Personalizing, not patronizing: the case for patient autonomy by unbiased presentation of management options in stage I testicular cancer.

Testicular cancer (TC) is the most common neoplasm in males aged 15-40 years. The majority of patients have no evidence of metastases at diagnosis and thus have clinical stage I (CSI) disease [Oldenburg J, Fossa SD, Nuver J et al. Testicular seminoma and non-seminoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2013; 24(Suppl 6): vi125-vi132; de Wit R, Fizazi K. Controversies in the management of clinical stage I testis cancer. J Clin Oncol 2006; 24: 5482-5492.]. Management of CSI TC is controversial and options include surveillance and active treatment. Different forms of adjuvant therapy exist, including either one or two cycles of carboplatin chemotherapy or radiotherapy for seminoma and either one or two cycles of cisplatin-based chemotherapy or retroperitoneal lymph node dissection for non-seminoma. Long-term disease-specific survival is ∼99% with any of these approaches, including surveillance. While surveillance allows most patients to avoid additional treatment, adjuvant therapy markedly lowers the relapse rate. Weighing the net benefits of surveillance against those of adjuvant treatment depends on prioritizing competing aims such as avoiding unnecessary treatment, avoiding more burdensome treatment with salvage chemotherapy and minimizing the anxiety, stress and life disruption associated with relapse. Unbiased information about the advantages and disadvantages of surveillance and adjuvant treatment is a prerequisite for informed consent by the patient. In a clinical scenario like CSI TC, where different disease-management options produce indistinguishable long-term survival rates, patient values, priorities and preferences should be taken into account. In this review, we provide an overview about risk factors for relapse, potential benefits and harms of adjuvant chemotherapy and active surveillance and a rationale for involving patients in individualized decision making about their treatment rather than adopting a uniform recommendation for all.

A DNA Extraction Method for Insects From Sticky Traps: Targeting a Low Abundance Pest, Phthorimaea absoluta (Lepidoptera: Gelechiidae), in Mixed Species Communities.

Invasive insects can cause catastrophic damage to ecosystems and cost billions of dollars each year due to management expenses and lost revenue. Rapid detection is an important step to prevent invasive insects from spreading, but improvements in detection capabilities are needed for bulk collections like those from sticky traps. Here we present a bulk DNA extraction method designed for the detection of Phthorimaea absoluta Meyrick (Lepidoptera: Gelechiidae), an invasive moth that can decimate tomato crops. We test the extraction method for insect specimens on sticky traps, subjected to different temperature and humidity conditions, and among mock insect communities left in the field for up to 21 d. We find that the extraction method yielded high success (>92%) in recovering target DNA across field and lab trials, without a decline in recovery after three weeks, across all treatments. These results may have a large impact on tomato growing regions where P. absoluta is in the early stages of invasion or not yet present. The extraction method can also be used to improve detection capabilities for other bulk insect collections, especially those using sticky traps, to the benefit of pest surveys and biodiversity studies.

A multiplex real-time polymerase chain reaction assay to diagnose Epiphyas postvittana (Lepidoptera: Tortricidae).

A molecular assay for diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in North America is reported. The assay multiplexes two TaqMan real-time polymerase chain reaction (RT-PCR) probe systems that are designed to target DNA segments of the internal transcribed spacer region 2 (ITS2) and 18S rRNA gene. The RT-PCR probe designed for the 18S target recognizes a DNA sequence conserved in all of the moths included in the study and functions as a control in the assay. The second probe recognizes a segment of the ITS2 specifically found in E. postvittana and not found in the other moths included in the study, i.e., this segment is not conserved. Inclusion of the two markers in a single multiplex reaction did not affect assay performance. The assay was tested against 637 moths representing > 90 taxa in 15 tribes in all three subfamilies in the Tortricidae. The assay generated no false negatives based on analysis of 355 E. postvittana collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 282 moths representing 41 genera generated no false positives. Only three inconclusive results were generated from the 637 samples. Spike experiments demonstrated that DNA contamination in the assay can affect samples differently. Contaminated samples analyzed with the ITS2 RT-PCR assay and DNA barcode methodology by using the cytochrome oxidase I gene can generate contradictory diagnoses.

Molecular identification of the light brown apple moth (Lepidoptera: Tortricidae) in California using a polymerase chain reaction assay of the internal transcribed spacer 2 locus.

A molecular protocol using a hemi-nested polymerase chain reaction (PCR) of the internal transcribed spacer region 2 (ITS2) is reported for the diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in California. This protocol distinguishes the light brown apple moth from other moths in California based on size differences of PCR amplicons that are visualized on agarose gels. The molecular diagnostic tool generated no false negatives based on analysis of 337 light brown apple moths collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 424 moths representing other tortricid species generated correct identification for >95% of the samples and only two false positives. Of the 761 moths tested only fourteen produced no PCR amplicons and five generated inconclusive data.

In vitro sperm quality and DNA integrity of SexedULTRA™ sex-sorted sperm compared to non-sorted bovine sperm.

SexedULTRA™ is an improved method of sex-sorting sperm creating a less damaging environment to retain sperm integrity through the sorting process. The aim of this study was to evaluate the in vitro characteristics of fresh and frozen bovine sperm using the SexedULTRA™ method, and compare it to conventional (non-sorted) sperm. For both methods, percent total sperm motility was estimated visually and also classified into total and progressively motile using a computer assisted sperm analyzer (CASA). Percent sperm with intact plasma membranes (VIA) and acrosomes (PIA) were assessed using flow cytometry and sperm DNA fragmentation index (DFI) was estimated using the Bull sperm Halomax® Kit. Two contemporaneous ejaculates from 10 bulls were processed and cryopreserved using one of the two procedures (SexedULTRA™ and conventional). Sperm motility, VIA and PIA were assessed post-thaw (0 h) and post-incubation (3 h at 37 °C, 8 h and 24 h at 18 °C). DFI was analyzed post-thaw (0 h) and after 6, 24, 48 and 72 h of incubation at 37 °C. In a second experiment, ejaculates from 7 bulls were split sampled into the two types of processing (SexedULTRA™ and conventional) and diluted using a fresh semen extender developed for sex-sorted bovine sperm. Sperm quality was assessed after dilution (0 h) and after incubation for 12, 24, 48, 72 h at 18°, and the same time points of incubation at 37 °C for DFI. Frozen-thawed SexedULTRA™ sperm was significantly (P < 0.05) better than conventional semen after a 3 h incubation at 37 °C for PIA, and after a 24 h incubation at 18 °C for percent visual motility and PIA. DFI was significantly lower for SexedULTRA™ compared to conventional at all time points of incubation (37 °C). Fresh SexedULTRA™ sperm showed improved quality compared to conventional at all time points of incubation at 18 °C for percent visual and total motile sperm, VIA, PIA, and DFI. Significant differences were also found in progressive motile sperm immediately after dilution (0 h), but not at any time point after incubation. The results show that the SexedULTRA™ process maintains the quality of sex-sorted sperm and, in many cases, has better in vitro longevity than conventional semen.

Diagnosis of Lobesia botrana (Lepidoptera: Tortricidae) Using Real-Time PCR.

A real-time PCR assay is reported for identification of Lobesia botrana (Denis and Schiffermüller) collected in California. This assay multiplexes two independent TaqMan probe systems in a single reaction tube to reduce handling time and sample exposure to environmental contaminants. One probe system targets a segment of DNA located in the internal transcribed spacer region 2 (ITS2) that is present in the L. botrana genome but absent in native North American Tortricidae. The second probe system serves as a control for DNA quality by targeting a segment of the 18S rDNA gene that is conserved in L. botrana and all of the tested nontarget species. The assay successfully diagnosed 70 Lobesia botrana specimens and 95 nontarget specimens. No false-positive or false-negative results were observed supporting its application for identification of this pest in California.

A cleaved form of prolactin in the mouse pituitary gland: identification and comparison of in vitro synthesis and release in strains with high and low incidences of mammary tumors.

A form of rat PRL with a clip in its large disulfide loop, the so-called cleaved PRL, has been reported to have a greater mammogenic activity than the intact molecule. This study was undertaken to investigate the presence of cleaved PRL in the mouse pituitary gland with the purpose of correlating its concentrations with the incidence of mammary tumors. We could identify this molecule in the mouse pituitary by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, although its concentration was not high enough for ready detection in crude extracts of pituitary tissues. Cleaved mouse PRL and its 16 K and 8 K fragments cross-reacted with antibodies to the intact molecule and, thus, cannot be differentiated from the latter by RIA. When pituitaries were incubated for 6 h with 14C-labeled amino acids, cleaved PRL from the pituitaries of male mice incorporated more radioactive amino acids than the corresponding molecules from female mice. However, treatments such as ovariectomy, ovariectomy plus estradiol benzoate, perphenazine, and 2-Br-alpha-ergocryptine affected the concentration of labeled cleaved PRL in the same manner as they did that of the intact molecule. The ratio of labeled cleaved PRL to the labeled intact molecule in mice with a high incidence of mammary tumors (C3H/St) was not much different from that in mice with a low incidence (C57BL/6J) in pituitary plus medium. However, this ratio was slightly but consistently higher in medium from the C3H/St strain, raising the interesting question whether such a preponderance prevails in the circulation of this and other mammary tumor-prone strains as well.

Estrogen in high doses inhibits perphenazine-induced prolactin release.

We investigated the effects of high doses of estrogen on the basal and stimulated secretion of PRL. Daily administration of 0.1 micrograms estradiol benzoate to female mice for 4 weeks significantly increased the amplitude of PRL release induced by perphenazine. A dose of 1.0 micrograms had no stimulatory effect, while doses of 10, 20, and 50 micrograms completely prevented the release of PRL in response to perphenazine. The rise in basal serum PRL concentrations seen at lower doses of estradiol benzoate was also minimized with 20- and 50-micrigrams doses. At the same time, PRL concentrations within the pituitary gland as well as pituitary weight consistently increased in response to all high doses of estradiol benzoate tested, although the increment at the highest dose (50 micrograms) was somewhat smaller. These results suggest that prolonged administration of estrogen in large amounts may be detrimental to the natural, acute release of PRL. We postulate that this effect may account, in part, for the observed mammostatic and antigalactic effects of high doses of estrogen.

Identification of a less immunoreactive form of prolactin in the rat pituitary.

Disc electrophoretic analysis of prolactin (PRL) in the rat pituitary is usually performed using a 7.5% concentration of acrylamide gel. Re-electrophoresis of the eluate of the PRL band from such a gel on polyacrylamides of serially increasing concentrations revealed that the PRL band of 7.5% gel actually contains at least two additional proteins. One of these, whose apparent mol. wt. is 31,000, has significant pigeon crop-sac stimulating activity but very little or no cross-reactivity in a RIA for rat PRL. It also has a rapid turnover rate in short-term in vitro incubations. This newly-found protein seems to be a nonimmunoreactive molecular variant of rat PRL.

Detection of a high molecular weight variant of prolactin in human plasma by a combination of electrophoretic and immunologic techniques.

We have developed a method for detecting in human plasma a form of prolactin (PRL) with an apparent molecular weight (MW) of approximately 25,000, distinct from traditional human PRL (hPRL) which has a MW of 23,000. The method is based upon the higher MW of the variant and its ability to partially cross-react with antibody to 23K hPRL. It consists of retrieval of the protein from plasma by immunoprecipitation, its separation from 23K hPRL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer onto nitrocellulose paper by electroblotting, followed by detection with immunostaining and autoradiography. The method is sensitive enough to detect hPRL and its 25K variant in 100 microliter of human plasma. All plasma tested so far has shown the presence of the new form. The method has also revealed another protein in human plasma that cross-reacts with a specific antibody to 23K hPRL. This protein may constitute an additional post-translational variant of hPRL. The 25K variant reported here most likely represents the glycosylated form of the hormone recently detected in ovine and human pituitary glands. The method described can be adapted to detect other plasma components for which an antibody is available.

Cleaved prolactin: evidence for its occurrence in human pituitary gland and plasma.

A form of PRL that is cleaved in its large disulfide loop has been reported in rat and mouse pituitary glands, but it is not known whether it exists in the human pituitary gland and whether it circulates in blood. Recently, we developed an immunoelectrophoretic technique which is capable of measuring immunoreactive variants of PRL and other hormones in plasma. We describe here detection of cleaved PRL in normal and tumor pituitary tissue from a prolactinoma patient and in the plasma of pregnant women.

The self-association of adenosine-5'-triphosphate studied by circular dichroism at low ionic strengths.

The self-association of adenosine-5'-triphosphate (ATP) was studied as a function of pH, additional counterions, concentration and temperature. Circular dichroism measurements were employed as a measure of the base-stacking. The self-association of ATP is pH dependent with the protonation of the adenine ring helping stabilize the association. Highly charged counterions alter this aggregation. At pH 2.8 and 20 degrees C, a dimerization constant of 88 M-1 is obtained, while an isodesmic model leads to an equilibrium constant of 158 M-1. With increasing pH, the association constants decrease. At pH 2.8 there is a very strong temperature dependence of the CD amplitude. These results indicate the existence of additional electrostatic stabilization for the stacking of the adenine rings. At acidic pHs, models are proposed to explain this high degree of stability and a calculation of the approximate electrostatic contribution to the aggregation shows it to be of the proper magnitude.

Management of localized seminoma, stage I-II: SIU/ICUD Consensus Meeting on Germ Cell Tumors (GCT), Shanghai 2009.

The treatment of patients with Stage I-II seminoma has changed considerably in the past decade, and in November 2009, an International Consensus meeting was held under the sponsorship of the Union for International Cancer Control (UICC), Société Internationale d'Urologie (SIU), and International Consultation on Urological Diseases (ICUD) to review recent updates in the published data and develop international consensus guidelines on the treatment of this group of patients. In Stage I disease, the consensus conference recommended that patients should be informed of all treatment options, including the potential benefits and side effects of each treatment. It was agreed that this discussion should include a review of the possible salvage treatment effects. In addition, in patients willing and able to adhere to a surveillance program, this should be considered the management option of choice (assuming facilities are available for suitable monitoring). For Stage IIA disease, the consensus conference recommended that radiotherapy should be considered the standard treatment in the absence of contraindications. For Stage IIB disease, chemotherapy or radiotherapy were considered reasonable treatment approaches, and for Stage IIC disease, chemotherapy should be considered the standard treatment approach. For patients with a residual mass after chemotherapy, the consensus conference noted that patients with masses <3 cm in diameter could likely be safely observed, and patients with residual masses >3 cm in diameter could be considered for immediate surgery or close observation. It was also noted that surgery in this setting is technically challenging and could be associated with greater morbidity than in patients with nonseminomatous tumors.

Production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile by the combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328.

A new soil isolate, tentatively identified as Rhodococcus equi TG328, was found to be effective in the production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile. The conversion is catalysed by two enzymes. First, a nitrile hydratase converts the (R,S)-nitrile to (R,S)-2-phenylpropionamide. Second, a stereoselective amidase converts the S-(+)-amide to S-(+)-2-phenylpropionic acid. Conditions for optimal enzyme production and accumulation of S-(+)-2-phenylpropionic acid by resting cells were studied. The reaction of resting cells for 30 h at 10 degrees C with (R,S)-2-phenylpropionitrile resulted in the production of 100 g of S-(+)-2-phenylpropionic acid per litre of reaction mixture. The enantiometric excess of the purified S-(+)-2-phenylpropionic acid was 99.4%. The amount of S-(+)-2-phenylpropionic acid accumulated was enhanced by lower reaction temperatures. In addition, unreacted R-(-)-2-phenylpropionamide with 99.0% enantiometric excess was isolated.