Genome-wide association research on the reproductive traits of Qianhua Mutton Merino sheep.

Objective: Qianhua Mutton Merino sheep is a new breed of meat wool sheep cultivated independently in China. In 2018, it was approved by the state and brought into the national list of livestock and poultry genetic resources. Qianhua Mutton Merino sheep has the common characteristics of typical meat livestock varieties with rapid growth and development in the early stage and high meat production performance.The objective of this research is to investigate the Genome-wide association of the reproductive traits of Qianhua Mutton Merino sheep. Methods: Qianhua Mutton Merino sheep from the breeding core group were selected as the research object, GWAS was conducted on genes associated with the reproductive traits (singleton or twins, birth weight, age [in days] for sexual maturity, weaning weight, and daily gain from birth to weaning) of Qianhua mutton merino. Results: Our study findings showed that 151 loci of SNPs were detected, among which 3 SNPs related to birth weight and weaning weight occupied a significant portion of the wide genome. The candidate genes preliminarily obtained were SYNE1, SLC12A4, BMP2K, CAMK2D, IMMP2L, DMD, and BCL2. Conclusion: 151 SNP loci were detected for five traits related to reproduction (including singleton or twins, birth weight, age [in days] at sexual maturity, weaning weight, and daily weight gain from birth to weaning). The functions of these candidate genes were mainly enriched in nucleotide metabolism, metal ion binding, oxytocin signaling pathway, and neurotrophin signaling pathway.

Investigation of the antibacterial activity of benziothiazolinone against Xanthomonas oryzae pv. oryzae.

Plant pathogenic bacteria can cause numerous diseases for higher plants and result in severe reduction of crop yield. Introduction of new bactericides can always effectively control these plant diseases. Benziothiazolinone (BIT) is a novel fungicide registered in China for the control of plant fungal diseases, however, its anti-bacterial activity is not well studied. The results of activity tests showed that BIT exhibited stronger inhibitory activity against bacteria, particularly for Xanthomonas oryzae pv. oryzae (Xoo) (EC50 = 0.17 µg/mL), which was superior than that of the tested fungi in vitro. BIT also exhibited excellent protective and curative activity against rice bacterial leaf blight (BLB) caused by Xoo with the control efficacies of 71.37% and 91.64% at 600 µg/mL, respectively. After treatment with BIT, Xoo cell surface became wrinkled and the cell shape was distorted with extruding cellular content. It was also found that BIT decreased DNA synthesis and affected the biofilm formation and motility of Xoo cells. However, no significant change in the protein content was observed. Moreover, the results of quantitative real-time PCR also showed that expressions of several genes related to DNA synthesis, biofilm formation and motility of Xoo cells were down- or up-regulated, which further proved the anti-bacterial activity of BIT in influencing the biological properties of Xoo. Additionally, BIT also enhanced the activity of phenylalanine ammonia lyase (PAL), a plant defense enzyme. Taken together, benziothiazolinone might be served as an alternative candidate for the control of BLB.

Large-scale production of MXenes as nanoknives for antibacterial application.

Antimicrobial resistance of existing antibacterial agents has become a pressing issue for human health and demands effective antimicrobials beyond conventional antibacterial mechanisms. Two-dimensional (2D) nanomaterials have attracted considerable interest for this purpose. However, obtaining a high yield of 2D nanomaterials with a designed morphology for effective antibacterial activity remains exceptionally challenging. In this study, an efficient one-step mechanical exfoliation (ECO-ME) method has been developed for rapidly preparing Ti3C2 MXenes with a concentration of up to 30 mg mL-1. This synthetic pathway involving mechanical force endows E-Ti3C2 MXene prepared by the ECO-ME method with numerous irregular sharp edges, resulting in a unique nanoknife effect that can successfully disrupt the bacterial cell wall, demonstrating better antibacterial activity than the MXenes prepared by conventional wet chemical etching methods. Overall, this study provides a simple and effective method for preparing MXenes on a large scale, and its antibacterial effects demonstrate great potential for E-Ti3C2 in environmental and biomedical applications.

A Specific High Toxicity of Xinjunan (Dioctyldiethylenetriamine) to Xanthomonas by Affecting the Iron Metabolism.

Xanthomonas spp. encompass a wide range of phytopathogens that brings great economic losses to various crops. Rational use of pesticides is one of the effective means to control the diseases. Xinjunan (Dioctyldiethylenetriamine) is structurally unrelated to traditional bactericides, and is used to control fungal, bacterial, and viral diseases with their unknown mode of actions. Here, we found that Xinjunan had a specific high toxicity toward Xanthomonas spp., especially to the Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial leaf blight. Transmission electron microscope (TEM) confirmed its bactericidal effect by morphological changes, including cytoplasmic vacuolation and cell wall degradation. DNA synthesis was significantly inhibited, and the inhibitory effect enhanced with the increase of the chemical concentration. However, the synthesis of protein and EPS was not affected. RNA-seq revealed differentially expressed genes (DEGs) particularly enriched in iron uptake, which was subsequently confirmed by siderophore detection, intracellular Fe content and iron-uptake related genes transcriptional level. The laser confocal scanning microscopy and growth curve monitoring of the cell viability in response to different Fe condition proved that the Xinjunan activity relied on the addition of iron. Taken together, we speculated that Xinjunan exerted bactericidal effect by affecting cellular iron metabolism as a novel mode of action. IMPORTANCE Sustainable chemical control for rice bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae need to be developed due to limited bactericides with high efficiency, low cost, and low toxicity in China. This present study verified a broad-spectrum fungicide named Xinjunan possessing a specific high toxicity to Xanthomonas pathogens, which were further confirmed by affecting the cellular iron metabolism of Xoo as a novel mode of action. These findings will contribute to the application of the compound in the field control of Xanthomonas spp.-caused diseases, and be directive for future development of novel specific drugs for the control of severe bacterial diseases based on this novel mode of action.

Investigation of the antifungal activity of the dicarboximide fungicide iprodione against Bipolaris maydis.

Southern corn leaf blight (SCLB), mainly caused by Bipolaris maydis, is a destructive disease of maize worldwide. Iprodione is a widely used dicarboximide fungicide (DCF); however, its antifungal activity against B. maydis has not been well studied until now. In this study, the sensitivity of 103 B. maydis isolates to iprodione was determined, followed by biochemistry and physiology assays to ascertain the fungicide's effect on the morphology and other biological properties of B. maydis. The results indicated that iprodione exhibited strong inhibitory activity against B. maydis, and the EC50 values in inhibiting mycelial growth ranged from 0.088 to 1.712 µg/mL, with a mean value of 0.685 ± 0.687 µg/mL. After treatment with iprodione, conidial production of B. maydis was decreased significantly, and the mycelia branches increased with obvious shrinkage, distortion and fracture. Moreover, the expression levels of the osmotic pressure-related regulation genes histidine kinase (hk) and Ssk2-type mitogen-activated protein kinase (ssk2) were upregulated, the glycerin content of mycelia increased significantly, the relative conductivity of mycelia increased, and the cell wall membrane integrity was destroyed. The in vivo assay showed that iprodione at 200 µg/mL provided 79.16% protective efficacy and 90.92% curative efficacy, suggesting that the curative effect was better than the protective effect. All these results proved that iprodione exhibited strong inhibitory activity against B. maydis and provided excellent efficacy in controlling SCLB, indicating that iprodione could be an alternative candidate for the control of SCLB in China.

In Vitro Determination of Sensitivity of Fusarium fujikuroi to Fungicide Azoxystrobin and Investigation of Resistance Mechanism.

Rice bakanae disease (RBD) caused by Fusarium fujikuroi is a widespread and destructive disease of rice. It is urgent to introduce a new class of fungicide to manage the fungicidal resistance problem and effectively control the disease. Azoxystrobin (AZO) is an active fungicide with a broad antifungal spectrum, while its activity against F. fujikuroi is not well investigated. In this study, the baseline sensitivity of F. fujikuroi to AZO was established by testing the sensitivity of 100 isolates, collected from Anhui Province of China. The mechanism of resistance to AZO was also investigated. AZO exhibited a strong activity against the 100 F. fujikuroi isolates with EC50 values of 0.822 ± 0.285 and 0.762 ± 0.283 µg/mL for mycelial growth and conidial germination, respectively, and both of the baseline sensitivity curves were validated as unimodal curves. To investigate the resistance mechanism, six mutants with resistance factor (RF) values >50 were generated from wild-type sensitive strains through UV mutagenesis, and sequence analysis showed that mutation G143A in cyt b conferred the resistance to AZO. Mycelial growth, conidia production, pathogenicity, and ATP production were decreased in all six resistant mutants as compared to the parental strains, indicating the fitness penalties in this phenotype of resistance mutation. In addition, the cross-resistance assay showed that there was no cross-resistance between AZO and carbendazim, prochloraz, phenamacril, or pydiflumetofen. AZO can be an efficient candidate to control RBD in China with moderate to low fungal resistance risk, but continuous resistance monitoring should be performed during the application of this fungicide.

Biological Activity of Pyraclostrobin Against Coniella granati Causing Pomegranate Crown Rot.

Pomegranate crown rot caused by Coniella granati is one of the most severe diseases of pomegranate. No fungicides have been registered for controlling this disease in China. Pyraclostrobin, belonging to strobilurin fungicides, has a broad spectrum of activity against many phytopathogens. In this study, based on the mycelial growth and conidial germination inhibition methods, we investigated the biological activity of pyraclostrobin against C. granati in the presence of 50 µg/ml of salicylhydroxamic acid using 80 isolates collected from different orchards in China from 2012 to 2018. The EC50 (50% effective concentration) values ranged from 0.040 to 0.613 µg/ml for mycelial growth and 0.013 to 0.110 µg/ml for conidium germination. Treated with pyraclostrobin, the hyphae morphology changed and conidial production of C. granati decreased significantly. The result of transmission electron microscope showed that treatment of pyraclostrobin could make the cell wall thinner and lead to ruptured cell membrane and formation of intracellular organelle autophagosomes. The pyraclostrobin showed good protective and curative activities against C. granati on detached pomegranate fruits. In field trials, pyraclostrobin showed excellent control efficacy against this disease, in which the treatment of 25% pyraclostrobin EC 1,000× provided 92.25 and 92.58% control efficacy in 2019 and 2020, respectively, significantly higher than that of other treatments. Therefore, pyraclostrobin could be a candidate fungicide for the control of pomegranate crown rot.

DCs-based therapies: potential strategies in severe SARS-CoV-2 infection.

Pneumonia caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is spreading globally. There have been strenuous efforts to reveal the mechanisms that the host defends itself against invasion by this virus. The immune system could play a crucial role in virus infection. Dendritic cell as sentinel of the immune system plays an irreplaceable role. Dendritic cells-based therapeutic approach may be a potential strategy for SARS-CoV-2 infection. In this review, the characteristics of coronavirus are described briefly. We focus on the essential functions of dendritic cell in severe SARS-CoV-2 infection. Basis of treatment based dendritic cells to combat coronavirus infections is summarized. Finally, we propose that the combination of DCs based vaccine and other therapy is worth further study.

A Guanidyl-Based Bivalent Peptidomimetic Inhibits K-Ras Prenylation and Association with c-Raf.

Unusual lipid modification of K-Ras makes Ras-directed cancer therapy a challenging task. Aiming to disrupt electrostatic-driven protein-protein interactions (PPIs) of K-Ras with FTase and GGTase I, a series of bivalent dual inhibitors that recognize the active pocket and the common acidic surface of FTase and GGTase I were designed. The structure-activity-relationship study resulted in 8 b, in which a biphenyl-based peptidomimetic FTI-277 was attached to a guanidyl-containing gallate moiety through an alkyl linker. Cell-based evaluation demonstrated that 8 b exhibited substantial inhibition of K-Ras processing without apparent interference with Rap-1A processing. Fluorescent imaging showed that 8 b disrupts localization of K-Ras to the plasma membrane and impairs interaction with c-Raf, whereas only FTI-277 was found to be inactive. These results suggest that targeting the PPI interface of K-Ras may provide an alternative method of inhibiting K-Ras.

Systematic drug safety evaluation based on public genomic expression (Connectivity Map) data: myocardial and infectious adverse reactions as application cases.

Adverse drug reaction (ADR) is of great importance to both regulatory agencies and the pharmaceutical industry. Various techniques, such as quantitative structure-activity relationship (QSAR) and animal toxicology, are widely used to identify potential risks during the preclinical stage of drug development. Despite these efforts, drugs with safety liabilities can still pass through safety checkpoints and enter the market. This situation raises the concern that conventional chemical structure analysis and phenotypic screening are not sufficient to avoid all clinical adverse events. Genomic expression data following in vitro drug treatments characterize drug actions and thus have become widely used in drug repositioning. In the present study, we explored prediction of ADRs based on the drug-induced gene-expression profiles from cultured human cells in the Connectivity Map (CMap) database. The results showed that drugs inducing comparable ADRs generally lead to similar CMap expression profiles. Based on such ADR-gene expression association, we established prediction models for various ADRs, including severe myocardial and infectious events. Drugs with FDA boxed warnings of safety liability were effectively identified. We therefore suggest that drug-induced gene expression change, in combination with effective computational methods, may provide a new dimension of information to facilitate systematic drug safety evaluation.

Matrix-induced autologous chondrocyte implantation for the treatment of chondral defects of the knees in Chinese patients.

Articular cartilage injury is the most common type of damage seen in clinical orthopedic practice. The matrix-induced autologous chondrocyte implant (MACI) was developed to repair articular cartilage with an advance on the autologous chondrocyte implant procedure. This study aimed to evaluate whether MACI is a safe and efficacious cartilage repair treatment for patients with knee cartilage lesions. The primary outcomes were the Knee Injury and Osteoarthritis Outcome Score (KOOS) domains and magnetic resonance imaging (MRI) results, compared between baseline and postoperative months 3, 6, 12, and 24. A total of 15 patients (20 knees), with an average age of 33.9 years, had a mean defect size of 4.01 cm(2). By 6-month follow-up, KOOS results demonstrated significant improvements in symptoms and knee-related quality of life. MRI showed significant improvements in four individual graft scoring parameters at 24 months postoperatively. At 24 months, 90% of MACI grafts had filled completely and 10% had good-to-excellent filling of the chondral defect. Most (95%) of the MACI grafts were isointense and 5% were slightly hyperintense. Histologic evaluation at 15 and 24 months showed predominantly hyaline cartilage in newly generated tissue. There were no postoperative complications in any patients and no adverse events related to the MACI operation. This 2-year study has confirmed that MACI is safe and effective with the advantages of a simple technique and significant clinical improvements. Further functional and mechanistic studies with longer follow-up are needed to validate the efficacy and safety of MACI in patients with articular cartilage injuries.

Synthesis and antibacterial activities of acylide derivatives bearing an aryl-tetrazolyl chain.

Seventeen acylides bearing an aryl-tetrazolyl alkyl-substituted side chain were synthesized, starting from clarithromycin, via several reactions including hydrolysis, acetylating, esterification, carbamylation, and Michael addition. The structures of all new compounds were confirmed by (1)H nuclear magnetic resonance spectroscopy, (13)C nuclear magnetic resonance spectroscopy, and mass spectrometry. All these synthesized acylides were evaluated for in vitro antimicrobial activities against gram-positive pathogens (Staphylococcus aureus, Staphylococcus epidermidis) and gram-negative pathogens (Pseudomonas aeruginosa, Escherichia coli), using the broth microdilution method. Results showed that compounds 10 e, 10 f, 10 g, 10 h, 10 o have good antibacterial activities.

Prediction of the likelihood of drug interactions with kinase inhibitors based on in vitro and computational studies.

Kinase inhibitors (KIs) represent an important group of anticancer drugs, and many of them are substrates and inhibitors of human cytochrome P450s (CYPs), raising the potential of harmful drug interactions. This study investigated the effect of a library of KIs (n = 91) including 11 FDA-approved KIs on human CYP1A2, 2D6, 2C9, and 3A4 using high-throughput screening kits and the binding modes with CYPs using the Discovery Studio program 3.1. The KIs exhibited differential inhibitory effect on CYP1A2, 2D6, 2C9, and 3A4, while some of them showed activating effect on CYP2C9 and 3A4. For example, SP 600125 was a potent inhibitor for CYP1A2, but enhanced the activity of CYP2C9 fourfolds. Among the 80 KIs that are not used clinically, about 13% showed significant inhibition to CYPs. Nilotinib, sunitinib, and imatinib were found to be potent CYP1A2 inhibitor. Our docking studies have demonstrated the importance of multiple amino acid residues in the active sites of CYP1A2, 2C9, 2D6, and 3A4 in binding with various KIs. Finally, the in vitro data were used to predict potential KI-drug interactions. These findings indicate that many KIs can serve as CYP inhibitors, and further studies are needed to examine the clinical impact.

Prediction of drug-target interactions for drug repositioning only based on genomic expression similarity.

Small drug molecules usually bind to multiple protein targets or even unintended off-targets. Such drug promiscuity has often led to unwanted or unexplained drug reactions, resulting in side effects or drug repositioning opportunities. So it is always an important issue in pharmacology to identify potential drug-target interactions (DTI). However, DTI discovery by experiment remains a challenging task, due to high expense of time and resources. Many computational methods are therefore developed to predict DTI with high throughput biological and clinical data. Here, we initiatively demonstrate that the on-target and off-target effects could be characterized by drug-induced in vitro genomic expression changes, e.g. the data in Connectivity Map (CMap). Thus, unknown ligands of a certain target can be found from the compounds showing high gene-expression similarity to the known ligands. Then to clarify the detailed practice of CMap based DTI prediction, we objectively evaluate how well each target is characterized by CMap. The results suggest that (1) some targets are better characterized than others, so the prediction models specific to these well characterized targets would be more accurate and reliable; (2) in some cases, a family of ligands for the same target tend to interact with common off-targets, which may help increase the efficiency of DTI discovery and explain the mechanisms of complicated drug actions. In the present study, CMap expression similarity is proposed as a novel indicator of drug-target interactions. The detailed strategies of improving data quality by decreasing the batch effect and building prediction models are also effectively established. We believe the success in CMap can be further translated into other public and commercial data of genomic expression, thus increasing research productivity towards valid drug repositioning and minimal side effects.

The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner.

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.

Apogossypol derivatives as antagonists of antiapoptotic Bcl-2 family proteins.

Guided by a combination of nuclear magnetic resonance binding assays and computational docking studies, we synthesized a library of 5,5' substituted Apogossypol derivatives as potent Bcl-XL antagonists. Each compound was subsequently tested for its ability to inhibit Bcl-XL in an in vitro fluorescence polarization competition assay and exert single-agent proapoptotic activity in human cancer cell lines. The most potent compound BI79D10 binds to Bcl-XL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively, and potently inhibits cell growth in the H460 human lung cancer cell line with an EC50 value of 680 nmol/L, expressing high levels of Bcl-2. BI79D10 also effectively induces apoptosis of the RS11846 human lymphoma cell line in a dose-dependent manner and shows little cytotoxicity against bax-/-bak-/- mouse embryonic fibroblast cells, in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that BI79D10 has little off-target effects. BI79D10 displays in vivo efficacy in transgenic mice, in which Bcl-2 is overexpressed in splenic B cells. Together with its improved plasma and microsomal stability relative to Apogossypol, BI79D10 represents a lead compound for the development of novel apoptosis-based therapies for cancer.

Bcl-XL-templated assembly of its own protein-protein interaction modulator from fragments decorated with thio acids and sulfonyl azides.

Protein-protein interactions have key importance in various biological processes and modulation of particular protein-protein interactions has been shown to have therapeutic effects. However, disrupting or modulating protein-protein interactions with low-molecular-weight compounds is extremely difficult due to the lack of deep binding pockets on protein surfaces. Herein we describe the development of an unprecedented lead synthesis and discovery method that generates only biologically active compounds from a library of reactive fragments. Using the protein Bcl-XL, a central regulator of programmed cell death, we demonstrated that an amidation reaction between thio acids and sulfonyl azides is applicable for Bcl-XL-templated assembly of inhibitory compounds. We have demonstrated for the first time that kinetic target-guided synthesis can be applied not only on enzymatic targets but also for the discovery of small molecules modulating protein-protein interactions.

Structure-based design of imidazole-containing peptidomimetic inhibitors of protein farnesyltransferase.

A series of imidazole-containing peptidomimetic PFTase inhibitors and their co-crystal structures bound to PFTase and FPP are reported. The structures reveal that the peptidomimetics adopt a similar conformation to that of the extended CVIM tetrapeptide, with the imidazole group coordinating to the catalytic zinc ion. Both mono- and bis-imidazole-containing derivatives, 13 and 16, showed remarkably high enzyme inhibition activity against PFTase in vitro with IC50 values of 0.86 and 1.7 nM, respectively. The peptidomimetics were also highly selective for PFTase over PGGTase-I both in vitro and in intact cells. In addition, peptidomimetics and were found to suppress tumor growth in nude mouse xenograft models with no gross toxicity at a daily dose of 25 mg kg(-1).

Inhibiting angiogenesis and tumorigenesis by a synthetic molecule that blocks binding of both VEGF and PDGF to their receptors.

Angiogenesis depends on vascular endothelial growth factor (VEGF) for initiation and platelet-derived growth factor (PDGF) for maintenance of blood vessels. We have designed a targeted library of compounds from which we identified a novel molecule, GFB-204, that binds PDGF and VEGF, blocks binding of PDGF and VEGF to their receptors (200-500 nM) and subsequently inhibits PDGFR and Flk-1 tyrosine phosphorylation and stimulation of the protein kinases Erk1, Erk2 and Akt and the signal transducer and activator of transcription STAT3. GFB-204 is selective for PDGF and VEGF and does not inhibit EGF, IGF-1 and FGF stimulation of Erk1/2, Akt and STAT3. GFB-204 inhibits endothelial cell migration and capillary network formation in vitro. Finally, treatment of mice with GFB-204 suppresses human tumor growth and angiogenesis. Thus, inhibition of VEGF and PDGF receptor binding with a synthetic molecule results in potent inhibition of angiogenesis and tumorigenesis.