Application of environmental DNA to detect an endangered marine skate species in the wild.

Environmental DNA (eDNA) techniques have only recently been applied in the marine environment to detect the presence of marine species. Species-specific primers and probes were designed to detect the eDNA of the endangered Maugean skate (Zearaja maugeana) from as little as 1 L of water collected at depth (10-15 m) in Macquarie Harbour (MH), Tasmania. The identity of the eDNA was confirmed as Z. maugeana by sequencing the qPCR products and aligning these with the target sequence for a 100% match. This result has validated the use of this eDNA technique for detecting a rare species, Z. maugeana, in the wild. Being able to investigate the presence, and possibly the abundance, of Z. maugeana in MH and Bathurst harbour (BH), would be addressing a conservation imperative for the endangered Z. maugeana. For future application of this technique in the field, the rate of decay was determined for Z. maugeana eDNA under ambient dissolved oxygen (DO) levels (55% saturation) and lower DO (20% saturation) levels, revealing that the eDNA can be detected for 4 and 16 hours respectively, after which eDNA concentration drops below the detection threshold of the assay. With the rate of decay being influenced by starting eDNA concentrations, it is recommended that samples be filtered as soon as possible after collection to minimize further loss of eDNA prior to and during sample processing.

Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death.

Leydig cells are crucial to the production of testosterone in males. It is unknown if the cancer chemotherapeutic drug, 6-mercaptopurine (6 MP), produces Leydig cell failure among adult survivors of childhood acute lymphoblastic leukemia. Moreover, it is not known whether Leydig cell failure is due to either a loss of cells or an impairment in their function. Herein, we show, in a subset of childhood cancer survivors, that Leydig cell failure is related to the dose of 6 MP. This was extended, in a murine model, to demonstrate that 6 MP exposure induced caspase 3 activation, and the loss of Leydig cells was independent of Bak and Bax activation. The death of these non-proliferating cells was triggered by 6 MP metabolism, requiring formation of both cytosolic reactive oxygen species and thiopurine nucleotide triphosphates. The thiopurine nucleotide triphosphates (with physiological amounts of dATP) uniquely activated the apoptosome. An ABC transporter (Abcc4/Mrp4) reduced the amount of thiopurines, thereby providing protection for Leydig cells. The studies reported here demonstrate that the apoptosome is uniquely activated by thiopurine nucleotides and suggest that 6 MP induced Leydig cell death is likely a cause of Leydig cell failure in some survivors of childhood cancer.

Cyclic nucleotide compartmentalization: contributions of phosphodiesterases and ATP-binding cassette transporters.

Cyclic nucleotides [e.g., cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)] are ubiquitous second messengers that affect multiple cell functions from maturation of the egg to cell division, growth, differentiation, and death. The concentration of cAMP can be regulated by processes within membrane domains (local regulation) as well as throughout a cell (global regulation). The phosphodiesterases (PDEs) that degrade cAMP have well-known roles in both these processes. It has recently been discovered that ATP-binding cassette (ABC) transporters contribute to both local and global regulation of cAMP. This regulation may require the formation of macromolecular complexes. Some of these transporters are ubiquitously expressed, whereas others are more tissue restricted. Because some PDE inhibitors are also ABC transporter inhibitors, it is conceivable that the therapeutic benefits of their use result from the combined inhibition of both PDEs and ABC transporters. Deciphering the individual contributions of PDEs and ABC transporters to such drug effects may lead to improved therapeutic benefits.

Back to the future: reducing reliance on torso computed tomography in the initial evaluation of blunt trauma.

BACKGROUND: Reliance on chest-abdomen-pelvis computed tomography (CAP) in the initial evaluation of blunt trauma is a major source of patient radiation exposure. Our trauma surgeon group (TSG) modified its practice to limit the use of CAP. We evaluated the effect of this practice change on patient radiation exposure and diagnostic accuracy. METHODS: We compared data on blunt injury trauma activations evaluated by the five-member TSG for two 6-month intervals, before (T1) and after (T2) instituting the practice change. Patient demographic and injury data, complications, torso imaging and radiation dosage were collected. Following analysis of T1, the surgeon with the lowest CAP use was identified and found to have no errors or delays in diagnosis. The TSG agreed to adopt that surgeon's focus on findings of the physical examination and Focused Assessment Sonography for Trauma to reduce CAP use in the initial evaluation. T2 was analyzed to assess the effect of implementation of this guideline. RESULTS: There were 897 patients in T1 and 948 in T2. In the two intervals, patients did not differ by age, sex, mortality, or probability of survival. CAP use decreased by 38.5% with a significant drop in mean patient radiation exposure (p < 0.001). There were no missed injuries or delays in diagnosis in either interval. CONCLUSION: The use of CAP and its associated radiation burden in the initial evaluation of blunt trauma can be reduced without diagnostic errors by comparing use and identifying best practice. This process has implications for optimal trauma care. LEVEL OF EVIDENCE: Diagnostic study, level IV; case management study, level IV.

Deregulated hepatic metabolism exacerbates impaired testosterone production in Mrp4-deficient mice.

The physiological role of multidrug resistance protein 4 (Mrp4, Abcc4) in the testes is unknown. We found that Mrp4 is expressed primarily in mouse and human Leydig cells; however, there is no current evidence that Mrp4 regulates testosterone production. We investigated its role in Leydig cells, where testosterone production is regulated by cAMP, an intracellular messenger formed when the luteinizing hormone (LH) receptor is activated. Because Mrp4 regulates cAMP, we compared testosterone levels in Mrp4(-/-) and Mrp4(+/+) mice. Young Mrp4(-/-) mice had significantly impaired gametogenesis, reduced testicular testosterone, and disruption of Leydig cell cAMP homeostasis. Both young and adult mice had impaired testosterone production. In Mrp4(-/-) primary Leydig cells treated with LH, intracellular cAMP production was impaired and cAMP-response element-binding protein (CREB) phosphorylation was strongly attenuated. Notably, expression of CREB target genes that regulate testosterone biosynthesis was reduced in Mrp4(-/-) Leydig cells in vivo. Therefore, Mrp4 is required for normal Leydig cell testosterone production. However, adult Mrp4(-/-) mice are fertile, with a normal circulating testosterone concentration. The difference is that in 3-week-old Mrp4(-/-) mice, disruption of gonadal testosterone production up-regulates hepatic Cyp2b10, a known testosterone-metabolizing enzyme. Therefore, defective testicular testosterone production de-regulates hepatic Cyp-mediated testosterone metabolism to disrupt gametogenesis. These findings have important implications for understanding the side effects of therapeutics that disrupt Mrp4 function and are reported to alter androgen production.

Caribbean corals in crisis: record thermal stress, bleaching, and mortality in 2005.

BACKGROUND: The rising temperature of the world's oceans has become a major threat to coral reefs globally as the severity and frequency of mass coral bleaching and mortality events increase. In 2005, high ocean temperatures in the tropical Atlantic and Caribbean resulted in the most severe bleaching event ever recorded in the basin. METHODOLOGY/PRINCIPAL FINDINGS: Satellite-based tools provided warnings for coral reef managers and scientists, guiding both the timing and location of researchers' field observations as anomalously warm conditions developed and spread across the greater Caribbean region from June to October 2005. Field surveys of bleaching and mortality exceeded prior efforts in detail and extent, and provided a new standard for documenting the effects of bleaching and for testing nowcast and forecast products. Collaborators from 22 countries undertook the most comprehensive documentation of basin-scale bleaching to date and found that over 80% of corals bleached and over 40% died at many sites. The most severe bleaching coincided with waters nearest a western Atlantic warm pool that was centered off the northern end of the Lesser Antilles. CONCLUSIONS/SIGNIFICANCE: Thermal stress during the 2005 event exceeded any observed from the Caribbean in the prior 20 years, and regionally-averaged temperatures were the warmest in over 150 years. Comparison of satellite data against field surveys demonstrated a significant predictive relationship between accumulated heat stress (measured using NOAA Coral Reef Watch's Degree Heating Weeks) and bleaching intensity. This severe, widespread bleaching and mortality will undoubtedly have long-term consequences for reef ecosystems and suggests a troubled future for tropical marine ecosystems under a warming climate.

The cost of pursuing a medical career in the military: a tale of five specialties.

PURPOSE: The physician payment system is a focus of potential reform in the United States. The authors explored the effects of the military's method of physician payment on physicians' returns on educational investment for several specialties. METHOD: This retrospective, observational study used national data from 2003 and standard financial techniques to calculate the net present value-the current value of an expected stream of cash flows at a particular rate of interest-of the educational investments of medical students in ten 30-year career paths: either military or civilian careers in internal medicine, psychiatry, gastroenterology, general surgery, or orthopedics. RESULTS: At a 5% discount rate, in the civilian world, the lowest return on an educational investment accrued to psychiatrists ($1.136 million) and the highest to orthopedists ($2.489 million), a range of $1.354 million. In the military, the lowest returns accrued to internists ($1.377 million) and the highest to orthopedists ($1.604 million); however, the range was only $0.227 million, one-sixth that found in the civilian sector. The authors also found that most military physicians do not remain in the military for their full careers. CONCLUSIONS: Choosing a military career substantially decreases the net present value of an educational investment for interventionalists, but it does so only modestly for primary care physicians. Further, a military career path markedly diminishes specialty-specific variation in the net present values of educational investment. Adopting a military structure for engaging medical students might help reverse the current trend of declining interest in primary care.

Substrate overlap between Mrp4 and Abcg2/Bcrp affects purine analogue drug cytotoxicity and tissue distribution.

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2'-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [(125)I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy.