Assessing Microbial Metabolic and Biological Diversity to Inform Natural Product Library Assembly.

The pressing need for novel chemical matter to support bioactive compound discovery has led natural product researchers to explore a wide range of source organisms and environments. One of the implicit guiding principles behind those efforts is the notion that sampling different environments is critical to accessing unique natural products. This idea was tested by comparing fungi from disparate biomes: aquatic sediments from Lake Michigan (USA) and terrestrial samples taken from the surrounding soils. Matched sets of Penicillium brevicompactum, Penicillium expansum, and Penicillium oxalicum from the two source environments were compared, revealing modest differences in physiological performance and chemical output. Analysis of LC-MS/MS-derived molecular feature data showed no source-dependent differences in chemical richness. High levels of scaffold homogeneity were also observed with 78-83% of scaffolds shared among the terrestrial and aquatic Penicillium spp. isolates. A comparison of the culturable fungi from the two biomes indicated that certain genera were more strongly associated with aquatic sediments (e.g., Trichoderma, Pseudeurotium, Cladosporium, and Preussia) versus the surrounding terrestrial environment (e.g., Fusarium, Pseudogymnoascus, Humicola, and Acremonium). Taken together, these results suggest that focusing efforts on sampling the microbial resources that are unique to an environment may have a more pronounced effect on enhancing the sought-after natural product diversity needed for chemical discovery and screening collections.

Systematic review: dorsal bridge plating in distal radius fractures.

PURPOSE: Distal radius fractures are the most common upper limb fractures in adults (up to 18% of all fractures in the Emergency Department). Conservative management is possible for the majority, the preferred surgical technique being volar plate fixation. Dorsal bridge plating (DBP) is an alternative method of treatment for complex fractures. DBP acts as an internal fixator and can be used in patients needing early rehabilitation. This systematic review assesses the demographics, functional and radiological outcomes and complications of using DBP in patients with distal radius fractures compared to volar plate fixation. METHODS: A literature search of PubMed, Cochrane, EMBASE and Google Scholar was performed according to PRISMA guidelines. Seven hundred and sixty-one articles were found; 11 articles met the inclusion criteria. Cadaveric studies and case studies of less than five patients were excluded. Primary outcome measures were functional and radiological outcomes. Complications were recorded as secondary outcomes. RESULTS: Three hundred and ninety-four patients were included in the study with an average age of 54.8 years (53.9% male and 46.1% female). Weighted mean follow-up was 55.2 weeks; the mean time to plate removal was 17.3 weeks with a mean DASH score of 25.7. The weighted range of movement was 46.9° flexion, 48.8° extension, 68.4° pronation and 67.5° supination. The radiological parameters show satisfactory outcomes with a mean radial height of 10mm, volar tilt of 3.1°, ulnar variance of 0.5mm and radial inclination of 18.8°. The complication rate was 11.4%. Digital stiffness was the most common complication but improved if tenolysis was performed at plate removal. CONCLUSIONS: DBP is a good alternative to volar plating for complex distal radius fractures. The functional outcomes showed a slight loss of range of movement, whereas the radiological outcomes were within recommended limits. A significant disadvantage of the plate is the need for further surgical removal.

Seeking the ghost of worms past.

The mechanisms of protective immunity to parasite infections in humans are still elusive. Here, Woolhouse and Hagan discuss new evidence suggesting that the extremely slow development of acquired immunity to human schistosomes may depend on exposure to antigens from these worms after they die.

Gentamicin-attenuated Leishmania infantum: cellular immunity production and protection of dogs against experimental canine leishmaniasis.

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. Here, we show that L. infantum H-line induced significantly higher levels of IFN-γ and lower levels of IL-10 compared with those in dogs infected with L. infantum wild type (WT). Anti-Leishmania-specific total IgG, IgG1, and IgG2 antibodies were present in the serum of all infected dogs, with levels of IgG2 subclass highest in the sera of dogs inoculated with L. infantum H-line. Relatively high levels of IgG1 were found in the sera of dogs infected with L. infantum WT. Six of seven dogs immunized intradermally (i.d.) with the attenuated line later showed a positive skin test to leishmanin, whereas the dogs infected with L. infantum WT did not. No clinical abnormalities were observed, and no parasites found in the visceral organs of the dogs inoculated intravenously (i.v.) with L. infantum H-line over 24 months post-inoculation. Dogs which had been immunized with L. infantum H-line i.d. 12 months previously were protected against challenge with L. infantum WT. These data suggest that the L. infantum H-line was safe and induced a protection which is correlated with cellular immunity in dogs.

Praziquantel: its use in control of schistosomiasis in sub-Saharan Africa and current research needs.

Treatment with praziquantel (PZQ) has become virtually the sole basis of schistosomiasis control in sub-Saharan Africa and elsewhere, and the drug is reviewed here in the context of the increasing rate that it is being used for this purpose. Attention is drawn to our relative lack of knowledge about the mechanisms of action of PZQ at the molecular level, the need for more work to be done on schistosome isolates that have been collected recently from endemic areas rather than those maintained in laboratory conditions for long periods, and our reliance for experimental work mainly on Schistosoma mansoni, little work having been done on S. haematobium. There is no evidence that resistance to PZQ has been induced in African schistosomes as a result of its large-scale use on that continent to date, but there is also no assurance that PZQ and/or schistosomes are in any way unique and that resistant organisms will not be selected as a result of widespread drug usage. The failure of PZQ to produce complete cures in populations given a routine treatment should therefore solicit considerable concern. With few alternatives to PZQ currently available and/or on the horizon, methods to monitor drug-susceptibility in African schistosomes need to be devised and used to help ensure that this drug remains effective for as long a time as possible.

Breakthrough cancer pain: The importance of the right treatment at the right time.

BACKGROUND: Confusion remains over the definition of breakthrough cancer pain (BTcP) potentially leading to delayed diagnosis and treatment. METHODS: An on-line survey was conducted in four EU countries among relevant healthcare professionals and cancer patients diagnosed with BTcP. The roles of healthcare professionals (HCPs) were examined and their knowledge and use of available medications recorded. Patients were questioned on how BTcP affected their lives and on the medications they had received/were receiving. RESULTS: There was a 'time lag' of 58 and 13 weeks in Germany and Spain respectively between the initial diagnosis of BTcP and its treatment. Four in ten oncologists across the four countries considered themselves not fully confident in their choice of the appropriate therapy. A quarter of patients in Germany, Italy and Spain and four in ten in France were treated only with increased dosages of the therapy already prescribed for their background pain - often morphine. Almost another quarter received morphine in addition to their treatment for background pain. Oncologists indicated a need for faster-acting treatments revealing a potential lack of awareness of rapid onset oral opioids and patients expressed a desire for more effective pain relief and better psychological support. CONCLUSIONS: There is a need for a universal definition of BTcP to facilitate earlier and more accurate diagnosis. It is essential that BTcP is treated immediately on diagnosis with therapies that more closely mirror its temporal characteristics to ensure that patients' desire for more effective pain relief is fulfilled. SIGNIFICANCE: Many cancer patients suffered episodes of BTcP needlessly over many months due to missed diagnosis. Even after diagnosis, many physicians were not fully confident in their choice of 'rescue' therapy which perhaps is not surprising given the very low level of awareness of treatment guidelines, both national and international.

Cytotoxic activity, tumor accumulation, and tissue distribution of ruthenium-103-labeled bleomycin.

Bleomycin (BLM) was labeled with gamma-emitting 103Ru. Yields of 103Ru-labeled BLM as high as 50.6% were attained. 103Ru-labeled BLM was stable in vitro and the 103ru label was not displaced by large excesses of Cu (II) and Co (II) or Fe (III). Chromatography of the urine following 103Ru-labeled BLM injection indicated no in vivo decomposition. Pharmacokinetic studies in healthy inbred SD and tumor-bearing inbred BUF rats demonstrated tumor accumulations, tissue distributions, and clearance nearly identical with those reported for 3H-labeled BLM. Cytotoxicity studies on a WI-L2 human B-cell line showed that BLM labeled with nonradioactive Ru retained 100% of the activity demonstrated by native BLM. Thus BLM may be labeled with isotopes of Ru to form stable complexes by a simple, rapid reaction without loss of its chemotherapeutic properties or variations in its in vivo distribution. BLM labeled with the proper Ru isotope should prove useful as a gamma-emitting tracer for BLM or a beta-emitting compound capable of providing combination chemotherapy and radiotherapy of tumors.

Stability, characterization, and kinetics of 111In-labeled monoclonal antitumor antibodies in normal animals and nude mouse-human tumor models.

Monoclonal antibodies (MoAbs) against carcinoembryonic antigen were successfully radiolabeled with 111In, and the radiopharmaceutical was characterized in vitro and in normal and tumor-bearing mice. The 111In-MoAb proved to be stable in vitro and in vivo under normal conditions, although instability could be induced in vitro with large quantities of iron-free transferrin. Animal distribution studies with 111In-MoAb demonstrated tumor localization superior to 67Ga and pharmacokinetics that were highly similar to those of endogenously labeled 75Se-MoAb. The 111In-MoAb followed first-order kinetics and fit a two-compartmental model when studied in nude mice bearing human colon tumors known to express carcinoembryonic antigen. Significant quantities of radiolabel appeared in tissues other than tumor, with liver and skin having the highest concentrations. Sufficient tumor/background ratios were formed for scanning purposes. The data indicate that 111In-MoAb may prove to be effective as a radiopharmaceutical for tumor imaging.

Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells.

Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with 111In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity.

Athymic mouse model of a human T-cell tumor.

Because of the large number of different immunoconjugates which can be produced from monoclonal antibody-directed anti-cancer therapy, it would be useful to have in vivo tumor models to compare such preparations. Although historically human leukemias-lymphomas have been difficult to establish in athymic mice we have succeeded in establishing human T-cell tumors from primary MOLT-4 cultures in 290 of 353 animals and have successfully transferred tumors in 42 of 45 animals during ten serial passages. The potential utility of this model for testing immunoconjugates of murine monoclonal antibody T101 have been confirmed by: (a) in all 148 tumors sampled including all passaged tumors the human T-cell antigen, T65, was expressed in a manner identical to that of cultured cells; (b) 111In-T101 was concentrated preferentially in the tumor; and (c) T101 injected by both the i.p. and i.v. routes bound to tumor and induced antigenic modulation to the same extent as that observed previously in vitro and in human studies.

New multi-scale perspectives on the stromatolites of Shark Bay, Western Australia.

A recent field-intensive program in Shark Bay, Western Australia provides new multi-scale perspectives on the world's most extensive modern stromatolite system. Mapping revealed a unique geographic distribution of morphologically distinct stromatolite structures, many of them previously undocumented. These distinctive structures combined with characteristic shelf physiography define eight 'Stromatolite Provinces'. Morphological and molecular studies of microbial mat composition resulted in a revised growth model where coccoid cyanobacteria predominate in mat communities forming lithified discrete stromatolite buildups. This contradicts traditional views that stromatolites with the best lamination in Hamelin Pool are formed by filamentous cyanobacterial mats. Finally, analysis of internal fabrics of stromatolites revealed pervasive precipitation of microcrystalline carbonate (i.e. micrite) in microbial mats forming framework and cement that may be analogous to the micritic microstructures typical of Precambrian stromatolites. These discoveries represent fundamental advances in our knowledge of the Shark Bay microbial system, laying a foundation for detailed studies of stromatolite morphogenesis that will advance our understanding of benthic ecosystems on the early Earth.

Inner workings of thrombolites: spatial gradients of metabolic activity as revealed by metatranscriptome profiling.

Microbialites are sedimentary deposits formed by the metabolic interactions of microbes and their environment. These lithifying microbial communities represent one of the oldest ecosystems on Earth, yet the molecular mechanisms underlying the function of these communities are poorly understood. In this study, we used comparative metagenomic and metatranscriptomic analyses to characterize the spatial organization of the thrombolites of Highborne Cay, The Bahamas, an actively forming microbialite system. At midday, there were differences in gene expression throughout the spatial profile of the thrombolitic mat with a high abundance of transcripts encoding genes required for photosynthesis, nitrogen fixation and exopolymeric substance production in the upper three mm of the mat. Transcripts associated with denitrification and sulfate reduction were in low abundance throughout the depth profile, suggesting these metabolisms were less active during midday. Comparative metagenomics of the Bahamian thrombolites with other known microbialite ecosystems from across the globe revealed that, despite many shared core pathways, the thrombolites represented genetically distinct communities. This study represents the first time the metatranscriptome of living microbialite has been characterized and offers a new molecular perspective on those microbial metabolisms, and their underlying genetic pathways, that influence the mechanisms of carbonate precipitation in lithifying microbial mat ecosystems.

Endotoxin-stimulated production of rat hypothalamic interleukin-1 beta in vivo and in vitro, measured by specific immunoradiometric assay.

Regulation of a number of aspects of the acute-phase response, including induction of fever and activation of the hypothalamo-pituitary-adrenal axis, occurs within the hypothalamus. The acute-phase response appears to be co-ordinated by the inflammatory cytokine interleukin-1 (IL-1). A number of studies using hybridization techniques to measure IL-1 gene expression and immunocytochemistry to localize immunoactive IL-1 have established the concept that the central nervous system, and in particular the hypothalamus, is a site of IL-1 production, and that levels increase in response to inflammatory stimuli. In this report we present data on the levels of IL-1 beta produced in the rat hypothalamus using quantitative immunoassay techniques. Bacterial endotoxin, administered to rats in vivo, evoked increases in hypothalamic IL-1 beta levels which were significant within 1 h, and reached maximum levels at 5-10 h. The response to endotoxin was dose-related, and levels reached in hypothalamic extracts corresponded to intrahypothalamic levels of the order of 20 ng/ml. During short-term in-vitro culture of rat hypothalami, endotoxin stimulated a dose-related increase in both the synthesis and the secretion of IL-1 beta, which reached similar levels to those seen after in-vivo stimulation. Hypothalami obtained from animals stimulated with endotoxin in vivo did not, however, show any evidence of persistent stimulation of IL-1 beta production when subsequently cultured in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a two-site immunoradiometric assay for rat/human corticotrophin-releasing factor. Application to the measurement of interleukin-1 beta-stimulated production of hypothalamic CRF in vitro.

The hypothalamic hormone corticotrophin-releasing factor (CRF) is a highly conserved, 41-residue peptide, the N terminal region of which rarely induces antibody production, which has hindered the development of two-site immunometric assays. A synthetic N terminal peptide, CRF1-20-Cys-Tyr-NH2, was conjugated to bovine serum albumin through the cysteine thiol group, and used to prepare N terminal directed CRF-specific antibodies. The same peptide, conjugated through the cysteine thiol group to activated thiol-Sepharose, was used to affinity purify N terminal CRF-specific antibodies, and these were used in conjunction with a radioiodinated C terminal directed monoclonal anti-CRF antibody for the development of a specific, sensitive two-site immunoradiometric assay for CRF. To test the utility of the assay, hypothalami were stimulated in vitro with interleukin-1 beta, a putative regulator of CRF secretion, and CRF was measured in hypothalamic homogenates and conditioned media. Interleukin-1 beta dose-dependently stimulated synthesis and secretion of CRF, demonstrating the applicability of the immunoradiometric assay, and confirming previous reports that interleukin-1 beta can directly stimulate CRF secretion from the rat hypothalamus in vitro.

Molecular cloning and characterisation of the 22-kilodalton adult Schistosoma mansoni antigen recognised by antibodies from mice protectively vaccinated with isolated tegumental surface membranes.

A cDNA clone from an adult Schistosoma mansoni lambda gt11 expression library (A12) encoding an antigenic polypeptide of 22 kDa is described. A12 is 797 bp long and has one open reading frame encoding a protein of 190 amino acids which does not contain a signal sequence or membrane anchor motif and has no homologies with any sequences on the currently available data bases. Its product (sm22.6) is recognised by antibodies from mice protectively vaccinated with purified adult S. mansoni tegumental membranes and by serum from S. mansoni-infected Brazilians. It is present in all post-snail life cycle stages except the egg, is not sex-specific, and is found in 9 species of Schistosoma, but not in a range of other helminths. Data are presented which suggest that sm22.6 is a soluble, peripheral membrane protein.

Adult schistosome cDNA libraries as a source of antigens for the study of experimental and human schistosomiasis.

Protective immunity has been demonstrated in experimental schistosomiasis and is also believed to occur in man. It can be mediated by antibodies from infected animals or animals immunized with attenuated organisms. Recombinant Escherichia coli synthesizing antigenic polypeptides from the three principal species of schistosome that infect man, Schistosoma mansoni, S. japonicum and S. haematobium, have been constructed. Libraries of adult worm cDNA were prepared from each species in the expression vector lambda gt 11 and directly screened with antibodies from animals experimentally immunized with S. mansoni and S. japonicum and from humans infected with S. haematobium. The S. mansoni clones have been analysed in greatest detail. At least four different types of clones were identified. All the detected recombinant polypeptide antigens were recognised by antibodies from chronically infected mice and most were also recognised by antibodies from mice immunized with attenuated cercariae and anti-surface membrane antibodies. Clones synthesizing species-specific antigens for both S. mansoni and S. japonicum were identified by simultaneous screening of both libraries. At least three types of S. haematobium clones were identified by screening with human infection serum, most of which were species-specific. All the antigens were in the form of fusion peptides with E. coli beta-galactosidase and their expression was induced by isopropylthiogalactopyranoside. Since known protective monoclonal antibodies recognise highly glycosylated membrane proteins which cannot be identified in the form of nascent polypeptides, the direct identification of polypeptide antigens defined by their reactivity, as reported here, is an essential step in producing reagents by recombinant DNA technology, suitable for vaccination and diagnosis.

Structure of the gene encoding a putative Schistosoma mansoni tegumental antigen precursor.

In order to obtain the complete gene encoding the putative precursor of a 15-kDa Schistosoma mansoni tegumental antigen (Sm15), two cDNAs (A70 and A184) and two fragments of independent genomic clones were subcloned and sequenced. The collated sequence contains 4700 nucleotides and represents the full length open reading frame of the gene, encoding a protein of 1032 amino acids with a calculated molecular mass of 116,900. Thus, the gene encodes a much longer protein than that identified in the tegumental membranes, suggesting that it encodes a precursor that is subsequently highly processed. A 964-bp region composed of 5 closely related repeats was found to be present within the translated frame. The predicted protein is highly acidic and there is no indication of hydrophobic domains that may represent transmembrane regions or indicate attachment of a GPI anchor. The coding region has no homologies in the currently available data bases. In the 5' non-transcribed area a copy of the SM alpha repeat family is present. The coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression.

Adenovirus-mediated over-expression of interleukin-1 receptor antagonist reduces susceptibility to excitotoxic brain injury in perinatal rats.

In seven-day-old rats, intracerebral injection of N-methyl-D-aspartate transiently stimulates expression of Interleukin-1 beta messenger RNA. To evaluate the role of Interleukin-1 beta in the pathogenesis of excitotoxic injury, we sought to determine if Interleukin-1 receptor antagonist, an endogenous competitive inhibitor of Interleukin-1 beta, could attenuate N-methyl-D-aspartate-induced injury. To induce sustained over-expression of Interleukin-1 receptor antagonist in the brain, a recombinant adenovirus encoding Interleukin-1 receptor antagonist was administered by intracerebroventricular injection into three-day-old rats. Increased brain concentrations of Interleukin-1 receptor antagonist two to six days later were documented by assays of tissue homogenates and by immunocytochemistry. To evaluate the impact of Interleukin-1 receptor antagonist on N-methyl-D-aspartate neurotoxicity, three-day-old animals received intracerebroventricular injections of either adenovirus encoding Interleukin-1 receptor antagonist or a control adenovirus encoding beta-galactosidase, followed four days later by right intrastriatal injections of N-methyl-D-aspartate (10 nmol/0.5 microliter), a dose that typically elicits excitotoxic injury in the ipsilateral striatum and adjacent hippocampus, or saline. Animals were killed five days later, and brain damage was quantitated by measurement of bilateral cross-sectional areas of the striatum and anterior hippocampus. In three independent experiments, in N-methyl-D-aspartate-lesioned animals, both striatal and hippocampal injuries were reduced in animals that had been infected with adenovirus that encoded Interleukin-1 receptor antagonist, in comparison with littermates infected with the control adenovirus (right striatal volume loss ranged from 16 to 24%, compared with 54-65% volume loss in control). There was no striatal atrophy in adenovirus-infected saline-injected animals. These results provide strong support for the hypothesis that Interleukin-1 beta is a mediator of excitotoxic brain injury in perinatal rats.

Optimal handling of dimercaptosuccinic acid for quantitative renal scanning.

Methods of optimizing quantitative renal imaging with Tc-99m dimercaptosuccinic acid (DSMA) were investigated. Rats were injected with DMSA (one kit per rat) and sacrificed at 0.5, 2.0, and 24 hr after injection. Fifty percent of the injected dose localized in the kidneys at 0.5, 2, and 24 hr after injection while background activity peaked at 0.5 hr and then declined to give substantially higher kidney-to-background ratios at 24 hr. Delayed scanning should increase the accuracy of clinical studies in patients with low kidney-to-background ratios at 1-2 hr. After injection of DMSA, 1 ml of air was introduced into the reaction vials and incubated 20 min. Kidney uptake decreased from 50 to 40% and liver uptake increased from 7.5 to 17%. If multiple doses must be drawn from a single vial, air should not be introduced, and the doses should be drawn together and administered immediately to minimize radiopharmaceutical deterioration.

Tissue distribution of 203 Pb-acetate: comparison with 67 Ga-citrate as an abscess-localizing agent.

Since 203Pb-acetate accumulates in necrotic tumor tissue, the possibility was raised that it might also accumulate in other necrotic tissue such as abscess. We first studied the tissue distribution and excretion of 203Pb-acetate in control rats at 4, 24, 48, 72, and 96 hr. An enterohepatic circulation for lead is suggested. We then compared the uptakes of 203Pb-acetate and 67Ga-citrate in experimental abscesses in rats. The mean gallium accumulation in the abscess was 10 times that of lead at 24 hr and 12 times that of lead at 72 hr. The abscess-to-tissue ratios were greater for gallium for every tissue examined, although the abscessed areas were clearly visualized by scanning at 24 and 72 hr with both agents. With the exception of blood, abscess-to-tissue ratios for 67Ga at 24 hr were higher than or equal to those at 72 hr. However, the 67Ga ratios for the inflamed tissue surrounding the abscess to muscle and blood were higher at 72 hr than at 24 hr, which suggests that inflammation without abscess might be better identified by gallium scanning at 72 hr.