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Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators. Keywords: Whole cell biosensor, Aromatic catabolism, Transcription factor, PcaU, Shikimate

Contains fulltext : 221797.pdf (Publisher’s version ) (Open Access) Background: Paro and other robot animals can improve wellbeing for older adults and people with dementia, through reducing depression, agitation and medication use. However, nursing and care staff we contacted expressed infection control concerns. Little related research has been published. We assessed (i) how microbiologically contaminated robot animals become during use by older people within a care home and (ii) efficacy of a cleaning procedure. Methods: This study had two stages. In stage one we assessed microbial load on eight robot animals after interaction with four care home residents, and again following cleaning by a researcher. Robot animals provided a range of shell-types, including fur, soft plastic, and solid plastic. Stage two involved a similar process with two robot animals, but a care staff member conducted cleaning. The cleaning process involved spraying with anti-bacterial product, brushing fur-type shells, followed by vigorous top-to-tail cleaning with anti-bacterial wipes on all shell types. Two samples were taken from each of eight robots in stage one and two robots in stage two (20 samples total). Samples were collected using contact plate stamping and evaluated using aerobic colony count and identification (gram stain, colony morphology, coagulase agglutination). Colony counts were measured by colony forming units per square centimetre (CFU/cm2). Results: Most robots acquired microbial loads well above an acceptable threshold of 2.5 CFU/cm2 following use. The bacteria identified were micrococcus species, coagulase negative staphylococcus, diptheriods, aerobic spore bearers, and staphylococcus aureus, all of which carry risk for human health. For all devices the CFU/cm2 reduced to well within accepted limits following cleaning by both researcher and care staff member. Conclusions: Companion robots will acquire significant levels of bacteria during normal use. The simple cleaning procedure detailed in this study reduced microbial load to acceptable levels in controlled experiments. Further work is needed in the field and to check the impact on the transmission of viruses. 17 p.

Despite being an autosomal dominant disorder caused by a known coding mutation in the gene HTT, Huntington's disease (HD) patients with similar trinucleotide repeat mutations can have an age of onset that varies by decades. One likely contributing factor is the genetic heterogeneity of patients that might modify their vulnerability to disease. We report that although the heterozygous depletion of the autophagy adaptor protein Alfy/Wdfy3 has no consequence in control mice, it significantly accelerates age of onset and progression of HD pathogenesis. Alfy is required in the adult brain for the autophagy-dependent clearance of proteinaceous deposits, and its depletion in mice and neurons derived from patient fibroblasts accelerates the aberrant accumulation of this pathological hallmark shared across adult-onset neurodegenerative diseases. These findings indicate that selectively compromising the ability to eliminate aggregated proteins is a pathogenic driver, and the selective elimination of aggregates may confer disease resistance.

SummaryThe biophysical and biochemical properties of membrane lipids can alter the conformation and function of membrane-spanning proteins, yet the specific, physiological consequence in vivo of changing the membrane milieu for a specific protein has been rarely investigated. Using various genetic approaches to eliminate expression of the membrane-associated protein Flotillin-1, we have found that the lipid environment of the dopamine transporter (DAT) is necessary for mice to respond to amphetamine but not cocaine, because the localization of DAT to cholesterol-rich membranes is required for a DAT conformation that is essential for reverse transport of dopamine. Furthermore, a conditional rather than constitutive loss-of-function approach was necessary to reveal this phenotype, indicating a broader role for membrane-protein interactions that are modulated by Flotillin-1. Taken together, these findings demonstrate how interaction of a transmembrane protein with its membrane environment can regulate distinct events in the vertebrate brain that give rise to specific behavioral outcomes.

Mammals maintain a nearly constant core body temperature (T(b)) by balancing heat production and heat dissipation. This comes at a high metabolic cost which is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering T(b) [1–6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7–10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR associated with increased hypothalamic levels of the endogenous opioid leu-enkephalin; which is derived from the KOR agonist precursor, dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independently of calorie intake. Similar results were seen with diet-induced obese mice subjected to CR. In contrast, inhibiting KOR did not change T(b) in animals fed ad libitum. Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, while chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nuclei lowered T(b). These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating T(b) and energy expenditure.

Summary: Hot flushes are a sudden feeling of warmth commonly associated with the decline of gonadal hormones at menopause. Neurons in the arcuate nucleus of the hypothalamus that express kisspeptin and neurokinin B (Kiss1ARH neurons) are candidates for mediating hot flushes because they are negatively regulated by sex hormones. We used a combination of genetic and viral technologies in mice to demonstrate that artificial activation of Kiss1ARH neurons evokes a heat-dissipation response resulting in vasodilation (flushing) and a corresponding reduction of core-body temperature in both females and males. This response is sensitized by ovariectomy. Brief activation of Kiss1ARH axon terminals in the preoptic area of the hypothalamus recapitulates this response, while pharmacological blockade of neurokinin B (NkB) receptors in the same brain region abolishes it. We conclude that transient activation of Kiss1ARH neurons following sex-hormone withdrawal contributes to the occurrence of hot flushes via NkB release in the rostral preoptic area. : The underlying cause of hot flushes is poorly understood. Padilla et al. provide evidence that a subpopulation of hypothalamic neurons can generate hot flush symptoms in mice. Establishing the mechanism of hot flush generation may allow for the development of therapies. Keywords: hot flashes, temperature regulation, neurokinin B, kisspeptin, menopause, estrogen, optogenetics, chemogenetics

cis,cis-Muconic acid is a polyunsaturated dicarboxylic acid that can be produced renewably via the biological conversion of sugars and lignin-derived aromatic compounds. Subsequently, muconic acid can be catalytically converted to adipic acid – the most commercially significant dicarboxylic acid manufactured from petroleum. Nylon-6,6 is the major industrial application for adipic acid, consuming 85% of market demand; however, high purity adipic acid (99.8%) is required for polymer synthesis. As such, process technologies are needed to effectively separate and catalytically transform biologically derived muconic acid to adipic acid in high purity over stable catalytic materials. To that end, this study: (1) demonstrates bioreactor production of muconate at 34.5 g L−1 in an engineered strain of Pseudomonas putida KT2440, (2) examines the staged recovery of muconic acid from culture media, (3) screens platinum group metals (e.g., Pd, Pt, Rh, Ru) for activity and leaching stability on activated carbon (AC) and silica supports, (4) evaluates the time-on-stream performance of Rh/AC in a trickle bed reactor, and (5) demonstrates the polymerization of bio-adipic acid to nylon-6,6. Separation experiments confirmed AC effectively removed broth color compounds, but subsequent pH/temperature shift crystallization resulted in significant levels of Na, P, K, S and N in the crystallized product. Ethanol dissolution of muconic acid precipitated bulk salts, achieving a purity of 99.8%. Batch catalysis screening reactions determined that Rh and Pd were both highly active compared to Pt and Ru, but Pd leached significantly (1–9%) from both AC and silica supports. Testing of Rh/AC in a continuous trickle bed reactor for 100 h confirmed stable performance after 24 h, although organic adsorption resulted in reduced steady-state activity. Lastly, polymerization of bio-adipic acid with hexamethyldiamine produced nylon-6,6 with comparable properties to its petrochemical counterpart, thereby demonstrating a path towards bio-based nylon production via muconic acid.

Background Extended-release naltrexone (XR-NTX) is an injectable monthly sustained-release mu opioid receptor antagonist, which blocks the typical effects of heroin and other opioid agonists. Use of XR-NTX among opioid dependent persons leaving jails and prisons is increasing despite scant high-quality evidence regarding XR-NTX's effectiveness at re-entry. Methods This 24-week, open-label randomized controlled trial examines the effectiveness of XR-NTX as opioid relapse prevention at release from jail (N = 85) compared to enhanced treatment as usual (ETAU, N = 85). A third, non-randomized, quasi-experimental naturalistic arm of participants who have newly initiated a jail-to-community methadone treatment program (MTP, N = 85) allows for comparisons to a methadone standard-of-care. Results We describe the rationale, design, and primary and secondary outcomes of the study. The primary outcome is an opioid relapse event; the primary contrast is a time-to-relapse comparison of XR-NTX and ETAU over a 24-week treatment phase. Secondary outcomes are rates of: (a) post-release opioid treatment participation, (b) opioid, alcohol, and cocaine use, (c) injection drug use and HIV sexual risk behaviors, (d) overdose (fatal and non-fatal) and all-cause mortality, and, (e) re-incarceration. Conclusions XR-NTX is a potentially important, effective treatment and relapse prevention option for a large US population of persons with opioid use disorders leaving jails. This study will estimate XR-NTX's effectiveness relative to existing standards of care, including counseling-only treatment-as-usual and methadone maintenance.

OBJECTIVES More elderly patients (80 years of age) are being referred for aortic valve replacement (AVR) with or without CABG. Current risk stratification models may not accurately predict the preoperative risk in these patients. We sought to determine which perioperative variables were relevant in determining short-term (30-day to in-hospital) outcomes in our intuition's series of consecutive AVR and AVR+CABG surgeries. We constructed a novel variable, patient-prosthesis mismatch (PPM) in the presence of diminished functional status (NYHA) classification, and studied its role as a predictor of mortality risk. METHODS From 2006 to 2010, 509 patients undergoing AVR or AVR+CABG were evaluated. We created four groups based on the age and procedure (AVR80, AVR+CABG80, AVR80 and AVR+CABG80). PPM was defined as a calculated effective orifice area index value of ≤ 0.85, and it was calculated from manufacturer-generated charts. In-hospital and 30-day outcomes were assessed using the Chi-square and logistic regression analyses. RESULTS Overall observed 30-day mortality for all groups was lower (n = 8, 1.6%) than the STS-predicted mortality. Reoperation and PPM+NYHA class III-IV were associated with short-term mortality, but age80 years was not. Octogenarians referred for surgery often had advanced heart failure. CONCLUSIONS Overall, short-term outcomes after AVR with or without CABG were excellent and lower than predicted by the STS model. The low risk of AVR with CABG supports the consideration for earlier surgical referral and intervention for patients with a high likelihood of aortic stenosis progression before the onset of advanced heart failure ensues, regardless of the age. This should help further decrease the already very low mortality observed in these series. Efforts to avoid PPM in the setting of advanced heart failure may improve short-term results in this subset of patients.

A two-vessel umbilical cord (or single umbilical artery) is a perinatal finding that may be detected in infants with congenital or chromosomal abnormalities. Its presence has been associated with an increased risk of genitourinary defects. The urologic anomalies that typically are associated with a two-vessel cord often are minor and self-limiting. These defects usually can be detected perinatally in routine prenatal screening ultrasonographic examinations. The neonatal physical examination, an additional postnatal ultrasonography, and possibly a voiding cystourethrogram will be sufficient to complete the screening for genitourinary abnormalities in children with a single umbilical artery. Infants with two-vessel umbilical cords do not require additional screening for genitourinary defects, unless the imaging or amniocentesis detects a genitourinary abnormality. This review details the history of two-vessel cords and the associated genitourinary anomalies.

Nicotine binds to and activates a family of ligand-gated ion channels, neuronal nicotinic acetylcholine receptors (nAChRs). Chronic nicotine exposure alters the expression of various nAChR subtypes, which likely contributes to nicotine dependence; however, the underlying mechanisms regulating these changes remain unclear. A growing body of evidence indicates that microRNAs (miRNAs) may be involved in nAChR regulation. Using bioinformatics, miRNA library screening, site-directed mutagenesis, and gene expression analysis, we have identified a limited number of miRNAs that functionally interact with the 3′-untranslated regions (3′ UTRs) of mammalian neuronal nAChR subunit genes. In silico analyses revealed specific, evolutionarily conserved sites within the 3′ UTRs through which the miRNAs regulate gene expression. Mutating these sites disrupted miRNA regulation confirming the in silico predictions. In addition, the miRNAs that target nAChR 3′ UTRs are expressed in mouse brain and are regulated by chronic nicotine exposure. Furthermore, we show that expression of one of these miRNAs, miR-542-3p, is modulated by nicotine within the mesocorticolimbic reward pathway. Importantly, overexpression of miR-542-3p led to a decrease in the protein levels of its target, the nAChR β2 subunit. Bioinformatic analysis suggests that a number of the miRNAs play a general role in regulating cholinergic signaling. Our results provide evidence for a novel mode of nicotine-mediated regulation of the mammalian nAChR gene family.

Platelet-activating factor (PAF) is an early product of the inflammatory environment, influencing development and resolution of inflammation. Its production is greater in neutrophils and macrophages, which predominantly synthesize 1-alkyl sn-2 acetyl glycerophosphocholine (GPC) than in non-granulocytes (B cells and endothelial cells), which lack a respiratory burst and synthesize 1-acyl sn-2 acetyl GPC as their major PAF species. This study investigated whether the respiratory burst was responsible for the quantitative and qualitative differences in sn-2 acetyl GPC species generation by neutrophils and macrophages versus those cells lacking the NADPH oxidase complex. The myeloid cell line PLB-985 (capable of differentiation into neutrophils) was used to test this hypothesis, since these cells had previously been generated with a non-functional respiratory burst (X-CGD PLB-985). Differentiated PLB-985 cells underwent a large respiratory burst in response to PMA (phorbol ester), and smaller respiratory bursts in response to A23187 (calcium ionophore), and the bacterial polypeptide fMLP (receptor mediated activation). Concurrently, treated cells were assessed for production of 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC species by gas chromatography/mass spectrometry. Neither cell type generated these lipid species in response to PMA, but both cell types generated equal levels of sn-2 acetyl GPC in response to A23187, with five times more 1-hexadecyl than 1-palmitoyl species. Upon fMLP activation, X-CGD PLB-985 cells produced significantly less 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC in comparison to the wild-type PLB-985 cells. These findings suggest phagocytic oxidant production by NADPH oxidase is not essential for sn-2 acetyl GPC generation, but appears important for optimal production of PAF in response to some stimuli.

Purpose: Cryosurgical ablation of the prostate has been reported as potential treatment for radioresistant clinically localized prostate cancer. We report our experience with the safety and efficacy of salvage cryosurgery using the argon based CRYOCare system (Endocare, Inc, Irvine, California).Materials and Methods: Between October 1997 and September 2000, 38 men with a mean age of 71.9 years underwent salvage cryosurgery for recurrent prostate cancer after radiation therapy failed. All patients had biochemical disease recurrence, defined as an increase in prostate specific antigen (PSA) of greater than 0.3 ng./ml. above the post-radiation PSA nadir. Subsequently prostate biopsy was positive for cancer. Pre-cryosurgery bone scan demonstrated no evidence of metastatic disease. In addition, these patients received 3 months of neoadjuvant androgen deprivation therapy before cryotherapy.Results: The PSA nadir was 0.1 or less, 1 or less and greater than 1 ng./ml. in 31 (81.5%), 5 (13.2%) and 2 (5.3%) patients...

Clinically relevant varicoceles require surgical management through one of several techniques. We introduce an innovative technique for varicocelectomy via a subinguinal approach that allows identification and preservation of the arteries and lymphatics. This method allows the safe excision of the varicocele, while minimizing the risk of complications.

Over this past decade, macroautophagy has gained prominence in the field of adult-onset neurodegeneration: from sporadic disorders such as Alzheimer’s and Parkinson’s disease, to genetic disorders such as Huntington’s disease and frontotemporal dementia, the influence of this fundamental pathway has become an important topic of discussion. While there has been particular emphasis on the potential benefits of macroautophagy, there is growing literature that also suggests that macroautophagy contributes towards neurotoxicity. In this review, we discuss the molecular mechanism of macroautophagy and the currently available pharmacological tools, with special emphasis on mammalian macroautophagy in adult brain. Studies indicate that neuronal context strongly influences the role macroautophagy plays in maintaining cellular health, reflecting an ongoing need for better understanding of how macroautophagic regulation is achieved in the heavily differentiated and polarized neurons if we are to effectively manipulate it to treat neurodegenerative disease.

Invertebrate and vertebrate vestigial (vg) and vestigial-like (VGLL) genes are involved in embryonic patterning and cell fate determination. These genes encode cofactors that interact with members of the Scalloped/TEAD family of transcription factors and modulate their activity. We have previously shown that, in mice, Vgll2 is differentially expressed in the developing facial prominences. In this study, we show that the zebrafish ortholog vgll2a is expressed in the pharyngeal endoderm and ectoderm surrounding the neural crest derived mesenchyme of the pharyngeal arches. Moreover, both the FGF and retinoic acid (RA) signaling pathways, which are critical components of the hierarchy controlling craniofacial patterning, regulate this domain of vgll2a expression. Consistent with these observations, vgll2a is required within the pharyngeal endoderm for NCC survival and pharyngeal cartilage development. Specifically, knockdown of Vgll2a in zebrafish embryos using Morpholino injection results in increased cell death within the pharyngeal arches, aberrant endodermal pouch morphogenesis, and hypoplastic cranial cartilages. Overall, our data reveal a novel non-cell autonomous role for Vgll2a in development of the NCC-derived vertebrate craniofacial skeleton.

BACKGROUND: Frontline treatment of small cell lung carcinoma (SCLC) relies heavily on chemotherapeutic agents and radiation therapy. Though SCLC patients respond well to initial cycles of chemotherapy, they eventually develop resistance. Identification of novel therapies against SCLC is therefore imperative. METHODS AND FINDINGS: We have designed a bioluminescence-based cell viability assay for high-throughput screening of anti-SCLC agents. The assay was first validated via standard pharmacological agents and RNA interference using two human SCLC cell lines. We then utilized the assay in a high-throughput screen using the LOPAC(1280) compound library. The screening identified several drugs that target classic cancer signaling pathways as well as neuroendocrine markers in SCLC. In particular, perturbation of dopaminergic and serotonergic signaling inhibits SCLC cell viability. CONCLUSIONS: The convergence of our pharmacological data with key SCLC pathway components reiterates the importance of neurotransmitter signaling in SCLC etiology and points to possible leads for drug development.

Nicotine dependence is a psychiatric disorder characterized by a recurrent, periodic compulsion to use tobacco due to neurophysiological, psychological, and social factors. This disorder has behavioral and physiological characteristics that are similar to those of other addictions, but also unique aspects that require special attention because of its ubiquity on a global scale, its staggering effects on rates of morbidity and mortality, and its high prevalence of psychological comorbidities, including psychiatric disorders. Our chapter focuses on the epidemiology of comorbid nicotine dependence and psychiatric disorders, the underlying biobehavioral mechanisms of comorbidity, cultural factors, psychiatric disorders most often associated with nicotine dependence, and treatment approaches. While there is a need for ongoing studies to more clearly delineate the best treatment interventions for persons with comorbid psychiatric disorders and nicotine dependence, the evidence to date suggests use of all of the medication, behavioral, and peer-support approaches available. Culture changes are needed in mental health and addiction treatment settings to continue to improve access to effective interventions for persons with nicotine dependence and psychological comorbidities.

Nkx2.2 is an essential regulator of pancreatic endocrine differentiation. Nkx2.2-null mice are completely devoid of β-ells and have a large reduction of α- and PP cells. In the place of these islet populations, there is a corresponding increase in the ghrelin-positive ε-cells. Molecular studies have indicated that Nkx2.2 functions as an activator and repressor to regulate islet cell fate decisions. To determine whether Nkx2.2 is solely important for islet cell fate decisions or also has the capability to control ghrelin at the promoter level, we studied the transcriptional regulation of the ghrelin promoter within the pancreas, in vitro and in vivo. These studies demonstrate that both of the previously identified transcriptional start sites in the ghrelin promoter are active within the embryonic pancreas; however, the long transcript is preferentially up-regulated in the Nkx2.2-null pancreas. We also show that the promoter region between −619 and −488 bp upstream of the translational start site is necessary for repression of ghrelin in αTC1 and βTC6 cells. Surprisingly, we also show that Nkx2.2 is able to bind to and activate the ghrelin promoter in several cell lines that do or do not express endogenous ghrelin. Together, these results suggest that the up-regulation of ghrelin expression in the Nkx2.2-null mice is not due to loss of repression of the ghrelin promoter in the nonghrelin islet populations. Furthermore, Nkx2.2 may contribute to the activation of ghrelin in mature islet ε-cells.

OBJECTIVES To report our experience with transurethral resection of the ejaculatory ducts (TURED) in infertile men with symptomatic ejaculatory duct obstruction (EDO). PATIENTS AND METHODS We retrospectively reviewed the operative cases of one urologist from 1995 to 2001, identifying 15 patients with symptomatic EDO who underwent TURED. These men had normal hormone levels and no other known genitourinary dysfunction. Investigations included a history, physical examination, semen analysis, semen culture, and high-resolution transrectal ultrasonography. Responses to focused questions and semen analysis were evaluated after surgery. RESULTS Before surgery, all patients complained of a decrease in the volume of their ejaculate, 14 of 15 had a non-projectile ejaculation, nine had a genitourinary infection necessitating antibiotic treatment, and five had pain with orgasm. The mean ejaculate volume and total motile sperm count was 1.1 mL and 8.1 million sperm per ejaculate. After surgery, at a mean follow-up of 2 months, 10 men reported having projectile ejaculation, and eight reported a marked improvement in their sensation of orgasm. Overall, 14 men reported a subjective improvement in their ejaculation. The average postoperative ejaculate volume was 2.3 mL and the total motile sperm count was 38.1 million per ejaculate. CONCLUSIONS Men with symptomatic EDO who underwent TURED showed improvements in their ejaculation, sensation of orgasm, semen analysis values and fertility.

Objectives To evaluate and study the factors for progression after radical prostatectomy with seminal vesicle invasion in a cohort of European and American patients. Methods The data of 137 patients with isolated seminal vesicle invasion who underwent radical retropubic prostatectomy between 1988 and 2001 were analyzed. The Gleason score of the specimen, presence of capsular invasion, and surgical margin status were noted. Follow-up data were obtained through routine serum prostate-specific antigen (PSA) assays and digital rectal examinations. A defining criterion for progression was a postoperative serum PSA level greater than 0.2 ng/mL or any postoperative radiotherapy or hormonal treatment. Kaplan-Meier analysis was used to determine the actuarial biochemical recurrence-free likelihood, and the log-rank test was used to compare these results. Differences were considered statistically significant when the P value was less than 0.05. Results After a mean follow-up of 4.9 years (range 0.9 to 13.4), 70 patients (51.9%) had progression. The biochemical 5-year progression-free survival rate was 33.8%. In univariate and multivariate analyses, only preoperative PSA level ( P = 0.001) and Gleason score of the specimen ( P = 0.01) were independent predictors of progression. Neither capsular invasion nor positive surgical margins predicted progression. When an analysis was performed according to the major Gleason grade of the radical prostatectomy specimen, Gleason grade 5 was associated with a worse prognosis compared with Gleason grade 3 and 4 ( P = 0.01). The mean time to progression was 20.5, 17.1, and 10.1 months for Gleason grade 3, 4, and 5, respectively. Conclusions Seminal vesicle invasion after radical prostatectomy has historically been associated with a poor prognosis. However, in the present study, seminal vesicle invasion was associated with a 34% rate of freedom from progression at 5 years after surgery alone. Preoperative PSA and Gleason score of the radical prostatectomy specimen were independent factors for progression in the present study, which described the largest patient group to date. The Gleason grade of the radical prostatectomy specimen distinguished among different times to progression.

We surveyed the growing literature on ejaculatory duct obstruction and provide suggestions regarding its diagnosis and management.Ejaculatory duct obstruction is a rare cause of male infertility. With the advent of the high resolution transurethral ultrasound (TRUS) technology, there has been an increase in diagnosis of this disorder. As for the treatment, it appears that central cystic lesions and partial obstructions respond best to transurethral resection of the ejaculatory ducts (TURED).Ejaculatory duct obstruction is a rare but surgically correctable cause of male infertility. Although there are no pathognomonic findings associated with ejaculatory duct obstruction, the diagnosis should be suspected in an infertile male with oligospermia or azoospermia with low ejaculate volume, normal secondary sex characteristics, testes, and hormonal profile, and dilated seminal vesicles, midline cyst, or calcifications on TRUS. In select cases, TURED has resulted in marked improvement in semen parameters, and pregnancies have been achieved. More studies are needed in the areas of diagnosis and long-term surgical outcome.

Chronic granulomatous disease is the manifestation of genetic defects of the leukocyte NADPH oxidase resulting in the absence of a respiratory burst. Patients with chronic granulomatous disease can develop chronic granulomas in many locations of the body, including the skin. Using an established murine model of X-linked chronic granulomatous disease (X-CGD) created by homologous recombinant disruption of the gene encoding the gp91phox component of the NADPH oxidase, in this study we examined cutaneous reactivity to sterile Aspergillus fumigatus hyphae. Injection of Aspergillus fumigatus into the dorsal ears of X-CGD mice resulted in an enhanced inflammatory response by 24 h, consisting of neutrophils, which developed into suppurative granulomas by 10 d. Intradermal injection of Aspergillus fumigatus into wild-type mice only resulted in a transient inflammatory response that resolved by 10 d. Injection of Aspergillus fumigatus into female carrier mice resulted in an acute inflammatory response that was similar to that of wild-type mice, but, at higher doses of Aspergillus fumigatus, many carriers subsequently developed granulomatous lesions that were qualitatively similar but smaller than those seen in X-CGD mice by 30 d. Consistent with the ability of X-CGD mice to mount an enhanced neutrophil-rich inflammatory response to Aspergillus fumigatus, significant levels of the potent neutrophil activator/chemoattractant leukotriene B4 were measured by mass spectrometry in skin biopsies at 24 and 72 h. In contrast to the exaggerated inflammatory response to intradermal Aspergillus fumigatus in X-CGD mice compared to their wild-type counterparts, similar levels of inflammation were seen in a model of delayed-type hypersensitivity using 2,4-dinitrofluorobenzene. This study represents the first report of a cutaneous granuloma model in mice with X-CGD, which may also prove useful as a functional test to evaluate the efficacy of gene therapy protocols being developed for chronic granulomatous disease.

Unilateral ureteral obstruction (UUO) is characterized by decreases in renal function and increases in interstitial fibrosis. Previous studies have indicated that pharmacologic manipulations that increase nitric oxide (NO) are beneficial to the obstructed kidneys. NO is produced from arginine by nitric oxide synthase (NOS), an enzyme that exists in both constitutive and inducible (iNOS) forms. To determine the role of the inducible form of NOS in UUO, we used mice with a targeted deletion of iNOS (iNOS -/- mice) and compared them with wild-type (WT) mice. Kidneys were obstructed for 2 weeks in both WT and iNOS -/- mice, and were then removed and bisected. Half of the kidney was embedded in paraffin and tissue sections were examined for interstitial volume or the presence of macrophages. The remainder was flash-frozen and samples were used to measure tissue collagen (hydroxyproline) or transforming growth factor-beta (TGF-beta). This study demonstrates that both cortex and medulla of obstructed kidneys of iNOS -/- mice exhibit significantly increased interstitial volume and interstitial macrophages as compared with their WT counterparts. Furthermore tissue collagen was increased to 9.2+/-1.3 microg/mg tissue in WT obstructed kidneys, whereas in iNOS -/- kidneys, collagen was increased to 13.2+/-0.8 microg/mg tissue. The profibrotic cytokine TGF-beta was also significantly increased in obstructed kidneys of iNOS -/- mice, as compared with WT mice. No differences were noted between the unobstructed kidneys of iNOS -/- mice compared with WT mice in any of the parameters examined. These results demonstrate that targeted deletion of the iNOS results in exacerbation of fibrotic events in the obstructed kidney. These results confirm previous pharmacologic studies, and suggest that NO produced via the inducible NOS normally serves a protective function in UUO.

Evidence is accumulating suggesting that platelet-activating factor plays a role in inflammatory dermatoses. Mass spectrometric methods were used to examine the molecular species of sn-2 acetyl glycerophosphocholines (GPC) synthesized by primary cultures of human neonatal foreskin-derived keratinocytes. Ionophore-stimulated keratinocytes synthesize both 1-alkyl and 1-acyl sn-2 acetyl-GPC, and the relative amounts were as follows: hexadecyl > palmitoyl > octadecyl > stearoyl at the sn-1 position. PAF synthesis in the keratinocyte-derived cell line HaCaT was inhibited by dexamethasone, suggesting that the anti-inflammatory effects of glucocorticosteroids in inflammatory dermatoses might be in part related to the inhibition of the synthesis of mediators such as PAF.