A contemporary single-institution retrospective cohort study comparing perioperative outcomes between robotic and open partial nephrectomy for poorly functioning renal moieties in children with duplex collecting systems.

INTRODUCTION: Studies evaluating robotic-assisted laparoscopic partial nephrectomy (RALPN) are limited to case series, amounting to a lack of studies directly comparing perioperative outcomes for RALPN to open partial nephrectomy (OPN). OBJECTIVE: To compare perioperative outcomes between RALPN and OPN. METHODS: A single-institution, retrospective cohort study was performed to compare perioperative outcomes (operative time (OT), length of stay (LOS), complications, readmissions, postoperative interventions, loss in size of the residual moiety, and the presence of postoperative 'contained fluid collections') for RALPN and OPN between February 2007 and July 2014. Descriptive statistics were calculated and unadjusted analyses performed, including Chi-squared/Fisher's exact for categorical variables and Wilcoxon rank sum for continuous variables. RESULTS: During the study period, 43 partial nephrectomies were performed for benign disease: 27 (63%) RALPN and 16 (37%) OPN. The RALPN cohort was significantly older than the OPN cohort (3.5 vs. 0.8 years; P = 0.003). The cohorts were otherwise similar regarding race, American Society of Anesthesiologist score (ASA), affected moiety, laterality, associated anomalies, moiety function, and surgical indication. Robotic-assisted laparoscopic partial nephrectomy was associated with a significantly shorter LOS (1 vs. 3 days; P = 0.002). Operative time and complication rates were comparable. The OPN group had a longer median follow-up (2.7 years vs. 1.1; P = 0.03). No differences were observed between the cohorts for readmissions, postoperative interventions, loss in size of residual moiety, or development of 'contained fluid collections'. These outcomes are reported in the Summary Table. 'Contained fluid collections' occurred more frequently after lower pole resections (73% vs. 30%; P = 0.009). DISCUSSION: This study is one of the few to directly compare RALPN to OPN, and demonstrated that RALPN has comparable (if not better) outcomes than OPN. In particular, RALPN provides the advantage of a shorter LOS. Avoiding the flank incision used in OPN may also offer reduced pain; however, this was not studied here and the literature provides weak evidence for this effect. This unadjusted analysis may have been confounded by its short median follow-up and significantly younger OPN cohort. CONCLUSION: In this contemporary comparative analysis, RALPN predicted a similar median OT and safety profile to OPN while offering the advantage of a shorter LOS. Regardless of surgical cohort, there were no adverse effects on the residual renal moieties and postoperative 'contained fluid collections' occurred with notable frequency and were independently associated with lower pole pathology.

Robotic-assisted laparoscopic bladder augmentation in the pediatric patient.

INTRODUCTION: Bladder augmentation is a common surgical intervention for neuropathic bladder dysfunction, and has conventionally been an open procedure. We present a robotic ileocystoplasty to demonstrate the feasibility of an entirely intracorporeal approach in a pediatric patient. METHODS: The patient was a 6 year old (18.5 kg) boy with a neurogenic bladder secondary to lumbar myelomeningocele. Urodynamics revealed a small capacity and poorly compliant bladder and he was incontinent between frequent catheterizations. A robotic augmentation cystoplasty was performed. RESULTS: At one-month postoperatively, a cystogram revealed no urine leak, and the suprapubic tube was removed. The patient resumed CIC every 3 h during the day and once overnight until postoperative urodynamic studies confirmed safe dynamics, after which the CIC interval was lengthened. CONCLUSION: Robotic bladder augmentation is safe and feasible in a select pediatric population. The entire procedure including preparation of the bowel segment can be completed intracorporeally, even in smaller children.

Prepubertal presentation of varicocele does not affect outcomes.

INTRODUCTION: Varicoceles in prepubertal boys are uncommon and little is known of the natural history. Historically, a large percentage of these boys have undergone surgical repair with the belief that such early presentation carried a worse prognosis, making assessment of longitudinal outcomes difficult. OBJECTIVE: While there may be concern that varicocele could represent a progressive disease and therefore prepubertal presentation would portend a worse prognosis, we hypothesized that there would be no difference between the prepubertal boys and other adolescents with varicocele. STUDY DESIGN: We retrospectively reviewed a database of boys at a single institution with a documented left-sided varicocele between 1995 and 2011. Inclusion criteria were one or more of the following: 1. Clinician-documented Tanner 1 status, 2. Right testis orchidometric or ultrasound calculated volume of ≤3 cc's. Patients were drawn from a prospectively maintained database of all boys presenting to the outpatient urology clinic receiving a diagnosis of varicocele. A cohort of adolescent boys was assembled by matching as closely as possible with respect to testis volume disparity and grade of varicocele. All matches were within 2% of volume difference. Volume was calculated using the length*width*height*0.71 formula. Testis size disparity was set to a threshold of ≥20% using the Lambert formula: (VolumeRight - VolumeLeft)/VolumeRight*100%. Our primary outcome was defined as hypotrophy or the need for surgery for hypotrophy at the termination of the study. We planned a single subgroup analysis of boys based on presentation with or without hypotrophy. The decision for surgery or observation was made by the individual clinician at the time of patient assessment. RESULTS: On presentation, the prepubertal cohort was younger (10.8 vs 14.1 years), and with smaller left (2.4 vs 11.6 cc) and right (2.4 vs 11.6 cc) testis volume. There were no significant differences with respect to varicocele grade and volume differentials at presentation. At the end of the study, 76% of the prepubertal cohort had neither hypotrophy nor the requirement for operation, compared with 83% of the matched cohort (P = 0.71, Fisher's exact test). Similarly, there were no significant differences in outcome when comparing prepubertal boys with initial symmetry or hypotrophy to their matched cohort of older adolescents. DISCUSSION: The prepubertal varicocele is a rare clinical problem for which little data exists to guide the clinician. In a review of Pubmed indexed English language manuscripts, we were only able to find five papers with information on Tanner stage; only 31 prepubertal boys have longitudinal data reported. This study approximately doubles the number of boys for whom such data is available in the literature. Our chief limitation was sample size. A power analysis indicated that a final-analysis cohort of 90 prepubertal boys would be required to detect a 20% difference in outcome between that group and a matched cohort of pubertal or post-pubertal boys. We propose that given the lack of evidence for worse outcomes in prepubertal boys with varicocele that prepubertal status, in and of itself, not be considered an additional indication for correction of varicocele. CONCLUSION: In our retrospective cohort of prepubertal boys with left testis varicocele and their matched cohort, we did not detect a difference in the rate of good outcomes, defined as the absence of hypotrophy and lack of need for surgical intervention. While we may have suspected, as have others, that prepubertal presentation would have conveyed a more pressing need to intervene, it is likely that these boys represent the very same patients that we see more commonly later in their adolescence, and should thus be managed in a similarly conservative fashion.

Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1.

DAX-1 [dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] is an orphan nuclear receptor that represses transcription by steroidogenic factor-1 (SF-1), a factor that regulates expression of multiple steroidogenic enzymes and other genes involved in reproduction. Mutations in the human DAX1 gene (also known as AHC) cause the X-linked syndrome AHC, a disorder that is associated with hypogonadotropic hypogonadism also. Characterization of Dax1-deficient male mice revealed primary testicular defects that included Leydig cell hyperplasia (LCH) and progressive degeneration of the germinal epithelium, leading to infertility. In this study, we investigated the effect of Dax1 disruption on the expression profile of various steroidogenic enzyme genes in Leydig cells isolated from Dax1-deficient male mice. Expression of the aromatase (Cyp19) gene, which encodes the enzyme that converts testosterone to estradiol, was increased significantly in the Leydig cells isolated from mutant mice, whereas the expression of other proteins (e.g., StAR and Cyp11a) was not altered. In in vitro transfection studies, DAX-1 repressed the SF-1-mediated transactivation of the Cyp19 promoter but did not inhibit the StAR or Cyp11a promoters. Elevated Cyp19 expression was accompanied by increased intratesticular levels of estradiol. Administration of tamoxifen, a selective estrogen-receptor modulator, restored fertility to the Dax1-deficient male mice and partially corrected LCH, suggesting that estrogen excess contributes to LCH and infertility. Based on these in vivo and in vitro analyses, aromatase seems to be a physiologic target of Dax-1 in Leydig cells, and increased Cyp19 expression may account, in part, for the infertility and LCH in Dax1-deficient mice.

Sertoli cell-specific rescue of fertility, but not testicular pathology, in Dax1 (Ahch)-deficient male mice.

DAX1 is an orphan member of the nuclear hormone receptor superfamily of transcription factors. Our recent characterization of Dax1 (Ahch)-deficient male mice revealed a primary testicular defect resulting in hypogonadism and sterility. The progressive degeneration of the germinal epithelium, independent of abnormal gonadotropin and testosterone production, suggested an intrinsic loss of Dax1 function in the Sertoli cells. To test this hypothesis, we assessed the effect of Sertoli cell-specific expression of a human DAX1 (AHC) transgene driven using the promoter of the Müllerian inhibiting substance (MIS) gene. The MIS-DAX1 transgene partially rescued the mutant phenotype of the Dax1-deficient male mice. Although testicular morphology remained abnormal, fertility was restored to levels matching that of wild-type littermates. Examination of several markers of sperm fertilizing capability revealed significant improvements in MIS-DAX1-rescued mice. Epididymal sperm count and sperm motility were greater in 12-week-old rescued mice than in age-matched Dax1-deficient mice. The ability of sperm to undergo an immediate acrosome reaction was impaired in Dax1-deficient animals, and sperm from Dax1-deficient mice fertilized only 8.2 +/- 6.8% of eggs in vitro, significantly less than rescue (67.8 +/- 19.1%) and wild-type (88.9 +/- 3.9%) sperm. These results indicate that Dax1 expression in Sertoli cells is adequate to overcome crucial thresholds related to sperm production and function. However, the failure to completely rescue the testicular pathology of Dax1-deficient mice suggests that Dax1 expression in other somatic cells is essential for normal testicular development.

Mutant vasopressin precursors that cause autosomal dominant neurohypophyseal diabetes insipidus retain dimerization and impair the secretion of wild-type proteins.

Autosomal dominant familial neurohypophyseal diabetes insipidus is caused by mutations in the arginine vasopressin (AVP) gene. We demonstrated recently that mutant AVP precursors accumulate within the endoplasmic reticulum of neuronal cells, leading to cellular toxicity. In this study, the possibility that mutant AVP precursors interact with wild-type (WT) proteins to alter their processing and function was explored. WT and mutant precursors were epitope-tagged to allow them to be distinguished in transfected cells. An in vivo cross-linking reaction revealed homo- and heterodimer formation between WT and mutant precursors. Mutant precursors were also shown to impair intracellular trafficking of WT precursors from the endoplasmic reticulum to the Golgi apparatus. In addition to the cytotoxicity caused by mutant AVP precursors, the interaction between the WT and mutant precursors suggests that a dominant-negative mechanism may also contribute to the pathogenesis of familial neurohypophyseal diabetes insipidus.

Role of Ahch in gonadal development and gametogenesis.

Ahch (also known as Dax1) encodes a transcription factor that has been implicated in sex determination and gonadal differentiation. Mutations in human AHC cause X-linked, adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Duplication of the Xp21 dosage-sensitive sex reversal (DSS) region, which contains the Ahch locus, and transgenic overexpression of Ahch cause male-to-female sex reversal. Using Cre-mediated disruption of Ahch, we have generated a mouse model of AHC-HH that allows the function of Ahch to be examined in both males and females. Although Ahch has been postulated to function as an ovarian determination gene, the loss of Ahch function in females does not affect ovarian development or fertility. Ahch is instead essential for the maintenance of spermatogenesis. Lack of Ahch causes progressive degeneration of the testicular germinal epithelium independent of abnormalities in gonadotropin and testosterone production and results in male sterility. Ahch is thus not an ovarian determining gene, but rather has a critical role in spermatogenesis.

The murine Dax-1 promoter is stimulated by SF-1 (steroidogenic factor-1) and inhibited by COUP-TF (chicken ovalbumin upstream promoter-transcription factor) via a composite nuclear receptor-regulatory element.

The Dax-1 gene encodes a protein that is structurally related to members of the orphan nuclear receptor superfamily. Dax-1 is coexpressed with another orphan nuclear receptor, steroidogenic factor-1 (SF-1), in the adrenal, gonads, hypothalamus, and pituitary gland. Mutations in Dax-1 cause adrenal hypoplasia congenita, a disorder that is characterized by adrenal insufficiency and hypogonadotropic hypogonadism. These developmental and endocrine abnormalities are similar to those caused by disruption of the murine Ftz-F1 gene (which encodes SF-1), suggesting that these nuclear receptors act along the same developmental cascade. Cloning of the murine Dax-1 gene revealed a candidate SF-1-binding site in the Dax-1 promoter. In transient expression assays in SF-1-deficient JEG-3 cells, SF-1 stimulated expression of the Dax-1 promoter. However, deletion or mutation of the consensus SF-1-binding site did not eliminate SF-1 stimulation. Further analyses revealed the presence of a cryptic SF-1 site that creates an imperfect direct repeat of the SF-1 element. When linked to the minimal thymidine kinase promoter, each of the isolated SF-1 sites was sufficient to mediate transcriptional regulation by SF-1. Mutation of both SF-1 sites eliminated SF-1 binding and stimulation of the Dax-1 promoter. Unexpectedly, mutation of either half of the composite SF-1 sites increased basal activity in JEG-3 cells, suggesting interaction of a repressor protein. Gel shift analyses of the composite response element revealed an additional complex that was not supershifted by SF-1 antibodies. This complex was eliminated by mutation of either half-site, and it was supershifted by antibodies against chicken ovalbumin upstream promoter-transcription factor (COUP-TF). We propose that Dax-1 is stimulated by SF-1, and that SF-1 and COUP-TF provide antagonistic pathways that converge upon a common regulatory site.

Steroidogenic factor-1 contains a carboxy-terminal transcriptional activation domain that interacts with steroid receptor coactivator-1.

The orphan nuclear receptor, steroidogenic factor-1 (SF-1), plays an important role in the development of the adrenal gland and in sexual differentiation. SF-1 regulates the transcription of variety of genes, including several steroidogenic enzymes, Müllerian inhibiting substance, and gonadotropin genes. In this report, we sought to identify domains in SF-1 that are required for transactivation and to determine whether SF-1 interacts with a subset of known coactivators. Natural variants of the FTZ-F1 locus include embryonal long terminal repeat-binding protein (ELP)-1, ELP-2, and SF-1, which share the DNA-binding domain. Analyses of the transcriptional activity of these variants revealed that the activity of ELP-2 and SF-1 was much greater than ELP-1, which contains a distinct carboxy terminus. Further studies were performed using GAL4-SF-1 fusion proteins that were constructed by replacement of the zinc finger region and FTZ-F1 box of SF-1 with the DNA-binding domain of GAL4. Elimination of the putative AF-2 domain at the carboxy terminus of GAL4-SF-1 proteins resulted in a complete loss of transactivation. Several lines of evidence demonstrated that SF-1 interacts with steroid receptor coactivator-1 (SRC-1). Full-length SRC-1 enhanced GAL4-SF-1-mediated transactivation, whereas a dominant negative form of SRC-1, consisting of its interaction domain alone, inhibited the activity of GAL4-SF-1. In mammalian two-hybrid assays, fusion of the VP16 activation domain to the interaction domain of SRC-1 confirmed the interaction between SRC-1 and GAL4-SF-1 and demonstrated that the AF-2 domain is required for interaction with SRC-1. Furthermore, SRC-1, together with the cAMP responsive element binding protein (CBP) or a closely related factor, p300, synergistically enhanced transcriptional activity of GAL4-SF-1. We conclude that the carboxy-terminal AF-2 region of SF-1 functions as an activation domain and that SRC-1 and CBP/p300 are components of the coactivator complex with SF-1.

The Role of DAX-1 in Reproduction.

Mutations in a gene referred to as Dax-1 cause an X-linked form of adrenal hypoplasia congenita (AHC). The disorder is limited to males and is characterized by neonatal adrenal insufficiency and failure to undergo puberty because of hypogonadotropic hypogonadism. Consistent with these clinical manifestations, the Dax-1 gene is expressed in the adrenal gland, gonads, hypothalamus and pituitary gland. The DAX-1 protein is structurally related to orphan nuclear receptors, although it lacks the characteristic zinc finger DNA-binding domain that is highly conserved in other members of this family. Dax-1 has been shown to repress the transcription of genes that are regulated by another nuclear receptor, steroidogenic factor-1 (SF-1). AHC mutations in Dax-1 eliminate its repressive activity. Genetic testing for Dax-1 mutations will enhance our ability to diagnose and treat AHC. Studies of the biological role of Dax-1 will provide new insights into the development and function of the adrenal gland and the reproductive axis.

The first human alpha-spectrin structural domain begins with serine.

The 106-amino acid sequence motifs of spectrin have been suggested to fold into stable structural domains, consisting mostly of coiled coils of triple helices. With the advent of molecular biology and biophysical techniques, structural studies of these spectrin 106-amino acid structural domains became approachable. However, one of the difficulties in such an approach is determination of the correct phasing of the structural domains, which may or may not coincide with the phasing of the sequence motifs. Proper identification of the domain phasing is vital to the construction of stable spectrin domains for molecular studies. A previously published phasing shift for Drosophila alpha-spectrin indicated a downstream phase-shift of 26 amino acids for the structural domain (Winograd, E., Hume, D., and Branton, D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10788-10791). Using this phase-shift, we prepared a recombinant spectrin peptide with the sequence from residue 49 to residue 155 of human erythrocyte alpha-spectrin and found this peptide to be unstable relative to other peptides that we prepared. Using several other recombinant alpha-spectrin peptides and following the protease digestion approach, we digested spectrin peptides with elastase and chymotrypsin and analyzed the amino acid sequence of the digestive products. We provide the first experimental evidence in identifying the first amino acid residue of the first spectrin domain in human erythrocyte alpha-spectrin as residue 52 (Ser).

The behavior of L-ascorbic acid in the healing process of dorsal wounds in guinea pigs.

The behavior of L-ascorbic acid (AsA) in the healing process of wounds in guinea pigs was investigated by determining AsA and dehydroascorbic acid (DAsA) levels. Dorsal skin wounds of guinea pigs fed AsA-deficient diets for 10 days were surgically induced, and the animals were intraperitoneally supplemented with 0, 0.5, 5, 50 mg/day of AsA for 4 days, respectively. The animals were sacrificed, and the amounts of AsA and DAsA in healing skin wound and intact skin were determined by high-performance liquid chromatography (HPLC). It was found that total AsA levels in the healing wound of the animals in AsA-supplemented groups were almost similar to those in intact skin, respectively. However, DAsA levels in the healing wounds on day 4 of the healing period are significantly higher than those not only in the intact skin but also in the completely regenerated skin on day 14 of the healing period. From these results, it was suggested that the wound healing process was accompanied by the oxidation of AsA, moreover, the reduction of DAsA to AsA did not sufficiently occur in vivo.