Discovery and multimerization of cross-reactive single-domain antibodies against SARS-like viruses to enhance potency and address emerging SARS-CoV-2 variants.

Coronaviruses have been the causative agent of three epidemics and pandemics in the past two decades, including the ongoing COVID-19 pandemic. A broadly-neutralizing coronavirus therapeutic is desirable not only to prevent and treat COVID-19, but also to provide protection for high-risk populations against future emergent coronaviruses. As all coronaviruses use spike proteins on the viral surface to enter the host cells, and these spike proteins share sequence and structural homology, we set out to discover cross-reactive biologic agents targeting the spike protein to block viral entry. Through llama immunization campaigns, we have identified single domain antibodies (VHHs) that are cross-reactive against multiple emergent coronaviruses (SARS-CoV, SARS-CoV-2, and MERS). Importantly, a number of these antibodies show sub-nanomolar potency towards all SARS-like viruses including emergent CoV-2 variants. We identified nine distinct epitopes on the spike protein targeted by these VHHs. Further, by engineering VHHs targeting distinct, conserved epitopes into multi-valent formats, we significantly enhanced their neutralization potencies compared to the corresponding VHH cocktails. We believe this approach is ideally suited to address both emerging SARS-CoV-2 variants during the current pandemic as well as potential future pandemics caused by SARS-like coronaviruses.

Hexamerization of Anti-SARS CoV IgG1 Antibodies Improves Neutralization Capacity.

The SARS-CoV-2 pandemic and particularly the emerging variants have deepened the need for widely available therapeutic options. We have demonstrated that hexamer-enhancing mutations in the Fc region of anti-SARS-CoV IgG antibodies lead to a noticeable improvement in IC50 in both pseudo and live virus neutralization assay compared to parental molecules. We also show that hexamer-enhancing mutants improve C1q binding to target surface. To our knowledge, this is the first time this format has been explored for application in viral neutralization and the studies provide proof-of-concept for the use of hexamer-enhanced IgG1 molecules as potential anti-viral therapeutics.

Optimization of factors affecting the rooting of pine wilt disease resistant Masson pine (Pinus massoniana) stem cuttings.

Pine wilt disease (PWD) is a devastating disease affecting trees belonging to the genus Pinus. To control the spread of PWD in the Masson pine forest in China, PWD resistant Masson pine clones have been selected by the Anhui Academy of Forestry. However, because Masson pine is a difficult-to-root species, producing seedlings is challenging, especially from trees older than 5 years of age, which impedes the application of PWD resistant clones. In this study, we investigated the factors affecting rooting of PWD resistant clones and established a cheap, reliable, and simple method that promotes rooting. We tested the effects of three management methods, four substrates, two cutting materials, two cutting treatments, and three collection times on the rooting of cuttings obtained from 9-year-old PWD resistant clones. Rooting was observed only in stem cuttings treated with the full-light automatic spray management method. Additionally, stem cuttings showed a significantly higher rooting rate and root quality than needles cuttings. Compared with other substrates, stem cuttings planted in perlite produced the longest adventitious root and the highest total root length and lateral root number. Moreover, stem cuttings of PWD resistant clones collected in May showed a significantly higher rooting rate and root quality than those collected in June and July. Moreover, stem cuttings prepared with a horizontal cut while retaining the needles showed significantly higher rooting rate and root quality than those prepared with a diagonal cut while partly removing the needles. This study promotes the reproduction of seedlings of PWD-resistant Masson pine clones which helps control the spread of PWD, meanwhile, provides a technical reference for the propagation of mature pine trees via cuttings.

TaWRKY74 participates copper tolerance through regulation of TaGST1 expression and GSH content in wheat.

Glutathione S-transferase (GST) is the key enzyme in glutathione (GSH) synthesis, and plays a crucial role in copper (Cu) detoxification. Nonetheless, its regulatory mechanisms remain largely unclear. In this study, we identified a Cu-induced glutathione S-transferase 1 (TaGST1) gene in wheat. Yeast one-hybrid (Y1H) screened out TaWRKY74, which was one member from the WRKY transcription factor family. The bindings between TaGST1 promoter and TaWRKY74 were further verified by using another Y1H and luciferase assays. Expression of TaWRKY74 was induced more than 30-folds by Cu stress. Functions of TaWRKY74 were tested by using transiently silence methods. In transiently TaWRKY74-silenced wheat plants, TaWRKY74 and TaGST1 expression, GST activity, and GSH content was significantly inhibited by 25.68%, 19.88%, 27.66%, and 12.68% in shoots, and 53.81%, 52.11%, 23.47%, and 17.11% in roots, respectively. However, contents of hydrogen peroxide, malondialdehyde, or Cu were significantly increased by 2.58%, 12.45%, or 37.74% in shoots, and 25.24%, 53.84%, and 103.99% in roots, respectively. Notably, exogenous application of GSH reversed the adverse effects of transiently TaWRKY74-silenced wheat plants during Cu stress. Taken together, our results suggesting that TaWRKY74 regulated TaGST1 expression and affected GSH accumulation under Cu stress, and could be useful to ameliorate Cu toxicity for crop food safety.

Exogenous Glutathione Alleviates Cadmium Toxicity in Wheat by Influencing the Absorption and Translocation of Cadmium.

Cadmium (Cd), a toxic heavy metal, is harmful to plants and human health. Glutathione (GSH) could alleviate Cd toxicity of plant species, whereas its mechanism responsible for wheat remains poorly understood. Here, we found that exogenous GSH application significantly increased the fresh and dry weight, root elongation, chlorophyll contents, while decreased the contents of malondialdehyde (MDA) and GSH, and translocation factor of Cd compared with Cd treatment. Moreover, GSH application significantly increased activities of antioxidant enzymes and expression of related genes, which involved in GSH synthesis, especially in roots. In addition, we found that GSH application suppressed Cd-induced expression of metal transporter genes TaNramp1, TaNramp5, TaHMA2, TaHMA3, TaLCT1 and TaIRT2 in roots. Taken together, our results suggested that GSH could alleviate Cd toxicity in wheat by increasing GSH synthesis gene expression or suppressing Cd transporter genes expression, and further affecting Cd uptake and translocation in wheat plants.

Melatonin promotes potassium deficiency tolerance by regulating HAK1 transporter and its upstream transcription factor NAC71 in wheat.

Melatonin (MT) is involved in various physiological processes and stress responses in animals and plants. However, little is known about the molecular mechanisms by which MT regulates potassium deficiency (DK) tolerance in crops. In this study, an appropriate concentration (50 µmol/L) was found to enhance the tolerance of wheat plants against DK. RNA-seq analysis showed that a total of 6253 and 5873 differentially expressed genes (DEGs) were separately identified in root and leaf tissues of the DK + MT-treated wheat plants. They functionally involved biological processes of secondary metabolite, signal transduction, and transport or catabolism. Of these, an upregulated high-affinity K transporter 1 (TaHAK1) gene was next characterized. TaHAK1 overexpression markedly enhanced the K absorption, while its transient silencing exhibited the opposite effect, suggesting its important role in MT-mediated DK tolerance. Moreover, yeast one-hybrid (Y1H) was used to screen the upstream regulators of TaHAK1 gene and the transcription factor TaNAC71 was identified. The binding between TaNAC71 and TaHAK1 promoter was evidenced by using Y1H, LUC, and EMSA assays. Transient overexpression of TaNAC71 in wheat protoplasts activated the TaHAK1 expression, whereas its transient silencing inhibited the TaHAK1 expression and aggravated the sensitivity to DK. Exogenous MT application greatly upregulated the expression of TaHAK1 in both transient overexpression and silencing systems. Our findings revealed some molecular mechanisms underlying MT-mediated DK tolerance and helped broaden its practical application in agriculture.

[Theoretical study of the influence of different substituents on the electron-photon spectra of quinoline].

Different substituents result in different changes in electron-photon spectra, and to reveal the relationship between substituents and spectra, a theoretical investigation was elaborated via quantum chemical calculations. Density functional theory and single excitation configuration interaction were respectively employed in optimizing geometric and electronic structures of ground and excited states, and the absorption and emission spectra were studied by time-dependent density functional theory methods. The results show that all the different substituents bring on different geometric and electronic structures of ground and excited states, different energies of frontier molecular orbitals as well as different pi-conjugated systems, the spectra change with all the differences, and relationships are brought out in this paper, which gives theoretical reference for identifying different derivatives from electron-photon spectra.

Comparative sperm ultrastructure of three species in Siniperca (Teleostei: Perciformes: Sinipercidae).

Siniperca chuatsi, Siniperca kneri, and Siniperca scherzeri are three of the most economically important sinipercid species. The ultrastructure and morphology of the mature spermatozoa of them are examined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The sperm consists of an acrosome-less head, a short midpiece and a long flagellum. Ultrastructurally, it has a homogeneously electron-dense nucleus in a granular pattern with nuclear lucent and a nuclear fossa excluding the centriolar complex. One to four mitochondria with lamellar cristae encircle the basal body of the flagellum in the midpiece. The cytoplasm surrounding the centrioles and the cylindric cytoplasmic channel contains glycogen granules and vesicles. Comprising the conventional 9+2 axoneme, vesicles and lateral fins, the sperm flagellum is inserted laterally on the nucleus, therefore the spermatozoon is asymmetrical. All of the spermatozoa of the three species are of the primitive or ect-aquasperm form and conform to the teleostean type II spermatozoa instead of the previously supposed type I. Variations in the shape of the heads, angles between the two centrioles, location of the cytoplasmic vesicles, mitochondrial number and structural characteristics of the lateral fins are notable among the three species. S. chuatsi is a sister-group of the other two species and is the most differentiated. The present study provides fresh insights to the comparative spermatology of Siniperca fishes and will be useful to the existing knowledge of the sinipercid fishes in systematic characters, biodiversity conservation and reproduction.

Anti-IL-17A therapy protects against bone erosion in experimental models of rheumatoid arthritis.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine secreted by a subset of memory T cells and other innate immune cells. It is associated with rheumatoid arthritis (RA) due to IL-17A expression in RA synovial fluid. The severe bone erosive rat adjuvant-induced arthritis (rAIA) and mouse collagen-induced arthritis (mCIA) models were used to address the therapeutic efficacy of anti-IL-17A treatment with a focused investigation on bone protection. In the rAIA model, treatment with anti-IL-17A completely alleviated arthritis, lowered the level of receptor activator of NFκB ligand (RANKL), and inhibited structural damage to the bones. In the mCIA model, IL-17A neutralization coincident with arthritis development or in mice with established arthritis diminished joint swelling by inhibiting disease initiation and progression. Intriguingly, even the few joints that became outwardly severely inflamed in the presence of an anti-IL-17A antagonist had diminished joint histopathology scores compared to severely inflamed, control-treated mice. The bone-preserving property correlated with decreased RANKL message in severely inflamed paws of arthritic mice. These data identify IL-17A as a key factor in inflammation-mediated bone destruction and support anti-IL-17A therapy for the treatment of inflammatory bone diseases such as RA.

Structural, cellular, and molecular evaluation of bone erosion in experimental models of rheumatoid arthritis: assessment by µCT, histology, and serum biomarkers.

Bone erosion is a clinical endpoint for various diseases including rheumatoid arthritis. In this paper, we used rodent arthritis models with severe bone erosion to examine the structural, cellular, and molecular aspects of the inflammation-driven bone resorption process. Our data show that bone loss is observed only in chronically, severely inflamed joints. The most severely affected anatomic sites were the metatarsal phalangeal joint and tarsal bones of the paw. The magnitude of the inflammation-driven bone erosion was dependent on both the duration of inflammatory response and the severity of the joint swelling response. The application of micro-computed tomography well demonstrated the therapeutic benefit of anti-IL-17A in protection of bones from erosion. Alterations in the cellular profile of the joint occurred prior to any major structural deterioration of the bone. Receptor activator for nuclear factor κB ligand, a potent inducer of osteoclast differentiation and bone resorption, was elevated in animals coincident with severe arthritis initiation. The experimental approaches and concepts outlined in this paper provide a valuable process to evaluate and quantify therapies that modulate rodent arthritis-associated bone-erosion models.

Neutralization or absence of the interleukin-23 pathway does not compromise immunity to mycobacterial infection.

Interleukin-23 (IL-23), a member of the IL-12 family, is a heterodimeric cytokine that is composed of the p40 subunit of IL-12 plus a unique p19 subunit. IL-23 is critical for autoimmune inflammation, in part due to its stimulation of the proinflammatory cytokine IL-17A. It is less clear, however, if IL-23 is required during the immune response to pathogens. We examined the role of IL-23 during Mycobacterium bovis BCG infection. We found that IL-23 reduces the bacterial burden and promotes granuloma formation when IL-12 is absent. However, IL-23 does not contribute substantially to host resistance when IL-12 is present, as the ability to control bacterial growth and form granulomata is not affected in IL-23p19-deficient mice and mice treated with a specific anti-IL-23p19 antibody. IL-23p19-deficient mice are also able to mount an effective memory response to secondary infection with BCG. While IL-23p19-deficient mice do not produce IL-17A, this cytokine is not necessary for effective control of infection, and antibody blocking of IL-17A in both wild-type and IL-12-deficient mice also has little effect on the bacterial burden. These data suggest that IL-23 by itself does not play an essential role in the protective immune response to BCG infection; however, the presence of IL-23 can partially compensate for the absence of IL-12. Furthermore, neutralization of IL-23 or IL-17A does not increase susceptibility to mycobacterial BCG infection.

Dietary salt supplementation selectively downregulates NPR-C receptor expression in kidney independently of ANP.

Atrial natriuretic peptide (ANP) has negative modulatory effects on a variety of pathophysiological mechanisms; i.e., it inhibits hypoxia-induced pulmonary vasoconstriction and vascular remodeling and facilitates natriuresis and vasorelaxation in NaCl-supplemented subjects. We have previously demonstrated organ-selective potentiation of ANP in the pulmonary circulation of hypoxia-adapted animals by local downregulation of its clearance receptor (NPR-C; Li H, Oparil S, Meng QC, Elton T, and Chen Y-F. Am J Physiol Lung Cell Mol Physiol 268: L328-L335, 1995). The present study tested the hypothesis that NPR-C expression is attenuated selectively in kidneys of NaCl-supplemented subjects. Adult male wild-type (ANP+/+) and homozygous mutant (ANP-/-) mice were studied after 5 wk of normal or high-salt diets. Mean arterial pressure (MAP) and left (LV) and right ventricular (RV) mass were greater in ANP-/- mice than in ANP+/+ mice fed the normal-salt diet; salt supplementation induced increases in plasma ANP in ANP+/+ mice and in MAP and LV, RV, and renal mass in ANP-/- mice but not in ANP+/+ mice. NPR-C mRNA levels were selectively and significantly reduced (>60%) in kidney, but not in lung, brain, LV, or RV, by dietary salt supplementation in both genotypes. NPR-A mRNA levels did not differ among diet-genotype groups in any organ studied. cGMP content was significantly increased in kidney, but not in lung or brain, by dietary salt supplementation in both genotypes. These findings suggest that selective downregulation of NPR-C in the kidney in response to dietary salt supplementation may contribute to local elevation in ANP levels and may be functionally significant in attenuating the development of salt-sensitive hypertension.