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Dysregulated oncogenic serine/threonine kinases play a pathological role in diverse forms of malignancies, including multiple myeloma (MM), and thus represent potential therapeutic targets. Here, we evaluated the biological and functional role of p21-activated kinase 4 (PAK4) and its potential as a new target in MM for clinical applications. PAK4 promoted MM cell growth and survival via activation of MM survival signaling pathways, including the MEK-extracellular signal-regulated kinase pathway. Furthermore, treatment with orally bioavailable PAK4 allosteric modulator (KPT-9274) significantly impacted MM cell growth and survival in a large panel of MM cell lines and primary MM cells alone and in the presence of bone marrow microenvironment. Intriguingly, we have identified FGFR3 as a novel binding partner of PAK4 and observed significant activity of KPT-9274 against t(4;14)-positive MM cells. This set of data supports PAK4 as an oncogene in myeloma and provide the rationale for the clinical evaluation of PAK4 modulator in myeloma.

Introduction: To compare oncological and functional results of robot-assisted radical prostatectomy (RARP) and brachytherapy (BT) with a single-center prospective randomized study., Materials and Methods: From January 2012 to January 2014, 165 patients with low risk prostate cancer, prostate volume ≤ 50 g, normal urinary (IPSS ≤ 7 and mean flow rate ≥ 15 mL/sec) and erectile functions (IIEF-5 > 17) were enrolled and randomly assigned to the RARP or BT group. Our end points included the comparison of biochemical recurrence-free survival rates, urinary function (IPSS and EPIC scores) and potency rates (IIEF-5 score) at different time points during the first 2 years after surgery between the two groups., Results: The biochemical recurrence-free survival rates were 96.1% and 97.4% for the BT and RARP groups, respectively (p = 0.35). Significantly higher IPSS scores were assessed in the BT than in the RARP group at all the postoperative time points (p < 0.05). Significantly higher continence rates were assessed in the BT than in the RARP group during only the first 6 months of follow up (p < 0.05). Significantly lower potency rates were assessed in the BT than in the RARP group at all the postoperative time points (p < 0.05)., Conclusions: Our data showed similar biochemical recurrence-free survival rates after BT and RARP. BT patients confirmed constantly higher rates of urinary symptoms while only reporting better continence rates for the first 6 months after surgery. RARP patients reported higher potency rates than BT patients during all the follow up period.

In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753-65. ©2016 AACR., Competing Interests: STATEMENT W.C.H. is a consultant for and receives research support from Novartis., (©2016 American Association for Cancer Research.)

Significant insights into disease pathogenesis have been gleaned from population-level genetic studies; however, many loci associated with complex genetic disease contain numerous genes, and phenotypic associations cannot be assigned unequivocally. In particular, a gene-dense locus on chromosome 11 (61.5-61.65 Mb) has been associated with inflammatory bowel disease, rheumatoid arthritis, and coronary artery disease. Here, we identify TMEM258 within this locus as a central regulator of intestinal inflammation. Strikingly, Tmem258 haploinsufficient mice exhibit severe intestinal inflammation in a model of colitis. At the mechanistic level, we demonstrate that TMEM258 is a required component of the oligosaccharyltransferase complex and is essential for N-linked protein glycosylation. Consequently, homozygous deficiency of Tmem258 in colonic organoids results in unresolved endoplasmic reticulum (ER) stress culminating in apoptosis. Collectively, our results demonstrate that TMEM258 is a central mediator of ER quality control and intestinal homeostasis., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Obesity is associated with vitamin D deficiency, which can lead to serious problems during pregnancy. However, the mechanisms of the deficiency and guidelines for vitamin D supplementation during pregnancy are not established yet, and variations in environmental exposures combined with the difficulties of performing research in pregnant women are obstacles in the evaluation of vitamin D metabolism. Baboons (Papio spp.) are an excellent, well-established model for reproductive research and represent a unique opportunity to study vitamin D metabolism in a controlled environment. This study used secondary data and specimen analysis as well as a novel experimental design to evaluate pregnant and nonpregnant baboons that were or were not exposed to sunlight while they were obese and after weight reduction. Daily D3 intake was 71% higher in nonpregnant obese baboons than in their nonobese counterparts, but serum vitamin D concentrations did not differ between these populations. In addition, serum 25-hydroxyvitamin D concentrations correlated negatively with the obesity index. This report is the first to show the effect of obesity and pregnancy on vitamin D concentrations in a NHP population. These data underline the importance of adequate vitamin D supplementation in obese animals.

Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8--whose expression was increased in mutant erythroblasts, monocytes and macrophages--is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.

High cancer death rates indicate the need for new anticancer therapeutic agents. Approaches to discovering new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds through phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the gene PDE3A, encoding phosphodiesterase 3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells, whereas others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggestive of a neomorphic activity. Coexpression of SLFN12 with PDE3A correlates with DNMDP sensitivity, whereas depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.

The polymorphism ATG16L1 T300A, associated with increased risk of Crohn's disease, impairs pathogen defense mechanisms including selective autophagy, but specific pathway interactions altered by the risk allele remain unknown. Here, we use perturbational profiling of human peripheral blood cells to reveal that CLEC12A is regulated in an ATG16L1-T300A-dependent manner. Antibacterial autophagy is impaired in CLEC12A-deficient cells, and this effect is exacerbated in the presence of the ATG16L1(∗)300A risk allele. Clec12a(-/-) mice are more susceptible to Salmonella infection, supporting a role for CLEC12A in antibacterial defense pathways in vivo. CLEC12A is recruited to sites of bacterial entry, bacteria-autophagosome complexes, and sites of sterile membrane damage. Integrated genomics identified a functional interaction between CLEC12A and an E3-ubiquitin ligase complex that functions in antibacterial autophagy. These data identify CLEC12A as early adaptor molecule for antibacterial autophagy and highlight perturbational profiling as a method to elucidate defense pathways in complex genetic disease., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic β-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote β-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat β cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation., Competing Interests: Notes The authors declare no competing financial interest.

Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced interleukin-2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a previously unknown mechanism of action for a therapeutic agent: alteration of the activity of an E3 ubiquitin ligase, leading to selective degradation of specific targets.

Introduction: Percutaneous fetoscopic endoluminal reversible tracheal occlusion (FETO) was developed to prevent the pulmonary complications of fetal congenital diaphragmatic herniation. There is an urgent need to establish the closest to human translational model of FETO in order to improve fetal outcomes and to determine new clinical approaches and applications., Material and Methods: Seven non-human primates underwent two subsequent surgeries: the first, the FETO in the experimental group (n = 3) or sham operation in the control animals (S-FETO, n = 4) at 132-142 days of gestation (dGA); the second, the reversal of occlusion or sham operation at 162 ± 5 dGA. Maternal stress axis, complete blood count, and biochemical parameters were evaluated and newborn tracheal radiography was performed., Results: The average pregnancy duration and neonatal weights in the FETO group did not differ from the animals in the S-FETO group. There was no bleeding or premature fetal membrane rupture during the procedures in any of the baboons. The maximal tracheal width was 7.02 ± 0.6 mm in the FETO versus 5.46 ± 0.6 mm in S-FETO group., Discussion: This is the very first report of a successful FETO model in non-human primates. Similarities to human tracheomegaly were for the first time documented in any model studied., (© 2014 S. Karger AG, Basel.)

Introduction: The aim of this paper is to report the outcomes of 30 (first) robot-assisted pyeloplasties (RPs) performed at our institute for the treatment of ureteropelvic junction obstruction (UPJO).
, Methods: In the period between March 2005 and September 2012, 30 RPs due to UPJO have been performed at our institute using a three arms Da Vinci Robot, standard version. All the procedures were performed transperitoneally with Anderson-Hynes technique. 29/30 (97%) patients were affected by primary UPJO and 1/30 (3%) reported a recurrent disease. The access foresaw the positioning of three robotic trocars (one 12 mm trocar for the camera and two 8-mm trocars for the robotic arms) and one assistant 12 mm trocar. The anastomosis was confectioned on a double-J ureteral tutor with 4/0 monocryl running sutures. Outcome success was defined as resolution of preoperative symptoms and ureteropelvic junction obstruction, confirmed by the improvement of the diuretic renogram. Mean follow-up was 48.3 months (range 5-96).
, Results: All procedures have been completed robotically and no conversion was needed. 
Mean operative times, blood losses and hospital stay were respectively 189 minutes (range 105-420), 60 mL (range 25-100) and 4.2 days (range 2-14).
 29/30 patients were cured, with a success rate of 97%.
 As regards mild complications, 3/30 patients (10%) reported a moderate abdominal pain a few hours after surgery, which was successfully treated with common analgesic drugs.
 A s for major complications, one patient (3%) developed ileus, probably due to a mild urine extravasation through the ureteropelvic suture, which spontaneously disappeared three days after surgery.
, Conclusion: RP seems to be a "new gold standard" in the treatment of UPJO.
 The three-dimensional versatility and the outstanding accuracy of robot-assisted intracorporeal suturing produced excellent results after the first procedures without requiring a previous 'sound' laparoscopic experience.

Objectives: The aim of this study was to verify the efficacy and safety of intravesical treatment combining sodium hyaluronate (HA) and chondroitin sulfate (CS) in patients with interstitial cystitis/bladder pain syndrome (IC/BPS)., Methods: Between February 2010 and May 2011, 20 consecutive women with IC/BPS were treated with intravesical instillations containing sodium HA (1.6%; 800 mg/50 ml) and sodium CS (2%; 1 g/50 ml) weekly for the first month, biweekly for the second month, and then monthly for at least 3 months. Before and after treatment, all patients filled in the Interstitial Cystitis Symptom Index and Problem Index (ICSI/ICPI), the Patient Health Questionnaire 9 and the Pelvic Pain and Urgency/Frequency Patient Symptom Scale (PUF). Treatment efficacy was assessed by comparing the pre- and post-treatment mean scores of the three questionnaires using Student's t test (p value <0.05 was considered significant)., Results: Statistically significant mean decreases in ICSI (from 13.0 to 9.3; p = 0.0003), ICPI (from 11.35 to 8.85; p = 0.0078) and PUF (from 20.0 to 15.75; p = 0.0007) questionnaire scores were seen. No cases of side effects or complications were observed. The mean follow up was 5 months., Conclusions: Despite the limitations of this study, the outcomes confirmed the role of combination therapy with HA and CS as a safe and effective option for the treatment of IC/BPS. Further randomized controlled studies with a higher number of patients and a longer follow-up period are needed to confirm these results.

Target-identification and mechanism-of-action studies have important roles in small-molecule probe and drug discovery. Biological and technological advances have resulted in the increasing use of cell-based assays to discover new biologically active small molecules. Such studies allow small-molecule action to be tested in a more disease-relevant setting at the outset, but they require follow-up studies to determine the precise protein target or targets responsible for the observed phenotype. Target identification can be approached by direct biochemical methods, genetic interactions or computational inference. In many cases, however, combinations of approaches may be required to fully characterize on-target and off-target effects and to understand mechanisms of small-molecule action.

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: Placental abruption is a serious condition that increases perinatal morbidity and mortality. Clinical prevention and treatment options are limited, especially in human preterm deliveries. Knowledge of the mechanisms that keep the placenta in place during pregnancy is critical for developing strategies for the prevention of abruption. Failure of physiological transformation of spiral arteries has been described as a major contributing factor of the placental abruption development. Baboons (Papio spp.) share striking similarities with humans in regard to placental structure, utero-placental blood flow, and fetal development; however, the mode of trophoblast invasion is shallow in baboons. This fact prompted the hypothesis that the incidence of placental abruption will be increased in baboons compared to humans., Material and Methods: Baboon placentas were collected between 2002 and 2008. Two independent veterinary pathologists evaluated the slides. A certified physician pathologist performed additional histology., Results: Placental abruption was diagnosed in 22 baboons among 2423 live births during the study period (0.9% prevalence). The most common clinical presentations were fetal demise and vaginal bleeding. The most common pathological findings were intraplacental hemorrhages with or without hematoma formation (86.4%). Other findings consisted of neutrophil infiltration (50%), decidual necrosis (22.7%), decidual vascular congestion and inflammation, villous congestion and retroplacental hemorrhage/hematoma (each 18.2%). These pathologic findings were the same for term and preterm deliveries., Conclusion: This is the first systematic study of placental abruption in non-human primates, analyzing a large colony of baboons. Despite differences in trophoblast invasion, the clinical features observed in placental abruption affecting baboons resembled those reported in humans. The cluster of placental pathological findings in baboons also agreed with clinical reports, but the prevalence of these findings differed between baboons and humans. We discuss a mechanism of anti-abruption forces that offset shallow trophoblast invasion observed in baboons., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sequencing of the human genome in the last decade has not yet led to a concomitant increase in the numbers of novel drug targets. While the pharmaceutical industry has invested heavily in improving drugs for existing protein targets, it has not tended toward a similar investment in experimental approaches to identify cellular targets of drugs. It is striking that the targets of numerous widely used FDA-approved drugs remain unknown. The development of robust, unbiased methods for target identification would greatly enhance our understanding the mechanisms-of-action of small molecules. Cell-based phenotypic screens followed by unbiased target identification have the potential to identify novel combinations of small molecules and their protein targets, shed light on drug polypharmacology, and enable unbiased screening approaches to drug discovery. Classical biochemical enrichment with immobilized small molecules has been used for over four decades but has been limited by issues concerning specificity and sensitivity. The application of mass spectrometry-based quantitative proteomics in combination with these affinity reagents has proven to be especially useful in addressing these common issues in affinity purification experiments. We describe the use of SILAC in identifying proteins that bind small-molecule probes and drugs in a cellular context.

Background: Injury of the ureter is a potential complication of any difficult abdominopelvic surgical procedure, with an incidence ranging between 0.5% and 10% in most series. The treatment depends on the severity and the place of the lesion. The severe mid and upper ureteral injuries usually require complex treatment procedures, which can be contraindicated in case of severe comorbidities with high anesthesiologic risk. We report our experience in the endoscopic treatment with Wallgraft stenting of complete iatrogenic iliac ureteral injury in a high-risk surgical patient., Methods: A 74-year-old female patient was admitted at our department due to a complete lesion of the right iliac ureter following right iliac artery aneurysm repair with iatrogenic ileal injury. The patient was in coma, affected by severe respiratory insufficiency, sepsis and uroperitoneum. These conditions contraindicated a general anesthesia encumbering the positioning of a nephrostomy, and influenced our indication of Wallgraft stent placement. The Wallgraft endoprosthesis (10 x 50 mm) is a self-expanding super-alloy metallic high flexible stent covered by PET, which can be placed at the level of the lesion using fluoroscopy after introducing an angiographic catheter and guidewire. This procedure is usually indicated in the treatment of vascular lesions., Results: After positioning the Wallgraft stent, pyelography showed a complete closure of the lesion. The patient's clinical condition improved quickly and remained stable at a follow-up of 45 months., Conclusions: Although the Wallgraft stent positioning cannot be considered as a standard treatment for all the complete ureteral lesions, it can be proposed in the management of some patients with severe comorbidities, which do not allow any other approach.

Objective: • To retrospectively report the objective and subjective outcomes of suburethral tension adjustable sling (Remeex system) implantation for stress urinary incontinence (SUI) caused by 'true' intrinsic sphincter deficiency (ISD) with fixed urethra., Patients and Methods: • Thirty female patients with severe SUI, mainly because of iatrogenic ISD, underwent Remeex system positioning between May 2002 and July 2008 (mean follow-up 60.6 months, range 22-96 months). • Before surgery, patients were evaluated by physical examination, translabial ultrasonography, flexible cystoscopy, urodynamics, 1-hour pad test and compilation of a quality-of-life questionnaire. • Postoperatively, based on the physical examination and pad test, patients were stratified into three groups: (i) Cured: perfectly dry patients at stress test, pad weight 0-1 g; (ii) Improved: patients with mild to moderate incontinence, pad weight 2-50 g; and (iii) Failed: unchanged or worsened patients, pad weight >50 g., Results: • At the final follow-up visit, 26 (86.0%) patients were cured, 2 (7.0%) were improved and 2 (7.0%) had failed. • In particular, the total mean pad weight decreased to 33.2 ± 15.6 (71%) and the total mean questionnaire score significantly increased to 86.9 ± 6.7 (74.0%). • Sling tension readjustment was needed during follow-up in two patients (7%). • Among the complications, persistent urinary retention (10%), seroma formation (3%) and de novo urgency (7%) were easily treated., Conclusion: • The Remeex system produced remarkable 5-year results with a low complication rate. These outcomes have also been confirmed in a worse prognosis patient group as reported in the present study.

Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress., (©2011 Macmillan Publishers Limited. All rights reserved)

Objective: Oxidative stress (OS) is an important mechanism of teratogenesis. Recent work suggests increased OS in males. We evaluated whether male gender increased the risk of cyanotic congenital heart defects (CCHD) whose development is linked to OS and other common congenital anomalies (CA) in non-diabetic pregnancies., Methods: CDC-National Center for Health Statistics data for 19 states in 2006 were reviewed. CCHD, anencephaly, spina bifida, congenial diaphragmatic hernia (CDH), omphalocele, gastroschisis, limb defects, cleft lip with or without cleft palate (CL/P) and isolated cleft palate were evaluated. Adjusted odds ratio (OR) (95% CI) were calculated for CA in males with females as the reference group., Results: Of 1,194, 581, cases analyzed after exclusions, 3037 (0.25%) had major CA. Males had elevated adjusted OR (95% CI) for CCHD: 1.198 (1.027, 1.397), CDH: 1.487 (1.078, 2.051), and CL/P: 1.431 (1.24, 1.651). There was a significant interaction between cigarette use and (male) fetal gender and also with maternal age in the CL/P group., Conclusions: In non-diabetic pregnancies, male gender appears to be an independent risk factor for some types of CA believed to be associated with OS. Cigarette smoking, a well recognized source of OS only increased the risk of CL/P in males.

Objective: To evaluate the dose-response relationship (D90 >144 Gy: probable absence of biochemical failure) in patients with prostate cancer treated by iodine-125 (I-125) brachytherapy., Material and Methods: From May 1999 to December 2006, 150 patients were treated by I-125 brachytherapy. The median follow-up was 60 months. All patients had clinical stage T1-T2, PSA < or =10 ng/ml, Gleason Scores < or = 3+3=6, IPSS >14 ml/sec. and prostate weight <50 gr. Implantation was ultrasound-guided, using a real-time technique and loose seeds of I-125 (dose 160 Gy). After 30 days, a post-implantation assessment was performed by pelvic CT scan for a definitive evaluation of the D90. All patients were subjected to clinical evaluation, PSA dosage and compilation of IPSS and IEFF questionnaires. In the event of biochemical failure (ASTRO), a prostate biopsy was performed. A D90 >144 Gy was considered the cut-off in order to predict the absence of biochemical failure., Results: Biochemical failure was observed in 9 patients: 5 with positive and 4 with negative prostate biopsies. The D90 >144 Gy cut-off was not achieved in 18 patients at the post-implantation assessment, however only 2 of them (one of whom had a positive biopsy) had biochemical failure (11.1%). On the other hand, only 2 of the 9 patients with biochemical failure had a D90 < 144 Gy while 6 patients had D90 >150 Gy, 5 with positive prostate biopsies., Conclusions: In our experience, the D90 >144 Gy cut-off does not seem to predict, in a reliable way, the control of prostate cancer following brachytherapy. Limitations of the analysis were the number of the patients, the learning curve, dosimetry processing and the relatively short follow-up.

Background: Advances in mass spectrometry-based proteomics have enabled the incorporation of proteomic data into systems approaches to biology. However, development of analytical methods has lagged behind. Here we describe an empirical Bayes framework for quantitative proteomics data analysis. The method provides a statistical description of each experiment, including the number of proteins that differ in abundance between 2 samples, the experiment's statistical power to detect them, and the false-positive probability of each protein., Methodology/principal Findings: We analyzed 2 types of mass spectrometric experiments. First, we showed that the method identified the protein targets of small-molecules in affinity purification experiments with high precision. Second, we re-analyzed a mass spectrometric data set designed to identify proteins regulated by microRNAs. Our results were supported by sequence analysis of the 3' UTR regions of predicted target genes, and we found that the previously reported conclusion that a large fraction of the proteome is regulated by microRNAs was not supported by our statistical analysis of the data., Conclusions/significance: Our results highlight the importance of rigorous statistical analysis of proteomic data, and the method described here provides a statistical framework to robustly and reliably interpret such data.

The aim of this review is to critically compare the different procedures performed for the treatment of ureteropelvic junction obstruction (UPJO) in order to identify, currently, the best treatment that a urologist should propose to patients with this condition. Three different types of procedures were assessed: open pyeloplasty (OP), endopyelotomy, and laparoscopic pyeloplasty (LP). Regarding efficacy, success rates of 94.1, 62-83, and 95.9-97.2% were reported for OP, endopyelotomy, and LP, respectively. Concerning operative time and length of hospital stay, no extensive data are available in the literature, although endopyelotomy seems to provide shorter times with respect to those reported after OP and LP. Regarding the complication rate, it was very similar after the different techniques and due to the respective approaches. Overall, our data support the conclusion that LP provided a balance between the highly successful technique reported by OP and the quick postoperative recovery provided by the endoscopic approach. Anyway, in spite of these clear advantages, the reproducibility of LP is still strongly limited by the challenge of the learning curve. The da Vinci robot (Intuitive Surgical, Inc., Sunnyvale, CA), providing an extraordinary vision and precision of surgical movement, appears to be changing this scenario, allowing naïve surgeons to achieve very good results after few procedures. In this setting, robot-assisted pyeloplasty seems to be emerging as the new standard of care in the patients with UPJO, which will further take place over the other techniques once its costs decrease.

Laparoscopic pyeloplasty (LP) has proved to be an effective minimally invasive treatment for ureteropelvic junction obstruction (UPJO). However, its application is still limited by the challenge of the laparoscopic learning curve, which seems to be overcome by the recent introduction of robot assistance. The aim of this manuscript is to show our outcomes after the first robot-assisted laparoscopic pyeloplasties (RP) and critically evaluate the feasibility of this technique when performed by a surgical team without any previous laparoscopic experience. Between March 2005 and July 2008, 16 patients with UPJO underwent transperitoneal RPs. Before and after surgery patients were evaluated by ultrasonography, intravenous urography or retrograde pyelography, computed tomography (CT) scan, and/or diuretic renography. Mean follow-up was 16.8 months. The assessed outcomes were mean operative time (OT), mean estimated blood loss (EBL), mean length of hospital stay (LOS), success (SR), and complication rates (CR). OT, EBL, LOS, and SR were 202 min, 60 ml, 5.2 days, and 94%, respectively. Among the minor complications, two patients (12%) reported moderate abdominal pain while, concerning major complications, one patient (6%) developed ileus. Robot assistance was confirmed as a special tool for laparoscopic treatment of UPJO with excellent outcomes after a shorter learning curve. As shown by our results, the feasibility of RP may also be extended to naïve surgeons who can approach this technique even in the absence of previous laparoscopic training, rapidly attaining results similar to those reported by both laparoscopically experienced and expert robotic surgeons.

Purpose: We retrospectively report objective and subjective outcomes in 40 male patients who underwent bone anchored suburethral synthetic sling positioning for stress urinary incontinence due to intrinsic sphincter deficiency., Materials and Methods: Patients with stress urinary incontinence due to radical retropubic prostatectomy (32), robot assisted laparoscopic prostatectomy (3) and transurethral prostate resection (5) underwent bone anchored suburethral synthetic sling positioning between December 2002 and December 2007. Mean followup was 35.2 months (range 2 to 62). Previous anti-incontinence procedures, radiotherapy and transurethral procedures due to urethral stricture were performed in 5, 11 and 5 patients, respectively. Before and after surgery patients were evaluated by physical examination, urethral cystoscopy, urodynamics, a 1-hour pad test and a quality of life questionnaire. Patients were stratified into 3 groups, including group 1-cured (dry with a pad weight of 0 to 1 gm), group 2-improved (mild to moderate incontinence with a pad weight of 2 to 50 gm) and group 3-failed (patient condition unchanged with a pad weight of greater than 50 gm)., Results: At the final followup visit 22 (55%), 5 (12.5%) and 13 patients (32.5%) were cured, improved and failed, respectively. Mean pad weight significantly decreased to 51.3 gm in 54% of cases, while the mean total questionnaire score significantly increased to 72.9 in 65% and abdominal leak point pressure significantly increased to 92.5 cm H(2)O in 52%. Statistical analysis showed a significant association between preoperative radiotherapy and treatment failure (85% of patients). Complications were perineal pain in 73% of cases, detrusor overactivity in 5% and sling infection in 15%., Conclusions: The bone anchored suburethral synthetic sling is a simple and attractive procedure that can produce immediate good results with low morbidity, especially when strictly selected patients are treated. Radiotherapy remains a strong predictor of failure.

The RALP is the most modern technology available for the treatment of intracapsular prostate cancer (CaP), which can produce a shorter learning curve and better results than the traditional techniques. METHODS. Between March 2005 and March 2008, 90 patients (64.3 ys, range 52-71) with intracapsular CaP underwent RALP at our institute. Before surgery the patients underwent routine examinations and filled in IIEF, IPSS and EORTC-QLQC30/PR 25 questionnaires. Patients were followed up with PSA assay, physical examination and compilation of the questionnaires. Median follow-up was 12.5 months (range 1-35 months). RESULTS. Mean operative time was 230 min. Discharge and catheter removal were at day 7.4 and 8.2, respectively, after surgery. Pathological staging reported pT2 and pT3 in 57 (63%) and 33 patients (37%), respectively. Positive surgical margins were assessed in 30 patients (33%), particularly 8.7% in pT2 tumors. The one-year biochemical disease-free survival rate was 90%. Regarding the functional results, 81 patients (90%) were perfectly continent while a mild and a moderate incontinence were reported in 7 (8%) and 2 (2%) patients, respectively. Mean IPSS score decreased from 8 to 4; among the patients who underwent bilateral nerve sparing RALP and no adjuvant therapy, 31 (70.4%) reported satisfactory sexual intercourses. Concerning postoperative quality of life, mean EORTC-QLQC30/PR 25 questionnaires scores were very similar before and after RALP. CONCLUSIONS. After 90 cases of RALP the oncological and functional results are definitely promising. However, a wider number of patients and a greater follow-up are needed to confirm these data particularly as regards the functional results.

Most small-molecule probes and drugs alter cell circuitry by interacting with 1 or more proteins. A complete understanding of the interacting proteins and their associated protein complexes, whether the compounds are discovered by cell-based phenotypic or target-based screens, is extremely rare. Such a capability is expected to be highly illuminating--providing strong clues to the mechanisms used by small-molecules to achieve their recognized actions and suggesting potential unrecognized actions. We describe a powerful method combining quantitative proteomics (SILAC) with affinity enrichment to provide unbiased, robust and comprehensive identification of the proteins that bind to small-molecule probes and drugs. The method is scalable and general, requiring little optimization across different compound classes, and has already had a transformative effect on our studies of small-molecule probes. Here, we describe in full detail the application of the method to identify targets of kinase inhibitors and immunophilin binders.

Objectives: To evaluate retrospectively the objective and subjective parameters in 42 male patients who underwent bone anchored sub-urethral sling positioning (BAUS) for SUI (stress urinary incontinence) due to ISD (intrinsic sphincter deficiency)., Methods: Patients with SUI due to radical retropubic prostatectomy (36 patients), transurethral resection of prostate (5 patients) and open simple prostatectomy (1 patient) underwent BAUS positioning between July 1999 and September 2005 (mean FU = 41 months). Before and after surgery, the patients were evaluated by physical examination, urethrocystoscopy, urodynamics, 1 h pad test and QoL questionnaire. Surgical technique involved perineal implantation to the pubic rami using four anchors of a sub-urethral sling made of synthetic (26 patients), biological (4 patients) or mixed (12 patients) material. Patients were stratified into three groups: (1) Cured: dry patients at stress test, pad weight 0-1 g. (2) Improved: patients with mild-moderate incontinence, pad weight 2-50 g. (3) Failed: unchanged patients, pad weight > 50 g., Results: At the final follow-up visit cured, improved and failed patients were 26 (62%), 4 (8%) and 12 (30%), respectively. Mean pad weight significantly decreased from 104.6 to 47.3 g (55%) and mean total questionnaire score significantly increased to 50.7 (66%). Mean ALPP significantly increased to 50.4 cmH2O (44.8%). Better results were seen with synthetic slings. Main complications were perineal pain (76%), detrusor overactivity (12%) and sling infection (4.8%)., Conclusions: BAUS implantation is a safe, effective, minimally invasive option for iatrogenic male incontinence due to ISD. It compares favourably with AUS.

Objectives: To verify the objective and subjective outcomes of transobturator tape (TOT) in the treatment of female stress urinary incontinence due to urethral hypermobility., Methods: A total of 108 consecutive patients with stress urinary incontinence (mean age 58 +/- 4.5 years), who underwent the TOT procedure (43 patients received the ObTape, 55 patients the Monarc, and 10 patients the I-STOP sling) from June 2002 to December 2004, were assessed in December 2005. Before surgery, the patients were evaluated by history, physical examination, stress test, cotton swab test (Q-Tip test), and ultrasonography. After surgery, the compilation of a specific quality-of-life questionnaire was also included. Of the 108 patients, 35 had previously undergone urogynecologic surgery; associated prolapse was repaired simultaneously in 45 patients. The outcomes were analyzed considering five postoperative aspects: obstructive symptoms, irritative symptoms, urinary continence, pain, and satisfaction., Results: Postoperatively, 74% reported minimal obstructive symptoms, 78.7% had no urge symptoms, and de novo urgency occurred in 14.8%. Objective continence rates were increased significantly (80%), although the subjective rate was significantly greater (92%). Also, 88% of patients reported no pelvic pain, and only 7.3% reported dyspareunia; 88% of patients were significantly satisfied with the TOT procedure. Regarding complications, vaginal erosions were reported, using the ObTape, in 6.4% of patients, sling rejection in 3.8%, and incorrect positioning or sliding of the sling in 6.4%. Morbidity did not seem to be affected by previous or associated surgery., Conclusions: The TOT procedure is a simple, safe, and effective technique for the treatment of stress urinary incontinence due to urethral hypermobility. Our data have demonstrated good global success with a low rate of minor complications.

Purpose of Review: The biochemistry of blood coagulation has been well defined over the past 50 years. Although much is known about the sequence of the proteolytic cascade and its regulation in the pathway to fibrin generation, many important questions remain unsolved about the mechanism of initiation and the structure of the protein complexes that form during blood coagulation., Recent Findings: This article summarizes some of the advances that have been made in this field from the last quarter of 2002 and during 2003. The papers, which vary in rigor and content, have been selected on the basis of their interest and possible contribution to knowledge in this field. Summaries are given of new findings on the source of factor V and the synthesis of factor VIII, the mechanism of tissue factor action in the initiation of blood coagulation, the structure and membrane-binding properties of the protein complexes formed, and regulation of the blood coagulation cascade., Summary: Continued progress in this field offers opportunity for understanding the basis of thrombotic diseases and bleeding disorders, with the potential for defining novel targets for therapeutic applications. Some of the conclusions reviewed are conflicting, and further work will be necessary to place the results in the context of what has already been established. The structural biology of the coagulation proteins and understanding of hemostasis and thrombosis in a physiologic context have important implications for future work.

A case of a asymptomatic paraurethral leiomyoma in a young female patient is presented. Paraurethral leiomyoma is a benign hormone-dependent tumor of mesenchymal origin. Up today only 7 true paraurethral leiomyomas have been reported in the literature. The tumor can be asymptomatic, but can cause dysuria, urinary frequency, urinary retention and dispareunia. simple excision is usually the adequate treatment.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.

The target of this work is to evaluate the translabial ultrasonography (US) reliability as valid alternative to chain cystography in the pre and post operative assessment of patients with stress urinary incontinence (SUI). From June 1996 to May 1999, we studied 448 patients ranging in age from 35 to 90 years old with SUI from defect of anatomic support. Patients underwent translabial ultrasonography. The translabial US was performed with the patient in lithotomic position using a linear 7.5 MHz probe. The bladder was slightly filled, and the probe positioned longitudinally at the introitus to evaluate downwards and posterior rotation of bladder neck and urethra in basal conditions as well as during the abdominal strain. The evaluation of the anterior urethral angle of the 448 patients who underwent translabial US showed that, during the abdominal strain, all the patients with SUI had a very significative rotation of the urethral axis compared to continent women. Translabial US is a quick, simple, reliable non-invasive procedure. It may be used routinely for the pre and post-operative evaluation of anti-incontinence surgery.

Objectives: Anterolateral transabdominal incisions provide good exposure for supramesocolonic and inframescolonic surgery. However, these incisions section and denervate the rectus abdominis, oblique and transversus abdominis muscles with marked loss of active muscle control in a large number of patients. In 1974, Giuliani described an anterolateral transbdominal approach for renal tumours, which provides good visualization and good access to the renal pedicle, as well as good exposure caudally as far as the aortic bifurcation and cranially as far as the diaphragm. The authors report a new anatomical technique using this incision, which splits the muscles and preserves the nerves thereby avoiding the abdominal muscle hypotonia., Material and Methods: From March 1996 to March 1998, Giuliani's surgical incision was performed in 35 patients undergoing radical nephrectomy for renal cancer (24 on the left side and 11 on the right side). The mean age of the patients was 63.2 years (range: 42 to 80 years) and the mean follow-up was 11.6 months., Results: Tone and active control of muscles of the abdominal wall were completely preserved in all of these 35 patients. However, all patients presented a slight sensory loss in the low portion of the transverse skin incision close to the umbilicus, which improved with time and resolved completely in about 50% of cases., Conclusion: The mahor advantage of this anatomical incision compared to the conventional technique is to eliminate permanent functional deficits and hypotonia of the abdominal wall. This anatomical approach also allows easy and perfectly safe wound closure in layers, by reconstructing the anterior abdominal wall.

A total of 12 patients with completely resected, recurrent papillary tumors of the bladder were entered into a dose-finding study using intravesical idarubicin, a new anthracycline agent that has been shown in vitro to be more active than doxorubicin or daunorubicin, its parental compound. Patients were scheduled to receive eight weekly instillations with the following dose levels: 6.5, 12.5, and 20 mg, all of them diluted in 50 ml saline. Each dose level was initially studied in 3 patients. Dose escalation in the individual patients was not allowed so as to avoid undue toxicity and to evaluate the cumulative toxicity induced by each dose level. Overall, 4 patients were withdrawn due to severe local toxicity (chemocystitis) after a median of 2 instillations (range 1-3) and 3 more patients refused to continue treatment due to mild to moderate toxicity after a median of 4 instillations (range 2-4). Both the patients treated with 20 mg idarubicin and 2 of the 6 patients treated with 12.5 mg were withdrawn due to local toxicity. In contrast, no systemic toxicity was encountered at any dose level. We conclude that doses ranging from 6.5 to 12.5 mg and concentrations varying between 0.125 and 0.250 mg/ml are more appropriate for phase II studies, implying repeated instillations. At these doses and concentrations, however, it is unlikely that idarubicin might be more active than doxorubicin or epirubicin, whereas it might be more toxic.