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Cyanobacterial phycobiliproteins, such as phycoerythrin (PE) and phycocyanin (PC), are colored potential bioactive proteins that have antioxidant and antimicrobial properties. In this study, we formulated a new food prototype based on PE and PC-fortified low-fat yogurt and cream cheese. Four distinct low-fat yogurt and cream cheese products were manufactured, including a control group (No PE and PC), samples produced with phycoerythrin (+ PE), samples produced with phycocyanin (+ PC), and samples produced with both phycoerythrin and phycocyanin (PC + PE). Afterwards statistically compared the physicochemical composition, colorimetric properties, antioxidant and antimicrobial activities, and sensory profile of the fortified foods at 4 °C and 8 °C for 28 and 42 days. Additionally, we confirmed that PE and PC are not toxic to Caenorhabditis elegans at concentrations up to 1 mg/mL. The results showed that the MIC of PE and PC against E. coli was significantly higher than against S. aureus (3.12 ± 0.05 µg/mL vs. 1.56 ± 0.01 µg/mL, respectively; p ≤ 0.05). Additionally, the maximum diameter of the inhibition zone of PE and PC against S. aureus was significantly higher than against E. coli (6.6 ± 0.011 mm vs. 11.66 ± 0.02 mm, respectively; p ≤ 0.05). Results of color parameters showed that the control group had significantly higher L* values than the samples enriched with PE and PC. Moreover PE and PC significantly increased the a* and b* values respectively. The amount of ΔE in the control yogurts and cream cheese was higher than in the samples with PE and PC. Overall, the results showed that adding PE and PC had a significant effect on all measured factors (p < 0.01). Cream cheeses and low-fat yogurts enriched with either PE or PE + PC had the greatest antioxidant activity and the lowest number of psychrophilic bacteria and mold, and yeast counts at the end of the test period. Therefore, low-fat yogurt and cream cheese containing cyanobacterial PE and PC can be considered an innovative dairy product for the food industry. This study marks the initial effort to employ PE and PC derived from Nostoc sp. and Spirulina sp. as antioxidant and antimicrobial agents in the food industry., Competing Interests: Competing interests The authors declare no competing interests. Ethical statement All methods were carried out following relevant guidelines and regulations. The authors did not do any experiments on humans and/or the use of human tissue samples. All experimental protocols and panelists involved in the study were approved by the ethics committee of Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. Participants gave their consent to take part and use their information. The full name of the ethics committee that approved the study is Dr Fahimeh Nemati and Dr Sarvenaz Falsafi., (© 2024. The Author(s).)

The present study was performed to evaluate the effect of phycoerythrin (PE) treatment extracted from Nostoc sp. on the shelf-life extension of the Nile Tilapia ( Oreochromis niloticus ) fillet at 4°C and 8°C. After extraction and purification of pigment in BG-110 medium, the pigment PE was extracted and purified with 56% ammonium sulfate followed by dialysis. After that, the effect of pigment on Escherichia coli and Staphylococcus aureus were investigated. The fillet samples were immersed in pigment solution, and their physicochemical, microbiological and sensory properties were examined. The results showed that the concentration and purity of the pigments increased after the dialysis. The results from performed chemical tests and total number of living mesophilic bacteria, psychrotrophic bacteria, Staphylococcus aureus coagulase positive, and coliform bacteria of the samples compared to the blank sample showed that sample treated with algae extracts were able to control the increase in these parameters. In these tests, the highest levels belonged to Nile Tilapia fillet sample Nile Tilapia fillet coated with PE solution at a temperature 8°C and the lowest amount was observed with fillet coated with PE solution at a temperature of 4˚C (P≤0.05). The results of sensory evaluation showed that the highest score of taste, texture, color, and total acceptance were observed for Nile Tilapia fillet coated with PE solution at temperature 8°C. In conclusion, the extract pigments from Nostoc sp. has strong antimicrobial activity and can maintain the quality parameters for controlling of spoilage bacteria and extend the shelf-life of Oreochromis niloticus ., Competing Interests: Authors have no conflict of interest on this work.

Porphyridium purpureum is a well-known Rhodophyta that recently has attracted enormous attention because of its capacity to produce many high-value metabolites such as the pigment phycoerythrin and several high-value fatty acids. Phycoerythrin is a fluorescent red protein-pigment commercially relevant with antioxidant, antimicrobial activity, and fluorescent properties. The volumetric mass transfer coefficient (k L a) was kept constant within the different scaling-up stages in the present study. This scaling-up strategy was sought to maintain phycoerythrin production and other high-value metabolites by Porphyridium purpureum , using hanging-bag photobioreactors. The k L a was monitored to ensure the appropriate mixing and CO 2 diffusion in the entire culture during the scaling process (16, 80, and 400 L). Then, biomass concentration, proteins, fatty acids, carbohydrates, and phycoerythrin were determined in each step of the scaling-up process. The k L a at 16 L reached a level of 0.0052 s -1 , while at 80 L, a value of 0.0024 s -1 was achieved. This work result indicated that at 400 L, 1.22 g L -1 of biomass was obtained, and total carbohydrates (117.24 mg L -1 ), proteins (240.63 mg L -1 ), and lipids (17.75% DW) were accumulated. Regarding fatty acids production, 46.03% palmitic, 8.03% linoleic, 22.67% arachidonic, and 2.55% eicosapentaenoic acid were identified, principally. The phycoerythrin production was 20.88 mg L -1 with a purity of 2.75, making it viable for food-related applications. The results of these experiments provide insight into the high-scale production of phycoerythrin via the cultivation of P. purpureum in an inexpensive and straightforward culture system.

The nutritional value of the marine cryptophyte Rhodomonas lens for the filter feeder Brachionus plicatilis as well as its biotechnological potential as a source of phycoerythrin (PE) and polyunsaturated fatty acids (PUFA) were evaluated in semi-continuous cultures maintained with different daily renewal rates (RR), from 10% (R10) to 50% (R50) of the total volume. Steady-state cell density decreased from 22 to 7 × 10 6  cells mL -1 with increasing RR, with the maximum cell productivity, nearly 0.4 g L -1  day -1 , observed with R40. PE cell content attained the highest values with the highest RR (circa 9 pg cell -1 ). All treatments of R. lens maintained under nitrate-saturated conditions (R20-R50) showed a similar high content of PUFAs, > 60% of total fatty acids (FA), with linolenic acid (18:3n-3) and 18:4n-3, representing 12 and 29% of total FA respectively. The PUFA level in the nitrogen-limited R10 cultures was significantly lower (37%). R. lens promoted higher weight gain in the rotifer B. plicatilis than Tisochrysis lutea (T-ISO), a species commonly used for rotifer culture and enrichment. Significant differences were found in the protein content and in the ratio n-3/n-6 fatty acids among rotifers fed with R. lens from different RRs, with higher values being found in those fed with R. lens from higher RRs. The enrichment of the rotifers for short periods of 3 h was sufficient to modify the biochemical composition of the rotifers, but it was evidenced as too short for the accumulation of PUFAs, when compared to long-term (24 h) enrichment. The rotifers reflected the higher protein and PUFA content of R. lens cultivated with nutrient sufficient microalgae (R40) after only 3 h of enrichment. These results demonstrate that semi-continuous culture of R. lens under appropriate conditions can strongly enhance the nutritional value of this species, being reflected in the growth and biochemical composition of the filter feeder, even in short exposure periods.

The effect of oven-drying at 25, 40 and 60 °C was evaluated on three macroalgae of relevance in Europe, namely Ulva rigida , Gracilaria sp. and Fucus vesiculosus , with respect to quality aspects, including their potential to be exploited as a source of valuable compounds. Notably, as compared to freeze-drying, oven-drying at 25 °C promoted the extraction of chlorophylls and carotenoids from U. rigida , as well as those of phycoerythrin and chlorophyll a from Gracilaria sp., while 40 °C favored the recovery of fucoxanthin and pheophytin a from F. vesiculosus . On the other hand, the use of oven-drying had a negative impact on the extraction of phenolic compounds from this alga, also diminishing the antioxidant activity of the resulting extracts. Instead, the impact of oven-drying of raw material on the recovery of specific polysaccharides differed among the macroalgae. While the amounts of ulvans and fucoidans obtained from macroalgae dried at higher temperatures tended to be superior, the recovery of agar was not affected with the drying temperatures applied to Gracilaria sp. The overall results showed that oven-drying might serve as a good alternative to stabilize Ulva rigida , Gracilaria sp. and Fucus vesiculosus , especially if extraction of pigments and polysaccharides is aimed, thought the appropriate temperature applied must be adapted for each macroalgae.

Surface capture assays can measure fluorescently labeled analytes across a 1000-fold concentration range and at the sub-nanomolar level, but many biological molecules exhibit 1,000,000-fold variations in abundance down to the femtomolar level. The goal of this work is to expand the dynamic range of fluorescence assays by using imaging to combine molecular counting with single-molecule calibration of ensemble intensities. We evaluate optical limits imposed by surface-captured fluorescent labels, compare performances of different fluorophore classes, and use detector acquisition parameters to span wide ranges of fluorescence irradiance. We find that the fluorescent protein phycoerythrin provides uniquely suitable properties with exceptionally intense and homogeneous single-fluorophore brightness that can overcome arbitrary spot detection threshold biases. Major limitations imposed by nonspecifically bound fluorophores were then overcome using rolling circle amplification to densely label cancer-associated miRNA biomarkers, allowing accurate single-molecule detection and calibration across nearly 5 orders of magnitude of concentration with a detection limit of 29 fM. These imaging and molecular counting strategies can be widely applied to expand the limit of detection and dynamic range of a variety of surface fluorescence assays.

Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6-8 K MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique. In the present study, the method was successfully applied for separation and purification of R-phycoerythrin (R-PE), a protein widely used as a fluorescent probe, from the red alga Gracilaria lemaneiformis. The separation was performed with the elution of ammonium sulfate from 50% to 0% in 21.5 h at a flow rate of 0.5 ml/min, while the lower channel was eluted with water at a flow rate of 0.05 ml/min after sample charge, and the column was rotated at 200 rpm. After a single run, the absorbance ratio A 565 /A 280 (a criterion for the purity of R-PE) was increased from 0.5 of the crude to 6.5. The purified R-PE exhibited a typical "three peaks" spectrum with absorbance maximum at 497, 538 and 565 nm. The Native-PAGE showed one single protein band and 20 kDa (subunits α and β) and 30 kDa (subunit γ) can be observed in SDS-PAGE analysis which were consistent with the (αβ) 6 γ subunit composition of R-PE. The results indicated that CpC is an efficient method to obtain protein with the high purity from a complex source., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Allo-immunizations against HLA antigens are known to be deleterious in transfusion and organ transplantation. The development of new tests based on solid phase assays for screening and identification of HLA antibodies in particular those using Luminex ® bead based technology has completely changed the way of allo-immunization monitoring because of their extreme sensitivity. They allow a better characterization of these antibodies, identification of acceptable antigens and the use of virtual cross-matches. All these new possibilities improve the managing of patients before and after platelets transfusion or organ transplantation. However, this technology displays some limits that should be known in order to interpret correctly the results. Beside these bead based assays, cellular cross-matches based on Complement Dependent Cytotoxicity (CDC) and flow cytometry are still used and useful in organ transplantation since beads are produced in vitro and do not reflected exactly what happens physiologically. Moreover, differences of sensitivity between these methods make results interpretation and decision making difficult in some cases., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

The widespread unicellular cyanobacteria Synechococcus are major contributors to global marine primary production. Here, we report their abundance, phylogenetic diversity (as assessed using the RNA polymerase gamma subunit gene rpoC1) and pigment diversity (as indirectly assessed using the laterally transferred cpeBA genes, encoding phycoerythrin-I) in surface waters of the northwestern Pacific Ocean, sampled over nine distinct cruises (2008-2015). Abundance of Synechococcus was low in the subarctic ocean and South China Sea, intermediate in the western subtropical Pacific Ocean, and the highest in the Japan and East China seas. Clades I and II were by far the most abundant Synechococcus lineages, the former dominating in temperate cold waters and the latter in (sub)tropical waters. Clades III and VI were also fairly abundant in warm waters, but with a narrower distribution than clade II. One type of chromatic acclimater (3dA) largely dominated the Synechococcus communities in the subarctic ocean, while another (3dB) and/or cells with a fixed high phycourobilin to phycoerythrobilin ratio (pigment type 3c) predominated at mid and low latitudes. Altogether, our results suggest that the variety of pigment content found in most Synechococcus clades considerably extends the niches that they can colonize and therefore the whole genus habitat., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

R-Phycoerythrin is one of the phycobiliproteins widely found in seaweeds. In this study, we have shown to extract and purify R-Phycoerythrin from the south east cost Indian red seaweed Halymenia floresia. R-Phycoerythrin was extracted in 50mM phosphate buffer (pH 7.0). The preparative native PAGE purification was employed alternative to the chromatography and therefore can be scaled up efficiently. Both the yield and the purity of R-Phycoerythrin are very effective. The purified R-Phycoerythrin showed a single band on the examination by native PAGE electrophrosis. SDS-PAGE analysis showed five bands at 16kDa, 21kDa, 30kDa, 39kDa and 47kDa which corresponds to the α, β and γ', γ and αβ subunits. This preparative method for R-Phycoerythrin purification can offer a reference for R-Phycoerythrin purification from other marine red macro algae., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The cyanobacterium Microcystis aeruginosa is a mesophilic freshwater organism, which cannot tolerate sulphide. However, it was possible to isolate a sulphide-resistant (S(r)) mutant strain that was able to survive in a normally lethal medium sulphide. In order to evaluate the cost of the mutation conferring sulphide resistance in the S(r) strain of M. aeruginosa, the morphology and the photosynthetic performance were compared to that found in the wild-type, sulphide-sensitive (S(s)) strain. An increase in size and a disrupted morphology was observed in S(r) cells in comparison to the S(s) counterpart. Phycoerythrin and phycocyanin levels were higher in the S(r) than in the S(s) cells, whereas a higher carotenoid content, per unit volume, was found in the S(s) strain. The irradiance-saturated photosynthetic oxygen-production rate (GPR max) and the photosynthetic efficiency (measured both by oxygen production and fluorescence, α(GPR) and α(ETR)) were lower in the S(r) strain than in the wild-type. These results appear to be the result of package effect. On the other hand, the S(r) strain showed higher quantum yield of non-photochemical quenching, especially those regulated mechanisms (estimated throughout qN and Y(NPQ)) and a significantly lower slope in the maximum quantum yield of light-adapted samples (Fv'/Fm') compared to the S(s) strain. These findings point to a change in the regulation of the quenching of the transition states (qT) in the S(r) strain which may be generated by a change in the distribution of thylakoidal membranes, which somehow could protect metalloenzymes of the electron transport chain from the lethal effect of sulphide.

The ability to enumerate, classify, and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function and their role in the aquatic community and microbial food web. Traditional micro-phytoplankton quantification methods using microscopic techniques require preservation and are slow, tedious and very laborious. The availability of more automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and overall phytoplankton biomass. The FlowCam(®) is an imaging cytometry tool that was originally developed for use in aquatic sciences and provides a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments.

Detection of Hendra viral IgG antibody in animal sera is useful for surveillance following a virus outbreak. The commonly used enzyme-linked immunosorbent assay and fluorescence-based Luminex assay typically consist of three steps and take at least several hours to complete. We have simplified the procedure to two steps in an effort to develop a rapid procedure for IgG antibody, but not IgM antibody, detection. This is achieved by conjugating the fluorescence label R-phycoerythrin directly onto the IgG binding protein Protein G. The use of magnetic nanoparticles, due to their large specific surface area, has helped reduce each of the binding steps to 20 min. As a result, the whole assay can be completed in 60 min. We also demonstrate a method to quickly estimate IgG antibody titres by assaying the sera at only two dilutions (i.e. 1:20 and 1:1000) and using the fluorescence ratio at these dilutions as an indicator of antibody titre. The results of this approach correlated well with the well-regarded serum neutralization test in virus antibody assays. This protocol reported here can be adopted in Luminex assays, fluorescence-linked immunosorbent assays and assays on microfluidics platforms for rapid antibody surveillance of Hendra and other viruses., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Solenopora jurassica is a fossil calcareous alga that functioned as an important reef-building organism during the Palaeozoic. It is of significant palaeobiological interest due to its distinctive but poorly understood pink and white banding. Though widely accepted as an alga there is still debate over its taxonomic affinity, with recent work arguing that it should be reclassified as a chaetetid sponge. The banding is thought to be seasonal, but there is no conclusive evidence for this. Other recent work has, however demonstrated the presence of a unique organic boron-containing pink/red pigment in the pink bands of S. jurassica. We present new geochemical evidence concerning the seasonality and pigmentation of S. jurassica. Seasonal growth cycles are demonstrated by X-ray radiography, which shows differences in calcite density, and by varying δ13C composition of the bands. Temperature variation in the bands is difficult to constrain accurately due to conflicting patterns arising from Mg/Ca molar ratios and δ18O data. Fluctuating chlorine levels indicate increased salinity in the white bands, when combined with the isotope data this suggests more suggestive of marine conditions during formation of the white band and a greater freshwater component (lower chlorinity) during pink band precipitation (δ18O). Increased photosynthesis is inferred within the pink bands in comparison to the white, based on δ13C. Pyrolysis Gas Chromatography Mass Spectrometry (Py-GCMS) and Fourier Transform Infrared Spectroscopy (FTIR) show the presence of tetramethyl pyrrole, protein moieties and carboxylic acid groups, suggestive of the presence of the red algal pigment phycoerythrin. This is consistent with the pink colour of S. jurassica. As phycoerythrin is only known to occur in algae and cyanobacteria, and no biomarker evidence of bacteria or sponges was detected we conclude S. jurassica is most likely an alga. Pigment analysis may be a reliable classification method for fossil algae.

A one-step chromatographic method for the purification of R-phycoerythrin (R-PE) of Grateloupia turuturu Yamada is described. Native R-PE was obtained with a purity index of 2.89 and a recovery yield of 27% using DEAE-Sepharose Fast Flow chromatography with a three-step increase in ionic strength. The analysis by SDS electrophoresis showed a broad band between 18 and 21kDa in size corresponding to subunits α and β and a low intensity band of 29kDa corresponding to the γ subunit. Two forms of R-PE were identified by gel filtration chromatography: a native form with a molecular weight of 260±5kDa and a dissociated form with a molecular weight of 60±2kDa. The native form presented the characteristic absorption spectrum of R-PE with three absorbance maxima at 498, 540 and 565nm, whereas the dissociated form presented only the 498 and 540nm peaks. Moreover, the two forms displayed two different fluorescence maxima., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This work characterizes a unicellular cyanobacterium with nearly spherical cells and thin-outlined sheaths that divide irregularly, forming small packets immersed in a diffluent mucilaginous layer. It was isolated growing on calcite speleothems and walls in a show cave in Collbató (Barcelona, Spain). Spectral confocal laser and transmission electron microscopy were used to describe the morphology, fine structure and thylakoid arrangement. The pigments identified were phycoerythrin, phycocyanin, allophycocyanin and chlorophyll a. Three-dimensional reconstructions, generated from natural fluorescence z-stacks, revealed a large surface area of nearly flat, arm-like thylakoidal membranes connected to each other and forming a unified structure in a way that, to our knowledge, has never been described before. Phylogenetic analyses using the 16S rRNA gene sequence showed 95% similarity to strain Chroococcus sp. JJCM (GenBank accession no. AM710384). The diacritical phenotypic features do not correspond to any species currently described, and the genetic traits support the strain being classified as the first member of an independent genus in the order Chroococcales and the family Chroococcaceae. Hence, we propose the name Chalicogloea cavernicola gen. nov., sp. nov. under the provisions of the International Code of Nomenclature for Algae, Fungi and Plants. The type strain of Chalicogloea cavernicola is COLL 3(T) (=CCALA 975(T) =CCAP 1424/1(T)).

In marine Synechococcus there is evidence for the adaptive evolution of spectrally distinct forms of the major light harvesting pigment phycoerythrin (PE). Recent research has suggested that these spectral forms of PE have a different evolutionary history than the core genome. However, a lack of explicit statistical testing of alternative hypotheses or for selection on these genes has made it difficult to evaluate the evolutionary relationships between spectral forms of PE or the role horizontal gene transfer (HGT) may have had in the adaptive phenotypic evolution of the pigment system in marine Synechococcus. In this work, PE phylogenies of picocyanobacteria with known spectral phenotypes, including newly co-isolated strains of marine Synechococcus from the Gulf of Mexico, were constructed to explore the diversification of spectral phenotype and PE evolution in this group more completely. For the first time, statistical evaluation of competing evolutionary hypotheses and tests for positive selection on the PE locus in picocyanobacteria were performed. Genes for PEs associated with specific PE spectral phenotypes formed strongly supported monophyletic clades within the PE tree with positive directional selection driving evolution towards higher phycourobilin (PUB) content. The presence of the PUB-lacking phenotype in PE-containing marine picocyanobacteria from cyanobacterial lineages identified as Cyanobium is best explained by HGT into this group from marine Synechococcus. Taken together, these data provide strong examples of adaptive evolution of a single phenotypic trait in bacteria via mutation, positive directional selection and horizontal gene transfer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The Nobel Prize in Chemistry was awarded in 2000 for the discovery of conductive organic polymers, which have subsequently been adapted for applications in ultrasensitive biological detection. Here, we report the first use of this new class of fluorescent probes in a diverse range of cytometric and imaging applications. We demonstrate that these "Brilliant Violet" reporters are dramatically brighter than other UV-violet excitable dyes, and are of similar utility to phycoerythrin (PE) and allophycocyanin (APC). They are thus ideally suited for cytometric assays requiring high sensitivity, such as MHC-multimer staining or detection of intracellular antigens. Furthermore, these reporters are sensitive and spectrally distinct options for fluorescence imaging, two-photon microscopy and imaging cytometry. These ultra-bright materials provide the first new high-sensitivity fluorescence probes in over 25 years and will have a dramatic impact on the design and implementation of multicolor panels for high-sensitivity immunofluorescence assays., (Published 2012 Wiley Periodicals, Inc.)

In this study, we explored the relationship between the circulating tumor cells (CTC) and the CTC-cancer stem cells (CSC) in the patients with breast cancer. The magnetic-activated cell separation (MACS) method and flow cytometry (FCM) for selection of epithelial cells from the peripheral blood mononuclear cells (PBMC) were used to analyze the enriched epithelial cells that were labeled with anti-cytokeratin(CK)-fluorescein isothiocyanate, anti-CD44-phycoerythrin (PE) and anti-CD24-PE, respectively. The CK+ cells were attributed to CTC and the CK+CD44+ CD24-/low cells were thought as to CTC-CSC in 26 breast cancer patients, respectively. Our results showed the CK+ tumor cells were detected in 19 of 26 patients, with the CK+ tumor cells varying from 0.11% to 5.42 %. The CTC-CSC were identified in 18 of the 19 patients with CTC and the percentage of CTC-CSC in CTC was 19.01%. The results yet suggested the breast cancer patients with high-rate CK+ tumor cells were at the advanced tumor node metastases (TNM) stage III, and the patients with low-rate CK+ cells were at the modest TNM stage I. The difference between the two groups was statistically significant (p<0.001). We concluded that there is a significant relationship between CTC and CTC-CSC, but not among TNM stages, in breast cancer metastasis.

High-quality NaYF₄:Yb/Er/Gd up-conversion nanoparticles (UCNPs) were first synthesized by a solvothermal method using rare earth stearate, sodium fluoride, ethanol, water, and oleic acid as precursors. Doped Gd³⁺ ions can promote the transition of NaYF₄ from cubic to hexagonal phase, shorten the reaction time, and reduce the reaction temperature without reducing the luminescence intensity of NaYF₄:Yb/Er UCNPs. X-ray diffraction, infrared spectroscopy, transmission electron microscopy, and luminescence spectroscopy were applied to characterize the UCNPs. The nanoparticles exhibited small size and excellent green up-conversion photoluminescence, making them suitable for biological applications. After the surfaces of NaYF₄:Yb/Er/Gd UCNPs were modified with amino groups through the Stöber method, they could be brought close enough to the analytically important protein called R-phycoerythrin (R-PE) bearing multiple carboxyl groups so that energy transfer could occur. A luminescence resonance energy transfer (LRET) system was developed using NaYF₄:Yb/Er/Gd UCNPs as an energy donor and R-PE as an energy acceptor. As a result, a detection limit of R-PE of 0.5 μg/ml was achieved by the LRET system with a relative standard deviation of 2.0%. Although this approach was first used successfully to detect R-PE, it can also be extended to the detection of other biological molecules., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The isolated cyanobacterium containing biopigments like chlorophyll-a, phycoerythrin, phycocyanin, and carotenoid was cultured under different quality of light modes to ascertain biomass and pigment productivity. On the basis of 16S rRNA gene sequence, the isolate was identified as Pseudanabaena sp. Maximum biomass concentration obtained in white-, blue-, and green-light was 0.82, 0.94, and 0.89 g/L, respectively. It was observed that maximum phycoerythrin production was in green light (39.2 mg/L), ensued by blue light (32.2 mg/L), while phycocyanin production was maximum in red light (10.9 mg/L). In yellow light, pigment production as well as the growth rate gradually declined after 12 days. Carotenoid production decreased in blue-, white-, and red-light after 15 days, while in green light it had increased gradually. The present communication suggests that Pseudanabaena sp. can be used for commercial production of phycoerythrin when grown under green light., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study explored the chronic and harmful effects of different doses of the triazole fungicide, tebuconazole, on the growth, and metabolic and enzymatic variations in the filamentous paddy field cyanobacterium, Westiellopsis prolifica Janet. The growth of the cyanobacterium was determined by an estimation of the change in pigment contents. Chlorophyll-a, carotenoids and accessory pigments such as phycocyanin, allophycocyanin and phycoerythrin, were shown to decline over a 16-day period by a factor of 92%, 93%, 83%, 95% and 100%, respectively, with increasing doses of the fungicide. Metabolic and enzymatic activities were also adversely affected. Over the 16 days, a gradual rise in the total phenol content was recorded when Westiellopsis prolifica Janet was treated with 60 ppm of the fungicide, despite the reduction in carbohydrates, proteins and amino acids by 96%, 92% and 90%, respectively. Moreover, the enzymes nitrate reductase (NR), glutamine synthetase (GS) and succinate dehydrogenase (SDH) also registered reductions of 93%, 90% and 98%, respectively. This study indicates that tebuconazole, although an important fungicide used extensively in rice fields, exhibits an inhibitory effect on the growth and metabolic activities of Westiellopsis prolifica Janet and hence possibly on other varieties as well.

A cyanobacterium, semi-filamentous multicellular strain ABRG5-3, was isolated and its unique nature was characterized. This axenic strain formed colonies and was motile on an agarose plate. The 16S rRNA gene of ABRG5-3 exhibited similarities to those of the Limnothrix and Pseudanabaena strains, which are known as filamentous and nonheterocystous cyanobacteria. Peaks in absorbance for the accumulation of chlorophyll a, phycocyanin, and phycoerythrin were observed in the cell extract. Natural separation of the pigments occurred in the supernatant of the autolysed cells. The cell lysis was promoted by osmotic shocks and lysozyme treatments. Chlorophyll a and total DNA were abundantly recovered from the cells. Analysis of the restriction-modification system for genomic DNA revealed novel diversity. Moreover, we made a successful attempt to create antibiotic-resistant strains by conjugation with a foreign plasmid, which indicates that strain ABRG5-3 is transformable.

The study showed that the picocyanobacteria community of the Great Mazurian Lakes system (GML) was dominated by phycoerythrin-rich (PE) ecotypes and demonstrated a gradual decrease of the ratio between PE and phycocyanin-rich (PC) ecotypes. The Great Mazurian Lakes offer better conditions for the PE ecotype than for the PC one, despite the considerably high trophic status, probably thanks to low turbidity and attenuation of light in the water column. The successful isolation of PE and PC picocyanobacteria was achieved by two methods: the classic plate method and a modified flow-cytometry method. The modified flow-cytometry method proved to be superior: being more selective for PE picocyanobacteria as well as less time consuming and less laborious. The modifications introduced to the method, such us concentration of cyanobacterial cells by centrifugation to the density required by the flow cytometer, did not hinder the isolation while allowing to skip an intermediate phase of enrichment cultures that had been formerly proposed. The first phylogenetic analyses based on cpcBA operon and 16S rRNA gene demonstrated that picocyanobacteria isolates from GML could, with a high bootstrap support, be grouped into five and four clusters, respectively. Based on a cpcBA-IGS analysis and IGS length the study suggests that at least one of the clusters is new and has not been previously described.

A microflow cytometer was developed that ensheathed the sample (core) fluid on all sides and interrogated each particle in the sample stream at four different wavelengths. Sheathing was achieved by first sandwiching the core fluid with the sheath fluid laterally via fluid focusing. Chevron-shaped groove features fabricated in the top and bottom of the channel directed sheath fluid from the sides to the top and bottom of the channel, completely surrounding the sample stream. Optical fibers inserted into guide channels provided excitation light from diode lasers at 532 and 635 nm and collected the emission wavelengths. Two emission collection fibers were connected to PMTs through a multimode fiber splitter and optical filters for detection at 635 nm (scatter), 665 nm and 700 nm (microsphere identification) and 565 nm (phycoerythrin tracer). The cytometer was capable of discriminating microspheres with different amounts of the fluorophores used for coding and detecting the presence of a phycoerythrin antibody complex on the surface of the microspheres. Assays for Escherichia coli were compared with a commercial Luminex flow cytometer.

A rosy discoloration partly masking the Luca Signorelli frescoes in St. Brizio Chapel (Orvieto Cathedral, Italy) for many years proved to be a biological alteration, so the present research focused on investigating biodeteriogens and selecting an appropriate biocide to treat them. Optical epifluorescence and electronic microscopic observations of the rosy powder revealed a prevalent autofluorescent coccoid form with a diameter bigger than 5 microm. Chlorophylls a and b were extracted, suggesting the presence of cyanobacteria, a thesis subsequently confirmed by flow cytometry. Cultural media were inoculated with the rosy powder, and microorganisms grew as a green patina in phototrophic conditions and as a rosy patina when organic compounds were added to the mineral medium. The rosy discoloration was most likely caused by the presence of phycoerythrin. The sequencing of the cyanobacteria-specific polymerase chain reaction (PCR)-DGGE bands matched, with a similarity percentage >94, uncultured cyanobacteria, and the sequences were deposited in the GenBank under EU874241, EU874242, EU874243, EU874244, EU874245, EU874246, and EU874247. Finally, the efficiency of the two biocides Neo Desogen and Metatin 5810-101, both based on benzalkonium chloride, was evaluated using adenosine triphosphate measurements and PCR-based detection of cyanobacteria. Metatin, used in situ at 2% of the trade product, proved to be the better biocide, no cyanobacteria being detected after the Metatin treatment.

Electric field gradient focusing (EFGF) is a technique used to simultaneously separate and concentrate biomacromolecules, such as proteins, based on the opposing forces of an electric field gradient and a hydrodynamic flow. Recently, we reported EFGF devices fabricated completely from copolymers functionalized with poly(ethylene glycol), which display excellent resistance to protein adsorption. However, the previous devices did not provide the predicted linear electric field gradient and stable current. To improve performance, Tris-HCl buffer that was previously doped in the hydrogel was replaced with a phosphate buffer containing a salt (i.e., potassium chloride, KCl) with high mobility ions. The new devices exhibited stable current, good reproducibility, and a linear electric field distribution in agreement with the shaped gradient region design due to improved ion transport in the hydrogel. The field gradient was calculated based on theory to be approximately 5.76 V/cm(2) for R-phycoerythrin when the applied voltage was 500 V. The effect of EFGF separation channel dimensions was also investigated; a narrower focused band was achieved in a smaller diameter channel. The relationship between the bandwidth and channel diameter is consistent with theory. Three model proteins were resolved in an EFGF channel of this design. The improved device demonstrated 14,000-fold concentration of a protein sample (from 2 ng/mL to 27 microg/mL).

The indicator function of the fluorescence signals of the cyanopigments phycocyanin and phycoerythrin as early warning parameters against the microcystins in drinking water was investigated by lab- and pilot-scale studies. The early warning function of the fluorescence signals was examined with regard to the signals' real-time character, their sensitivity and the behaviour of the cyanopigments in different treatment stages in comparison to microcystins. Fluorescence measurements confirmed the real-time character, since they can be carried out on-site without the pre-concentration of pigments. The limit of detection of phycoerythrin is determined at 0.7 microg/L and of phycocyanin at 5.3 microg/L respectively. If the pigment/microcystin ratio is known and calculated to be higher than 1, very low microcystin concentrations can be estimated by the fluorescence signals. The compared behaviour of both pigments and selected microcystins (MC-LR and MC-RR) during water treatment shows that pigments have an early warning function against microcystins in conventional treatment stages using pre-oxidation with permanganate, powdered-activated carbon and chlorination. In contrast, cyanopigments do not have an early warning function if chlorine dioxide is used as a pre-oxidant or final disinfection agent. In order to use pigment control measurements in drinking water treatment the initial pigment/toxin ratio of the raw water must be known.

Pseudanabaena species are poorly known filamentous bloom-forming cyanobacteria closely related to Limnothrix. We isolated 28 Pseudanabaena strains from the Baltic Sea (BS) and the Albufera de Valencia (AV; Spain). By combining phenotypic and genotypic approaches, the phylogeny, diversity and evolutionary diversification of these isolates were explored. Analysis of the in vivo absorption spectra of the Pseudanabaena strains revealed two coexisting pigmentation phenotypes: (i) phycocyanin-rich (PC-rich) strains and (ii) strains containing both PC and phycoerythrin (PE). Strains of the latter phenotype were all capable of complementary chromatic adaptation (CCA). About 65 kb of the Pseudanabaena genomes were sequenced through a multilocus sequencing approach including the sequencing of the16 and 23S rRNA genes, the ribosomal intergenic spacer (IGS), internal transcribed spacer 1 (ITS-1), the cpcBA operon encoding PC and the IGS between cpcA and cpcB. In addition, the presence of nifH, one of the structural genes of nitrogenase, was investigated. Sequence analysis of ITS and cpcBA-IGS allowed the differentiation between Pseudanabaena isolates exhibiting high levels of microdiversity. This multilocus sequencing approach revealed specific clusters for the BS, the AV and a mixed cluster with strains from both ecosystems. The latter comprised exclusively CCA phenotypes. The phylogenies of the 16 and 23S rRNA genes are consistent, but analysis of other loci indicated the loss of substructure, suggesting that the recombination between these loci has occurred. Our preliminary results on population genetic analyses of the PC genes suggest an evolutionary diversification of Pseudanabaena through purifying selection.

An innovative nanoprobe-based device that can measure and adjust the pH, can mimic biochemistry, can create microscale vortices in water, and can be used to trap single molecules is presented. Because the analytes in question to trap and detect are small in dimensions, we start by presenting scaling issues and challenging limitations for miniaturized chemical nanosensors. Advantages of using nanoprobes e.g., isolated nanowires, as the components in chemical sensing are discussed. How the observation of the physical property can beneficially change with isomorphic scaling is highlighted. Some of the technology-related constrains are presented for specific sensors. Solutions to overcome such problems are also given. Different aspects, e.g., sample size and sensitivity, for chemical sensing at the nanoscale are highlighted.

Kleptoplasty is the retention of plastids obtained from ingested algal prey, which can remain temporarily functional and be used for photosynthesis by the predator. With a new approach based on cell cycle analysis, we have addressed the question of whether the toxic, bloom-forming dinoflagellate Dinophysis norvegica practice kleptoplasty or if they replicate their own plastid DNA. Dividing (G2) and non-dividing (G1) D. norvegica cells from a natural population were physically separated with a flow cytometer based on their DNA content. Average numbers of nuclear and plastid rDNA copies were quantified with real-time PCR both in the G1 and G2 group. Cells from the G1 group contained 5800 +/- 340 copies of nuclear rDNA and 1300 +/- 200 copies of plastid rDNA; cells from the G2 group contained 9700 +/- 58 copies of nuclear rDNA and 1400 +/- 220 copies of plastid rDNA (mean +/- SD, n = 3). The ratio G2/G1 in average rDNA copies per cell was 1.67 for nuclear DNA and 1.07 for plastid DNA. These ratios show that plastid acquisition in D. norvegica is either uncoupled with the cell cycle, or plastids accumulate rapidly in the beginning of the cell cycle owing to feeding, as would be expected in a protist with kleptoplastic behaviour but not in a protist with own plastid replication. In addition, flow cytometry measurements on cells from the same population used for real-time PCR showed that when kept without plastidic prey, live Dinophysis cells lost on average 36% of their plastid phycoerythrin fluorescence in 24 h. Together these findings strongly suggest that D. norvegica does not possess the ability for plastid replication.

A preliminary study was carried out on a picocyanobacterial mixed culture harvested from the Gulf of Trieste (Northern Adriatic) and identified as Synechococcus spp. both by transmission electron microscopy observations, biliprotein composition and molecular analyses. Absorption and fluorescence spectra revealed phycourobilin and phycoerythrobilin chromophores, suggesting the presence of both CU- and C-phycoerythrin, besides phycocyanobilin chromophores typical for phycocyanins and allophycocyanins. Both biliprotein analyses and molecular identification indicated the presence of at least two Synechococcus subgroups presumably differing either in phycoerythrin type or in physiological traits. Among the exoenzymatic activities acting on different substrates, only aminopeptidase showed high hydrolysis rates and the uptake of organic molecules was positive for leucine but not for thymidine. The protein carbon mobilized was high compared with the leucine incorporation rates, resulting in low percentages of newly mobilized carbon utilized by cultures. The organic carbon incorporated as leucine was compared with the photosynthetically produced one, and the balance between the phototrophic- and heterotrophic-like processes was c. 3 : 1. Our findings suggest that the Synechococcus heterotrophy plays an important role in cell's metabolism, and that the photoheterotrophic behavior, together with their chromatic adaptation capability, might represent the key for the absolute dominance of this genus in the Adriatic Sea.

Aims: To test Fountain Flow Cytometry (FFC) for the rapid and sensitive detection of Naegleria lovaniensis amoebae (an analogue for Naegleria fowleri) in natural river waters., Methods and Results: Samples were incubated with one of two fluorescent labels to facilitate detection: ChemChrome V6, a viability indicator, and an R-phycoerytherin (RPE) immunolabel to detect N. lovaniensis specifically. The resulting aqueous sample was passed as a stream in front of a light-emitting diode, which excited the fluorescent labels. The fluorescence was detected with a digital camera as the sample flowed toward the imager. Detections of N. lovaniensis were made in inoculated samples of natural water from eight rivers in France and the United States. FFC enumeration yielded results that are consistent with other counting methods: solid-phase cytometry, flow cytometry, and hemocytometry, down to concentrations of 0.06 amoebae ml(-1), using a flow rate of 15 ml min(-1)., Conclusions: This study supports the efficacy of using FFC for the detection of viable protozoa in natural waters and indicates that use of RPE illuminated at 530 nm and detected at 585 nm provides a satisfactory means of attenuating background., Significance and Impact of the Study: Because of the severe global public health issues with drinking water and sanitation, there is an urgent need to develop a technique for the real-time detection of viable pathogens in environmental samples at low concentrations. FFC addresses this need.

The performance of two homemade fluorescence-induced capillary electrophoresis detectors, one based on light-emitting diode (LED) as the excitation source and a charge-coupled device (CCD) photodetector and the other based on a commercial luminescence spectrometer (Xe lamp) as the excitation source and a photomultiplier tube as a detector, were compared for the determination of fluorescent proteins R-phycoerythrin and B-phycoerythrin. Both devices use commercially available, reasonably priced optical components that can be used by nonexperts. After fine optimization of several optical and separation parameters in both devices, a zone capillary electrophoresis methodology was achieved with 50mM borate buffer (pH 8.4) and 10mM phytic acid for the determination of two phycobiliproteins. Detection limits of 0.50 and 0.64microg/ml for R-phycoerythrin and B-phycoerythrin, respectively, were achieved by using the LED-induced fluorescence capillary electrophoresis (LED-IF-CE) system, and corresponding detection limits of 2.73 and 2.16microg/ml were achieved by using the Xe lamp-IF-CE system. Analytical performance and other parameters, such as cost and potential to miniaturization, are compared for both devices.

This paper reports both the experimental application and 2-D simulation of ITP of proteins in a networked microfluidic chip. Experiments demonstrate that a mixture of three fluorescent proteins can be concentrated and stacked into adjacent zones of pure protein under a constant voltage of 100 V over a 2 cm long microchannel. Measurements of the isotachophoretic velocity of the moving zones demonstrates that, during ITP under a constant voltage, the zone velocity decreases as more of the channel is occupied by the terminating electrolyte. A 2-D ITP model based on the Nernst-Planck equations illustrates the stacking and separation features of ITP using simulations of three virtual proteins. The self-sharpening behavior of ITP zones dispersed by a T-junction is clearly demonstrated both by experiment and by simulation. Comparison of 2-D simulations of ITP and zone electrophoresis (ZE) confirms that ZE lacks the ability to resharpen protein zones after they pass through a T-junction.

An integrated and simplified microfluidic device using a 250 microm x 1-4 cm of organic light emitting diode (OLED) array as a two-dimensional light source for single-channel and multichannel whole-column imaging detection was developed. This fluorescence detection system was used for isoelectric focusing (IEF) of R-phycoerythrin in a microchip. The IEF conditions were optimized, and the total analysis time was extremely reduced to 30 s for 2-cm-long microchannels at 700 V/cm of electric field strength without the presence of electroosmotic flow. The compression of pH gradient caused by electrolytes drawing into the microchannels was efficiently restrained when 1% hydroxylpropylmethyl cellulose in 2% ampholyte was used as the carrier for IEF. Under optimized IEF conditions, the detection limit of this system was approximately 0.6 microg/mL or 45 pg at 75 nL/column injection of R-phycoerythrin. This OLED-induced fluorescence detection system for WCID provides a high-speed IEF technique with quantitative ability and the potential for high integration and throughput microchip systems.

The influence of high light intensity on the growth and pigment accumulating ability of Anabaena azollae was investigated. A. azollae responded positively to high light intensity (6 klx) and was further evaluated at higher intensities (10 and 15 klx), in the presence of glucose, sucrose and jaggery +/- DCMU. Significant enhancement in phycobiliproteins and carotenoids was observed in the sugar supplemented cultures at high light intensities. SDS-PAGE profiles of whole cell proteins revealed the presence of unique bands in such treatments. Sucrose supplementation induced a 30-90 % increase in carotenoids, phycocyanin and phycoerythrin content at 10 klx. Molecular analysis of the stimulatory and interactive role of sugars on pigment enhancement at high light intensity may aid in better exploitation of cyanobacteria as a source of pigments.

Introduction: Blue-green 488-nm laser sources are widespread in flow cytometry but suffer some drawbacks for cell analysis, including their excitation of endogenous proteins (resulting in high cellular autofluorescence) and their less-than-optimal coincidence with the excitation maxima of commonly used fluorochromes, including the phycoerythrins (PE). Longer wavelength lasers such as green helium-neons and, more recently, diode-pumped solid state (DPSS) 532-nm sources have previously been employed to overcome these difficulties and improve overall sensitivity for PE. In this study, we evaluate an even longer wavelength DPSS 561-nm for its ability to improve PE and DsRed fluorescent protein detection sensitivity., Methods: A DPSS 561-nm laser emitting at 10 mW was mounted onto a BD LSR II. Mouse thymoma cells labeled with cell surface marker antibodies conjugated to the R- and B-forms of PE were analyzed and compared with conventional 488-nm excitation using the same bandpass filters and signal travel distances. A similar analysis was carried out with cell lines expressing the red fluorescent protein DsRed, several green-yellow excited low molecular weight fluorochromes, and a rhodamine-based caspase substrate. Additionally, cells labeled with PE and co-labeled with fluorescein or simultaneously expressing green fluorescent protein (GFP) were analyzed to determine if PE excitation at 561 nm with simultaneous fluorescein/GFP detection was feasible., Results: The DPSS 561-nm laser gave a several-fold improvement in the fluorochrome to autofluorescence ratios between PE-labeled cells and unlabeled controls. Analysis of cells expressing the fluorescent protein DsRed with the DPSS 561-nm source gave a 6-7-fold improvement in sensitivity over 488-nm excitation, and gave excellent excitation of yellow-green excited fluorochromes and rhodamine-based physiological probes. Yellow-green laser light also caused virtually no impingement on the spatially separated fluorescein/GFP detector, a significant problem with green laser sources, and also allowed simultaneous analysis of GFP and PE with virtually no signal overlap or requirement for color compensation., Conclusions: DPSS 561-nm laser excitation gave significantly improved sensitivity for both PE-labeled and DsRed expressing cells, with little contamination of a typical fluorescein/GFP detector.

A high-throughput method for measuring single-cell fluorescence spectra is presented. Upon excitation with a 488 nm argon-ion laser many bacterial cells were imaged by a 20x microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grating, and an intensified charge-coupled device (ICCD) camera simultaneously recorded the zero and the first orders of the fluorescence from each cell. Single-cell fluorescence spectra were reconstructed from the distance between zero-order and first-order maxima as well as the length and the pixel intensity distribution of the first-order images. By using this approach, the emission spectrum of E. coli cells expressing green fluorescent protein (GFP) was reconstructed. Also, fluorescence spectra of E. coli cells expressing non-fluorescent apo-subunits of R-phycoerythrin (R-PE) were recorded after incubation of the cells with phycoerythrobilin (PEB) chromophore. The fluorescence spectra are in good agreement with results obtained on the same cells using a fluorescence spectrometer or a fluorescence microscope. When spectra are to be acquired, this approach has a higher throughput, better sensitivity, and better spectral resolution compared to flow cytometry.

The dazzling diversity of the phytoplankton has puzzled biologists for decades. The puzzle has been enlarged rather than solved by the progressive discovery of new phototrophic microorganisms in the oceans, including picocyanobacteria, pico-eukaryotes, and bacteriochlorophyll-based and rhodopsin-based phototrophic bacteria. Physiological and genomic studies suggest that natural selection promotes niche differentiation among these phototrophic microorganisms, particularly with respect to their photosynthetic characteristics. We have analysed competition for light between two closely related picocyanobacteria of the Synechococcus group that we isolated from the Baltic Sea. One of these two has a red colour because it contains the pigment phycoerythrin, whereas the other is blue-green because it contains high contents of the pigment phycocyanin. Here we report theory and competition experiments that reveal stable coexistence of the two picocyanobacteria, owing to partitioning of the light spectrum. Further competition experiments with a third marine cyanobacterium, capable of adapting its pigment composition, show that this species persists by investing in the pigment that absorbs the colour not used by its competitors. These results demonstrate the adaptive significance of divergence in pigment composition of phototrophic microorganisms, which allows an efficient utilization of light energy and favours species coexistence.

Ozone, the main component of photochemical smog and air pollution, can damage the skin by oxidizing stratum corneum enzymes, lipids and structural proteins. We have developed a rapid screening assay to determine free radical scavenging capacity of various active ingredients that are frequently used in personal care products. Several known antioxidants including vitamin C, vitamin E analog Trolox, walnut seed extract, lipoic acid and ergothioneine inner salt were assayed for their ability to neutralize ozone-induced oxidation of beta-phycoerythrin, a fluorescent reporter protein derived from algae. The free radical scavenging capacities of these antioxidants were quantified and compared. The results demonstrate that this assay is a valuable primary screening tool for identifying antioxidant activity of natural or synthetic substrates that can be used in personal care products to protect the uppermost layer of our skin from oxidizing damage induced by O3.

Flow cytometric studies suggest that platelets are activated in ischaemic stroke or transient ischaemic attack (TIA). However, few studies have measured circulating leucocyte-platelet complexes in this patient population. Whole blood flow cytometry was used to quantify the expression of CD62P-, CD63-, and PAC1-binding, and the percentages of leucocyte-platelet complexes in acute (1-27 d, n = 79) and convalescent (79-725 d, n = 70) ischaemic cerebrovascular disease (CVD) patients compared with controls without CVD (n = 27). We performed a full blood count, and measured plasma levels of soluble P-selectin, soluble E-selectin, and von Willebrand factor antigen (VWF:Ag) as additional markers of platelet and/or endothelial cell activation. The median percentage CD62P expression and the median percentage monocyte-platelet complexes were higher in both acute and convalescent CVD patients than controls (P

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system is designed to provide early warning to civilians in the event of a terrorist attack. The final APDS will be completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. The system performance (current capabilities include aerosol collection, multiplexed immunoassays, sample archiving, data reporting, and alarming) was evaluated in a field test conducted in a Biosafety Level 3 facility, where the system was challenged with, and detected, a series of aerosolized releases containing two live, virulent biological threat agents (Bacillus anthracis and Yersinia pestis). Results presented here represent the first autonomous, simultaneous measurement of these agents.

As well as classically defined switched immunoglobulin isotype-expressing B cells, memory B cells are now thought to include IgM-expressing cells and memory cells that lack B cell lineage markers, such as B220 or CD19. We set out to compare the relative importance of memory B cell subsets with an established flow cytometry method to identify antigen-specific cells. After immunization with PE, we could detect B220+ and, as reported previously, B220- antigen-binding cells (McHeyzer-Williams, L.J., M. Cool, and M.G. McHeyzer-Williams. 2001. J. Immunol. 167:1393-1405). The B220-PE+ cells bore few markers typical of B cells, but resembled myeloid cells. Further analysis of the antigen-binding characteristics of these cells showed that, upon immunization with two fluorescent proteins, the B220- cells could bind both. Furthermore, this subpopulation was detected in RAG1-/- mice after transfer of anti-PE mouse serum. These data strongly suggest that these cells capture serum Ig, via Fc receptors, and thus appear antigen-specific. Investigation of these antigen-capturing cells in a variety of knockout mice indicates that they bind monomeric IgG in an FcgammaR1 (CD64)-dependent manner. We find no evidence of a B220- memory B cell population that is not explicable by antigen-capturing cells, and warn that care must be taken when using antigen-specificity or surface IgG as an indicator of B cell memory.

The algologically pure cultures of the green-brown cyanobacterium Chroococcidiopsis sp. and three cyanobacteria of the genus Gloeocapsa, the blue-green Gloeocapsa sp.1, the brown Gloeocapsa sp.2, and the red-orange Gloeocapsa sp.3, were isolated from sandstones and rock fissures in the high-polar regions of Antarctica. These cyanobacteria are the most widespread phycobionts of cryptoendolithic lichens in these regions. The comparative analysis of the absorption and the second-derivative absorption spectra of the cyanobacteria revealed considerable differences in the content of chlorophyll a and in the content and composition of carotenoids and phycobiliproteins. In addition to phycocyanin, allophycocyanin, and allophycocyanin B, which were present in all of the cyanobacteria studied, Gloeocapsa sp.2 also contained phycoerythrocyanin and Gloeocapsa sp.3 phycoerythrocyanin and C-phycoerythrin (the latter pigment is typical of nitrogen-fixing cyanobacteria). The fluorescence spectra of Gloeocapsa sp.2 and Gloeocapsa sp.3 considerably differed from the fluorescence spectra of the other cyanobacteria as well. The data obtained suggest that various zones of the lichens may be dominated either by photoheterotrophic or photoautotrophic cyanobacterial phycobionts, which differ in the content and composition of photosynthetic pigments.

Isolation of cyanobacteria was attempted from herbicide applied rice soils. The predominant genera were Westiellopsis followed by Anabaena, Nostoc and Oscillatoria. The herbicide tolerance was further tested by growing the cyanobacterial cultures in BG-11 medium supplemented with varying concentrations of the commonly used rice herbicide, viz butachlor under in vitro condition. The chlorophyll-a, phycobiliproteins and ammonia excretion were assessed at periodic intervals. Westiellopsis showed the maximum tolerance followed by Anabaena, Nostoc and Oscillatoria.

Background: Immunotoxicological studies in humans are usually carried out via the determination of some selected immune parameters in subjects occupationally and/or environmentally exposed to immunotoxic substance. One of the most often measured parameters is the determination of lymphocyte subsets, which needs to be carried out in a very short time (a few hours) after blood collection. This is the major problem limiting the determination of lymphocyte subpopulations in field studies, where samples are usually collected directly at the workplace, and very often at the end of the workshift. Unfortunately, these collection modalities significantly prolong the time between collection and analysis. The problem is more evident in multicentric studies, where a further problem is represented by the time needed to send samples to the laboratory., Objective: Since an immune evaluation was planned, including the determination of lymphocyte subpopulations CD4 (T-helper), CD8 (T-suppressor cytotoxic) and CD16/CD56 (natural killer) in the project "Assessing health effects in man from exposure to low doses of inorganic mercury in environmental and occupational settings", a method was developed for performing cytofluorimetric analysis in "field studies"., Methods: The method is based on commercially-available kits, and involves in loco treatment. Whole blood is labeled with monoclonal antibodies, and fixed samples immediately after collection. After the treatment, the samples are ready for flow cytometric analysis, which may be performed after a two-day period from sample collection., Results and Conclusion: The method described is adequate for immunotoxicity testing in field studies because it prolongs the maximum latency time from collection and cytofluorimetric analysis up to 48 hours. A second interesting characteristic of the method is the possibility of using whole blood, without any need of either complex manipulations or particular equipment.

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.

Effects of nitrogen limitation on Photosystem II (PSII) activities and on phycoerythrin were studied in batch cultures of the marine oxyphotobacterium Prochlorococcus marinus. Dramatic decreases in photochemical quantum yields (F(V)/F(M)), the amplitude of thermoluminescence (TL) B-band, and the rate of Q(A) reoxidation were observed within 12 h of growth in nitrogen-limited conditions. The decline in F(V)/F(M) paralleled changes in the TL B-band amplitude, indicative of losses in PSII activities and formation of non-functional PSII centers. These changes were accompanied by a continuous reduction in D1 protein content. In contrast, nitrogen deprivation did not cause any significant reduction in phycoerythrin content. Our results refute phycoerythrin as a nitrogen storage complex in Prochlorococcus. Regulation of phycoerythrin gene expression in Prochlorococcus is different from that in typical phycobilisome-containing cyanobacteria and eukaryotic algae investigated so far.

The formal description of Prochlorococcus marinus Chisholm et al. 1992, 299 was based on the non-axenic nomenclatural type, strain CCMP 1375T. The purification and properties of the axenic strain PCC 9511, derived from the same primary culture (SARG) as the type species, are reported here. Prochlorococcus PCC 9511 differs from the latter in possessing horseshoe-shaped thylakoids, exhibiting a low chlorophyll b2 content and lacking phycoerythrin, but shares these phenotypic properties with Prochlorococcus strain CCMP 1378. This relationship was confirmed by 16S rRNA sequence analyses, which clearly demonstrated that the axenic isolate is not co-identic with the nomenclatural type. Strain PCC 9511 has a low mean DNA base composition (32 mol% G+C) and harbours the smallest genome of all known oxyphotobacteria (genome complexity 1.3 GDa = 2 Mbp). Urea and ammonia are the preferred sources of nitrogen for growth, whereas nitrate is not utilized. Several different organic phosphorus compounds efficiently replace phosphate in the culture medium, indicative of ecto-phosphohydrolase activity. In order to distinguish strain PCC 9511 from the nomenclatural type, a new subspecies is proposed, Prochlorococcus marinus Chisholm et al. 1992 subsp. pastoris subsp. nov.