Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and flourescence evaluation

Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative Bmax values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, Kd and relative B max, which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Influence of environmental factors on zoospores of Saprolegnia diclina

The numbers of zoospores produced by a pathogenic strain of Saprolegnia diclina and their behaviour are markedly influenced by a variety of environmental variables including temperature, pH, oxygen tension and the presence of biocides. The use of the latter is not recommended, as fish readily succumb to equivalent concentrations of biocides. Analysis of the pattern of distribution of resulting zoospore cysts demonstrates that zoospores become dispersed by random movement even while in the proximity of the parent colony’s nutrient source. However, the presence of amino acids, in particular aspartic and glutamic acid, at concentrations which occur in fish tissue promotes the directed movement of zoospores towards the nutrient source thereby encouraging the colonization of fresh sites.

Acanthamoebal surface properties and the modulation of phagocytosis

The surface nature of Acanthamoeba trophozoites and cysts was investigated with respect to cell surface charge, hydrophobicity and surface carbohydrate composition. Particulate microelectrophoresis revealed a marked negative charge for both morphological forms, though less for cyst surfaces. Hydrophobicity was determined by adhesion to n-hexadecane and indicated a relatively low hydrophobic nature of both forms, though less so for cysts. Surface carbohydrate composition was studied by the use of fluorescent lectins and flow cytometry, using a ligand-receptor approach for further in depth analysis of binding of particular lectins. These studies showed trophozoite and cyst surfaces to be rich in N-acetylglucosamine, N-acteylneuraminic acid, mannose and glucose, with the addition of N-acetylgalactosamine on cysts. The importance of such surface properties was investigated with respect to phagocytosis of polystyrene latex microspheres, of different surface types and size. Investigations into the optimum conditions of uptake of beads indicated a preference for a medium devoid of nutrients, such as saline, though temperature was not a factor. An amoebal predilection for beads of lower charge and greater hydrophobicity was demonstrated. Furthermore, a preference for the largest bead size used (2.0 m) was observed. The influence of either Con A or mannose or glucose on bead association was apparently limited. The fate of foreign DNA ingested by Acanthamoeba appeared to indicate that such DNA was destroyed, as it could not be detected following extraction procedures and PCR amplification.

Some clinical aspects of the eye in extended contact lens wear

The limbal vascular response to extended contact lens wear was examined in a group comparative study initially intended to last eighteen months. After six months all patients wearing contact lenses had presented with micro-epithelial cysts. This unanticipated occurrence of the micro-epithelial-cysts necessitated termination of the study, and limited the quantity of data collected. However, sufficient results were available to allow a limited description of •the vascular response to this form of contact lens wear. Interpretations of the date collected ore discussed in relation to suggested vasostimulating factors in the cornea. The micro-epithelial cysts observed after extended wear were classified and their rate of recovery recorded. A further clinical study was undertaken to observe cysts in both contact lens - and non contact lens-wearing eyes. Cysts were observed in every category of patient, although the characteristic patterns varied. These observations of micro-epithelial cysts are discussed with respect to the aetiopathogeneses of corneal epithelial cystic disorders. Subsequently, attempts were made to induce cysts in rabbit corneae by extended contact lens wear. Clinical observations revealed cyst-like appearances. Histological sections did not contain cysts but did exhibit signs characteristic •of cystic disorders of the corneal epithelium. In general, the results from the study indicate that extended wear is subjectively acceptable to contact lens wearers. However, the objective findings of significant vascular changes, micro-epithelial cysts and cases of acute red eye response cast considerable doubt on the recommendation of extended wear contact lenses for purely cosmetic applications.

OCT retinal thickness ratios and frequency of follow-up in Wet AMD

Poster Introduction: In neovascular age-related macular degeneration (nAMD), optical coherence tomography (OCT) is an important tool to determine when intravitreal injections of ranibizumab should be administered. Current guidelines recommend that patients should be reviewed four weekly and OCT indications for further treatment include subretinal fluid and intraretinal fluid or cysts. Purpose: We have reviewed the OCT scans of subjects who have successfully responded to ranibizumab to look for factors that might predict which patients will not require injection and could have extended appointments. Method: This was a prospective study in which we observed for 6 consecutive months the OCT images of 28 subjects who had received intravitreal ranibizumab for nAMD and were judged to be clinically inactive at recruitment to the study. Ratios between full retinal thickness (FRT = neurosensory retina + outer reflective band) and outer reflective band (ORB) thickness at the fovea were calculated for each subject at the moment of entering the study and at each successive visit for 6 consecutive months. Results: Patients with lower FRT/ORB ratios were found to be less likely to require an additional injection of ranibizumab and no subject with a ratio of 1.75 or less needed further injections. Conclusion: This small pilot study suggests that on macular OCT, the FRT/ORB ratio, and in particular values of 1.75 or less, may prove to be a useful, practical tool when deciding the follow up period for subjects undergoing treatment with intravitreal ranibizumab for nAMD.

Statnote 34 : the Kolmogorov-Smirnov (KS) test

The Kolmogorov-Smirnov (KS) test is a non-parametric test which can be used in two different circumstances. First, it can be used as an alternative to chi-square (?2) as a ‘goodness-of-fit’ test to compare whether a given ‘observed’ sample of observations conforms to an ‘expected’ distribution of results (KS, one-sample test). An example of the use of the one-sample test to determine whether a sample of observations was normally distributed was described previously. Second, it can be used as an alternative to the Mann-Whitney test to compare two independent samples of observations (KS, two-sample test). Hence, this statnote describes the use of the KS test with reference to two scenarios: (1) to compare the observed frequency (Fo) of soil samples containing cysts of the protozoan Naegleria collected each month for a year with an expected equal frequency (Fe) across months (one-sample test), and (2) to compare the abundance of bacteria on cloths and sponges sampled in a domestic kitchen environment (two-sample test).

Spectrum of morphological and visual changes due to vitreomacular interface disorders encountered in a large consecutive cohort of patients

Aim: Identify the incidence of vitreomacular traction (VMT) and frequency of reduced vision in the absence of other coexisting macular pathology using a pragmatic classification system for VMT in a population of patients referred to the hospital eye service. Methods: A detailed survey of consecutive optical coherence tomography (OCT) scans was done in a high-throughput ocular imaging service to ascertain cases of vitreomacular adhesion (VMA) and VMT using a departmental classification system. Analysis was done on the stages of traction, visual acuity, and association with other macular conditions. Results: In total, 4384 OCT scan episodes of 2223 patients were performed. Two hundred and fourteen eyes had VMA/VMT, with 112 eyes having coexisting macular pathology. Of 102 patients without coexisting pathology, 57 patients had VMT grade between 2 and 8, with a negative correlation between VMT grade and number of Snellen lines (r= -0.61717). There was a distinct cutoff in visual function when VMT grade was higher than 4 with the presence of cysts and sub retinal separation and breaks in the retinal layers. Conclusions: VMT is a common encounter often associated with other coexisting macular pathology. We estimated an incidence rate of 0.01% of VMT cases with reduced vision and without coexisting macular pathology that may potentially benefit from intervention. Grading of VMT to select eyes with cyst formation as well as hole formation may be useful for targeting patients who are at higher risk of visual loss from VMT.

Age determination of sediment cores of the Golf of Lions

Siliciclastic turbidites represent huge volumes of sediments, which are of particular significance for (1) petroleum researchers, interested in their potential as oil reservoirs and (2) sedimentologists, who aim at understanding sediment transport processes from continent to deep-basins. An important challenge when studying marine turbidites has been to establish a reliable chronology for the deposits. Indeed, conventional marine proxies applied to hemipelagic sediments are often unreliable in detrital clays. In siliciclastic turbidites, those proxies can be used only in hemipelagic intervals, providing a poor constraint on their chronology. In this study, we have used sediments from the Rhône Neofan (NW Mediterranean Sea) to demonstrate that pollen grains can provide a high-resolution chronostratigraphical framework for detrital clays in turbidites. Vegetation changes occurring from the end of Marine Isotopic Stage 3 to the end of Marine Isotopic Stage 2 (from ~30 to ~18 ka cal. BP) are clearly recorded where other proxies have failed previously, mainly because the scarcity of foraminifers in these sediments prevented any continuous Sea Surface Temperature (SST) record and radiocarbon dating to be obtained. We show also that the use of palynology in turbidite deposits is able to contribute to oceanographical and sedimentological purposes: (1) Pinus pollen grains can document the timing of sea-level rise, (2) the ratio between pollen grains transported from the continent via rivers and dinoflagellate cysts (elutriating) allows us to distinguish clearly detrital sediments from pelagic clays. Finally, taken together, all these tools show evidence that the Rhône River disconnected from the canyon during the sea-level rise and thus evidence the subsequent rapid starvation of the neofan at 18.5 ka cal. BP. Younger sediments are hemipelagic: the frequency of foraminifers allowed to date sediments with radiocarbon. First results of Sea Surface Temperature obtained on foraminifers are in good agreement with the dinoflagellate cysts climatic signal. Both provide information on the end of the deglaciation and the Holocene.

Expressão de MMPs, marcadores angiogênicos e proliferação celular em tumores odontogênicos

INTRODUCTION AND OBJECTIVE: The study of biological behavior of odontogenic lesions is essential to the establishment of appropriate therapeutic approach and prognosis. The production of extracellular matrix metalloproteinases (MMPs), angiogenesis and cell proliferation contribute to tumor growth. This paper aims to review the literature on odontogenic tumors (OT) selected according to the new World Health Organization classification (WHO- 2005) by evaluating the expression of MMPs, angiogenic and cell proliferation. Furthermore, it aims to verify the relation between these markers and the biological behavior of these lesions. RESULTS: it was found that MMPs -1, -2, -7, -9 and -26 had a higher expression in both epithelial component and stroma, and 13 particularly in the stroma. Increased angiogenesis was observed in more aggressive OT. CD105 expression was higher in keratocystic odontogenic tumour (KOT) and CD34 in solid ameloblastomas (SA). It was observed a higher expression of Ki-67 and p53 in SA and KOT and a low cell proliferation rate in the adenomatoid odontogenic tumour (AOT). CONCLUSION: These results show that MMPs are involved in invasion and recurrence of some odontogenic lesions and are associated with the biological behavior of these tumors. Angiogenesis is critical to provide support to cell proliferation and these concomitant events are correlated with different levels of biological behavior in OT when compared to odontogenic cysts, hence the use of angiogenic inhibitors may be a potential therapeutic approach in these lesions.

Expressão de MMPs, marcadores angiogênicos e proliferação celular em tumores odontogênicos

INTRODUCTION AND OBJECTIVE: The study of biological behavior of odontogenic lesions is essential to the establishment of appropriate therapeutic approach and prognosis. The production of extracellular matrix metalloproteinases (MMPs), angiogenesis and cell proliferation contribute to tumor growth. This paper aims to review the literature on odontogenic tumors (OT) selected according to the new World Health Organization classification (WHO- 2005) by evaluating the expression of MMPs, angiogenic and cell proliferation. Furthermore, it aims to verify the relation between these markers and the biological behavior of these lesions. RESULTS: it was found that MMPs -1, -2, -7, -9 and -26 had a higher expression in both epithelial component and stroma, and 13 particularly in the stroma. Increased angiogenesis was observed in more aggressive OT. CD105 expression was higher in keratocystic odontogenic tumour (KOT) and CD34 in solid ameloblastomas (SA). It was observed a higher expression of Ki-67 and p53 in SA and KOT and a low cell proliferation rate in the adenomatoid odontogenic tumour (AOT). CONCLUSION: These results show that MMPs are involved in invasion and recurrence of some odontogenic lesions and are associated with the biological behavior of these tumors. Angiogenesis is critical to provide support to cell proliferation and these concomitant events are correlated with different levels of biological behavior in OT when compared to odontogenic cysts, hence the use of angiogenic inhibitors may be a potential therapeutic approach in these lesions.

(Supplement) Concentration in palynomorphs per gram of dry sediment in ANDRILL core AND-2A

An exceptional triple palynological signal (unusually high abundance of marine, freshwater, and terrestrial palynomorphs) recovered from a core collected during the 2007 ANDRILL (Antarctic geologic drilling program) campaign in the Ross Sea, Antarctica, provides constraints for the Middle Miocene Climatic Optimum. Compared to elsewhere in the core, this signal comprises a 2000-fold increase in two species of dinoflagellate cysts, a synchronous five-fold increase in freshwater algae, and up to an 80-fold increase in terrestrial pollen, including a proliferation of woody plants. Together, these shifts in the palynological assemblages ca. 15.7 Ma ago represent a relatively short period of time during which Antarctica became abruptly much warmer. Land temperatures reached 10 °C (January mean), estimated annual sea-surface temperatures ranged from 0 to 11.5 °C, and increased freshwater input lowered the salinity during a short period of sea-ice reduction.

(Table 1) Abundance of unicellular eukaryotes in ice cores collected during the July 2008 CCGS Amundsen cruise in Darnley Bay

Influence of methanogenic populations in Holocene lacustrine sediments revealed by clone libraries and fatty acid biogeochemistry.Biological characteristics of ice-associated algal communities were studied in Darnley Bay (western Canadian Arctic) during a 2-week period in July 2008 when the landfast ice cover had reached an advanced stage of melt. We found two distinct and separate algal communities: (1) an interior ice community confined to brine channel networks beneath white ice covers; and (2) an ice melt water community in the brackish waters of both surface melt ponds and the layer immediately below the ice cover. Both communities reached maximum chlorophyll a concentrations of about 2.5 mg/m**3, but with diatoms dominating the interior ice while flagellates dominated the melt water community. The microflora of each community was diverse, containing both unique and shared algal species, the latter suggesting an initial seeding of the ice melt water by the bottom ice community. Absorption characteristics of the algae indicated the presence of mycosporine-like amino acids (MAAs) and carotenoid pigments as a photoprotective strategy against being confined to high-light near-surface layers. Although likely not contributing substantially to total annual primary production, these ice-associated communities may play an important ecological role in the Arctic marine ecosystem, supplying an accessible and stable food source to higher trophic levels during the period of ice melt.

Dinocyst assemblages of site HM52-43

Past sea-surface conditions over the northern North Atlantic during the last glacial maximum were examined from the study of 61 deep-sea cores. The last glacial maximum time slice studied here corresponds to an interval between Heinrich layers H2 and H1, and spanning about 20-16 ka on a 14C time scale. Transfer functions based on dinocyst assemblages were used to reconstruct sea-surface temperature, salinity, and sea-ice cover. The results illustrate extensive sea-ice cover along the eastern Canadian margins and sea-ice spreading, only during winter, over most of the northern North Atlantic. On the whole, much colder winter prevailed, despite relatively mild conditions in August (10-15°C at most offshore sites), thus suggesting a larger seasonal contrast of temperatures than today. Lower salinity than at present is reconstructed, especially along the eastern Canadian and Scandinavian margins, likely because of meltwater supply from the surrounding ice sheets. These reconstructions contrast with those established by CLIMAP on the basis of planktonic foraminifera. These differences are discussed with reference to the stratigraphical frame of the last glacial maximum, which was not the coldest phase of the last glacial stage. The respective significance of dinocyst and foraminifer records is also examined in terms of the thermohaline characteristics of surface waters and the vertical structure of upper water masses, which was apparently much more stratified than at present in the northern North Atlantic, thus preventing deep-water formation.

Tab. 1: Radiocarbon ages of samples from the Canadian archipelago

A sediment-sampling program was carried out in the Nares Strait region during the Nares 2001 Expedition to obtain cores for high-resolution palaeoceanographic studies of late Pleistocene-Holocene climate change. Long cores (>4 m) were obtained from basins near Coburg Island, Jones Sound, John Richardson Fiord off Kane Basin, and in northeastern Hall Basin. Short cores and grab samples were taken on shelves east and west of northern Smith Sound and in Kennedy Channel. Detailed studies of sediment texture, stable isotopes, microfossils and palynomorphs were made on the longest cores from Jones Sound and Hall Basin at the southern and northern ends of the Nares Strait region.

Pollen profile and age determination for sediment core M72/5_619-1 (22-GC3)

Sediments from the Black Sea, a region historically dominated by forests and steppe landscapes, are a valuable source of detailed information on the changes in regional terrestrial and aquatic environments at decadal to millennial scales. Here we present multi-proxy environmental records (pollen, dinoflagellate cysts, Ca, Ti and oxygen isotope data) from the uppermost 305 cm of the core 22-GC3 (42°13.53' N, 36°29.55' E) collected from a water depth of 838 m in the southern part of the Black Sea in 2007. The records span the last ~ 18 kyr (all ages are given in cal kyr BP). The pollen data reveal the dominance of the Artemisia-steppe in the region, suggesting rather dry/cold environments ~ 18-14.5 kyr BP. Warming/humidity increase during melt-water pulses (~ 16.1-14.5 kyr BP), indicated by d18O records from the 22-GC3 core sediment and from the Sofular Cave stalagmite, is expressed in more negative d13C values from the Sofular Cave, usually interpreted as the spreading of C3 plants. The records representing the interstadial complex (~ 14.5-12.9 kyr BP) show an increase in temperature and moisture, indicated by forest development, increased primary productivity and reduced surface run-off, whereas the switch from primary terrigenous to primary authigenic Ca origin occurs ~ 500 yr later. The Younger Dryas cooling is clearly demonstrated by more negative d13C values from the Sofular Cave and a reduction of pines. The early Holocene (11.7-8.5 kyr BP) interval reveals relatively dry conditions compared to the mostly moist and warm middle Holocene (8.5-5 kyr BP), which is characterized by the establishment of the species-rich warm mixed and temperate deciduous forests in the low elevation belt, temperate deciduous beech-hornbeam forests in the middle and cool conifer forest in upper mountain belt. The border between the early and middle Holocene in the vegetation records coincides with the opening of the Mediterranean corridor at ~ 8.3 kyr BP, as indicated by a marked change in the dinocyst assemblages and in the sediment lithology. Changes in the pollen assemblages indicate a reduction in forest cover after ~ 5 kyr BP, which was likely caused by increased anthropogenic pressure on the regional vegetation.