagnosis and surgical planning for complex cloacal malformations.

INTRODUCTION: Cloacal malformations, a confluence of the urinary tract, vagina and rectum into a single common channel, has a broad and complex anatomic spectrum requiring an imaging tool for visualization, measurement, and surgical planning for the reconstruction of these structures. We evaluated the role of 3-D fluoroscopy for this purpose, as it offers a combination of spatial correlation with precise anatomic measurements. METHODS: We examined our imaging protocol for patients with a cloacal malformation and report our experience with rotational fluoroscopy and 3-D reconstruction in 16 consecutive patients referred for cloacal reconstruction. The length of the common channel (CC), the length of the urethra from the bladder neck to the common channel, and the height (and existence or absence) of a vagina or vaginas were determinants of the surgical procedures used for the repair. RESULTS: We performed 16 consecutive 3-D cloacagrams (age range 4 months to 9 years) using a new protocol (Figure 1) that provided the following data which helped with surgical planning: Gynecologic: 3 cases with a single vagina, 5 cases with a duplicated Mullerian system (3 of which were asymmetric) and 2 cases with high vaginas requiring vaginal replacement. Colorectal: Four had a high rectum requiring an abdominal approach, and 6 had a rectum reachable via a posterior sagittal approach. Urologic: Two ectopic ureters requiring reimplantation, 3 patients had vesicoureteral reflux (1 bilateral, 2 unilateral), 1 patient had no bladder, and 7 had a normal sized bladder. Common channel length and urethral length were demonstrated in all cases and used to decide between a total urogenital mobilization or a separation of vagina(s) from the common channel, urogenital separation. CONCLUSION: The 3-D cloacagram can help predict the surgical plan for urologic, gynecologic, and colorectal components of the cloacal repair. It can predict the CC length as well as the length of the urethra. It helps with predicting the need for vaginal replacement and whether an abdominal approach is needed for the rectum. Its effectiveness is based on the ability to adequately distend structures and see their distal most extent, an advantage over other modalities such as MRI. Added benefits (particularly from the 3D view) include a better spatial understanding of the defect and the diagnosis of concomitant urological abnormalities such as vesicoureteral reflux and ectopic ureters. Disadvantages to this procedure include the need for general anesthesia and a higher exposure to radiation. LEVEL OF EVIDENCE: 3.

Gene Delivery for Limb-Girdle Muscular Dystrophy Type 2D by Isolated Limb Infusion.

In a previous limb-girdle muscular dystrophy type 2D (LGMD2D) clinical trial, robust alpha-sarcoglycan gene expression was confirmed following intramuscular gene (SGCA) transfer. This paved the way for first-in-human isolated limb infusion (ILI) gene transfer trial to the lower limbs. Delivery of scAAVrh74.tMCK.hSGCA via an intravascular route through the femoral artery predicted improved ambulation. This method was initially chosen to avoid safety concerns required for large systemic vascular delivery viral loads. ILI methods were adopted from the extensive chemotherapy experience for treatment of malignancies confined to the extremities. Six LGMD2D subjects were enrolled in a dose-ascending open-label clinical trial. Safety of the procedure was initially assessed in the single limb of a non-ambulant affected adult at a dose of 1 × 1012 vg/kg. Subsequently, ambulatory children (aged 8-13 years) were enrolled and dosed bilaterally with either 1 × 1012 vg/kg/limb or 3 × 1012 vg/kg/limb. The six-minute walk test (6MWT) served as the primary clinical outcome; secondary outcomes included muscle strength (maximum voluntary isometric force testing) and SGCA expression at 6 months. All ambulatory participants except one had pre- and post-treatment muscle biopsies. All four subjects biopsied had confirmed SGCA gene delivery by immunofluorescence, Western blot analysis (14-25% of normal), and vector genome copies (5.4 × 103-7.7 × 104 vg/µg). Muscle strength in the knee extensors (assessed by force generation in kilograms) showed improvement in two subjects that correlated with an increase in fiber diameter post gene delivery. Six-minute walk times decreased or remained the same. Vascular delivery of AAVrh74.tMCK.hSGCA was effective at producing SGCA protein at low doses that correlated with vector copies and local functional improvement restricted to targeted muscles. Future trials will focus on systemic administration to enable targeting of proximal muscles to maximize clinical benefit.

Pediatric Percutaneous Nephrostomy: A Multicenter Experience.

PURPOSE: To analyze technique, outcomes, and complications of a large series of pediatric percutaneous nephrostomy (PCN) procedures performed at 4 tertiary pediatric centers. MATERIALS AND METHODS: Retrospective multicenter study of PCNs performed during an 11-year period. Six hundred seventy-five PCNs were performed on 441 patients (median age: 4 y, range: 1 d-18 y, median weight: 17 kg, range: 0.7-112 kg); 31% were younger than 1 year. The most frequent indications for PCN procedures included hydronephrosis (57%), calculus (14%), and infection (12%). Forty-five percent of patients had severe and 32% had moderate hydronephrosis. RESULTS: Technical success was 99% (n = 668); 7 failures occurred from lost access, during tract dilatation (n = 5) and during staghorn calculi without dilatation (n = 2). General anesthesia was used in 73% of procedures. Combined ultrasound and fluoroscopy was used in 98% of procedures. Of the 668 procedures, 561 (84%) were primary nephrostomy insertions, and 107 (16%) were a variety of exchanges (secondary catheter insertions). Twenty-four of 675 (4%) were transplanted kidneys. Access sites included lower (47%), mid (28%), and upper (12%) poles and pelvis (11%). Catheters were predominantly 7-8 French (n = 352). The mean catheter dwell time was 25 days (0-220 d). Total primary catheter days were 14,482, with an additional 2,241 days after secondary procedures. Follow-up in 653/668 (98%) procedures documented elective removal (79%) and salvage procedures (21%), which included wire exchange (8.7%), nephroureteral stent/catheter conversion (8.8%), and tube upsizing (3.5%). Periprocedural complications occurred in 30/668 (4.5%) procedures: 1 major (0.1%) self-limiting hematuria requiring transfusion and 29 (4.4%) minor complications. CONCLUSIONS: PCN is safe and successful in children of all ages, with few major complications. PCN in children is associated with specific technical challenges and requires ongoing management tailored to the very young to achieve good outcomes.

Image-guided percutaneous removal of ballistic foreign bodies secondary to air gun injuries.

BACKGROUND: Ballistic injuries with retained foreign bodies from air guns is a relatively common problem, particularly in children and adolescents. If not removed in a timely fashion, the foreign bodies can result in complications, including pain and infection. Diagnostic methods to identify the presence of the foreign body run the entire gamut of radiology, particularly radiography, ultrasound (US) and computed tomography (CT). Removal of the foreign bodies can be performed by primary care, emergency, surgical, and radiologic clinicians, with or without imaging guidance. OBJECTIVE: To evaluate the modalities of radiologic detection and the experience of image-guided ballistic foreign body removal related to air gun injuries within the interventional radiology department of a large pediatric hospital. MATERIALS AND METHODS: A database of more than 1,000 foreign bodies that were removed with imaging guidance by the interventional radiologists at our institution was searched for ballistic foreign bodies from air guns. The location, dimensions, diagnostic modality, duration, complications and imaging modality used for removal were recorded. In addition, the use of sedation and anesthesia required for the procedures was also recorded. RESULTS: Sixty-one patients with ballistic foreign bodies were identified. All foreign bodies were metallic BBs or pellets. The age of the patients ranged from 5 to 20 years. The initial diagnostic modality to detect the foreign bodies was primarily radiography. The primary modality to assist in removal was US, closely followed by fluoroscopy. For the procedure, 32.7% of the patients required some level of sedation. Only two patients had an active infection at the time of the removal. The foreign bodies were primarily in the soft tissues; however, successful removal was also performed from intraosseous, intraglandular and intratendinous locations. All cases resulted in successful removal without complications. CONCLUSION: Image-guided removal of ballistic foreign bodies secondary to air guns is a very effective procedure that can obviate the need for open surgical procedures in children.

Sclerotherapy for splenic cysts in children.

BACKGROUND: Sclerotherapy has been described as a treatment option for nonparasitic pediatric splenic cysts; however, there are limited data on its long-term effectiveness. METHODS: We performed a retrospective review and prospective follow-up imaging study of children treated for nonparasitic splenic cysts at our institution during 2006-2015. Included patients had International Classification of Disease, Ninth Revision, Clinical Modification diagnosis code 289.59 or 759.0 and underwent either sclerotherapy or a partial splenic operation (partial splenectomy, cyst excision, or marsupialization). Charts were reviewed for demographics, imaging, treatments, and complications. Identified patients were contacted and asked to return for splenic ultrasonography. RESULTS: Six surgical patients and 19 sclerotherapy patients were identified. Sclerotherapy patients underwent a median of four treatments with a decrease in the size of the cyst or complete ablation in 89.5% (17/19). Of the two patients who underwent unsuccessful sclerotherapy, one patient had sclerotherapy with sotradecol and ethanol for eight treatments, developed infection, and underwent total splenectomy. The other patient did not improve with sclerotherapy and underwent partial splenectomy. Nine patients (eight patients treated with sclerotherapy and one patient treated with partial splenectomy) were successfully contacted and underwent follow-up imaging at a median follow-up of 2.4 y since last treatment. Of the eight patients treated with sclerotherapy, four patients remained cyst-free, three had small residual asymptomatic cysts stable in size, and one developed a recurrent small asymptomatic cyst; the patient treated with surgery had no recurrence. CONCLUSIONS: Sclerotherapy appears to be an effective and durable alternative treatment for children with splenic cysts.

Ion Motion Induced Emittance Growth of Matched Electron Beams in Plasma Wakefields.

Plasma-based acceleration is being considered as the basis for building a future linear collider. Nonlinear plasma wakefields have ideal properties for accelerating and focusing electron beams. Preservation of the emittance of nano-Coulomb beams with nanometer scale matched spot sizes in these wakefields remains a critical issue due to ion motion caused by their large space charge forces. We use fully resolved quasistatic particle-in-cell simulations of electron beams in hydrogen and lithium plasmas, including when the accelerated beam has different emittances in the two transverse planes. The projected emittance initially grows and rapidly saturates with a maximum emittance growth of less than 80% in hydrogen and 20% in lithium. The use of overfocused beams is found to dramatically reduce the emittance growth. The underlying physics that leads to the lower than expected emittance growth is elucidated.

Follistatin Gene Therapy for Sporadic Inclusion Body Myositis Improves Functional Outcomes.

Sporadic inclusion body myositis, a variant of inflammatory myopathy, has features distinct from polymyositis/dermatomyositis. The disease affects men more than women, most commonly after age 50. Clinical features include weakness of the quadriceps, finger flexors, ankle dorsiflexors, and dysphagia. The distribution of weakness is similar to Becker muscular dystrophy, where we previously reported improvement following intramuscular injection of an isoform of follistatin (FS344) by AAV1. For this clinical trial, rAAV1.CMV.huFS344, 6 × 1011 vg/kg, was delivered to the quadriceps muscles of both legs of six sporadic inclusion body myositis subjects. The primary outcome for this trial was distance traveled for the 6-min walk test. The protocol included an exercise regimen for each participant. Performance, annualized to a median 1-year change, improved +56.0 m/year for treated subjects compared to a decline of -25.8 m/year (p = 0.01) in untreated subjects (n = 8), matched for age, gender, and baseline measures. Four of the six treated subjects showed increases ranging from 58-153 m, whereas two were minimally improved (5-23 m). Treatment effects included decreased fibrosis and improved regeneration. These findings show promise for follistatin gene therapy for mild to moderately affected, ambulatory sporadic inclusion body myositis patients. More advanced disease with discernible muscle loss poses challenges.