TAQing2.0 for genome reorganization of asexual industrial yeasts by direct protein transfection.

Genomic rearrangements often generate phenotypic diversification. We previously reported the TAQing system where genomic rearrangements are induced via conditional activation of a restriction endonuclease in yeast and plant cells to produce mutants with marked phenotypic changes. Here we developed the TAQing2.0 system based on the direct delivery of endonucleases into the cell nucleus by cell-penetrating peptides. Using the optimized procedure, we introduce a heat-reactivatable endonuclease TaqI into an asexual industrial yeast (torula yeast), followed by a transient heat activation of TaqI. TAQing2.0 leads to generation of mutants with altered flocculation and morphological phenotypes, which exhibit changes in chromosomal size. Genome resequencing suggested that torula yeast is triploid with six chromosomes and the mutants have multiple rearrangements including translocations having the TaqI recognition sequence at the break points. Thus, TAQing2.0 is expected as a useful method to obtain various mutants with altered phenotypes without introducing foreign DNA into asexual industrial microorganisms.

Improvement of production yield of l-cysteine through in vitro metabolic pathway with thermophilic enzymes.

The demand for the amino acid l-cysteine is increasing in the food, cosmetic, and pharmaceutical industries. Conventionally, the commercial production of l-cysteine is achieved by its extraction from the acid hydrolysate of hair and feathers. However, this production method is associated with the release of environmentally hazardous wastewater. Additionally, l-cysteine produced from animal sources cannot be halal-certified, which limits the market size. Although recent studies have developed an alternative commercial l-cysteine production method based on microbial fermentation, the production yield was insufficient owing to the cytotoxicity of l-cysteine against the host cells. In a previous study, we had developed an in vitrol-cysteine production method with a combination of 11 thermophilic enzymes, which yielded 10.5 mM l-cysteine from 20 mM glucose. In this study, we performed re-screening for enzymes catalyzing the rate-limiting steps of the in vitro pathway. Subsequently, the genes encoding enzymes necessary for the in vitro synthesis of l-cysteine were assembled in an expression vector and co-expressed in a single strain. To prevent the synthesis of hydrogen peroxide (H2O2), which is a byproduct and inhibits the enzyme activity, the redox balance in this biosynthetic pathway was maintained by replacing the H2O2-forming NADH oxidase with another enzymatic reaction in which pyruvate was used as a sacrificial substrate. The re-designed in vitro synthetic pathway resulted in the production of 28.2 mM l-cysteine from 20 mM glucose with a molar yield of 70.5%.

Comparison of metabolic profiles of yeasts based on the difference of the Crabtree positive and negative.

The Crabtree effect involves energy management in which yeasts utilize glycolysis as the terminal electron acceptor instead of oxygen, despite the presence of sufficient dissolved oxygen, when oxygen concentrations exceed a certain limit. The Crabtree effect is detrimental to bakery yeast production, because it results in lower cellular glucose yields. Batch culture of Saccharomyces cerevisiae, a Crabtree positive yeast, decreased the cell yield of glucose and produced large amounts of ethanol despite a high specific glucose consumption rate compared to Candida utilis, a Crabtree negative yeast. This study investigated the effect of these characteristics on metabolite levels. We performed metabolome analysis of both yeasts during each growth phase of batch culture using liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Principle component analysis of metabolome data indicated that the Crabtree effect affected metabolites related to NADH synthesis in central metabolism. The amount of these metabolites in S. cerevisiae was lower than that in C. utilis. However, to maintain the specific glucose consumption rate at high levels, yeasts must avoid depletion of NAD+, which is essential for glucose utilization. Our results indicated that NADH was oxidized by converting acetaldehyde to ethanol in S. cerevisiae, which is in accordance with previous reports. Therefore, the specific NADH production rates of S. cerevisiae and C. utilis did not show a difference. This study suggested that NAD+/NADH ratio is disrupted by the Crabtree effect, which in turn influenced central metabolism and that S. cerevisiae maintained the NAD+/NADH ratio by producing ethanol.

In vitro production of cysteine from glucose.

Cysteine is a commercially valuable amino acid with an increasing demand in the food, cosmetic, and pharmaceutical industries. Although cysteine is conventionally manufactured by extraction from animal proteins, this method has several problems, such as troublesome waste-water treatment and incompatibility with some dietary restrictions. Fermentative production of cysteine from plant-derived substrates is a promising alternative for the industrial production of cysteine. However, it often suffers from low product yield as living organisms are equipped with various regulatory systems to control the intracellular cysteine concentration at a moderate level. In this study, we constructed an in vitro cysteine biosynthetic pathway by assembling 11 thermophilic enzymes. The in vitro pathway was designed to be insensitive to the feedback regulation by cysteine and to balance the intra-pathway consumption and regeneration of cofactors. A kinetic model for the in vitro pathway was built using rate equations of individual enzymes and used to optimize the loading ratio of each enzyme. Consequently, 10.5 mM cysteine could be produced from 20 mM glucose through the optimized pathway. However, the observed yield and production rate of the assay were considerably lower than those predicted by the model. Determination of cofactor concentrations in the reaction mixture indicated that the inconsistency between the model and experimental assay could be attributed to the depletion of ATP and ADP, likely due to host-derived, thermo-stable enzyme(s). Based on these observations, possible approaches to improve the feasibility of cysteine production through an in vitro pathway have been discussed.

Developing a single strain for in vitro salvage synthesis of NAD+ at high temperatures and its potential for bioconversion.

BACKGROUND: Thermostable enzymes have several advantages over their mesophilic counterparts for industrial applications. However, trade-offs such as thermal instability of enzyme substrates or co-factors exist. Nicotinamide adenine dinucleotide (NAD+) is an important co-factor in many enzyme-catalyzed oxidation-reduction reactions. This compound spontaneously decomposes at elevated temperatures and basic pH, a property that limits catalysis of NAD+/NADH-dependent bioconversions using thermostable enzymes to short timeframes. To address this issue, an "in vitro metabolic pathway" for salvage synthesis of NAD+ using six thermophilic enzymes was constructed to resynthesize NAD+ from its thermal decomposition products at high temperatures. RESULTS: An integrated strain, E. coli DH5α (pBR-CI857, pGETS118-NAD+), that codes for six thermophilic enzymes in a single operon was constructed. Gene-expression levels of these enzymes in the strain were modulated by their sequential order in the operon. An enzyme solution containing these enzymes was prepared by the heat purification from the cell lysate of the integrated strain, and used as an enzyme cocktail for salvage synthesis of NAD+. The salvage activity for synthesis of NAD+ from its thermal decomposition products was found to be 0.137 ± 0.006 µmol min-1 g-1 wet cells. More than 50% of this initial activity remained after 24 h at 60 °C. The enzyme cocktail could maintain a NAD+ concentration of 1 mM for 12 h at 60 °C. Furthermore, this enzyme cocktail supported continuous NAD+/NADH-dependent redox reactions using only NAD+/NADH derived from host cells, without the need for addition of external NAD+. CONCLUSIONS: The integrated strain allows preparation of an enzyme cocktail that can solve the problem of NAD+ instability at high temperatures. The strain simplifies preparation of the enzyme cocktail, and thus expands the applicability of the in vitro metabolic engineering method using thermostable enzymes. Further optimization of gene expressions in the integrated strain can be achieved by using various types of ribosome binding sites as well as promoters.

Metabolomics approach to reduce the Crabtree effect in continuous culture of Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is an important microorganism for fermentation and the food industry. However, during production, S. cerevisiae commonly uses the ethanol fermentation pathway for glucose utilization if excess sugar is present, even in the presence of sufficient oxygen levels. This aerobic ethanol fermentation, referred to as the Crabtree effect, is one of the most significant reasons for low cell yield. To weaken the Crabtree effect in fed-batch and continuous culture, sugar flow should be limited. In addition, in continuous culture, the dilution rate must be reduced to avoid washing out cells. However, under such conditions, production speed might be sacrificed. It is difficult to solve this problem with the tradeoff between cell yield and production speed by using conventional tactics. However, a metabolomics approach may be an effective way to search for clues regarding metabolic modulation. Therefore, the purpose of this study was to reduce ethanol production in continuous culture of S. cerevisiae at a higher dilution rate through a metabolomics approach. We used a metabolomics analysis to identify metabolites that were drastically increased or decreased in continuous culture when the dilution rate shifted from biomass formation to ethanol fermentation. The individual addition of two of the selected metabolites, fumaric acid and malic acid, reduced ethanol production at a higher dilution rate. This result demonstrates the potential for using metabolomics approaches to identify metabolites that reduce ethanol production in continuous culture at high dilution rates.

Interleukin-18 may lead to benign prostatic hyperplasia via thrombospondin-1 production in prostatic smooth muscle cells.

BACKGROUND: Although inflammation plays an important role in the development of benign prostatic hyperplasia (BPH), little is known about the exact mechanism underlying this pathogenesis. Here, we investigated the relationship between the inflammatory reaction and BPH. METHODS: cDNA microarray analysis was used to identify changes in inflammation-related gene expression in a recently established rat model that mimics human BPH. To investigate the genes identified in the analysis, quantitative (q)RT-PCR, Western blotting, immunostaining, and a cell proliferation assay were conducted using BPH model tissues, human prostate tissues, and normal human prostate cultured cells. RESULTS: Of the 31,100 genes identified in the cDNA analysis, seven inflammatory-response-related genes were expressed at a >2-fold higher level in rat BPH tissues than in normal rat prostate tissues. The levels of the most commonly expressed pro-inflammatory cytokine, IL-18, significantly increased in rat BPH tissues. In humans, IL-18 was localized in the epithelial and stromal components, while its receptor was strongly localized in smooth muscle cells. Furthermore, in human prostate smooth muscle cell line (PrSMC), IL-18 effected dose-dependent increases in the phosphorylated Akt and thrombospondin-1 (TSP-1) levels. TSP-1 promoted proliferation of the human prostate stromal cells (PrSC). CONCLUSIONS: IL-18 may act directly in BPH pathogenesis by inducing TSP-1 production in prostatic smooth muscle cells via Akt phosphorylation.

Identification of the replication region of a 111-kb circular plasmid from Rhodococcus opacus B-4 by λ Red recombination-based deletion analysis.

The replication region of the 111-kb circular plasmid pKNR from Rhodococcus opacus B-4 was identified. A PCR-based deletion analysis using the λ Red recombination technique followed by restriction digestion and PCR-amplification analyses revealed that a 2.5-kb fragment covering one putative open reading frame (ORF) was involved in the replication of pKNR. The product of this ORF showed significant similarity to a functionally unknown protein encoded in the replication region of the 70-kb circular plasmid of Clavibacter michiganensis and to ones in other bacterial large circular plasmids. These observations suggest that the product of the identified ORF and its orthologs can serve as novel replication proteins for large circular bacterial plasmids.

Regulation of cell proliferation through a KIT-mediated mechanism in benign prostatic hyperplasia.

BACKGROUND: We investigated the role of the KIT-mediated mechanism in benign prostatic hyperplasia (BPH), and discuss the pathophysiology of BPH and a candidate target of BPH medical therapy. METHODS: We performed RT-PCR, Western blotting, and immunohistochemistry to examine the expression of KIT in the prostate using a human prostate stromal cell line (PrSC) and human prostate. To investigate the pathophysiological function of KIT, the effects of KIT ligand, stem cell factor (SCF), and imatinib mesylate on cell proliferation were investigated using PrSC. Additionally, we compared the expression level and distribution of KIT in normal prostate and BPH of humans to clarify the contribution of KIT to the pathogenesis of BPH. RESULTS: KIT was expressed in PrSC and human prostate, indicating that these samples are suitable for examining the function of KIT. Immunohistochemical analysis demonstrated that KIT was localized in interstitial cells (ICs) of the stromal component in human prostate. Administration of imatinib mesylate dose-dependently inhibited cell proliferation of PrSC with downregulation of JAK2 and STAT1, which are the main pathways downstream of SCF/KIT signal. SCF promoted cell proliferation of PrSC with upregulation of JAK2 and STAT1. KIT expression and the number of KIT-positive ICs in BPH were found to be significantly larger than in normal prostate. CONCLUSIONS: This is the first report to suggest that KIT regulates cell proliferation in the prostate and plays a significant role in the pathophysiology of BPH. Our study may lead to a greater understanding of the mechanism of BPH and provide a therapeutic target.

Attenuation of spermatogonial stem cell activity in cryptorchid testes.

PURPOSE: To elucidate the mechanism of infertility caused by cryptorchidism we focused on early stage spermatogenesis and spermatogonial stem cell activity in undifferentiated spermatogonia in cryptorchid testes. MATERIALS AND METHODS: Histological findings and expression patterns of the stem cell marker undifferentiated embryonic cell transcription factor 1 were examined in a unilateral cryptorchid rat model. We removed unilateral descended testis and contralateral descended testis from cryptorchid and normal rats (control), respectively, 18 days postcoitum to 144 days postpartum. RESULTS: In descended testes gonocyte differentiation into early A spermatogonia occurred at 9 days postpartum. However, this transformation was altered in undescended testes. Furthermore, the undifferentiated embryonic cell transcription factor 1 negative early A spermatogonia-to-positive early A spermatogonia ratio was significantly higher in the undescended testis group (mean ± SD 0.69 ± 0.04) than in the control (0.46 ± 0.10, p = 0.037) and descended testis (0.44 ± 0.05, p = 0.022) groups, indicating decreased early A spermatogonia with spermatogonial stem cell activity in cryptorchid testes. CONCLUSIONS: In cryptorchid testes the differentiation from gonocytes into early A spermatogonia and the stem cell activity of early A spermatogonia were altered during the early stage of spermatogenesis, suggesting that the loss of spermatogonial stem cell activity in cryptorchid rats resulted in altered spermatogenesis, thus interfering with fertility.

Tamsulosin reduces nighttime urine production in benign prostatic hyperplasia patients with nocturnal polyuria: a prospective open-label long-term study using frequency-volume chart.

AIMS: The effects of tamsulosin treatment on changes in frequency-volume chart (FVC) data, especially nighttime urine production, over time were assessed, and the mechanisms underlying the improvement of nocturia in benign prostatic hyperplasia (BPH) patients with nocturnal polyuria (NP) are discussed. METHODS: A total of 104 patients with lower urinary tract symptoms secondary to BPH were enrolled. After enrollment in the study, the patients were treated with tamsulosin (0.2 mg) once daily. Visits were scheduled every 4 weeks until week 12 (month 3) after study entry, and then every 12 weeks subsequently. All patients completed the International Prostate Symptom Score (IPSS), quality of life (QOL) index, and 3-day FVC, and underwent uroflowmetry at enrollment and on each visit. RESULTS: Eighty-two patients (mean age: 70.9 ± 7.1 years) were analyzed for 24 months after treatment. Patients were divided into two groups, NP and nonNP, based on FVC outcome. The IPSS, QOL index, and maximum flow rate improved during the 24-month period after treatment in both groups. Mean daytime urine volume significantly increased in the NP group, but no changes were detected in the nonNP group. Mean nighttime urine frequency significantly decreased in the NP group over a 24-month period, and was associated with a significant decrease in nighttime urine volume that was not found in the nonNP group. Maximum voided volume increased most months after treatment in both groups. CONCLUSIONS: The present long-term prospective study using FVC demonstrated that tamsulosin reduced nighttime urine production in BPH patients with NP.

Up-regulation of α1a and α1d-adrenoceptors in the prostate by administration of subtype selective α1-adrenoceptor antagonist tamsulosin in patients with benign prostatic hyperplasia.

PURPOSE: We examined the change in α(1)-adrenoceptor subtype expression in the prostate due to chronic tamsulosin administration in a benign prostatic hyperplasia rat model and in patients. MATERIALS AND METHODS: We measured α(1)-adrenoceptor subtype expression after tamsulosin administration in the prostate of the benign prostatic hyperplasia rat model using TaqMan® reverse transcriptase-polymerase chain reaction. We also measured expression before and after 12-week tamsulosin treatment in the prostate of patients with benign prostatic hyperplasia. We examined the correlation between the change in α(1)-adrenoceptor expression due to tamsulosin treatment and acute urinary retention during long-term followup. RESULTS: The expression of α(1a) and α(1d)-adrenoceptors was significantly increased in dose dependent fashion by tamsulosin in the benign prostatic hyperplasia rat model. Median mRNA expression of subtypes α(1a) and α(1d)-adrenoceptors was 1.4 (IQR 0.6, 3.0) and 1.7 × 1,000 copies per 1 ng ß-actin (IQR 0.9, 2.4) before treatment, and 6.0 (IQR 2.0, 8.0) and 2.2 × 1,000 copies per 1 ng ß-actin (IQR 1.7, 3.6), respectively, after treatment. The expression of α(1a) and α(1d)-adrenoceptors significantly increased after tamsulosin treatment (p <0.01 and <0.05, respectively). This increase was observed in 10 patients in whom acute urinary retention did not develop during long-term followup but not in 4 in whom acute urinary tract retention developed. CONCLUSIONS: Tamsulosin up-regulated α(1a) and α(1d)-adrenoceptors, suggesting that it has clinical selectivity for α(1a) and α(1d)-adrenoceptors. Up-regulation of α(1)-adrenoceptors subtype expression is considered an adaptive response to chronic tamsulosin administration. The difference in the response to α(1)-adrenoceptors antagonists among patients may contribute to the diversity in the long-term efficiency of α(1)-adrenoceptor antagonists.

Role of KIT-Positive Interstitial Cells of Cajal in the Urinary Bladder and Possible Therapeutic Target for Overactive Bladder.

In the gastrointestinal tract, interstitial cells of Cajal (ICCs) act as pacemaker cells to generate slow wave activity. Interstitial cells that resemble ICCs in the gastrointestinal tract have been identified by their morphological characteristics in the bladder. KIT is used as an identification marker of ICCs. ICCs in the bladder may be involved in signal transmission between smooth muscle bundles, from efferent nerves to smooth muscles, and from the urothelium to afferent nerves. Recent research has suggested that not only the disturbance of spontaneous contractility caused by altered detrusor ICC signal transduction between nerves and smooth muscle cells but also the disturbance of signal transduction between urothelial cells and sensory nerves via suburothelial ICC may induce overactive bladder (OAB). Recent reports have suggested that KIT is not only a detection marker of these cells, but also may play a crucial role in the control of bladder function. Research into the effect of a c-kit receptor inhibitor, imatinib mesylate, on bladder function implies that KIT-positive ICCs may be therapeutic target cells to reduce bladder overactivity and that the blockage of c-kit receptor may offer a new therapeutic strategy for OAB treatment, although further study will be needed.

Correlation between improvements in Overactive Bladder Symptom Score and health-related quality of life questionnaires in overactive bladder patients treated with an antimuscarinic drug.

AIMS: To evaluate the relationship between treatment-related changes in Overactive Bladder Symptom Scores (OABSS) and health-related quality of life (HRQOL) questionnaires. METHODS: Ninety-five patients with OAB symptoms were enrolled. All patients completed the OABSS, International Prostate Symptom Score (IPSS)-Quality Of Life (QOL) index and King's Health Questionnaire (KHQ) at enrollment and then again 4, 8, and 12 weeks after treatment with propiverine hydrochloride 10 mg twice daily. We evaluated the relationship between treatment-related changes in the OABSS, IPSS-QOL, and KHQ. RESULTS: Statistically significant improvements were observed in all 4 OABSS subscales and total OABSS from baseline to 4 weeks with further improvements occurring at 12 weeks (all P < 0.01). The OABSS after antimuscarinic treatment correlated positively with both the IPSS-QOL index and KHQ domain scores. There was a moderate but statistically significant correlation between the change in total OABSS and 2 OABSS subscales (urinary urgency and urge incontinence) and improvement in the IPSS-QOL index (P < 0.01). Treatment-related changes in total OABSS were significantly correlated with changes in six KHQ domains. Moderate but statistically significant correlations were observed between the change in total OABSS and impact on life, physical limitations, emotions, and severity measures (r > 0.30, P < 0.05). Small but statistically significant correlations were observed between the change in total OABSS and role limitations or social limitations (P < 0.05). CONCLUSIONS: Improvement in the OABSS correlated with improvements in HRQOL after treatment. The OABSS is a useful tool to evaluate OAB symptom severity after medical treatment.

Correlation between expression of α1-adrenoceptor subtype mRNA and severity of lower urinary tract symptoms or bladder outlet obstruction in benign prostatic hyperplasia patients.

OBJECTIVE: To examine whether the direct correlation between the expression of α1-adrenoceptor (AR) subtype mRNA and severity of lower urinary tract symptoms (LUTS) or bladder outlet obstruction (BOO) in the prostate exists in benign prostatic hyperplasia (BPH) patients. PATIENTS AND METHODS: Sixty-eight patients with LUTS and BOO secondary to BPH were enrolled. Four prostate needle biopsy specimens were obtained from the transition zone to examine the expression level of α1-AR subtypes by Taqman reverse-transcription polymerase chain reaction. The correlation and regression between each expression level of α1-AR subtype and clinical findings such as patient age, prostate volume, International Prostate Symptom Score (IPSS), quality of life (QOL) index, maximum flow rate in uroflowmetry (Qmax) and post-void residual urine volume (PVR) were assessed by stepwise multiple regression analysis. The correlation and regression between this expression level and individual symptoms of IPSS were assessed by Pearson's correlation coefficient and multiple regression analyses. RESULTS: Stepwise multiple regression analysis showed that the expression levels of α(1a) -AR, α(1b) -AR, α(1d) -AR and total α(1) -AR mRNA showed a significant regression with patient age, but not with prostate volume, IPSS, QOL index, Qmax and PVR. Pearson's correlation coefficient and multiple regression analyses demonstrated no correlation and regression between each α(1) -AR subtype mRNA expression level and individual symptoms of IPSS. CONCLUSIONS: There was no direct correlation between the expression of α1-AR subtype mRNA in the prostate and severity of LUTS or BOO in BPH patients, although the significant regression of this expression with patient age existed. LUTS and BOO may be associated with multiple factors and several other conditions may contribute to LUTS and BOO.

TRPM8-expressing dorsal root ganglion neurons project dichotomizing axons to both skin and bladder in rats.

To reveal the mechanism of urinary urgency evoked by cold sensation, we investigated the convergence of the primary sensory afferents of the skin and bladder. Dichotomizing afferents of L6-S1 dorsal root ganglion neurons that innervate the skin and bladder was constantly observed with retrograde neuron tracers in rats. In-situ hybridization revealed that approximately 8.0% of the double-labelled cells expressed transient receptor potential channel melastatin member 8 (TRPM8) transcripts in the dorsal root ganglions. Cold and menthol stimuli to the skin generated bladder nerve responses conducted through dichotomizing axons, which significantly decreased in the presence of the TRPM8 blocker BCTC. Taken together, TRPM8-expressing sensory neurons with dichotomizing axons projecting to the skin and bladder may be responsible for the urinary urgency evoked by cold sensation.

Spermatogenesis after 1-stage fowler-stephens orchiopexy in experimental cryptorchid rat model.

PURPOSE: To evaluate the validity of 1-stage Fowler-Stephens orchiopexy, we performed this procedure for intra-abdominal testes in an experimental cryptorchid rat model and assessed postoperative spermatogenesis. MATERIALS AND METHODS: Cryptorchidism in rats was induced by injecting flutamide into the abdomen of pregnant Sprague Dawley rats for 7 days (days 14 to 20 of gestation). Four-week-old cryptorchid rats were divided into the 4 groups of sham operation (group 1, 7 rats), orchiopexy only (group 2, 7), spermatic vessel ligation only (group 3, 7), and spermatic vessel ligation and orchiopexy (1-stage Fowler-Stephens orchiopexy, group 4, 7). The testes were removed 12 weeks after birth in all groups, and testicular weight, testicular histopathological findings and presence of sperm in the epididymis were examined. RESULTS: All operated testes had reticulated vessels on the tunica albuginea. There were no statistically significant differences in the testicular weight between groups 1 (mean +/- SD 0.47 +/- 0.04 gm) and 4 (0.30 +/- 0.19), suggesting that Fowler-Stephens orchiopexy did not induce significant testicular atrophy. However, seminiferous tubule diameters in group 4 were significantly smaller than in group 1 (p <0.001), and no sperm was observed in the epididymis of group 4 rats, suggesting that Fowler-Stephens orchiopexy reduced seminiferous tubule diameters and did not improve spermatogenesis. CONCLUSIONS: Although Fowler-Stephens orchiopexy is a good procedure to maintain testicular size and correct cosmetic deformity of an absent scrotum, it may not significantly contribute to the improvement of spermatogenesis.

Is antibiotic prophylaxis effective in preventing urinary tract infections in patients with vesicoureteral reflux?

The main concern in the management of children with vesicoureteral reflux (VUR) is the prevention of urinary tract infections and avoidance of renal damage. Medical management has been recommended or preferentially suggested over surgery in all but a few select clinical situations. Prophylactic antibiotics are prescribed routinely in the management of young children with radiological evidence of VUR following an episode of acute pyelonephritis. Prophylaxis is generally maintained until the VUR resolves spontaneously or is corrected surgically. Although the administration of prophylactic antibiotics has been universal in children with VUR, some authors have reported that long-term antibiotic prophylaxis does not fully prevent urinary tract infections or scarring, that antibiotic-related adverse events are known to occur, and that the incidence of pyelonephritis does not increase in spite of prophylactic antibiotic cessation. Recently, four prospective, randomized, controlled trials of antibiotic prophylaxis for preventing pyelonephritis and renal scarring were reported and some placebo-controlled, double-blind prospective studies are ongoing. The goal of this review is to evaluate the treatment of VUR using antibiotic prophylaxis, and its advantages and disadvantages based on appropriate descriptions and studies in the literature.

Laparoscopic orchiectomy and subsequent internal ring closure for extra-abdominal testicular nubbin in children.

OBJECTIVES: To present our preliminary experience with laparoscopic groin exploration and subsequent laparoscopic orchiectomy and internal ring closure for testicular nubbin in children and discuss the usefulness of our new treatment strategy. The advantages of laparoscopic orchiopexy for intra-abdominal testis are the ability to start treatment as soon as a diagnosis has been made and to permit minimally invasive surgery. These advantages can apply to laparoscopic orchiectomy for a testicular nubbin. METHODS: A total of 6 boys with a testicular nubbin (age range 14-76 months, mean age 27.3 months) underwent laparoscopic orchiectomy at our institution from June 2007 to June 2008. We opened the posterior parietal peritoneum by incising the peritoneum lateral to the spermatic vessel, distal to the patent processus vaginalis and medial and distal to the vas deferens (laparoscopic groin exploration). Next, the testicular nubbin was pulled out into the abdomen and was then removed laparoscopically after identification and division of the gubernaculums. Finally, laparoscopic complete internal ring and peritoneal defect closure was performed with 5-0 Vicryl suture. RESULTS: The average operative time, including the diagnostic time, was 64 minutes. No intraoperative or postoperative complications developed. In 1 boy with a testicular nubbin, an open processus vaginalis was present, and simultaneous laparoscopic transection of the processus vaginalis and subsequent internal ring closure were performed. CONCLUSIONS: All patients with a testicular nubbin could be treated as soon as the diagnosis was made using only laparoscopic management, with minimal morbidity and good short-term results.