Mediatorless Glucose Biosensor and Direct Electron Transfer Type Glucose/Air Biofuel Cell Enabled with Carbon Nanodots

Utilization of carbon nanodots (CNDs), newcomers to the world of carbonaceous nanomaterials, in the electrochemistry realm has rarely been reported so far. In this study, CNDs were used as immobilization supports and electron carriers to promote direct electron transfer (DET) reactions of glucose oxidase (GOx) and bilirubin oxidase (BOD). At the CNDs electrode entrapped with GOx, a high rate constant (<i>k</i>_s) of 6.28 ± 0.05 s^–1 for fast DET and an apparent Michaelis–Menten constant (<i>K</i>_M^app) as low as 0.85 ± 0.03 mM for affinity to glucose were found. By taking advantage of its excellent direct bioelectrocatalytic performances to glucose oxidation, a DET-based biosensor for glucose detection ranging from 0 to 0.64 mM with a high sensitivity of 6.1 μA mM^–1 and a limit of detection (LOD) of 1.07 ± 0.03 μM (S/N = 3) was proposed. Additionally, the promoted DET of BOD immobilized on CNDs was also observed and effectively catalyzed the reduction of oxygen to water at the onset potential of +0.51 V (vs Ag/AgCl). On the basis of the facilitated DET of these two enzymes at CNDs electrodes, a mediator-free DET-type glucose/air enzymatic biofuel cell (BFC), in which CNDs electrodes entrapped with GOx and BOD were employed for oxidizing glucose at the bioanode and reducing oxygen at the biocathode, respectively, was successfully fabricated. The constructed BFC displayed an open-circuit voltage (OCV) as high as 0.93 V and a maximum power density of 40.8 μW cm^–2 at 0.41 V. These important features of CNDs have implied to be promising materials for immobilizing enzymes and efficient platforms for elaborating bioelectrochemical devices such as biosensors and BFCs

Table_1_Rouxiella badensis, a new bacterial pathogen of onion causing bulb rot.xlsx

Onion bulb rot can be caused by multiple plant pathogens including bacterial pathogens. During our routine survey of commercial onion farms in 2014, 2020, and 2021, seven putative Rouxiella spp. strains were isolated from symptomatic onion samples in Georgia, United States. Upon fulfilling Koch’s postulates on onion, a genome analysis was conducted. Whole-genome indices (ANI and dDDH) showed that the strains belonged to Rouxiella badensis. Although the seven R. badensis strains were not pathogenic on onion foliage, the strains were able to cause bulb rot and could also produce necrotic lesions in a red onion scale assay. R. badensis populations increased significantly and to a level comparable to P. ananatis PNA 97-1R in a red onion scale infection assay. The core-genome analysis grouped all onion R. badensis strains from Georgia together, and distinct from R. badensis strains isolated from other sources and locations. Based on the genome analysis of strains (from the current study and available genomes in the repository), type I, III (Ssa-Esc and Inv-Mxi-Spa types), and V secretion systems are present in R. badensis genomes, while type II, IV, and VI secretion systems are absent. However, various secondary metabolite gene clusters were identified from R. badensis genomes, and a thiol/redox-associated enzyme gene cluster similar to the Pantoea alt cluster mediating thiosulfinate tolerance was also present in onion strains of R. badensis. This is the first report of R. badensis as a plant pathogen.</p

Identification of Tetraazacyclic Compounds as Novel Potent Inhibitors Antagonizing RORγt Activity and Suppressing Th17 Cell Differentiation

<div><p>CD4⁺ T-helper cells that produce interleukin-17 (Th17 cells) are characterized as pathological T-helper cells in autoimmune diseases. Differentiation of human and mouse Th17 cells requires a key transcription regulator, retinoic acid receptor-related orphan receptor γt (RORγt), which is a potential therapeutic target for autoimmune diseases. To develop a therapeutic agent for Th17-mediated autoimmune diseases, we have established a high-throughput screening (HTS) assay for candidate screening, in which the luciferase activity in RORγt-LBD positive and negative Jurkat cells were analyzed to evaluate induction of RORγt activity by compounds. This technique was applied to screen a commercially-available drug-like chemical compound library (Enamine) which contains 20155 compounds. The screening identified 17 compounds that can inhibit RORγt function in the HTS screen system. Of these, three tetraazacyclic compounds can potently inhibit RORγt activity, and suppress Th17 differentiation and IL-17 production. These three candidate compounds could significantly attenuate the expression of the <i>Il17a</i> by 65%- 90%, and inhibit IL-17A secretion by 47%, 63%, and 74%, respectively. These compounds also exhibited a potent anti-RORγt activity, with EC₅₀ values of 0.25 μM, 0.67 μM and 2.6 μM, respectively. Our data demonstrated the feasibility of targeting the RORγt to inhibit Th17 cell differentiation and function with these tetraazacyclic compounds, and the potential to improve the structure of these compounds for autoimmune diseases therapeutics.</p></div

The structures of compound 7, 11, and 14.

<p>The structures of compound 7, 11, and 14.</p

EC₅₀ and CC₅₀ of novel tetraazacyclic compounds.

<p>RORγt⁺-Jurkat cells were seeded onto 96-well round-bottom plates (2x10⁴) overnight and incubated with the compounds titrated at 5-fold gradient final concentrations of 5 μM to 8 nM. 6 h later, relative luciferase activity was recorded to obtain the EC₅₀ values of compounds 7, 11 and 14 (A, B, C, respectively). Jurkat cells (wild type) were seeded onto 96-well round-bottom plates (2x10⁴) overnight and incubated with the compounds at 5-fold gradient concentrations. 48 h treatment later, MTT was added onto 96-well plate and incubated at 37°C for 4 h. MTT was cleaved by live cells to a colored formazan product. After centrifugation (1500rpm, 5min), the supernatant was discarded. 100 μl DMSO was added to dissolve the formazan product and the solution was gently shaken for 10 min. Absorbance at 570 nm wavelength was recorded to identify CC₅₀ values of compounds 7, 11 and 14 (D, E, F, respectively). The results are shown as mean ± SEM.</p

Purification of the CD4⁺CD25^- T cells.

<p>Single-cell suspensions from spleens of 12-week-old C57BL/6 were made by crushing the spleen through a cell strainer, and red blood cells (RBCs) were lysed with an RBC lysis buffer. CD4⁺ T cells were then purified using MACS magnetic cell column with a CD4⁺ T cell isolation kit. And about 90% cells were CD4⁺ CD25^- T cell after isolation. The proportion was significantly higher than before separation, which was only about 32%.</p

Establishment of the 293T-RORγt and Jurkat-RORγt reporter cell lines.

<p>The pGL4.31 reporter plasmids were transfected into 293T cells. The transfected cells were then selected in medium containing hygromycin B to obtain a stable clone which express pGL4.31 reporter gene. Then pBIND-RORγtLBD-IRES-GFP plasmids were also transfected into the stable clone. The second transfected cells were sorted by GFP. GFP⁺-293T stable cell purity was determined >96% by flow cytometry analysis, which yielded a RORγt⁺-293T (RORγt-LBD positive 293T cells) stable reporter cell line, and GFP^--293T stable cells were also sorted and used as a RORγt negative control cell line (Fig 1 top panel). Similar to this procedure, Jurkat cells were also transfected with pGL4.31 reporter plasmids by electroporation and selected in RPMI 1640 medium containing hygromycin B. And then the PBIND-RORγtLBD-IRES-GFP plasmids were transfected into the stable Jurkat clone and were sorted by GFP. GFP⁺-Jurkat stable cell purity was determined >96% by flow cytometry analysis and GFP^--Jurkat stable cells were also sorted (Fig 1 bottom panel).</p

Three candidate compounds inhibit mouse Th17 cell differentiation.

<p>CD4⁺CD25^- T cells were isolated from spleens of 8–12 week old mice using MACS magnetic cell column with a CD4⁺ T cell isolation kit. CD4⁺CD25^- T cells were cultured under Th17 polarizing conditions with vehicle control and hit compounds (5 μM) as described in the methods section. RORγt (A), IL-17A (B) and IL-17F (C) expression was quantified and normalized to GAPDH. The results are shown as mean ± SEM; ** <i>P</i>< 0.01.</p

Validation of the Jurkat-RORγt-LBD stable cell lines.

<p>RORγt⁺-Jurkat (RORγt-LBD positive Jurkat cells) and RORγt^--Jurkat (RORγt-LBD negative Jurkat cells) stable cells (1x10⁵) were seeded onto 12-well plates and incubated in 1 mL RPMI 1640 complete medium overnight. The cells were cultured with different concentrations of PMA (100 nM, 10 nM, 1 nM) or DMSO (vehicle control) for 4hr. The total protein was extracted to determine luciferase activity (A). The fold changes in luciferase activity compared to the DMSO control were determined in RORγt⁺-Jurkat cells and RORγt^--Jurkat cells, when the cells were cultured with different concentrations of PMA (100 nM, 10 nM, 1 nM) or DMSO for 4hr (B, C). RORγt⁺ and RORγt^--Jurkat cells (1 × 10⁵) were seeded onto 12-well plates and incubated in 1 mL RPMI 1640 complete medium overnight. Then the cells were cultured with different concentrations of PMA (100 nM, 10 nM, 1 nM) or DMSO (vehicle control) for 6 hr, at which point total protein was extracted to determine luciferase activity (D). The fold changes in luciferase activity upon exposure to compounds vs DMSO were determined in RORγt⁺-Jurkat cells and RORγt^--Jurkat cells using different concentrations of PMA (100 nM, 10 nM, 1 nM) or DMSO (vehicle control) for 6hr (E, F). The results are shown as means ± SEM.</p

Image_2_A putative multi-sensor hybrid histidine kinase, BarAAc, inhibits the expression of the type III secretion system regulator HrpG in Acidovorax citrulli.JPEG

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, severely damages watermelon, melon, and other cucurbit crops worldwide. Although many virulence determinants have been identified in A. citrulli, including swimming motility, twitching motility, biofilm formation, and the type III secretion system (T3SS), research on their regulation is lacking. To study virulence regulation mechanisms, we found a putative histidine kinase BarAAc that may be related to the T3SS regulator HrpG in A. citrulli. We deleted and characterized barAAc (Aave_2063) in A. citrulli Aac5 strain. Compared to the wild-type Aac5, virulence and early proliferation of barAAc mutant in host watermelon cotyledons were significantly increased, and induction of hypersensitive response in non-host tobacco was accelerated, while biofilm formation and swimming motility were significantly reduced. In addition, the transcriptomic analysis revealed that the expression of many T3SS-related genes was upregulated in the ΔbarAAc deletion mutant when cultured in KB medium. Meanwhile, the ΔbarAAc deletion mutant showed increased accumulation of the T3SS regulator HrpG in KB medium, which may account for the increased deployment of T3SS. This suggests that the putative histidine kinase BarAAc is able to repress the T3SS expression by inhibiting HrpG in the KB medium, which appears to be important for rational energy allocation. In summary, our research provides further understanding of the regulatory network of A. citrulli virulence.</p