A highly multiplexed melt-curve assay for detecting the most prevalent carbapenemase, ESBL, and AmpC genes.

Resistance to third-generation cephalosporins and carbapenems in Gram-negative bacteria is chiefly mediated by beta-lactamases including extended-spectrum beta-lactamase (ESBL), AmpC, and carbapenemase enzymes. Routine phenotypic detection methods do not provide timely results, and there is a lack of comprehensive molecular panels covering all important markers. An ESBL/carbapenemase high-resolution melt analysis (HRM) assay (SHV, TEM, CTX-M ESBL families, and NDM, IMP, KPC, VIM and OXA-48-like carbapenemases) and an AmpC HRM assay (16S rDNA control, FOX, MOX, ACC, EBC, CIT, and DHA) were designed and evaluated on 111 Gram-negative isolates with mixed resistance patterns. The sensitivity for carbapenemase, ESBL, and AmpC genes was 96.7% (95% confidence interval [CI]: 82.8-99.9%), 93.6% (95% CI: 85.7-97.9%), and 93.8% (95% CI: 82.8-98.7%), respectively, with a specificity of 100% (95% CI: 95.6-100%), 93.9% (95% CI: 79.8-99.3%), and 93.7% (95% CI: 84.5-98.2%). The HRM assays enable the simultaneous detection of the 14 most important ESBL, carbapenemase, and AmpC genes and could be used as a molecular surveillance tool or to hasten detection of antimicrobial resistance for treatment management.

Emergence of carriage of CTX-M-15 in faecal Escherichia coli in horses at an equine hospital in the UK; increasing prevalence over a decade (2008-2017).

BACKGROUND: This study investigated changes over time in the epidemiology of extended-spectrum ß-lactamase (ESBL) producing Escherichia coli within a single equine referral hospital in the UK. Faecal samples were collected from hospitalised horses in 2008 and 2017, processed using selective media and standard susceptibility laboratory methods. A novel real-time PCR with high resolution melt analysis was used to distinguish blaCTX-M-1 and blaCTX-M-15 within CTX-M-1 group. RESULTS: In 2008, 457 faecal samples from 103 horses were collected, with ESBL-producing E. coli identified in 131 samples (28.7, 95% CI 24.6-33.1). In 2017, 314 faecal samples were collected from 74 horses with ESBL-producing E. coli identified in 157 samples (50.0, 95% CI 44.5-55.5). There were 135 and 187 non-duplicate ESBL-producing isolates from 2008 and 2017, respectively. In 2008, 12.6% of isolates belonged to CTX-M-1 group, all carrying blaCTX-M-1, whilst in 2017, 94.1% of isolates were CTX-M-1 group positive and of these 39.2 and 60.8% of isolates carried blaCTX-M-1 and blaCTX-M-15, respectively. In addition, the prevalence of doxycycline, gentamicin and 3rd generation cephalosporin resistance increased significantly from 2008 to 2017 while a decreased prevalence of phenotypic resistance to potentiated sulphonamides was observed. CONCLUSIONS: The real-time PCR proved a reliable and high throughput method to distinguish between blaCTX-M-1 and blaCTX-M-15. Furthermore, its use in this study demonstrated the emergence of faecal carriage of CTX-M-15 in hospitalised horses, with an increase in prevalence of ESBL-producing E. coli as well as increased antimicrobial resistance to frequently used antimicrobials.

Acute phase proteins and IP-10 as triage tests for the diagnosis of tuberculosis: systematic review and meta-analysis.

OBJECTIVES: We examined the data reported in studies for diagnostic purposes and to discuss whether their intended use could be extended to triage, as rule-in or rule-out tests to select individuals who should undergo further confirmatory tests. METHODS: We searched Scopus, PubMed and Web of Science with the terms 'acute phase proteins,' 'IP-10,' 'tuberculosis,' 'screening' and 'diagnosis,' extracted the sensitivity and specificity of the biomarkers and explored methodologic differences to explain performance variations. Summary estimates were calculated using random-effects models for overall pooled accuracy. The hierarchical summary receiver operating characteristic model was used for meta-analysis. RESULTS: We identified 14, four and one studies for C-reactive protein (CRP), interferon γ-induced protein 10 (IP-10) and alpha-1-acid glycoprotein (AGP). The pooled CRP sensitivity/specificity (95% confidence interval) was 89% (80-96) and 57% (36-65). Sensitivity/specificity were higher in high-tuberculosis-burden countries (90%/64%), HIV-infected individuals (91%/61%) and community-based studies (90%/62%). IP-10 sensitivity/specificity in TB vs. non-TB studies was 85%/63% and in TB and HIV coinfected vs. other lung conditions 94%/21%. However, IP-10 studies included diverse populations and a high risk of bias, resulting in very low-quality evidence. AGP had 86%/93% sensitivity/specificity. CONCLUSIONS: Few studies have evaluated CRP, IP-10 and AGP for the triage of symptomatic patients. Their high sensitivity and moderate specificity warrant further prospective studies exploring whether their combined use could optimize performance.

Monitoring the response of patients with cutaneous leishmaniasis to treatment with pentamidine isethionate by quantitative real-time PCR, and identification of Leishmania parasites not responding to therapy.

BACKGROUND: Leishmania (Viannia) guyanensis is believed to be the principal cause of cutaneous leishmaniasis (CL) in Suriname. This disease is treated with pentamidine isethionate (PI), but treatment failure has increasingly been reported. AIM: To evaluate PI for its clinical efficacy, to compare parasite load, and to assess the possibility of treatment failure due to other infecting Leishmania species. METHODS: Parasite load of patients with CL was determined in skin biopsies using real-time quantitative PCR before treatment and 6 and 12 weeks after treatment. Clinical responses were evaluated at week 12 and compared with parasite load. In parallel, molecular species differentiation was performed. RESULTS: L. (V.) guyanensis was the main infecting species in 129 of 143 patients (about 90%). PI treatment led to a significant decrease (P < 0.001) in parasite counts, and cured about 75% of these patients. Treatment failure was attributable to infections with Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis and L. (V.) guyanensis (1/92, 1/92 and 22/92 evaluable cases, respectively). There was substantial agreement beyond chance between the parasite load at week 6 and the clinical outcome at week 12, as indicated by the κ value of 0.61. CONCLUSIONS: L. (V.) guyanensis is the main infecting species of CL in Suriname, followed by L. (V.) braziliensis and L. (L.) amazonensis. Furthermore, patient response to PI can be better anticipated based on the parasite load 6 weeks after the treatment rather than on parasite load before treatment.

Leishmania donovani infection drives the priming of human monocyte-derived dendritic cells during Plasmodium falciparum co-infections.

Functional impairment of dendritic cells (DCs) is part of a survival strategy evolved by Leishmania and Plasmodium parasites to evade host immune responses. Here, the effects of co-exposing human monocyte-derived DCs to Leishmania donovani promastigotes and Plasmodium falciparum-infected erythrocytes were investigated. Co-stimulation resulted in a dual, dose-dependent effect on DC differentiation which ranged from semi-mature cells, secreting low interleukin(-12p70 levels to a complete lack of phenotypic maturation in the presence of high parasite amounts. The effect was mainly triggered by the Leishmania parasites, as illustrated by their ability to induce semi-mature, interleukin-10-producing DCs, that poorly responded to lipopolysaccharide stimulation. Conversely, P. falciparum blood-stage forms failed to activate DCs and only slightly interfered with lipopolysaccharide effects. Stimulation with high L. donovani concentrations triggered phosphatidylserine translocation, whose onset presented after initiating the maturation impairment process. When added in combination, the two parasites could co-localize in the same DCs, confirming that the leading effects of Leishmania over Plasmodium may not be due to mutual exclusion. Altogether, these results suggest that in the presence of visceral leishmaniasis-malaria co-infections, Leishmania-driven effects may overrule the more silent response elicited by P. falciparum, shaping host immunity towards a regulatory pattern and possibly delaying disease resolution.

Molecular tools for diagnosis of visceral leishmaniasis: systematic review and meta-analysis of diagnostic test accuracy.

Molecular methods have been proposed as highly sensitive tools for the detection of Leishmania parasites in visceral leishmaniasis (VL) patients. Here, we evaluate the diagnostic accuracy of these tools in a meta-analysis of the published literature. The selection criteria were original studies that evaluate the sensitivities and specificities of molecular tests for diagnosis of VL, adequate classification of study participants, and the absolute numbers of true positives and negatives derivable from the data presented. Forty studies met the selection criteria, including PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), and loop-mediated isothermal amplification (LAMP). The sensitivities of the individual studies ranged from 29 to 100%, and the specificities ranged from 25 to 100%. The pooled sensitivity of PCR in whole blood was 93.1% (95% confidence interval [CI], 90.0 to 95.2), and the specificity was 95.6% (95% CI, 87.0 to 98.6). The specificity was significantly lower in consecutive studies, at 63.3% (95% CI, 53.9 to 71.8), due either to true-positive patients not being identified by parasitological methods or to the number of asymptomatic carriers in areas of endemicity. PCR for patients with HIV-VL coinfection showed high diagnostic accuracy in buffy coat and bone marrow, ranging from 93.1 to 96.9%. Molecular tools are highly sensitive assays for Leishmania detection and may contribute as an additional test in the algorithm, together with a clear clinical case definition. We observed wide variety in reference standards and study designs and now recommend consecutively designed studies.

LATENT CLASS ANALYSIS IN DIAGNOSTIC TESTS EVALUATION FOR CANINE LEISHMANIA INFANTUM INFECTION.

Accurate assessment of diagnostic tests may be biased if an imperfect reference test is used for comparison; such a situation exists for the diagnosis of canine leishmaniasis. We compared classical diagnostic tests for Leishmania infantum with Latent Class Analysis (LCA), to assess whether we could make a more accurate calculation of diagnostic accuracy. Microscopy (Lymph node aspirate), serological test (IFAT), and molecular tests (LAMP and PCR) data were recorded for 75 dogs captured in Tunisian endemic area and suspected of leishmaniasis. Sensitivity and specificity estimates with the 2 x 2 contingency tables (Microscopy as gold standard) were broadly corroborated by LCA. However, the LCA provided a way to control the study limitations (small sample size) as well as for confounding factors. It also produces consistent estimates of the test characteristics. LCA estimation of the sensitivity and specifcity of the LAMP cpb assay (se: 68.7% [95% CI 573-80%] and sp: 86.2 [95% CI 749-975%]) is higher as compared to classical calculations (se: 54.2% [95% CI 38.2-69.5%] and sp: 80% [95% CI 65.2-89.5%). Considering the lack of an adequate reference standard, LCA proved to be a useful tool to independently evaluate diagnostic methods.

Size and albedo of Kuiper belt object 55636 from a stellar occultation.

The Kuiper belt is a collection of small bodies (Kuiper belt objects, KBOs) that lie beyond the orbit of Neptune and which are believed to have formed contemporaneously with the planets. Their small size and great distance make them difficult to study. KBO 55636 (2002 TX(300)) is a member of the water-ice-rich Haumea KBO collisional family. The Haumea family are among the most highly reflective objects in the Solar System. Dynamical calculations indicate that the collision that created KBO 55636 occurred at least 1 Gyr ago. Here we report observations of a multi-chord stellar occultation by KBO 55636, which occurred on 9 October 2009 ut. We find that it has a mean radius of 143 +/- 5 km (assuming a circular solution). Allowing for possible elliptical shapes, we find a geometric albedo of in the V photometric band, which establishes that KBO 55636 is smaller than previously thought and that, like its parent body, it is highly reflective. The dynamical age implies either that KBO 55636 has an active resurfacing mechanism, or that fresh water-ice in the outer Solar System can persist for gigayear timescales.

New Trypanosoma (Duttonella) vivax genotypes from tsetse flies in East Africa.

Salivarian trypanosomes pose a substantial threat to livestock, but their full diversity is not known. To survey trypanosomes carried by tsetse in Tanzania, DNA samples from infected proboscides of Glossina pallidipes and G. swynnertoni were identified using fluorescent fragment length barcoding (FFLB), which discriminates species by size polymorphisms in multiple regions of the ribosomal RNA locus. FFLB identified the trypanosomes in 65 of 105 (61.9%) infected proboscides, revealing 9 mixed infections. Of 7 different FFLB profiles, 2 were similar but not identical to reference West African Trypanosoma vivax; 5 other profiles belonged to known species also identified in fly midguts. Phylogenetic analysis of the glycosomal glyceraldehyde phosphate dehydrogenase gene revealed that the Tanzanian T. vivax samples fell into 2 distinct groups, both outside the main clade of African and South American T. vivax. These new T. vivax genotypes were common and widespread in tsetse in Tanzania. The T. brucei-like trypanosome previously described from tsetse midguts was also found in 2 proboscides, demonstrating a salivarian transmission route. Investigation of mammalian host range and pathogenicity will reveal the importance of these new trypanosomes for the epidemiology and control of animal trypanosomiasis in East Africa.

Phylogenetic analysis reveals the presence of the Trypanosoma cruzi clade in African terrestrial mammals.

Despite the impact of some trypanosome species on human and livestock health, the full diversity of trypanosomes in Africa is poorly understood. A recent study examined the prevalence of trypanosomes among a wide variety of wild vertebrates in Cameroon using species-specific PCR tests, but six trypanosome isolates remained unidentified. Here they have been re-examined using fluorescent fragment length barcoding (FFLB) and phylogenetic analysis of glycosomal glyceraldehyde phosphate dehydrogenase gGAPDH and 18S ribosomal RNA (rDNA) genes. Isolates from a monkey (Cercopithecus nictitans) and a palm civet (Nandinia binotata) belonged to the Trypanosoma cruzi clade, known previously only from New World and Australian terrestrial mammals, and bats from Africa, Europe and South America. Of the four other isolates, three from antelope were identified as Trypanosoma theileri, and one from a crocodile as T. grayi. This is the first report of trypanosomes of the T. cruzi clade in African terrestrial mammals and expands the clade's known global distribution in terrestrial mammals. Previously it has been hypothesized that African and New World trypanosomes diverged after continental separation, dating the divergence to around 100 million years ago. The new evidence instead suggests that intercontinental transfer occurred well after this, possibly via bats or rodents, allowing these trypanosomes to establish and evolve in African terrestrial mammals, and questioning the validity of calibrating trypanosome molecular trees using continental separation.

New molecular tools for the identification of trypanosome species.

Trypanosomes are the causative agents of many diseases of medical and veterinary importance, including sleeping sickness and nagana in Africa, and Chagas disease in South America. Accurate identification of trypanosome species is essential, as some species are morphologically indistinguishable, yet differ greatly in their pathogenicity. A range of molecular tools has been developed for identification of species and strains of trypanosomes. PCR, using primer sets designed to amplify a specific DNA fragment from each trypanosome species, is frequently used. More recently, generic systems have been developed that can potentially recognize all trypanosome species, such as amplification of the internal transcribed spacer and fluorescent fragment length barcoding, both of which use interspecies size variation in PCR fragments amplified from the ribosomal RNA locus. Loop-mediated isothermal amplification is a promising technique and is able to detect trypanosomes in blood, serum and cerebrospinal fluid. The advantages of these techniques for high-throughput and sensitive molecular identification will be discussed.

The identification, diversity and prevalence of trypanosomes in field caught tsetse in Tanzania using ITS-1 primers and fluorescent fragment length barcoding.

We report on the development of two generic, PCR-based methods, which replace the multiple species-specific PCR tests used previously to identify the trypanosome species carried by individual tsetse flies. The first method is based on interspecies size variation in the PCR product of the ITS-1 region of the ribosomal RNA (rRNA) locus. In the second approach, length variation of multiple fragments within the 18S and 28S rRNA genes is assayed by PCR amplification with fluorescent primers; products are subsequently sized accurately and rapidly by the use of an automated DNA sequencer. Both methods were used to identify samples collected during large-scale field studies of trypanosome-infected tsetse in Tanzania in the National Parks of Tarangire and Serengeti, and the coastal forest reserve of Msubugwe. The fluctuations of trypanosome prevalence over time and two different field seasons are discussed. As well as facilitating the identification of trypanosome species with increased speed, precision and sensitivity, these generic systems have enabled us to identify two new species of trypanosome.

A novel, high-throughput technique for species identification reveals a new species of tsetse-transmitted trypanosome related to the Trypanosoma brucei subgenus, Trypanozoon.

We describe a novel method of species identification, fluorescent fragment length barcoding, based on length variation in regions of the 18S and 28Salpha ribosomal DNA. Fluorescently tagged primers, designed in conserved regions of the 18S and 28Salpha ribosomal DNA, were used to amplify fragments with inter-species size variation, and sizes determined accurately using an automated DNA sequencer. By using multiple regions and different fluorochromes, a barcode unique to each species was generated. The technique was developed for the identification of African tsetse-transmitted trypanosomes and validated using DNA from laboratory isolates representing known species, subspecies and subgroups. To test the methodology, we examined 91 trypanosome samples from infected tsetse fly midguts from Tanzania, most of which had already been identified by species-specific and generic PCR tests. Identifications were mainly in agreement, but the presence of an unknown trypanosome in several samples was revealed by its unique barcode. Phylogenetic analyses based on 18S rDNA and glycosomal glyceraldehyde phosphate dehydrogenase gene sequences confirmed that this trypanosome is a new species and it is within the Trypanosoma brucei clade, as a sister group of subgenus Trypanozoon. The overall identification rate of trypanosome-infected midgut samples increased from 78 to 96% using FFLB instead of currently available PCR tests. This was due to the high sensitivity of FFLB as well as its capacity to identify previously unrecognised species. FFLB also allowed the identification of multiple species in mixed infections. The method enabled high-throughput and accurate species identification and should be applicable to any group of organisms where there is length variation in regions of rDNA.

Trypanosome identification in wild tsetse populations in Tanzania using generic primers to amplify the ribosomal RNA ITS-1 region.

Tsetse flies transmit many species of trypanosomes in Africa, some of which are human and livestock pathogens of major medical and socio-economic impact. Identification of trypanosomes is essential to assess the disease risk posed by particular tsetse populations. We have developed a single generic PCR test to replace the multiple species-specific PCR tests used previously to identify the trypanosome species carried by individual tsetse flies. In the generic PCR test, inter-species size variation in the PCR product of the internal transcribed spacer (ITS-1) region of the ribosomal RNA repeat region enables species identification. The test was applied to identify trypanosomes in midgut samples stored on FTA cards from wild-caught flies in two regions of Tanzania. Identifications were verified by sequencing the amplified ITS-1 region and/or species-specific PCR tests. The method facilitated the identification of large numbers of field samples quickly and accurately. Whereas species-specific tests are incapable of recognising previously unknown species, the generic test enabled a new species to be identified by the unique size of the amplified product. Thus, even without access to any isolate of this new species, we could collect data on its distribution, prevalence and co-occurrence with other trypanosomes. The combined molecular and ecological profiles should facilitate the isolation and full biological characterization of this species in the future.

Charon's radius and atmospheric constraints from observations of a stellar occultation.

The physical characteristics of Pluto and its moon, Charon, provide insight into the evolution of the outer Solar System. Although previous measurements have constrained the masses of these bodies, their radii and densities have remained uncertain. The observation of a stellar occultation by Charon in 1980 established a lower limit on its radius of 600 km (ref. 3) (later refined to 601.5 km; ref. 4) and suggested a possible atmosphere. Subsequent, mutual event modelling yielded a range of 600-650 km (ref. 5), corresponding to a density of 1.56 +/- 0.22 g cm(-3) (refs 2, 5). Here we report multiple-station observations of a stellar occultation by Charon. From these data, we find a mean radius of 606 +/- 8 km, a bulk density of 1.72 +/- 0.15 g cm(-3), and rock-mass fraction 0.63 +/- 0.05. We do not detect a significant atmosphere and place 3sigma upper limits on atmospheric number densities for candidate gases. These results seem to be consistent with collisional formation for the Pluto-Charon system in which the precursor objects may have been differentiated, and they leave open the possibility of atmospheric retention by the largest objects in the outer Solar System.

In vivo effect of 5-ethynyluracil on 5-fluorouracil metabolism determined by 19F nuclear magnetic resonance spectroscopy.

Biochemical modulation of 5-fluorouracil (5-FU) has been used over the past 20 years to improve the therapeutic efficacy of this antineoplastic agent. Recently, modulation of the catabolic pathway of this fluoropyrimidine has been the focus of extensive preclinical and clinical investigation. Dihydropyrimidine dehydrogenase catalyzes the rate-limiting step in the catabolism of 5-FU and rapidly degrades 60-90% of the drug. An irreversible inactivating inhibitor of this enzyme, 5-ethynyluracil (EU), markedly improves the antitumor effect of 5-FU in animal models. Early clinical studies have shown a substantial alteration of the systemic disposition of 5-FU with an increase in 5-FU terminal half-life and have also indicated that EU allows safe oral administration of 5-FU by improving the oral bioavailability of the fluoropyrimidine, which is otherwise too erratic and unpredictable for a drug with such a limited therapeutic window. We evaluated the effect of EU on the metabolism of 5-FU in mice bearing colon 38 tumors using 19F nuclear magnetic resonance spectroscopy. Ex vivo measurements of tissue extracts from liver, kidney, and tumor indicated a >95% elimination of alpha-fluoro-beta-ureidopropionic acid and a-fluoro-beta-alanine signals in the tissues of mice that received 2 mg/kg of EU before administration of 5-FU. The spectra also showed an increased formation of fluoronucleotides in both normal and tumor tissues, a prolonged presence of 5-FU, and the accumulation of 5-fluorouridine that otherwise is undetectable, particularly in normal tissues. The in vivo NMR experiments on colon 38 tumors confirmed these findings, showing a complete elimination of the a-fluoro-beta-ureidopropionic acid and a-fluoro-beta-alanine signals in tumors treated with EU and a dramatic formation and accumulation of 5-fluorouridine mono-, di-, and triphosphates and 5-fluorouridine. Thus, by inactivating dihydropyrimidine dehydrogenase, EU prolonged the half-life for 5-FU, almost completely eliminated its catabolism for 4-6 h, which led to an increased accumulation of 5-fluorouridine mono-, di-, and triphosphates in both normal and tumor tissues.

19F NMR monitoring of in vivo tumor metabolism after biochemical modulation of 5-fluorouracil by the uridine phosphorylase inhibitor 5-benzylacyclouridine.

A uridine phosphorylase inhibitor, 5-benzylacyclouridine (BAU), has been utilized as biochemical modulator of 5-fluorouracil (5-FU) anti-tumor activity in a murine tumor model. The effect of BAU on 5-FU metabolism has been evaluated using in vitro and in vivo 19F NMR spectroscopy. The analysis of the NMR data revealed an increased formation and retention of fluorouracil nucleotides and fluorouridine in colon 38 tumors treated with the regimen containing BAU and a reduction in 5-FU catabolites (alpha-fluoro-beta-ureidopropionic acid and alpha-fluoro-beta-alanine). In the normal tissues evaluated, the presence of BAU did not significantly alter the metabolism and presence of fluoropyrimidine species, indicating a more selective effect on tumor tissues. Therapy experiments on C57/BL6 mice bearing colon 38 tumor showed that the administration of 120 mg/kg BAU 30 min before 5-FU at 85 mg/kg, on a weekly basis, resulted in an increased antineoplastic effect compared to the same dose of 5-FU alone. A smaller dose of 5-FU (60 mg/kg) also administered 30 min after 120 mg/kg BAU caused a reduction in tumor growth similar to 5-FU alone. The addition of BAU to 5-FU (85 mg/kg) resulted in a slight increase, although statistically nonsignificant, in host toxicity without causing any toxic death during the chemotherapeutic treatment. 19F NMR spectroscopy is here shown to be a powerful technique to evaluate changes in the metabolism of fluoropyrimidines after the use of biochemical modulator and to allow a correlation between improved therapeutic response with the biochemical effects generated in tissues.

Interaction of human neutrophil flavocytochrome b with cytosolic proteins: transferred-NOESY NMR studies of a gp91phox C-terminal peptide bound to p47phox.

During activation of the neutrophil NADPH oxidase, cytosolic p47(phox) is translocated to the membrane where it associates with flavocytochrome b via multiple binding regions, including a site in the C-terminus of gp91(phox). To investigate this binding site further, we studied the three-dimensional structure of a gp91(phox) C-terminal peptide (551SNSESGPRGVHFIFNKEN568) bound to p47(phox) using transferred nuclear Overhauser effect spectroscopy (Tr-NOESY) NMR. Using MARDIGRAS analysis and simulated annealing, five similar sets of structures of the p47(phox)-bound peptide were obtained, all containing an extended open bend from Ser5 to Phe14 (corresponding to gp91(phox) residues 555-564). The ends of the peptide were poorly defined, however, suggesting they were more flexible. Therefore further refinement was performed on the Ser5-Phe14 region of the peptide after omitting the ends of the peptide from consideration. In this case, two similar structures were obtained. Both structures again exhibited extended open-bend conformations. In addition, the amino acid side chains that showed evidence of immobilization on binding to p47(phox) correlated directly with those that were found previously to be essential for biological activity. Thus during NADPH oxidase assembly, the C-terminus of gp91(phox) binds to 47(phox) in an extended conformation between gp91(phox) residues 555 and 564, with immobilization of all of the amino acid side chains in the 558RGVHFIF564 region except for His561.

Preclinical toxicology studies with the new dopamine agonist pergolide. Acute, subchronic, and chronic evaluations.

Pergolide (LY127809, CAS 66104-23-2), a dopamine agonist for the treatment of Parkinson's disease, was evaluated for toxicity in acute, subchronic, and chronic studies. Acute toxicity tests using oral, intravenous and intraperitoneal routes were conducted in rats, mice, rabbits, and dogs. The acute oral median lethal doses (MLD) ranged from 8.4 to 33.6 mg/kg in Wistar and Fischer 344 rats, and from 54.0 to 87.2 mg/kg in ICR mice. Oral doses of 20 and 25 mg/kg produced no mortality in rabbits or dogs, respectively. The MLD by the iv route ranged from 0.59 to 0.87 mg/kg for Fischer 344 rats and from 11.6 to 37.1 mg/kg for ICR mice. The predominant signs of toxicity in the acute studies included hyperactivity, poor grooming, ptosis, aggressive behavior, increased gnawing activity, tremors, convulsions, and emesis. In the subchronic and chronic studies, Fischer 344 rats, B6C3F1 mice, and beagle dogs were administered pergolide either by gavage or in the diet for up to 1 year. Daily doses in these studies ranged up to 20 mg/kg for rats, 45 mg/kg for mice, and 5 mg/kg for dogs. The predominant treatment-related effects seen in these studies were attributable to the pharmacologic activity of pergolide. These consisted primarily of CNS-mediated clinical signs in rats and dogs, weight loss or decreased weight gain, emesis in dogs, and inhibition of lysis of corpora lutea with a corresponding increase in the weight of the uterus and ovaries. Pergolide treatment was not associated with any specific target organ toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinogenicity studies of fluoxetine hydrochloride in rats and mice.

The antidepressant drug fluoxetine HCl was tested for carcinogenicity in three well designed and controlled studies in Fischer rats and C57BL/6 x C3H F1 mice. The compound was administered to the animals for 24 months at dietary doses of approximately 0, 0.5, 2.0, or 10.0 mg/kg body weight in rats and 1.0, 5.0, or 10.0 mg/kg in mice. The highest dose tested was a maximum tolerated dose for both species as evidenced by clinical signs (rats and mice) and some mortality (mice) referable to central nervous system pharmacological effects, decreased weight gain (rats), and histopathological changes of phospholipidosis (rats) and hepatic fatty change (mice). There was no evidence of an increased incidence of any type of unusual or commonly occurring spontaneous neoplasm in either rats or mice. There were statistically significant decreases in a few commonly occurring neoplasms. The data reported herein provide convincing evidence that fluoxetine is neither a complete carcinogen nor a tumor promoter.