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1.  Phenotypic and genetic characterization of Dunaliella (Chlorophyta) from Indian salinas and their diversity 
Aquatic Biosystems  2012;8:27.
Background
The genus Dunaliella (Class – Chlorophyceae) is widely studied for its tolerance to extreme habitat conditions, physiological aspects and many biotechnological applications, such as a source of carotenoids and many other bioactive compounds. Biochemical and molecular characterization is very much essential to fully explore the properties and possibilities of the new isolates of Dunaliella. In India, hyper saline lakes and salt pans were reported to bloom with Dunaliella spp. However, except for the economically important D. salina, other species are rarely characterized taxonomically from India. Present study was conducted to describe Dunaliella strains from Indian salinas using a combined morphological, physiological and molecular approach with an aim to have a better understanding on the taxonomy and diversity of this genus from India.
Results
Comparative phenotypic and genetic studies revealed high level of diversity within the Indian Dunaliella isolates. Species level identification using morphological characteristics clearly delineated two strains of D. salina with considerable β-carotene content (>20 pg/cell). The variation in 18S rRNA gene size, amplified with MA1-MA2 primers, ranged between ~1800 and ~2650 base pairs, and together with the phylogeny based on ITS gene sequence provided a pattern, forming five different groups within Indian Dunaliella isolates. Superficial congruency was observed between ITS and rbcL gene phylogenetic trees with consistent formation of major clades separating Indian isolates into two distinct clusters, one with D. salina and allied strains, and another one with D. viridis and allied strains. Further in both the trees, few isolates showed high level of genetic divergence than reported previously for Dunaliella spp. This indicates the scope of more numbers of clearly defined/unidentified species/sub-species within Indian Dunaliella isolates.
Conclusion
Present work illustrates Indian Dunaliella strains phenotypically and genetically, and confirms the presence of not less than five different species (or sub-species) in Indian saline waters, including D. salina and D. viridis. The study emphasizes the need for a combined morphological, physiological and molecular approach in the taxonomic studies of Dunaliella.
doi:10.1186/2046-9063-8-27
PMCID: PMC3598838  PMID: 23114277
Dunaliella; Diversity; India; 18S rDNA; ITS; rbcL gene
2.  The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA 
BMC Plant Biology  2010;10:83.
Background
Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri.
Results
The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA.
Conclusions
These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable plastid transformation system for this model alga, and they will complement the forthcoming D. salina nuclear genome sequence, placing D. salina in a group of a select few photosynthetic eukaryotes for which complete genome sequences from all three genetic compartments are available.
doi:10.1186/1471-2229-10-83
PMCID: PMC3017802  PMID: 20459666
3.  The Selectivity of Milking of Dunaliella salina 
The process of the simultaneous production and extraction of carotenoids, milking, of Dunaliella salina was studied. We would like to know the selectivity of this process. Could all the carotenoids produced be extracted? And would it be possible to vary the profile of the produced carotenoids and, consequently, influence the type of carotenoids extracted? By using three different D. salina strains and three different stress conditions, we varied the profiles of the carotenoids produced. Between Dunaliella bardawil and D. salina 19/18, no remarkable differences were seen in the extraction profiles, although D. salina 19/18 seemed to be better extractable. D. salina 19/25 was not “milkable” at all. The milking process could only be called selective for secondary carotenoids in case gentle mixing was used. In aerated flat-panel photobioreactors, extraction was much better, but selectiveness decreased and also chlorophyll and primary carotenoids were extracted. This was possibly related to cell damage due to shear stress.
doi:10.1007/s10126-009-9195-0
PMCID: PMC2816252  PMID: 19475448
Dunaliella salina; Carotenoids; Extraction; Selectivity; Stress conditions
4.  Oxidative Stress Is a Mediator for Increased Lipid Accumulation in a Newly Isolated Dunaliella salina Strain 
PLoS ONE  2014;9(3):e91957.
Green algae offer sustainable, clean and eco-friendly energy resource. However, production efficiency needs to be improved. Increasing cellular lipid levels by nitrogen depletion is one of the most studied strategies. Despite this, the underlying physiological and biochemical mechanisms of this response have not been well defined. Algae species adapted to hypersaline conditions can be cultivated in salty waters which are not useful for agriculture or consumption. Due to their inherent extreme cultivation conditions, use of hypersaline algae species is better suited for avoiding culture contamination issues. In this study, we identified a new halophilic Dunaliella salina strain by using 18S ribosomal RNA gene sequencing. We found that growth and biomass productivities of this strain were directly related to nitrogen levels, as the highest biomass concentration under 0.05 mM or 5 mM nitrogen regimes were 495 mg/l and 1409 mg/l, respectively. We also confirmed that nitrogen limitation increased cellular lipid content up to 35% under 0.05 mM nitrogen concentration. In order to gain insight into the mechanisms of this phenomenon, we applied fluorometric, flow cytometric and spectrophotometric methods to measure oxidative stress and enzymatic defence mechanisms. Under nitrogen depleted cultivation conditions, we observed increased lipid peroxidation by measuring an important oxidative stress marker, malondialdehyde and enhanced activation of catalase, ascorbate peroxidase and superoxide dismutase antioxidant enzymes. These observations indicated that oxidative stress is accompanied by increased lipid content in the green alga. In addition, we also showed that at optimum cultivation conditions, inducing oxidative stress by application of exogenous H2O2 leads to increased cellular lipid content up to 44% when compared with non-treated control groups. Our results support that oxidative stress and lipid overproduction are linked. Importantly, these results also suggest that oxidative stress mediates lipid accumulation. Understanding such relationships may provide guidance for efficient production of algal biodiesels.
doi:10.1371/journal.pone.0091957
PMCID: PMC3961284  PMID: 24651514
5.  DNA fingerprinting differentiation between β-carotene hyperproducer strains of Dunaliella from around the world 
Saline Systems  2009;5:5.
Background
Dunaliella salina is the most important species of the genus for β-carotene production. Several investigations have demonstrated that D. salina produces more than 10% dry weight of pigment and that the species grows in salt saturated lagoons. High plasticity in the green stage and the almost indistinguishable differences in the red phase make identification and differentiation of species and ecotypes very difficult and time consuming.
Results
In this work, we applied our intron-sizing method to compare the 18S rDNA fingerprint between D. salina (CCAP 19/18), D. salina/bardawil (UTEX LB2538) and β-carotene hyperproducing strains of Dunaliella isolated from salt saturated lagoons in Baja, Mexico. All hyperproducer strains reached β-carotene levels of about 10 pg/cell. Optical microscopy did not allow to differentiate between these Dunaliella strains; however, 18S rDNA fingerprinting methodology allowed us to differentiate D. salina from D. salina/bardawil.
Conclusion
In Baja Mexico we found D. salina and D. salina/bardawil species by using intron-sizing-method. The National Center for Biotechnology Information (NCBI) Dunaliella 18S rDNA gene sequences were analyzed with our methodology and extraordinary correlation was found with experimental results.
doi:10.1186/1746-1448-5-5
PMCID: PMC2710335  PMID: 19563682
6.  DsHsp90 Is Involved in the Early Response of Dunaliella salina to Environmental Stress† 
Heat shock protein 90 (Hsp90) is a molecular chaperone highly conserved across the species from prokaryotes to eukaryotes. Hsp90 is essential for cell viability under all growth conditions and is proposed to act as a hub of the signaling network and protein homeostasis of the eukaryotic cells. By interacting with various client proteins, Hsp90 is involved in diverse physiological processes such as signal transduction, cell mobility, heat shock response and osmotic stress response. In this research, we cloned the dshsp90 gene encoding a polypeptide composed of 696 amino acids from the halotolerant unicellular green algae Dunaliella salina. Sequence alignment indicated that DsHsp90 belonged to the cytosolic Hsp90A family. Further biophysical and biochemical studies of the recombinant protein revealed that DsHsp90 possessed ATPase activity and existed as a dimer with similar percentages of secondary structures to those well-studied Hsp90As. Analysis of the nucleotide sequence of the cloned genomic DNA fragment indicated that dshsp90 contained 21 exons interrupted by 20 introns, which is much more complicated than the other plant hsp90 genes. The promoter region of dshsp90 contained putative cis-acting stress responsive elements and binding sites of transcriptional factors that respond to heat shock and salt stress. Further experimental research confirmed that dshsp90 was upregulated quickly by heat and salt shock in the D. salina cells. These findings suggested that dshsp90 might serve as a component of the early response system of the D. salina cells against environmental stresses.
doi:10.3390/ijms13077963
PMCID: PMC3430215  PMID: 22942684
Dunaliella salina; gene structure; haloadaption; heat shock; Hsp90; osmotic stress; structural feature
7.  The Carotenogenesis Pathway via the Isoprenoid-β-carotene Interference Approach in a New Strain of Dunaliella salina Isolated from Baja California Mexico 
Marine Drugs  2009;7(1):45-56.
D. salina is one of the recognized natural sources to produce β-carotene, and an useful model for studying the role of inhibitors and enhancers of carotenogenesis. However there is little information in D. salina regarding whether the isoprenoid substrate can be influenced by stress factors (carotenogenic) or selective inhibitors which in turn may further contribute to elucidate the early steps of carotenogenesis and biosynthesis of β-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-β-carotene interference in order to promote the interruption or accumulation of the programmed biosynthesis of carotenoids. When Carotenogenic and non-carotenogenic cells of D. salina BC02 were grown under photoautotrophic growth conditions in the presence of 200 µM fosmidomycin, carotenogenesis and the synthesis of β-carotene were interrupted after two days in cultured D. salina cells. This result is an indirect consequence of the inhibition of the synthesis of isoprenoids and activity of the recombinant DXR enzyme thereby preventing the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol (MEP) and consequently interrupts the early steps of carotenogenesis in D. salina. The effect at the level of proteins and RNA was not evident. Mevinolin treated D. salina cells exhibited carotenogenesis and β-carotene levels very similar to those of control cell cultures indicating that mevinolin not pursued any indirect action in the biosynthesis of isoprenoids and had no effect at the level of the HMG-CoA reductase, the key enzyme of the Ac/MVA pathway.
doi:10.3390/md7010045
PMCID: PMC2666888  PMID: 19370170
bioactive compounds
8.  Molecular Fingerprinting of Fish-Pathogenic Lactococcus garvieae Strains by Random Amplified Polymorphic DNA Analysis 
Journal of Clinical Microbiology  2003;41(2):751-756.
In this work, we used the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Lactococcus garvieae, an important pathogen for fish. Fifty-seven strains with different hosts and geographical origins, including Japan and several countries of the Mediterranean area such as Spain, Portugal, France, Italy, England, and Turkey, were analyzed. Two primers, oligonucleotides 5 and 6 (Pharmacia Biotech) were utilized; primer 5 was the most discriminative, since allowed us to differentiate 10 RAPD -types related to the origin of the strains. Regardless of the oligonucleotide primer employed, the 57 isolates of L. garvieae studied were separated into three genetic groups, composed of the Spanish, Portuguese, English, and Turkish strains (group A), the Italian and French strains (group B), and the Japanese strains (group C). The similarity of isolates within each group, estimated on the basis of the Dice coefficient, ranged from 75 to 100%. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.
doi:10.1128/JCM.41.2.751-756.2003
PMCID: PMC149703  PMID: 12574277
9.  Complete genome sequence of Halomicrobium mukohataei type strain (arg-2T) 
Standards in Genomic Sciences  2009;1(3):270-277.
Halomicrobium mukohataei (Ihara et al. 1997) Oren et al. 2002 is the type species of the genus Halomicrobium. It is of phylogenetic interest because of its isolated location within the large euryarchaeal family Halobacteriaceae. H. mukohataei is an extreme halophile that grows essentially aerobically, but can also grow anaerobically under a change of morphology and with nitrate as electron acceptor. The strain, whose genome is described in this report, is a free-living, motile, Gram-negative euryarchaeon, originally isolated from Salinas Grandes in Jujuy, Andes highlands, Argentina. Its genome contains three genes for the 16S rRNA that differ from each other by up to 9%. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence from the poorly populated genus Halomicrobium, and the 3,332,349 bp long genome (chromosome and one plasmid) with its 3416 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
doi:10.4056/sigs.42644
PMCID: PMC3035239  PMID: 21304667
extreme halophile; mesophile; free-living; motile; non-pathogenic; facultatively anaerobic; rod-shaped; Halobacteriaceae
10.  Multilocus Sequence Typing Confirms the Close Genetic Interrelatedness of Three Distinct Flavescence Dorée Phytoplasma Strain Clusters and Group 16SrV Phytoplasmas Infecting Grapevine and Alder in Europe▿  
Applied and Environmental Microbiology  2007;73(12):4001-4010.
Vineyards of southern France and northern Italy are affected by the flavescence dorée (FD) phytoplasma, a quarantine pathogen transmitted by the leafhopper of Nearctic origin Scaphoideus titanus. To better trace propagation of FD strains and identify possible passage between the vineyard and wild plant compartments, molecular typing of phytoplasma strains was applied. The sequences of the two genetic loci map and uvrB-degV, along with the sequence of the secY gene, were determined among a collection of FD and FD-related phytoplasmas infecting grapevine, alder, elm, blackberry, and Spanish broom in Europe. Sequence comparisons and phylogenetic analyses consistently indicated the existence of three FD phytoplasma strain clusters. Strain cluster FD1 (comprising isolate FD70) displayed low variability and represented 17% of the disease cases in the French vineyard, with a higher incidence of the cases in southwestern France. Strain cluster FD2 (comprising isolates FD92 and FD-D) displayed no variability and was detected both in France (83% of the cases) and in Italy, whereas the more-variable strain cluster FD3 (comprising isolate FD-C) was detected only in Italy. The clonal property of FD2 and its wide distribution are consistent with diffusion through propagation of infected-plant material. German Palatinate grapevine yellows phytoplasmas (PGY) appeared variable and were often related to some of the alder phytoplasmas (AldY) detected in Italy and France. Finally, phylogenetic analyses concluded that FD, PGY, and AldY were members of the same phylogenetic subclade, which may have originated in Europe.
doi:10.1128/AEM.02323-06
PMCID: PMC1932733  PMID: 17468266
11.  On the mechanism of rapid plasma membrane and chloroplast envelope expansion in Dunaliella salina exposed to hypoosmotic shock 
The Journal of Cell Biology  1986;102(1):289-297.
Dunaliella salina cells rapidly diluted from their normal 1.71 M NaCl- containing growth medium into medium containing 0.86 M NaCl swelled within 2--4 min to an average volume 1.76 X larger and a surface area 1.53 X larger than found in control cells. Morphometric analysis of thin section electron micrographs revealed that certain organelles, including the chloroplast, nucleus, and some types of vacuoles, also expanded in surface area as much or more than did the entire cell. It is likely that glycerol, the most important osmotically active intracellular solute, was present in high concentration within these organelles as well as in the cytoplasm itself. Thin section and freeze- fracture electron microscopy were utilized to trace the origin of membrane material whose addition permitted the large increase in plasma membrane surface area and the equally large growth of the chloroplast outer envelope. The findings indicated that the plasma membrane's expansion resulted from its selective fusion with numerous small (less than or equal to 0.25 micron diam) vesicles prevalent throughout the cytoplasm. In contrast, new membrane added to the chloroplast outer envelope was drawn from an entirely different source, namely, elements of the endoplasmic reticulum.
PMCID: PMC2114047  PMID: 3941156
12.  Molecular Clone and Expression of a NAD+-Dependent Glycerol-3-Phosphate Dehydrogenase Isozyme Gene from the Halotolerant alga Dunaliella salina 
PLoS ONE  2013;8(4):e62287.
Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.
doi:10.1371/journal.pone.0062287
PMCID: PMC3633914  PMID: 23626797
13.  High-throughput sequencing of Bacillus anthracis in France: investigating genome diversity and population structure using whole-genome SNP discovery 
BMC Genomics  2014;15:288.
Background
Single nucleotide polymorphisms (SNPs) are ideal signatures for subtyping monomorphic pathogens such as Bacillus anthracis. Here we report the use of next-generation sequencing technology to investigate the historical, geographic and genetic diversity of Bacillus anthracis in France. 122 strains isolated over a 60-years period throughout the country were whole-genome sequenced and comparative analyses were carried out with a focus on SNPs discovery to discriminate regional sub-groups of strains.
Results
A total of 1581 chromosomal SNPs precisely establish the phylogenetic relationships existing between the French strains. Phylogeography patterns within the three canSNP sub-lineages present in France (i.e. B.Br.CNEVA, A.Br.011/009 and A.Br.001/002) were observed. One of the more remarkable findings was the identification of a variety of genotypes within the A.Br.011/009 sub-group that are persisting in the different regions of France. The 560 SNPs defining the A.Br.011/009- affiliated French strains split the Trans-Eurasian sub-group into six distinct branches without any intermediate nodes. Distinct sub-branches, with some geographic clustering, were resolved. The 345 SNPs defining the major B.Br CNEVA sub-lineage clustered three main phylogeographic clades, the Alps, the Pyrenees, and the Massif Central, with a small Saône-et-Loire sub-cluster nested within the latter group. The French strains affiliated to the minor A.Br.001/002 group were characterized by 226 SNPs. All recent isolates collected from the Doubs department were closely related. Identification of SNPs from whole-genome sequences facilitates high-resolution strain tracking and provides the level of discrimination required for outbreak investigations. Eight diagnostic SNPs, representative of the main French-specific phylogeographic clusters, were therefore selected and developed into high-resolution melting SNP discriminative assays.
Conclusions
This work has established one of the most accurate phylogenetic reconstruction of B. anthracis population structure in a country. An extensive next-generation sequencing (NGS) dataset of 122 French strains have been created that allowed the identification of novel diagnostic SNPs useful to rapidly determine the geographic origin of any strain found in France.
doi:10.1186/1471-2164-15-288
PMCID: PMC4023602  PMID: 24734872
Bacillus anthracis; Whole genome sequencing; Single nucleotide polymorphism; Molecular typing; Comparative genomics; HRM
14.  Primary Screening of the Bioactivity of Brackishwater Cyanobacteria: Toxicity of Crude Extracts to Artemia salina Larvae and Paracentrotus lividus Embryos 
Marine Drugs  2010;8(3):471-482.
Cyanobacteria are a diverse group of Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against vertebrates, invertebrates, plants, microalgae, fungi, bacteria, viruses and cell lines. The aim of this study was to assess the toxic effects of aqueous, methanolic and hexane crude extracts of benthic and picoplanktonic cyanobacteria isolated from estuarine environments, towards the nauplii of the brine shrimp Artemia salina and embryos of the sea urchin Paracentrotus lividus. The A. salina lethality test was used as a frontline screen and then complemented by the more specific sea urchin embryo-larval assay. Eighteen cyanobacterial isolates, belonging to the genera Cyanobium, Leptolyngbya, Microcoleus, Phormidium, Nodularia, Nostoc and Synechocystis, were tested. Aqueous extracts of cyanobacteria strains showed potent toxicity against A. salina, whereas in P. lividus, methanolic and aqueous extracts showed embryo toxicity, with clear effects on development during early stages. The results suggest that the brackishwater cyanobacteria are producers of bioactive compounds with toxicological effects that may interfere with the dynamics of invertebrate populations.
doi:10.3390/md803471
PMCID: PMC2857359  PMID: 20411110
brackishwater cyanobacteria; sea urchin Paracentrotus lividus embryo larval bioassay; brine shrimp Artemia salina lethality test; benthic habitats
15.  Effect of Ca2+ Channel Block on Glycerol Metabolism in Dunaliella salina under Hypoosmotic and Hyperosmotic Stresses 
PLoS ONE  2011;6(12):e28613.
The effect of Ca2+ channel blockers on cytosolic Ca2+ levels and the role of Ca2+ in glycerol metabolism of Dunaliella salina under hypoosmotic or hyperosmotic stress were investigated using the confocal laser scanning microscope (CLSM). Results showed that intracellular Ca2+ concentration increased rapidly when extracellular salinity suddenly decreased or increased, but the increase could be inhibited by pretreatment of Ca2+ channel blockers LaCl3, verapamil or ruthenium red. The changes of glycerol content and G3pdh activity in D. salina to respect to hypoosmotic or hyperosmotic stress were also inhibited in different degrees by pretreatment of Ca2+ channel blockers, indicating that the influx of Ca2+ via Ca2+ channels are required for the transduction of osmotic signal to regulate osmotic responses of D. salina to the changes of salinity. Differences of the three blockers in block effect suggested that they may act on different channels or had different action sites, including influx of Ca2+ from the extracellular space via Ca2+ channels localized in the plasma membrane or from intracellular calcium store via the mitochondrial. Other Ca2+-mediated or non-Ca2+-mediated osmotic signal pathway may exist in Dunaliella in response to hypoosmotic and hyperosmotic stresses.
doi:10.1371/journal.pone.0028613
PMCID: PMC3237467  PMID: 22194866
16.  Comparative Analysis on the Key Enzymes of the Glycerol Cycle Metabolic Pathway in Dunaliella salina under Osmotic Stresses 
PLoS ONE  2012;7(6):e37578.
The glycerol metabolic pathway is a special cycle way; glycerol-3-phosphate dehydrogenase (G3pdh), glycerol-3-phosphate phosphatase (G3pp), dihydroxyacetone reductase (Dhar), and dihydroxyacetone kinase (Dhak) are the key enzymes around the pathway. Glycerol is an important osmolyte for Dunaliella salina to resist osmotic stress. In this study, comparative activities of the four enzymes in D. salina and their activity changes under various salt stresses were investigated, from which glycerol metabolic flow direction in the glycerol metabolic pathway was estimated. Results showed that the salinity changes had different effects on the enzymes activities. NaCl could stimulate the activities of all the four enzymes in various degrees when D. salina was grown under continuous salt stress. When treated by hyperosmotic or hypoosmotic shock, only the activity of G3pdh in D. salina was significantly stimulated. It was speculated that, under osmotic stresses, the emergency response of the cycle pathway in D. salina was driven by G3pdh via its response to the osmotic stress. Subsequently, with the changes of salinity, other three enzymes started to respond to osmotic stress. Dhar played a role of balancing the cycle metabolic pathway by its forward and backward reactions. Through synergy, the four enzymes worked together for the effective flow of the cycle metabolic pathways to maintain the glycerol requirements of cells in order to adapt to osmotic stress environments.
doi:10.1371/journal.pone.0037578
PMCID: PMC3366960  PMID: 22675484
17.  Protective effects of Dunaliella salina – a carotenoids-rich alga – against ultraviolet B-induced corneal oxidative damage in mice 
Molecular Vision  2012;18:1540-1547.
Purpose
Ultraviolet B (UVB) radiation from sunlight is known to be a risk factor for human corneal damage. The purpose of this study was to investigate the protective effects of Dunaliella salina (D. salina) on UVB radiation-induced corneal oxidative damage in male imprinting control region (ICR) mice.
Methods
Corneal oxidative damage was induced by exposure to UVB radiation at 560 μW/cm2. Animals were orally administered (gavage) D. salina at doses of 0, 123, and 615 mg/kg bodyweight/day for eight days. Corneal surface damages were graded according to smoothness and the extent of lissamine green staining. Corneal glutathione (GSH) and malondialdehyde (MDA) levels, as well as the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), and glutathione reductase (GSH-Rd) in cornea were measured to monitor corneal injury.
Results
UVB irradiation caused significant damage to the corneas, including apparent corneal ulcer and severe epithelial exfoliation, leading to decrease in the activities of SOD, catalase, GSH-Px, GSH-Rd, and GSH content in cornea, whereas there was increased corneal MDA content as compared with the control group. Treatment with D. salina could significantly (p<0.05) ameliorate corneal damage and increase the activities of SOD, catalase, GSH-Px, GSH-Rd, and GSH content, and decrease the MDA content in corneas when compared with the UVB-treated group.
Conclusions
The studies demonstrate that D. salina exhibits potent protective effects on UVB radiation-induced corneal oxidative damage in mice, likely due to both the increase of antioxidant enzyme activity and the inhibition of lipid peroxidation.
PMCID: PMC3380915  PMID: 22736944
18.  Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution 
There is a particularly high interest to derive carotenoids such as β-carotene and lutein from higher plants and algae for the global market. It is well known that β-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased β-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-012-4502-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-012-4502-5
PMCID: PMC3586100  PMID: 23095941
Dunaliella salina; Adaptive laboratory evolution; β-carotene and lutein; Carotenoid metabolism; LED-based photobioreactor
19.  Centaurea Revisited: A Molecular Survey of the Jacea Group 
Annals of Botany  2006;98(4):741-753.
• Background and Aims The genus Centaurea has traditionally been considered to be a complicated taxon. No attempt at phylogenetic reconstruction has been made since recent revisions in circumscription, and previous reconstructions did not include a good representation of species. A new molecular survey is thus needed.
• Methods Phylogenetic analyses were conducted using sequences of the internal transcribed spacers (ITS) 1 and 2 and the 5·8S gene. Parsimony and Bayesian approaches were used.
• Key Results A close correlation between geography and the phylogenetic tree based on ITS sequences was found in all the analyses, with three main groups being resolved: (1) comprising the most widely distributed circum-Mediterranean/Eurosiberian sections; (2) the western Mediterranean sections; and (3) the eastern Mediterranean and Irano-Turanian sections. The results show that the sectional classification in current use needs major revision, with many old sections being merged into larger ones. A large polytomy in the eastern Mediterranean clade suggests a rapid and recent speciation in this group. Some inconsistencies between morphology and molecular phylogeny may indicate that hybridization has played a major role in the evolution of the genus.
• Conclusions Phylogenetic analysis of ITS has been useful in identifying the major lineages in the group, and unraveling many inconsistencies in the sectional classification. However, most recent groups in the eastern Mediterranean clade are not resolved and reticulation in the western Mediterranean group of sections makes phylogenetic relationships within these two groups somewhat obscure.
doi:10.1093/aob/mcl157
PMCID: PMC2806161  PMID: 16873424
Cardueae; Centaureinae; Centaurea; Centaurea jacea group; hybridization; ITS sequences; phylogeny; systematics
20.  The alternative oxidase mediated respiration contributes to growth, resistance to hyperosmotic media and accumulation of secondary metabolites in three species 
Plant respiration, similar to respiration in animal mitochondria, exhibits both osmosensitive and insensitive components with the clear distinction that the insensitive respiration in plants is quantitatively better described as ‘less’ sensitive rather than ‘insensitive’. Salicylic hydroxamic acid (SHAM)-sensitive respiration was compared with the respiration sensitive to other inhibitors in rice, yeast and Dunaliella salina. The influence of SHAM was largely in the osmotically less sensitive component and enhanced with external osmotic pressure unlike other inhibitors that inhibited the osmotically sensitive component. SHAM inhibited germination and root growth but not shoot growth. Osmotic remediation of respiration that developed in due course of time with rice seedlings was abolished by SHAM and was not due to water and ionic uptake mechanisms. Yeast and Dunaliella also showed susceptibility of growth and respiration to SHAM. Glycerol retention was influenced by all inhibitors, while growth was inhibited demonstrably by SHAM in Dunaliella. Respiration in plants needs to be seen as a positive contribution to overall growth and not merely for burning away of the biomass.
doi:10.1007/s12298-008-0023-1
PMCID: PMC3550620  PMID: 23572891
SHAM; inhibitors of respiration; yeast; rice; Dunaliella; osmotic sensitivity; voids
21.  Phylogenetic Relationships among the Colobine Monkeys Revisited: New Insights from Analyses of Complete mt Genomes and 44 Nuclear Non-Coding Markers 
PLoS ONE  2012;7(4):e36274.
Background
Phylogenetic relationships among Asian and African colobine genera have been disputed and are not yet well established. In the present study, we revisit the contentious relationships within the Asian and African Colobinae by analyzing 44 nuclear non-coding genes (>23 kb) and mitochondrial (mt) genome sequences from 14 colobine and 4 non-colobine primates.
Principal Findings
The combined nuclear gene and the mt genome as well as the combined nuclear and mt gene analyses yielded different phylogenetic relationships among colobine genera with the exception of a monophyletic ‘odd-nosed’ group consisting of Rhinopithecus, Pygathrix and Nasalis, and a monophyletic African group consisting of Colobus and Piliocolobus. The combined nuclear data analyses supported a sister-grouping between Semnopithecus and Trachypithecus, and between Presbytis and the odd-nosed monkey group, as well as a sister-taxon association of Pygathrix and Rhinopithecus within the odd-nosed monkey group. In contrast, mt genome data analyses revealed that Semnopithecus diverged earliest among the Asian colobines and that the odd-nosed monkey group is sister to a Presbytis and Trachypithecus clade, as well as a close association of Pygathrix with Nasalis. The relationships among these genera inferred from the analyses of combined nuclear and mt genes, however, varied with the tree-building methods used. Another remarkable finding of the present study is that all of our analyses rejected the recently proposed African colobine paraphyly and hybridization hypothesis and supported reciprocal monophyly of the African and Asian groups.
Significance
The phylogenetic utility of large-scale new non-coding genes was assessed using the Colobinae as a model, We found that these markers were useful for distinguishing nodes resulting from rapid radiation episodes such as the Asian colobine radiation. None of these markers here have previously been used for colobine phylogenetic reconstruction, increasing the spectrum of molecular markers available to mammalian systematics.
doi:10.1371/journal.pone.0036274
PMCID: PMC3338693  PMID: 22558416
22.  Comparisons between Geographically Diverse Samples of Carried Staphylococcus aureus▿  
Journal of Bacteriology  2009;191(18):5577-5583.
Approximately one-third of the human population is asymptomatically colonized by Staphylococcus aureus. However, much of the global diversity within the carriage populations remains uncharacterized, and it is unclear to what degree the variation is geographically partitioned. We isolated 300 carriage isolates from 1,531 adults contemporaneously in four countries: France, Algeria, Moldova, and Cambodia. All strains were characterized by multilocus sequence typing. Six clonal complexes (CCs) were present in all four samples (CC30, -45, -121, -15, -5, and -8). Analyses based on the genotype frequencies revealed the French and Algerian samples to be most similar and the Cambodian sample to be most distinct. While this pattern is consistent with likely rates of human migration and geographic distance, stochastic clonal expansion also contributes to regional differences. Phylogenetic analysis revealed a highly divergent and uncharacterized genotype (ST1223) within Cambodia. This lineage is related to CC75, which has previously been observed only in remote aboriginal populations in northern Australia.
doi:10.1128/JB.00493-09
PMCID: PMC2737961  PMID: 19592594
23.  Screening of a Protein That Interacts with the Matrix Attachment Region-Binding Protein from Dunaliella salina 
BioMed Research International  2013;2013:862450.
We isolated the matrix attachment region-binding protein (MBP) DMBP-1 from Dunaliella salina in our previous studies. MBPs are part of the cis-acting protein family cluster. The regulatory function possibly works through the interaction of the MBPs with each other. In the present study, DMBP-1 was used as the bait in screening the D. salina cDNA library for DMBP-1 interactors that could potentially mediate the DMBP-1-regulated functions. A novel MBP, namely, DMBP-2, was identified as a DMBP-1 binding partner. The cDNA of DMBP-1 was 823 bp long and contained a 573 bp open reading frame, which encoded a polypeptide of 191 amino acids. The interaction between DMBP-2 and DMBP-1 was further confirmed through glutathione S-transferase pull-down assays.
doi:10.1155/2013/862450
PMCID: PMC3777206  PMID: 24093104
24.  Hepatoprotective and Antioxidant Activity of Dunaliella salina in Paracetamol-induced Acute Toxicity in Rats 
Paracetamol has a reasonable safety profile when taken in therapeutic doses. However, it could induce hepatotoxicity and even more severe fatal acute hepatic damage when taken in an overdose. The green alga, Dunaliella salina was investigated for hepatoprotective and antioxidant activity against paracetamol-induced liver damage in rats. Male albino Wistar rats overdosed with paracetamol showed liver damage and oxidative stress as indicated by significantly (P<0.05) increased serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total and direct bilirubin, malondialdehyde, cholesterol and nitric oxide. At the same time, there were decreased activities of serum superoxide dismutase and total antioxidant capacity compared with the control group. Treatment with D. salina methanol extract at doses of 500 and 1000 mg/kg body weight or silymarin could significantly (P<0.05) decrease the liver damage marker enzymes, total and direct bilirubin, malondialdehyde, cholesterol and nitric oxide levels and increase the activities of superoxide dismutase and total antioxidant capacity in serum when compared with paracetamol intoxicated group. Liver histopathology also showed that D. salina reduced the centrilobular necrosis, congestion and inflammatory cell infiltration evoked by paracetamol overdose. These results suggest that D. salina exhibits a potent hepatoprotective effect on paracetamol-induced liver damage in rats, which may be due to both the increase of antioxidant enzymes activity and inhibition of lipid peroxidation.
PMCID: PMC3928727  PMID: 24591738
Dunaliella salina; paracetamol; hepatotoxicity; antioxidant; reactive oxygen species
25.  Corky Root of Lettuce Caused by Strains of a Gram-Negative Bacterium from Muck Soils of Florida, New York, and Wisconsin 
Applied and Environmental Microbiology  1989;55(10):2635-2640.
Slow-growing bacteria similar to the bacterium causing lettuce corky root (CR) in California (strain CA1) were isolated from muck soils of Florida, New York, and Wisconsin, using lettuce seedlings as bait. All strains were tested for reaction with polyclonal antibodies produced against strain CA1 and for pathogenicity on CR-susceptible (Salinas) and CR-resistant (Green Lake) lettuce cultivars in a greenhouse. Five strains from Florida, three from New York, and three from Wisconsin induced severe CR symptoms on Salinas and mild symptoms on Green Lake. All strains were gram-negative, aerobic, oxidase positive, and catalase positive and reduced nitrate to ammonia. Whole-cell fatty acid compositions were similar for all strains and resembled that of Pseudomonas paucimobilis. Since this fatty acid pattern is unique, it is suggested that CR of lettuce is caused by strains of the same bacterium in Florida, New York, Wisconsin, and California.
Images
PMCID: PMC203136  PMID: 16348032

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