Polar Microalgae: New Approaches towards Understanding Adaptations to an Extreme and Changing Environment

Polar Regions are unique and highly prolific ecosystems characterized by extreme environmental gradients. Photosynthetic autotrophs, the base of the food web, have had to adapt physiological mechanisms to maintain growth, reproduction and metabolic activity despite environmental conditions that would shut-down cellular processes in most organisms. High latitudes are characterized by temperatures below the freezing point, complete darkness in winter and continuous light and high UV in the summer. Additionally, sea-ice, an ecological niche exploited by microbes during the long winter seasons when the ocean and land freezes over, is characterized by large salinity fluctuations, limited gas exchange, and highly oxic conditions. The last decade has been an exciting period of insights into the molecular mechanisms behind adaptation of microalgae to the cryosphere facilitated by the advancement of new scientific tools, particularly “omics” techniques. We review recent insights derived from genomics, transcriptomics, and proteomics studies. Genes, proteins and pathways identified from these highly adaptable polar microbes have far-reaching biotechnological applications. Furthermore, they may provide insights into life outside this planet, as well as glimpses into the past. High latitude regions also have disproportionately large inputs into global biogeochemical cycles and are the region most sensitive to climate change

Characterization of a trimeric light-harvesting complex in the diatom Phaeodactylum tricornutum built of FcpA and FcpE proteins

Fucoxanthin chlorophyll proteins (Fcps), the light-harvesting antennas of heterokont algae, are encoded by a multigene family and are highly similar with respect to their molecular masses as well as to their pigmentation, making it difficult to purify single Fcps. In this study, a hexa-histidine tag was genetically added to the C-terminus of the FcpA protein of the pennate diatom Phaeodactylum tricornutum. A transgenic strain expressing the recombinant His-tagged FcpA protein in addition to the endogenous wild type Fcps was created. This strategy allowed, for the first time, the purification of a specific, stable trimeric Fcp complex. In addition, a pool of various trimeric Fcps was also purified from the wild-type cells using sucrose density gradient ultracentrifugation and gel filtration. In both the His-tagged and the wild-type Fcps, excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated using circular dichroism spectroscopy. Mass spectrometric analyses of the trimeric His-tagged complex indicated that it is composed of FcpA and FcpE polypeptides. It is confirmed here that a trimer is the basic organizational unit of Fcps in P. tricornutum. From circular dichroism spectra, it is proposed that the organization of the pigments on the polypeptide backbone of Fcps is a conserved feature in the case of chlorophyll a/c containing algae

In Silico and In Vivo Investigations of Proteins of a Minimized Eukaryotic Cytoplasm

Algae with secondary plastids such as diatoms maintain two different eukaryotic cytoplasms. One of them, the so-called periplastidal compartment (PPC), is the naturally minimized cytoplasm of a eukaryotic endosymbiont. In order to investigate the protein composition of the PPC of diatoms, we applied knowledge of the targeting signals of PPC-directed proteins in searches of the genome data for proteins acting in the PPC and proved their in vivo localization via expressing green fluorescent protein (GFP) fusions. Our investigation increased the knowledge of the protein content of the PPC approximately 3-fold and thereby indicated that this narrow compartment was functionally reduced to some important cellular functions with nearly no housekeeping biochemical pathways

СИНДРОМ «ЗАПЕРТОГО ЧЕЛОВЕКА» ВСЛЕДСТВИЕ МНОЖЕСТВЕННЫХ ИНФАРКТОВ СТВОЛА ГОЛОВНОГО МОЗГА И ОЧАГОВ ДЕМИЕЛИНИЗАЦИИ ПОСЛЕ ЛУЧЕВОЙ ТЕРАПИИ АДЕНОМЫ ГИПОФИЗА С АКРОМЕГАЛИЕЙ

Locked-in syndrome (LIS) is a rare neurological disorder, usually appears as a result of the pons cerebellar damage, mostly after the brain stroke. Locked-in syndrome is characterized by the paralysis of skeletal muscles (respiratory, facial, pharyngeal, lingual and muscles of the extremities). Patient is unable to speak and breath, facial expressions and voluntary movements are also impossible. Acromegaly is a disease that can be described by the increase of the growth hormone (GH) and Insulin-like growth factor (IGF-1) and develops in most cases due to the pituitary adenomas. Pituitary adenoma (PA) can be treated by neurosurgical techniques, pharmaceutical and radiation therapy (RT). We present a clinical case of 33-year-old woman with PA-caused acromegaly, that developed muscle weakness, nausea, vomit and respiratory disturbance in a 2 months after the radiation therapy. Subacute comatose state was developed in the patient. MRI of the brain revealed a multi-focal lesion of the media-basal regions on both sides, frontal corpus callosum and brain stem. Differential diagnosis included an acute demyelination (SD, PML), viral encephalitis and vasculitis. Treatment included methylprednisolone pulse therapy and plasmapheresis. The consciousness cleared up, but there was no spontaneous breathing, tetraplegia persisted. Autoimmune and infectious diseases was excluded. The homozygous mutation PAI-1-675 4G/4G was found. In this case, acromegaly induced endothelial dysfunction was the pathogenesis factor of multiple cerebral infarctions and demyelinating lesions, as well as RT and its proven pathological influence on the vascular wall and the fibrinolytic system. The revealed thrombophilia was also a factor of multiple cerebral infarctions. A Potential combination of pathogenic factors in the development of cerebral should be taken into account in predicting complications of RT. Cиндром «запертого человека» (СЗЧ) является редким неврологическим расстройством, обычно возникающим в результате поражения моста мозга, чаще всего вследствие инсульта. При СЗЧ возникает паралич скелетных мышц (дыхательные, лицевые, глотки, языка, а также конечностей), т. е. утрачивается способность к речи, мимике, произвольным движениям, дыханию. Акромегалия – заболевание, при котором наблюдается повышение уровня гормона роста (GH), инсулиноподобного фактора роста 1 (IGF-1), и развивается в большинстве случаев при аденомах гипофиза. При лечении аденомы гипофиза применяют нейрохирургические методики, медикаментозную и лучевую терапию (ЛТ). В представленном клиническом наблюдении у 33-летней женщины через 2 месяца после дистанционной ЛТ опухоли гипофиза, проявлявшейся акромегалией, появилась мышечная слабость, тошнота, рвота, нарушения дыхания. Развилось подостро коматозное состояние. При магнитно-резонансной томографии головного мозга выявлено многоочаговое поражение медиобазальных отделов обоих полушарий, передних отделов мозолистого тела и ствола. Дифференциальный диагноз включал острую демиелинизацию (SD, PML), вирусный энцефалит и васкулит. Пациентка получила пульс-терапию Метилпреднизолоном, плазиообмен. Сознание прояснилось, но самостоятельное дыхание не восстановилось, оставалась тетраплегия. Аутоиммунные заболевания и инфекционные болезни были исключены. Обнаружена мутантная гомозигота PAI-1-675 4G\4G. В данном случае, вероятно, патогенетическими факторами развития множественных инфарктов и очагов демиелинизации выступили как сама акромегалия, приводящая к эндотелиальной дисфункции, так и ЛТ с ее доказанным патологическим воздействием на сосудистую стенку и фибринолитическую систему. Множественным инфарктам способствовала и выявленная тромбофилия. Возможное сочетание патогенетических факторов развития инфарктов головного мозга следует учитывать при прогнозировании осложнений ЛТ

A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1

СЛУЧАЙ НЕЙРОБЛАСТОМЫ У ВЗРОСЛОГО: ОБЗОР ЛИТЕРАТУРЫ И ОПИСАНИЕ КЛИНИЧЕСКОГО СЛУЧАЯ

Neuroblastoma is a malignant tumor derived from the neuroblasts of the sympathetic nervous system, which develop in any region of the nervous system. Usually, neuroblastoma is detected in children aged 1–2 years. About 90% of cases are diagnosed before the age of 5 years. The incidence of adult neuroblastoma is only 0.3 cases per million people per year. The clinical course and biological activity of adult neuroblastoma is different than children neuroblastoma. Early diagnosis of this disease in adults is necessary for timely start of treatment and increasing life expectancy. In this clinical observation, we present a detailed description of the course of this rare disease in the 34-year-old male and literature review on adult neuroblastoma.Нейробластома – злокачественная опухоль, происходящая из нейробластов симпатической нервной системы, которая может развиваться в любом регионе нервной системы. Обычно нейробластома обнаруживается у детей в возрасте 1–2 лет, около 90 % случаев заболевания диагностируется в возрасте до 5 лет. Частота случаев нейробластомы у взрослых составляет всего 0,3 случая на 1 млн человек в год. Клиническое течение и биологическая активность нейробластомы у взрослых отличаются по сравнению с детьми. Ранняя диагностика заболевания у взрослых необходима для своевременного начала лечения и увеличения продолжительности жизни. В представленном клиническом наблюдении мы делимся подробным описанием течения этого редкого заболевания у мужчины 34 лет, а также приводим обзор литературы по нейробластоме у взрослых.

Genome-Wide Transcriptome Analyses of Silicon Metabolism in Phaeodactylum tricornutum Reveal the Multilevel Regulation of Silicic Acid Transporters

BACKGROUND:Diatoms are largely responsible for production of biogenic silica in the global ocean. However, in surface seawater, Si(OH)(4) can be a major limiting factor for diatom productivity. Analyzing at the global scale the genes networks involved in Si transport and metabolism is critical in order to elucidate Si biomineralization, and to understand diatoms contribution to biogeochemical cycles. METHODOLOGY/PRINCIPAL FINDINGS:Using whole genome expression analyses we evaluated the transcriptional response to Si availability for the model species Phaeodactylum tricornutum. Among the differentially regulated genes we found genes involved in glutamine-nitrogen pathways, encoding putative extracellular matrix components, or involved in iron regulation. Some of these compounds may be good candidates for intracellular intermediates involved in silicic acid storage and/or intracellular transport, which are very important processes that remain mysterious in diatoms. Expression analyses and localization studies gave the first picture of the spatial distribution of a silicic acid transporter in a diatom model species, and support the existence of transcriptional and post-transcriptional regulations. CONCLUSIONS/SIGNIFICANCE:Our global analyses revealed that about one fourth of the differentially expressed genes are organized in clusters, underlying a possible evolution of P. tricornutum genome, and perhaps other pennate diatoms, toward a better optimization of its response to variable environmental stimuli. High fitness and adaptation of diatoms to various Si levels in marine environments might arise in part by global regulations from gene (expression level) to genomic (organization in clusters, dosage compensation by gene duplication), and by post-transcriptional regulation and spatial distribution of SIT proteins

Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching

Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms. NPQ depends on the conversion of diadinoxanthin (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms

Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana

The potential use of algae in biofuels applications is receiving significant attention. However, none of the current algal model species are competitive production strains. Here we present a draft genome sequence and a genetic transformation method for the marine microalga Nannochloropsis gaditana CCMP526. We show that N. gaditana has highly favourable lipid yields, and is a promising production organism. The genome assembly includes nuclear (~29 Mb) and organellar genomes, and contains 9,052 gene models. We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis. Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed. The availability of a genome sequence and transformation methods will facilitate investigations into N. gaditana lipid biosynthesis and permit genetic engineering strategies to further improve this naturally productive alga

De Novo Transcriptomic Analysis of an Oleaginous Microalga: Pathway Description and Gene Discovery for Production of Next-Generation Biofuels

Background: Eustigmatos cf. polyphem is a yellow-green unicellular soil microalga belonging to the eustimatophyte with high biomass and considerable production of triacylglycerols (TAGs) for biofuels, which is thus referred to as an oleaginous microalga. The paucity of microalgae genome sequences, however, limits development of gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for a non-model microalgae species, E. cf. polyphem, and identify pathways and genes of importance related to biofuel production. Results: We performed the de novo assembly of E. cf. polyphem transcriptome using Illumina paired-end sequencing technology. In a single run, we produced 29,199,432 sequencing reads corresponding to 2.33 Gb total nucleotides. These reads were assembled into 75,632 unigenes with a mean size of 503 bp and an N50 of 663 bp, ranging from 100 bp to.3,000 bp. Assembled unigenes were subjected to BLAST similarity searches and annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology identifiers. These analyses identified the majority of carbohydrate, fatty acids, TAG and carotenoids biosynthesis and catabolism pathways in E. cf. polyphem. Conclusions: Our data provides the construction of metabolic pathways involved in the biosynthesis and catabolism of carbohydrate, fatty acids, TAG and carotenoids in E. cf. polyphem and provides a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character o