A broad diversity of sex-determining systems has evolved in eukaryotes. However, information on the mechanisms of sex determination for unicellular microalgae is limited, including for diatoms, ...key-players of ocean food webs. Here we report the identification of a mating type (MT) determining gene for the diatom Pseudo-nitzschia multistriata. By comparing the expression profile of the two MTs, we find five MT-biased genes, of which one, MRP3, is expressed exclusively in MT+ strains in a monoallelic manner. A short tandem repeat of specific length in the region upstream of MRP3 is consistently present in MT+ and absent in MT- strains. MRP3 overexpression in an MT- strain induces sex reversal: the transgenic MT- can mate with another MT- strain and displays altered regulation of the other MT-biased genes, indicating that they lie downstream. Our data show that a relatively simple genetic program is involved in defining the MT in P. multistriata.
Flagella have been lost in the vegetative phase of the diatom life cycle, but they are still present in male gametes of centric species, thereby representing a hallmark of sexual reproduction. This ...process, besides maintaining and creating new genetic diversity, in diatoms is also fundamental to restore the maximum cell size following its reduction during vegetative division. Nevertheless, sexual reproduction has been demonstrated in a limited number of diatom species, while our understanding of its different phases and of their genetic control is scarce.
In the transcriptome of Leptocylindrus danicus, a centric diatom widespread in the world's seas, we identified 22 transcripts related to the flagella development and confirmed synchronous overexpression of 6 flagellum-related genes during the male gamete formation process. These transcripts were mostly absent in the closely related species L. aporus, which does not have sexual reproduction. Among the 22 transcripts, L. danicus showed proteins that belong to the Intra Flagellar Transport (IFT) subcomplex B as well as IFT-A proteins, the latter previously thought to be absent in diatoms. The presence of flagellum-related proteins was also traced in the transcriptomes of several other centric species. Finally, phylogenetic reconstruction of the IFT172 and IFT88 proteins showed that their sequences are conserved across protist species and have evolved similarly to other phylogenetic marker genes.
Our analysis describes for the first time the diatom flagellar gene set, which appears to be more complete and functional than previously reported based on the genome sequence of the model centric diatom, Thalassiosira pseudonana. This first recognition of the whole set of diatom flagellar genes and of their activation pattern paves the way to a wider recognition of the relevance of sexual reproduction in individual species and in the natural environment.
•Microfluidic platform allowing fast RT-qPCR analysis on continuously flowing droplets.•Screening of multiple combinations of samples and target genes in the same run.•The platform is optimized for ...both small and large number of samples.•It is perfectly suitable for diagnostic applications.•Automated system for generation and merging of pairs of droplets.
RT-qPCR represents a key method in cancer diagnostic, however the constant increase in patients and cancer biomarker panels to screen requires the implementation of faster approaches allowing smaller reagents and samples volumes consumption. To fulfil these needs, we present here a fully automated droplet microfluidics platform that couples a specifically designed thermal system with a fluorescence excitation/detection module. Additionally, the droplet generation and merging approaches allows the analysis of multiple samples and genes with no risk of contamination. This platform has been initially validated by investigating HER2 overexpression in two cancer cell lines starting from total RNA samples; these results have been successfully compared with those obtained by a commercially available machine, showing no limitation in terms of number of processed samples as well as reducing of 200 times the analysis costs per patient. Finally, we have demonstrated its capability in performing fast RT-qPCR, raising the throughput of analysis to a hundred samples in less than 20 min.
Diatoms are a fundamental microalgal phylum that thrives in turbulent environments. Despite several experimental and numerical studies, if and how diatoms may profit from turbulence is still an open ...question. One of the leading arguments is that turbulence favours nutrient uptake. Morphological features, such as the absence of flagella, the presence of a rigid exoskeleton and the micrometre size would support the possible passive but beneficial role of turbulence on diatoms. We demonstrate that in fact diatoms actively respond to turbulence in non-limiting nutrient conditions. TURBOGEN, a prototypic instrument to generate natural levels of microscale turbulence, was used to expose diatoms to the mechanical stimulus. Differential expression analyses, coupled with microscopy inspections, enabled us to study the morphological and transcriptional response of Chaetoceros decipiens to turbulence. Our target species responds to turbulence by activating energy storage pathways like fatty acid biosynthesis and by modifying its cell chain spectrum. Two other ecologically important species were examined and the occurrence of a morphological response was confirmed. These results challenge the view of phytoplankton as unsophisticated passive organisms.
Transposable elements (TEs) widely contribute to the evolution of genomes allowing genomic innovations, generating germinal and somatic heterogeneity, and giving birth to long non-coding RNAs ...(lncRNAs). These features have been associated to the evolution, functioning, and complexity of the nervous system at such a level that somatic retrotransposition of long interspersed element (LINE) L1 has been proposed to be associated to human cognition. Among invertebrates, octopuses are fascinating animals whose nervous system reaches a high level of complexity achieving sophisticated cognitive abilities. The sequencing of the genome of the Octopus bimaculoides revealed a striking expansion of TEs which were proposed to have contributed to the evolution of its complex nervous system. We recently found a similar expansion also in the genome of Octopus vulgaris. However, a specific search for the existence and the transcription of full-length transpositionally competent TEs has not been performed in this genus.
Here, we report the identification of LINE elements competent for retrotransposition in Octopus vulgaris and Octopus bimaculoides and show evidence suggesting that they might be transcribed and determine germline and somatic polymorphisms especially in the brain. Transcription and translation measured for one of these elements resulted in specific signals in neurons belonging to areas associated with behavioral plasticity. We also report the transcription of thousands of lncRNAs and the pervasive inclusion of TE fragments in the transcriptomes of both Octopus species, further testifying the crucial activity of TEs in the evolution of the octopus genomes.
The neural transcriptome of the octopus shows the transcription of thousands of putative lncRNAs and of a full-length LINE element belonging to the RTE class. We speculate that a convergent evolutionary process involving retrotransposons activity in the brain has been important for the evolution of sophisticated cognitive abilities in this genus.
Current information on the response of phytoplankton to turbulence is linked to cell size and nutrient availability. Diatoms are considered to be favored by mixing as dissolved nutrients are more ...easily accessible for non-motile cells. We investigated how diatoms exploit microscale turbulence under nutrient repletion and depletion conditions. Here, we show that the chain-forming diatom Chaetoceros decipiens, continues to take up phosphorus and carbon even when silicon is depleted during turbulence. Our findings indicate that upon silica depletion, during turbulence, chain spectra of C. decipiens remained unchanged. We show here that longer chains are maintained during turbulence upon silica depletion whereas under still conditions, shorter chains are enriched. We interpret this as a sign of good physiological state leading to a delay of culture senescence. Our results show that C. decipiens senses and responds to turbulence both in nutrient repletion and depletion. This response is noteworthy due to the small size of the species. The coupling between turbulence and biological response that we depict here may have significant ecological implications. Considering the predicted increase of storms in Northern latitudes this response might modify community structure and succession. Our results partly corroborate Margalef's mandala and provide additional explanations for that conceptualization.
Lactic acidosis is a buildup of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase ...complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1α subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57BL/6 wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1α in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1α through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noa(m631) zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis.
The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low ...concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO
. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.
In humans, OFD1 is mutated in oral-facial-digital type I syndrome leading to prenatal death in hemizygous males and dysmorphic faces and brain malformations, with polycystic kidneys presenting later ...in life in heterozygous females. To elucidate the function of Ofd1, we have studied its function during zebrafish embryonic development. In wild-type embryos, ofd1 mRNA is widely expressed and Ofd1-green fluorescent protein (GFP) fusion localizes to the centrosome/basal body. Disrupting Ofd1 using antisense morpholinos (MOs) led to bent body axes, hydrocephalus and oedema. Laterality was randomized in the brain, heart and viscera, likely a consequence of shorter cilia with disrupted axonemes and perturbed intravesicular fluid flow in Kupffer's vesicle. Embryos injected with ofd1 MOs also displayed convergent extension (CE) defects, which were enhanced by loss of Slb/Wnt11 or Tri/Vangl2, two proteins functioning in a non-canonical Wnt/Planar Cell Polarity (PCP) pathway. Pronephric glomerular midline fusion was compromised in vangl2 and ofd1 loss of function embryos and we suggest this anomaly may be a novel CE defect. Thus, Ofd1 is required for ciliary motility and function in zebrafish, supporting data showing that Ofd1 is essential for primary cilia function in mice. In addition, our data show that Ofd1 is important for CE during gastrulation, consistent with data linking primary cilia and non-canonical Wnt/PCP signalling.
•Microvalve prepared as monolithic PDMS device showing negligible back-flow volume.•The fabricated microvalve can sustain high pressure operations (up to 4bar).•Fabrication combines 3D printing, PDMS ...replica molding and wire-removal strategy.•Droplets can pass through the valve without been damaged or perturbed.•Valve is suitable for Droplet-on-Demand generation and droplet sorting.
Droplet microfluidics allows the implementation of several biochemical and bioanalytical protocols in microfluidic devices. However, this integration typically requires the use of microfluidic valves, which allow the control of different operations on the same channels network. Unfortunately, the valves commercially available are almost exclusively suitable for-single phase flow, and only one valve system compatible with multi-phase systems has been presented so far in the literature. We present here a new pinch-like valve that withstands high pressures (tested up to 4bar), allows droplet manipulation and combines the advantages of conventional pinch valves regarding the absence of dead volume, while making negligible the backflow generated (less than 2nL). The conception and design of the presented valve, based on numerical simulations, is performed by an optimized microfabrication strategy, combining PDMS soft-lithography and 3D printing technologies. The valves had been tested regarding their mechanical stability and proving their capability of allowing droplets passing through them, without any perturbation. Finally, the very small back-flow volume generated during the actuation, has been exploited for various applications in droplets microfluidics, like droplet handling, droplet-on-demand generation and sorting.
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