Inspiringly, the hollow spherical structures show enhanced capacitance performance with a specific capacitance of 251 F g−1 at 0.5 A g−1, which compares favorably with the vast majority of two-dimensional COFs and other porous electrode materials.Covalent organic frameworks (COFs) have served as a family of porous crystalline molecules for various promising applications. However, controllable synthesis of COFs with uniform morphology is paramount yet still remains quite challenging. Herein, we report self-templated synthesis of uniform and unique hollow spheres based on highly conjugated three-dimensional (3D) COFs with diameters of 500–700 nm. A detailed time-dependent study reveals the continuous transformation from initial nano sphere-like particles into uniform hollow spherical structures with Ostwald ripening mechanism. Particularly, the resulting 3D COF (3D-Sp-COF) is prone to transport ions more efficiently and the lithium-ion transference number  are promising porous crystalline materials but controllable synthesis of COFs with uniform morphology remains challenging. Here, the authors report a self-templated synthesis of uniform and unique hollow spheres based on highly conjugated three-dimensional COFs. Despite their crystalline features, COFs are usually obtained as microcrystalline morphologies and their long-range ordered growth is limited in the nano/micro domain because of the internal defects and kinetic trapping of smaller crystallites13. The morphology of the crystallites is an important factor for their applications, such as molecular absorption and catalysis15, drug delivery17, energy storage19, etc. In general, the geometry of the building blocks plays a key role to determine the morphology of the crystallites22. Based on the geometry of the building blocks, COFs are mainly classified as two-dimensional (2D) COFs and three-dimensional (3D) COFs25. Since the first report on 2D COFs presented by Yaghi and co-workers in 2005 (ref. 26), extensive efforts have been devoted to this fast developing area28. Controllable synthesis of COF crystallites with uniform morphology, which is beneficial to fine modulating the material performance and better understanding the forming mechanisms of COF crystallites30, is highly desirable yet quite challenging32. Various COFs with uniform morphology such as free-standing films36, nanosheets39, tubes40, shuttle41, belts42, fibers44, and spheres46 have been recently reported mainly based on 2D COFs with fully exploring the potential of the 2D-layered structures. As compared with 2D COFs, 3D COFs with extended networks are generally able to achieve intrinsically large surface areas48, low density of open sites50, and fascinating confinement effects53, which have recently received increasing interest. However, the synthesis of 3D COFs with uniform morphologies remains unexplored. It is quite challenging to obtain 3D COF crystallite structures with uniform morphology mainly due to the “unavoidable” existence of cross-linked networks53. Considering their unique chemical and structural characteristics, it is charming to construct 3D COFs with uniform morphologies via controllable synthesis.Covalent organic frameworks (COFs), as an important type of crystalline porous materials with well-defined and predictable structures, demonstrate extensive applications in molecule storage and separation2 g−1) and high ion mobility, which serve as attractive candidates as electrode materials for energy storage applications. Among these, the well-defined hollow spherical 3D-Sp-COF@96 h shows enhanced specific capacitance (251 F g−1 at 0.5 A g−1) as compared with the 3D COFs with defective intermediate morphology, revealing that the characteristics of 3D COFs can be facilely regulated by exploring the potential of the microstructures. According to our preliminary specific capacitance studies, overwhelming high specific capacitance of 251 F g−1 has been achieved for 3D COFs by facilely modulating the morphological features.Herein, we report the controllable synthesis of 3D COFs (3D-Sp-COF) with uniform and unique hollow spherical morphology based on highly conjugated building blocks via self-templated synthetic methods. Their formation mechanism of hollow spheres based on 3D COFs has been investigated by a detailed time-dependent study and the growth process has been dominated by Ostwald ripening. Interestingly, all the 3D COFs show large surface area (up to 1016 m2) was synthesized by Suzuki cross-coupling reaction  and terephthalaldehyde (Ph-2CHO) with an acetic acid catalyst at 120 °C  and 2,5-dihydroxyterephthalaldehyde was also conducted under the same conditions to afford 3D-SpOH-COF  at 4.73°, 9.52°, 12.36°, and 14.13° corresponding to the (200), (400), (411), and (501) Bragg planes of the space group I41/AMD, respectively, indicating high crystallinity of the resulting 3D COF. 3D-SpOH-COF exhibited intense diffraction peaks (2θ) at 4.17°, 8.46°, 10.07°, 12.76°, 13.92°, 19.78°, and 14.13° corresponding to the (200), (400), (211), (411), (431), and (631), respectively  compared favorably with the experimental patterns, which shows that the obtained crystal structure is very close to the predicted structure. To illustrate the lattice packing, the crystal models were constructed by employing Materials Studio 8.0 software package. Considering that 3D-Sp-COF was composed of tetragonal disphenoid-shaped spirobifluorene core and linear imine bonds, the possible lattice structures with different interpenetration degrees were proposed to demonstrate a fold-interpenetrated diamond (dia) topology similar to the reported SP-3D-COF 1 and SP-3D-COF 2 (ref. 53). According to the detailed simulation, the calculated PXRD pattern of 3D-Sp-COF  and 3D-SpOH-COF , Ph-2CHO, and 3D-Sp-COF were tested  more efficiently. As predicted, the lithium-ion transference number (t+) of 3D-Sp-COF of 0.7 is recorded  were studied by means of field emission scanning electron microscopy, transmission electron microscopy (TEM), and SEM-energy-dispersive spectrum (EDS) mapping , demonstrating as a typical property of microporous molecule. The nonlocal density functional theory showed pore size distributions with a major pore width of 1.86 nm for 3D-Sp-COF@72 h and 3D-Sp-COF@96 h  increased from 0.24 to 0.47 cm3 g−1 (P/P0 = 0.99). Based on these data, the specific surface area of hollow microspheres increased significantly compared with that of the solid microspheres.The permanent porosity of 3D-Sp-COFs at various reaction time was characterized by the nitrogen adsorption–desorption isotherms at 77 K  and galvanostatic charge–discharge (GCD) experiments for 3D-Sp-COFs were performed to explore the electrochemical characteristics  were tested , which were proved by SEM, TEM, EDS, and BET investigations. The mechanism of hollow spheres formation was studied using a detailed real-time tracking method, indicating that the formation of hollow spheres is the result of Ostwald ripening mechanism. In the Li/3D-COF/Li symmetric cells, ion conduction pathways provided by 3D COF are beneficial for enhancing the ion transport and the t+ of 3D-Sp-COF approaches 0.7. The value overwhelms most of the typical PEO-based polymer electrolytes and even comparable to the signal-ion electrolyte systems. Because of the large surface area and hollow spherical architecture, the 3D COF shows a high specific capacitance of 251 F g−1 at a current density of 0.5 A g−1, which is superior to the vast majority of 2D COFs and other porous electrode materials. Remarkably, the specific capacitance increases dramatically with the numbers of cycles, achieving a maximum of 364 F g−1 at the 6000th cycle due to the wetting activation of the hollow spheres. This study provides a feasible morphological modulation strategy to improve the electrochemical characteristics, which provides a deep insight into the design and synthesis of efficient and stable electrode materials for energy storage applications.In summary, a set of highly conjugated 3D-Sp-COFs was designed and synthesized, beginning from tetrahedral tetraamine and terephthalaldehyde with spirobifluorene core structure as building blocks by means of imine condensation reactions. PXRD patterns and modeling studies demonstrated these 3D COFs are seven-fold-interpenetrated microporous diamond (dia-c7) net with I41/AMD symmetry. Remarkably, 3D-Sp-COF crystallites self-assembled into uniform hollow microspheres with narrow size distribution (500–700 nm) and large surface area . This mixture was sonicated for 5 min in order to get a homogeneous dispersion. The tube was then flash frozen at 77 K (liquid N2 bath), degassed by freeze–pump–thaw technique for three times. The tube was sealed off and then placed in an oven at 120 °C for a certain period of time . The resulting precipitate was filtered, washed with anhydrous tetrahydrofuran, and extracted by Soxhlet extractor for 24 h. The solid was dried at 80 °C under vacuum for 12 h to afford yellow powder .A Pyrex tube was charged with 4,4′,4″,4‴-tetraaniline , terephthalaldehyde , and solvent mixture of 1,2-dichlorobenzene/n-butanol/6 M aqueous acetic acid . This mixture was sonicated for 5 min in order to get a homogeneous dispersion. The tube was then flash frozen at 77 K (liquid N2 bath), degassed by a freeze–pump–thaw technique for three times. The tube was sealed off and then placed in an oven at 120 °C for a certain period of time . The resulting precipitate was filtered, washed with anhydrous tetrahydrofuran, and extracted by a Soxhlet extractor for 24 h. The solid was dried at 80 °C under vacuum for 12 h to afford orange powder .A Pyrex tube was charged with 4,4′,4″,4‴-tetraaniline , 2,5-dihydroxyterephthalaldehyde , and solvent mixture of 1,2-dichlorobenzene/Supplementary Information


Neisseria gonorrhoeae with acquired antibiotic resistance are urgently needed. Gonococcal infection of the human cervix is initiated by an interaction between a galactose modification made to its surface appendages, pili, and the I-domain region of (host) complement receptor 3 (CR3). By targeting this crucial gonococcal–I-domain interaction, it may be possible to prevent cervical infection in females. To this end, we identified the I-domain galactose-binding epitope of CR3 and characterized its galactose lectin activity. Moreover, we identified two drugs, carbamazepine and methyldopa, as effective host-targeted therapies for gonorrhea treatment. At doses below those currently used for their respective existing indications, both carbamazepine and methyldopa were more effective than ceftriaxone in curing cervical infection ex vivo. This host-targeted approach would not be subject to N. gonorrhoeae drug resistance mechanisms. Thus, our data suggest a long-term solution to the growing problem of multidrug-resistant N. gonorrhoeae infections.Novel therapies that avert the problem of  Neisseria gonorrhoeae has emerged as a major human health threat, and new approaches to treat gonorrhea are urgently needed. N. gonorrhoeae pili are posttranslationally modified by a glycan that terminates in a galactose. The terminal galactose is critical for initial contact with the human cervical mucosa via an interaction with the I-domain of complement receptor 3 (CR3). We have now identified the I-domain galactose-binding epitope and characterized its galactose-specific lectin activity. Using surface plasmon resonance and cellular infection assays, we found that a peptide mimic of this galactose-binding region competitively inhibited the N. gonorrhoeae-CR3 interaction. A compound library was screened for potential drugs that could similarly prohibit the N. gonorrhoeae-CR3 interaction and be repurposed as novel host-targeted therapeutics for multidrug-resistant gonococcal infections in women. Two drugs, methyldopa and carbamazepine, prevented and cured cervical cell infection by multidrug-resistant gonococci by blocking the gonococcal-CR3 I-domain interaction.In the absence of a vaccine, multidrug-resistant  Neisseria gonorrhoeae (the gonococcus) is an exclusive human pathogen that causes the sexually transmitted infection, gonorrhea. Infections caused by N. gonorrhoeae continue to be a global intractable problem , which are crucial to interactions with epithelial cells , or a disaccharide, Gal(α1-3)diNAcBac (–pgl) gene products. For example, PglD is essential for diNAcBac biosynthesis, whereas the glycosyltransferase PglA adds the terminal galactose to Gal(α1-3)diNAcBac  .al cells –11. ThesilE gene . Pilin cdiNAcBac  13–15).–15.N. godiNAcBac . Expressression)  17–19).–19.N. goMβ2, CD11b/CD18, and Mac-1) is an innate immune pattern recognition receptor. Expression of CR3 has historically been limited to cells of monocytic lineage; however, CR3 is also expressed on the apical surface of the human cervix  use CR3 as a mechanism to promote disease  is 77% similar to the mouse I-domain . *, identical amino acids; •, similar amino acids. The G2 peptide region is boxed in red. Download FIG S1, PDF file, 0.08 MB.Complement receptor 3 I-domain sequence. Complement receptor 3 is a heterodimer composed of an alpha subunit, CD11b, and a beta subunit, CD18. CD11b contains an ∼200-amino-acid insertion domain, or I-domain, and a lectin domain. The human I-domain of CD11b  and both human rI-domain  and rCR3 protein . This high-affinity interaction was dependent upon the terminal galactose of Gal(α1-3)diNAcBac, as no interaction was detected for MS11 pglA pili, which have diNAcBac, in the concentration range tested  or wild-type  pilin were flowed over immobilized human rI-domain. The sensorgrams show a negative response with every pglA pilin injection, whereas positive response units (RU) and an RU build are seen with WT pilin injections. (b) Five dilutions (0.125 to 2 μM) of pglA or WT pilin were flowed over a flow cell to which rCR3 was immobilized. (c) Five dilutions (0.125 to 2 μM) of pglA or WT pilin were flowed over mouse rI-domain immobilized to a flow cell. Note that although an RU build (shown by the black arrows) was detected for pili in the upper concentration ranges shown in panel C, recombinant pili could not be maintained in solution at these higher concentrations; therefore, it was not possible to approach saturation. As a result of this issue and with the negative RU response observed during pilin injections (shown by green arrows), we could not determine a KD for recombinant pilin with the mouse rI-domain. (d) Nine dilutions (0.0039 to 1 μM) of α1-3,β1-4,α1-3 galactotetraose over immobilized I-domain. (e) Nine dilutions (0.0039 to 1 μM) of linear β-2 trisaccharide over immobilized I-domain. (f) Nine dilutions (0.0039 to 1 μM) of P1 antigen over immobilized I-domain. (g) Nine dilutions (0.0039 to 1 μM) of α1-3 galactobiose over immobilized I-domain. (h) Nine dilutions (0.0039 to 1 μM) of lactose over immobilized I-domain. (i) Nine dilutions (0.0039 to 1 μM) of α-lactose over immobilized I-domain. (j) Nine dilutions (0.0039 to 1 μM) of methyl-α-d-galactopyranoside over immobilized I-domain. (k) Eight dilutions (0.0078 to 1 μM) of sucrose (negative control) over immobilized I-domain. The RUs are due to bulk shift. There is no build in RU after injections as with the other carbohydrates. (l) Nine dilutions (0.0039 to 1 μM) of α1-3,β1-4,α1-3 galactotetraose over immobilized rCR3. (m) Nine dilutions (0.0039 to 1 μM) of linear β-2 trisaccharide over immobilized rCR3. (n) Nine dilutions (0.0039 to 1 μM) of P1 antigen over immobilized rCR3. (o) Nine dilutions (0.0039 to 1 μM) of α1-3 galactobiose over immobilized rCR3. (p) Nine dilutions (0.0039 to 1 μM) of lactose over immobilized rCR3. (q) Nine dilutions (0.0039 to 1 μM) of α-lactose over immobilized rCR3. (r) Nine dilutions (0.0039 to 1 μM) of methyl-α-d-galactopyranoside over immobilized rCR3. (s) Eight dilutions (0.0078 to 1 μM) of sucrose (negative control) over immobilized rCR3. The RUs are due to bulk shift. There is no build in RU after injections as with the other carbohydrates. (t) Five dilutions (0.0625 to 1 μM) of α1-3 galactobiose over immobilized biotin-AhxG2-streptavidin complex. (u) Five dilutions (0.0625 to 1 μM) of methyl-α-d-galactopyranoside over immobilized biotin-AhxG2-streptavidin complex. (v) Five dilutions (0.0625 to 1 μM) of sucrose (negative control) over immobilized biotin-AhxG2-streptavidin complex. The RUs are due to bulk shift. Download FIG S2, PDF file, 0.7 MB.Blank- and reference-subtracted SPR sensorgrams for results in Copyright © 2020 Poole et al.2020Poole et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the D], 229 to 320 nM) and rCR3 . We then investigated the effects of linkage and anomeric configuration on galactose recognition. Synthetic glycans with a terminal α1-4 galactose bound with similar affinity to α1-3 galactose: rI-domain  and rCR3 . Synthetic glycans linked with a terminal β1-3 or β1-4 galactose also bound with high affinity   . These p00623.2)  as well 00623.2) . The G2 00623.2) .10.1128/mBio.03046-19.3FIG S3FIG S3, PDF file, 0.6 MB.I-domain peptide library competition SPR screen sensorgrams with the carbohydrate galactobiose  and I-domain flow cell. (a) I-domain library peptide A1 only. (b) I-domain library peptide A1+1-3Gal. (c) I-domain library peptide A2 only. (d) I-domain library peptide A2+1-3Gal. (e) I-domain library peptide A3 only. (f) I-domain library peptide A3+1-3Gal. (g) I-domain library peptide A4 only. (h) I-domain library peptide A4+1-3Gal. (i) I-domain library peptide A5 only. (j) I-domain library peptide A5+1-3Gal. (k) I-domain library peptide B1 only. (l) I-domain library peptide B1+1-3Gal. (m) I-domain library peptide B2 only. (n) I-domain library peptide B2+1-3Gal. (o) I-domain library peptide B3 only. (p) I-domain library peptide B3+1-3Gal. (q) I-domain library peptide B4 only. (r) I-domain library peptide B4+1-3Gal. (s) I-domain library peptide B5 only. (t) I-domain library peptide B5+1-3Gal. (u) I-domain library peptide C1 only. (v) I-domain library peptide C1+1-3Gal. (w) I-domain library peptide C2 only. (x) I-domain library peptide C2+1-3Gal. (y) I-domain library peptide C3 only. (z) I-domain library peptide C3+1-3Gal. (aa) I-domain library peptide C4 only. (ab) I-domain library peptide C4+1-3Gal. (ac) I-domain library peptide C5 only. (ad) I-domain library peptide C5+1-3Gal. (ae) I-domain library peptide D1 only. (af) I-domain library peptide D1+1-3Gal. (ag) I-domain library peptide D2 only. (ah) I-domain library peptide D2+1-3Gal. (ai) I-domain library peptide D3 only. (aj) I-domain library peptide D3+1-3Gal. (ak) I-domain library peptide D4 only.  I-domain library peptide D4+1-3Gal. (am) I-domain library peptide D5 only. (an) I-domain library peptide D5+1-3Gal. (ao) I-domain library peptide E1 only. (ap) I-domain library peptide E1+1-3Gal. (aq) I-domain library peptide E2 only. (ar) I-domain library peptide E2+1-3Gal. (as) I-domain library peptide E3 only. (at) I-domain library peptide E3+1-3Gal. (au) I-domain library peptide E4 only. (av) I-domain library peptide E4+1-3Gal. (aw) I-domain library peptide E5 only. (ax) I-domain library peptide E5+1-3Gal. (ay) I-domain library peptide F1 only. (az) I-domain library peptide F1+1-3Gal. (ba) I-domain library peptide F2 only. (bb) I-domain library peptide F2+1-3Gal. (bc) I-domain library peptide F3 only. (bd) I-domain library peptide F3+1-3Gal. (be) I-domain library peptide F4 only. (bf) I-domain library peptide F4+1-3Gal. (bg) I-domain library peptide F5 only. (bh) I-domain library peptide F5+1-3Gal. (bi) I-domain library peptide G1 only. (bj) I-domain library peptide G1+1-3Gal. (bk) I-domain library peptide G2 only. (bl) I-domain library peptide G2+1-3Gal. (bm) I-domain library peptide G3 only. (bn) I-domain library peptide G3+1-3Gal. (bo) I-domain library peptide G4 only. (bp) I-domain library peptide G4+1-3Gal. (bq) I-domain library peptide G5 only. (br) I-domain library peptide G5+1-3Gal. (bs) I-domain library peptide H1 only. (bt) I-domain library peptide H1+1-3Gal. (bu) I-domain library peptide H2 only. (bv) I-domain library peptide H2+1-3Gal. (bw) I-domain library peptide H3 only. (bx) I-domain library peptide H3+1-3Gal. (by) I-domain library peptide H4 only. (bz) I-domain library peptide H4+1-3Gal. (ca) I-domain library peptide H5 only. (cb) I-domain library peptide H5+1-3Gal. Download Copyright © 2020 Poole et al.2020Poole et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03046-19.7TABLE S1Table S1, DOCX file, 0.01 MB.Peptide list. Peptides from the I-domain peptide library screen, the biotin-labelled G2 peptide, and two control peptides are listed. For the biotinylated G2 peptide, Ahx is a six-carbon spacer. Control P1 and P2 are peptides from an unrelated gonococcal protein called AniA. They have similar solubility to G2 peptide and were included as a non-I-domain peptide control. Download Copyright © 2020 Poole et al.2020Poole et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the d-galactose, were flowed over immobilized biotinylated G2 peptide (biotin-AhxG2) complexed with streptavidin. The reference cell contained streptavidin complexed with biotin. Both galactobiose and methyl-α-d-galactose interacted with the G2 peptide with high affinity  as a st10.1128/mBio.03046-19.4FIG S41MF7 was used for docking studies using AutoDock Vina implemented in the YASARA software suit. More-positive energies indicate stronger binding. Percentages of the populated docked structures are also shown, indicating that cluster A1, which includes the G2 peptide region, is the preferred binding galactose-α-OH conformation. Download FIG S4, PDF file, 0.05 MB.AutoDock Vina experiment of galactose-α-OH. Molecular docking identified four possible galactose-binding sites within the area of the G2 peptide. (a) AutoDock Vina experiment of galactose-α-OH into the G2 region (marked in red) of the human CR3 I-domain results in 4 distinct clusters: A1, B1, C1, and D1, with a ligand population of 70%, 10%, 15%, and 5%, respectively. (b) A total of 999 runs were docked. PDB structure Copyright © 2020 Poole et al.2020Poole et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the N. gonorrhoeae cells are associated with the female uterine cervix via an interaction with CR3  but not control (P1 and P2 nonblocking) peptides  different from that to uninfected cells with the use of 100 μM G2 peptide. Only background fluorescence was recorded for assays performed using CHO-neo cells or uninfected cells and for wells devoid of Pex or CHO cells incubated with MS11 gfp. Thus, these data confirmed the lectin function of the CR3 I-domain, and they highlight the importance of the pilin Gal(α1-3)diNAcBac glycan-CR3 I-domain interaction to cervical infection.Analysis of cervical biopsy specimens from women with active gonococcal infections showed that more than 92% of with CR3 . The N. al pilus , and is al pilus . Therefoal pilus . Pex celpeptides  and b. TN. gonorrhoeae MS11 wild-type pilin from binding to human rI-domain in direct competition SPR experiments. Only six compounds bound with high affinity to rI-domain and completely blocked the interaction of N. gonorrhoeae MS11 pilin with rCR3 or rI-domain. Two of these blocking compounds met our criteria for current use and safety in humans, carbamazepine (KD = 2.12 nM ± 0.24) and methyldopa (KD = 1.01 nM ± 0.09), which were further evaluated for their potential utility in treating N. gonorrhoeae cervical infection  Carbamazepine identification in SPR screen. Highlighted spot is carbamazepine . (b) Methyldopa identification in SPR screen. Highlighted spot is methyldopa. (c) Single-cycle kinetics curve of carbamazepine against the rI-domain of CD11b surface plasmon resonance assay. Maximum concentration was 10 nM with a 1:2 dilution down to 0.625 nM. (d) Single-cycle kinetics curve of methyldopa against the rI-domain of CD11b surface plasmon resonance assay. Maximum concentration was 10 nM with a 1:2 dilution down to 0.625 nM. Download Copyright © 2020 Poole et al.2020Poole et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the N. gonorrhoeae adherence to CHO-neo, CHO-CR3, and Pex cells was evaluated using a fluorometric adherence assay (The ability of carbamazepine and methyldopa to block ce assay . Gonococ10.1128/mBio.03046-19.6FIG S6N. gonorrhoeae. (a) Fluorometric adherence assays performed using CHO-neo and CHO-CR3 cells show that a CR3- and dose-dependent reduction in N. gonorrhoeae strain MS11 gfp adherence occurred in the presence of methyldopa (Md). Fluorescence (y axis), indicative of adherence, was recorded following a 1-h infection. Carbamazepine (b) and methyldopa (c) treatments resulted in significant reductions in the numbers of viable gonococci at 24 h posttreatment, which were further reduced by 48 h. Both drugs were more effective than ceftriaxone (Cfx), the current recommended therapy, in treating established Pex cell infections with the noted low-passage-number gonococcal isolates. (d) Well diffusion assays showed that methyldopa had no effect on the N. gonorrhoeae strains tested in the absence of human cells. Each assay was performed in triplicates on 3 separate occasions. Data are presented as the means and variances of the average values obtained for each assay. *, P ≤ 0.0001 versus vehicle control; Cip, ciprofloxacin. Download FIG S6, PDF file, 0.2 MB.The effect of repurposed drugs on Copyright © 2020 Poole et al.2020Poole et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To assess the ability of either drug to cure an established gonococcal infection, Pex cells were challenged with the noted gonococcal strains for 90 min and then treated with carbamazepine, methyldopa, or ceftriaxone. In these treatment assays, less than 99.95% of viable gonococci remained after a 24-h treatment with ≥10 μM carbamazepine or ≥10 μM methyldopa for all strains tested, including multidrug-resistant gonococci  and c. KNeisseria gonorrhoeae strains 1291 and MS11  bacteria were shown to bind the CR3 I-domain. Using SPR, we found that a high-affinity  interaction with the I-domain occurred for pili with a Gal(α1-3)diNAcBac disaccharide, whereas no concentration-dependent interaction (defined herein as no binding of ≤ 2 μM glycan to rI-domain or rCR3) was recorded for pili isolated from MS11 pglA (diNAcBac monosaccharide). These data suggest that, whereas gonococcal pili with both a di- and monosaccharide are capable of binding to the CR3 I-domain, a high-affinity pilin glycan-CR3 I-domain interaction requires the terminal galactose. That is, the diNAcBac present on pglA pilin, by itself, was not sufficient to allow a high-affinity interaction with the CR3 I-domain.Gonococcal pili are phase-variably modified with a monosaccharide or a disaccharide , 16. In D values in the low to mid nanomolar range) interactions with human rI-domain and rCR3. Notably, this galactose-binding activity was limited to the human CR3 I-domain; mouse I-domain was unable to bind any of the glycans tested. Thus, although murine and human I-domain share 77% similarity at the amino acid level, the functional galactose-specific lectin activity that we observed may be limited to humans or higher primates. In this regard, the production  of natural anti-galactose antibodies is also limited to higher primates  that governs the interaction between gonococcal pilin and the human CR3 I-domain, which is critical to infection of the female cervix. A further goal was to determine whether we could target the gonococcus-CR3 interaction as a potential novel approach to the growing problem of untreatable gonorrhea. To this end, we identified a peptide receptor mimic, G2, which replicates the high-affinity terminal galactose binding activity observed for I-domain and CR3. Both CHO-CR3 and Pex cells were tested, and with both cell types, the G2 peptide displayed dose-dependent blocking. The ability of the G2 peptide to block gonococcal adhesion in these models validates prophylactic and therapeutic strategies targeting CR3 as a novel approach to combat this multidrug-resistant pathogen.N. gonorrhoeae. Additionally, targeting CR3 may exist as a viable option to treat other antibiotic-resistant human pathogens that use this receptor to initiate infection/disease.Repurposing existing drugs is the most rapid path to clinical intervention. Carbamazepine and methyldopa are widely prescribed orally administered drugs and are available in mucosal secretions and in serum  and used to procured Pex cells as described previously . CHO-neoN. gonorrhoeae strains used in this study included the laboratory strains 1291 and MS11 -expressing gonococci were made by transforming the noted strains with the plasmid pCmGFP (GenBank accession number FJ172221) , GC-IsoVitaleX agar plate cultures and enumerated spectrophotometrically, as previously described  , 50. Thecassette , 52 intolsewhere . For useescribed .Mβ2) was purchased from R&D Systems .Recombinant human and mouse I-domain proteins were made and purified as previously described . Human rg, 15 min). The supernatant, containing pili, was transferred to a new tube and incubated at 56°C for 1 h. This crude pilus preparation was separated on a 4% to 12% polyacrylamide gradient gel. Pilin was isolated by electroelution  into SDS buffer using a Mini Whole Gel Eluter . To verify pilin isolation, different mass fractions were collected, and 20 μl of each fraction was analyzed by Western blotting using an anti-pilin antibody  . Samples were analyzed at 25°C in phosphate-buffered saline (PBS) at a flow rate of 10 μl/min and by using single-cycle kinetics. Human rI-domain, mouse rI-domain, and human rCR3 were immobilized onto separate cells of a Series S CM5 sensor chip using an NHS capture kit  for purified pilin experiments. For experiments in which pilin-glycan-like structures were examined, human rI-domain and rCR3 were immobilized onto separate cells of a CM5 chip. A blank immobilization was used as a control/reference on all chips. MS11 wild-type and MS11 ing, UK) , resemblNP_000623.2, amino acids Gly127 to Ala325 of CD11b) was used to generate a peptide library . Each peptide comprised 15 amino acids, with a 5-amino-acid sliding window, to give a 10-amino-acid overlap down the sequence, for a total of 40 peptides  , was inc1MF7 [All molecular modeling was performed using YASARA . A recta1MF7 ). The coN. gonorrhoeae MS11 gfp was used to challenge (1 h) Pex or CHO cells simultaneously with peptide or drug competitor. Infected (devoid of peptide or drug) and uninfected  control cell assays were treated in parallel with competitive peptide/drug inhibition assays. Fluorescence  intensity, corresponding to bacterial adherence, was recorded using a Synergy HT multimode microplate reader . Blank wells, devoid of Pex or CHO cells, were inoculated with bacteria and served as a control for nonspecific binding. Each assay was performed in triplicates on 3 separate occasions. Peptides used are described in Fluorometric adherence assays were performed essentially as described previously . In brieD of each interaction. For carbamazepine and methyldopa, saturation occurred between 1.6 nM and 8 nM, and so a new concentration range (10 nM to 0.625 nM) was tested. Any drug with a KD of >1 μM was discarded from further analyses. For competitive SPR assays, N. gonorrhoeae pilin, drug, or drug plus pilin were flowed at 1 μM over human rI-domain, and the response units of each interaction were recorded. Any drug that could not compete with pilin for binding  to the I-domain was also omitted from further analyses. All SPR sensorgrams and result plots described above were analyzed with Biacore S200 evaluation software . A literature review was used to evaluate the remaining drugs for known long-term safety in humans and known therapeutic concentrations.Human rI-domain was immobilized onto a CM5 sensor chip with a blank control flow cell as described above. A combination of two FDA-approved drug libraries, Microsource-CPOZ  and ML Drug (741 drugs), was purchased from Compounds Australia. Each drug was made up to 1 μM in 10% DMSO in a 384-well plate, with a new chip for every plate screened, just before use in the Biacore S200. A single-concentration injection screen (yes/no) binding assay was performed. Binding was determined based on the response unit shift  of the stability of the binding phase of the dissociation cycle. Binding “hits” were rescreened across a concentration range of 1.6 nM to 1 μM to define the KN. gonorrhoeae for 90 min. The infection medium was then removed, the cells were rinsed thrice, and fresh medium containing 0.1% DMSO (vehicle control), carbamazepine (100 pM to 100 μM), methyldopa (100 pM to 100 μM), or ceftriaxone  was added. Infections then proceeded for an additional 24 h or 48 h, after which, the infection medium was removed. Pex cell monolayers were subsequently lysed, serial dilutions of the Pex cell lysates were plated, and viable gonococci were enumerated by counting CFU after 48 h of incubation . For sequential infection assays, viable bacterial colonies that survived a treatment assay were harvested from the enumeration (CFU) plates and used to inoculate new Pex cell monolayers. For these sequential assays, each infection  proceeded for 24 h. Pex cells were then processed, and bacteria were enumerated, as described above. For all assays, the percentage of N. gonorrhoeae that survived carbamazepine, methyldopa, or ceftriaxone treatment was determined as a function of bacteria that survived DMSO treatment (set to 100%). All assays were performed in triplicates on 3 separate occasions. A nonparametric ANOVA was used to determine the statistical significance of bacterial survival.Infection studies were performed as previously described with modification using a multiplicity of infection of 100 . To estaN. gonorrhoeae strains were spread uniformly across the surfaces of GC agar plates at a culture density of 107 bacteria per ml. Wells were then punctured within the agar surface to which carbamazepine or methyldopa (100 pM to 100 μM), 0.2 μg/ml ceftriaxone, 10 μg/ml ciprofloxacin, or 1% DMSO (vehicle control) was added. Following an overnight incubation , inhibition of N. gonorrhoeae growth was measured as the diameter (in millimeters) of the area of clearing surrounding (and inclusive of) each well on each agar plate, i.e., the zone of inhibition (ZOI). For agar plates in which a ZOI was not visible, data were recorded as the diameter of the well (6 mm). Assays were performed in triplicates on 3 separate occasions. Statistical significance of data obtained was determined using a Student's t test.Well diffusion assays were performed essentially as described previously . Briefly


Environmental contamination by lead (Pb) and other toxic metals is of significant environmental and human health concern. Heavy metals are not readily eliminated by degradation, and thus remediation of contaminated media  requires the outright removal or cleanup of these metals. Evaluation of the performance and cost efficiency of various remediation methods has led to the development of bioremediation as an inexpensive, innovative and environmentally friendly cleanup strategy.Senna obtusifolia (Sicklepod), in Dareta Village, Zamfara, Nigeria.The present study was designed to assess the Pb remediation potential of wild Senna obtusifolia samples were collected from established plots and Pb content was determined using a Shimadzu atomic absorption spectrophotometer  after wet digestion.Soil and The mean concentrations of Pb (mg/kg) in soil, roots, stems and leaves, respectively, were 130.68±5.2, 61.33±17.86, 66.64±18.10 and 173.39±13.73 for plot 1, 287.84±6.5, 69.42±11.62, 123.4±3.67 and 294.28±4.38 for plot 2, 315.73±4.13, 68.42±10.22, 86.89±6.08 and 290.61±7.47 for plot 3, 396.86±5.48, 91.64±2.87, 150.58±2.21 and 282.53±5.69 for plot 4 and 264.23±8.02, 72.71±2.18, 124.60±2.27 and 282.40±3.79 for plot 5. Average values for the translocation factor, bioaccumulation factor and bioconcentration factor were 3.65±0.66, 1.01±0.23 and 0.29±0.10, respectively.Senna obtusifolia leaves was found to be higher than the Pb content of the stem and root, indicating relatively low restriction and the efficiency of internal transport of the toxic metal from the roots towards the aerial parts. High translocation and bioaccumulation factors indicate that the plant has vital characteristics for phytoextraction of Pb. The mean Pb concentration of Senna obtusifolia leaves was found to be far above Codex general standards and the European Union (EU) maximum levels for Pb in leafy vegetables.Soil Pb levels in the present study were found to be within the United States Environmental Protection Agency (USEPA) standards and the Dutch Intervention Values for Pb in soil. Lead content of Senna obtusifolia has significant characteristics for phytoextraction of Pb and that consumption of Senna obtusifolia leaves from the study area would pose a serious risk of Pb intoxication.The study concludes that wild The authors declare no competing financial interests. Environmental contamination by heavy metals such as lead (Pb) or cadmium (Cd) is of significant concern. These metals cannot be destroyed by degradation, and thus remediation of contaminated medium requires outright removal or cleanup.ANOVAAnalysis of varianceBACbioaccumulation factorBCFBiological concentration factorTFTranslocation factorMelastoma melabathricum for instance has been shown to accumulate up to 13 800 mg/kg in the root of the plant, but only a small amount of the Pb could be translocated from the root to the above ground part (translocation factor (TF) <1), indicating that it is a good bioaccumulator (bioavailability factor >1) of the toxic metal and that phytostabilization is the mechanism at work in the uptake of Pb.Noccaea praecox has been reported to accumulate over 4000 mg/kg Pb in the shoot, while Noccaea rotundifolia and Noccaea caerulescens have recorded Pb uptake greater than 28 700 mg/kg and 65 631 mg/kg, respectively.9Plants with exceptional metal accumulating capacity (hyperaccumulators) occur throughout the plant kingdom.3) and galena (PbS). Other ore minerals such as pyromorphite (Pb5(PO4)3Cl) and anglesite (PbSO4) also occur frequently, but are less important.15Once introduced into the soil, Pb is difficult to remove. The heavy metal is strongly bounded to soil and all interactions within the soil matrix are pH dependent. Under acidic conditions (pH < 5.5) Pb is more mobile and readily available to plants. Lead is one of the constituents that makes up the earth's crust in nature. The metal is commonly found in water, soil and plants at barely detectable levels.et al. recorded soil Pb levels ranging from 1029.42±98.50 mg/kg to 6724.68±184.00 mg/kg in the area and observed that the old grinding mill and other areas where ore processing took place were eventually covered with wild Senna obtusifolia growing aggressively and stifling the growth of preexisting plants.20Mass acute Pb pollution and poisoning was reported in Zamfara state in 2010. The source of the widespread poisoning was traced to artisanal gold mining and ore processing in the villages. Lead concentration exceeding 100 000 mg/kg, far above 400 mg/kg considered acceptable for residential areas, was reported in Dareta common areas including residential compounds. Over 735 children were reported to have died and thousands sickened by the toxic metal in what is believed to be the worst Pb poisoning worldwide in the last forty years.Senna obtusifolia, in Dareta Village, Zamfara State, Nigeria. Despite the high soil Pb concentrations recorded, areas covered with wild Senna obtusifolia were found to be within permissible limits, hence the need to investigate the phytoremediation potentials of the plant. A fast growth rate, high above ground biomass, tolerance to Pb pollution, survival and adaptability to prevailing environmental conditions exhibited by wild Senna obtusifolia in Dareta village are some of the important characteristics to consider when choosing a plant to phytoremediate Pb in soil.Senna obtusifolia. Putshaka et al. has previously reported Senna obtusifolia to accumulate 81.50 mg/kg Pb, a value that is greater than the corresponding soil Pb concentration in the flood plain of a tannery wastewater stream in Challawa industrial estate, Kano.23The present study was designed to assess the Pb remediation potentials of wild .24Zamfara State is located in the northwest geopolitical zone of Nigeria. It occupies a land mass of about 39 762 sq km and Gusau is its capital. The state shares boundaries with Sokoto, Niger, Kebbi, Katsina and Kaduna State within the country and an international boundary with Niger Republic The climate of this northwestern Nigerian State is warm and tropical, with temperatures sometimes rising up to 38°C at the peak of the dry season (between March and April). The rains begin between mid-March and May, lasting for about six months up until end of October, while the extremely dry, cold and dusty wind that blows from the Sahara towards the western coast of Africa (Harmattan) lasts from December to February. The mean annual rainfall in the area fluctuates between 36 and 80 mm, varying slightly, from the northern to the southern parts of the state. The vegetation of the state is made up of Sudan and Guinea Savannah. The Sudan Savannah is found in the northern, western and eastern part of the state. The Northern Guinea Savannah is found in the southern part of the state. This vegetation type is largely found in the Gusau, Tsafe and Anka Local Government Areas.25Dareta village (the study area) is in the Anka Local Government Area, located on 12°06′30′ N 5°56′00′ E with total area of about 2746 sq km and a human population of about 142 280.Senna obtusifolia was harvested at the control site in Zaria, Kaduna State and soil samples collected from the same points. Samples were preserved in black polyethene bags and transported to the Environmental Laboratory, National Research Institute for Chemical Technology, Zaria, Nigeria. Leaves, stems and roots of Senna obtusifolia were thoroughly washed to remove all adhered soil, cut into pieces and air dried for 5 days in the laboratory. The dried leaves samples were pulverized, and stems and roots were pounded before sieving through 1 mm mesh and digested. The digestion of 1g was carried out using 5 ml of concentrated nitric acid according to Awofolu.27Five different plots were carefully selected for the study and designated as plots 1, 2, 3, 4 and 5. Three sampling points were established in each plot. The plants were uprooted carefully and soil samples collected from the same points. Soil samples were collected at the surface level (0–10 cm depth) in duplicates using a soil auger. Additionally, some Soil samples from each plot were thoroughly mixed to obtain a representative sample, air dried, crushed and sieved with 2 mm mesh before wet digestion. One (1) g of a well mixed sample from each sampling point was taken into a 250 ml glass beaker and digested with 10 ml of concentrated nitric acid, perchloric acid and hydrofluoric acid in the ratio 3:1:1 on a hot plate. After evaporating to near dryness, 10 ml of 2% nitric acid was added, filtered into a 50 ml-volumetric flask and then made up to mark with distilled deionized water.18Lead concentrations in the digests were determined using a Shimadzu atomic absorption spectrophotometer  equipped with Zeaman background correction and graphite furnace at the National Research Institute for Chemical Technology, Zaria, Nigeria. The instrument was then set to 0 by running the respective reagent blanks and Pb concentration was determined at a wavelength of 283.3 nm. An average value of three replicates was taken for each determination. Data obtained were subjected to statistical analysis.Appropriate quality assurance procedures and precautions were taken to ensure the authenticity of the results. Samples were carefully handled to avoid cross-contamination. Glassware was properly cleaned and deionized water was used throughout the study. All of the reagents, including nitric acid , hydrofluoric acid  and perchloric acid  were of analytical grade. In order to check the reliability of the analytical method employed for Pb determination, one blank and combine standards were run with every batch of 12 samples to detect background contamination and monitor consistency between batches. The result of the analysis was validated by digesting and analyzing standard reference materials (Lichen coded IAEA-336) following the same procedure. The analyzed values and the certified reference values of the elements determined were compared to determine the reliability of the analytical method employed.Test for normality was carried out using the Shapiro-Wilks test, and the Z-score test was used to check for outliers.Senna obtusifolia roots, stem and leaves across the five different plots under study. Probabilities less than 5% (p < 0.05) were considered to be statistically significant. Independent t-test was used to compare Pb content of Senna obtusifolia from the study area and the Pb content of Senna obtusifolia from Zaria (control). Probabilities less than 5% (p < 0.05) were considered to be statistically significant.Having passed the test for normality and outliers, data collected were subjected to statistical test of significance using the analysis of variance (ANOVA) test to assess significant variation in Pb concentration in the soil, Senna obtusifolia at α = 0.05.Pearson product-moment correlation coefficient was used to determine the association between Pb levels in the soil, stem, roots and leaves of All the above-mentioned statistical analyses were performed by SPSS software 17.00 for Windows.et al., and expressed in L is mg of heavy metal per kg of plant leaves and HMS is total content of heavy metal per kg of soil.The bioavailability factor of heavy metals in plants, also known as the bioavailability index, was calculated according to Malik The biological concentration factor (BCF) was calculated as a metal concentration ratio of plant roots to soil, and is given in The TF was described as the ratio of heavy metals in plant shoots to that in the roots, as given in The bioaccumulation factor (BAC) was calculated as the ratio of a heavy metal in plant shoots to that in soil, as shown in .To evaluate the accuracy and precision of the employed analytical procedure, standard reference materials of Lichen coded IAEA-336 was analyzed in a like manner to the samples in the present analysis. The analyzed values were found to be within the ranges of the certified reference values for the elements in the present study, suggesting the reliability of the employed methods Senna obtusifolia tissues across sampling plots in Dareta village are presented in Senna obtusifolia tissues are presented in Senna obtusifolia tissues are shown in Lead content of soil and Senna obtusifolia roots, Senna obtusifolia stem and Senna obtusifolia leaves in the study area ranged from 125.41–402.45 mg/kg, 49.88–94.56 mg/kg, 55.54–152.42 mg/kg and 163.81–297.82 mg/kg, respectively. The highest soil Pb concentration (402.45 mg/kg) was recorded in plot 4, while the lowest (125.41 mg/kg) was recorded in plot 1. The highest and lowest concentration of Pb in Senna obtusifolia roots and stem were also recorded in plot 4 and 1, respectively .Senna obtusifolia leaves recorded the highest Pb concentration (297.82 mg/kg) in plot 2 and the lowest Pb concentration (163.81 mg/kg) in plot 1. The mean soil Pb level of soil, Senna obtusifolia roots, stem and leaves from Zaria (control) were 67.84±6.09 mg/kg, 3.92±1.82 mg/kg, 4.48±2.28 mg/kg and 4.64±1.7 mg/kg, respectively.Senna obtusifolia plots in the order of: soil > Senna obtusifolia leaves > Senna obtusifolia stem > Senna obtusifolia roots.Senna obtusifolia roots, Senna obtusifolia stem and Senna obtusifolia leaves, and Pb concentrations in soil were significantly higher than the Pb concentration in roots and stem. The Pb concentration in Senna obtusifolia leaves was also found to be significantly (p < 0.05) higher than in Senna obtusifolia stem and Senna obtusifolia roots. The Pb concentration in Senna obtusifolia stems was found to be significantly (p < 0.05) higher than in the root. The difference in overall Pb concentration between in soil and Senna obtusifolia leaves was not statistically significant (p > 0.05). The differences in Pb concentrations between soil and Senna obtusifolia tissues across the plots were found to be statistically significant  (Table 2).Statistical analysis revealed a significant  difference in overall Pb concentration between the soil, Senna obtusifolia leaves (r = 0.829), concentration of Pb in soil and Senna obtusifolia stem (r= 0.776), concentration of Pb in soil and Senna obtusifolia roots (r = 0.659), concentration of Pb in Senna obtusifolia roots and Senna obtusifolia stem (r = 0.760) and the concentration of Pb in Senna obtusifolia stem and Senna obtusifolia leaves (r = 0.690).Senna obtusifolia plot 1, plot 2, plot 3, plot 4 and plot 5, respectively. The highest TF obtained for Pb in Senna obtusifolia was found in plot 2, and plot 1 recorded the lowest TF for Pb. The translocation factor followed the trend: Senna obtusifolia plot 2 > plot 3 > plot 5 > plot 4 > plot 1. The BAC of 1.33, 1.02, 0.92, 0.71 and 1.07 was computed for Senna obtusifolia plot 1, plot 2, plot 3, plot 4, and plot 5, respectively. The BCF values were 0.47, 0.24, 0.22. 0.23 and 0.28 for plot 1, plot 2, plot 3, plot 4 and plot 5, respectively. Bioaccumulation factor followed the trend: Senna obtusifolia plot 1 > plot 5 > plot 2 > plot 3 > plot 4, while the BCF followed the trend: Senna obtusifolia plot 1 > plot 5 > plot 2 > plot 4 > plot 3.Senna obtusifolia roots, stem and leaves  showed that the soil Pb levels across the five plots were all below the United States Environmental Protection Agency (USEPA) guideline for soil Pb levels (400 mg/kg). Nigeria has no standards for soil Pb levels, but the Department of Petroleum Resources adopts the Dutch standards for the assessment of soil pollution in the country. The Dutch soil remediation policy uses target and intervention values to assess soil contamination. The remediation intervention value, used to indicate the Pb level at which the functional properties of the soil to support human, animal and plant life are seriously threatened or impaired, is 530 mg/kg for Pb.Lead concentrations (mg/kg) in soil, This value represents the soil Pb concentration above which the soil is said to be seriously contaminated. The target value (85 mg/kg) indicates a soil Pb level below which there is sustainable soil quality. It is the soil Pb level that must be attained to fully recover all the functional properties of the soil for humans, animal and plant life, and is thus the bench mark for environmental quality on the assumption of negligible risk to the ecosystem.18Senna obtusifolia leaves, stem and roots,  despite the fact that soil Pb levels were within the USEPA acceptable limit and Dutch soil remediation intervention values, may suggest that a good proportion of Pb in Dareta soils is present in the mobile phase, thus readily available for uptake by plants. Plants growing in a Pb-enriched environment are able to accumulate large quantities of the metal in their tissues depending on the percentage bioavailabilty/mobility of the toxic metal. Susceptible plant species that are not resistant to the metal usually die off, allowing the resistant ones to thrive. This could possibly explain the fact that the old grinding mill and other areas where Pb processing had taken place in Dareta were overgrown with Senna obutsifolia at the time of this study. The concentration of Pb in Senna obtusifolia leaves did not follow the trend of soil Pb level across the studied plots .Senna obtusifolia plot 3 recorded the highest mean Pb level in leaves, while Senna obtusifolia plot 5 recorded the lowest mean concentration. The observation may be attributed to differences in the chemical form of Pb present in soils at the different Senna obtusifolia plots and differences in soil chemistry. It also suggests that the bioavailable percentage of total Pb has a significant effect on Pb uptake. Bano reported that Senna obtusifolia growing by the road side accumulates up to 300 mg/g dry weight of Pb.Cassia occidentalis in Ghana.Senna obtusifolia leaves was found to be higher than the Pb content of the stem and the roots, indicating significant accumulation of the metal in leaves. Leaves may therefore be considered to be the major storage organ for Pb in Senna obtusifolia. The large difference in Pb concentrations between roots and leaves indicate relatively low restriction, hence the efficiency of the internal transport of the toxic metal from the roots towards the aerial parts.Senna obtusifolia from root to leaves is reflected by the high TF . Transfer factors across the Senna obtusifolia plots were all greater than 2. Plant species with TF values greater than 1 are considered suitable for phytoextraction which generally requires translocation of the heavy metals to easily harvestable parts such as shoots.Senna obtusifolia is characterized by a widely distributed and highly branched root system and has vital characteristics for phytoextraction of the toxic metal.38The observed high Pb levels in Andropogon gerardii V.) and switchgrass (Panicum virgatum L.), Levy et al., reported normal concentrations of Pb in plants with a range from 0.5 mg/kg to 10 mg/kg and phytotoxic concentration from 30 mg/kg to 300 mg/kg.Senna obtusifolia in the present study showed Pb concentrations far higher than the normal concentration, but within the phytotoxic levels. The results indicate that Senna obtusifolia is tolerant to the toxic effect of the target metal. Given its fast growth rate, high above ground biomass, tolerance to Pb pollution, survival and adaptability to prevailing environmental conditions, widely distributed and highly branched root system, accumulation of the metal from soil and high translocation from root to shoot exhibited by wild Senna obtusifolia in Dareta village, the plant could be considered a green alternative method for the mitigation of Pb pollution and poisoning in Zamfara.The phytoextraction potential of a given plant species is essentially determined not only by its TF, but also by the BAC and BCF.Senna obtusifolia from Dareta village compared to Senna obtusifolia from Bassawa village, Zaria may be attributed to elevated soil Pb levels due the influence of mining and processing of Pb-rich gold ore by artisans in Dareta village. The significant (p < 0.01) positive correlations observed between concentrations of Pb in soil and Senna obtusifolia leaves (r = 0.829), concentrations of Pb in soil and Senna obtusifolia stem (r= 0.776), and concentrations in soil and Senna obtusifolia roots (r = 0.659) indicate that as Pb concentration in soil increases, Pb contents of the leaves, stem and roots of Senna obtusifolia also increases, suggesting that same source may be responsible for the presence of the toxic metal at the determined concentrations. The significant difference of Pb contents in Senna obtusifolia tissues (roots stem and leaves) may be a reflection the preferred storage organ for the metal in the plant.The significantly higher Pb levels observed in tissues of Senna obtusifolia is a green leafy vegetable and an important delicacy, commonly called ‘tapassa’ among the Hausa of northern Nigeria. The lead content of the leaves recorded in this study (Table 2) was found to be generally higher than the permissible levels by the Food and Agriculture Organization of the United Nations and World Health Organization (FAO/WHO) and the European Union (EU) maximum levels of 0.30 mg/kg for Pb in leafy vegetables.Senna obtusifolia leaves were also higher than the reported human toxicity levels of 1.00 mg Pb/day.47Senna obtusifolia leaves was found to be higher than the Pb content of the stem and root, indicating significant accumulation of the toxic metal in the leaves. Senna obtusifolia leaves were therefore noted as the major storage organ for Pb in the plant. The high mobility of Pb in Senna obtusifolia from root to leaves was reflected by a high TF. The high translocation factor and BAC recorded in this study indicates that Senna obtusifolia has vital characteristics which can be used for phytoextraction of the metal. Significantly higher Pb levels in Senna obtusifolia from Dareta village compared to Senna obtusifolia leaves from Bassawa village, Zaria (control) can be attributed to the influence of mining and processing of Pb-rich gold ore by artisans in Dareta. The range of Pb concentrations of Senna obtusifolia leaves was found to be far above the Codex general standards and the EU maximum levels for Pb in leafy vegetables. The Pb content of Senna obtusifolia leaves was also higher than the reported human toxicity levels of 1.00 mg Pb/day. Consumption of Senna obtusifolia leaves from the study area thus poses serious risk of Pb intoxication. The authors recommend a mass literacy campaign to educate the local population on the inherent dangers of consumption of Senna obtusifolia from the area. Optimizing the remediation potentials of wild Senna obtusifolia growing in Dareta is also recommended.The ability of plants to clean up Pb contaminated sites depends on the amount of the metal that can be accumulated by them, soil Pb concentration, rate of uptake, and translocation and accumulation in harvestable tissues. Soil Pb levels in this study were found to be within the USEPA standards as well as the Dutch intervention values. The lead content of


To achieve the robust high-performance computer-aided diagnosis systems for lymph nodes, CT images may be typically collected from multicenter data, which cause the isolated performance of the model based on different data source centers. The variability adaptation problem of lymph node data which is related to the problem of domain adaptation in deep learning differs from the general domain adaptation problem because of the typically larger CT image size and more complex data distributions. Therefore, domain adaptation for this problem needs to consider the shared feature representation and even the conditioning information of each domain so that the adaptation network can capture significant discriminative representations in a domain-invariant space. This paper extracts domain-invariant features based on a cross-domain confounding representation and proposes a cycle-consistency learning framework to encourage the network to preserve class-conditioning information through cross-domain image translations. Compared with the performance of different domain adaptation methods, the accurate rate of our method achieves at least 4.4% points higher under multicenter lymph node data. The pixel-level cross-domain image mapping and the semantic-level cycle consistency provided a stable confounding representation with class-conditioning information to achieve effective domain adaptation under complex feature distribution. Novel techniques for medical image analysis, computer-aided diagnosis algorithms, and related topics have been recently emerging. The research in these directions depends not only on the development of artificial intelligence methods but also on the promotion of big data techniques. In fact, deep learning algorithms for computer-aided diagnosis typically use realistic big medical datasets that are manually labeled. Problems pertaining to medical image analysis are complicated by the fact that clinical images of the same modality may differ if these images are generated by devices of different models, manufacturers, or imaging parameters. For big-data-driven medical diagnosis algorithms, a model trained only by one centrally acquired dataset cannot typically generalize well, a shortcoming which limits the application scope of such model , 2. HoweThere are two types of methods in domain adaption: data-centric methods and subspace-centric methods . On the In domain adaption problems, the most commonly used methods are the adversarial training ones such as the gradient reversal layer (GRL) method or the maximum mean discrepancy (MMD) method. Ganin et al.  proposedThe unsupervised image translation problem is similar to the domain adaption problem. For a data-centric problem, whether it is an unsupervised domain-adaption problem or a style translation problem, the main purpose is to find a domain-invariant representation across the two domains. In comparison with the methods that use specific constraint losses such as the MMD or CORAL methods, a more flexible implicit-loss scheme named cross-domain image mapping is proposed for unsupervised image style translation , 14, by The biggest difference between the domain-adaptation problem and the image translation problem is that the former problem is characterized by a weakening of image reconstruction quality and an enhancement of the class-conditioning information in the domain-invariant space. As mentioned above, the domain-invariant space in the domain-adaptation problem should have the characteristics of being classifiable and domain invariant. The features extracted from the two domains can be reconstructed across domains, and a generated image cannot be distinguished as to which domain it originates from. The GTA  uses theIn another main contribution of this paper, a domain-adaptation network is built based on the cross-domain confounding representation to solve the multicenter problem of computer-aided diagnosis data. Inspired by the unsupervised image translation problem, a cross-domain confounding representation is used to implicitly force the network to extract domain-invariant features through cross-domain mapping. The network architecture consists of a pair of encoders and decoders, as well as a discriminator and a classifier. The pair of encoders has common parameters for feature extraction across the two domains, while the classifier achieves the final classification in the domain-invariant space. Because of the complex feature distribution of medical images and the requirement of class-conditioning information in the domain-invariant space, the classification cycle consistency is added to the loss function after the cross-domain mapping to help the network update the gradient with class-conditioning information. We introduce here our approach in three stages. In the first stage, we introduce the network architecture and the main functions of each component. In the second stage, we combine the functions of all parts to describe the construction of the loss function. In the third stage, we introduce how the whole network uses loss function to achieve the main functions of each component and the verification of network.G has six deconvolution layers to reconstruct an image through the domain-invariant features. The network function structure is mainly composed of two parts. One part extracts the cross-domain confounding representation through the loss of GAN, and the other part guarantees the consistency of the class condition information in the codec process through the classification cycle consistency. Our network architecture consists of pairs of decoders and discriminators as shown in S and T, respectively, and S′ and T′ be the generate image, respectively. Also, let the source and target images be xS and xT. In order to achieve cross-domain confounding representation and classification cycle consistency, we optimize the domain discriminator D, decoder G, classifier C, and encoder F as follows.Let the source and target distributions be D in each domain so that the discriminator discriminates the domain other than the two domains. The loss function of D is shown in equations ..D in eacG is to maximize the domain discriminator confusion by generating images that fool this domain discriminator as much as possible. This confusion constitutes a part of the adversarial loss. As the training progresses, the decoder G generates images with more domain characteristics. The classification cycle consistency (C(F(xS⟶S′)) and C(F(xS⟶T))) is introduced to update G. The generated images are reclassified through the encoder F and the discriminator D. By minimizing the classification loss, the decoding network captures more flexible class structure changes, retaining the class-conditioning information in the generated image. The loss function of G is given by equations ..G is to C is applied to the domain-invariant space. The goal of the classifier is to correctly classify the source and target images, xS and xT. For image classification, we directly optimize the classifier C by minimizing a binary cross-entropy loss. For the generated image, the classifier also minimizes the classification loss of the cross-domain reconstructed image in the target domain. So, the label information can be introduced in the target domain to improve the classification performance of the target-domain-specific features. It should be noted that when we use cross-domain-generated images to update C, we do not use the gradient information of F and G. The loss function of C is shown in the following equation:The classifier F is the same as the decoder G. By minimizing the loss of the classifier C, classification cycle consistency (C(F(xS⟶S′)) and C(F(xS⟶T))) enhances the ability to extract certain domain-specific features. The GAN adversarial loss combines this encoder loss with the domain discriminant loss. In addition, the cross-domain discriminant loss is introduced to ensure the extraction of domain-invariant features. The purpose of the generator G is only to generate an image of each domain to fool the corresponding domain discriminator D without cross-domain mapping, while the purpose of the encoder F is mainly to map the image to the domain-invariant space that maximizes the domain discriminator confusion during cross-domain generation. We adjust the proportion of the same-domain reconstruction loss and cross-domain reconstruction loss in F by the parameters α (0.1) and β (0.1). The functions of the module F and module G are different from each other in terms of the optimization process, implicitly enforcing domain invariance of the extracted features. At the same time, the goal of the encoder network F is to minimize the label prediction loss with L2 regularization in the cross-domain confounding representation. Equation  dataset  and MNISBecause the target-domain labels are not available in practical domain-adaptation problems, the final domain-adaptation model is generally obtained as the end-of-training model. Indeed, the model stability during training is crucial for reaching a highly optimal model in practical applications. We mainly evaluate the model performance in terms of stability and accuracy. For experimental setup, batch normalization was employed prior to the rectified linear-unit (ReLU) activation function in network training. Moreover, the Adam optimizer with a momentum of 0.99 was adopted. This framework was executed using PyTorch under the NVIDIA TITAN V 12-GB GPU.We demonstrate experimentally the classification performance based on our data and assess the subsequent domain adaptation effects on the associated source domain. In particular, we use the CT data of each single center to learn the parameters of the network architecture. The results are derived for the validation set and are shown in We report here the experimental validation results of our approach. We use the commonly used domain-adaptation methods for comparison. All the mentioned procedures are reproduced in the same deep learning framework according to the reference papers. In addition to the medical dataset, we also validate our method on the simple data distributions of the digit datasets to demonstrate the universality of our method.We test the three common domain adaptation settings. The first setting, SVHN MNIST, refers to domain adaption with SVHN as the source domain and MNIST as the target domain, while the setting MNIST SVHN refers to opposite domain-adaptation process. In the same way, the two settings, plain enhanced and enhanced plain, represent the domain adaptation on our medical dataset of the plain and enhanced CT scans. F and discriminator C networks. From The source-only method means that the model was trained with the source-domain dataset and directly tested on the target-domain dataset. The network architecture is the combination of the encoder For the complex data distribution of the CT images, our method achieves the best performance . The worse performance of DSN  shows the complexity between domain-invariant features and domain-specific features under complex data distributions. GRL  and MMD  do not perform well on complex data distributions compared with the source-only performance, which demonstrate the effectiveness of class-conditioning information on domain-invariant spaces. The other methods show large fluctuations in network performance stability when the features become complex. Meanwhile, the performance of our method is the most stable and is also robust to the shift of the class-conditioning information in the complex feature distribution. This stability makes our method more applicable in practice for domain adaptation. Compared to the results of F and G, constrains the class-conditioning feature in domain invariant space. Compared with the result from GTA (We run additional experiments to assess the effect of the classification cycle consistency on domain adaptation and thus verify the importance of the class-conditioning information in domain-invariant spaces. Specifically, we test the proposed framework with and without the classification cycle consistency loss. The results are shown in from GTA , if it wTo solve the computer-aided diagnosis for muticenter lymph node data, a domain adaptation network used for complex data distribution is proposed. Unlike other domain adaptation methods, the proposed unsupervised domain adaptation method extracts domain-invariant feature through cross-domain confounding representations so that it can get a domain-invariant space for target and source data. Moreover, the classification cycle consistency captures the class-conditioning information in domain-invariant space in order to solve the difficult-to-perceive-detail class-conditioning information. The effectiveness of the proposed method is proved through the experimental results. Meanwhile, the proposed method achieves the more robust performance under both simple data distribution and complex data distribution, which may be because of the available constraint of the class-conditioning feature in domain invariant space. Moreover, it is found that the cross-domain confounding representations based on adversarial training are also robust for the change of updata sequence. Although the accuracy rates are higher than 70% through domain adaptation, it is much lower than what it should be. Maybe it is because the weight for each loss constraint needs to be well selected to balance the effect of various constraints on different parts. Furthermore, the domain adaptation performance is also needed to be demonstrated under 3D image application.This paper proposes a domain adaptation method to solve the multicenter problem of the computer-aided diagnosis for lymph nodes. Aiming at the high-resolution and complex feature distribution of the lymph CT images, this paper constructs a confounding representation of domain features based on a cross-domain mapping to achieve domain-invariant feature extraction. For the complex data distribution of lymph CT images, the classification cycle consistency guides the proposed model to perceive significant classifiable representations in a domain-invariant space. Through the above method, our model achieves a stable domain adaptation of high-resolution images in complex medical field. The experimental results for simple data distributions also show the versatility of the proposed method. The training stability also allows us to simply get the optimal model under the target domain, which can further achieve the multidomain medical data integration.


While GCS proteins have been demonstrated to regulate O2-dependent pathways, the mechanism by which the O2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O2-dependent GCS activity.The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O Since the discovery of cyclic dimeric guanosine monophosphate (c-di-GMP) as a bacterial second messenger and its effects on biofilm formation and antibiotic resistance, the proteins involved in c-di-GMP pathways have been of considerable interest as possible targets for the development of new antibiotics . To date2) levels have been demonstrated to regulate biofilm formation and to affect virulence, making it an important external signal, likely due to its importance in metabolism triphosphate, sodium salt (GTP-α-S), and guanosine-5′-(α-thio)-triphosphate, sodium salt (GpCpp); Jena Biosciences) were dissolved in 100 mM potassium phosphate, pH 7.0, buffer to a stock concentration of 600 μM. The cell was maintained at 25°C, stirred at 750 rpm, and contained 200 μl of protein. Twenty injections of 2 μl of GTP/analog were made over ∼1.5 h.Substrate affinity was determined using a Microcal Auto-iTC200 within the Pennsylvania State University Huck Institute Automated Biological Calorimetry Core facility. PccGCS WT (Uniprot accession number: C6D9C2) consists of four domains, based on sequence alignments and domain homology, with the following approximate boundaries: HisTag (residues 1–11), globin domain (residues 12–181), middle domain (residues 182–331) and DGC domain (residues 332–481)   and 2. S  consists of four segments (HisTag (residues 1–11), globin domain (residues 12–166), middle domain (residues 167–306), and DGC domain (residues 307–485); BpeGReg was accomplished in the same manner as described above for PccGCS. BpeGReg contains 17 lysine residues throughout the 485 amino acid sequence  to disrupt the π-helical nature of helix αB ; this mutation led to a construct with undetectable c-di-GMP production from GTP . Ta. TaPccGC2-binding signal. π-Helices are defined by the occurrence of multiple hydrogen bonds between the amide backbone that reside in amino acids i+5 and i within an α-helix and require a bulge or insertion within a typical α-helix sequence. Mutation of residues within the bulge can alter the π-helix structure and function, but without significantly disrupting the α-helix [Shigella passing through the human small intestine. In response to high concentrations of the bile salt deoxycholate (DOC), which binds to invasion plasmid antigen D (IpaD) of the type three secretion system of Shigella, a π-helical shift within IpaD leads to association with invasion plasmid antigen B (IpaB) and eventually invasion of host cells [EcDosC and 78 DGC-containing GCS homologues, the π-helical kink of EcDosC  is highly conserved [BpeGReg  and PccGCS   .BpeGReg, cross-links were observed between the globin αG/αH loop and the π-helical kink within helix αB of the middle domain. As the αG/αH loop in the globin was observed to become rigid after gas ligand binding to the heme within the histidine kinase-containing GCS from Anaeromyxobacter sp. Fw109-5 [BpeGReg, which could result in signal transduction through a π-helical shift in the middle domain and increased DGC activity. Cross-links also show that PccGCS globin αF helix  interacts with the π-helical αB helix of the middle domain, suggesting that the middle domain π-helix is involved in signal transduction pathways in both BpeGReg and PccGCS. The similar secondary structures (PccGCS WT and π-helix mutant (H237A/K238A construct) but major differences in DGC activities and oligomerization -O2 vs. Fe(II)) [To further probe the role of direct globin-DGC interactions, isolated d excess . These r Fe(II)) .PccGCS and BpeGReg was used to identify sequence characteristics that could result in differences in crosslinking patterns and globin-dependent MBP-PccDGC activity. The alignment indicates that the overall sequences are approximately 35% identical and 52% similar  than the globin domain of BpeGReg, while BpeGReg has a larger DGC domain when compared to PccGCS (17 residue extension at the C-terminus). One possibility is a role for the 12-residue N-terminal extension of the PccGCS globin domain; however, cross-links were not observed between the globin extension and the DGC domain, possibly due to the absence of a lysine within the extension. The data did identify cross-links between the globin domain (helix αB) and the DGC (helix αD) of PccGCS, suggesting that interactions between the globin and DGC helices could be mediating the cognate interactions that allow for globin-specificity in activating MBP-PccDGC.A pairwise sequence alignment of the amino acid sequences of  similar ; PccGCS BpeGReg and PccGCS cross-linking analysis, π-helix mutations, and the globin-DGC activity assays suggest that DGC-containing GCS proteins can form relatively compact structures, with the N-terminal globin domain directly interacting with the C-terminal diguanylate cyclase domain. These GCSs have a globular shape based on analytical gel filtration experiments [PccGCS, suggesting that globin domain helices that undergo conformational changes due to ligand binding can directly transduce signal through interactions near the DGC active site. The middle domain αB helix/π-helical region has emerged as a central component of DGC-containing GCS proteins and appears to be crucial in maintaining the correct quaternary structure for function, even in the presence of direct globin-DGC domain interactions, as evidenced by the lack of O2-dependent changes in activity in the studies on isolated domains. Cross-linking interactions between the DGC output domain and the sensor globin domain within PccGCS, and the activity of the DGC domain of PccGCS being influenced by the cognate PccGCS globin domain, but not the BpeGReg globin domain, are likely due to sequence differences. Based on the results of the present study, the proper quaternary structures of DGC GCSs that effectively convert GTP into c-di-GMP appear dependent on the middle domain/π-helical region for correct positioning of subunits within oligomers. Future structural studies will add to our understanding of how GCS structure leads to function and the mechanism of signal transduction within GCS proteins, and may allow for targeting GCS activity as a method to modulate bacterial biofilm formation and virulence.eriments , and theClick here for additional data file.


The involvement of different structural domains of the coat protein (CP) of turnip mosaic virus, a potyvirus, in establishing and/or maintaining particle assembly was analyzed through deletion mutants of the protein. In order to identify exclusively those domains involved in protein–protein interactions within the particle, the analysis was performed by agroinfiltration “in planta”, followed by the assessment of CP accumulation in leaves and the assembly of virus-like particles lacking nucleic acids, also known as empty virus-like particles (eVLP). Thus, the interactions involving viral RNA could be excluded. It was found that deletions precluding eVLP assembly did not allow for protein accumulation either, probably indicating that non-assembled CP protein was degraded in the plant leaves. Deletions involving the CP structural core were incompatible with particle assembly. On the N-terminal domain, only the deletion avoiding the subdomain involved in interactions with other CP subunits was incorporated into eVLPs. The C-terminal domain was shown to be more permissive to deletions. Assembled eVLPs were found for mutants, eliminating the whole domain. The C-terminal domain mutants were unusually long, suggesting some role of the domain in the regulation of particle length. The identification of the CP domains responsible for eVLP formation will allow for new approaches to protein stretch replacement with peptides or proteins of nanobiotechnological interest. Finally, specific cases of application are considered. As a relevant step in the viral life cycle, the assembly of the viral particle is a crucial process. In the case of plant viruses, our present knowledge about the mechanisms involved is rather limited. Considering plant viruses with tubular virions, all of them non-enveloped ss(+)RNA viruses, tobacco mosaic virus (rigid virions) is the most studied and best known ,2. PotatConversely, the coat proteins (CP) of other plant viruses are able to give rise to particles in the absence of a guiding RNA, the so-called empty virus-like particles . In theWithin plant tubular viruses able to form eVLPs, potyviruses have a prominent position. With members described for most botanical plant families, the potyvirus genus (flexuous virions) contains over one third of all described plant virus species ,22. TheiThe detailed structural knowledge about potyvirus virions and eVLPs ) is facilitating their nanobiotechnological exploitation. Thus far, most developments have been carried out in TuMV VNPs. For instance, selected peptides have been genetically fused between the first and second amino acid at the exposed N-terminal region, obtaining VNPs functionalized for different biotechnological applications ,20,26. CUntil more detailed structural studies of these regions are available, an approach involving deletion mutants of the CP, followed by the examination of their ability to form particles, can provide answers to those questions. Such a study is presented in this work, whose results are also discussed under the perspective of the virus structure.®PCR System 9700, Applied Biosystems, Foster City, CA, USA) was performed on this plasmid, amplifying the sequence encoding the truncated versions, in addition to the sequences corresponding to the initiation codon, the STOP codon and the sequence in 5’ CACC for directional cloning into the pENTRTM/D-TOPOTM vector . Once in the pENTR vector, the insert was moved to the expression vector pEAQ-HT-Dest1 by Gateway cloning, using the enzyme LR clonase (ThermoFisher), and following the manufacturer’s instructions. Information regarding the cloning process and plasmids appears in For plasmid construction to express different truncated versions of the capsid protein, a previous construct of the expression plasmid pEAQ-HT-Dest1 with the coding sequence of the complete capsid protein, was used . A PCR , were incubated overnight at 4 °C. After incubation, the plates were washed intensively and then incubated with the primary antibody, diluted in conjugate buffer , 2% Polyvinylpyrrolidone (PVP) 40 (w/v)) following the manufacturer’s specifications, for 1 h at room temperature. Then, secondary antibodies, diluted 1:1500 in the same buffer, were added and incubated for 1 h at room temperature. The color was developed by an alkaline phosphatase reaction and detected after the addition of p-nitrophenylphosphate. Absorbance was measured at 405 nm .To evaluate the production and accumulation of the viral CP in agroinfiltrated plant tissue, an indirect ELISA was performed using the anti-poty antibody , a monoclonal antibody directed against a conserved epitope in potyvirus CPs, which was still present in all the deleted constructs. The plates  coated with 100 µL plant extract, obtained by homogenizing 1 cmThe eVLP assembly was assessed by TEM, performed as previously described .As a first step for deletion design, we located the TuMV CP domains to be interrogated, within the recently published virus structure (PBD ID 6T34 ). In thiEvaluating the possible implications of the solvent-exposed N-terminal domain in the assembly, the NS region shows an important implication in virion integrity, interacting with the core domain of two other subunits, one located in the same turn (subunit +1) and another one in a different turn (subunit +10). Regarding the NNS region, investigations related to immunogenicity of different CP sequences , as wellAt the C-terminal domain, the CS region forms an internal structure around the particle pore, sustained by small and local interactions with upper and lower subunits. According to the available structural model, CS presents an electronegative potential, which could generate repulsion forces between different subunits, thus providing turgor to the inner wall of the particle and contributing to channel formation. Despite the absence of a well-defined CNS structure in the TuMV model, the solved structure of another potyvirus, PVY , shows tPotyviridae have claimed the implication of regions of different lengths in particle assembly, but these studies were conducted before the structural characterizations, consequently they were not structure-based, with the exception of PVY [To assess the implication of the different domains in particle assembly, CP deletion mutants were designed to generate symmetric constructs: one pair of mutants in NS and CS, another pair in NNS and CNS, and two additional deletions implicating part of the core domain from each end. Previous CP deletion studies in the n of PVY ,35,36,37The first construct is just wild type CP, whose plant-made eVLPs have already shown their usefulness in previous nanobiotechnological applications ,20. In tIn TuMV deletion mutants, the ability to accumulate protein and assemble particles was evaluated, so as to identify indispensable domains for assembly. Production and accumulation of the CP truncated versions was evaluated by an anti-poty ELISA on agroinfiltrated plant tissue, 12 days after infiltration, for the detection of potyviral CP. The ELISA results are shown in According to the results obtained, the NNS, CS and CNS regions can be deleted without affecting CP production and accumulation. No accumulation of the protein was found after deleting the NS domain or stretches of the core region. These results indicate that either the truncated protein is not produced adequately, or there is no accumulation due to its rapid degradation. We favor the degradation interpretation and assume that the protein is not capable of forming stable high-ordered structures, i.e., particles with a certain level of assembly, since there is no obvious reason why the deleted protein could not be correctly translated. This interpretation makes sense if we consider the interactions taking place between the core domain and the NS, very important for the assembly of viral particles ,24,28.In order to assess expendability of the CS, CNS and NNS regions for particle assembly, constructs WT, Δ1-54, Δ273-288 and Δ245-288, giving a high level of production and accumulation, were purified. All of these constructs gave rise to eVLPs, clearly indicating that the deleted regions do not play an essential role in assembly. Particles were then analyzed by TEM to determine the stability of their structural integrity, as well as possible modifications. Some of the obtained micrographs are shown in Potyviridae identified stretches in the N-terminal domain that still allowed assembly, whereas deletions affecting larger parts prevented assembly [Potyviridae is highly variable [Mutant particles Δ1-54 tend to form visible and insoluble aggregates that remain in suspension briefly before depositing and appear in the micrographs as intricate protein nets , V, but assembly ,35,36,37variable ,39, thusA peculiar characteristic of the Δ1-54 particles is their tendency to form aggregates. Considering the structural position of the NNS region, this portion of the N-terminal domain could, in fact, be considered as a functional ‘spacer’, such that its absence would allow new lateral interactions between individual nanoparticles and induce aggregation into nets. These nanoparticles could be deployed when a large protein accumulation is necessary. This type of application would be similar to that obtained by adding glutaraldehyde to the viral nanoparticles , but forRegarding the C-terminal domain, Δ273-288 eVLPs appear as highly elongated nanoparticles, some of them reaching more than six microns. These long nanoparticles are brittle as shown in Finally, Δ245-288 eVLPs are nanoparticles much longer than WT-eVLPs, with lengths of several microns. Furthermore, these particles are more flexuous, although with a similar diameter to unmodified eVLPs. Over time, these nanoparticles tend to fragment into small segments  are unusually long compared to WT-eVLPs. This observation suggests that the domain  could have a role in the regulation of the length of the assembled particles. In the case of PVY eVLPs , the particles show significant differences compared to those of TuMV and, as described above, the deletions at the C-terminal region do not increase their length. For PVY, the whole C-terminal domain has been solved, showing that it is fully luminal and, in the case of virions, involved in interactions with the viral RNA. This type of precise structural information is not available for TuMV. The differential results obtained for eVLPs of viruses so similar could be the reflection of subtle differences between different potyviruses in this region. Again, more structural studies should shed light about this point.All the results obtained show that the NNS and CNS regions are not essential for CP self-assembly, since they do not seem to generate indispensable interactions for particle formation. The implication of these domains in the infectious process of potyviruses and viruses with similar shape was previously studied ,37,40,41