Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

Molecular evidence for Acanthocephala as a subtaxon of Rotifera

Rotifers are free-living animals usually smaller than 1 mm that possess a characteristic wheel organ. Acanthocephalans (thorny-headed worms) are larger endoparasitic animals that use vertebrates and arthropods to complete their life cycle. The taxa Acanthocephala and Rotifera are considered separate phyla, often within the taxon Aschelminthes. We have reexamined the relationship between Rotifera and Acanthocephala using 18S rRNA gene sequences. Our results conclusively show that Acanthocephala is the sister group of the rotifer class Bdelloidea. Rotifera was nonmonophyletic in all molecular analyses, which supports the hypothesis that the Acanthocephala represent a taxon within the phylum Rotifera and not a separate phylum. These results agree with a previous cladistic study of morphological characters. Correspondence to: J.R. Garey     

Gene mapping in marsupials and monotremes

Somatic cell genetic mapping of marsupial and monotreme species will greatly extend the power of comparative gene mapping to detect ancient mammalian gene arrangements. The use of eutherian-marsupial cell hybrids for such mapping is complicated by the frequent retention of deleted and rearranged marsupial chromosomes. We used staining techniques, involving the fluorochromes Hoechst 33258 and chromomycin A₃, to facilitate rapid and unequivocal identification of marsupial chromosomes and chromosome segments and to make chromosome assignment and regional localization of marsupial genes possible. Chromosome segregation in rodent-macropod hybrids was consistent with preferential loss of the marsupial complement. The extent of loss was very variable. Some hybrids retained 30% of the marsupial complement; some retained small centric fragments; and some, no cytologically identifiable marsupial material. We examined the chromosomes and gene products of a number of rodent-grey kangaroo Macropus giganteus hybrids, and have assigned the genes Pgk-A (phosphoglycerate kinase-A), Hpt (Hypoxanthine phosphoribosyl transferase), and Gpd (Glucose-6-phosphate dehydrogenase) to the long arm of the kangaroo X chromosome, and provisionally established the gene order Pgk-A -Hpt -Gpd.     

Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae. HEM12 gene sequence and evidence for two conserved glycines essential for enzymatic activity.

The HEM12 gene from Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase which catalyzes the sequential decarboxylation of the four acetyl side chains of uroporphyrinogen to yield coproporphyrinogen, an intermediate in protoheme biosynthesis. The gene was isolated by functional complementation of a hem12 mutant. Sequencing revealed that the HEM12 gene encodes a protein of 362 amino acids with a calculated molecular mass of 41,348 Da. The amino acid sequence shares 50% identity with human and rat uroporphyrinogen decarboxylase and shows 40% identity with the N-terminus of an open reading frame described in Synechococcus sp. We determined the sequence of two hem12 mutations which lead to a totally inactive enzyme. They correspond to the amino acid changes Gly33----Asp and Gly300----Asp, located in two evolutionarily conserved regions. Each of these substitutions impairs binding of substrates without affecting the overall conformation of the protein. These results argue that a single active center exists in uroporphyrinogen decarboxylase.     

Relationship between aquifer biofilms and unattached microbial indicators of urban groundwater contamination

Aquifers, springs and other groundwater‐dependent ecosystems are threatened by urban land use, which causes water quality deterioration through nutrient loading, sewage infiltration, groundwater extraction and, along coasts, seawater intrusion. The presence of certain microbes in groundwater can indicate that an aquifer is anthropogenically contaminated. Interpretations made from observations of indicator microbes in groundwater are limited because the relationship between the presumably allochthonous indicator microbes and relevant autochthonous microbial communities has not been characterized. This study addressed whether autochthonous aquifer biofilms can influence the presence of presumed microbial indicators in groundwater, and simultaneously used microbial indicators to trace sources of urban contamination at a karst spring of conservation concern. These questions were approached using a 17‐month time series analysis of attached biofilm and adjacent unattached bacteria in the submerged karst aquifer conduit associated with this spring. Environmental 16S rRNA gene sequencing was performed to characterize these communities, and community structure data were contextualized with groundwater geochemical and hydrogeological measurements. Linear regression models were developed to explain the relative abundance patterns of indicator microbes and other unattached microbes at this site. The results of this study suggest that dominant aquifer biofilms do not influence the presence of unattached microbial taxa that are presumed to be indicators of groundwater contamination, and generated new information about the origin of coliform bacteria at the study site. These results build confidence in the use of microbial indicators in groundwater‐dependent ecosystem conservation strategies and inform future management plans for urban aquifers and springs worldwide.     

Immunomodulatory Agents as Adjunctive Therapy for the Treatment of Resistant Candida Species

Invasive Candida infections have increased fivefold over the past 20 years. During this time, the incidence of antifungal resistance and infection due to non-albicans species has risen with the increasing use of broad spectrum antifungals. As few new antifungal agents are in development, strategies to improve outcomes in the treatment of Candida infections are sorely needed. The use of immunotherapy to augment the host immune response as an adjunctive treatment for Candida infections is a potentially robust and promising approach. The purpose of this review is to focus on new developments in the use of adjunctive immunotherapy for the treatment of Candida infections, and discuss the potential impact of antifungal resistance on the host immune response.     

Impact of New Diagnostic Approaches for Invasive Candidiasis on Antifungal Stewardship

Background

Invasive candidiasis (IC) including candidemia and deep-seated candidiasis is associated with up to 50 % overall mortality and up to $80,000 in attributable cost (2015 US$). Rapid diagnostic tests (RDTs) for Candida have been developed. However, whether RDTs along with real-time decision support translate to better attainment of stewardship goals—improve clinical outcomes, minimize unintended consequences of antifungal use, and reduce healthcare costs—is unknown. The purpose of this systematic review was to provide an up-to-date review of recently published studies that have assessed how RDTs for IC impact attainment of these goals.

Methods

Three electronic bibliographic databases were searched using a pre-defined search strategy evaluating the impact of RDTs for IC on attainment of antifungal stewardship goals. Quality assessments were performed by two reviewers using established study methodology metrics.

Results and Conclusions

Eight studies were identified of which five had sufficient information to be included in the review. Despite the limitations of the various studies and the different methodologies employed, the studies all produced similar conclusions. Compared to conventional methods and baseline stewardship activities, the integration of RDTs for IC and real-time decision support, mainly through antifungal stewardship, was associated with decreased mortality, more optimal use of antifungals, and reduced healthcare costs. However, larger clinical studies are needed to confirm these trends.

     

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources

AIMS: The genus Enterococcus includes opportunistic pathogens such as E. faecalis and E. faecium, and is also used to assess water quality. Speciation of enterococci in environmental studies can be particularly problematic, therefore protocols for unambiguous, DNA-based analysis could receive wide use in applications ranging from water quality monitoring to microbial source tracking. The goal of this work was to investigate the usefulness of PCR for speciation of putative, biochemically identified E. faecalis and E. faecium isolated from water, faeces and sewage. METHODS AND RESULTS: Putative enterococci (n = 139) were isolated on mEI agar from dog, human, gull and cow faeces, and from sewage, freshwaters and marine waters. A total of 128 isolates passed standard physiological tests for the genus, and were speciated by the API 20 Strep (APIStrep) biochemical test system. 42.2% were identified as E. faecalis, and all were confirmed by PCR. 19.5% were biochemically identified as E. faecium, but only seven were PCR-positive. CONCLUSIONS: The 16S rDNA of PCR-positive and PCR-negative E. faecium, including isolates that were inconclusively identified by APIStrep, was sequenced. All formed a monophyletic clade with E. faecium sequences in Genbank. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical identification of E. faecalis agreed 100% with PCR assays, therefore a simple protocol of isolation on mEI followed by PCR should be useful for environmental studies. Discrepancies among biochemical identification, PCR confirmation and DNA sequencing were noted for E. faecium, indicating that routine isolation/identification of E. faecium from environmental samples is a much more difficult task.