Use of Coniothyrium minitans transformed with the hygromycin B resistance gene to study survival and infection of Sclerotinia sclerotiorum sclerotia in soil

A Coniothyrium minitans strain (T3) co-transformed with the genes for β-glucuronidase (uidA) and hygromycin phosphotransferase (hph), the latter providing resistance to the antibiotic hygromycin B, was used to investigate the survival and infection of sclerotia of Sclerotinia sclerotiorum by C. minitans over time in four different soils. Infection of sclerotia was rapid in all cases, with the behaviour of transformant T3 and wild type parent A69 being similar. Differences were seen between the soils in the rate of infection of sclerotia by C. minitans and in their indigenous fungal populations. Amendment of agar with hygromycin B enabled the quantification of C. minitans in soil by dilution plating where there was a high background of other microorganisms. In Lincoln soil from New Zealand, which had a natural but low population of C. minitans the hygromycin B resistance marker allowed the umambiguous discrimination of the applied transformed isolate from the indigenous hygromycin B sensitive one. In this soil, although the indigenous C. minitans population was detected from sclerotia, none were recovered on the dilution plates, indicating the increased sensitivity of C. minitans detection from soil using sclerotial baiting. C. minitans was a very efficient parasite, being able to infect a large proportion of sclerotia within a relatively short time from an initially low soil population. The addition of hygromycin B to agar also allowed the detection of C. minitans from decaying sclerotia by inhibiting secondary fungal colonisers. This is the first report to show that fungi colonising sclerotia already infected by C. minitans mask the detection of C. minitans from sclerotia rather than displacing the original parasite

Inter-Relationship between Testicular Dysgenesis and Leydig Cell Function in the Masculinization Programming Window in the Rat

The testicular dysgenesis syndrome (TDS) hypothesis proposes that maldevelopment of the testis, irrespective of cause, leads to malfunction of the somatic (Leydig, Sertoli) cells and consequent downstream TDS disorders. Studies in rats exposed in utero to di(n-butyl) phthalate (DBP) have strongly supported the TDS concept, but so far no direct evidence has been produced that links dysgenesis per se to somatic cell dysfunction, in particular to androgen production/action during the ‘masculinization programming window’ (MPW; e15.5–e18.5). Normal reproductive tract development and anogenital distance (AGD) are programmed within the MPW, and TDS disorders arise because of deficiencies in this programming. However, DBP-induced focal testicular dysgenesis (Leydig cell aggregation, ectopic Sertoli cells, malformed seminiferous cords) is not evident until after the MPW. Therefore, we used AGD as a read-out of androgen exposure in the MPW, and investigated if this measure was related to objectively quantified dysgenesis (Leydig cell aggregation) at e21.5 in male fetuses exposed to vehicle, DBP (500 or 750 mg/kg/day) or the synthetic glucocorticoid dexamethasone (Dex; alone or plus DBP-500) from e15.5–e18.5 (MPW), e13.5–e20.5 or e19.5–e20.5 (late window). Dysgenesis was found only in animals exposed to DBP during the MPW, and was negatively correlated (R2 = −0.5) with AGD at e21.5 and at postnatal day 8, irrespective of treatment period. Dysgenesis was also negatively correlated (R2 = –0.5) with intratesticular testosterone (ITT) at e21.5, but only when treatments in short windows (MPW, late window) were excluded; the same was true for correlation between AGD and ITT. We conclude that AGD, reflecting Leydig cell function solely within the MPW, is strongly related to focal dysgenesis. Our results point to this occurring because of a common early mechanism, targeted by DBP that determines both dysgenesis and early (during the MPW) fetal Leydig cell dysfunction. The findings provide strong validation of the TDS hypothesis

Parental occupational exposure to endocrine disrupting chemicals and male genital malformations: A study in the danish national birth cohort study

<p>Abstract</p> <p>Background</p> <p>Sex hormones closely regulate development of the male genital organs during fetal life. The hypothesis that xenobiotics may disrupt endogenous hormonal signalling has received considerable scientific attention, but human evidence is scarce.</p> <p>Objectives</p> <p>We analyse occurrence of hypospadias and cryptorchidism according to maternal and paternal occupational exposure to possible endocrine disrupting chemicals.</p> <p>Methods</p> <p>We conducted a follow-up study of 45,341 male singleton deliveries in the Danish National Birth Cohort during 1997-2009. Information on work during pregnancy was obtained by telephone interviews around gestational week 16. Parents' job titles were classified according to DISCO-88. A job exposure matrix for endocrine disrupting chemicals (EDCs) was implemented to assess occupational exposures. The Medical Birth and National Hospital Register provided data on congenital anomalies diagnosed at birth or during follow-up, which ended in 2009. Crude and adjusted hazard ratios (HR) were obtained from Cox regression models.</p> <p>Results</p> <p>Among all pregnancies, 6.3% were classified as possibly or probably exposed to EDCs. The most prevalent occupations conferring possible exposure were cleaners, laboratory technicians, hairdressers and agricultural workers (58% of all potentially exposed). The final cumulative incidence of cryptorchidism in boys was 2.2% (1002 cases), and of hypospadias 0.6% (262 cases). The occurrence of hypospadias increased when mothers were probably [HRa = 1.8 (95% CI 1.0-2.6)] or possibly exposed to one or more EDCs [HRa = 2.6 (95% CI 1.8-3.4). Possible paternal exposure to heavy metals increased the risk of hypospadias [HRa 2.2 (95% CI: 1.0-3.4)] and cryptorchidism [HRa 1.9 (95% CI: 1.1-2.7)]. None of the exposure groups reached statistical significance.</p> <p>Conclusion</p> <p>The study provides some but limited evidence that occupational exposure to possible endocrine disrupting chemicals during pregnancy increases the risk of hypospadias.</p

Masking effect of anti-androgens on androgenic activity in European river sediment unveiled by effect-directed analysis

This study shows that the androgen receptor agonistic potency is clearly concealed by the effects of androgen receptor antagonists in a total sediment extract, demonstrating that toxicity screening of total extracts is not enough to evaluate the full in vitro endocrine disrupting potential of a complex chemical mixture, as encountered in the environment. The anti-androgenic compounds were masking the activity of androgenic compounds in the extract with relatively high anti-androgenic potency, equivalent to 200 nmol flutamide equivalents/g dry weight. A two-step serial liquid chromatography fractionation of the extract successfully separated anti-androgenic compounds from androgenic compounds, resulting in a total androgenic potency of 3,820 pmol dihydrotestosterone equivalents/g dry weight. The fractionation simplified the chemical identification analysis of the original complex sample matrix. Seventeen chemical structures were tentatively identified. Polyaromatic hydrocarbons, a technical mixture of nonylphenol and dibutyl phthalate were identified to contribute to the anti-androgenic potency observed in the river sediment sample. With the GC/MS screening method applied here, no compounds with AR agonistic disrupting potencies could be identified. Seventy-one unidentified peaks, which represent potentially new endocrine disrupters, have been added to a database for future investigation

Prostaglandins, masculinization and its disorders:effects of fetal exposure of the rat to the cyclooxygenase inhibitor- indomethacin

Recent studies have established that masculinization of the male reproductive tract is programmed by androgens in a critical fetal ‘masculinization programming window’ (MPW). What is peculiar to androgen action during this period is, however, unknown. Studies from 20 years ago in mice implicated prostaglandin (PG)-mediation of androgen-induced masculinization, but this has never been followed up. We therefore investigated if PGs might mediate androgen effects in the MPW by exposing pregnant rats to indomethacin (which blocks PG production by inhibiting cyclooxygenase activity) during this period and then examining if androgen production or action (masculinization) was affected. Pregnant rats were treated with indomethacin (0.8 mg/kg/day; e15.5–e18.5) to encompass the MPW. Indomethacin exposure decreased fetal bodyweight (e21.5), testis weight (e21.5) and testicular PGE2 (e17.5, e21.5), but had no effect on intratesticular testosterone (ITT; e17.5) or anogenital index (AGI; e21.5). Postnatally, AGI, testis weight and blood testosterone were unaffected by indomethacin exposure and no cryptorchidism or hypospadias occurred. Penis length was normal in indomethacin-exposed animals at Pnd25 but was reduced by 26% (p&lt;0.001) in adulthood, an effect that is unexplained. Our results demonstrate that indomethacin can effectively decrease intra-testicular PGE2 level. However, the resulting male phenotype does not support a role for PGs in mediating androgen-induced masculinization during the MPW in rats. The contrast with previous mouse studies is unexplained but may reflect a species difference

Anogenital distance in human male and female newborns: a descriptive, cross-sectional study

BACKGROUND: In animal studies of the effects of hormonally active agents, measurement of anogenital distance (AGD) is now routine, and serves as a bioassay of fetal androgen action. Although measurement of AGD in humans has been discussed in the literature, to our knowledge it has been measured formally in only two descriptive studies of females. Because AGD has been an easy-to-measure, sensitive outcome in animals studies, we developed and implemented an anthropometric protocol for measurement of AGD in human males as well as females. METHODS: We first evaluated the reliability of the AGD measures in 20 subjects. Then measurements were taken on an additional 87 newborns (42 females, 45 males). All subjects were from Morelos, Mexico. RESULTS: The reliability (Pearson r) of the AGD measure was, for females 0.50, and for males, 0.64. The between-subject variation in AGD, however, was much greater than the variation due to measurement error. The AGD measure was about two-fold greater in males (mean, 22 mm) than in females (mean, 11 mm), and there was little overlap in the distributions for males and females. CONCLUSION: The sexual dimorphism of AGD in humans comprises prima facie evidence that this outcome may respond to in utero exposure to hormonally active agents

A clinical pathway for community-acquired pneumonia: an observational cohort study

<p>Abstract</p> <p>Background</p> <p>Six hospitals instituted a voluntary, system-wide, pathway for community acquired pneumonia (CAP). We proposed this study to determine the impact of pathway antibiotics on patient survival, hospital length of stay (LOS), and total hospital cost.</p> <p>Methods</p> <p>Data were collected for adults from six U.S. hospitals with a principal CAP discharge diagnosis code, a chest infiltrate, and medical notes indicative of CAP from 2005-2007. Pathway and non-pathway cohorts were assigned according to antibiotics received within 48 hours of admission. Pathway antibiotics included levofloxacin 750 mg monotherapy or ceftriaxone 1000 mg plus azithromycin 500 mg daily. Multivariable regression models assessed 90-day mortality, hospital LOS, total hospital cost, and total pharmacy cost.</p> <p>Results</p> <p>Overall, 792 patients met study criteria. Of these, 505 (64%) received pathway antibiotics and 287 (36%) received non-pathway antibiotics. Adjusted means and p-values were derived from Least Squares regression models that included Pneumonia Severity Index risk class, patient age, heart failure, chronic obstructive pulmonary disease, and admitting hospital as covariates. After adjustment, patients who received pathway antibiotics experienced lower adjusted 90-day mortality (<it>p </it>= 0.02), shorter mean hospital LOS (3.9 vs. 5.0 days, <it>p </it>< 0.01), lower mean hospital costs ($2,485 vs.$3,281, <it>p </it>= 0.02), and similar mean pharmacy costs ($356 vs.$442, <it>p </it>= 0.11).</p> <p>Conclusions</p> <p>Pathway antibiotics were associated with improved patient survival, hospital LOS, and total hospital cost for patients admitted to the hospital with CAP.</p