The development of logic gate-based fluorescent probes that respond to intracellular hydrogen peroxide and pH in tandem.

Logic gate-based fluorescent probes are powerful tools for the discriminative sensing of multiple signaling molecules that are expressed in concert during the progression of many diseases such as inflammation, cancer, aging, and other disorders. To achieve logical sensing, multiple functional groups are introduced to the different substitution sites of a single fluorescent dye, which increases the complexity of chemical synthesis. Herein, we report a simple strategy that incorporates just one responsive unit into a hemicyanine dye achieving the logic gate-based sensing of two independent analytes. We introduce boronic acid to hemicyanine to quench the fluorescence, and in the presence of hydrogen peroxide (H2O2), the fluorescence is recovered due to removal of the boronate. Interestingly, the subsequent decrease in pH turned the red fluorescence of hemicyanine to green emissive because of protonation of the phenolic alcohol. This unique feature of the probe enables us to construct "INHIBIT" and "AND" logical gates for the accurate measuring of intracellular H2O2 and acidic pH in tandem. This study offers insight into the simple construction of logic-gate based fluorescent probes for the tandem sensing of multiple analytes that are correlatively produced during disease progression.

Physiological responses of the diatoms Thalassiosira weissflogii and Thalassiosira pseudonana to nitrogen starvation and high light.

As oceans warm, the depth of the upper mixed layer is predicted to decrease, resulting in insufficient nutrient supply and higher solar radiation for phytoplankton. In order to understand the photophysiological responses of the key eukaryotic phytoplankton diatoms to high light and nutrient limitation, we grew two diatoms, Thalassiosira weissflogii and Thalassiosira pseudonana under N starvation conditions and exposed them to high visible light. It showed that the large-sized diatom T. weissflogii can maintain photosynthetic activity for a longer period of time under nitrogen starvation as compared with the small-sized diatom T. pseudonana. The electron transfer reaction was inhibited in both diatoms and the fast closing of reaction centers promoted the development of QB non-reducing PSII centers, thus facilitated the rapid induction of NPQ, however, the induction of NPQ depended on the degree of N starvation. N starvation exacerbated the photoinhibition caused by high light. The smaller-sized T. pseudonana had a higher σi value and was more sensitive to high-light, but its PSII repair rate was also higher. In contrast, T. weissflogii was more tolerant to high light with a lower σi value, but the tolerance was severely reduced under N-starvation. This study provides helpful insight into how climate change variables impact diatom's photosynthetic physiology.

Complete mitochondrial genome of the sea slug, Placida sp., contains unusual noncoding regions.

The complete sequence (14,751 bp) of the mitochondrial DNA (mtDNA) of the opisthobranch gastropod Placida sp. was determined using long PCR and genome-walking techniques. The genome contains 13 protein-coding genes (PCG), 2 ribosomal RNA (rRNA), 22 transfer RNA (tRNA) genes that are typical of metazoan mtDNA, and 2 lengthy noncoding regions with a total length of 1441 bp. All the tRNA genes have general secondary structures without reduced T or D stems. The mitochondrial gene arrangement of Placida sp. is almost identical to that of sea slugs, whereas the tRNA gene arrangement is different from other animals outside Sacoglossa in the Euthyneura. A phylogenetic analysis based on the mitochondrial genome of Euthyneura indicated that Placida sp. and Placida dendritica are closely related to Elysia chlorotica as a sister taxon, which strongly supports their close phylogenetic affinity.

Immobilization of Chlorella sorokiniana GXNN 01 in alginate for removal of N and P from synthetic wastewater.

High costs and issues such as a high cell concentrations in effluents are encountered when utilizing microalgae for wastewater treatment. The present study analyzed nitrogen and phosphate removal under autotrophic, heterotrophic, mixotrophic and micro-aerobic conditions by Chlorella sorokiniana GXNN 01 immobilized in calcium alginate. The immobilized cells grew as well as free-living cells under micro-aerobic conditions and better than free-living cells under the other conditions. The immobilized cells had a higher ammonium removal rate (21.84%, 43.59% and 41.46%) than free living cells (14.35%, 38.57% and 40.59%) under autotrophic, heterotrophic, and micro-aerobic conditions, and higher phosphate removal rate (87.49%, 88.65% and 84.84%) than free living cells (20.21%, 42.27% and 53.52%) under heterotrophic, mixotrophic and micro-aerobic conditions, respectively. The data indicate that immobilized Chlorella sorokiniana GXNN 01 is a suitable species for use in wastewater treatment.

Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus.

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca(2+) binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln(122)-Pro(123)-Asn(124)) in LvLectin-1, but QPD (Gln(128)-Pro(129)-Asp(130)) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P<0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P<0.01) and 12 h (P<0.05), but the expression level of LvLectin-1 down-regulated significantly (P<0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus.

SHORT-TERM EFFECTS OF ACETATE AND MICROAEROBIC CONDITIONS ON PHOTOSYNTHESIS AND RESPIRATION IN CHLORELLA SOROKINIANA GXNN 01 (CHLOROPHYTA)(1).

The culture of microalgae using organic carbon sources decreases the cost of operation in closed systems. The effect of carbon sources on microalgae is thus an interesting problem in not only theoretical research but also practical production. The short-term effects of acetate and microaerobic conditions on the growth, photosynthesis, and respiration of the green microalga Chlorella sorokiniana I. Shihira & R.W. Krauss GXNN 01 were described after acetate addition to autotrophic cultures. As the acetate concentration increased, cells needed a longer lag phase to grow, and 243.8 mM acetate completely inhibited growth. Acetate addition induced an immediate response in photosynthesis and respiration. The activity of PS II and PS I were impaired and declined with different rates, and then recovered compared with autotrophic cells. Carbonic anhydrase and Rubisco activities were also inhibited at the beginning, and respiration was increased. We propose that ATP consumption for acetate assimilation results in surplus NADPH, and then accumulated reducing power over-reduces inter-photosystem components and raises the transthylakoid proton gradient, which redistributes energy between PS I and PS II, and leads to a decrease in the PS II/PS I ratio and O2 evolution. An apparent cyclic electron flow was also observed, which may be mainly mediated by NAD(P)H dehydrogenase-dependent pathway since NADPH was in excess. These observations pointed to an acclimation process after acetate addition, and suggested the interaction between photosynthesis and respiration involving ATP and reducing power.

ASSESSMENT OF PHOTOSYNTHETIC PERFORMANCE OF PORPHYRA YEZOENSIS (BANGIALES, RHODOPHYTA) IN CONCHOCELIS PHASE(1).

Photosynthetic characteristics of four Porphyra yezoensis Ueda [a taxonomic synonym of Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi] strains in conchocelis phase were investigated and compared with one wildtype of P. yezoensis and two strains of Porphyra haitanensis T. J. Chang et B. F. Zheng [a taxonomic synonym of Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata]. Results showed that experimental strains had higher contents of chl a and carotenoids, but a lower content of total phycobiliproteins than the wildtype. Meanwhile, photochemical efficiency of PSII was measured using pulse amplitude modulation (PAM) fluorometry technology. The value of PSII photosynthetic parameters of P. yezoensis strains were all higher than the wild strain, and the maximal quantum yields (Fv /Fm ), effective quantum yields Y(II), and relative photosynthetic electron transport rates (rETR) of P. haitanensis were higher than those of P. yezoensis. The present study verified the possibility of selective breeding of P. yezoensis using the filamentous sporophyte instead of the gametophytic thallus, the advantages being (i) nonrequirement of control of life cycle and (ii) direct and rapid cultivar improvement by artificial selection. We consider the method to be a promising technique for selective breeding of P. yezoensis cultivars.

Molecular cloning and mRNA expression of two peptidoglycan recognition protein (PGRP) genes from mollusk Solen grandis.

Peptidoglycan recognition proteins (PGRPs) play crucial role in innate immunity for both invertebrates and vertebrates, owing to their prominent ability in detecting and eliminating invading bacteria. In the present study, two short PGRPs from mollusk Solen grandis (designated as SgPGRP-S1 and SgPGRP-S2) were identified, and their expression patterns, both in tissues and toward three PAMPs stimulation, were then characterized. The full-length cDNA of SgPGRP-S1 and SgPGRP-S2 was 1672 and 1285 bp, containing an open reading frame (ORF) of 813 and 426 bp, respectively, and deduced amino acid sequences showed high similarity to other members of PGRP superfamily. Both SgPGRP-S1 and SgPGRP-S2 encoded a PGRP domain. The motif of Zn(2+) binding sites and amidase catalytic sites were well conserved in SgPGRP-S1, but partially conserved in SgPGRP-S2. The two PGRPs exhibited different tissue expression pattern. SgPGRP-S1 was highly expressed in muscle and hepatopancreas, while SgPGRP-S2 was highly in gill and mantle. The mRNA expression of SgPGRP-S1 could be induced acutely by stimulation of PGN, and also moderately by ß-1,3-glucan, but not by LPS, while expression of SgPGRP-S2 was significantly up-regulated (P < 0.01) when S. grandis was stimulated by all the three PAMPs, though the expression levels were relatively lower than SgPGRP-S1. Our results suggested SgPGRP-S1 and SgPGRP-S2 could serve as pattern recognition receptors (PRRs) involved in the immune recognition of S. grandis, and they might perform different functions in the immune defense against invaders.

Comparison of chlorophyll and photosynthesis parameters of floating and attached Ulva prolifera.

In mid-May 2008 a serious green tide caused mainly by floating Ulva prolifera (Müller) J. Agardh (Chlorophyta, Ulvales) thalli struck the coastal area of Qingdao, China. To understand the present physiological conditions of the floating alga, in this work both laboratory and field investigations were conducted on the floating U. prolifera thalli in comparison with the attached U. prolifera thalli collected from the area. The floating thalli of three distinctively different colors and attached thalli at three different stages of sporangium formation process were characterized under a microscope, while their photosynthetic parameters were determined with chlorophyll fluorescence technology. On the other hand, the sporangium formation status of the floating U. prolifera thalli was surveyed both in the laboratory and in the field. Comparisons showed that both of the paired morphological characteristics and the paired physiological parameters of the floating and attached U. prolifera thalli were consistent. Furthermore, some spores were confirmed in the field and some motile particles were found within the floating thalli. These results suggest that the floating U. prolifera thalli with different colors could be at different stages of sporangium formation. However, our results also showed that the floating alga thalli have only a limited reproductive potential. This might limit the duration and the further geographic expansion of the green algal bloom.

ISOLATION AND CHARACTERIZATION OF CHLORELLA SOROKINIANA GXNN01 (CHLOROPHYTA) WITH THE PROPERTIES OF HETEROTROPHIC AND MICROAEROBIC GROWTH(1).

Heterotrophic and anaerobic microalgae are of significance in both basic research and industrial application. A microalga strain was isolated from a wastewater treatment pond and identified as Chlorella sorokiniana Shihira et W. R. Krauss GXNN01 in terms of morphology, physiology, and phylogeny. The strain grows rapidly in heterotrophic or mixotrophic conditions with addition of various carbon sources, and even in anaerobic conditions. The maximum growth rate reached 0.28 d(-1) when using d,l-malate as the carbon source, and the protein content of the microalgae was 75.32% in cell dry weight. The strain was shown to be capable of (1) utilizing d,l-malate only with light, (2) inhibiting photosynthesis in mixotrophic growth, and (3) growing in anaerobic conditions with regular photosynthesis and producing oxygen internally. This study demonstrates the influence of oxygen (aerobic vs. anaerobic) and metabolic regime (autotrophy, mixotrophy, heterotrophy) on the physiological state of the cell.