scholarly journals An optimised method for intact nuclei isolation from diatoms

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rossella Annunziata ◽  
Cecilia Balestra ◽  
Pina Marotta ◽  
Antonella Ruggiero ◽  
Francesco Manfellotto ◽  
...  
Keyword(s):  
Phaeodactylum Tricornutum ◽  
High Sensitivity ◽  
Centric Diatom ◽  
Diatom Species ◽  
Isolated Nuclei ◽  
Nuclei Isolation ◽  
Low Degree ◽  
Speed Up ◽  
Peculiar Nature ◽  
Degree Of Fragmentation

AbstractDue to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH4F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell (“single nuclei”) genomics, transcriptomics and proteomics in different diatom species.

scholarly journals Identification, characterization, and application of a highly sensitive lactam biosensor from Pseudomonas putida

2019 ◽  
Author(s):  
Mitchell G. Thompson ◽  
Allison N. Pearson ◽  
Jesus F. Barajas ◽  
Pablo Cruz-Morales ◽  
Nima Sedaghatian ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
High Sensitivity ◽  
Biological Pathways ◽  
Biological Synthesis ◽  
Polymer Precursor ◽  
Development Times ◽  
Highly Sensitive ◽  
Speed Up ◽  
Exogenous Ligand

ABSTRACTCaprolactam is an important polymer precursor to nylon traditionally derived from petroleum and produced on a scale of 5 million tons per year. Current biological pathways for the production of caprolactam are inefficient with titers not exceeding 2 mg/L, necessitating novel pathways for its production. As development of novel metabolic routes often require thousands of designs and result in low product titers, a highly sensitive biosensor for the final product has the potential to rapidly speed up development times. Here we report a highly sensitive biosensor for valerolactam and caprolactam from Pseudomonas putida KT2440 which is >1000x more sensitive to exogenous ligand than previously reported sensors. Manipulating the expression of the sensor oplR (PP_3516) substantially altered the sensing parameters, with various vectors showing Kd values ranging from 700 nM (79.1 μg/L) to 1.2 mM (135.6 mg/L). Our most sensitive construct was able to detect in vivo production of caprolactam above background at ~6 μg/L. The high sensitivity and range of OplR is a powerful tool towards the development of novel routes to the biological synthesis of caprolactam.

scholarly journals NEW METHOD OF ESTIMATION OF THE RELATIVE AREA OF PERFORATIONS ON VALVES OF CENTRIC DIATOMS USING SEM IMAGES ON THE EXAMPLE OF MINIDISUS VODYANITSKIYI LYAKH ET BEDOSHVILI

2019 ◽  
Vol 21 ◽  
pp. 46-52
Author(s):  
Anton M. Lyakh ◽  
Yekaterina D. Bedoshvili ◽  
Olga V. Shikhat
Keyword(s):  
Land Plants ◽  
New Method ◽  
Centric Diatom ◽  
Relative Area ◽  
Diatom Species ◽  
Sem Images ◽  
Centric Diatoms ◽  
Diatom Valve ◽  
Small Valve ◽  
Valve Area

The diatoms interact with the environment through the siliceous frustule. The total area of frustule perforations determines the ability of diatom to exchange nutrients, gases and other matters. The aim of the present study was to estimate the area of perforations on the valve surface of a centric diatom. In the paper we describe a method for the estimation of the area of perforations on a diatom valve using SEM images. The method is tested on valves of centric diatom Minidiscus vodyanitskiyi Lyakh et Bedoshvili. The results show that the total area of cribral pores is less than 5% of the total valve area. This value is consistent with the relative perforation of land plants leaves, which is less than 3%. We hypothesize that such small valve area occupied by perforations is usual for many other centric diatom species. To verify this hypothesis additional researches are necessary.

scholarly journals Levels of Diatom Minor Sterols Respond to Changes in Temperature and Salinity

2020 ◽  
Vol 8 (2) ◽  
pp. 85 ◽  
Author(s):  
Ana Cristina Jaramillo-Madrid ◽  
Justin Ashworth ◽  
Peter J. Ralph
Keyword(s):  
Human Health ◽  
Phaeodactylum Tricornutum ◽  
Thalassiosira Pseudonana ◽  
Eukaryotic Cells ◽  
Effect Of Temperature ◽  
Diatom Species ◽  
Adaptive Traits ◽  
Chaetoceros Muelleri ◽  
Diverse Range ◽  
The Relationship

Diatoms are a broadly distributed and evolutionarily diversified group of microalgae that produce a diverse range of sterol compounds. Sterols are triterpenoids that play essential roles in membrane-related processes in eukaryotic cells. Some sterol compounds possess bioactivities that promote human health and are currently used as nutraceuticals. The relationship between sterol diversity in diatoms and their acclimation to different environments is not well understood. In this study, we investigated the occurrence of different sterol types across twelve diatom species, as well as the effect of temperature reduction and changes in salinity on the sterol contents of three model diatom species. In the diatoms Thalassiosira pseudonana, Phaeodactylum tricornutum and Chaetoceros muelleri, we found that changes in the relative contents of minor sterols accompanied shifts in temperature and salinity. This may be indicative of acquired adaptive traits in diatom metabolism.

New records of centric diatoms from Yakutia (Bolshoe Toko Lake): SEM morphology, ecology and distribution

2018 ◽  
Vol 52 (2) ◽  
pp. 245-252 ◽  
Author(s):  
S. I. Genkal ◽  
V. A. Gabyshev
Keyword(s):  
Species Composition ◽  
River Basin ◽  
New Records ◽  
Water Bodies ◽  
Centric Diatom ◽  
Diatom Species ◽  
Centric Diatoms ◽  
Sem Study ◽  
First Time ◽  
Sem Morphology

As a result of a SEM study of phytoplankton, the first data on centric diatom species composition in Bolshoye Toko Lake, Yakutia, were obtained. Ten species (Aulacoseira — 5, Cyclotella — 1, Discostella — 1, Handmannia — 1, Pliocaenicus — 1) were found, and one taxon from the genus Pliocaenicus was identified to the genus level. Of these, nine taxa are reported for the first time in the water bodies of the Aldan River basin and Discostella guslyakovyi in Yakutia.

scholarly journals Landscape Fragmentation around Ferruginous Caves of the Iron Quadrangle, Minas Gerais, Brazil

2019 ◽  
Vol 28 (1) ◽  
pp. 89-101 ◽  
Author(s):  
Mauro Gomes ◽  
Úrsula De Azevedo Ruchkys ◽  
Rodrigo Lopes Ferreira ◽  
Fernando Figueiredo Goulart
Keyword(s):  
Edge Effects ◽  
Landscape Fragmentation ◽  
Minas Gerais ◽  
Environmental Stability ◽  
Vegetation Coverage ◽  
Map Algebra ◽  
Research Article ◽  
Iron Quadrangle ◽  
Low Degree ◽  
Degree Of Fragmentation

The aim of this article is to analyse the landscape fragmentation around ferruginous caves in the Iron Quadrangle (Minas Gerais, Brazil) and the conditions of environmental stability of the caves inserted in vegetal remnants under edge effects caused by anthropic activities. The methodology applied involved the implementation of three different landscape metrics to stablish the total area of patches, the area of the patches under edge effects (core area), and the distance from the nearest neighbour. The measurements were calculated considering classes of vegetal coverage (herbaceous, shrub, and arboreal vegetation) and then processed and combined by using map algebra to obtain the fragmentation degree, which was classified into three classes: high, moderate, and low. Results reveal that 62.88% of the vegetation coverage of the study area presents a low degree of fragmentation. Among the caves under edge effects, 15% obtained negative indices of environmental stability. Although most of the analysed caves are located in areas with a low degree of fragmentation, the proximity of anthropized areas and the risk they represent for the speleological heritage reinforce the need to create strategies focused on the conservation of the caves.Featured ideas: research article on the results of research carried out regarding the composition and configuration of the landscape surrounding ferruginous caves located in the Iron Quadrangle, in the state of Minas Gerais, Brazil.

scholarly journals A whole-lake experiment confirms a small centric diatom species as an indicator of changing lake thermal structure

2016 ◽  
Vol 1 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Jasmine E. Saros ◽  
Robert M. Northington ◽  
Dennis S. Anderson ◽  
Nicholas John Anderson
Keyword(s):  
Thermal Structure ◽  
Centric Diatom ◽  
Diatom Species

Genetic poverty of an extremely specialized wetland species, Nehalennia speciosa: implications for conservation (Odonata: Coenagrionidae)

2009 ◽  
Vol 100 (4) ◽  
pp. 405-413 ◽  
Author(s):  
R. Bernard ◽  
T. Schmitt
Keyword(s):  
Isolation By Distance ◽  
Habitat Type ◽  
Genetic Distances ◽  
Allozyme Electrophoresis ◽  
Low Degree ◽  
Conservation Effort ◽  
Size Population ◽  
Wetland Species ◽  
Degree Of Isolation ◽  
Degree Of Fragmentation

AbstractOligo- and mesotrophic wetlands, such as bogs, fens and swamps, have become more and more restricted in Europe, and wetland species related to them have increasingly been threatened. Due to increasing habitat fragmentation, the exchange of individuals of these species among sites and, as a consequence, gene flow has been reduced or even eliminated. Therefore, we analysed the genetic structure of 11 populations of an endangered stenotopic damselfly, Nehalennia speciosa (Odonata: Coenagrionidae), in Poland and Lithuania by means of allozyme electrophoresis of 14 gene loci. The overall genetic diversity of all populations was low (A: 1.32; H: 2.6%; Ptot: 29.2%), and no significant differences were observed among the different groupings of populations (degree of fragmentation, habitat type and size, population size). The genetic differentiation among populations was also low (FST: 2.0%) and no regional groups were detected. A low degree of isolation by distance was observed for genetic distances. Taking into account these results, the conservation effort for this species should be focused on large local populations and not necessarily on metapopulation structures. Furthermore, N. speciosa could be (re-)introduced in extinct patches and seemingly suitable localities. Genetically, such relocations should be feasible due to the generally high genetic homogeneity of populations.

scholarly journals Identification and functional characterization of RNA silencing key-genes in the model pennate diatom species Phaeodactylum tricornutum

2019 ◽  
Author(s):  
Αιμιλία Γρυπιώτη
Keyword(s):  
In Silico ◽  
Small Rnas ◽  
Rna Silencing ◽  
Phaeodactylum Tricornutum ◽  
Functional Characterization ◽  
Diatom Species ◽  
Rna Dependent Rna Polymerase ◽  
Pennate Diatom ◽  
Key Genes

Ο μηχανισμός της RNA Σίγησης, γνωστός και ως μονοπάτι RNA παρεμβολής (RNA interference, RNAi), αποτελεί ένα συντηρημένο μηχανισμό ρύθμισης της γονιδιακής έκφρασης με τη διαμεσολάβηση μικρών RNA μορίων (sRNA), (Fire et al., 1998). Στα διάτομα, και στο διάτομο οργανισμό μοντέλο Phaeodactylum tricornutum, έχει παρατηρηθεί η σίγηση εξωγενών και ενδογενών γονιδίων μετά την εισαγωγή ανάστροφων επαναλήψεων, συμπληρωματικών μεταγράφων και τεχνητών miRNAs (De Riso et al., 2009; Kaur and Spillane, 2015). Στο P. tricornutum έχει προταθεί η παρουσία ενός ενδογενούς RNAi μονοπατιού μετά από μελέτες των sRNAs, της γονιδιακής έκφρασης και της μεθυλίωσης DNA του (Veluchamy et al., 2013; Rogato et al., 2014). Αυτό το RNAi μονοπάτι μπορεί να παίζει σημαντικό ρόλο στη ρύθμιση της έκφρασης γονιδίων που κωδικοποιούν πρωτεΐνες και μεταθετά στοιχεία (Transposable Elements,TEs) και πιθανώς να επηρεάζει την απόκριση προσαρμογής σε συνθήκες περιορισμένων θρεπτικών (Maumus et al., 2009). Ομόλογα των βασικών RNAi γονιδίων DICER (DCR), ARGONAUTE (AGO) και RNA-Dependent RNA polymerase (RDR) έχουν αναγνωριστεί με ανάλυση in silico (De Riso et al., 2009). Ωστόσο, η εξακρίβωση του γονιδιακού μοντέλου τους, ο χαρακτηρισμός της λειτουργίας τους και ο ρόλος τους σε επίπεδο φυσιολογίας παραμένουν άγνωστα. Στην παρούσα διατριβή, επιβεβαιώνεται η ύπαρξη ενός μοναδικού γονιδίου PtDCR, PtAGO και PtRDR στο P. tricornutum μετά από εκτεταμένη ανάλυση in silico των διαθέσιμων γονιδιωματικών και μεταγραφικών δεδομένων του. Η ανεύρεση και φυλογενετική ανάλυση των DCR, AGO και RDR ομόλογων γονιδίων στα διάτομα από όλες τις διαθέσιμες βάσεις δεδομένων παρουσιάζουν μια μη αναμενόμενη διαφοροποίηση του RNAi μονοπατιού σε αυτούς τους οργανισμούς. Το cDNA των PtDCR/AGO/RDR κλωνοποιήθηκε και αναγνωρίστηκαν μετάγραφα εναλλακτικού ματίσματος των PtDCR και PtAGO. Ο υποκυτταρικός εντοπισμός των PtDCR-/AGO-/RDR-YFP διερευνήθηκε με συνεστιακή μικροσκοπία. Για τον χαρακτηρισμό της λειτουργίας των PtDCR και PtAGO γονιδίων δοκιμάστηκε αρχικά η έκφρασή τους στα ετερόλογα συστήματα της ζύμης Saccharomyces cerevisiae και του φυτού Nicotiana bethamiana. Στη συνέχεια, με την εφαρμογή της CRISPR/Cas9-καθοδηγούμενης μεταλλαξιγένεσης, πρόσφατα προσαρμοσμένη στο P. tricornutum, δημιουργήθηκαν επιτυχώς οι μεταλλαγμένες σειρές με εκτοπισμό PtDCR-KO και PtAGO-KO (KnockOut). Η μελέτη του φαινοτύπου στην ανάπτυξη των PtDCR-KO σειρών έγινε σε καλλιέργειες υπό φυσιολογικές συνθήκες, υπό συνθήκες περιορισμένων νιτρικών και μετά από UV-επαγόμενο στρες. Παράλληλα, διεξήχθη αλληλούχιση νέας γενιάς (NGS) και ανάλυση των ολικών μεταγράφων mRNA και small RNAs. Τα πειράματα των καλλιεργειών υποδεικνύουν ότι η PtDCR ίσως παίζει ρόλο στην απόκριση υπό την έλλειψη νιτρικών. Η μεταγραφική ανάλυση αποκάλυψε ότι και τα sRNA και τα mRNA μετάγραφα επηρεάστηκαν στη DCR-KO σειρά. Συνολικά, η κατανομή του μεγέθους των sRNA μετατοπίστηκε προς μεγαλύτερου μεγέθους μόρια στην PtDCR-KO σειρά. Επίσης, ο αριθμός των sRNA που εντοπίζονται σε TEs ήταν δραματικά μειωμένος στη PtDCR-KO σειρά και παρατηρήθηκε μία συνακόλουθη αύξηση του αριθμού των mRNA κάποιων TEs. Ενδιαφέρον παρουσιάζει ότι στα PtDCR-KO sRNA μετάγραφα παρατηρούνται επίσης αλλαγές στους tRNA-παραγόμενους sRNA πληθυσμούς υποδεικνύοντας ένα πιθανό ρόλο της DCR στην επεξεργασία αυτών των μορίων στα διάτομα. Έχει προταθεί ότι η κινητοποίηση των TE παίζει σημαντικό ρόλο στη εξελικτική διαφοροποίηση των διατόμων και στην ικανότητά τους για γρήγορη απόκριση και προσαρμογή σε ποικίλα περιβάλλοντα. Συμπερασματικά, τα αποτελέσματα αυτής της διατριβής δείχνουν ότι το μοναδικό γονίδιο DCR στο P. tricornutum παίζει σημαντικό ρόλο στην παραγωγή των TE-παραγόμενων sRNAs και πιθανά στην κινητοποίηση των TEs, επηρεάζοντας σημαντικά την απόκριση προσαρμογής των διατόμων και την εξέλιξή τους.

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