Mannitol Does Not Enhance Tobramycin Killing of Pseudomonas aeruginosa in a Cystic Fibrosis Model System of Biofilm Formation

Cystic Fibrosis (CF) is a human genetic disease that results in the accumulation of thick, sticky mucus in the airways, which results in chronic, life-long bacterial biofilm infections that are difficult to clear with antibiotics. Pseudomonas aeruginosa lung infection is correlated with worsening lung disease and P. aeruginosa transitions to an antibiotic tolerant state during chronic infections. Tobramycin is an aminoglycoside currently used to combat lung infections in individuals with CF. While tobramycin is effective at eradicating P. aeruginosa in the airways of young patients, it is unable to completely clear the chronic P. aeruginosa infections in older patients. A recent report showed that co-addition of tobramycin and mannitol enhanced killing of P. aeruginosa grown in vitro as a biofilm on an abiotic surface. Here we employed a model system of bacterial biofilms formed on the surface of CF-derived airway cells to determine if mannitol would enhance the antibacterial activity of tobramycin against P. aeruginosa grown on a more clinically relevant surface. Using this model system, which allows the growth of robust biofilms with high-level antibiotic tolerance analogous to in vivo biofilms, we were unable to find evidence for enhanced antibacterial activity of tobramycin with the addition of mannitol, supporting the observation that this type of co-treatment failed to reduce the P. aeruginosa bacterial load in a clinical setting.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Recognition of the Trachypetidae stat.n. as a new extant family of Ichneumonoidea (Hymenoptera), based on molecular and morphological evidence

The Trachypetinae (type genus Trachypetus Guérin de Méneville) comprise seven species of large-bodied wasps in three genera (Cercobarcon Tobias, Megalohelcon Turner and Trachypetus) endemic to continental Australia. Historically they have been variously treated, as members of the Helconinae in the case of Megalohelcon, or as separate subfamilies (Cercobarconinae and Trachypetinae). Some 25 years ago they were united in a single subfamily, the Trachypetinae, based on a number of characters. Although there has been conflicting evidence from morphological and molecular phylogenetic studies as to how best to treat the group, there has been a growing consensus that they fall outside the rest of the Braconidae, although taxon sampling has been a limiting factor for molecular studies. We generated a molecular dataset comprising five gene fragments (nuclear 28S ribosomal rDNA, nuclear 18S, elongation factor 1-alpha, mitochondrial 16S rDNA, and mitochondrial cytochrome oxidase subunit 1) for a taxonomically broad range of Braconidae, Ichneumonidae, trachypetines and outgroup hymenopterans including the first molecular data for the trachypetines Cercobarcon and Trachypetus obtained using specially designed internal primers. Molecular and combined molecular and morphological analyses confirm the monophyly of the Trachypetinae and robustly place them as sister to the Braconidae. Detailed morphological analysis including newly recognized characters shows that trachypetines lack several synapomorphies that define the Braconidae, and that they possess a number of symplesiomorphies absent from this family but found in some ichneumonids. We conclude that family-level status is warranted for the group based on both molecular and morphological criteria, and hence we propose the new family, Trachypetidae Schulz stat.n. (type genus Trachypetus Guérin de Méneville), for it. As a result, the remaining extant Braconidae become clearly defined based on synapomorphies not present in Trachypetidae stat.n. This published work has been registered on ZooBank, http://zoobank.org/urn:lsid:urn:lsid:zoobank.org:pub:5418F709-D724-4F14-89D8-1E054D1D27D0 .     

BIOGEOGRAPHY OF A WIDESPREAD FRESHWATER CRUSTACEAN: PSEUDOCONGRUENCE AND CRYPTIC ENDEMISM IN THE NORTH AMERICAN DAPHNIA LAEVIS COMPLEX

The lack of morphological variation in many freshwater invertebrates over vast distances has been cited as evidence for their frequent, long-distance dispersal. This scenario implies that vicariance will be an insignificant determinant of species distributions or diversity. We carried out a phylogeographic and population genetics study of one widespread crustacean group, the North American Daphnia laevis complex. Allozyme and sequence variation of two mtDNA genes (12S and 16S rRNA) clearly indicates the existence of five morphologically cryptic, largely allopatric groups (Daphnia dubia, D. laevis laevis, D. laevis gessneri, D. magniceps magniceps, and D. magniceps pacifica ssp. n.). Within each of these groups, there is weak or no genetic differentiation over large geographic areas suggesting their recent long-distance dispersal. The present-day distributions and phylogeography of the regional groups suggests the occurrence of both deep and shallow vicariance events. Although divergence times from mtDNA sequences do indicate both deep and shallow divergences, these estimates are incongruent with their proposed vicariance times. The results show that even within closely related freshwater invertebrates, a complex biogeography exists, whose analysis is made difficult by long-distance dispersal, cryptic endemism, and pseudocongruence.     

Ant predation on red oak borer confirmed by field observation and molecular gut-content analysis

1 Populations of an indigenous longhorn beetle, the red oak borer Enaphalodes rufulus (Haldeman) (Coleoptera: Cerambycidae), recently reached epidemic levels in the Ozark National Forests of Arkansas and Missouri, resulting in extensive tree mortality.
2 The factors regulating E. rufulus populations are largely unknown. Ants appear to be the most abundant potential predators in the Ozarks and have been shown to play a role in regulating populations of other forest insects.
3 The main objective of the present study was to determine whether ants are predators of early E. rufulus life stages by direct observation of E. rufulus eggs artificially placed on tree trunks and by development of molecular tools to detect E. rufulus DNA within ant gut contents.
4 Three hundred and eighty E. rufulus eggs were applied to ten northern red oaks. Ants ate or carried away 72% of the eggs within 1 h. Camponotus pennsylvanicus (De Geer) and Aphaenogaster tennesseensis (Mayr) were identified as the two primary ant species observed in the study.
5 A portion of the mitochondrial 16S rRNA gene of E. rufulus was sequenced and polymerase chain reaction primers were developed to detect E. rufulus DNA in the guts of C. pennsylvanicus . Enaphalodes rufulus DNA persisted in ant gut contents for at least 24 h after ingestion under laboratory conditions, and E. rufulus DNA was detected in field-collected ant populations, suggesting the natural occurrence of ant predation on this insect.     

Phylogenetic relationships among four members of Stizostedion (Percidae) determined by mitochondrial DNA and allozyme analyses

Phylogenetic relationships among four Stizostedion species were examined using mitochondrial DNA (mtDNA) and allozyme analyses. Twenty-six allozyme loci were scored, and mtDNA variation was examined using 24 restriction endonucleases, yielding 48–57 restriction sites among the species. Genetic distance analyses show that the two North American species ( S. canadense and S. vitreum ) cluster in one group, while the two European species ( S. hciopercu and S. vogense ) form a second group. Nei's genetic distance between these two groups was 0.7 ± 0.2 for allozymes, while the corresponding mtDNA sequence divergence was 14.8 ± 2.0%, suggesting that these two groups diverged approximately 10 million years ago. Thus, these data are consistent with the hypothesis that Stizostedion colonized North America during the Pliocene.     

Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648‐bp segment near the 5′ terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus, the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5′ region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus. Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.     

A Quick Embedding Method for Light and Electron Microscopy of Textile Fibers

A quick embedding method employing UV polymerization reactions has been devised for embedding fibers in acrylic and meth-acrylate media. The resultant thin, flat embed-dings are suitable for both light and electron microscopy.