Enzymatic Assembly of Small Synthetic Genes with Repetitive Elements.

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences, which results in synthesis failure or delays by DNA synthesis vendors. This represents a major obstacle for the development of synthetic biology since repetitive elements are increasingly being used in the design of genetic circuits and design of biomolecular nanostructures. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden-Gate-compatible overhangs. Then, synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate Assembly. We demonstrate the method by constructing eight challenging genes with repetitive elements, e.g., multiple repeats of RNA aptamers and RNA origami scaffolds with multiple identical aptamers. The genes range in size from 133 to 456 base pairs and are assembled with fidelities of up to 87.5%. The method was developed to facilitate our own specific research but may be of general use for constructing challenging and repetitive genes and, thus, a valuable addition to the molecular cloning toolbox.

Nature and nurture: Comparing mouse behavior in classic versus revised anxiety-like and social behavioral assays in genetically or environmentally defined groups.

Widely used rodent anxiety assays like the elevated plus maze (EPM) and the open field test (OFT) are conflated with rodents' natural preference for dark over light environments or protected over open spaces. The EPM and OFT have been used for decades but are often criticized by behavioral scientists. Years ago, two revised anxiety assays were designed to improve upon the "classic" tests by excluding the possibility to avoid or escape aversion. The 3-D radial arm maze (3DR) and the 3-D open field test (3Doft) utilize continual motivational conflict to better model anxiety; each consist of an open space connected to ambiguous paths toward uncertain escape. Despite their utility, the revised assays have not caught on. This could be because no study yet has directly compared classic and revised assays in the same animals. To remedy this, we contrasted behavior from a battery of assays (EPM, OFT, 3DR, 3Doft and a sociability test) in mice defined genetically by isogenic strain, or environmentally by postnatal experience. One major motivation for this work is to inform future studies by offering a transparent look at individual outcomes on these assays, as there is no one-size-fits-all test to assess rodent anxiety-like behavior. Findings suggest that classic assays may sufficiently characterize differences across genetically defined groups, but the revised 3DR may be advantageous for investigating more nuanced behavioral differences such as those stemming from environmental factors. Finally, exposure to multiple assays significantly affected sociability, highlighting concerns for designing and interpreting batteries of rodent behavioral tests.

Nature and nurture: comparing mouse behavior in classic versus revised anxiety-like and social behavioral assays in genetically or environmentally defined groups.

Widely used rodent anxiety assays like the elevated plus maze (EPM) and the open field test (OFT) are often conflated with rodents' natural preference for dark over light environments or protected over open spaces. The EPM and OFT have been used for many decades, yet have also been criticized by generations of behavioral scientists. Several years ago, two revised anxiety assays were designed to improve upon the "classic" tests by excluding the possibility to avoid or escape aversive areas of each maze. The 3-D radial arm maze (3DR) and the 3-D open field test (3Doft) each consist of an open space connected to ambiguous paths toward uncertain escape. This introduces continual motivational conflict, thereby increasing external validity as an anxiety model. But despite this improvement, the revised assays have not caught on. One issue may be that studies to date have not directly compared classic and revised assays in the same animals. To remedy this, we contrasted behavior in a battery of assays (EPM, OFT, 3DR, 3Doft, and a sociability test) in mice defined either genetically by isogenic strain, or environmentally by postnatal experience. Findings indicate that the optimal assay to assess anxiety-like behavior may depend upon grouping variable (e.g. genetic versus environment). We argue that the 3DR may be the most ecologically valid of the anxiety assays tested, while the OFT and 3Doft provided the least useful information. Finally, exposure to multiple assays significantly affected sociability measures, raising concerns for designing and interpreting batteries of behavioral tests in mice.

Structure, folding and flexibility of co-transcriptional RNA origami.

RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the method further, an improved understanding of RNA structural properties and folding principles is required. Here we use cryogenic electron microscopy to study RNA origami sheets and bundles at sub-nanometre resolution revealing structural parameters of kissing-loop and crossover motifs, which are used to improve designs. In RNA bundle designs, we discover a kinetic folding trap that forms during folding and is only released after 10 h. Exploration of the conformational landscape of several RNA designs reveal the flexibility of helices and structural motifs. Finally, sheets and bundles are combined to construct a multidomain satellite shape, which is characterized by individual-particle cryo-electron tomography to reveal the domain flexibility. Together, the study provides a structural basis for future improvements to the design cycle of genetically encoded RNA nanodevices.

Bilateral Testicular Burkitt's Lymphoma in a Child.

Primary non-Hodgkin's lymphoma (NHL) represents less than 5% of testicular malignancies and most often occurs in elderly men (>60 years old). We present a case of a 6-year-old boy who presented with bilateral, painless testicular swelling. Imaging studies showed bilateral heterogenous infiltrative process without focal mass. Serum testicular tumor markers were negative. Incisional testicular biopsies revealed a highly aggressive metastatic Burkitt's lymphoma with involvement of the pancreas and bone marrow. Chemotherapy was initiated with good response and showed resolution of the testicular lesions.

Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control.

Designer RNA scaffolds constitute a promising tool for synthetic biology, as they can be genetically expressed to perform specific functions in vivo such as scaffolding enzymatic cascades and regulating gene expression through CRISPR-dCas9 applications. RNA origami is a recently developed RNA design approach that allows construction of large RNA nanostructures that can position aptamer motifs to spatially organize other molecules, including proteins. However, it is still not fully understood how positioning multiple aptamers on a scaffold and the orientation of a scaffold affects functional properties. Here, we investigate fusions of single-guide RNAs and RNA origami scaffolds (termed sgRNAO) capable of recruiting activating domains for control of gene expression in yeast. Using MS2 and PP7 as orthogonal protein-binding aptamers, we observe a gradual increase in transcriptional activation for up to four aptamers. We demonstrate that different aptamer positions on a scaffold and scaffold orientation affect transcriptional activation. Finally, sgRNAOs are used to regulate expression of enzymes of the violacein biosynthesis pathway to control metabolic flux. The integration of RNA origami nanostructures at promoter sites achieved here, can in the future be expanded by the addition of functional motifs such as riboswitches, ribozymes and sensor elements to allow for complex gene regulation.

Synthetic Translational Regulation by Protein-Binding RNA Origami Scaffolds.

Rational design approaches for the regulation of gene expression are expanding the synthetic biology toolbox. However, only a few tools for regulating gene expression at the translational level have been developed. Here, we devise an approach for translational regulation using the MS2 and PP7 aptamer and coat-protein pairs in Escherichia coli. The aptamers are used as operators in transcription units that encode proteins fused to their cognate coat proteins, which leads to self-repression. RNA origami scaffolds that contain up to four aptamers serve as an alternate binder to activate translation. With this system, we demonstrate that the increase in expression of a reporter protein is dependent on both the concentration and number of aptamers on RNA origami scaffolds. We also demonstrate regulation of multiple proteins using a single MS2 coat protein fusion and apply this method to regulate the relative expression of enzymes of the branched pathway for deoxyviolacein biosynthesis.

Sinonasal Chondrosarcoma Presenting With Isolated Severe Vision Loss.

ABSTRACT: A 24-year-old man presented with a 2-month history of progressive, painless vision loss in the right eye, with no history of headache, nasal congestion, rhinorrhea, or epistaxis. His visual acuity was counting fingers at 1 ft in the right eye and 20 of 20 in the left eye with a right relative afferent pupillary defect and mild temporal optic disc pallor. MRI of the brain and orbits showed a mass involving bilateral ethmoid and sphenoid sinuses and right nasal cavity. He underwent urgent extended endoscopic endonasal transsphenoidal approach for resection of the sinonasal skull base tumor and photon radiation therapy. Pathology revealed a well-differentiated cartilaginous neoplasm with focal areas of entrapped native bone, consistent with a chondrosarcoma WHO grade I/III. At 6-month follow-up after surgery, he had a visual acuity of 20/40 in the right eye and 20/20 in the left eye. Malignant tumors from the sinonasal area should be kept in the differential diagnosis for compressive optic neuropathies and may present with vision loss even in the absence of nasal or sinus symptoms.

Apraclonidine for the pharmacologic confirmation of acute Horner syndrome.

Apraclonidine is the most widely used pharmacologic agent to confirm Horner syndrome. It is a strong α-2 and a weak α-1 adrenergic agonist and reversal of anisocoria is considered a positive test. The utility of apraclonidine in acute Horner syndrome remains controversial as the exact timing for denervation sensitivity to develop remains unknown. The goal of this study was to describe the use of apraclonidine in the diagnosis of acute Horner syndrome in patients with an unequivocal onset within 7 days. We identified 3 patients who were referred to ophthalmology/neuro-ophthalmology service and had reversal of anisocoria within 7 days. Two cases of second-order Horner syndrome after cardiac surgery and a case of a third-order Horner syndrome from a carotid cavernous sinus fistula resulted in reversal of anisocoria 72 h, 48 h, and 5 days after onset. Photographic documentation was provided for all cases. Our results suggest that apraclonidine has utility in the acute period and positive results can be seen as early as 48 h after onset. Apraclonidine should therefore still be considered to confirm the presence of acute Horner syndrome before extensive neuroimaging is performed.

EyePrintPRO therapeutic scleral contact lens: indications and outcomes.

OBJECTIVE: To describe indications and outcomes of patients fitted with the EyePrintPRO therapeutic scleral lens. METHODS: A database search of patients fitted with the EyePrintPRO from 2014 to 2016. Fourteen eyes of 10 patients were reviewed retrospectively. Patient demographics, medical and ocular history, indications for fitting, duration of wear, symptoms, and best-corrected visual acuity (BCVA) were analyzed. RESULTS: Mean age at lens fitting was 49 years (range, 21-67 years). The average duration of wear was 12 months (range, 7-17 months). Indications for fitting included limbal stem cell deficiency, post-photorefractive keratectomy (PRK) decentred ablation, pellucid marginal degeneration, Stevens-Johnson syndrome, keratoconus, dry eye, neurotrophic keratitis, exposure keratitis from facial nerve paralysis, and post-radial keratotomy (RK) symptoms. Mean BCVA was 20/36 (range, 20/20-20/200). After the fitting, mean BCVA was 20/21 (range 20/10-20/60, p = 0.001). Nine patients reported resolution of their blurry vision, and all reported improvement of dry eye, eye redness, and pain symptoms. Six of 7 previous lens wearers reported significantly greater comfort with EyePrintPRO wear and the ability to wear the lens throughout the day; only 2 experienced fogging and needed to clean the lens after 4-6 hours of wear. CONCLUSIONS: A variety of indications for the EyePrintPRO scleral lens exist, and patients experience resolution of major symptoms. The ophthalmologist should be aware that therapeutic scleral lenses, including the EyePrintPRO, exist for patients for whom there is no surgical intervention or who want to delay or obviate the need for surgery.

Kinetics of Trifluoromethane Clathrate Hydrate Formation from CHF3 Gas and Ice Particles.

We report the formation kinetics of trifluoromethane clathrate hydrate (CH) from less than 75 µm diameter ice particles and CHF3 gas. As previously observed for difluoromethane and propane hydrate formation, the initial stages of the reaction exhibit a strong negative correlation of the reaction rate with temperature, consistent with a negative activation energy of formation. The values obtained for trifluoromethane, ca. -6 kJ/mol (H2O), are similar to those for difluoromethane, even though the two molecules have different intermolecular interactions and sizes. The activation energy is lesser per mole of H2O, but greater per mole of guest molecule, than for propane hydrate, which has a different crystal structure. We propose a possible explanation for the negative activation barrier based on the stabilization of metastable structures at low temperature. A pronounced dependence of the formation kinetics on the gas flow rate into the cell is observed. At 253 K and a flow rate of 15 mmol/h, the stage II enclathration of trifluoromethane proceeds so quickly that the overpressure, the difference between the gas cell pressure and the hydrate vapor pressure, is only 0.06 MPa.

Gene expression alterations in cryptorchid males using spermatozoal microarray analysis.

OBJECTIVE: To identify differential spermatozoal gene expression patterns using microarray between controls and patients with cryptorchidism. DESIGN: Prospective laboratory analysis. SETTING: Pediatric urology clinic and laboratory of an academic hospital in the United States. PATIENT(S): Ten control patients and 12 cryptorchid males (8 unilateral, 4 bilateral). INTERVENTION(S): Ejaculates were collected and motile sperm were isolated by Percoll centrifugation, total RNA was extracted and verified using sperm-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Biotin-labeled amplified RNA was hybridized to Affymetrix Human Genome Focus arrays. Differentially expressed genes were identified using permutation t-test. MAIN OUTCOME MEASURE(S): Semen analyses and differential spermatozoal gene expression patterns measured by microarray. RESULT(S): Mean semen volume was not different between control and patients with cryptorchidism. Mean sperm density was significantly decreased between control, unilateral, and bilateral cryptorchid samples (110 vs. 87 vs. 16 million/mL). From the microarray expression data, we identified 43 genes differentially expressed between the two groups. Thirty-eight genes were significantly underexpressed in the cryptorchid samples including many transcriptional factors (cul3, prm1, hspcd35) and a testis-specific cell-adhesion gene (tpx-1) involved in germ cell maturation and sperm tail formation. An antiapoptotic gene (TNFAIP3) was highly overexpressed in the cryptorchid samples. CONCLUSION(S): Gene expression profiles offer insight into the diverse alterations that occur in cryptorchidism. The observed changes in spermatozoal expression of transcriptional and antiapoptotic genes may result in poor seminal parameters in formerly cryptorchid males.

Usefulness of short-term retrievable ureteral stent in pediatric laparoscopic pyeloplasty.

PURPOSE: Methods of stenting after laparoscopic pyeloplasty have included indwelling Double-J stents and percutaneous nephrostomy tubes. The disadvantages of these methods are that they necessitate a second surgery for stent removal or require an external drainage bag. To circumvent these issues, the tolerance, safety and outcomes of using a Double-J ureteral stent with a dangler, permitting early office removal, was investigated in a series of pediatric laparoscopic pyeloplasties. MATERIALS AND METHODS: Medical records from a consecutive series of pediatric patients undergoing transperitoneal laparoscopic pyeloplasties were reviewed. Indications for surgery included ipsilateral flank pain with severe hydronephrosis (12 patients), recurrent pyelonephritis with severe hydronephrosis (2), and hematuria and flank pain (6). All patients were discharged home within 24 to 48 hours of the procedure with prophylactic oral antibiotics. The stent was removed by postoperative day 18 during a followup office visit. Patient tolerance of the indwelling stent, outpatient removal and success of pyeloplasty were assessed. RESULTS: A total of 20 patients underwent transperitoneal laparoscopic pyeloplasty by 1 surgeon (LAB) between 2001 and 2005. All patients underwent cystoscopy and retrograde Double-J ureteral stent placement before pyeloplasty under the same anesthesia. Mean patient age at operation was 11.3 years (median 11.3, range 4.6 to 17.2). Stents were left indwelling for a mean of 10.3 days (median 10, range 7 to 18). All patients tolerated the Double-J stent well, with 2 requiring anticholinergic therapy for mild urgency symptoms and 1 demonstrating urinary tract infection. All patients tolerated outpatient stent removal via the dangler at the office without discomfort. One patient was lost to followup. At a mean followup of 1.04 years (range 0.1 to 2.88) 17 of 19 patients (89%) had resolution of flank pain/urinary tract infections, with sonographic improvement in hydronephrosis with or without endoscopic intervention. Six patients (30%) had flank pain with or without continuous hydronephrosis and required re-stenting, and 3 also required balloon dilation. Of these 6 patients 2 (10%) had recurrent ureteropelvic junction obstruction and required open pyeloplasty. All patients are now clinically and radiologically unobstructed and asymptomatic. CONCLUSIONS: Pediatric transperitoneal laparoscopic pyeloplasty with indwelling Double-J ureteral stent with a dangler is successful and the stent is well tolerated. Whether the duration of ureteral stenting affects the surgical success will require further controlled long-term studies.

Overnight catheter drainage in children with poorly compliant bladders improves post-obstructive diuresis and urinary incontinence.

PURPOSE: Overnight catheter drainage (OCD) has been suggested as a treatment for boys with valve bladder syndrome as well as those with polyuric renal failure. We report our experience using overnight catheter drainage in children with poorly compliant bladders. MATERIALS AND METHODS: Between 1999 and 2004 OCD was initiated in 6 boys and 5 girls (median age 7.5 years) with poorly compliant bladders in whom aggressive daytime intermittent catheterization and anticholinergic use had failed. Etiologies of bladder dysfunction included posterior urethral valves in 1 case, The Hinman-Allen syndrome in 4, myelomeningocele in 2, neuroblastoma in 1, prune-belly syndrome in 1, transverse myelitis in 1 and sacral agenesis in 1. The amount of post-obstructive diuresis was calculated during urodynamics as the volume drained minus volume instilled divided by weight per hour. Other parameters evaluated included serum creatinine, degree of upper tract dilatation, and episodes of urinary tract infection (UTI) and incontinence. RESULTS: Median duration of OCD and followup was 28 months, respectively. One patient did not tolerate overnight drainage. Of 9 patients who were incontinent before OCD daytime urinary incontinence resolved in 6 and improved in 3. Recurrent febrile UTIs in 3 children were eliminated. Despite no change in serum creatinine with overnight drainage, 7 of 8 patients (88%) with upper tract dilatation improved on followup ultrasound. CONCLUSIONS: In select children with poorly compliant bladders OCD reduces diuresis, reduces the frequency of UTIs, improves upper tract dilatation and improves continence. Overnight drainage in conjunction with daytime clean intermittent catheterization may prove to be an alternative to bladder augmentation in select children with poorly compliant bladders and early stages of renal compromise.

Tubularized incised plate hypospadias reoperation.

PURPOSE: We report our updated experience with tubularized incised plate hypospadias reoperations in a series of patients of whom the majority had undergone prior urethral plate incision. MATERIALS AND METHODS: Records of 31 consecutive patients undergoing tubularized incised plate reoperation were reviewed. The decision for this repair was based on a preserved urethral plate that appeared supple despite prior surgery. RESULTS: Of the 31 patients the mean number of prior operations was 1.1, including 18 (58%) who had undergone primary repairs that involved midline plate incision. Overall, 28 (90%) patients had a successful outcome with a functional neourethra and vertical slit meatus. Complications occurred in 7 (23%) patients, consisting mostly of fistulas. Among 27 cases in which dartos was used as a barrier layer fistulas occurred in 1 (6%) of 18 when a ventral flap was placed over the neourethra versus 3 (33%) of 9 when adjacent tissues alongside the neourethra were approximated in the midline (p = 0.055). The rate of complications was not affected by history of urethral plate incision. In 3 patients partial or complete glans dehiscence or a large fistula occurred, and 2 subsequently required staged buccal graft urethroplasty. CONCLUSIONS: Tubularized incised plate reoperation results in a functional neourethra with a vertical slit meatus when the plate has been preserved and appears supple after prior surgery. Fistulas are less likely when a flap is interposed between the neourethra and skin. Complications are low despite previous urethral plate incision if there is no apparent scarring of the plate. An alternative technique for reoperative urethroplasty should be considered if the urethral plate has been excised or is grossly scarred.

Effect of urethral plate characteristics on tubularized incised plate urethroplasty.

PURPOSE: We determined outcomes of tubularized incised plate urethroplasty based on preoperative urethral plate configuration or width. MATERIALS AND METHODS: Records of consecutive prepubertal boys undergoing tubularized incised plate distal hypospadias repair were reviewed. The urethral plate was characterized as flat, cleft or deep, and results in each group were noted. In addition, the width of the plate after separation from the glans wings before midline incision was measured in some patients, with outcomes determined according to those less than 8 mm versus 8 mm or greater. RESULTS: Of 159 patients plate configuration was recorded in 143, widths in 48 and both in 46. Outcomes were determined at a mean of 8 months postoperatively. Overall, there were no cases of meatal stenosis and fistulas occurred in 3 patients (2%). No significant difference in results was predicted by plate configuration or width. CONCLUSIONS: Tubularized incised plate urethroplasty for distal hypospadias repair has a low complication rate regardless of urethral plate configuration or width. Therefore, this procedure is potentially applicable in all cases of primary distal hypospadias.

Dihydrotestesterone induction of EPHB2 expression in the female genital tubercle mimics male pattern of expression during embryogenesis.

PURPOSE: The Eph family constitutes the largest class of membrane bound tyrosine kinase receptors in higher vertebrates, being involved in complex cell-to-cell recognition events via their membrane anchored ligands, the ephrins. To uncover evidence for possible molecular involvement in genitourinary development, we evaluated the pattern of EphB2 expression in male and female genital tubercles (GTs) after culture with and without dihydrotestosterone (DHT). MATERIALS AND METHODS: GTs of embryos from time pregnant CD1 mice heterozygous for the EphB2lacZ mutation were explanted at 12.5 to 14.5 days after conception. In these mutant mice the exons encoding the EphB2 tyrosine kinase and C-terminal domains were replaced with bacterial lacZ sequence encoding beta-galactosidase to demonstrate EphB2 expression. The GTs were microdissected, cultured for 24 to 48 hours with or without addition of 10 nM DHT and then stained with X-gal to visualize the beta-galactosidase reporter of EphB2 expression. RESULTS: LacZ staining was detected along the borders of the urethral plate (UP) in males. Females expressed EphB2 at the GT base but DHT exposure induced EphB2 expression along the UP. Under culture conditions, progressive approximation of the UP borders was noted in male fetuses and androgen exposed females. UP approximation was not seen in the nonexposed female tubercles. CONCLUSIONS: EphB2 expression appears to be temporally and spatially regulated in the developing mouse GT and differs between sexes. Male pattern of expression can be induced in the female GT by DHT exposure. Given DHT exposure alters EphB2 expression, EphB2 is a candidate androgen regulated gene. These findings may give insight into UP closure in normal and pathological conditions.