An ancient, Antarctic-specific species complex: large divergences between multiple Antarctic lineages of the tardigrade genus Mesobiotus.

Antarctica has been isolated and progressively glaciated for over 30 million years, with only approximately 0.3 % of its area currently ice-free and capable of supporting terrestrial ecosystems. As a result, invertebrate populations have become isolated and fragmented, in some cases leading to speciation. Terrestrial invertebrate species currently found in Antarctica often show multi-million year, and even Gondwanan, heritage, with little evidence of recent colonisation. Mesobiotus is a globally distributed tardigrade genus. It has commonly been divided into two "groups", referred to as harmsworthi and furciger, with both groups currently considered cosmopolitan, with global reports including from both the Arctic and the Antarctic. However, some authors considered that Meb. furciger, as originally described, may represent an Antarctic-specific lineage. Using collections of tardigrades from across the Antarctic continent and publicly available sequences obtained from online databases, we use mitochondrial and nuclear ribosomal sequence data to clarify the relationships of Antarctic Mesobiotus species. Our analyses show that all Antarctic members belong to a single lineage, evolving separately from non-Antarctic representatives. Within this Antarctic lineage there are further deep divisions among geographic regions of the continent, consistent with the presence of a species complex. Based on our data confirming the deep divisions between this Antarctic lineage, which includes representatives of both groups, we recommend that the use of furciger and harmsworthi group terminology is now abandoned, as it leads to systematic and biogeographical confusion.

Tracheostomy in the critically ill: the myth of dead space.

Benefits and advantages of tracheostomy have been vigorously debated. There is a lack of consensus as to whether perceived clinical improvement is attributable to fundamental changes in respiratory dynamics. We compare the effect of tracheostomy versus endotracheal tube on dead space, airway resistance and other lung parameters in critically ill ventilated patients. Data collected included patients who were admitted to surgical, burn and neurosurgical intensive care units at the University of North Carolina. Twenty-four intubated patients were included in our analysis with various aetiologies of respiratory failure. Tracheostomy was deemed necessary either for severe neurological devastation or failure to wean from the ventilator. The diameter of the endotracheal tubes ranged from 6-8 mm and the tracheostomy tube diameters were from 6.4-8.9 mm. Internal diameters between endotracheal tube and tracheostomy tubes, ventilator settings and sedation were kept consistent throughout the study. Respiratory parameters were measured using the Respironics' non-invasive cardiac output 2 device (Phillips, Andover, MA) immediately prior to tracheostomy and repeated within 24 hours of tracheostomy. Only two (8%) of the patients had slight improvement (>6% decrease in dead space). The average dead space of endotracheal versus tracheostomy tubes was 41±12.6% and 40±14.6%, respectively (P=0.75). The remaining 22 patients (92%) had no significant change in dead space, compliance or other respiratory parameters. This study shows that there is no significant difference in respiratory mechanics and dead space with a tracheostomy versus endotracheal tube.

Microscopic polyarteritis presenting with skin necrosis in a patient with sickle-cell disease.

A 20-year-old Caribbean woman with sickle cell anaemia was admitted with a 4-day history of fever and a painful swollen right ankle. She rapidly developed skin necrosis. The differential diagnosis is discussed. This case illustrates the difficulty in identifying the cause of cutaneous necrosis in an acutely ill patient. In our patient, histopathology implicated a vasculitic process, which was subsequently identified as a manifestation of microscopic polyarteritis.

Vulval squamous cell carcinoma arising in chronic hidradenitis suppurativa.

We report a case of vulval squamous cell carcinoma (SCC) arising in chronic hidradenitis suppurativa (HS). The patient had a complex medical history including a 25-year-history of Crohn's disease. In addition she had recently received immunosuppressive therapy for nephrotic syndrome secondary to membranous glomerulonephritis. A painful nodule was noted on the vulva that was clinically very suspicious of SCC. An excision biopsy confirmed the diagnosis. There are few publications in the English literature citing association between HS and the development of SCC. The first report in the English literature of vulval SCC arising in chronic HS was published in 1999. We wish to draw attention to the possibility that patients with HS may develop SCC in lesional skin. A painful lump or ulcer could easily be mistaken for an inflammatory lesion and a low threshold for biopsy is warranted. We suggest constant vigilance with regard to malignant change in ano-genital HS as the diagnosis can be difficult.

Fixed drug eruption following metronidazole therapy and the use of topical provocation testing in diagnosis.

Fixed drug eruption is characterized by recurrent well-defined lesions appearing in the same location each time the drug responsible is taken. A number of agents have been implicated. Metronidazole, a nitroimidazole agent widely used for its antibacterial and antiprotozoal activity, has been reported only rarely as the causative agent. We describe a patient with FDE due to metronidazole in whom we were able to induce the clinical and histological features of FDE by topical provocation testing. In agreement with the published literature we commend the use of topical provocation testing as a possible first-line investigation in the diagnosis of FDE. This may avoid the need for subsequent oral provocation testing and therefore the prevention of possible adverse sequelae.

Haemorrhage into chronic plaque psoriasis as a consequence of disseminated intravascular coagulation.

We describe a patient with chronic plaque psoriasis who developed haemorrhage into pre-existing lesions during an episode of disseminated intravascular coagulation secondary to sepsis. Disseminated intravascular coagulation is a complex disorder characterized by widespread intravascular deposition of fibrin with consumption of coagulation factors and platelets and occurs as a consequence of many disorders that release procoagulant material into the circulation or cause widespread endothelial damage or platelet aggregation. As both disseminated intravascular coagulation and psoriasis occur relatively frequently in the general population we were surprised to find no previous reports of this phenomenon in the literature.

Relative expression and stability of a chromosomally integrated and plasmid-borne marker gene fusion in environmentally competent bacteria.

A xylE-iceC transcriptional fusion was created by ligatinga DNA fragment harboring the cloned xylE structural gene from the TOL plasmid of Pseudomonas putida mt-2 into the cloned iceC gene of Pseudomonas syringae Cit7. This fusion construct was integrated into the chromosome of Pseudomonas syringae Cit7 by homologous recombination. Both cis-merodiploid strain Cit7m17 and marker exchange strain Cit7h69 produced the XylE gene product, catechol2,3-dioxygenase. Strain Cit7m17, in which XylE was influenced by transcription initiated by the amp promoter on pBR322, exhibited XylE activity in stationary phase at levels about 45 times higher than strain Cit7h69, permitting detection of 10(7) Cit7m17 cells in the spectrophotometric assay and 10(3) cells in HPLC measurements. The stability of xylE in both Cit7m17 and Cit7h69 was compared with maintenance of xylE in several plasmid-borne constructs in P.aeruginosa, Erwinia herbicola, and Escherichia coli. Only the xylE-iceC fusion in the chromosome of Cit7h69 and Cit7m17was stable in plate assays over the course of these studies. Even though strain Cit7h69 stably expressed xylE, the low level of expression precludes its use in direct spectrophotometric or HPLC assays as a means for detecting cells in environmental samples. However, expression of xylEin Cit7h69 is sufficient for identification of colonies harboring this marker gene which is useful in laboratory plate assays, and as a marker gene system for the detection of environmentally-competent strains chromosomally taggedwith xylE for use in autecological studies.

Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2,4-dichlorophenoxyacetate.

The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 micrograms/g of a model xenobiotic, phenoxyacetic acid (PAA). P. putida PP0301(pR0103) is a constitutive degrader of 2,4-dichlorophenoxyacetate (2,4-D) and is also active on the non-inducing substrate, PAA. PAA is the parental compound of 2,4-dichlorophenoxyacetic acid (2,4-D) and whilst the indigenous soil microbiota degraded 500 micrograms/g 2,4-D to less than 10 micrograms/g, PAA degradation was insignificant during a 40-day period. No significant degradation of PAA occurred in soil inoculated with the parental strain P. putida PP0301 or the inducible 2,4-D degrader P. putida PP0301(pR0101). Moreover, co-amendment of soil with 2,4-D and PAA induced the microbiota to degrade 2,4-D; PAA was not degraded. P. putida PP0301-(pR0103) mineralized 500-micrograms/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence. In unamended soil, survival of the plasmid-free parental strain P. putida PP0301 was similar to the survival of the GEM strain P. putida PP0301(pR0103). However, in PAA amended soil, survival of the parent strain was over 10,000-fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.

Ecologically significant effects of Pseudomonas putida PPO301(pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, on microbial populations and processes in soil.

Pseudomonas putida PPO301 (pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, affected microbial populations and processes in a nonsterile xeric soil. In soil amended with 2,4-dichlorophenoxyacetate (500 micrograms/g soil) and inoculated with PPO301 (pRO103), the rate of evolution of carbon dioxide was retarded for approximately 35 days; there was a transient increase in dehydrogenase activity; and the number of fungal propagules decreased below detection after 18 days. In unamended soil inoculated with PPO301(pRO103), the rate of evolution of carbon dioxide and the dehydrogenase activity were unaffected, and the numbers of fungal propagules were reduced by about two orders of magnitude. The numbers of total, spore-forming, and chitin-utilizing bacteria were reduced transiently in soil either amended or unamended with 2,4-dichlorophenoxyacetate and inoculated with PPO301(pRO103). The activities of arylsulfatases and phosphatases in soil were not affected by the presence of PPO301(pRO103), either in the presence or absence of 2,4-dichlorophenoxyacetate. In soil amended with 2,4-dichlorophenoxyacetate and inoculated with the parental strain (PPO301) or not inoculated, the evolution of carbon dioxide, the numbers of fungal propagules and of total, spore-forming, and chitin-utilizing bacteria, and the dehydrogenase activity were not affected as in soil inoculated with PPO301(pRO103). These results demonstrated that a genetically engineered microorganism, in the presence of the substrate on which its novel genes can function, is capable of inducing measurable ecological effects in soil.

Assay for detection and enumeration of genetically engineered microorganisms which is based on the activity of a deregulated 2,4-dichlorophenoxyacetate monooxygenase.

An assay system was developed for the enumeration of genetically engineered microorganisms expressing a deregulated 2,4-dichlorophenoxyacetate (TFD) monooxygenase, which converts phenoxyacetate (PAA) to phenol. In PAA-amended cultures of Pseudomonas aeruginosa PAO1C(pRO103) and Pseudomonas putida PPO301(pRO103), strains which express a deregulated TFD monooxygenase, phenol production was proportional to cell number. Phenol was reacted, under specific conditions, with a 4-aminoantipyrine dye to form an intensely colored dye-phenol complex (AAPPC), which when measured spectrophotometrically could detect as few as 10(3) cells per ml. This assay was corroborated by monitoring the disappearance of PAA and the accumulation of phenol by high-performance liquid chromatography and gas chromatography. The AAPPC assay was modified for use with plate cultures and clearly distinguished colonies of PPO301(pRO103) and PAO1C(pRO103) from a strain expressing a regulated TFD monooxygenase. Colonies of P. putida PPO301(pRO101) remained cream colored, while colonies of PPO301(pRO103) and PAO1C(pRO103) turned a distinct red.

Effects of 2,4-dichlorophenol, a metabolite of a genetically engineered bacterium, and 2,4-dichlorophenoxyacetate on some microorganism-mediated ecological processes in soil.

A genetically engineered microorganism, Pseudomonas putida PPO301(pRO103), and the plasmidless parent strain, PPO301, were added at approximately 10 CFU/g of soil amended with 500 ppm of 2,4-dichlorophenoxyacetate (2,4-D) (500 mug/g). The degradation of 2,4-D and the accumulation of a single metabolite, identified by gas chromatography-mass spectrophotometry as 2,4-dichlorophenol (2,4-DCP), occurred only in soil inoculated with PPO301(pRO103), wherein 2,4-DCP accumulated to >70 ppm for 5 weeks and the concentration of 2,4-D was reduced to <100 ppm. Coincident with the accumulation of 2,4-DCP was a >400-fold decline in the numbers of fungal propagules and a marked reduction in the rate of CO(2) evolution, whereas 2,4-D did not depress either fungal propagules or respiration of the soil microbiota. 2,4-DCP did not appear to depress the numbers of total heterotrophic, sporeforming, or chitin-utilizing bacteria. In vitro and in situ assays conducted with 2,4-DCP and fungal isolates from the soil demonstrated that 2,4-DCP was toxic to fungal propagules at concentrations below those detected in the soil.

Periplasmic superoxide dismutases in Aquaspirillum magnetotacticum.

Aquaspirillum magnetotacticum MS-1 cells cultured microaerobically (dissolved O2 tension 1% of saturation), expressed proteins with superoxide dismutase (SOD) activity. The majority (roughly 95%) of total cell superoxide dismutase activity was located in the cell periplasm with little or no activity in the cell cytoplasm. Iron-type SOD (FeSOD) contributed 88% of the total activity activity detected, although a manganese-type SOD (MnSOD) was present in the periplasm as well. Cells cultured at a higher dissolved O2 tension (10% of saturation) expressed increased activity of the MnSOD relative to that of the FeSOD.

Freeze-Thawing of Aquaspirillum magnetotacticum Cells Selectively Releases Periplasmic Proteins.

Cells of the gram-negative bacterium Aquaspirillum magnetotacticum, when suspended in buffer and freeze-thawed, produced pinkish orange supernatant fluid. The fluid contained

Iron respiration-driven proton translocation in aerobic bacteria.

Washed cell suspensions of Aquaspirillum magnetotacticum MS-1, A. itersonii E12639, Bacillus subtilis 6633, and Escherichia coli CSH27 translocated protons in response to the added oxidant O2 or NO3-, with triphenylmethylphosphonium bromide as the permeant ion. Iron respiration-driven proton translocation was observed in A. magnetotacticum MS-1, B. subtilis, and E. coli but not in a nonmagnetic strain of A. magnetotacticum (strain NM-1A) or with A. itersonii. Proton translocation to Fe3+ was totally inhibited by 500 microM NaN3 or 0.5 microM carbonyl cyanide m-chlorophenylhydrazone.