The flavodiiron protein from Syntrophomonas wolfei has five domains and acts both as an NADH:O2 or an NADH:H2 O2 oxidoreductase.

Flavodiiron proteins (FDPs) are a family of enzymes with a significant role in O2 /H2 O2 and/or NO detoxification through the reduction of these species to H2 O or N2 O, respectively. All FDPs contain a minimal catalytic unit of two identical subunits, each one having a metallo-ß-lactamase-like domain harboring the catalytic diiron site, and a flavodoxin-like domain. However, more complex and diverse arrangements in terms of domains are found in this family, of which the class H enzymes are among the most complex. One of such FDPs is encoded in the genome of the anaerobic bacterium Syntrophomonas wolfei subsp. wolfei str. Goettingen G311. Besides the core domains, this protein is predicted to have three additional ones after the flavodoxin core domain: two short-chain rubredoxins and a NAD(P)H:rubredoxin oxidoreductase-like domain. This enzyme, FDP_H, was produced and characterized and the presence of the predicted cofactors was investigated by a set of biochemical and spectroscopic methodologies. Syntrophomonas wolfei FDP_H exhibited a remarkable O2 reduction activity with a kcat = 52.0 ± 1.2 s-1 and a negligible NO reduction activity (~ 100 times lower than with O2 ), with NADH as an electron donor, that is, it is an oxygen-selective FDP. In addition, this enzyme showed the highest turnover value for H2 O2 reduction (kcat = 19.1 ± 2.2 s-1 ) ever observed among FDPs. Kinetic studies of site-directed mutants of iron-binding cysteines at the two rubredoxin domains demonstrated the essential role of these centers since their absence leads to a significant decrease or even abolishment of O2 and H2 O2 reduction activities.

Ecological Dichotomies Arise in Microbial Communities Due to Mixing of Deep Hydrothermal Waters and Atmospheric Gas in a Circumneutral Hot Spring.

Little is known of how the confluence of subsurface and surface processes influences the assembly and habitability of hydrothermal ecosystems. To address this knowledge gap, the geochemical and microbial composition of a high-temperature, circumneutral hot spring in Yellowstone National Park was examined to identify the sources of solutes and their effect on the ecology of microbial inhabitants. Metagenomic analysis showed that populations comprising planktonic and sediment communities are archaeal dominated, are dependent on chemical energy (chemosynthetic), share little overlap in their taxonomic composition, and are differentiated by their inferred use of/tolerance to oxygen and mode of carbon metabolism. The planktonic community is dominated by putative aerobic/aerotolerant autotrophs, while the taxonomic composition of the sediment community is more evenly distributed and comprised of anaerobic heterotrophs. These observations are interpreted to reflect sourcing of the spring by anoxic, organic carbon-limited subsurface hydrothermal fluids and ingassing of atmospheric oxygen that selects for aerobic/aerotolerant organisms that have autotrophic capabilities in the water column. Autotrophy and consumption of oxygen by the planktonic community may influence the assembly of the anaerobic and heterotrophic sediment community. Support for this inference comes from higher estimated rates of genome replication in planktonic populations than sediment populations, indicating faster growth in planktonic populations. Collectively, these observations provide new insight into how mixing of subsurface waters and atmospheric oxygen create dichotomy in the ecology of hot spring communities and suggest that planktonic and sediment communities may have been less differentiated taxonomically and functionally prior to the rise of oxygen at ∼2.4 billion years ago (Gya). IMPORTANCE Understanding the source and availability of energy capable of supporting life in hydrothermal environments is central to predicting the ecology of microbial life on early Earth when volcanic activity was more widespread. Little is known of the substrates supporting microbial life in circumneutral to alkaline springs, despite their relevance to early Earth habitats. Using metagenomic and informatics approaches, water column and sediment habitats in a representative circumneutral hot spring in Yellowstone were shown to be dichotomous, with the former largely hosting aerobic/aerotolerant autotrophs and the latter primarily hosting anaerobic heterotrophs. This dichotomy is attributed to influx of atmospheric oxygen into anoxic deep hydrothermal spring waters. These results indicate that the ecology of microorganisms in circumneutral alkaline springs sourced by deep hydrothermal fluids was different prior to the rise of atmospheric oxygen ∼2.4 Gya, with planktonic and sediment communities likely to be less differentiated than contemporary circumneutral hot springs.

The multidomain flavodiiron protein from Clostridium difficile 630 is an NADH:oxygen oxidoreductase.

Flavodiiron proteins (FDPs) are enzymes with a minimal core of two domains: a metallo-ß-lactamase-like, harbouring a diiron center, and a flavodoxin, FMN containing, domains. FDPs are O2 or NO reducing enzymes; for many pathogens, they help mitigate the NO produced by the immune system of the host, and aid survival during fluctuating concentrations concentrations of oxygen. FDPs have a mosaic structure, being predicted to contain multiple extra domains. Clostridium difficile, a threatening human pathogen, encodes two FDPs: one with the two canonical domains, and another with a larger polypeptide chain of 843 amino acids, CD1623, with two extra domains, predicted to be a short-rubredoxin-like and an NAD(P)H:rubredoxin oxidoreductase. This multi-domain protein is the most complex FDP characterized thus far. Each of the predicted domains was characterized and the presence of the predicted cofactors confirmed by biochemical and spectroscopic analysis. Results show that this protein operates as a standalone FDP, receiving electrons directly from NADH, and reducing oxygen to water, precluding the need for extra partners. CD1623 displayed negligible NO reductase activity, and is thus considered an oxygen selective FDP, that may contribute to the survival of C. difficile in the human gut and in the environment.

The use of 1% toluidine blue eye drops in the diagnosis of ocular surface squamous neoplasia.

PURPOSE: To evaluate the use of in vivo toluidine blue (TB) dye in the diagnosis of ocular surface squamous neoplasia and to correlate staining intensity with the histopathological diagnosis. METHODS: A prospective study was performed at the Federal University of Sao Paulo, Brazil. Patients with conjunctival epithelial lesions were examined by slit-lamp biomicroscopy, with and without 1% TB eye drops, and the results were photo documented. Before the instillation of the dye, 1% proxymetacaine HCl eye drops were used. All patients were submitted to surgery and histopathological analysis to confirm the diagnosis. The patients were grouped according to the histopathological aspects of the lesions into 3 groups: group 1-patients with ocular surface squamous neoplasia; group 2-patients with premalignant lesions; and group 3-patients with pterygium. The digital images were analyzed by 2 masked examiners who had no previous access to the histopathological results. The photographs were classified according to the positivity and intensity of the staining. The statistical analysis method chosen depended on the type of data, with the level of significance set as P < 0.05. RESULTS: Forty-seven patients were included in the study: 10 had benign lesions (pterygium), 10 had premalignant lesions (actinic keratosis), and 27 had malignant lesions (conjunctival intraepithelial neoplasia and conjunctival squamous cell carcinoma). Agreement between observers regarding the analysis of the digital photographs was 100% for positivity and 82.9% for intensity of staining (κ = 0.938). Ninety percent of patients with premalignant lesions and all patients with malignant lesions showed positive staining with 1% TB. One patient had positive staining, but histopathological examination revealed a benign lesion (false positive). CONCLUSIONS: The use of 1% TB eye drops is an efficient method for the clinical diagnosis of ocular surface squamous neoplasia and premalignant lesions. Nevertheless, the intensity of the staining does not correlate with the degree of malignancy of these tumors.

Extramedullary plasmacytoma of the third eyelid gland in a dog.

A case of extramedullary plasmacytoma of the third eyelid gland in a 7-year-old American Cocker Spaniel is reported. An enlargement of the third eyelid gland, abundant mucopurulent discharge, mild hyperemia and corneal pigmentation in the OD was present. Excisional biopsy of the mass revealed the gland was infiltrated and partially destroyed by a uniform population of neoplastic plasma cells. The neoplastic cells were positive for CD138, Ki-67 and lambda light chain. CD20, CD3, kappa light chain and cytokeratin were negative. Twelve months following surgery, no recurrence was observed. To the authors' knowledge, this is the first extramedullary plasmacytoma of the third eyelid gland reported in dogs.

The expression of cyclooxygenase 2 in retinoblastoma: primary enucleated eyes and enucleation after conservative treatment.

PURPOSE: To provide insight into the relationship between cyclooxygenase-2 (COX-2) expression and histopathologic features of retinoblastoma specimens treated either by primary or secondary enucleation. DESIGN: Laboratory investigation. METHODS: Twenty-five retinoblastoma specimens received between 1994 and 2003 were retrieved for this study from the Ocular Pathology Registry, Federal University of São Paulo, Brazil and the Henry C. Witelson Ocular Pathology Laboratory and Registry, McGill University, Montreal, Canada. The specimens retrieved were divided into two groups: Group I, enucleation was performed as a form of primary treatment (n = 15) and Group II, enucleation after failure of conservative treatment (n = 10). Patient information and formalin-fixed, paraffin-embedded specimens were obtained. New sections of these blocks were used for hematoxylin and eosin stain and for immunoassay using a monoclonal mouse anti-COX-2 antibody. Two independent ophthalmic pathologists reviewed all of the microslides. RESULTS: Twenty-three specimens (92%) presented a high expression of COX-2 (15 in Group I; eight in Group II) and there was no statistical difference in COX-2 expression between the two groups (P = .07). However, all specimens expressed COX-2 to a different degree. The areas of tumor invasion were positive for COX-2 in 87.5% of the two groups. CONCLUSION: Most retinoblastoma specimens revealed a high COX-2 expression. Future studies will be necessary to correlate the high expression of COX-2 in retinoblastoma and a possible applicability of anti-COX-2 medications in the treatment of these tumors.

Relationship between histopathological features of chemotherapy treated retinoblastoma and P-glycoprotein expression.

BACKGROUND: P-glycoprotein (P-gp) has been identified as a possible mediator of chemoresistance in retinoblastoma. The aim of this study was to determine the expression of P-gp in retinoblastoma treated with chemotherapy prior to enucleation. METHODS: Seventeen enucleated specimens of retinoblastoma from 16 patients were studied. Nine had been treated with chemotherapy alone, and eight had been treated with chemotherapy and other forms of local treatment. Tumour differentiation as well as choroidal and optic nerve invasion were assessed. P-gp immunohistochemical staining was performed and evaluated as negative, low or high. RESULTS: Histopathological assessment of the cases showed that 14 of 17 eyes (82.3%) had viable retinoblastoma cells. Nine retinoblastomas were considered regressed with a well-differentiated component, five regressed retinoblastomas had viable cells with poor differentiation and three retinoblastomas had regressed leaving no viable cells. Sixteen of 17 retinoblastomas were P-gp positive. In the one case with optic nerve invasion and the three cases with massive choroidal invasion, P-gp expression was found in invading retinoblastoma cells. CONCLUSION: Almost all retinoblastomas expressed P-gp. High levels of P-gp expression might play a role in chemotherapy resistance of retinoblastoma or, conversely, chemotherapy might induce P-gp expression. These results might have an impact on management of bilateral retinoblastoma.

Immunohistochemical profile of lymphoid lesions of the orbit.

BACKGROUND: Orbital idiopathic inflammation, lymphoid hyperplasia, and lymphoma may all present clinically in the same manner. Histopathology and especially immunohistochemistry play a major role in the differential diagnosis. The purpose of this study was to determine the immunophenotypic features of these lesions. METHODS: Fifty-five orbital lymphoid lesions were retrieved from the ophthalmic pathology registries at McGill University, Montreal, Canada, and the Federal University of São Paulo, São Paulo, Brazil. Formalin-fixed, paraffin-embedded, histopathologic sections were stained with hematoxylin and eosin and periodic acid-Schiff. The sections were also immunostained for B-cell (CD20) and T-cell (CD43) markers and for immunoglobulin light chains kappa and lambda. Two pathologists determined the histopathologic and immunohistochemical pattern of each lesion in a masked fashion. RESULTS: Of the 55 lesions, 11 (20%) were idiopathic chronic inflammations, 22 (40%) were lymphoid hyperplasias and 22 (40%) were lymphomas. Idiopathic inflammation displayed a predominance of T cells and all lesions expressed polyclonal light chains. Lymphoid hyperplasia displayed a mixture of B cells and T cells, with a slight predominance of the former and all lesions expressed polyclonal light chains. Lymphoma showed a striking predominance of B cells and all lesions expressed monoclonal light chains, usually kappa (63.7%). The differences in the mean percentages of B cells among the orbital lymphoid lesions (inflammation, 35%; hyperplasia, 65.9%; lymphoma, 87.3%) were statistically significant (p < 0.001). INTERPRETATION: Orbital lymphoid lesions can be differentiated based on the percentages of B cells and T cells and the monoclonal or polyclonal expression of immunoglobulin light chains.

An outbreak of Mycobacterium chelonae infection after LASIK.

OBJECTIVE: To describe an outbreak of mycobacterial keratitis after laser in situ keratomileusis (LASIK), including the microbiologic investigation, clinical findings, treatment response, and outcome. DESIGN: Retrospective, noncomparative, interventional case series. PARTICIPANTS: Patients (n = 10) who underwent LASIK surgery between August 22 and September 4, 2000, and developed mycobacterial infection. METHODS: Patients were prospectively followed in relation to microbiologic investigation, clinical findings, treatment response, and outcome. MAIN OUTCOME MEASURES: Most patients underwent bilateral simultaneous LASIK. Postoperative infection was signaled by the appearance of corneal infiltrates in the third postoperative week. The microbiologic workup was performed on cultures obtained either by direct scraping of the cornea or by lifting the flap. Medical therapy was instituted based on drug susceptibility testing. Surgical interventions such as corneal debridement and flap removal were performed during recurrences or when there was no satisfactory clinical response. RESULTS: Cultures revealed Mycobacterium subspecies chelonae. Patients were treated with topical clarithromycin (1%), tobramycin (1.4%), and ofloxacin (0.3%). Oral clarithromycin (500 mg twice a day) was prescribed for those patients who did not respond clinically to topical treatment. Four eyes healed on this regimen. Flap removal was necessary in seven eyes. CONCLUSIONS: This report highlights mycobacteria as an etiologic infectious agent after LASIK. Diagnosis can be difficult and is often delayed. The treatment mainstay is prolonged antibiotic therapy. Surgical debridement and flap removal may shorten the disease course.

Ocular and central nervous system paracoccidioidomycosis in a pregnant woman with acquired immunodeficiency syndrome.

PURPOSE: To describe an atypical case of central nervous system and ocular paracoccidioidomycoses simulating ocular toxoplasmosis in a pregnant woman with acquired immunodeficiency syndrome (AIDS). DESIGN: Interventional case report. METHODS: Case report. RESULTS: A 25-year-old pregnant woman with AIDS, presented with a severe ocular inflammation in the right eye involving the choroid, retina, and the optic disk, which rapidly progressed to retinal detachment, iris neovascularization, and neovascular glaucoma. The left eye was normal. Magnetic resonance imaging (MRI) showed a focal hypodense contrast-enhanced ring lesion in the brain. Serum antibody titers were negative for Toxoplasma gondii, but the polymerase chain reaction was positive for the parasite in the vitreous sample. The patient responded partially to specific treatment for toxoplasmosis, and there was a small reduction in size of the brain lesion. She progressed to a blind painful eye, which was enucleated. Paracoccidioides brasiliensis was found in the histopathological studies of the eye and oropharynx. With the diagnosis of disseminated ocular paracoccidioidomycoses, the patient was treated with trimethoprim-sulfamethoxazole with a satisfactory outcome and reduction in size of the brain lesion. CONCLUSION: Although ocular infection with ocular paracoccidioidomycoses is rare, this diagnosis should be considered when investigating ocular inflammation in a patient with AIDS.

Fordyce nodules in a buccal membrane graft to the ocular surface.

PURPOSE: To present a case of epibulbar Fordyce nodules, with a referral diagnosis of primary tumor. METHODS: Case report. RESULTS: A 38-year-old woman was referred for ocular oncology consultation because of a conjunctival lesion in the right eye. She had had a buccal mucous graft to treat recurrent pterygium 18 years earlier. The lesion consisted of multiple small, yellow granules over a pink, thickened mucosa from the 12 to 3 o'clock meridians. Excisional biopsy revealed multiple subepithelial sebaceous glands consistent with Fordyce nodules. CONCLUSIONS: Fordyce nodules are a possible late benign complication of buccal mucous grafts.

Cystine: a promoter of the growth and aggregation of calcium oxalate crystals in normal undiluted human urine.

PURPOSE: Many variables are known to be associated with the formation of calcium oxalate stones. We noted that on analysis a number of patients with calcium oxalate calculi also had cystine in the stones. Some but not all of these patients showed urinary cystine excretion slightly above the normal limits, resembling heterozygous carriers. This finding raised the question of whether some recurrent stone formers may be at risk for calcium oxalate calculi when they excrete cystine in above normal concentrations. MATERIALS AND METHODS: Pooled urine obtained from 3 pairs of age and sex matched controls was independently analyzed. Each urine sample was divided into spun and filtered, and ultrafiltered urine. A Multisizer II (Coulter Electronics Ltd, Beds, England) was used to measure particle number, diameter and volume. The metastable limit of each specimen was determined. Promotion activity was measured in spun and filtered, and ultrafiltered urine using 3 concentrations of cystine (80, 160 and 320 micromol./l.). Results were confirmed by measuring the incorporation of (14)C-oxalate into the crystals. Scanning electron microscopy was performed to study further the agglomerates as well as exclude cystine crystals. Each experiment was repeated 6 times. Crystalline material was collected for x-ray powder diffraction analysis. RESULTS: The urine metastable limit did not change with increasing cystine concentrations. Particle diameter increased significantly from 10.6 microm in ultrafiltered urine alone to 11.6 and 13.5 microm (p < 0.05) at 160 and 320 micromol/l. cystine, respectively. In addition, particle volume also increased proportionally in a dose response manner to cystine concentration. The dose of 320 micromol/l. cystine increased the crystal growth rate 52%. 14C-oxalate experiments confirmed these results. Scanning electron microscopy at 500x magnification revealed no cystine crystals in any experiments performed. Furthermore, x-ray powder diffraction analysis of samples revealed that experimentally determined parameters matched reference values for calcium oxalate trihydrate but not for cystine, again confirming absent cystine in the samples. CONCLUSIONS: Adding cystine to undiluted human urine resulted in the marked enhancement of calcium oxalate crystal precipitation. When considered with the finding of cystine in calcium oxalate stones in the noncystinuric population, this result implies that urinary cystine may be a risk factor for calcium oxalate calculi. Cystine was not observed in any calcium oxalate crystals, suggesting that the mechanism of crystal formation was a salting out effect.