Influence of a low-head dam on water quality of an urban river system.

Dam removal in the United States is becoming a common practice for stream restoration as these structures age, climate driven precipitation patterns change, and ecological uplift becomes desirable. Yet in highly urbanized watersheds, these dams may operate as retention basins removing pollutants and mitigating hydrological change. While elimination may be ecologically and economically advantageous, sediment and pollutant removal processes may be better protective of water quality and damaging flooding. In Central Virginia, we compared a watershed split between an urbanized subwatershed (>20% impervious surface encompassing 37.8% of the total watershed land surface) flowing through a 18 Ha reservoir with a rural subwatershed (<5% impervious encompassing 63.2% of the total watershed land surface) located in the James River and Chesapeake Bay watersheds. This reservoir is scheduled for removal in the near future. Comparisons of data suggest that while portions of the urbanized watershed are degraded, this condition is not reflected at the confluence where water quality more closely resembles the rural and minimally impervious subwatershed. This conclusion was further strengthened from data collected following an unexpected dam overtopping in August 2018 where the reservoir was temporarily drained because of safety concerns. After the draining, water quality reversed with the confluence resembling the urbanized rather than the rural subwatershed. Most significantly, water quality flowing into the James River quickly and significantly shifted from a good to a degraded condition. This case study suggests reservoirs in highly urbanized watersheds may serve as critical water quality improvement structures and removal as part of a stream restoration strategy must be carefully considered.

First outbreak of linezolid-resistant vancomycin-resistant Enterococcus faecium in an Irish hospital, February to September 2014.

An outbreak of linezolid-resistant vancomycin-resistant Enterococcus faecium (LRVREfm) occurred in the hepatology ward of a tertiary referral hospital in Ireland between February and September 2014. LRVREfm was isolated from 15 patients; pulsed-field gel electrophoresis confirmed spread of a single clone. This is the first report of an outbreak of linezolid-resistant vancomycin-resistant enterococcus in Ireland.

Epidemiology and molecular typing of VRE bloodstream isolates in an Irish tertiary care hospital.

OBJECTIVES: Ireland has the highest rate of vancomycin-resistant Enterococcus faecium (VREfm) isolated from blood of nosocomial patients in Europe, which rose from 33% (110/330) in 2007 to 45% (178/392) in 2012. No other European country had a VREfm rate from blood cultures of >25%. Our aim was to elucidate the reasons for this significantly higher rate in Ireland. METHODS: The epidemiology and molecular typing of VRE from bloodstream infections (BSIs) was examined in a tertiary care referral hospital and isolates were compared with those from other tertiary care referral centres in the region. RESULTS: The most common source of VRE BSIs was intra-abdominal sepsis, followed by line-related infection and febrile neutropenia. Most of the isolates were positive for vanA; 52% (43/83) possessed the esp gene and 12% (10/83) possessed the hyl gene. Genotyping by SmaI macrorestriction analysis (PFGE) of isolates revealed clonal relatedness between bloodstream isolates and environmental isolates. VRE BSI isolates from two other tertiary care hospitals in the Dublin region showed relatedness by PFGE analysis. MLST revealed four STs (ST17, ST18, ST78 and ST203), all belonging to the clonal complex of hospital-associated strains. CONCLUSIONS: Irish VRE BSI isolates have virulence factor profiles as previously reported from Europe. Typing analysis shows the spread of individual clones within the hospital and between regional tertiary care hospitals. Apart from transmission of VRE within the hospital and transfer of colonized patients between Irish hospitals, no other explanation for the persistently high VREfm BSI rate in Ireland has been found.

Investigation of the first outbreak of OXA-48-producing Klebsiella pneumoniae in Ireland.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) strains are encountered with increasing frequency in Europe. In November 2010 the European Centre for Disease Control (ECDC) graded Ireland as only having sporadic occurrence of CPE. AIM: To describe the epidemiological and molecular typing analysis of the first outbreak of OXA-48-producing Klebsiella pneumoniae in an Irish tertiary care referral centre. METHODS: Sixteen OXA-48-producing K. pneumoniae isolates were detected, from both clinical and screening specimens, and analysed by pulsed-field gel electrophoresis and by multi-locus sequence typing. FINDINGS: Typing analysis revealed that two outbreak strains were circulating in the hospital, one among surgical patients and one among medical patients. The 'medical strain' ST13 had already been identified as an internationally disseminated clone, whereas the 'surgical strain' ST221 had not previously been reported as an OXA-48-carrying strain. CONCLUSION: Although the outbreak on surgical wards was successfully controlled by implementing strict infection control measures, intermittent detection of individual patients carrying the 'medical strain' of OXA-48 K. pneumoniae has persisted since then. The experience from this outbreak suggests that OXA-48 K. pneumoniae is endemic at low level in the healthcare setting in the Dublin region.

First isolation and outbreak of OXA-48-producing Klebsiella pneumoniae in an Irish hospital, March to June 2011.

Five OXA-48-producing Klebsiella pneumoniae were detected in a tertiary referral hospital in Ireland between March and June 2011. They were found in the clinical isolates of five cases that were inpatients on general surgical wards. None of the cases had received healthcare at a facility outside of Ireland in the previous 12 months. This is the first report of OXA-48-producing K. pneumoniae in Ireland.

Behavioral profiles of inbred strains on novel olfactory, spatial and emotional tests for reference memory in mice.

Studying the behavior of genetic background strains provides important information for the design and interpretation of cognitive phenotypes in mutant mice. Our experiments examined the performance of three commonly used strains (C57BL/6J, 129S6, DBA/2J) on three behavioral tests for learning and memory that measure very different forms of memory, and for which there is a lack of data on strain differences. In the social transmission of food preference test (STFP) all three strains demonstrated intact memory for an odor-cued food that had been sampled on the breath of a cagemate 24 hours previously. While C57BL/6J and 129S6 mice showed good trace fear conditioning, DBA/2J mice showed a profound deficit on trace fear conditioning. In the Barnes maze test for spatial memory, the 129S6 strain showed poor probe trial performance, relative to C57BL/6J mice. Comparison of strains for open field exploratory activity and anxiety-like behavior suggests that poor Barnes maze performance reflects low exploratory behavior, rather than a true spatial memory deficit, in 129S6 mice. This interpretation is supported by good Morris water maze performance in 129S6 mice. These data support the use of a C57BL/6J background for studying memory deficits in mutant mice using any of these tasks, and the use of a 129S6 background in all but the Barnes maze. A DBA/2J background may be particularly useful for investigating the genetic basis of emotional memory using fear conditioning.

Galanin peptide levels in hippocampus and cortex of galanin-overexpressing transgenic mice evaluated for cognitive performance.

Galanin-overexpressing transgenic mice (GAL-tg) generated on a dopamine beta-hydroxylase promoter were previously shown to express high levels of galanin mRNA in the locus coeruleus, and to perform poorly on challenging cognitive tasks. The present study employed radioimmunoassay to quantitate the level of galanin peptide overexpression in two brain regions relevant to learning and memory, the hippocampus and cerebral cortex. Approximately 4-fold higher levels of galanin were detected in the hippocampus of GAL-tg as compared to WT. Approximately 10-fold higher levels of galanin were detected in the frontal cortex of GAL-tg as compared to WT. A second cohort of GAL-tg and WT again showed high levels of galanin overexpression in GAL-tg as compared to WT in both brain regions. Correlation analyses were conducted between galanin peptide concentrations and behavioral scores on four learning and memory tasks: the Morris water maze, social transmission of food preference, standard delay fear conditioning, and trace fear conditioning. While some significant correlations were detected, neither hippocampal nor cortical galanin levels in the two cohorts of GAL-tg consistently correlated with performance across these diverse cognitive tasks. Several interpretations of these findings are discussed, including the possibility that a threshold level of galanin overexpression is sufficient to impair performance on learning and memory tasks in mice.

Lack of effect of moderate Purkinje cell loss on working memory.

192 immunoglobulin G-saporin (192-sap) is an immunotoxin which targets the cholinergic basal forebrain after injection into either the ventricular system or the parenchyma of the rat brain. When injected by the i.c.v. route, 192-sap kills some cerebellar Purkinje cells in addition to its more extensive killing of the cholinergic basal forebrain. Behaviorally, i.c.v. injections of 192-sap result in impaired performance in a variety of experimental paradigms of learning and memory including a working memory task in the radial maze. The current study examined the contribution, if any, of immunotoxin-induced Purkinje cell loss to impaired performance in the radial maze. To meet this aim, we used i.c.v. injection of another immunotoxin, OX7-saporin (OX7-sap), at a dose that produced Purkinje cell loss of similar extent to that produced by i.c.v. 192-sap. We then compared these OX7-sap-injected rats with 192-sap-injected rats in a radial maze working memory task. We found a working memory impairment only in the 192-sap-injected rats. These data show that moderate Purkinje cell loss alone is insufficient to impair working memory. Furthermore, the data are consistent with the idea that the working memory deficit observed in 192-sap-injected animals is likely due to lesioning of the cholinergic basal forebrain.

Pharmacological evidence supporting a role for galanin in cognition and affect.

1. Galanin is localized in brain pathways involved in both cognition and affect. 2. Galanin has inhibitory actions on a variety of memory tasks including the Morris water maze, delayed nonmatching to position, T-maze delayed alternation, starburst maze, passive avoidance, active avoidance, and spontaneous alternation. 3. Galanin may inhibit learning and memory by inhibiting neurotransmitter release and neuronal firing rate. 4. Two signal transduction mechanisms through which galanin exerts its inhibitory actions are the inhibition of phosphatidyl inositol hydrolysis and the inhibition of adenylate cyclase. 5. Galanin released during periods of burst firing from noradrenergic locus coeruleus terminals in the ventral tegmental area (VTA) may lead to symptoms of depression through inhibition of dopaminergic VTA neurons. 6. Intraventricular galanin has anxiolytic effects in a punished drinking test. Intra-amygdala galanin has anxiogenic effects in a punished drinking test.

Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer's disease.

Galanin is a neuropeptide with multiple inhibitory actions on neurotransmission and memory. In Alzheimer's disease (AD), increased galanin-containing fibers hyperinnervate cholinergic neurons within the basal forebrain in association with a decline in cognition. We generated transgenic mice (GAL-tg) that overexpress galanin under the control of the dopamine beta-hydroxylase promoter to study the neurochemical and behavioral sequelae of a mouse model of galanin overexpression in AD. Overexpression of galanin was associated with a reduction in the number of identifiable neurons producing acetylcholine in the horizontal limb of the diagonal band. Behavioral phenotyping indicated that GAL-tgs displayed normal general health and sensory and motor abilities; however, GAL-tg mice showed selective performance deficits on the Morris spatial navigational task and the social transmission of food preference olfactory memory test. These results suggest that elevated expression of galanin contributes to the neurochemical and cognitive impairments characteristic of AD.

Threshold relationship between lesion extent of the cholinergic basal forebrain in the rat and working memory impairment in the radial maze.

The cholinergic basal forebrain (CBF) degenerates in Alzheimer's Disease (AD), and the degree of this degeneration correlates with the degree of dementia. In the present study we have modeled this degeneration in the rat by injecting various doses of the highly selective immunotoxin 192 IgG-saporin (192-sap) into the ventricular system. The ability of 192-sap-treated rats to perform in a previously learned radial maze working memory task was then tested. We report here that 192-sap created lesions of the CBF and, to a lesser extent, cerebellar Purkinje cells in a dose-dependent fashion. Furthermore, we found that rats harboring lesions of the entire CBF greater than 75% had impaired spatial working memory in the radial maze. Correlational analysis of working memory impairment and lesion extent of the component parts of the CBF revealed that high-grade lesions of the hippocampal-projecting neurons of the CBF were not sufficient to impair working memory. Only rats with high-grade lesions of the hippocampal and cortical projecting neurons of the CBF had impaired working memory. These data are consistent with other 192-sap reports that found behavioral deficits only with high-grade CBF lesions and indicate that the relationship between CBF lesion extent and working memory impairment is a threshold relationship in which a high degree of neuronal loss can be tolerated without detectable consequences. Additionally, the data suggest that the CBF modulates spatial working memory via its connections to both the hippocampus and cortex.

Selective lesion of the cholinergic basal forebrain causes a loss of cortical neuropeptide Y and somatostatin neurons.

Degeneration of the cholinergic basal forebrain (CBF) and changes in cortical neuropeptide levels have been reported in Alzheimer's disease. In the present study, we sought to determine if a selective cholinergic lesion of nucleus basalis magnocellularis (Nbm) could affect the number and distribution of neuropeptide Y (NPY) and somatostatin (SS) immunoreactive neurons in the frontoparietal and occipital cortices of rats. Brain sections were evaluated at survival times of 1, 2, 4, 8, 12, 24, 48, 78 and 100 weeks after intraventricular injection of 192-saporin, an immunotoxin directed at the low affinity neurotrophin receptor (p75NGFr), that selectively destroys the CBF. Following the immunotoxin lesion of the Nbm, the number of NPY-labeled neurons decreased 33% in the frontoparietal cortex and 60% in the occipital cortex compared to age-matched normal controls at most survival time points. A significant loss of SS-labeled neurons in both cortical regions was seen 12 weeks after 192-saporin injection with no further change up to 100-week survival time. The effect of age on neuropeptidergic populations was evaluated in normal control rats. The number of NPY and SS immunoreactive neurons in aged rats (21-26 months) decreased by 42% in the frontoparietal cortex and 27% in the occipital cortex when compared with young (3-6 months) and middle-age (9-14 months) rats. When both non-lesioned and lesioned animals with different ages were pooled for linear regression, a significant correlation was found between the number of cortical NPY- and SS-labeled neurons and cortical acetylcholinesterase (AChE) histochemical staining intensity. These findings indicate that: (1) cholinergic denervation of the Nbm is associated with an irreversible loss of neocortical NPY and SS immunoreactive neurons analogous to that observed in Alzheimer's disease and aging; (2) the degree of the loss of cortical NPY and SS immunoreactive neurons seems to be related to the extent of the reduction of cortical AChE intensity in both toxin-injected and normal aged rats. These findings may reflect a trophic dependence of NPY and SS neurons on cortical cholinergic input.

The behavioral functions of the cholinergic basal forebrain: lessons from 192 IgG-saporin.

Until recently our understanding of the functional neuroanatomy of the cholinergic basal forebrain (CBF) has been hindered by the lack of a lesioning technique that is truly selective. The development of the immunotoxin 192 IgG-saporin (192-sap) has greatly improved our ability to create specific lesions of the CBF. Rats with such lesions have been studied in a wide variety of behavioral paradigms of learning, memory, and attention. Complete or near-complete destruction of the CBF results in deficits in a variety of behavior paradigms including passive avoidance, spatial tasks (water and radial mazes), delayed matching to position/sample, and attentional tasks. However, interpretation of many experiments is hampered by incomplete lesions and/or concomitant damage to cerebellar Purkinje neurons. Future studies will need to address these issues. Recent development of a similar immunotoxin that is effective in primates should permit more sophisticated behavioral analysis of CBF function. Additionally, immunotoxins selective for other types of neurons, such as the noradrenergic selective anti-DBH-saporin, will permit analysis of the behavioral functions of other diffusely projecting systems and how these other systems may interact with the CBF.

Central noradrenergic lesioning using anti-DBH-saporin: anatomical findings.

The ability to create lesions of discrete neuronal populations is an important strategy for clarifying the function of these populations. The power of this approach is critically dependent upon the selectivity of the experimental lesioning technique. Anti-neuronal immunotoxins offer an efficient way to produce highly specific neural lesions. Two previous immunotoxins have been shown to be effective in both the CNS and PNS. They are OX7-saporin, which is targeted at Thy1, and 192-saporin, which is targeted at the low affinity neurotrophin receptor, p75NTR. In the present study, we sought to determine if an immunotoxin targeted at the neurotransmitter synthesizing enzyme, dopamine beta-hydroxylase (DBH), could selectively destroy central noradrenergic neurons after intraventricular administration. This immunotoxin, which consists of a monoclonal antibody to DBH coupled by a disulfide bond to saporin (a ribosome inactivating protein), has been shown to be selectively toxic to peripheral noradrenergic sympathetic neurons in rats after systemic injection. In the present study, immunohistochemical and Cresyl violet staining showed that the noradrenergic neurons of the locus coeruleus are destroyed bilaterally after intraventricular (i.c.v.) injection of 5, 10, and 20 micrograms of anti-DBH-saporin (alpha-DBH-sap) into rats. Complete bilateral lesioning of the A5 and A7 cell groups occurred at the two higher doses. Lesions of the A1/C1 and A2/C2/C3 cell groups were incomplete at all three doses. Dopaminergic neurons of the substantia nigra and ventral tegmental area and serotonergic neurons of the raphé, all monoaminergic neurons that do not express DBH, survived all alpha-DBH-sap doses. The cholinergic neurons of the basal forebrain, which are selectively killed by i.c.v. injection of 192-saporin, and cerebellar Purkinje cells which are killed by OX7-saporin, were not killed by alpha-DBH-sap. These results show that alpha-DBH-sap efficiently and selectively destroys CNS noradrenergic neurons after i.c.v. injection. The preferential destruction of locus coeruleus, A5, and A7 over A1/C1 and A2/C2/C3 may be due to more efficient access of the immunotoxin to these neurons and their terminals after i.c.v. injection.

Healing of chondral and osteochondral defects in a canine model: the role of cultured chondrocytes in regeneration of articular cartilage.

In this study a canine model was developed to investigate the nature of early healing responses to both chondral and osteochondral defects and to evaluate the tissue regenerative capacity of cultured autologous chondrocytes in chondral defects. The healing response to surgically created chondral defects was minor, with little cellular infiltration. In contrast, osteochondral defects exhibited a rapid cellular response, resulting ultimately in the formation of fibrous tissue. The lack of significant cellular activity in chondral defects suggests that an evaluation of the capacity of cultured autologous chondrocytes to regenerate articular cartilage is best studied in chondral defects using the canine model. When dedifferentiated cultured articular chondrocytes were implanted into chondral defects, islands of type II collagen staining were demonstrated in the regenerative tissue within 6 weeks. The relatively early expression of cartilage specific markers by the implanted chondrocytes, coupled with the inability of untreated chondral defects to repair or regenerate, demonstrates the utility of the canine model in evaluating novel materials for cartilage repair and regeneration.

Loss of nucleus basalis magnocellularis, but not septal, cholinergic neurons correlates with passive avoidance impairment in rats treated with 192-saporin.

Intraventricular injection of 192-saporin, an immunotoxin directed at the low affinity neurotrophin receptor (p75NGFr), selectively destroys cholinergic neurons in the basal forebrain (CBF). In the present study, we sought to determine if there was a correlation between degree of CBF neuron destruction and degree of passive avoidance behavioral impairment. 192-saporin caused a decrease in the number of p75NGFr + neurons in both nucleus basalis magnocellularis (Nbm) and medial septal nucleus/diagonal band of Broca (MS/DBB). All rats had >95% loss of the p75NGFr + cholinergic neurons in the MS/DBB, but there was variation in the extent of the Nbm cell loss. A significant correlation was found between the severity of impairment of passive avoidance learning and the magnitude of the loss in the number of p75NGFr + neurons in the Nbm. Step-through latency also correlated significantly with the magnitude of loss of AChE histochemical staining intensity in dorsolateral neocortex ipsilateral to the injection of 192-saporin. These data show that >95% loss of cholinergic neurons in MS/DBB is not sufficient to impair passive avoidance learning. However, in the presence of severe loss of cholinergic neurons from the MS/DBB, the resulting deficit in passive avoidance behavior is proportional to the degree of cholinergic neuron loss from the Nbm. These results are interpreted as support for the hypothesis that the cholinergic projection from Nbm to neocortex plays a role in passive avoidance behavior.

Hormone binding and transcription activation by estrogen receptors: analyses using mammalian and yeast systems.

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the human estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labeling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. C530 mutant), C381 becomes the site of estrogen-compatible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed mutagenesis of selected residues in the ER and region-specific chemical mutagenesis of the ER hormone binding domain with initial phenotypic screening in yeast have enabled the identification of a region near C530 important in discrimination between estrogens and antiestrogens and of other residues important in hormone-dependent transcriptional activation. Some ER mutants with alterations in the carboxy-terminal portion of the hormone binding domain are transcriptionally inactive yet bind hormone and also function as potent dominant negative ERs, suppressing the activity of wild-type ER at low concentrations. These studies reveal a separation of the hormone binding and transcription activation functions of the ER. They are also beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer. In addition, some dominant negative receptors may prove useful in examining ER mechanisms of action and in suppressing the estrogen-dependent growth of breast cancer cells.

Structure-function analysis of the hormone binding domain of the human estrogen receptor by region-specific mutagenesis and phenotypic screening in yeast.

To investigate the structural requirements for recognition and response to ligands by the human estrogen receptor (hER), a series of point mutations were generated in the hormone binding domain (HBD) of the receptor using a limited formic acid treatment of its cDNA. Receptors having a reduced sensitivity to the estrogen, 17 beta-estradiol (E2), or to the antiestrogen, trans-hydroxytamoxifen, were selected from a library of intact hER cDNAs containing these mutant HBDs by expression and phenotypic screening in yeast (Saccharomyces cerevisiae). Several were sequenced, and the encoded receptors were characterized in both yeast and mammalian (Chinese hamster ovary) cells using hormone-binding and transactivation assays. In general, parallel phenotypes were observed in yeast and in Chinese hamster ovary cells following estrogen exposure. We report on 15 receptors having point mutations located at various positions throughout the HBD. Four categories of mutants were identified: 1) those showing no change from wild type in their response to E2; 2) those showing a greatly reduced transactivation response over the range of ligand concentrations tested; 3) those requiring much higher concentrations of E2 for maximal transactivation, indicating a reduced sensitivity to ligand; and 4) those showing reduced response to E2, but little change in response to trans-hydroxytamoxifen in yeast. Two mutations in the carboxyl terminus of the HBD eliminated hormone-dependent transactivation despite the continued ability to bind E2 with high affinity. Therefore, our results show a separation of the transactivation and hormone-binding functions of the hER, and indicate that the integrity of many regions throughout the large, approximately 250-amino acid HBD is important for these functions. Our studies also demonstrate the advantages of using regional mutagenesis combined with phenotypic screening in yeast to complement site-directed mutagenesis when investigating a large, functionally complex region.