Evaluation of an ultrafiltration membrane for the removal of fish viruses and bacteria in aquaculture water

publishedVersio

Nasal responses to elevated temperature and Francisella noatunensis infection in Atlantic cod (Gadus morhua)

We report the histological and transcriptomic changes in the olfactory organ of Atlantic cod exposed to Francisella noatunensis. Experimental infection was performed at either 12oC or 17oC. Infected fish presented the classic gross pathologies of francisellosis. Nasal morpho-phenotypic parameters were not significantly affected by temperature and infection, except for the number of mucus cells in the 12oC group seven weeks after the challenge. A higher number of genes were altered through time in the group reared at 17oC. At termination, the nasal transcriptome of infected fish in both groups was similar to the control. When both infected groups were compared, 754 DEGs were identified, many of which were involved in signalling, defence, transmembrane and enzymatic processes. In conclusion, the study reveals that elevated temperature could trigger responses in the olfactory organ of Atlantic cod and shape the nasal response to F. noatunensis infection.Nasal responses to elevated temperature and Francisella noatunensis infection in Atlantic cod (Gadus morhua)publishedVersio

Sulforaphane enhances SPBP expression.

<p>(<b>A</b>) Schematic representation of the domain structure of human SPBP. TAD: trans-activation domain, DBD: DNA binding domain, NLS: Nuclear Localisation Signal, ePHD/ADD: extended PHD/ADD domain, Q1/Q2: Glutamine rich stretches. Numbers below indicate amino acid positions. (<b>B</b>) NRF2, SPBP and p62 display similar induction upon sulforaphane treatment. HeLa cells were exposed to 20 µM sulforaphane and cell extracts harvested for the indicated time points. Equivalent aliquots from the extracts were subjected to SDS-PAGE and western blot using specific anti-SPBP antibody, anti-p62 antibody, anti-NRF2 antibody or anti-actin as indicated. Fold induction calculated and correlated to actin in three independent experiments with standard deviations are shown to the right (**p<0.01, *p<0.05). (<b>C</b>) Control experiment showing that DMSO alone does not induce any changes in NRF2, SPBP or p62 expression levels. Equivalent aliquots of HeLa cell extracts exposed to DMSO and harvested for the indicated time points were subjected to SDS-PAGE and western blot using specific anti-SPBP antibody, anti-p62 antibody, anti-NRF2 antibody or anti-actin antibody as indicated. Fold induction calculated and correlated to actin are shown to the right.</p

SPBP and NRF2 cooperate to induce expression from the p62 promoter, and colocalize in nuclear speckles.

<p>(<b>A</b>) SPBP and NRF2 cooperate to enhance expression from the wild type p62 promoter. Transient transfections were carried out in HEK293 cells using 60 ng of the p62 promoter construct (−1781/+46), and 50 ng or 100 ng of an NRF2 expression vector, and 100 ng of a SPBP expression vector, as indicated. The data represent the mean of three independent experiments with standard deviations, each performed in triplicate (***p<0.001, *p<0.05). (<b>B</b>) SPBP recruits NRF2 to specific nuclear speckles. HeLa cells were transiently transfected with expression vectors for EGFP-NRF2 and mCherry-SPBP, and analysed 24 hours post transfection by live cell imaging using a confocal laser scanning fluorescence microscope. The Pearson's colocalisation scatter was generated using Volocity (Perkin Elmer).</p

Nucleosome positioning sequences impair the synergistic effect of NRF2 and SPBP on the p62 promoter.

<p>Reporter constructs (60 ng) containing the indicated nucleosome position sequences inserted downstream of the transcription start site of p62 promoter were transfected into HEK293 cells together with the indicated amounts of expression plasmids for SPBP and/or NRF2. The luciferase activity of the promoter constructs cotransfected with empty expression plasmids was set to 1. The data represent the mean of three independent experiments with standard deviations, each performed in triplicate (***p<0.001, **p<0.01, *p<0.05).</p

Knock down of SPBP impairs p62 expression and sulforaphane induced p62 body formation.

<p>(<b>A</b>) Endogenous SPBP and p62 are coexpressed in several cell lines. Extracts of the indicated human cell lines were analysed by western blotting using the indicated antibodies. (<b>B</b>) siRNA mediated knock down of SPBP reduces p62 expression level. HeLa cells transfected with two various SPBP siRNAs were analysed by western blotting using antibodies as indicated (left panel). The graph (right panel) shows the fold reduction calculated and correlated to actin in three independent experiments with standard deviations (*p<0.05, <i>n.s.</i> not significant). (<b>C</b>) Knock down of SPBP reduces the amount of p62 mRNA transcripts. The p62 mRNA levels were measured by quantitative RT-PCR. Hela cells were transfected with SPBP siRNAs or Control siRNA. RT-PCR reactions were run on p62, GADPH and β-actin mRNA. The average amount of p62 mRNA correlated to β-actin and GADPH mRNA based on two independent experiments are shown with standard deviations (*p<0.05, <i>n.s.</i> not significant). (<b>D</b>) p62 body formation upon sulforaphane treatment is reduced in SPBP siRNA knock down cells. HeLa cells were transfected with SPBP siRNA or Control siRNA and treated with 20 µM sulforaphane for 8 hours two days post transfection. Cells were fixed, stained with polyclonal antibodies against SPBP (green) and p62 (red) and analysed by confocal microscopy. The graph shows counting of p62 bodies in cells, each based on counting of more than 60 cells. Arrowheads indicate some of the p62 bodies in the cytoplasm.</p

SPBP mediates transcriptional activation via ARE elements.

<p>(<b>A</b>) Schematic representation of the human −1781/+46 p62 promoter construct in front of the Luciferase gene. Conserved transcription factor binding sites relevant for this study are indicated. (<b>B</b>) Mutation of the ARE elements impairs SPBP mediated activation of the p62 promoter. Transient transfections were carried out in HEK293 cells using 60 ng of wild-type or mutated reporter vectors, and 100 ng of expression vectors for SPBP (upper panel) or CBP (lower panel). The data represent the mean of three independent experiments with standard deviations, each performed in triplicate (**p<0.01, *p<0.05, <i>n.s.</i> not significant). (<b>C</b>) Chromatin immunoprecipitations show that SPBP is associated with the p62 promoter. HeLa cell extracts (1.5×10⁷ cells per antibody) were immunoprecipitated with preimmune serum, polyclonal anti-SPBP antibody or polyclonal anti-NRF2 antibody. Input Control (1:40) was included. PCR was performed on chromatin precipitated with each antibody using primers aligning to positions −1324/−1173 in the p62 promoter (upper panel). Primers aligning to positions −3351/−3069 of the cathepsin D promoter were used as control. (<b>D</b>) SPBP mediated enhancement of the NQO1 promoter is dependent on the ARE element. Transient transfections were carried out in HEK293 cells using 60 ng of wild-type or mutated reporter vectors, and 100 ng of SPBP expression vectors. The data shows the mean of three independent experiments with standard deviations, each performed in triplicate (**p<0.01, <i>n.s.</i> not significant).</p

NIPSNAP1 and NIPSNAP2 Act as “Eat Me” Signals for Mitophagy

The clearance of damaged or dysfunctional mitochondria by selective autophagy (mitophagy) is important for cellular homeostasis and prevention of disease. Our understanding of the mitochondrial signals that trigger their recognition and targeting by mitophagy is limited. Here, we show that the mitochondrial matrix proteins 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) and NIPSNAP2 accumulate on the mitochondria surface upon mitochondrial depolarization. There, they recruit proteins involved in selective autophagy, including autophagy receptors and ATG8 proteins, thereby functioning as an “eat me” signal for mitophagy. NIPSNAP1 and NIPSNAP2 have a redundant function in mitophagy and are predominantly expressed in different tissues. Zebrafish lacking a functional Nipsnap1 display reduced mitophagy in the brain and parkinsonian phenotypes, including loss of tyrosine hydroxylase (Th1)-positive dopaminergic (DA) neurons, reduced motor activity, and increased oxidative stress

SAMM50 acts with p62 in piecemeal basal- and OXPHOS-induced mitophagy of SAM and MICOS components

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy