Environmental DNA metabarcoding reveals estuarine benthic community response to nutrient enrichment - Evidence from an in-situ experiment.

Nutrient loading is a major threat to estuaries and coastal environments worldwide, therefore, it is critical that we have good monitoring tools to detect early signs of degradation in these ecologically important and vulnerable ecosystems. Traditionally, bottom-dwelling macroinvertebrates have been used for ecological health assessment but recent advances in environmental genomics mean we can now characterize less visible forms of biodiversity, offering a more holistic view of the ecosystem and potentially providing early warning signals of disturbance. We carried out a manipulative nutrient enrichment experiment (0, 150 and 600 g N fertilizer m-2) in two estuaries in New Zealand to assess the effects of nutrient loading on benthic communities. After seven months of enrichment, environmental DNA (eDNA) metabarcoding was used to examine the response of eukaryotic (18S rRNA), diatom only (rbcL) and bacterial (16S rRNA) communities. Multivariate analyses demonstrated changes in eukaryotic, diatom and bacterial communities in response to nutrient enrichment at both sites, despite differing environmental conditions. These patterns aligned with changes in macrofaunal communities identified using traditional morphological techniques, confirming concordance between disturbance indicators detected by eDNA and current monitoring approaches. Clear shifts in eukaryotic and bacterial indicator taxa were seen in response to nutrient loading while changes in diatom only communities were more subtle. Community changes were discernible between 0 and 150 g N m-2 treatments, suggesting that estuary health assessment tools could be developed to detect early signs of degradation. Increasing variation in community structure associated with nutrient loading could also be used as an indicator of stress or approaching tipping points. This work represents a first step towards the development of molecular-based estuary monitoring tools, which could provide a more holistic and standardized approach to ecosystem health assessment with faster turn-around times and lower costs.

The development of a national approach to monitoring estuarine health based on multivariate analysis.

New Zealand has a complex coastal environment spanning a large latitudinal gradient and three water masses. Here we assess whether multivariate analyses of benthic macrofaunal community composition can be a sensitive approach to assessing relative estuarine health across the country, negating the need for regional indices and reducing reliance on reference sites. Community data were used in separate canonical analyses of principal coordinates to create multivariate models of community responses to gradients in mud content and heavy metal contamination. Both models performed well (R2 = 0.81, 0.71), and were unaffected by regional and estuarine typology differences. The models demonstrate a sensitive and standardized approach to assessing estuarine health that allowed separation of the two stressors. This approach could be applied to other stressors, countries or regions.

A constitutively active ERBB4/HER4 allele with enhanced transcriptional coactivation and cell-killing activities.

In the normal breast, ERBB4 regulates epithelial differentiation and functions as a nuclear chaperone for signal transducer and activator of transcription (STAT) 5A, thereby stimulating milk-gene expression. In addition, ERBB4 functions as a proapoptotic protein, suppressing the growth of malignant cells. We hypothesize that these ERBB4 activities can be marshaled to suppress the growth of breast tumors. To this end, we have created an ERBB4 allele harboring an activating transmembrane mutation (ERBB4-CA) by substituting isoleucine 658 for glutamic acid. This base substitution forms a valine-glutamic acid-glycine activation domain first identified in oncogenic ERBB2/HER2/Neu. Ectopic expression of ERBB4-CA in HEK293T cells resulted in a fivefold increase in receptor tyrosine phosphorylation. Functionally, ERBB4-CA exhibited higher levels of nuclear translocation than wild-type ERBB4, leading to significantly enhanced ERBB4-induced STAT5A simulation of the beta-casein promoter. Activated ERBB4 has been demonstrated to induce cell killing of breast tumor cells. Significantly, ERBB4-CA potentiated the proapoptotic function of ERBB4 in each breast, prostate and ovarian cancer cell line tested. Untransformed cell lines were resistant to both ERBB4 and ERBB4-CA-mediated apoptosis underscoring the potential utility of active ERBB4 signaling for the therapeutic intervention of human cancer.

The serine/threonine protein kinase, p90 ribosomal S6 kinase, is an important regulator of prostate cancer cell proliferation.

An increase in the activity of mitogen-activated protein kinase (MAPK) has been correlated with the progression of prostate cancer to advanced disease in humans. The serine/threonine protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of MAPK but its role in prostate cancer has not previously been examined. Increasing RSK isoform 2 (RSK2) levels in the human prostate cancer line, LNCaP, enhanced prostate-specific antigen (PSA) expression, an important diagnostic marker for prostate cancer, whereas inhibiting RSK activity using a RSK-specific inhibitor, 3Ac-SL0101, decreased PSA expression. The RSK2 regulation of PSA expression occurred via a mechanism involving both RSK2 kinase activity and its ability to associate with the coactivator, p300. RNA interference of the androgen receptor (AR) showed that the AR was important in the RSK2-mediated increase in PSA expression. RSK levels are higher in approximately 50% of human prostate cancers compared with normal prostate tissue, which suggests that increased RSK levels may participate in the rise in PSA expression that occurs in prostate cancer. Furthermore, 3Ac-SL0101 inhibited proliferation of the LNCaP line and the androgen-independent human prostate cancer line, PC-3. These results suggest that proliferation of some prostate cancer cells is dependent on RSK activity and support the hypothesis that RSK may be an important chemotherapeutic target for prostate cancer.

Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT-1) pre-mRNA is important for the regulation of VEGF activity.

Angiogenesis is essential for normal mammalian development and is controlled by the local balance of pro- and antiangiogenic factors. Here we describe a novel mouse cDNA sequence encoding sFLT-1 that is a potent antagonist to vascular endothelial growth factor (VEGF) and show for the first time its in vivo production. In situ hybridization and Northern blot analysis with probes specific for sFLT-1 or FLT-1 showed that the relative abundance of their mRNAs changed markedly in spongiotrophoblast cells in the placenta as gestation progressed. On day 11 of pregnancy, sFLT-1 mRNA was undetectable but FLT-1 readily apparent, and by day 17 sFLT-1 mRNA was abundant but FLT-1 barely detectable. sFLT-1 was identified in conditioned medium of cultured placenta from day 17 pregnant mice and likely to be present in the circulation, as there is a substantial increase of VEGF-binding activity in the serum from day 13 of pregnancy, which coincides with the abundant sFLT-1 expression in placenta. Expression of sFLT-1 was also observed in adult lung, kidney, liver, and uterus. These data suggest a novel mechanism of regulation of angiogenesis by alternative splicing of FLT-1 pre-mRNA. Treatment of pregnant mice with exogenous VEGF from day 9 to 17 of pregnancy, which alters the ratio of VEGF to sFLT-1, resulted in an increase in the number of resorption sites and fibrin deposition in the placenta of ongoing pregnancies. These findings have important implications for understanding placental function and may be relevant in a range of disease states.

Psychologic and neurologic consequences of partial and complete cerebral commissurotomy.

Preliminary psychologic testing was carried out on four patients who had undergone surgical sectioning of a portion or all of the corpus callosum and anterior commissure as a treatment for uncontrollable seizures. Results confirm earlier findings indicating the importance of the forebrain commissures in the interhemispheric exchange of a variety of sensory and motor information, and demonstrate that particular portions of the commissural system are responsible for transferring the information of specific sensory modalities. The patients also showed surprising abilities in performing complex tasks assumed to require integration of information from both hemispheres.

PRO_LIGAND: an approach to de novo molecular design. 2. Design of novel molecules from molecular field analysis (MFA) models and pharmacophores.

A computational approach for molecular design, PRO_LIGAND, has been developed within the PROMETHEUS molecular design and simulation system in order to provide a unified framework for the de novo generation of diverse molecules which are either similar or complementary to a specified target. In this instance, the target is a pharmacophore derived from a series of active structures either by a novel interpretation of molecular field analysis data or by a pharmacophore-mapping procedure based on clique detection. After a brief introduction to PRO_LIGAND, a detailed description is given of the two pharmacophore generation procedures and their abilities are demonstrated by the elucidation of pharmacophores for steroid binding and ACE inhibition, respectively. As a further indication of its efficacy in aiding the rational drug design process, PRO_LIGAND is then employed to build novel organic molecules to satisfy the physicochemical constraints implied by the pharmacophores.

Comparison of expression patterns for placenta growth factor, vascular endothelial growth factor (VEGF), VEGF-B and VEGF-C in the human placenta throughout gestation.

Angiogenesis and vascular transformation are important processes in the normal development of the placenta. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor and is thought to be important for placental development. Recently several new members of this family have been described. In this study we used in situ hybridisation to localise which cells in the placenta expressed mRNA for VEGF, placenta growth factor (PlGF), VEGF-B and VEGF-C. We were unable to find any message for either VEGF-B or VEGF-C in the placenta, suggesting that only low levels are produced which this method was unable to detect. The mRNA encoding VEGF was found to be produced by cells within the villous mesenchyme, decidual macrophages and decidual glands but, in contrast to our previous findings, not by trophoblast. The mRNA encoding PlGF was produced in large amounts by villous cytotrophoblast, syncytiotrophoblast and extravillous trophoblast. The mRNAs encoding VEGF and PlGF were thus not co-localised and it appears that there is unlikely to be any significant production of VEGF/PlGF heterodimer in the placenta.

Hepatocyte growth factor/scatter factor and its receptor c-met: localisation and expression in the human placenta throughout pregnancy.

Hepatocyte growth factor (HGF), also known as scatter factor, acts via the c-met receptor resulting in pronounced effects on certain epithelial cells. We hypothesised that HGF would be important in placental development where the trophoblast represents a specialised barrier of epithelial origin. In this paper we examine the expression and production of HGF and its receptor in the human placenta throughout pregnancy. In addition, RT-PCR was undertaken on human embryos to ascertain whether pre-implantation embryonic or trophoblast cells were under the influence of this growth factor. In samples from the first trimester of pregnancy in situ hybridisation with a c-met antisense probe detected message expression in villous cytotrophoblast and in decidual glands but not in extravillous trophoblast. Some c-met expression was detected in cytotrophoblast from the second trimester placentae; this declined to negligible levels by term. Staining with an anti c-met antibody largely confirmed these findings but found relatively strong staining of cytotrophoblast at term. HGF was confined to the villous core throughout pregnancy when examined by both in situ hybridisation and immunohistochemistry. Trophoblast was consistently negative for HGF. Pre-implantation embryos examined by RT-PCR were negative for both c-met and HGF mRNA. These results indicate that the HGF may exert an important influence on cytotrophoblast throughout the process of placental formation and growth.

Hepatocyte growth factor levels during normal and intra-uterine growth-restricted pregnancies.

Hepatocyte growth factor (HGF), also known as scatter factor, binds the c-met receptor. It has been shown to be involved in mesenchyme-epithelial interactions. HGF is produced by the villous mesenchyme of the placenta throughout pregnancy and its receptor located on the villous cytotrophoblast cells. In this study the levels of HGF were measured in consecutive samples of plasma taken from pregnant women. Normal pregnancies were compared with intrauterine growth restricted (IUGR) pregnancies (below the third centile). In both groups, the levels of HGF were found to increase significantly as pregnancy progressed and then fall post partum. There was a considerable amount of variation found between individual women but no significant difference (P=0.65) between the normal and IUGR pregnancies.

Dietary lead increases ethanol consumption in the rat.

Rats fed either a diet containing 500 ppm (parts per million) Pb (as lead acetate) or an unadulterated control diet for 50 days were offered a 15% ethanol (ETOH) solution in a nonchoice (one-bottle) test situation. The results from this test indicated that Pb-diet animals consumed greater amounts of the ETOH solution than did controls. In a subsequent choice (three-bottle, two-fluid) test situation offering a nonpreferred ETOH solution or tap water as alternatives, Pb-diet animals once again ingested greater amounts of the ETOH solution. These findings are discussed in terms of possible Pb-induced increases in emotionality and the potential stress-reduction properties of ETOH.

Effects of acute trimethyltin exposure on appetitive acquisition and extinction performance in the adult rat.

Adult male rats were given one intragastric infusion of either 7 mg/kg trimethyltin chloride (dose calculated as the base of trimethyltin [TMT]) or physiological saline. Twenty-one days after dosing, subjects from each condition were divided into two equal-sized groups and trained with either partial (PRF) or continuous (CRF) reinforcement in a straight alley maze. The acquisition phase of training, lasting 40 trials (4 trials/day), was followed by 20 trials of extinction training (4 trials/day). Analyses performed on total speed revealed that TMT-treated subjects performed at lower levels during acquisition than controls regardless of schedule condition. Also, the rate of resistance to extinction was significantly reduced for treated subjects compared with that of controls regardless of the training schedules used during acquisition. A partial reinforcement extinction effect was observed for both control and TMT-treated subjects, that is, independent of dose regimen; PRF training occasioned greater persistence during extinction than did CRF training. These findings are discussed in terms of their implications for contemporary empirical and theoretical issues relating to TMT-induced hippocampal lesions.

Prediction of intestinal absorption and blood-brain barrier penetration by computational methods.

This review surveys the computational methods that have been developed with the aim of identifying drug candidates likely to fail later on the road to market. The specifications for such computational methods are outlined, including factors such as speed, interpretability, robustness and accuracy. Then, computational filters aimed at predicting "drug-likeness" in a general sense are discussed before methods for the prediction of more specific properties--intestinal absorption and blood-brain barrier penetration--are reviewed. Directions for future research are discussed and, in concluding, the impact of these methods on the drug discovery process, both now and in the future, is briefly considered.

Renal arterial injuries caused by blunt trauma.

Ten patients from the Maine Medical Center with renal arterial injuries caused by blunt trauma are presented and analyzed along with 240 other case reports from the literature. Most patients were under 30 years of age and many were children. Injuries to the left side were more frequent; bilateral injuries occurred in 22 patients. Motor vehicle accidents were responsible for the majority of cases. Arterial thrombosis was the most common finding. Associated abdominal injuries were frequent, but the kidney itself was usually spared. Physical findings, urinalysis, and exploratory laparotomy were not reliable in the detection of arterial injury, but excretory urography and angiography were very accurate. Untreated unilateral injuries usually led to renal atrophy, but most did not cause hypertension; untreated bilateral injuries usually led to chronic renal failure or death. Few attempts at revascularization were successful. Excretory urography is recommended for all patients with blunt abdominal trauma; nonfunction should be investigated by angiography. Immediate bilateral repair is indicated in all cases of bilateral renal arterial injury, but repair of a unilateral injury is indicated only if it is promptly diagnosed in a young, stable patient.

Traumatic rupture of the pericardium.

Patients with traumatic rupture of the pericardium rarely survive to reach a hospital. Ten cases from the Maryland Institute for Emergency Medical Services Systems and 132 previously published cases are reviewed. Patients were usually men who were victims of violent thoracic trauma. The median age was 40 years. Half of the patients had left pleuropericardial tears; tears of the diaphragmatic pericardium, right pleuropericardium, and superior mediastinal pericardium were less frequent. Associated injuries of the heart or left hemidiaphragm were common. Pericardial rupture was usually discovered during surgical exploration for other indications, but physical or radiographic signs were occasionally present. Repair is indicated for most pericardial tears to prevent herniation of the heart or abdominal viscera.

Blunt hepatic trauma. Nonoperative management in adults.

Fifty-six adults were identified with blunt hepatic trauma. Sixteen patients (29%) were treated successfully with nonoperative management. There were no delayed laparotomies or deaths in the nonoperative group. All patients required close observation. Eight (50%) of 16 patients required transfusion of no more than 3 U of packed red blood cells. There were two significant late complications requiring readmission. Four (25%) of 16 patients had complex fractures, yet were treated successfully without laparotomy. The quantity of fluid in the abdomen, as estimated by computed tomography, did not predict failure of treatment. Nonoperative management of blunt hepatic injuries is a useful alternative in selected patients when the course of therapy is based on the hemodynamic stability of the patient and supported by computed tomographic findings.

Selective nonoperative management of blunt splenic trauma in adults.

The use of selective nonoperative management of blunt splenic trauma in adults is based on the undeniable benefits of this approach in children. Proper patient selection requires hemodynamic stability, lack of generalized peritoneal irritation, and minimal blood transfusion needs. Computed tomography is now used to make the diagnosis, but the decision for laparotomy is based on clinical grounds. Forty-one (87%) of 47 patients selected for nonoperative management were treated successfully without laparotomy, while the remaining 6 patients underwent delayed operations for persistent splenic bleeding. Blood transfusion requirements were significantly less in the observed group than in the operative group for patients with isolated trauma and for patients with polytrauma. There were no known missed intra-abdominal injuries and no deaths with the nonoperative approach. Analysis of our results has confirmed that nonoperative management is a safe and effective alternative to immediate laparotomy in properly selected patients and it can result in splenic salvage without the need for an operation.

Moose-motor vehicle collisions. An increasing hazard in northern New England.

OBJECTIVE: To analyze the epidemiology and epizootiology of moose-motor vehicle collisions (MMVC) and outcomes in severely injured patients to identify variables that might be modified to reduce the impact of this mutually deleterious interspecies interaction. DESIGN: Wildlife and Traffic Safety databases permitted retrospective, population-based assessment of MMVC epidemiology. A case series compiled from hospital trauma registries characterized morbidity and mortality from MMVC. SETTING: New Hampshire and Maine area. PATIENTS: All victims of MMVC (1980 through 1991) were included in population-based analyses. Twenty-three patients hospitalized at three rural trauma centers (January 1990 through June 1994) were included in the case series. MAIN OUTCOME MEASURES: Location, time of day and seasonal occurrence of MMVC were determined. Injury patterns and Injury Severity Scores were analyzed in 23 representative patients. Maine's 1991 traffic and medical data were linked, and factors predictive of injury from MMVC were identified using multivariate logistics. RESULTS: Most MMVC occur from April through October after dark. Of 23 subjects, 70% sustained head and/or face injuries and 26%, cervical spine injuries. Mortality was 9%. Mean Injury Severity Score was 15.7 (SD=9.0). Safety belt use, rear seat location, and light truck occupancy were associated with reduced injury (p<.05). CONCLUSIONS: Moose-motor vehicle collisions are increasing in rural regions. Prevention programs should emphasize defensive driving and seat belt use, especially during high-risk periods. Injury patterns in MMVC suggest a need for automobile design modifications that better protect the passenger compartment form direct impact.

Survival following nonpenetrating traumatic rupture of cardiac chambers.

We report the cases of 3 patients who survived cardiac chamber rupture resulting from blunt external trauma. All were drivers in motor vehicle collisions. All were seen with signs of pericardial tamponade and were treated by pericardiocentesis followed by emergency thoracotomy performed in the operating room. Ruptures of the right atrium and right and left ventricles were repaired by manual suture techniques without cardiopulmonary bypass. The cases of 37 previously reported patients who survived this injury are reviewed. We believe that patients with cardiac rupture who reach the hospital alive can often be saved by prompt diagnosis and immediate surgical treatment.

Monitoring hospital trauma mortality using statistical process control methods.

BACKGROUND: We sought to develop a simple and effective way to monitor trends in trauma mortality, using objective clinical categories and methods of statistical process control. STUDY DESIGN: Control charts and Pareto analysis were applied to trauma mortality data at the Maine Medical Center. We collected data prospectively on patients who died in our hospital after acute injury during 1985-1996 (and retrospectively for 1975-1984) to identify cases requiring medical quality review. We excluded from this study patients older than 80 years, those whose Glasgow Coma Scale motor component was never > 3 at any time after admission, and those with pathologic fractures, carcinomatosis, high quadriplegia, or severe burns. The remaining deaths were classified as resulting from inability to resuscitate (mostly hemorrhage), neurologic deterioration, or organ failure. The annual numbers in each of these categories were evaluated under the hypothesis of stationary Poisson processes with mean values equal to those seen from 1975-1984. RESULTS: After the exclusions, annual mortality from trauma has remained within control limits consistent with the Poisson model. Death from neurologic deterioration has shown a trend consistent with significant improvement in the process mean. Transient peaks in the other categories did not exceed control limits, but Pareto analysis prompted detailed studies of aortic and liver trauma. CONCLUSIONS: Process control methodology is easy to apply and potentially useful in monitoring hospital trauma mortality.