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.     

Regionally Distinct Alterations in the Composition of the Gut Microbiota in Rats with Streptozotocin-Induced Diabetes

The aim of this study was to map the microbiota distribution along the gut and establish whether colon/faecal samples from diabetic rats adequately reflect the diabetic alterations in the microbiome. Streptozotocin-treated rats were used to model type 1 diabetes mellitus (T1D). Segments of the duodenum, ileum and colon were dissected, and the microbiome of the lumen material was analysed by using next-generation DNA sequencing, from phylum to genus level. The intestinal luminal contents were compared between diabetic, insulin-treated diabetic and healthy control rats. No significant differences in bacterial composition were found in the luminal contents from the duodenum of the experimental animal groups, whereas distinct patterns were seen in the ileum and colon, depending on the history of the luminal samples. Ileal samples from diabetic rats exhibited particularly striking alterations, while the richness and diversity obscured some of the modifications in the colon. Characteristic rearrangements in microbiome composition and diversity were detected after insulin treatment, though the normal gut flora was not restored. The Proteobacteria displayed more pronounced shifts than those of the predominant phyla (Firmicutes and Bacteroidetes) in the rat model of T1D. Diabetes and insulin replacement affect the composition of the gut microbiota in different, gut region-specific manners. The luminal samples from the ileum appear more suitable for diagnostic purposes than the colon/faeces. The Proteobacteria should be at the focus of diagnosis and potential therapy. Klebsiella are recommended as biomarkers of T1D.     

不同盐度胁迫下杜氏盐藻全转录组测序及注释

【目的】通过对杜氏盐藻的转录组进行测序和基因功能分析,阐明不同浓度盐胁迫对杜氏盐藻生长发育以及不同信号途径的影响。【方法】分别获取9%NaCl浓度和24%NaCl浓度培养下的杜氏盐藻转录组并通过Illumina平台进行测序。将所得的序列进行拼接、去冗余处理。【结果】获得40682个unigenes,其中注释到NR数据库的10905个,注释到NT数据库的2768个,注释到SWISS-PROT数据库的7261个,注释到COG/KOG数据库的6499个。受到高盐胁迫的杜氏盐藻细胞相比低盐环境下,有717个基因表达上调,1012个基因表达下调。进一步对60个显著差异基因进行了功能聚类,发现盐胁迫诱导了光合作用途径的基因表达。【结论】杜氏盐藻通过提高光合作用基因表达增强耐盐性。该研究最大范围上挖掘了杜氏盐藻在高盐和低盐环境的基因转录水平,为深入揭示杜氏盐藻盐胁迫下基因差异表达提供了平台,并为进一步研究杜氏盐藻耐盐机理提供理论依据。     

Oxaloacetate and malate production in engineered Escherichia coli by expression of codon-optimized phosphoenolpyruvate carboxylase2 gene from Dunaliella salina

A new phosphoenolpyruvate carboxylase (PEPC) gene of Dunaliella salina is identified using homology analysis was conducted using PEPC gene of Chlamydomonas reinhardtii and Arabidopsis thaliana. Recombinant E. coli SGJS115 with increased production of malate and oxaloacetate was developed by introducing codon-optimized phosphoenolpyruvate carboxylase2 (OPDSPEPC2) gene of Dunaliella salina. E. coli SGJS115 yielded a 9.9 % increase in malate production. In addition, E. coli SGJS115 exhibited two times increase in the yield of oxaloacetate over the E. coli SGJS114 having identified PEPC2 gene obtained from Dunaliella salina.     

Cloning and Sequence Analysis of the Gene Encoding (6-4)photolyase from Dunaliella salina

DNA photolyase can repair UV-induced DNA damage in a light-dependent manner. A cDNA of (6-4)photolyase from Dunaliella salina (GenBank accession number: AY845324) was cloned, sequenced and its amino acid sequence was deduced. The derived amino acid sequence showed high homology with other (6-4)photolyases and a predicted 3D model was constructed by homology modeling. Revisions requested 20 May 2005 and 18 August 2005; Revisions received 2 August 2005 and 28 November 2005     

Expression of soybean Kunitz trypsin inhibitor gene SKTI in Dunaliella salina

Recently, microalgae have attracted much attention as a new bioreactor system for producing high-value heterologous proteins. In this paper, we investigated the expression of soybean Kunitz trypsin inhibitor gene SKTI in the chlorophyte Dunaliella salina. Using D. salina genomic DNA as template, the entire coding region of SKTI was amplified by PCR as a 654-bp DNA fragment. It had 100 % identity with the published sequence of SKTI. The entire SKTI fragment was cloned into the expression vector pCAM2201 at a site just downstream of 35S promoter of to yield pCAMSKTI. D. salina cells transfected with pCAMSKTI by means of the lithium acetate/polyethylene glycol-mediated method expressed a protein of 20.1 kDa as detected by anti-SKTI antibody. In addition, SDS-PAGE analysis of the cell extract also revealed an intense protein band indicative of the recombinant SKTI. SKTI was therefore successfully expressed in D. salina, and the expression could be detected for at least 35 generations.     

Characterization of a novel Dunaliella salina (Chlorophyta) strain and the assessment of its cultivation parameters

Physiological characteristics of a novel strain of the halophilic alga Dunaliella salina IPPAS D-232 isolated from the hypersaline lake Razval (Sol-Iletsk, Orenburg region) were studied. It was shown that the extract from the cells of this strain manifested antioxidant properties and also antibacterial activity relative to opportunistic bacteria. The influence of medium stirring and salinity on the level of D. salina antioxidant activity was assessed: it was established that medium salinization (93 g/L) and continuous stirring favored the highest accumulation of D. salina IPPAS D-232 biomass. It was found that the extremely halophilic archaea Halorubrum tebenquichense and moderately halophilic bacterium Marinococcus halophilus stimulated D. salina development and increased its antioxidant activity. The results obtained expand our knowledge of specific ecological features of the studied microalgal species; they could form the basis for the development of new approaches to the mass cultivation of D. salina.     

Effects of Heat Stress on Gut Microbiome in Rats

Gut microbiome, as the largest and most important micro-ecosystem, plays a critical role in health. The purpose of this study was to evaluate whether heat stress modulates the composition and diversity of the gut microbiome in rats. The heat stress model was prepared in rats with the heating temperature maintained at 35–38°C. Cecum contents were collected after heat stress for 3 h and days 1, 3 and 7. Total DNA was extracted for 16 S rRNA sequencing and analysis of intestinal microbiome composition and diversity. The study showed that the composition of the intestinal microbiome of heat stress group was changed. And the heat stress modulated key phylotypes of gut microbiota at the level of phylum and genus. In particular, the genus of Lactobacillus and Bacteroides were significantly reduced, whereas the Oscillospira and Clostridium were increased by heat stress. Meanwhile, the rats under the heat stress encountered the change in carbohydrate metabolism, amino acid metabolism, and membrane transport to defense against stress. Taken together, the composition and structure of gut microbiome were affected by heat stress and some key phylotypes were also significantly altered. We conclude that the heat stress could impact multiple biological functions, via altering the gut microbiome.     

Determination of intracellular osmotic volume and sodium concentration in dunaliella

A new method to measure intracellular volume in Dunaliella was developed, where lithium ions are used as monitors of the extracellular volume. Li⁺ is shown to be impenetrable to the intracellular volume, insignificantly absorbed to the algae, and is rapidly and evenly distributed within the extracellular volume. The method is suggested to be free of several limitations and consistent errors present in several previously employed techniques.

Using the new technique it is shown that both Dunaliella salina and Dunaliella bardawil adjust to a constant cellular volume when grown in a medium containing salt concentrations ranging from 0.5 molar to 4 molar NaCl. That volume is 90 femtoliter per cell for D. salina and 600 femtoliter per cell for D. bardawil. Nonosmotic volume accounts for about 10% of the total cell volume.

The intracellular sodium concentration, as determined with the new technique, was under all experimental conditions tested below 100 millimolar. This was true both for cells grown on 0.5 to 4 molar NaCl, and during the osmoregulatory process. It is thus concluded that intracellular NaCl is a minor contributor to the overall intracellular osmotic pressure in Dunaliella.

     

短小芽孢杆菌对盐藻SZ-05的生物量和β-胡萝卜素产量的影响

研究了短小芽孢杆菌（Bacillus pumilus）对盐藻空间诱变株系SZ-05（Dunaliella salina SZ-05）的生物量及β-胡萝卜素积累的影响。结果表明,短小芽孢杆菌显著提高了盐藻SZ-05的生物量和β-胡萝卜素的产量,明显降低了培养体系中的溶解氧和胞外多糖的含量。溶解氧的减少,使得藻细胞的光呼吸作用下降,光合作用速率提高,使藻细胞生物量增加。胞外多糖具有抗氧化作用,胞外多糖的减少可能进一步增加了β-胡萝卜素的合成,从而使β-胡萝卜素在胁迫条件下大幅度增加。     

DNA fingerprinting differentiation between β-carotene hyperproducer strains of Dunaliella from around the world

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.     

Peroxiredoxin 1 is involved in disassembly of flagella and cilia

Cilia/flagella are evolutionarily conserved cellular organelles. In this study, we demonstrated that Dunaliella salina Peroxiredoxin 1 (DsPrdx1) localized to the flagella and basal bodies, and was involved in flagellar disassembly. The link between DsPrdx1 and flagella of Dunaliella salina (D. salina) encouraged us to explore the function of its human homologue, Homo sapiens Peroxiredoxin 1 (HsPrdx1) in development and physiology. Our results showed that HsPrdx1 was overexpressed, and cilia were lost in esophageal squamous cell carcinoma (ESCC) cells compared with the non-cancerous esophageal epithelial cells Het-1A. Furthermore, when HsPrdx1 was knocked down by short hairpin RNA (shRNA) lentivirus in ESCC cells, the phenotype of cilia lost can be reversed, and the expression levels of tumor suppressor genes LKB1 and p-AMPK were increased, and the activity of the oncogene Aurora A was inhibited compared with those in cells transfected with scrambe-shRNA lentivirus. These findings firstly showed that Prdx1 is involved in disassembly of flagella and cilia, and suggested that the abnormal expression of the cilia-related gene including Prdx1 may affect both ciliogenesis and cancernogenesis.     

Low Temperature-Induced Alterations in the Chloroplast and Microsomal Membranes of Dunaliella salina

The metabolic regulation of membrane lipid composition has been examined using the cell wall-less, unicellular green alga Dunaliella salina (UTEX 1644) as a model system. Low temperature stress was employed to initiate and study the regulatory response.     

Application of the microalga Plagioselmis prolonga for the assessment of water quality in the Amursky and Nakhodka Bays (Sea of Japan)

Based on biotesting, we carried out an estimation of the water quality in the Amursky and Nakhodka Bays (Sea of Japan) using Plagioselmis prolonga (Cryptophyta). The obtained data were compared with the data from water biotesting using Dunaliella salina (Chlorophyta). It was shown that water from the Amursky Bay produced more a negative effect on both microalgae than water from the Nakhodka Bay. It was established that sensitivity of the P. prolonga microalga exceeded that of D. salina. This was confirmed by a sharp decrease of the P. prolonga motile cell number in the studied water.     

A Longitudinal Study of the Feline Faecal Microbiome Identifies Changes into Early Adulthood Irrespective of Sexual Development

Companion animals provide an excellent model for studies of the gut microbiome because potential confounders such as diet and environment can be more readily controlled for than in humans. Additionally, domestic cats and dogs are typically neutered early in life, enabling an investigation into the potential effect of sex hormones on the microbiome. In a longitudinal study to investigate the potential effects of neutering, neutering age and gender on the gut microbiome during growth, the faeces of kittens (16 male, 14 female) were sampled at 18, 30 and 42 weeks of age. DNA was shotgun sequenced on the Illumina platform and sequence reads were annotated for taxonomy and function by comparison to a database of protein coding genes. In a statistical analysis of diversity, taxonomy and functional potential of the microbiomes, age was identified as the only factor with significant associations. No significant effects were detected for gender, neutering, or age when neutered (19 or 31 weeks). At 18 weeks of age the microbiome was dominated by the genera Lactobacillus and Bifidobacterium (35% and 20% average abundance). Structural and functional diversity was significantly increased by week 30 but there was no further significant increase. At 42 weeks of age the most abundant genera were Bacteroides (16%), Prevotella (14%) and Megasphaera (8%). Significant differences in functional potential included an enrichment for genes in energy metabolism (carbon metabolism and oxidative phosphorylation) and depletion in cell motility (flagella and chemotaxis). We conclude that the feline faecal microbiome is predominantly determined by age when diet and environment are controlled for. We suggest this finding may also be informative for studies of the human microbiome, where control over such factors is usually limited.     

The cervical microbiome over 7 years and a comparison of methodologies for its characterization

Background

The rapidly expanding field of microbiome studies offers investigators a large choice of methods for each step in the process of determining the microorganisms in a sample. The human cervicovaginal microbiome affects female reproductive health, susceptibility to and natural history of many sexually transmitted infections, including human papillomavirus (HPV). At present, long-term behavior of the cervical microbiome in early sexual life is poorly understood.

Methods

The V6 and V6–V9 regions of the 16S ribosomal RNA gene were amplified from DNA isolated from exfoliated cervical cells. Specimens from 10 women participating in the Natural History Study of HPV in Guanacaste, Costa Rica were sampled successively over a period of 5–7 years. We sequenced amplicons using 3 different platforms (Sanger, Roche 454, and Illumina HiSeq 2000) and analyzed sequences using pipelines based on 3 different classification algorithms (usearch, RDP Classifier, and pplacer).

Results

Usearch and pplacer provided consistent microbiome classifications for all sequencing methods, whereas RDP Classifier deviated significantly when characterizing Illumina reads. Comparing across sequencing platforms indicated 7%–41% of the reads were reclassified, while comparing across software pipelines reclassified up to 32% of the reads. Variability in classification was shown not to be due to a difference in read lengths. Six cervical microbiome community types were observed and are characterized by a predominance of either G. vaginalis or Lactobacillus spp. Over the 5–7 year period, subjects displayed fluctuation between community types. A PERMANOVA analysis on pairwise Kantorovich-Rubinstein distances between the microbiota of all samples yielded an F-test ratio of 2.86 (p<0.01), indicating a significant difference comparing within and between subjects’ microbiota.

Conclusions

Amplification and sequencing methods affected the characterization of the microbiome more than classification algorithms. Pplacer and usearch performed consistently with all sequencing methods. The analyses identified 6 community types consistent with those previously reported. The long-term behavior of the cervical microbiome indicated that fluctuations were subject dependent.     

Screening Currency Notes for Microbial Pathogens and Antibiotic Resistance Genes Using a Shotgun Metagenomic Approach

Fomites are a well-known source of microbial infections and previous studies have provided insights into the sojourning microbiome of fomites from various sources. Paper currency notes are one of the most commonly exchanged objects and its potential to transmit pathogenic organisms has been well recognized. Approaches to identify the microbiome associated with paper currency notes have been largely limited to culture dependent approaches. Subsequent studies portrayed the use of 16S ribosomal RNA based approaches which provided insights into the taxonomical distribution of the microbiome. However, recent techniques including shotgun sequencing provides resolution at gene level and enable estimation of their copy numbers in the metagenome. We investigated the microbiome of Indian paper currency notes using a shotgun metagenome sequencing approach. Metagenomic DNA isolated from samples of frequently circulated denominations of Indian currency notes were sequenced using Illumina Hiseq sequencer. Analysis of the data revealed presence of species belonging to both eukaryotic and prokaryotic genera. The taxonomic distribution at kingdom level revealed contigs mapping to eukaryota (70%), bacteria (9%), viruses and archae (~1%). We identified 78 pathogens including Staphylococcus aureus, Corynebacterium glutamicum, Enterococcus faecalis, and 75 cellulose degrading organisms including Acidothermus cellulolyticus, Cellulomonas flavigena and Ruminococcus albus. Additionally, 78 antibiotic resistance genes were identified and 18 of these were found in all the samples. Furthermore, six out of 78 pathogens harbored at least one of the 18 common antibiotic resistance genes. To the best of our knowledge, this is the first report of shotgun metagenome sequence dataset of paper currency notes, which can be useful for future applications including as bio-surveillance of exchangeable fomites for infectious agents.