BUSCADOR RECOLECTA

17 pages. -- Characterization. -- Electrode preparation and electrochemical measurements. -- Density functional theory calculations. -- Figure S1-S17. -- Table S1. Elemental composition of ZnCo1.26Ni0.73Ox –SO4 before and after OER. -- Table S2. Assignment of FTIR characteristics peaks. -- Table S3. Comparison of OER activity of amorphous ZnCoxNiyOy-SO4 nanosheets with recently reported electrocatalysts in alkaline electrolyte. -- Table S4. Cdl and ECSAs of various catalysts. -- Table S5. Samples produced and characterized, including name, metal precursors type and nominal amount., Peer reviewed

All images are super-resolution single confocal sections except B, which is a projection of super-resolution confocal sections. (A, B) In the trachea of wild-type embryos, Reb and Kkv do not colocalise, and they show a complementary pattern (A’-A”’) at the local subcellular level. (C) In salivary gland of embryos expressing Reb, the patterns of Kkv and Reb are complementary. (D) Models for the role of kkv and exp/reb in chitin deposition. Kkv oligomerises in complexes that localise to the apical membrane (as proposed in [2]). In the absence of exp/reb activity, Kkv can polymerise chitin from sugar monomers (discontinuous red lines), but it cannot translocate it because the channel is closed, and polymerised chitin remains in the cytoplasm. In addition, Kkv is not homogeneously distributed. Exp/Reb form a complex with other proteins, which localises to the apical membrane. The presence of Exp/Reb complex regulates Kkv apical distribution and activity. In model 1, we propose that a factor/s recruited by Exp/Reb (Factor X) can induce a posttranslation or conformational modification to Kkv protein that opens the channel promoting translocation of chitin fibers to the extracellular domain. In model 2, we propose that a factor/s recruited by Exp/Reb (Factor X’) can induce changes in membrane composition/curvature that will then promote a conformational change in Kkv that opens the channel to translocate chitin. These membrane changes lead to Kkv shedding extracellularly. In model 3, we propose that Exp/Reb complex can bind and relocalise Factor X”, which normally inhibits Kkv-translocating activity. This neutralises the activity of Factor X” allowing chitin translocation. Scale bars: 5 μm., Peer reviewed

8 pages. -- Figure S1. Schematic diagram describing integration of the MUA-functionalized electrode array with a microwell layer. -- Figure S2. Electrochemical characterization of a microtiter plate. -- Figure S3. Nanoparticle-enabled electrochemical immunoassay for detection of podocin and nephrin. -- Figure S4. Characterization of urinary EVs. -- Figure S5. Using SPR to characterize EV capture and immunoprobe binding. -- Figure S6. Representative SEM images of EVs captured on an electrode surface functionalized with anti-CD63 Abs. -- Figure S7. SPR analysis of clinical urine samples., Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted by cells. EVs contain biological information related to parental cells and provide biomarkers for disease diagnosis. We have previously shown that the levels of podocin and nephrin expression on urinary EVs may be used to diagnose renal injury associated with preeclampsia. This paper describes a nanoparticle-enabled immunoassay integrated with an electrochemical plate for quantifying podocin and nephrin expression in urinary EVs. The strategy entailed capturing EVs on an electrode surface and then labeling EVs with gold nanoparticles that are both functionalized with antibodies for target specificity and impregnated with redox-active metal ions for electrochemical detection. These immunoprobes produced an electrochemical redox signal proportional to the expression level of EV surface markers. Electrochemical immunoassays were carried out in a novel microtiter plate that contained 16 wells with working electrodes connected to onboard counter/reference electrodes via capillary valves. Upon validation with recombinant proteins, a microtiter plate was used for analysis of urinary EVs from healthy and preeclamptic pregnant women. This analysis revealed a higher podocin to nephrin ratio for preeclamptic women compared to healthy controls (4.31 vs 1.69) suggesting that this ratio may be used for disease diagnosis., Peer reviewed

All images are projections of confocal sections, of super-resolution microscopy. (A, B) Kkv localises apically in the trachea of wild-type embryos (A) and in absence of exp reb (B). (C, D) The localisation of Kkv is apical also in presence of exp ΔMH2 in trachea (C) and in presence of MH2-exp in salivary glands (D). (E, F) At stage 14, in wild-type embryos (E) and in embryos deficient for exp and reb (F), Kkv is present in the apical membrane and in many intracellular vesicles (yellow arrowheads). (G) At stage 16, in wild-type embryo, Kkv apical distribution follows the pattern of taenidial folds and intracellular vesicles are mostly absent. (H) At stage 16, in exp reb mutant embryos, Kkv is apical but shows altered distribution pattern. (I, J) At stage 15, in control embryos, Kkv pattern is apical and covers the whole membrane leaving minimal spatial gaps (I); instead, in exp reb mutant embryos, Kkv distribution changes to a less organised pattern at the apical membrane (J). (K) Three different types of spatial distribution within a selected area. The positions of the defined objects can be random and exhibit characteristics of attraction (clustered pattern) or repulsion (regular pattern). The F-Function tends to be larger (≈1) for clustered patterns and smaller (≈0) for regular. The G-Function tends to be smaller (≈0) for clustered and larger (≈1) for regular patterns. (L) Kkv punctae (magenta) on the apical cell area marked by Armadillo (green) in the trachea of a control embryo. (L’) Positions of Kkv punctae on the selected area marked by black dots. (L”) Random pattern of distribution for the same area created by the spatial statistics 2D/3D image analysis plugin. (M) The corresponding observed F and G functions (blue) are displayed above and below the reference simulated random distributions (black) and the 95% confidence interval (light gray), respectively, indicating a nonrandom spatial pattern. (N) SDI histogram for the F-Function of the control (blue) and the Df(exp reb) samples. A significant difference between the frequency distributions for each group of individuals has been observed. (Kolmogorov–Smirnov D = 0.5833, p < 0.05) (N’) SDI histogram for the G-Function of the control (blue) and the Df(exp reb) samples. Statistical analysis of the distributions did not reveal significant differences between the two groups of individuals for this parameter (Kolmogorov–Smirnov D = 0.25, p > 0.05). (O) Kkv punctae (magenta) on the apical cell area marked by Armadillo (green) in the trachea of a exp reb mutant embryo. (O’) Positions of Kkv punctae on the selected area marked by black dots. (O”) Random pattern of distribution for the same area created by the spatial statistics 2D/3D image analysis plugin. (P) The corresponding observed F and G functions (blue) are displayed above and below the reference simulated random distributions (black), respectively. Both curves largely overlap with the 95% confidence interval (light gray), indicating a tendency towards a random spatial pattern. (Q) Frequency distribution histograms for the nearest neighbour distances between Kkv punctae in control (blue) and exp reb mutant samples. The distribution of values between the two groups is found significantly different (Kolmogorov–Smirnov D = 0.2036, p < 0.005). The underlying data for quantifications can be found in the S1 Data. Scale bars A-J: 10 μm; L, O: 2 μm., Peer reviewed

63 pages. -- Section 1. Materials and methods. -- Section 2. Synthesis and characterization data of DPA-S-COOH and intermediates to obtain it. -- Section 2. Synthesis and characterization data of Pd-S-COOH and intermediates to obtain it. -- Section 3. Synthesis and characterization data of CAEBD-S-COOH and intermediates to obtain it. -- Section 4. Synthesis and characterization data of Os-S-COOH and intermediates to obtain it. -- Section 5: Preparation of nanoparticles and nanocomposite materials. -- Section 6: Supplementary Schemes, Tables, Figures and videos, Photon upconversion (UC) based on triplet–triplet annihilation is a very promising phenomenon with potential application in several areas, though, due to the intrinsic mechanism, the achievement of diffusion-limited solid materials with air-stable UC is still a challenge. Herein, we report UC coordination polymer nanoparticles (CPNs) combining sensitizer and emitter molecules especially designed with alkyl spacers that promote the amorphous character. Beyond the characteristic constraints of crystalline MOFs, amorphous CPNs facilitate high dye density and flexible ratio tunability. To show the universality of the approach, two types of UC-CPNs are reported, exhibiting highly photostable UC in two different visible spectral regions. Given their nanoscale, narrow size distribution, and good chemical/colloidal stability in water, the CPNs were also successfully printed as anticounterfeiting patterns and used to make highly transparent and photostable films for luminescent solar concentrators, both showing air-stable UC., Peer reviewed

1 video. -- Video showing the scanning with a UV beam over the VVUC1-CPNs-based spraycoated pattern deposited onto DPA-S-CPNs background. The emission does not change all over the scanning as DPA-S-COOH emission is always activated., Photon upconversion (UC) based on triplet–triplet annihilation is a very promising phenomenon with potential application in several areas, though, due to the intrinsic mechanism, the achievement of diffusion-limited solid materials with air-stable UC is still a challenge. Herein, we report UC coordination polymer nanoparticles (CPNs) combining sensitizer and emitter molecules especially designed with alkyl spacers that promote the amorphous character. Beyond the characteristic constraints of crystalline MOFs, amorphous CPNs facilitate high dye density and flexible ratio tunability. To show the universality of the approach, two types of UC-CPNs are reported, exhibiting highly photostable UC in two different visible spectral regions. Given their nanoscale, narrow size distribution, and good chemical/colloidal stability in water, the CPNs were also successfully printed as anticounterfeiting patterns and used to make highly transparent and photostable films for luminescent solar concentrators, both showing air-stable UC., Peer reviewed

All images are single confocal sections except A-C, which are projections of confocal sections. (A, A’) In trachea of wild-type embryos, Kkv is present in the apical region (blue arrows) and in intracellular vesicles (yellow arrowheads). (B) In kkv mutants unable to polymerise chitin, Kkv is not properly localised. (C) GFP-Kkv localises to the apical region (blue arrows) and in intracellular vesicles (yellow arrowheads). (D) When reb and GFP-kkv are coexpressed in salivary glands, Kkv is present in the apical membrane (blue arrow), in intracellular vesicles (yellow arrowhead), and also in punctae in the lumen (pink arrowheads). This is clearly observed in orthogonal sections (D’). (E) These luminal punctae corresponded to membranous structures. (F, F’) In contrast, when GFP-kkv is expressed alone, luminal punctae are absent, and Kkv is only found apically (blue arrow) and in intracellular vesicles (yellow arrowhead). (G) Luminal punctae (pink arrowheads) are also observed in the trachea of embryos overexpressing reb and GFP-kkv. (H) When endocytosis is prevented, the coexpression of reb and GFP-kkv in salivary glands still leads to formation of Kkv luminal punctae (pink arrowheads). (I) Quantifications of the number of intracellular Kkv vesicles in salivary glands when expressing GFP-kkv (I’), reb and GFP-kkv (I”), and GFP-kkvΔCC. n is the number of salivary glands analysed. The underlying data for quantifications can be found in the S1 Data. Scale bars: 10 μm., Peer reviewed

All images show salivary glands. (A, C, E-L, N, O) Show single confocal sections and (B, D, M) show projections of several sections. (A-A’) The concomitant expression of reb and GFP-kkv leads to luminal chitin deposition (blue arrow in orthogonal section in A’). (B, C) Coexpression of expΔMH2 and GFP-kkv produces intracellular chitin punctae, some of which partially colocalise with GFP-Kkv vesicles (yellow arrowhead) while others do not (red arrowhead). GFP-Kkv vesicles without chitin are also observed (green arrowhead). Note the accumulation of chitin in the apical domain (white arrow in C) that is not deposited extracellularly in the lumen (blue arrow in orthogonal section in C’). (D) In Rab5DN background, intracellular chitin punctae are still present (white arrowheads). (E-L) Analysis of the nature of GFP-Kkv vesicles and chitin punctae using markers Golgin245 (E-F), Hrs 27–4 (G-H), Arl8 (I-J), and Rab11 (K-L); arrowheads indicate colocalisation between Kkv and each specific marker. (M, N) All GFP-Kkv vesicles colocalise with the membrane marker CD4-mIFP (white arrowheads), and few of them also with chitin (orange arrowheads); single chitin punctae do not colocalise with CD4-mIFP (red arrowheads). (O-O”) Frames from live imaging movie show that partially colocalising GFP-Kkv and chitin punctae (yellow arrow) can separate from each other; however, many GFP-Kkv (green arrow) and chitin puncta (red arrow) do not colocalise. Scale bars A-D, M: 10 μm; E-L, N-N”‘: 1 μm; O-O”: 5 μm., Peer reviewed

(A) Schematic representation of Kkv protein (CD, catalytic domain; WGTRE; CC, coiled-coil domain). (B, C, G, I, J) Show projections of confocal sections and (D-F, H, K-N) show single confocal sections. (B) The overexpression of GFP-kkvΔWGTRE in a kkv mutant background does not rescue the absence of extracellular chitin deposition (white arrow, note the absence of CBP) and the protein accumulates in a generalised pattern. (C-D) The overexpression of GFP-KkvΔWGTRE does not produce intracellular chitin punctae, neither in trachea at early stages (C-C’) nor in salivary glands (D). (E-E”‘) GFP-kkvΔWGTRE colocalise with the ER marker KDEL. (F) GFP-KkvΔWGTRE does not colocalise with the marker FK2. (G) The overexpression of GFP-kkvΔCC in a kkv mutant background rescues the lack of extracellular chitin deposition in the trachea (note the presence of CBP staining). (H, I) The simultaneous expression of reb and GFP-kkvΔCC in salivary glands produces ectopic extracellular chitin (H), and no defects in trachea (I). (J) The overexpression of reb in trachea leads to morphogenetic defects. (K, L) Overexpressed GFP-Kkv localises mainly apically (orange arrowheads) although a bit of the protein can be detected in the basal region (yellow arrowheads). (M, N) Apical accumulation of overexpressed GFP-KkvΔCC is less conspicuous. (O) Quantifications of accumulation of GFP-Kkv and GFP-KkvΔCC in apical versus basal region. n corresponds to the number of ratios analysed (apical/basal ratio per cell), and brackets indicate the number of embryos used. Ratios were obtained from the apical (orange line in K) and basal (yellow line in K) domains of single cells in trachea and salivary glands. The underlying data for quantifications can be found in the S1 Data. Scale bars B, C, G, I, J: 25 μm; D-F, H, K-N: 10 μm., Peer reviewed

Supplementary Figure S1, S2, S3 and S4., Peer reviewed