Insights into the Binding Profile of Anti-chlorpyrifos Recombinant Antibodies: From Computational Simulation to Immunoassay Validation.

A novel virtual screening strategy was proposed for the profiling and discovery of active variable regions (VRs) that encode hapten-specific recombinant antibodies (rAbs). Chlorpyrifos, a hazardous organophosphorus pesticide, was selected as the target. First, a VR model-14G4 from anti-chlorpyrifos hybridoma was built via homology modeling. Its binding pattern toward seven organophosphorus analogues was assessed through virtual screening by performing molecular docking. Based on energy scoring, visual examination, and molecular interaction analysis, chlorpyrifos-methyl was also inferred as the high-affinity target for model-14G4 and was then confirmed via a non-competitive surface plasmon resonance (SPR) assay. Subsequently, we attempted to discover hapten-specific VRs by creating a collection of VR models for anonymous testing. Chlorpyrifos and model-14G4 were employed as the known hit and active VRs, respectively. After molecular docking, a novel anti-chlorpyrifos VR (model-1) was identified due to its satisfactory energy scoring and a similar binding pattern to the reference model-14G4. Expressed by HEK293(F) mammalian cells, the newly prepared full-length rAb-model-1 and rAb-14G4 exhibited high sensitivities for detecting chlorpyrifos by the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), with IC50 of 3.01 ng/mL and 42.82 ng/mL, respectively. They recognized chlorpyrifos-methyl with a cross-reactivity (CR) of 2.5-17.3%. Moreover, the binding properties of rAb-model-1 for recognizing chlorpyrifos and chlorpyrifos-methyl were confirmed via a non-competitive microscale thermophoresis (MST) method. Thus, the experimental results showed good agreement with computational outputs on antibody profiling. Furthermore, the recognition diversity of rAb-model-1 for chlorpyrifos and chlorpyrifos-methyl was studied via molecular dynamics simulation. Overall, the proposed study provides a versatile and economical strategy for antibody characterization and promotes the in vitro production of rAbs for pesticide monitoring.

Gold Nanoclusters Enhance the Efficacy of the Polymer-Based Chaperone in Restoring and Maintaining the Native Conformation of Human Islet Amyloid Polypeptide.

The misfolding and un-natural fibrillation of proteins/peptides are associated with many conformation diseases, such as human islet amyloid polypeptide (hIAPP) in type 2 diabetes (T2D). Inspired by molecular chaperones maintaining protein homeostasis in vivo, many polymer-based artificial chaperones were introduced to regulate protein/peptide folding and fibrillation. However, the pure polymer chaperones prefer to agglomerate into large-size micelles in the physiological environment and thus lose their chaperone functions, which greatly restricts the application of polymer-based chaperones. Here, we designed and prepared a core-shell artificial chaperone based on a dozen poly-(N-isopropylacrylamide-co-N-acryloyl-O-methylated-l-arginine) (PNAMR) anchored on a gold-nanocluster (AuNC) core. The introduction of the AuNC core significantly reduced the size and enhanced the efficacy and stability of polymer-based artificial chaperones. The PNAMR@AuNCs, with a diameter of 2.5 ± 0.5 nm, demonstrated exceptional ability in maintaining the natively unfolded conformation of protein away from the misfolding and the following fibrillation by directly binding to the natively unfolded monomolecular hIAPP and hence in preventing their conversion into toxic oligomers. More excitingly, the PNAMR@AuNCs were able to restore the natural unfolded conformation of hIAPP via dissolving the ß-sheet-rich hIAPP fibrils. Considering the uniform molecular mechanism of protein misfolding and fibrillation in conformation disorders, this finding provides a generic therapeutic strategy for neurodegenerative diseases and other conformation diseases by using PNAMR@AuNC artificial chaperones to restore and maintain the native conformation of amyloid proteins.

RT-PCR negative COVID-19.

BACKGROUND: COVID-19 is a multi-system infection with emerging evidence-based antiviral and anti-inflammatory therapies to improve disease prognosis. However, a subset of patients with COVID-19 signs and symptoms have repeatedly negative RT-PCR tests, leading to treatment hesitancy. We used comparative serology early in the COVID-19 pandemic when background seroprevalence was low to estimate the likelihood of COVID-19 infection among RT-PCR negative patients with clinical signs and/or symptoms compatible with COVID-19. METHODS: Between April and October 2020, we conducted serologic testing of patients with (i) signs and symptoms of COVID-19 who were repeatedly negative by RT-PCR ('Probables'; N = 20), (ii) signs and symptoms of COVID-19 but with a potential alternative diagnosis ('Suspects'; N = 15), (iii) no signs and symptoms of COVID-19 ('Non-suspects'; N = 43), (iv) RT-PCR confirmed COVID-19 patients (N = 40), and (v) pre-pandemic samples (N = 55). RESULTS: Probables had similar seropositivity and levels of IgG and IgM antibodies as propensity-score matched RT-PCR confirmed COVID-19 patients (60.0% vs 80.0% for IgG, p-value = 0.13; 50.0% vs 72.5% for IgM, p-value = 0.10), but multi-fold higher seropositivity rates than Suspects and matched Non-suspects (60.0% vs 13.3% and 11.6% for IgG; 50.0% vs 0% and 4.7% for IgM respectively; p-values < 0.01). However, Probables were half as likely to receive COVID-19 treatment than the RT-PCR confirmed COVID-19 patients with similar disease severity. CONCLUSIONS: Findings from this study indicate a high likelihood of acute COVID-19 among RT-PCR negative with typical signs/symptoms, but a common omission of COVID-19 therapies among these patients. Clinically diagnosed COVID-19, independent of RT-PCR positivity, thus has a potential vital role in guiding treatment decisions.

Effective Enrichment of Low-Concentration Rare-Earth Ions by Three-Dimensional Thiostannate K2Sn2S5.

Rare-earth elements (REEs) in industrial wastewaters have great value for recycling and reuse, but their characteristic of low concentration poses a challenge to an efficient enrichment from wastewaters. In recent years, thiometallates featuring two-dimensional layers have shown great potential in the enrichment of REEs via the ion-exchange process. However, investigations on thiometallates featuring three-dimensional anionic frameworks for the recovery of REEs have not been reported. Herein, K2Sn2S5 (KTS-2), a thiostannate possessing a three-dimensional porous framework, was chosen as an ion-exchange material for capturing REEs from an aqueous solution. Indeed, KTS-2 exhibited excellent ion-exchange performance for all 16 REEs (except Pm). Specifically, KTS-2 displayed a high capture capacity (232.7 ± 7.8 mg/g) and a short equilibrium time (within 10 min) for Yb3+ ions. In addition, KTS-2 had a high distribution coefficient for Yb3+ ions (Kd > 105 mL/g) in the presence of excessive interfering ions. Impressively, KTS-2 could reach removal rates of above 95% for all 16 REEs in a large quantity of wastewater with low initial concentration (∼7 mg/L). Moreover, KTS-2 could be used as an eco-friendly material for ion exchange of REEs, since the released K+ cations would not cause secondary pollution to the water solution.

CellO: comprehensive and hierarchical cell type classification of human cells with the Cell Ontology.

Cell type annotation is a fundamental task in the analysis of single-cell RNA-sequencing data. In this work, we present CellO, a machine learning-based tool for annotating human RNA-seq data with the Cell Ontology. CellO enables accurate and standardized cell type classification of cell clusters by considering the rich hierarchical structure of known cell types. Furthermore, CellO comes pre-trained on a comprehensive data set of human, healthy, untreated primary samples in the Sequence Read Archive. CellO's comprehensive training set enables it to run out of the box on diverse cell types and achieves competitive or even superior performance when compared to existing state-of-the-art methods. Lastly, CellO's linear models are easily interpreted, thereby enabling exploration of cell-type-specific expression signatures across the ontology. To this end, we also present the CellO Viewer: a web application for exploring CellO's models across the ontology.

Synthesizing Crystalline Chalcogenidoarsenates in Thiol-Amine Solvent Mixtures.

Thiol-amine solvent mixtures have been widely applied in the solution processing of binary chalcogenide thin films due to their excellent ability to dissolve various bulk binary chalcogenides. However, application of this solvent system in preparing new crystalline chalcogenidometalates has not been explored. In this work, by using a thiol-amine solvent mixture of n-butylamine (BA) and 1,2-ethanedithiol (EDT) as the reaction medium and protonated piperazine (pip) cation as the template, we synthesized a series of new chalcogenidoarsenates with structures ranging from discrete clusters to two-dimensional layers, namely, [pipH2][pipH][AsS4] (1), [pipH2][pipH][As(Se0.4S0.6)4] (2), [pipH2]2[pipH]2[In2AsIII2AsV2S13.3(S2)0.7] (3), [pipH2]2[pipH]2[In2AsIII2AsV2S10.2Se3.1(Se2)0.7] (4), [pipH2]0.5[AsS(S2)] (5), [pipH2]0.5[AsS2] (6), [pipH]2[AgAsS4] (7), [pipH2]1.5[GaAsIIIAsVS7] (8), and Cs2[pipH]2[InAs6S12]Cl (9). Particularly, compounds 3, 4, and 8 contain mixed-valent AsIII and AsV ions in their discrete clusters and one-dimensional chain. In addition, compound 5 could thermodynamically transform to compound 6 with increasing reaction temperature, which may be attributed to the thermodynamically unstable S-S species in the chains of 5. The BA-EDT solvent mixture was crucial to the synthesis of these compounds, since no title crystals can be prepared by replacing the BA-EDT solvent mixture with other conventional solvents or removing one component of the BA-EDT solvent mixture from the reaction system. Our research demonstrates that thiol-amine solvent systems could be promising reaction media for growing novel crystalline chalcogenidometalates.

Au23(CR)14 nanocluster restores fibril Aß's unfolded state with abolished cytotoxicity and dissolves endogenous Aß plaques.

The misfolding of amyloid-ß (Aß) peptides from the natural unfolded state to ß-sheet structure is a critical step, leading to abnormal fibrillation and formation of endogenous Aß plaques in Alzheimer's disease (AD). Previous studies have reported inhibition of Aß fibrillation or disassembly of exogenous Aß fibrils in vitro. However, soluble Aß oligomers have been reported with increased cytotoxicity; this might partly explain why current clinical trials targeting disassembly of Aß fibrils by anti-Aß antibodies have failed so far. Here we show that Au23(CR)14 (a new Au nanocluster modified by Cys-Arg (CR) dipeptide) is able to completely dissolve exogenous mature Aß fibrils into monomers and restore the natural unfolded state of Aß peptides from misfolded ß-sheets. Furthermore, the cytotoxicity of Aß40 fibrils when dissolved by Au23(CR)14 is fully abolished. More importantly, Au23(CR)14 is able to completely dissolve endogenous Aß plaques in brain slices from transgenic AD model mice. In addition, Au23(CR)14 has good biocompatibility and infiltration ability across the blood-brain barrier. Taken together, this work presents a promising therapeutics candidate for AD treatment, and manifests the potential of nanotechnological approaches in the development of nanomedicines.

Cichoric acid from witloof inhibit misfolding aggregation and fibrillation of hIAPP.

The misfolding, aggregation and fibrillation of human islet amyloid polypeptide (hIAPP) has been acknowledged as a hallmark event in type-II diabetes. Hence, inhibiting the misfolding, aggregation and fibrillation of hIAPP have been accepted as a vital factor to treat the disease. Here cichoric acid was extracted from witloof to explore its inhibition effects on misfolding, aggregation and fibrillation of hIAPP. Thioflavin-T (ThT) fluorescence assay, dynamic light scattering (DLS) and atomic force microscopy (AFM) images showed that cichoric acid inhibited the aggregation and fibrillation of hIAPP in a dosage-dependent manner. Circular dichroism (CD) spectra showed that cichoric acid inhibited the misfolding of hIAPP from unfolded to ß-sheet. Molecular docking and further experiments revealed interactions between hIAPP and cichoric acid. Cichoric acid could bind to K1 and R11 of hIAPP via electrostatic interaction. In addition, cichoric acid could form π-π stacking with hIAPP residues F15 and F23. These interactions inhibited the misfolding, aggregation and fibrillation of hIAPP. These results, together with cichoric acid's good cytocompatibility and significant protective effects in hIAPP lesioned cell models, not only showed that cichoric acid could be used to fight against amyloidosis, but also brought a new perspective for Chinese herbal medicine as natural compound's medical potential.

Chiral Effect at Nano-Bio Interface: A Model of Chiral Gold Nanoparticle on Amylin Fibrillation.

Protein/Peptide amyloidosis is the main cause of several diseases, such as neurodegenerative diseases. It has been widely acknowledged that the unnatural fibrillation of protein/peptides in vivo is significantly affected by the physical and chemical properties of multiscale biological membranes. For example, previous studies have proved that molecule chirality could greatly influence the misfolding, fibrillation and assembly of ß-Amyloid peptides at the flat liquid-solid surface. However, how the nanoscale chirality influences this process remains unclear. Here we used gold nanoparticles (AuNPs, d = 4 ± 1 nm)-modified with N-isobutyl-L(D)-cysteine (L(D)-NIBC) enantiomers-as a model to illustrate the chiral effect on the amylin fibrillation at nano-bio interface. We reported that both two chiral AuNPs could inhibit amylin fibrillation in a dosage-dependent manner but the inhibitory effect of L-NIBC-AuNPs was more effective than that of D-NIBC-AuNPs. In-situ real time circular dichroism (CD) spectra showed that L-NIBC-AuNPs could inhibit the conformation transition process of amylin from random coils to α-helix, while D-NIBC-AuNPs could only delay but not prevent the formation of α-helix; however, they could inhibit the further conformation transition process of amylin from α-helix to ß-sheet. These results not only provide interesting insight for reconsidering the mechanism of peptides amyloidosis at the chiral interfaces provided by biological nanostructures in vivo but also would help us design therapeutic inhibitors for anti-amyloidosis targeting diverse neurodegenerative diseases.

Chiral ß-HgS quantum dots: Aqueous synthesis, optical properties and cytocompatibility.

ß-HgS quantum dots (QDs) have drawn enormous attention due to the size-tunable bandgap and the lowest quantum state in conduction band which have been applied to semiconductor transistor and photodetector. Though ß-HgS is the essential component of Tibetan medicine, the potential toxicity of ß-HgS limits its applications, especially in bio-application. Herein, chiral biomolecule enantiomers N-isobutyryl-L(D)-cysteine (L(D)-NIBC) and L(D)-cysteine (L(D)-Cys) were introduced into HgCl2 and Na2S aqueous solution to synthesize chiral ß-HgS QDs in one-pot, which significantly improved their water-solubility and cytocompatibility. Notably, all chiral ß-HgS QDs showed none cytotoxicity even at high concentration (20 mg·L-1), and the cytocompatibility of D-ß-HgS QDs was better than corresponding L-ß-HgS QDs at the concentration of 20 mg·L-1. This cytotoxicity discrimination was associated with the chirality inversion of chiral ß-HgS QDs compared with the corresponding chiral ligands. In-situ real-time circular dichroism (CD) monitoring indicated that the chirality of ß-HgS QDs originated from the asymmetrical arrangement of chiral ligands on the achiral core surface. Their chiroptical activity, near-infrared optical absorption (800 nm), fluorescence emission (900-1000 nm), high-performance photothermal conversion and good cytocompatibility, implied chiral ß-HgS QDs could be used as a candidate material for photothermal therapy or a near-infrared fluorescent probe in organism, which brings a novel insight for bio-application of ß-HgS QDs.

Heterologous protein incites abnormal plasma cell accumulation and autoimmunity in MRL-MpJ mice.

Although it is evident that there is complex interplay among genetic and environmental factors contributing to systemic autoimmunity, the events inciting autoreactivity are incompletely understood. Previously we demonstrated that MRL-MpJ mice posses a genetic background susceptible to autoimmunity development under conditions of altered inhibitory signaling. To gain better understanding of the influence of exogenous factors on autoreactivity in susceptible individuals, young MRL-MpJ mice were challenged with a single injection of heterologous protein and evaluated for evidence of autoimmunity. We found that MRL-MpJ mice developed high titer serum reactivity to DNA within 1 week of protein administration reaching maximal levels within 1 month. Importantly, the level of autoimmunity was sustained for an extended period of time (6 months). This was accompanied by a substantial increase in germinal center B cell and plasma cell numbers. In contrast, control mice showed no change in autoreactivity or lymphocyte homeostasis. Autoimmunity was dependent on marginal zone B cells as their depletion reduced serum auto-reactivity after challenge, thus suggesting immune stimulation with heterologous proteins can precipitate loss of B cell tolerance and autoimmunity in genetically prone individuals. This model may provide an important tool to further investigate the mechanisms whereby environmental stimuli trigger autoimmune reactivity in susceptible hosts.

Increased angiogenesis in Cdk4(R24C/R24C):Apc(+/Min) intestinal tumors.

Cyclin dependent kinase 4 (Cdk4) is a cell cycle regulator involved in early G1 cell cycle progression and has been indirectly implicated in angiogenesis in the Min mouse system, a mouse that harbors a mutation in the Apc gene. Apc(+/Min) mice when crossed with Ink4a/arf-/- mice, exhibited increased angiogenesis of colorectal tumors suggesting that dysregulation of Cdk4 (due to loss of Ink4a-mediated suppression) may contribute to enhanced angiogenesis. To demonstrate a direct role for Cdk4 in angiogenesis, we crossed mice that have an activated Cdk4, Cdk4(R24C/R24C) mice, with Apc(+/Min) mice and examined levels of angiogenesis in intestinal tumors formed. Our results show an increase in the percentage of highly vascularized tumors in Cdk4(R24C/R24C):Apc(Min/+) and Cdk4(+/R24C):Apc(Min/+) mice compared to Cdk4(+/+):Apc(Min/+) mice. In addition immunohistochemical analysis showed an increase in CD-31 staining localized to endothelial cells of Cdk4(R24C/R24C):Apc(Min/+) mouse tumors, supporting the hypothesis of increased vasculature in these tumors. Further analysis showed an increase in the expression of the E2F1 target proteins Vegf-b and Cyclin A in Cdk4(R24C/R24C):Apc(+/Min) intestinal tumors. Together these data suggest that the dysregulated Cdk4 gene plays an important role in angiogenesis during intestinal tumor formation and may in part act via increasing E2F1 target proteins. This is the first report to show that Cdk4 has a direct role in angiogenesis in vivo and may be an important drug target to reduce or prevent angiogenesis during intestinal tumor formation.

Accelerated atherosclerosis in ApoE deficient lupus mouse models.

The accelerated development of atherosclerosis with increased risk of cardiovascular disease in systemic lupus erythematosus (SLE) patients is not well understood. An appropriate mouse model would greatly help to understand the mechanisms of this association. We have therefore combined the ApoE(-/-) model of atherosclerosis with three different murine models of SLE. We found that induction of cGVH in B6.ApoE(-/-) mice, breeding a Fas null gene onto the B6.ApoE(-/-) mice, and breeding the ApoE(-/-) defect onto MRL/lpr mice all caused a modest increase of atherosclerosis at 24 weeks of age compared to B6.ApoE(-/-) controls. B cells in B6.ApoE(-/-) mice had certain phenotypic differences compared to congenic C57BL/6 mice, as indicated by high expression of MHC II, Fas, CD86, and by increased number of cells bearing marginal zone phenotype. Furthermore, B6ApoE(-/-) mice had significant titers of anti-oxLDL and anti-cardiolipin autoantibodies compared to their B6 counterparts. Our studies also indicate that, following induction of cGVH, marginal zone B cells in B6.ApoE(-/-) are depleted, and there is considerable increase in anti-oxLDL and anti-cardiolipin abs along with secretion of lupus-specific autoantibodies, such as anti-dsDNA and anti-chromatin abs. Histological sections showed that cGVH and/or Fas deficiency could exacerbate atherosclerosis. The production of anti-oxLDL and anti-cardiolipin in ApoE(-/-) mice was also increased. These observations define a connection between induction of lupus-like symptoms and development of severe atherosclerosis in ApoE deficient lupus mouse models.

Modulation of autoimmunity by TLR9 in the chronic graft-vs-host model of systemic lupus erythematosus.

Chronic graft-vs-host (cGVH) disease is induced in nonautoimmune mice by the transfer of alloreactive T cells that recognize foreign MHC class II. It closely resembles systemic lupus erythematosus, with antinuclear Abs and immune-mediated nephritis. Recent work has implicated TLRs, particularly TLR9, in the recognition of certain autoantigens in vitro and in vivo. To explore further the role of TLR9 in systemic autoimmunity, we induced cGVH disease in C57BL/6 (B6) mice lacking TLR9, including B6 mice expressing the anti-DNA-encoding IgH transgenes 3H9 or 56R (B6.3H9.TLR9(-/-), B6.56R.TLR9(-/-)). We found that cGVH disease caused breakdown of B cell tolerance to chromatin and DNA in TLR9(-/-) recipients of alloreactive cells, yet that nephritis was less severe and that some autoantibody titers were lower compared with B6-cGVH controls. Spleen lymphocyte analysis showed that cGVH disease strikingly depleted marginal zone B cells in B6 mice, but did not influence T cell subsets in either B6 or B6-TLR9(-/-) hosts. B6.56R.TLR9(-/-) mice had less spontaneous production of autoantibodies than B6.56R mice, but there were no significant differences between B6.56R and B6.56R.TLR9(-/-) postinduction of cGVH disease. Taken together, these results suggested that TLR9 may worsen some aspects of systemic autoimmunity while alleviating others.