Statistical analysis of the individual variability of 1D protein profiles as a tool in ecology: an application to parasitoid venom.

Understanding the forces that shape eco-evolutionary patterns often requires linking phenotypes to genotypes, allowing characterization of these patterns at the molecular level. DNA-based markers are less informative in this aim compared to markers associated with gene expression and, more specifically, with protein quantities. The characterization of eco-evolutionary patterns also usually requires the analysis of large sample sizes to accurately estimate interindividual variability. However, the methods used to characterize and compare protein samples are generally expensive and time-consuming, which constrains the size of the produced data sets to few individuals. We present here a method that estimates the interindividual variability of protein quantities based on a global, semi-automatic analysis of 1D electrophoretic profiles, opening the way to rapid analysis and comparison of hundreds of individuals. The main original features of the method are the in silico normalization of sample protein quantities using pictures of electrophoresis gels at different staining levels, as well as a new method of analysis of electrophoretic profiles based on a median profile. We demonstrate that this method can accurately discriminate between species and between geographically distant or close populations, based on interindividual variation in venom protein profiles from three endoparasitoid wasps of two different genera (Psyttalia concolor, Psyttalia lounsburyi and Leptopilina boulardi). Finally, we discuss the experimental designs that would benefit from the use of this method.

Ram seminal plasma proteome and its impact on liquid preservation of spermatozoa.

Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC-MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24h in milk at 15°C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. BIOLOGICAL SIGNIFICANCE: Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24h preservation period.

[In vivo imaging of ram spermatozoa in the ewe genital tract using fibered confocal microscopy]. / Visualisation in vivo des spermatozoïdes de bélier dans le tractus génital de brebis par microscopie confocale fibrée.

Sperm transit in the female tract is one of the key factors in the success of fertilization after artificial insemination in sheep species. However, its study is limited by the absence of in vivo imaging methods. The imaging of ram sperm in the female genital tract was made possible using the confocal fibered microscopy and fluorescent stains adapted to spermatozoa. Our results show the active role of the uterotubal junction in the selection of sperm during their transit.

Post-testicular sperm environment and fertility.

When mammalian spermatozoa exit the testis, they show a highly specialized morphology; however, they are not yet able to carry out their task: to fertilize an oocyte. This property, that includes the acquisition of motility and the ability to recognize and to fuse with the oocyte investments, is gained only after a transit through the epididymis during which the spermatozoa from the testis travel to the vas deferens. The exact molecular mechanisms that turn these cells into fertile gametes still remain mysterious, but surface-modifying events occurring in response to the external media are key steps in this process. Our laboratory has established cartographies of secreted (secretomes) and present proteins (proteomes) in the epididymal fluid of different mammals and have shown the regionalized variations in these fluid proteins along the epididymis. We have found that the main secreted proteins are common in different species and that enzymatic activities, capable of controlling the sperm surface changes, are present in the fluid. Our studies also indicate that the epididymal fluid is more complex than previously thought; it contains both soluble and particulate compartments such as exosome-like vesicles (epididymosomes) and certainly specific glycolipid-protein micelles. Understanding how these different compartments interplay to modify sperm components during their transit will be a necessary step if one wants to control and to ameliorate sperm quality and to obtain valuable fertility markers helpful to establish a male fertility based genetic selection.

Prevalence of human cytomegalovirus infection in pregnant and non-pregnant women.

OBJECTIVES: To determine human cytomegalovirus (HCMV) cervical reactivation in both pregnant and non-pregnant women and to ascertain whether or not it occurs in conjunction with hematogenic dissemination. METHODS: Clinical specimens were obtained from 40 pregnant and 62 non-pregnant women attended at the Ambulatory of the Gynecology-Obstetrics Unit of the Federal University of Espírito Santo (UFES) in Southeastern Brazil. Specimens under investigation were blood samples submitted to seroprevalence determination, antigenemia assay, HCMV-DNA detection, and vaginal secretion, submitted to HCMV-DNA detection. RESULTS: Viral seroprevalence was found in 98% of the women investigated, two of whom were found to be IgM positive, while no difference could be determined between pregnant and non-pregnant women. Antigenemia assay was negative in all cases. HCMV gB gene amplification was found in 5.1 and 8.5% of WBCs and in 10 and 14.5% of vaginal secretion from pregnant and non-pregnant women, respectively. CONCLUSION: The high seroprevalence observed is in accordance with previous Brazilian surveys. Antigenemia assay was unable to detect the occurrence of active infection in the immunocompetent women studied, most likely because it either occurred in a viral load undetectable by this assay or did not occur at all. Although the highest incidence of positivity was observed by gene amplification both in WBCs and secretion from non-pregnant than in pregnant women, the rate of viral detection was statistically similar for both groups.

Physiological and enzymatic properties of the ram epididymal soluble form of germinal angiotensin I-converting enzyme.

The 94-kDa ram epididymal fluid form of the sperm membrane-derived germinal angiotensin I-converting enzyme (ACE) was purified by chromatography, and some of its enzymatic properties were studied. For the artificial substrate furanacryloyl-L-phenylalanylglycylglycine (FAPGG), the enzyme exhibited a Michaelis constant (K(m)) of 0.18 mM and a V(max) of 34 micromoles/(min x mg) and for hippuryl-L-histidyl-L-leucine a K(m) of 2.65 mM and a V(max) of 163 micromoles/(min x mg) under the defined standard conditions (300 mM NaCl and 50 mM Tris; pH 7.5 and 8.3, respectively). The FAPGG hydrolysis was decreased by 82.5% and 67.5% by EDTA and dithioerythritol, respectively, and was totally inhibited by specific ACE inhibitors such as captopril, P-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro, and lisinopril. Optimum activity for FAPGG was with pH 6.0, 50 mM chloride, and 500 microM zinc. Under the various conditions tested, bradykinin, angiotensin (Ang) I, Ang II, and LHRH were competitors for FAPGG. Bradykinin and angiotensin I were the best competitors. The enzyme cleaved Ang I into Ang II, and the optimal conditions were with pH 7.5 and 300 mM chloride. The relationship between the carboxypeptidase activity in seminal plasma and the prediction of fertility of young rams was also studied. These results indicated a correlation between sperm concentration and ACE activity in semen but showed no statistically significant correlation between such activity and fertility of the animal. Finally, we tested the role of ACE in fertilization; no difference in the in vitro fertilization rate was observed in the presence of 10(-4) M captopril.

Vaginal reconstruction using a vertical rectus abdominis myocutaneous flap in a child: a case report.

Rhabdomyosarcoma is the most common soft tissue sarcoma in infants and children younger than 15 years old. Multimodal treatment using chemotherapy, radiation, and surgery have increased survival. Because of the potential for long-term survival, the importance of organ preservation and vaginal and pelvic reconstruction has been recognized. The authors report a case in which special circumstances led to vaginal reconstruction concomitantly with exenteration surgery. The authors present the first reported use of a vertical rectus abdominis myocutaneous flap in a child and describe the technique and its potential advantages.

Antenatal hydronephrosis with postnatal resolution: how long are postnatal studies warranted?

We present 2 cases of antenatal hydronephrosis with initial normalization of postnatal studies. Both patients experienced late-onset (6 and 22 months) hydronephrosis secondary to ureteropelvic junction obstruction, necessitating surgical intervention. These cases raise questions about the need for late follow-up imaging in patients with apparent resolution of hydronephrosis diagnosed antenatally.

In utero intervention in a patient with prune-belly syndrome and severe urethral hypoplasia.

Prenatal ultrasound scanning of a 20-year-old woman at 17 weeks of gestation revealed findings suggestive of bladder outlet obstruction, including bladder distension, dilated bilateral ureters, urinary ascites, and oligohydramnios. Vesicoamniotic shunts were placed with decompression of the bladder and correction of the amniotic fluid levels. Labor was induced at 36 weeks' gestation. At birth, the infant was noted to have prune-belly syndrome with severe urethral hypoplasia, a variant usually associated with a poor prognosis, necessitating vesicostomy for bladder drainage. We present a case of a patient with prune-belly syndrome and bladder outlet obstruction in whom early intervention resulted in an excellent outcome with preservation of renal and pulmonary function.

Pseudohypoaldosteronism associated with ureterocele and upper pole moiety obstruction.

A 2-month-old girl was diagnosed with a febrile urinary tract infection. Subsequent studies revealed a right renal duplication anomaly with a poorly functioning upper pole moiety subtended by an intravesical ureterocele. The patient was also found to have hyperkalemia, hyponatremia, and elevated serum aldosterone. After antibiotic therapy, the urinary tract infection resolved, although the electrolyte and hormonal abnormalities persisted. At 4 months of age, a right upper pole heminephrectomy was performed. The patient's electrolyte and hormonal levels normalized. This case represents an unusual case of pseudohypoaldosteronism presenting with urinary tract infection and obstructed upper pole moiety that resolved after surgical intervention.

Increased incidence of hypospadias in small-for-gestational age infants in a neonatal intensive-care unit.

OBJECTIVE: To identify the incidence of hypospadias in children born prematurely and small-for-gestational age (SGA), and to compare this subgroup with infants of similar age and weight without hypospadias. PATIENTS AND METHODS: Records from the neonatal intensive-care unit (NICU) of a major metropolitan hospital active in labour and delivery were reviewed over a 3-year period, specifically examining newborns admitted with the diagnosis of SGA, defined as a birth weight of < 10th percentile for gestational age. In all, 154 patients were identified and their charts reviewed, recording the presence and severity of hypospadias, gestational age, birth weight, placental weight, cord length, cord vessels, maternal age, parity, multiple births, drug exposure and associated comorbidity. A control group of age- and weight-matched infants without hypospadias were also identified and compared. RESULTS: Of the 154 patients, 17 (11%) had hypospadias; the hypospadias was distal in nine, mid-shaft in four and proximal in four. The severity of hypospadias did not correlate with the degree of prematurity or weight for gestational age. Placental weight, fetal weight, fetal to placental weight ratio and cord length were all lower in the hypospadias group than in the control group, but the differences were not statistically significant. The maternal age was evenly distributed (median 32 years, range 20-43). Most mothers were multiparous and births were multiple in five of 17 (30%). Cryptorchidism (three) and inguinal hernia (three) were present in four of the infants. CONCLUSIONS: The incidence of hypospadias in SGA infants admitted to the NICU is > 10 times higher than that reported for the general population. There was a trend to lower placental and fetal weight in SGA infants with hypospadias than in the controls. This finding merits further evaluation using a larger population database and suggests that factors resulting in SGA infants occur at a critical point early in development, affecting both somatic and urethral development.

Laser tissue soldering for hypospadias repair: results of a controlled prospective clinical trial.

PURPOSE: Laser tissue soldering has been shown to provide safe and effective tissue closure by creating an immediate leak-free anastomosis with minimal scar formation. We compared the results of laser tissue soldering and conventional suturing for hypospadias repair. MATERIALS AND METHODS: A consecutive group of 138 boys 4 months to 8 years old (mean age 15 months) was divided into a standard suturing (84) and a sutureless laser (54) hypospadias repair group. Urethral repair was defined as simple (Thiersch-Duplay or Snodgrass) and complex (onlay island flap or tube) in 101 and 37 cases, respectively. Laser tissue soldering was performed with 50% human albumin solder doped with 2.5 mg./ml. indocyanine green dye using an 808 nm. diode laser at 0.5 W. In the laser group sutures were used for tissue alignment only. At surgery neourethral and penile length, operative time for neourethral construction and the number of sutures or throws were measured. Postoperatively patients were examined for complications of wound healing, stricture or fistula. RESULTS: Mean patient age, urethral defect severity, type of repair, neourethral length and stenting time plus or minus standard error of mean were not significantly different in the 2 groups. Mean operative time was a fifth as long for laser tissue soldering in simple and complex hypospadias repair compared to controls (1.5 +/- 0.1 and 5.1 +/- 0.3 versus 8.5 +/- 0.8 and 26.7 +/- 1.7 minutes, respectively, p <0.001). The mean number of sutures used for tissue alignment in the laser group for simple and complex repair was significantly less than in controls (3.0 +/- 0.2 and 8.2 +/- 0.6 versus 8.5 +/- 0.8 and 23.2 +/- 1.5, respectively, p <0.001). All patients were followed a mean of 12 months (minimum 6, maximum 22). The complication rate was 4.7% in the laser group and 10.7% in controls with fistula in 2 of 54 cases, and fistula and meatal stenosis in 7 and 2 of 84, respectively. CONCLUSIONS: These preliminary results indicate that laser tissue soldering for hypospadias repair may be performed in almost sutureless fashion and more rapidly than conventional suturing. The ease of the laser technique and the lower complication rate in the laser group indicate that laser tissue soldering is an acceptable means of tissue closure in hypospadias repair.

Acute urinary retention in children.

PURPOSE: Acute urinary retention is relatively infrequent in children. There are a variety of causes that are poorly defined in the literature, and they differ greatly from those seen most frequently in adults. We review our experience with pediatric patients presenting with urinary retention to 2 major metropolitan children's hospitals. MATERIALS AND METHODS: Records from Egleston and Scottish Rite Hospitals for Children were reviewed for a 6-year period for the diagnosis of urinary retention. Urinary retention was defined as inability to empty the bladder volitionally for greater than 12 hours with a volume of urine greater than expected for age ([age in years + 2] x 30 cc) or a palpably distended bladder. All cases resulting postoperatively and believed secondary to surgical dissection, narcotic use or immobility, and children previously diagnosed with chronic neurological disorders and voiding dysfunction or with reduced mental status were excluded from study. The majority of these encounters were emergency department visits specifically for acute urinary retention. RESULTS: We identified 53 children meeting these criteria, including 37 boys 6 months to 17 years old and 16 girls 1 to 17 years old. Etiologies included neurological processes in 17%, severe voiding dysfunction in 15%, urinary tract infection in 13%, constipation in 13%, adverse drug effect in 13%, local inflammatory causes in 7%, locally invading neoplasms in 6%, benign obstructing lesions in 6%, idiopathic in 6%, combined urinary tract infection and constipation in 2%, and incarcerated inguinal hernia in 2% of cases. Adverse drug effects and dysfunctional voiding were implicated 3 times as often in males than females (16% versus 6% and 19% versus 6%, respectively). Urinary tract infections were 6 times more common in females (31% versus 5%). Constipation and local inflammatory processes were twice as common in females than males (19% versus 11% and 12% versus 5%, respectively). Local neoplasms, benign obstruction and idiopathic causes were found exclusively in males. CONCLUSIONS: Urinary retention in children is a relatively rare entity but there is a significant incidence of neurological abnormalities in this population. If a clear reason for this condition cannot be delineated based on history, physical examination and laboratory assessment, these patients should undergo prompt neurological evaluation and appropriate imaging studies.

Posterior urethral valves: pre- and postnatal management.

Posterior urethral valves are a common problem encountered by pediatric urologists. The diagnosis is most frequently suggested by antenatal screening ultrasound. A variety of pre- and postnatal parameters have been identified to aid in predicting ultimate renal outcome. These prognostic tools are invaluable to the clinician for counseling parents and for choosing appropriate management. Several approaches to the treatment of patients with posterior urethral valves exist, and the ideal strategy is debatable. As technology evolves, more options for early intervention become available. Whether early detection and antenatal intervention improve patient outcome remains to be proven.

Oral sedation allows for comfort and amnesia during office surgery.

Surgical procedures commonly associated with parenteral sedation or general anesthesia were completed in an office operating room with local anesthesia and oral medication. Diazepam and alprazolam were used in combination to produce a relaxed state, allowing local anesthesia to be instilled. Surgery was completed successfully in all patients, and no untoward effects were seen in the patients. Amnesia was associated with the drug combination, and a protective action of the long-lived drugs against lidocaine toxicity is theorized.

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins.

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.

Comparison of laparoscopic versus open nephrectomy in the pediatric population.

PURPOSE: Laparoscopic renal surgery has become an accepted approach for benign disease in adults. We compare our experience with laparoscopic and open nephrectomy in a pediatric population. MATERIALS AND METHODS: A total of 10 pediatric patients underwent laparoscopic nephrectomy or nephroureterectomy and an additional 10 consecutive children underwent similar open procedures. All patients had benign disease and were treated at a single institution. Medical records were reviewed retrospectively for relevant clinical data. RESULTS: Planned surgery was completed in all cases. There were no conversions to open surgery in the laparoscopic group. Mean operative time was 175.6 versus 120.2 minutes (p = 0.01) and mean hospital stay was 22.5 versus 41.3 hours (p = 0.03) in the laparoscopic and open nephrectomy groups, respectively. Blood loss was not statistically different. Analgesic use was qualitatively less in the laparoscopic nephrectomy group. CONCLUSIONS: Laparoscopic nephrectomy and nephroureterectomy may be performed safely in children. While operative time was somewhat longer in our initial laparoscopic series, postoperative hospital stay was significantly shorter than for open surgery. Further experience with this technique is warranted.

Bacteria of asymptomatic periradicular endodontic lesions identified by DNA-DNA hybridization.

Possible inclusion of contaminant bacteria during surgery has been problematic in studies of periradicular lesions of endodontic origin. Therefore, in this study, two different surgical techniques were compared. A second problem is that some difficult to cultivate species may not be detected using bacteriological methods. Molecular techniques may resolve this problem. DNA-DNA hybridization technology has the additional advantage that DNA is not amplified. The purpose of this investigation was to determine if bacteria from periradicular endodontic lesions could be identified using DNA-DNA hybridization. A full thickness intrasulcular mucoperiosteal (IS) flap (n = 20) or a submarginal (SM) flap (n = 16) was reflected in patients with asymptomatic apical periodontitis. DNA was extracted and incubated with 40 digoxigenin-labeled whole genomic probes. Bacterial DNA was detected in all 36 lesions. Seven probes were negative for all lesions. In patients with sinus tract communication, in teeth lacking intact full coverage crowns, and in patients with a history of trauma 4-13 probes provided positive signals. Seven probes were positive in lesions obtained by the IS, but not the SM technique. Two probes were in samples obtained with the SM technique, but not the IS. Only Bacteroides forsythus and Actinomyces naeslundii genospecies 2 were present in large numbers using either the IS or the SM technique. The SM flap technique, in combination with DNA-DNA hybridization, appeared to provide excellent data pertaining to periradicular bacteria. These results supported other studies that provide evidence of a bacterial presence and persistence in periradicular lesions.