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.

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.

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 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.

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.

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.

Ethical decision-making in critical care in Hong Kong.

OBJECTIVES: Biomedical ethics has assumed an increasingly important role in medicine over the past 30 yrs, and its development has served the important goal of protecting patients' rights and interests. However, medical ethics has evolved within a Western tradition, and conflict often arises when trying to apply Western medical ethics to patients from other cultures. Using Hong Kong as an example, this article reviews the nature and sources of cross-cultural conflict in the intensive care unit setting that often arises between physicians trained in Western medicine and patients from a Chinese cultural background. DATA SOURCES: This article draws on the first author's experience as a critical care physician in Hong Kong, and on a review of the literature on cross-cultural interactions in medicine. STUDY SELECTION: Studies were selected that contrasted the approaches of different cultures to common ethical dilemmas in medicine. Review articles examining the relationship between culture and ethics were also selected. CONCLUSIONS: Hong Kong presents an interesting case study because of the coexistence of Western and Chinese medicine in a predominantly Chinese population that practices many Chinese cultural traditions. Whereas contemporary Western medical ethics focuses on individual rights, autonomy, and self-determination, traditional Chinese societies place greater emphasis on such community values as harmony, responsibility, and respect for parents and ancestors. Specific areas of cross-cultural conflict include: the role of the patient and family in medical decision-making; the disclosure of unfavorable medical information to critically ill patients; the discussion of advance directives or code status with patients; and the withholding or withdrawal of life support.

Physician virtues and communicating with patients.

The growth of profit-driven medicine and managed care as well as the increasingly technologic focus of Western medicine have stimulated much reflection on the fundamental values of the medical profession and on the meaning of being a "good doctor." Many patients and many in the medical community have grown concerned about the fate of the doctor-patient relationship. In practicing medicine, physicians must be guided both by the basic principles of biomedical ethics and by Beauchamp's and Childress's four fundamental virtues: compassion, trustworthiness, discernment, and moral integrity. In addition, physicians must make the commitment to develop strong communication skills, for it is through communicating with patients that we forge a relationship with them and make them feel cared for. Good communication skills not only improve patient satisfaction and facilitate resolving the difficult ethical problems that arise in critical care but have also been shown to improve certain health outcomes. Unfortunately, studies have repeatedly shown physicians to have poor communication skills. In this article we identify key elements in preserving medicine's "covenant of trust" and in establishing good communication and rapport in critical care settings. We identify specific obstacles to good communication and propose strategies for overcoming them.

Whose death is it, anyway?

As medicine has increasingly gained the power to prolong life in the face of devastating illness, patients have increasingly become concerned about maintaining some control over how and when death arrives. Competent patients have the legal right to refuse treatment, but critically ill patients are frequently unable to participate in decision making. Advance directives were designed to help patients establish the level of care they would receive if they were to be rendered incompetent; yet, as the case discussed in this essay shows, even a valid advance directive does not guarantee that unwanted medical interventions will not be forced on us. The problem of physicians ignoring their patients' wishes goes beyond issues of communication and reflects an ongoing ambivalence about power and control in the physician-patient relationship. Unfortunately, many physicians find it easier to define success in terms of life and death than to try to determine what sort of existence is meaningful to an individual patient.

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.