Identification, characterization, and cellular localization of Leishmania major CTP:phosphocholine cytidylyltransferase.

The eukaryotic parasite Leishmania is the causative agent of the disease leishmaniasis, the second largest parasitic killer in the world behind malaria. A large percentage of Leishmania membrane phospholipids is phosphatidylcholine (PC), formed via the Kennedy pathway, where the enzyme CTP: phosphocholine cytidylyltransferase (CCT) catalyzes the second, rate limiting step. Leishmania major CCT was expressed in non-pathogenic Leishmania tarentolae and exhibited activity that increased 10-fold in the presence of PC:oleate lipid vesicles. Confocal microscopy of L. tarentolae expressing L. major CCT fused to a red fluorescent protein revealed the enzyme is cytoplasmic but may associate with internal membranes. A truncated mutant of L. major CCT containing the catalytic domain was expressed in Escherichia coli and in vitro analysis of the enzyme showed catalysis was divalent cation-dependent and yielded a Vmax of 374 nmol/min/mg and Km values of 0.0648 mM and 3.74 mM, respectively, for the substrates CTP and phosphocholine.

Controlled expression of Drosophila homeobox loci using the Hostile takeover system.

BACKGROUND: Hostile takeover (Hto) is a Drosophila protein trapping system that allows the investigator to both induce a gene and tag its product. The Hto transposon carries a GAL4-regulated promoter expressing an exon encoding a FLAG-mCherry tag. Upon expression, the Hto exon can splice to a downstream genomic exon, generating a fusion transcript and tagged protein. RESULTS: Using rough-eye phenotypic screens, Hto inserts were recovered at eight homeobox or Pax loci: cut, Drgx/CG34340, Pox neuro, araucan, shaven/D-Pax2, Zn finger homeodomain 2, Sex combs reduced (Scr), and the abdominal-A region. The collection yields diverse misexpression phenotypes. Ectopic Drgx was found to alter the cytoskeleton and cell adhesion in ovary follicle cells. Hto expression of cut, araucan, or shaven gives phenotypes similar to those of the corresponding UAS-cDNA constructs. The cut and Pox neuro phenotypes are suppressed by the corresponding RNAi constructs. The Scr and abdominal-A inserts do not make fusion proteins, but may act by chromatin- or RNA-based mechanisms. CONCLUSIONS: Hto can effectively express tagged homeodomain proteins from their endogenous loci; the Minos vector allows inserts to be obtained even in transposon cold-spots. Hto screens may recover homeobox genes at high rates because they are particularly sensitive to misexpression.

The zinc finger homeodomain-2 gene of Drosophila controls Notch targets and regulates apoptosis in the tarsal segments.

The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.

abd-A regulation by the iab-8 noncoding RNA.

The homeotic genes in Drosophila melanogaster are aligned on the chromosome in the order of the body segments that they affect. The genes affecting the more posterior segments repress the more anterior genes. This posterior dominance rule must be qualified in the case of abdominal-A (abd-A) repression by Abdominal-B (Abd-B). Animals lacking Abd-B show ectopic expression of abd-A in the epidermis of the eighth abdominal segment, but not in the central nervous system. Repression in these neuronal cells is accomplished by a 92 kb noncoding RNA. This "iab-8 RNA" produces a micro RNA to repress abd-A, but also has a second, redundant repression mechanism that acts only "in cis." Transcriptional interference with the abd-A promoter is the most likely mechanism.

A novel ruthenium-silver based antimicrobial potentiates aminoglycoside activity against Pseudomonas aeruginosa.

The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments in novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX, a broad-spectrum antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to the connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX on several antibiotic classes. We found that the combination of AGXX and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored the sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with a significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials. IMPORTANCE The emergence of drug-resistant bacteria coupled with the decline in antibiotic development highlights the need for novel alternatives. Thus, new strategies aimed at repurposing conventional antibiotics have gained significant interest. The necessity of these interventions is evident especially in gram-negative pathogens as they are particularly difficult to treat due to their outer membrane. This study highlights the effectiveness of the antimicrobial AGXX in potentiating aminoglycoside activities against P. aeruginosa. The combination of AGXX and aminoglycosides not only reduces bacterial survival rapidly but also significantly re-sensitizes aminoglycoside-resistant P. aeruginosa strains. In combination with gentamicin, AGXX induces increased endogenous oxidative stress, membrane damage, and iron-sulfur cluster disruption. These findings emphasize AGXX's potential as a route of antibiotic adjuvant development and shed light on potential targets to enhance aminoglycoside activity.

The Novel Silver-Containing Antimicrobial Potentiates Aminoglycoside Activity Against Pseudomonas aeruginosa.

The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments into novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX®, a broad-spectrum silver containing antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX® could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX® on several antibiotic classes. We found that the combination of AGXX® and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX®/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX®/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX®/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials.

Rap-GEF signaling controls stem cell anchoring to their niche through regulating DE-cadherin-mediated cell adhesion in the Drosophila testis.

Stem cells will undergo self-renewal to produce new stem cells if they are maintained in their niches. The regulatory mechanisms that recruit and maintain stem cells in their niches are not well understood. In Drosophila testes, a group of 12 nondividing somatic cells, called the hub, identifies the stem cell niche by producing the growth factor Unpaired (Upd). Here, we show that Rap-GEF/Rap signaling controls stem cell anchoring to the niche through regulating DE-cadherin-mediated cell adhesion. Loss of function of a Drosophila Rap-GEF (Gef26) results in loss of both germline and somatic stem cells. The Gef26 mutation specifically impairs adherens junctions at the hub-stem cell interface, which results in the stem cells "drifting away" from the niche and losing stem cell identity. Thus, the Rap signaling/E-cadherin pathway may represent one mechanism that regulates polarized niche formation and stem cell anchoring.

Palynological evidence for pre-agricultural reindeer grazing and the later settlement history of the Lycksele region, northern Sweden.

Analyses of high-resolution pollen data, coprophilous fungal spores, microscopic charcoal and sedimentology, combined with radiocarbon dating, allow the assessment of the impact of Sami and Nordic land use in the region surrounding the winter market town of Lycksele in northern Sweden. Such winter markets were established by the Crown during the seventeenth century AD to control the semi-nomadic movements of the Sami who traded here with Finnish settlers and were also taxed and educated. Little is known about Sami and Nordic co-existence beyond these market places, mainly due to a lack of archaeological evidence relating to Sami activity. Vegetation and land-use changes in the region between ~ AD 250 and 1825 reveal no signal for pre-seventeenth century agricultural activity, but the coprophilous fungal spore records suggest the increased regional presence of grazing herbivores (possibly reindeer) between ~ AD 800 and 1100. Sami activity in the parish of Lycksele has been suggested by rich metal finds dated to ~ AD 1000-1350 and they may have been attracted by an abundance of reindeer.

Phagocytic B cells in a reptile.

Evidence for a developmental relationship between B cells and macrophages has led to the hypothesis that B cells evolved from a phagocytic predecessor. The recent identification of phagocytic IgM+ cells in fishes and amphibians supports this hypothesis, but raises the question of when, evolutionarily, was phagocytic capacity lost in B cells? To address this, leucocytes were isolated from red-eared sliders, Trachemys scripta, incubated with fluorescent beads and analysed using flow cytometry and confocal microscopy. Results indicate that red-eared slider B cells are able to ingest foreign particles and suggest that ectothermic vertebrates may use phagocytic B cells as part of a robust innate immune response.

Preparation and Logistic Considerations in Performing PET and PET/Computed Tomography in Pediatric Patients.

It is good for the nuclear medicine technologist to be aware of the normal variants and the appearance of a good-quality scan. The primary responsibility as a nuclear medicine technologist is to provide an optimal quality scan that maximizes the reading physician's ability to correctly interpret while using current radiation safety practices as the best possible experience is created for patients and families. Combining the radiation safety aspects (nuclear) with how care is provided (medicine) and the use of the most recent equipment (technology) is what defines a nuclear medicine technologist.

Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps.

The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.

Heat-Damaged Red Blood Cell Scintigraphy in Helping Interpretation of 68Ga-DOTATATE PET/CT.

Ga-DOTATATE PET/CT was performed to determine the cause of endogenous Cushing syndrome in a 10-year-old boy whose MRI studies did not reveal pituitary or adrenal gland abnormality. The PET/CT images demonstrated an intense activity in a small soft tissue nodule in the left upper abdomen where splenules are commonly located. Heat-damaged Tc-labeled red blood cell imaging showed that this soft tissue nodule did not have increased red blood cell activity, which indicated that this lesion was a true somatostatin receptor-rich lesion. The pathology result following surgical resection of the lesion confirmed adrenocorticotropic hormone-producing neuroendocrine tumor.

Innovative Partnership between a Rural Mental Health Center and Community Pharmacy: Integration of a Mental Health Pharmacist.

PURPOSE: The purpose of this article is to describe how an innovative partnership between a rural community mental health center, community independent pharmacy and College of Pharmacy and integration of a mental health pharmacist lead to identification of medication therapy problems (MTP's) and interprofessional team partnerships with center mental health professionals. METHODS: A contractual arrangement was initiated between Northern Pines Mental Health Center (NPMHC), GuidePoint Pharmacy Services GPS) and the University of Minnesota College of Pharmacy (UMN CoP) to place a PGY1 resident at NPMHC. The resident was assigned to work closely with the Chief Medical Officer and provide initial comprehensive medication management (CMM) services to individuals who were enrolled in Assertive Community Treatment (ACT). A retrospective chart review was conducted to evaluate the impact of services provided. Patient inclusion criteria included ACT enrollees 18 years or older, a diagnosis of SPMI, taking at least one psychotropic medication, and participation in at least one resident-led CMM visit. Additional findings included the relationship between the pharmacist, the psychiatric physician, and other members of the ACT team. Descriptive statistics were used to document the findings. FINDINGS: N = 30 met the inclusion criteria: 18 males and 12 females, age ranged from 24 - 69 with average of 44 years old. 110 MTPs were identified ranging from no MTPs to 10 MTPs per patient, with a mean of 4 MTPs/patient. There was an uneven distribution of MTPs between psychiatric and medical conditions, with a disproportionately high occurrence of "Needs Additional Drug Therapy" in medical conditions and "Adverse Drug Reaction" in psychiatric conditions. In addition, the services were valued by members on the ACT team. CONCLUSION: Rural residents with SPMI in intensive community treatment have complex medication needs that require the training and skills of a clinical pharmacist. Despite the inclusion of a medication list as part of the ACT fidelity standards MTPs may go unrecognized and unresolved without the services of a clinical pharmacist conducting CMM. The pharmacist and psychiatric physician formed a collaborative partnership to address medication issues. We conclude that there is a need for integrating clinical pharmacist services into rural mental health centers.

Archaeological assessment reveals Earth's early transformation through land use.

Environmentally transformative human use of land accelerated with the emergence of agriculture, but the extent, trajectory, and implications of these early changes are not well understood. An empirical global assessment of land use from 10,000 years before the present (yr B.P.) to 1850 CE reveals a planet largely transformed by hunter-gatherers, farmers, and pastoralists by 3000 years ago, considerably earlier than the dates in the land-use reconstructions commonly used by Earth scientists. Synthesis of knowledge contributed by more than 250 archaeologists highlighted gaps in archaeological expertise and data quality, which peaked for 2000 yr B.P. and in traditionally studied and wealthier regions. Archaeological reconstruction of global land-use history illuminates the deep roots of Earth's transformation and challenges the emerging Anthropocene paradigm that large-scale anthropogenic global environmental change is mostly a recent phenomenon.

Identification and characterization of the nuclear isoform of Drosophila melanogaster CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the conversion of phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the eventual synthesis of phosphatidylcholine (PC). The enzyme is regulated by reversible association with cellular membranes, with the rate of catalysis increasing following membrane association. Two isoforms of CCT appear to be present in higher eukaryotes, including Drosophila melanogaster, which contains the tandem genes Cct1 and Cct2. Before this study, the CCT1 isoform had not been characterized and the cellular location of each enzyme was unknown. In this investigation, the cDNA encoding the CCT1 isoform from D. melanogaster has been cloned and the recombinant enzyme purified and characterized to determine catalytic properties and the effect of lipid vesicles on activity. CCT1 exhibited a V max of 23904 nmol of CDP-choline min (-1) mg (-1) and apparent K m values for phosphocholine and CTP of 2.29 and 1.21 mM, respectively, in the presence of 20 muM PC/oleate vesicles. Cytidylyltransferases require a divalent cation for catalysis, and the cation preference of CCT1 was found to be as follows: Mg (2+) > Mn (2+) = Co (2+) > Ca (2+) = Ni (2+) > Zn (2+). The activity of the enzyme is stimulated by a variety of lipids, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol, and the fatty acid oleate. Phosphatidylethanolamine and phosphatidic acid, however, did not have a significant effect on CCT1 activity. The cellular location of both CCT1 and CCT2 isoforms was elucidated by expressing green fluorescent fusion proteins in cultured D. melanogaster Schneider 2 cells. CCT1 was identified as the nuclear isoform, while CCT2 is cytoplasmic.

Carbaporphyrin ketals as potential agents for a new photodynamic therapy treatment of leishmaniasis.

Dimethyl and diethyl carbaporphyrin ketals inhibit the growth of Leishmania tarentolae promastigotes in vitro. The concentration dependency of the inhibitory effect was tested using the MTT assay. The presence of reactive oxygen species, such as singlet oxygen and superoxide, was detected using electron paramagnetic resonance spectroscopy with selected spin traps and confocal microscopy in cultures exposed to these carbaporphyrin ketals. These unique porphyrinoids show promise as potent inhibitors of Leishmania.

Industrial-era lead and mercury contamination in southern Greenland implicates North American sources.

To study the long-range transport of atmospheric pollutants from lower latitude industrial areas to the Arctic, we analysed a peat core spanning the last ~700cal.yr (~1300-2000CE) from southern Greenland, an area sensitive to atmospheric pollution from North American and Eurasian sources. A previous investigation conducted in the same location recorded atmospheric lead (Pb) pollution after ~1845, with peak values recorded in the 1970s, and concluded that a North American source was most likely. To confirm the origin of the lead, we present new Pb isotope data from Sandhavn, together with a high-resolution record for mercury (Hg) deposition. Results demonstrate that the mercury accumulation rate has steadily increased since the beginning of the 19th century, with maximum values of 9.3µgm-2yr-1 recorded ~1940. Lead isotopic ratios show two mixing lines: one which represents inputs from local and regional geogenic sources, and another that comprises regional geogenic and pollution sources. Detrending the Pb isotopic ratio record (thereby extracting the effect of the geogenic mixing) has enabled us to reconstruct a detailed chronology of metal pollution. The first sustained decrease in Pb isotope signals is recorded as beginning ~1740-1780 with the lowest values (indicating the highest pollution signature) dated to ~1960-1970. The 206Pb/207Pb ratio of excess Pb (measuring 1.222, and reflecting pollution-generated Pb), when compared with the Pb isotopic composition of the Sandhavn peat record since the 19th century and the timing of Pb enrichments, clearly points to the dominance of pollution sources from North America, although it did not prove possible to further differentiate the emissions sources geographically.

The legacy of past manuring practices on soil contamination in remote rural areas.

This paper demonstrates that there can be a legacy of contamination on former arable land in remote rural areas as a result of past manuring practices. In the first part of the study four farms abandoned in the late 19th to mid-20th century were investigated with samples collected from residual material in domestic hearths, the midden heaps, kailyards (walled garden for vegetables), infields (intensively managed arable land) and outfields (less intensively managed land for cropping or grazing). Consistent sequences in concentration values were found for such elements as Pb, Zn, Cu and P in the order hearth>midden>kailyard>infield>outfield. Such patterns can in part be explained in terms of atmospheric deposition on peat and turf which were subsequently burnt in hearths to result in enhanced elemental concentrations. The ash then was deposited in midden heaps and subsequently on kailyards or infields. In the second part, microanalytical results from St. Kilda are discussed. Enhanced loadings of Pb and Zn were found in the old arable land. The highest levels of Zn were found in small fragments of carbonised and humified material and bone fragments; in contrast Pb tended to be more uniformly distributed. Seabird waste was extensively applied to the arable land and some of the Zn may have accumulated in the soil by this pathway. The retention of Zn in bone is likely to have been very minor given the rarity of bone fragments as evident in thin sections (0.3%); this compares with 6.8% for black carbonised particles which are likely to provide the main storage sites for Zn.

High-resolution palynology reveals the land use history of a Sami renvall in northern Sweden.

The limited availability of historical and archaeological evidence means that much is still unknown about the development of Sami reindeer herding in Fennoscandia in both the recent and more distant past. To address this problem, high-resolution palynological analyses, 14C and 210Pb dating were undertaken on two adjacent (<25 m apart) peat profiles collected at a recently abandoned reindeer gathering pen (renvall) near Jokkmokk (~66.6°N, 19.8°E) in the boreal forest of northern Sweden. The aim was to assess the impact of Sami reindeer herding on the local environment through a study of pollen, coprophilous fungal spores, microscopic charcoal and sedimentology. The samples collected from within an annex to the renvall indicate cycles of use and abandonment of the pen on a multi-decadal timescale between ~ad 1800-2008, most obviously in the coprophilous fungal spore archive. The pattern and timing of these cycles confirm events previously known only from oral histories. Although the local pollen assemblage zones associated with the phasing of activity were reproducible in a second peat core beyond the boundary of the renvall, the coprophilous fungal spore signal in this paired profile was much less distinctive, possibly due to the typically shorter dispersal distances for these microfossils in comparison to pollen grains.

Elevated MIBG Activity at the Site of Erythema of Unknown Etiology.

A 6-year-old boy underwent I-MIBG therapy for recurrent neuroblastoma. On posttherapy scan, there were new foci of increased MIBG activity in the anterior abdomen, which were not seen on pretherapy I-MIBG scan. Physical examination demonstrated that these new foci of activity corresponded to the sites of asymptomatic erythema, which had of unknown etiology and was spontaneously resolved without treatment.