BUSCADOR RECOLECTA

(A-C) Genomic organization of tandemly arranged aqp1ab2 and aqp1ab1 genes in seabream (A) and Atlantic halibut (B), and of the single aqp1ab2 gene in Senegalese sole and Common sole (C). The four exons (1–4) of both genes are represented by blue and red boxes, and the start codons are indicated by upper arrows. Lower arrows indicate the position of the oligonucleotide primers used for RT-PCR. (D-E) Representative RT-PCR analysis of aqp1ab1 and aqp1ab2 expression in different adult tissues from seabream (D) and halibut (E). A single aqp1ab2 mRNA species is expressed in seabream and halibut, whereas two or one splice variants (aqp1ab1_v1 and aqp1ab1_v2) in addition to aqp1ab1 are detected, respectively, as indicated. (F-G) RT-PCR of aqp1ab2 expression in Senegalese sole (F) and Common sole (G) where two splice forms (aqp1ab2_v1 and aqp1ab2 _v2) in addition to aqp1ab2 are amplified in the ovary and testis. In D-G, the sizes (kb) of molecular markers are indicated on the left., Peer reviewed

(A-C) Schematic diagram of the seabream and halibut aqp1ab1 and sole aqp1ab2 genes, and the different emerging transcript variants identified in each species. The numbers represent the exons, and the initiation and stop codons in each mRNA are indicated. (D-F) Integral membrane views (cartoon renders) of the wild type and variant channels. Canonical transmembrane domains are shown in cyan, loops in magenta and the two conserved Asn-Pro-Ala (NPA) motifs in dark blue. Variant regions are shown in red. In sole Aqp1ab2_v2 (C and F), the splicing of the distal region of intron 2 causes out-of-frame readthrough of exon 3 and the spliced exon 4 resulting in a premature stop codon and the truncated protein., Peer reviewed

(A-C) Pf of X. laevis oocytes injected with water (W, controls) or expressing seabream SaAqp1ab1-WT, SaAqp1ab1_v1 or SaAqp1ab1_v2 (A), Atlantic halibut HhAqp1ab1-WT or HhAqp1ab1_v1 (B), or sole SsAqp1ab2-WT, SsAqp1ab2_v1 or SsAqp1ab2_v2 (C). Oocytes injected with SsAqp1ab2-WT or splice forms were co-injected with halibut YwhazLb and exposed to FSK for 1 h prior to the swelling assay. The data are the mean ± SEM (n indicated above each bar) and were statistically analyzed by an unpaired Student’s t-test (***, P < 0.001; with respect to controls). (D-F) Double immunostaining of oocytes expressing untagged SaAqp1ab1-WT or HA-tagged SaAqp1ab1_v1 or HA-SaAqp1ab1_v2 (D), Flag-tagged HhAqp1ab1-WT or HA-tagged HhAqp1_v1 (E), or Flag-tagged SsAqp1ab2-WT or HA-tagged SsAqp1ab2_v1 or HA-SsAqp1ab2_v2 (F), and the ER marker protein disulfide isomerase (PDI). The plasma membrane is indicated by an arrowhead, whereas the co-localization of aquaporin channels and PDI in the cytoplasm is indicated by arrows. Scale bars, 10 μm (insets 5 μm)., Peer reviewed

(A-C) Changes in Pf of X. laevis oocytes injected with water or expressing SaAqp1ab1-WT (A), HhAqp1ab1-WT (B) or SsAqp1ab2-WT (C) alone or in combination with different amounts of the isoforms of each paralog. For SsAqp1ab2-WT and splice forms, oocytes were co-injected with halibut YwhazLb and exposed to FSK. (D) Effect of the inhibition of the oocyte Pf by the different Aqp1ab1 splice forms, at a concentrations that produced approximately half-maximal reduction (as observed in A-C), in SaAqp1ab1 or HhAqp1ab1 expressing oocytes in the presence or absence of YwhazLa, and exposed to FSK. The percentage of Pf inhibition elicited by each isoform under the two conditions is indicated in each plot. In all panels, data are the mean ± SEM (n indicated above each bar) and were statistically analyzed by an unpaired Student’s t-test (*, P < 0.05: **, P < 0.01; ***, P < 0.001; with respect to oocytes injected with the WT form alone)., Peer reviewed

Figure S1: UV-VIS spectra of the SERS substrates herein studied: Glass_ Ag, Glass_AuNPs, Glass_Ag_AuNPs, 1G_Ag_AuNPs, 3G_Ag_AuNPs; Figure S2: SERS spectra of 6 spots recorded on bottom and top of 1G_Ag_AuNPs sample upon exposure to 1.2 ppmV DMMP in gas phase., Peer reviewed

Additional proposed models and more STEM images along different crystallographic orientations together with the simulated data are included., Peer reviewed

(A-C) Double immunolocalization of SaAqp1ab1-WT, Flag-tagged HhAqp1ab1-WT or Flag-tagged SsAqp1ab2-WT (green) with the respective HA-tagged splice forms in X. laevis oocytes as indicated. Oocytes expressing SsAqp1ab2-WT and splice forms were co-injected with halibut YwhazLb and exposed to FSK. In all panels, the plasma membrane is indicated by an arrowhead, whereas the co-localization of WT aquaporins and splice forms is indicated by arrows. Scale bars, 10 μm. (D-F) Representative immunoblots of total (TM) and plasma (PM) membrane protein extracts from oocytes injected with water (W) or co-expressing the different aquaporin WT channels with increasing amounts of the splice forms as indicated. (G-I) Illustrative immunoblots of precipitated proteins from oocytes treated as in A-C using anti-SaAqp1ab1 or anti-Flag (for HhAqp1ab1 and SsAqp1ab2) antibodies, and revealed with antibodies against SaAqp1ab1, Flag or HA. In D-I, upper blots were probed with SaAqp1ab1 (D and G) or Flag (E, F, H and I) antibodies, whereas in all panels the lower blots were probed with HA antibodies. Molecular mass markers (kDa) are on the left. The arrowhead in F and I indicate potential post-translational modifications of the SsAqp1ab1_v2 isoform., Peer reviewed

1 file, Supplementary material for the article https://doi.org/10.3390/foods9091239, Figure S1: Culture kinetics of Lb 1.-- Figure S2: Culture kinetics of Ln.-- Figure S3: Culture kinetics of Lb 3.-- Figure S4: Economical evaluation of Lb 2 bioproduction costs.-- Figure S5: Economical evaluation of Ln bioproduction costs.-- Figure S6: Economical evaluation of Lb 3 bioproduction costs.-- Table S1: Parameter kinetics of Lb 2 cultures.-- Table S2: Continuation of Table S2.-- Table S3: Parameter kinetics of Ln cultures.-- Table S4: Continuation of Table S3.-- Table S5: Parameter kinetics of Lb 3 cultures.-- Table S6: Continuation of Table S5.-- Table S7: Ranges of productive yields.-- Table S8: Amino acids content of fish discard peptones.-- Table S9: Continuation of Table S8, Peer reviewed

(A) Amino acid sequence alignment of SsAqp1ab2-WT, SsAqp1ab2_v1 and SsAqp1ab2_v2 splice forms highlighting the sequences employed for the production of the sole-specific Aqp1ab2 and Aqp1ab2_v1 antibodies (α-Aqp1ab2-Nt and α-Aqp1ab2_v1, in red and blue color, respectively). The N- and C-termini of the WT channel, the six transmembrane helices (TM1-TM6) and the two conserved NPA motifs are also indicated. (B) Immunoblots of X. laevis oocytes injected with water or expressing Aqp1ab2-WT, SsAqp1ab2_v1 or SsAqp1ab2_v2 and probed with the α-Aqp1ab2-Nt or α-Aqp1ab2_v1 antisera separately. (C) Representative photomicrographs of sole ovarian follicles at different developmental stages during oocyte growth and maturation. PG, primary growth stage; CA, cortical alveoli stage. Scale bars, 50 and 250 μm. (D) Immunoblots of Aqp1ab2-WT, Aqp1ab2_v1 and Aqp1ab2_v2 in protein extracts from previtellogenic (Pv), vitellogenic (Vt), hydrating (H) and mature (Mt) follicle-enclosed oocytes, as well as from ovulated oocytes (Ov), using the generated antisera. Alpha tubulin (Tuba) was used as loading control. Duplicated blots (right) were run in parallel and incubated with the primary antibodies preadsorbed by the antigenic peptide to test for specificity. The brackets indicate potential post-translational modifications. (E) Hoechst staining of intact vitellogenic follicles and defolliculated oocytes. Scale bar, 100 μm. (F) Immunoblots of Aqp1ab2 isoforms in total protein extracts from intact or defolliculated vitellogenic follicles (+/- follicle cells, Fc). In B, D and E, the arrowheads indicate aquaporin monomers, whereas the asterisks in D and E indicate cross-reactive polypeptides revealed with the Aqp1ab2_v1 antiserum. In all blots, molecular mass markers (kDa) are on the left., Peer reviewed

(A, D, G. J) Representative histological sections of follicle-enclosed oocytes at previtellogenesis, including the primary growth (A) and cortical alveoli (D) stages, vitellogenic (G) and maturing and hydrating (J) stage stained with hematoxylin and eosin. (B-C, E-F, H-I, K-L) Bright field (BF) images and immunostaining of total Aqp1ab2 or Aqp1ab2_v1 (red) in follicles in each of the different stages using the sole-specific α-Aqp1ab2-Nt and α-Aqp1ab2_v1 antisera. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and wheat germ agglutinin (WGA; green). Control sections incubated with preabsorbed antisera were negative (S1 Fig). The arrows and arrowheads, indicate granulosa and theca cells, respectively, surrounding the oocyte. Abbreviations: o, oocyte; y, yolk globule; gv, germinal vesicle; ve, vitelline envelope; cp, capillary; ca, cortical alveoli. Scale bars, 10 μm (A-D, H, I, L, K, and inset in I and L), 50 μm (G, J), 20 μm (E, F), 5 μm (inset in E and F)., Peer reviewed