Functional Divergence in the Affinity and Stability of Non-Canonical Cysteines and Non-Canonical Disulfide Bonds: Insights from a VHH and VNAR Study.

Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.

Rational Design, Synthesis of Fluorescence Probes for Quantitative Detection of Amyloid-ß in Alzheimer's Disease Based on Rhodamine-Metal Complex.

The efficient detection and monitoring of amyloid-ß plaques (Aß42) can greatly promote the diagnosis and therapy of Alzheimer's disease (AD). Fluorescence imaging is a promising method for this, but the accurate determination of Aß42 still remains a challenge. The development of a reliable fluorescent probe to detect Aß42 is essential. Herein, we report a rational design strategy for Aß42 fluorescence probes based on rhodamine-copper complexes, Rho1-Cu-Rho4-Cu, among them Rho4-Cu exhibits the best performance including high sensitivity (detection limit = 24 nM), high affinity (Kd = 23.4 nM), and high selectivity; hence, Rho4-Cu is selected for imaging Aß42 in AD mice, and the results showed that this probe can differentiate normal mice and AD mice effectively.

A photo-responsive F-box protein FOF2 regulates floral initiation by promoting FLC expression in Arabidopsis.

Floral initiation is regulated by various genetic pathways in response to light, temperature, hormones and developmental status; however, the molecular mechanisms underlying the interactions between different genetic pathways are not fully understood. Here, we show that the photoresponsive gene FOF2 (F-box of flowering 2) negatively regulates flowering. FOF2 encodes a putative F-box protein that interacts specifically with ASK14, and its overexpression results in later flowering under both long-day and short-day photoperiods. Conversely, transgenic plants expressing the F-box domain deletion mutant of FOF2 (FOF2ΔF), or double loss of function mutant of FOF2 and FOL1 (FOF2-LIKE 1) present early flowering phenotypes. The late flowering phenotype of the FOF2 overexpression lines is suppressed by the flc-3 loss-of-function mutation. Furthermore, FOF2 mRNA expression is regulated by autonomous pathway gene FCA, and the repressive effect of FOF2 in flowering can be overcome by vernalization. Interestingly, FOF2 expression is regulated by light. The protein level of FOF2 accumulates in response to light, whereas it is degraded under dark conditions via the 26S proteasome pathway. Our findings suggest a possible mechanistic link between light conditions and the autonomous floral promotion pathway in Arabidopsis.

Quantitative classification evaluation model for tight sandstone reservoirs based on machine learning.

Tight sandstone reservoirs are a primary focus of research on the geological exploration of petroleum. However, many reservoir classification criteria are of limited applicability due to the inherent strong heterogeneity and complex micropore structure of tight sandstone reservoirs. This investigation focused on the Chang 8 tight reservoir situated in the Jiyuan region of the Ordos Basin. High-pressure mercury intrusion experiments, casting thin sections, and scanning electron microscopy experiments were conducted. Image recognition technology was used to extract the pore shape parameters of each sample. Based on the above, through grey relational analysis (GRA), analytic hierarchy process (AHP), entropy weight method (EWM) and comprehensive weight method, the relationship index Q1 between initial productivity and high pressure mercury injection parameters and the relationship index Q2 between initial productivity and pore shape parameters are obtained by fitting. Then a dual-coupled comprehensive quantitative classification prediction model for tight sandstone reservoirs was developed based on pore structure and shape parameters. A quantitative classification study was conducted on the target reservoir, analyzing the correlation between reservoir quality and pore structure and shape parameters, leading to the proposal of favourable exploration areas. The research results showed that when Q1 ≥ 0.5 and Q2 ≥ 0.5, the reservoir was classified as type I. When Q1 > 0.7 and Q2 > 0.57, it was classified as type I1, indicating a high-yield reservoir. When 0.32 < Q1 < 0.47 and 0.44 < Q2 < 0.56, was classified as type II. When 0.1 < Q1 < 0.32 and 0.3 < Q2 < 0.44, it was classified as type III. Type I reservoirs exhibit a zigzag pattern in the northwest part of the study area. Thus, the northwest should be prioritized in actual exploration and development. Additionally, the initial productivity of tight sandstone reservoirs showed a positive correlation with the porosity, permeability, sorting coefficient, coefficient of variation, and median radius. Conversely, it demonstrated a negative correlation with the median pressure and displacement pressure. The perimeters of pores, their circularity, and the length of the major axis showed a positive correlation with the porosity, permeability, sorting coefficient, coefficient of variation, and median radius. On the other hand, they exhibited a negative correlation with the median pressure and displacement pressure. This study quantitatively constructed a new classification and evaluation system for tight sandstone reservoirs from the perspective of microscopic pore structure, achieving an overall model accuracy of 93.3%. This model effectively predicts and evaluates tight sandstone reservoirs. It provides new guidance for identifying favorable areas in the study region and other tight sandstone reservoirs.

Active Ingredients of Reduning Injection Maintain High Potency against SARS-CoV-2 Variants.

OBJECTIVE: To investigate the anti-coronavirus potential and the corresponding mechanisms of the two ingredients of Reduning Injection: quercetin and luteolin. METHODS: A pseudovirus system was designed to test the efficacy of quercetin and luteolin to inhibit SARS-CoV-2 infection and the corresponding cellular toxicity. Luteolin was tested for its activities against the pseudoviruses of SARS-CoV-2 and its variants. Virtual screening was performed to predict the binding sites by Autodock Vina 1.1.230 and PyMol. To validate docking results, surface plasmon resonance (SPR) was used to measure the binding affinity of the compounds with various proteins of the coronaviruses. Quercetin and luteolin were further tested for their inhibitory effects on other coronaviruses by indirect immunofluorescence assay on rhabdomyosarcoma cells infected with HCoV-OC43. RESULTS: The inhibition of SARS-CoV-2 pseudovirus by luteolin and quercetin were strongly dose-dependent, with concentration for 50% of maximal effect (EC50) of 8.817 and 52.98 µmol/L, respectively. Their cytotoxicity to BHK21-hACE2 were 177.6 and 405.1 µmol/L, respectively. In addition, luetolin significantly blocked the entry of 4 pseudoviruses of SARS-CoV-2 variants, with EC50 lower than 7 µmol/L. Virtual screening and SPR confirmed that luteolin binds to the S-proteins and quercetin binds to the active center of the 3CLpro, PLpro, and helicase proteins. Quercetin and luteolin showed over 99% inhibition against HCoV-OC43. CONCLUSIONS: The mechanisms were revealed of quercetin and luteolin inhibiting the infection of SARS-CoV-2 and its variants. Reduning Injection is a promising drug for COVID-19.

Anti-Coronaviral Nanocluster Restrain Infections of SARS-CoV-2 and Associated Mutants through Virucidal Inhibition and 3CL Protease Inactivation.

Antivirals that can combat coronaviruses, including SARS-CoV-2 and associated mutants, are urgently needed but lacking. Simultaneously targeting the viral physical structure and replication cycle can endow antivirals with sustainable and broad-spectrum anti-coronavirus efficacy, which is difficult to achieve using a single small-molecule antiviral. Thus, a library of nanomaterials on GX_P2V, a SARS-CoV-2-like coronavirus of pangolin origin, is screened and a surface-functionalized gold nanocluster (TMA-GNC) is identified as the top hit. TMA-GNC inhibits transcription- and replication-competent SARS-CoV-2 virus-like particles and all tested pseudoviruses of SARS-CoV-2 variants. TMA-GNC prevents viral dissemination through destroying membrane integrity physically to enable a virucidal effect, interfering with viral replication by inactivating 3CL protease and priming the innate immune system against coronavirus infection. TMA-GNC exhibits biocompatibility and significantly reduces viral titers, inflammation, and pathological injury in lungs and tracheas of GX_P2V-infected hamsters. TMA-GNC may have a role in controlling the COVID-19 pandemic and inhibiting future emerging coronaviruses or variants.

Evolution of SARS-CoV-2 Spikes shapes their binding affinities to animal ACE2 orthologs.

IMPORTANCE: Spike-receptor interaction is a critical determinant for the host range of coronaviruses. In this study, we investigated the SARS-CoV-2 WHU01 strain and five WHO-designated SARS-CoV-2 variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and the early Omicron variant, for their Spike interactions with ACE2 proteins of 18 animal species. First, the receptor-binding domains (RBDs) of Alpha, Beta, Gamma, and Omicron were found to display progressive gain of affinity to mouse ACE2. More interestingly, these RBDs were also found with progressive loss of affinities to multiple ACE2 orthologs. The Omicron RBD showed decreased or complete loss of affinity to eight tested animal ACE2 orthologs, including that of some livestock animals (horse, donkey, and pig), pet animals (dog and cat), and wild animals (pangolin, American pika, and Rhinolophus sinicus bat). These findings shed light on potential host range shift of SARS-CoV-2 VOCs, especially that of the Omicron variant.

Parametric study of anterior percutaneous endoscopic cervical discectomy (APECD).

Anterior percutaneous endoscopic cervical discectomy (APECD) is a common treatment for cervical spondylotic radiculopathy (CSR). In this study, the effects of various channel diameters and approach angles on cervical vertebrae on postoperative outcomes in APECD surgery were explored. A finite element model of intact cervical C3-C7 was constructed and then modified to obtain six surgical models. Range of motion (ROM) and intradiscal pressure (IDP) were calculated under different conditions of flexion (Fle), extension (Ext), lateral bending, and axial rotation. During Fle and bending to the left (LB), the ROM was closer to the intact model when the angle of approach was 90°. During bending to the left (LB) and rotation to the left (LR), the ROM changed considerably (43.2%, 33.7%, respectively) where the angle of approach was 45°. As the surgical channel diameter increased, the extent of the change in ROM compared with the intact model also increased. IDP decreased by 48% and 49%, respectively, compared with the intact model at the C5-C6 segment where the angle of approach was 45° and 60° during Fle, while it changed little at 90°, by less than 10%. The IDP was increased noticeably by 117.6%, 82.1%, and 105.8%, for channel diameters of 2, 3 and 4 mm, respectively. And declined noticeably during LB and LR (LB: 27.1%, 27.1%, 38.5%; LR: 37.4%, 35.5%, 48.7%). The results demonstrated that the shorter the surgical path, the smaller surgical diameter, the less the biomechanical influence on the cervical vertebra.

Design and Synthesis of Oleanolic Acid Trimers to Enhance Inhibition of Influenza Virus Entry.

Influenza is a major threat to millions of people worldwide. Entry inhibitors are of particular interest for the development of novel therapeutic strategies for influenza. We have previously discovered oleanolic acid (OA) to be a mild influenza hemagglutinin (HA) inhibitor. In this work, inspired by the 3D structure of HA as a homotrimeric receptor, we designed and synthesized 15 OA trimers with different linkers and central region via the copper-catalyzed azide-alkyne cycloaddition reaction. All of the OA trimers were evaluated for their antiviral activities in vitro, and 12c, 12e, 13c, and 13d were observed to exhibit robust potency (IC50 in the submicromolar range) against influenza A/WSN/33 (H1N1) virus that was stronger than that observed with oseltamivir. In addition, these compounds also displayed strong biological activity against A/Hong Kong/4801/2014 and B/Sichuan/531/2018 (BV). The results of hemagglutination inhibition assays and surface plasmon resonance binding assays suggest that these OA trimers may interrupt the interaction between the HA protein of influenza virus and the host cell sialic acid receptor, thus blocking viral entry. These findings highlight the utility of multivalent OA conjugates to enhance the ligand-target interactions in anti-influenza virus drug design and are also helpful for studying antiviral drugs derived from natural products.

Effective inhibition of coronavirus replication by Polygonum cuspidatum.

Background: The coronavirus disease 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 210 million individuals globally and resulted in over 4 million deaths since the first report in December 2019. The early use of traditional Chinese medicine (TCM) for light and ordinary patients, can rapidly improve symptoms, shorten hospitalization days and reduce severe cases transformed from light and normal. Many TCM formulas and products have a wide application in treating infectious and non-infectious diseases. Polygonum cuspidatum Sieb. et Zucc. (P. cuspidatum), is an important Traditional Chinese Medicine with actions of clearing away heat and eliminating dampness, draining the gallbladder to relieve jaundice, removing blood stasis to alleviate pain, resolving phlegm and arrest cough. In the search for anti-SARS-CoV-2, P. cuspidatum was recommended as as a therapeutic drug of COVID-19 pneumonia.In this study, we aimed to identifies P. cuspidatum is the potential broad-spectrum inhibitor for the treatment of coronaviruses infections. Methods: In the present study , we infected human malignant embryonal rhabdomyoma (RD) cells with the OC43 strain of the coronavirus, which represent an alternative model for SARS-CoV-2 and then employed the cell viability assay kit for the antiviral activity. We combined computer aided virtual screening to predicte the binding site and employed Surface plasmon resonance analysis (SPR) to comfirm the interaction between drugs and coronavirus. We employed fluorescence resonance energy transfer technology to identify drug's inhibition in the proteolytic activity of 3CLpro and Plpro. Results: Based on our results, polydatin and resveratrol derived from P. cuspidatum significantly suppressed HCoV-OC43 replication. 50% inhibitory concentration (IC50) values of polydatin inhibited SARS-CoV-2 Mpro and Plpro, MERS Mpro and Plpro were 18.66, 125, 14.6 and 25.42 µm, respectively. IC50 values of resveratrol inhibited SARS-CoV-2 Mpro and Plpro, MERS Mpro and Plpro were 29.81 ,60.86, 16.35 and19.04 µM, respectively. Finally, SPR assay confirmed that polydatin and resveratrol had high affinity to SARS-CoV-2, SARS-CoV 3Clpro, MERS-CoV 3Clpro and PLpro protein. Conclusions: we identified the antiviral activity of flavonoids polydatin and resveratrol on RD cells. Polydatin and resveratrol were found to be specific and selective inhibitors for SARS-CoV-2, 3CLpro and PLpro, viral cysteine proteases. In summary, this study identifies P. cuspidatum as the potential broad-spectrum inhibitor for the treatment of coronaviruses infections.

Does oblique lumbar interbody fusion promote adjacent degeneration in degenerative disc disease: A finite element analysis.

BACKGROUND: The number of oblique lumbar interbody fusion (OLIF) procedures has continued to rise over recent years. Adjacent segment degeneration (ASD) is a common complication following vertebral body fusion. Although the precise mechanism remains uncertain, ASD has gradually become more common in OLIF. Therefore, the present study analyzed the association between disc degeneration and OLIF to explore whether adjacent degeneration was promoted by OLIF in degenerative disc disease. METHODS: A three-dimensional nonlinear finite element (FE) model of the L3-S1 lumbar spine was developed and validated. Three lumbar spine degeneration models with different degrees of degeneration (mild, moderate and severe) and a model of OLIF surgery were constructed at the L4-L5 level. When subjected to a follower compressive load (500 N), hybrid moment loading was applied to all models of the lumbar spine and the range of motion (ROM), intradiscal pressure (IDP), facet joint force (FJF), average mises stress in the annulus (AMSA), average tresca stress in the annulus (ATSA) and average endplate stress (AES) were measured. RESULTS: Compared with the healthy lumbar spine model, the ROM, IDP, FJF, AMSA, ATSA and AES of the segments adjacent to the degenerated segment increased in each posture as the degree of disc degeneration increased. In different directions of motion, the ROM, IDP, FJF, AMSA, ATSA and AES in the OLIF model in the L3-L4 and L5-S1 segments were higher than those of the healthy model and each degenerated model. Compared with the healthy model, the largest relative increase in biomechanical parameters above (ROM, IDP, FJF, AMSA, ATSA or AES) was observed in the L3-L4 segment in the OLIF model, of 77.13%, 32.63%, 237.19%, 45.36%, 110.92% and 80.28%, respectively. In the L5-S1 segment the corresponding values were 68.88%, 36.12%, 147.24%, 46.00%, 45.88% and 51.29%, respectively. CONCLUSIONS: Both degenerated discs and OLIF surgery modified the pattern of motion and load distribution of adjacent segments (L3-L4 and L5-S1 segments). The increases in the biomechanical parameters of segments adjacent to the surgical segment in the OLIF model were more apparent than those of the degenerated models. In summary, OLIF risked accelerating the degeneration of segments adjacent to those of a surgical segment.

Application of Simulation Methods in Cervical Spine Dynamics.

Neck injury is one of the most frequent spine injuries due to the complex structure of the cervical spine. The high incidence of neck injuries in collision accidents can bring a heavy economic burden to the society. Therefore, knowing the potential mechanisms of cervical spine injury and dysfunction is significant for improving its prevention and treatment. The research on cervical spine dynamics mainly concerns the fields of automobile safety, aeronautics, and astronautics. Numerical simulation methods are beneficial to better understand the stresses and strains developed in soft tissues with investigators and have been roundly used in cervical biomechanics. In this article, the simulation methods for the development and application of cervical spine dynamic problems in the recent years have been reviewed. The study focused mainly on multibody and finite element models. The structure, material properties, and application fields, especially the whiplash injury, were analyzed in detail. It has been shown that simulation methods have made remarkable progress in the research of cervical dynamic injury mechanisms, and some suggestions on the research of cervical dynamics in the future have been proposed.

The Arabidopsis F-box protein FOF2 regulates ABA-mediated seed germination and drought tolerance.

The phytohormone abscisic acid (ABA) plays a crucial role at various plant developmental stages, including seed germination and seedling development, and regulates stomatal aperture in response to drought. However, the underlying mechanisms are not well understood. Here, we showed that F-BOX OF FLOWERING 2 (FOF2) is induced by ABA and drought stress. Overexpression of FOF2 led to reduced ABA sensitivity during seed germination and early seedling development, whereas the fof2 mutant exhibited increased sensitivity to ABA. Molecular and genetic analyses revealed that FOF2 negatively affected ABA-mediated seed germination and early seedling development partially by repressing the expression of the ABA-signaling genes ABI3 and ABI5. Additionally, we found that FOF2-overexpressing plants exhibited increased ABA contents, enhanced ABA sensitivity during stomatal closure, and decreased water loss, thereby improving tolerance to drought stress, in contrast to the fof2 mutant. Consistent with a higher ABA content and enhanced drought tolerance, the expression of ABA- and drought-induced genes and the ABA-biosynthesis gene NCED3 was upregulated in the FOF2-overexpressing plants but downregulated in fof2 mutant in response to drought stress. Taken together, our findings revealed that FOF2 plays an important negative role in ABA-mediated seed germination and early seedling development, as well as a positive role in ABA-mediated drought tolerance.

Biomechanical Effect of L4 -L5 Intervertebral Disc Degeneration on the Lower Lumbar Spine: A Finite Element Study.

OBJECTIVE: To ascertain the biomechanical effects of a degenerated L4 -L5 segment on the lower lumbar spine through a comprehensive simulation of disc degeneration. METHODS: A three-dimensional nonlinear finite element model of a normal L3 -S1 lumbar spine was constructed and validated. This normal model was then modified such that three degenerated models with different degrees of degeneration (mild, moderate, or severe) at the L4 -L5 level were constructed. While experiencing a follower compressive load (500 N), hybrid moment loads were applied to all models to determine range of motion (ROM), intradiscal pressure (IDP), maximum von Mises stress in the annulus, maximum shear stress in the annulus, and facet joint force. RESULTS: As the degree of disc degeneration increased, the ROM of the L4 -L5 degenerated segment declined dramatically in all postures (flexion: 5.79°-1.91°; extension: 5.53°-2.62°; right lateral bending: 4.47°-1.46°; left lateral bending: 4.86°-1.61°; right axial rotation: 2.69°-0.74°; left axial rotation: 2.69°-0.74°), while the ROM in adjacent segments increased (1.88°-8.19°). The largest percent decrease in motion of the L4 -L5 segment due to disc degeneration was in right axial rotation (75%), left axial rotation (69%), flexion (67%), right lateral bending (67%), left lateral bending right (67%), and extension (53%). The change in the trend of the IDP was the same as that of the ROM. Specifically, the IDP decreased (flexion: 0.592-0.09 MPa; extension: 0.678-0.334 MPa; right lateral bending: 0.498-0.205 MPa; left lateral bending: 0.523-0.272 MPa; right axial rotation: 0.535-0.246 MPa; left axial rotation: 0.53-0.266 MPa) in the L4 -L5 segment, while the IDP in adjacent segments increased (0.511-0.789 MPa). The maximum von Mises stress and maximum shear stress of the annulus in whole lumbar spine segments increased (L4 -L5 segment: 0.413-2.626 MPa and 0.412-2.783 MPa, respectively; adjacent segment of L4 -L5 : 0.356-1.493 MPa and 0.359-1.718 MPa, respectively) as degeneration of the disc progressively increased. There was no apparent regularity in facet joint force in the degenerated segment as the degree of disc degeneration increased. Nevertheless, facet joint forces in adjacent healthy segments increased as the degree of disc degeneration increased (extension: 49.7-295.3 N; lateral bending: 3.5-171.2 N; axial rotation: 140.2-258.8 N). CONCLUSION: Degenerated discs caused changes in the motion and loading pattern of the degenerated segments and adjacent normal segments. The abnormal load and motion in the degenerated models risked accelerating degeneration in the adjacent normal segments. In addition, accurate simulation of degenerated facet joints is essential for predicting changes in facet joint loads following disc degeneration.

RPN1a negatively regulates ABA signaling in Arabidopsis.

The 26S proteasome selectively regulates key abscisic acid (ABA) signaling proteins, but the physiological functions and mechanisms of RPN1a (a subunit of the 26S proteasome) in ABA signaling remain largely unknown. In this study, we found that the mRNA expression of RPN1a was suppressed by ABA treatment, and that RPN1a protein was expressed abundantly in guard cells. In the presence of ABA, rpn1a mutants showed rapid stomatal closure, low water loss, delayed germination, and inhibited root elongation. In addition, the transcripts of key ABA signaling genes, including ABI5, RD22, RD29A, and RD29B, were upregulated in rpn1a mutant plants in response to ABA. Furthermore, the ABI5 protein level was higher in rpn1a mutants subjected to ABA treatment. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that RPN1a interacts with ABI1. Overall, these findings suggest that RPN1a negatively regulates ABA signaling in Arabidopsis.