Allosteric Inhibition of the ß2-Adrenergic Receptor: Design and Synthesis of Cmpd-15 Analogs.

We designed and synthesized 27 new amide and dipeptide derivatives containing a substituted phenylalanine as negative allosteric modulators (NAMs) for the beta-2 adrenergic receptor (ß2AR). These analogs aimed to improve the activity of our lead compound, Cmpd-15, by introducing variations in three key regions: the meta-bromobenzyl methylbenzamide (S1), para-formamidophenylalanine (S2), and 1-cyclohexyl-1-phenylacetyl (S3) groups. The synthesis involved the Pd-catalyzed ß-C(sp3)-H arylation of N-acetylglycine with 1-iodo-4-substituent-benzenes as the key step. GloSensor cAMP accumulation assay revealed that six analogs (A1, C5, C6, C13, C15 and C17) surpass Cmpd-15 in ß2AR allosteric function. This highlights the crucial role of the S1 region (meta-bromobenzyl methylbenzamide) in ß2AR allostery while suggesting potential replaceability of the S2 region (para-formamidophenylalanine). These findings serve as a valuable springboard for further optimizing Cmpd-15, potentially leading to smaller, more active, and more stable ß2AR-targeting NAMs.

IL-6 and IL-1ß upregulation and tau protein phosphorylation in response to chronic alcohol exposure in the mouse hippocampus.

Alcoholism and alcohol abuse can lead to memory loss and cognitive dysfunction. The neuroinflammatory response plays an important role in the neurotoxic mechanism of chronic alcohol exposure. Additionally, the phosphorylation status of the tau protein is closely related to neurotoxicity and synaptic function. As inflammatory cytokines have been shown to regulate tau phosphorylation, in the present study, the aim was to determine whether cognitive impairment caused by chronic alcohol exposure is associated with neuroinflammation and tau hyperphosphorylation in the hippocampus. We established a chronic alcohol exposure model of C57BL/6J mice. The Y maze was used to assess the spatial recognition ability of mice, and ELISA was used to detect the levels of inflammatory cytokines IL-1ß and IL-6 in the serum. Immunohistochemical and western blot assays were used to assess the expression levels of IL-1ß and IL-6, as well as tau protein and its phosphorylation status in the hippocampus. We also analyzed the mRNA and protein expression of the synapse-associated proteins PSD95 and synaptophysin in the hippocampus. Our results showed that chronic alcohol exposure impaired the spatial recognition ability of mice upregulated the expression of IL-1ß and IL-6 in the serum and hippocampus and increased the phosphorylation of tau protein in the hippocampus. In addition, chronic alcohol exposure downregulated PSD95 and synaptophysin protein levels. The present results indicate that hippocampal IL-1ß, IL-6, and phosphorylated tau proteins may be involved in the neurotoxic mechanism of chronic alcohol exposure by mediating synaptic dysfunction.

Analysis of Pathogenicity and Virulence Factors of Ageratum leaf curl Sichuan virus.

Ageratum leaf curl Sichuan virus (ALCScV) is a novel monopartite begomovirus, which was identified from Ageratum conyzoides plants in Sichuan Province, China. In this study, we showed that ALCScV can induce typical dwarf and downward leaf-curling symptoms in Ageratum conyzoides, Helianthus annuus, and Nicotiana benthamiana plants and that the noncognate betasatellite can enhance disease symptoms and increase viral accumulation. Expression of the ALCScV-encoded V2, C1, and C4 proteins through a Potato virus X (PVX) vector caused severe symptoms in N. benthamiana. Further study revealed no symptoms in N. benthamiana plants inoculated with infectious ALCScV clones lacking the C4 protein and that the relative viral DNA accumulation levels significantly decreased when compared with ALCScV-inoculated plants. Thus, our mutational analyses demonstrated that C4 is a pathogenicity determinant that plays key roles in symptom formation and virus accumulation. Furthermore, we also demonstrated that the second glycine of C4 was critical for ALCScV pathogenicity.

The Malvastrum Yellow Vein Virus C4 Protein Promotes Disease Symptom Development and Enhances Virus Accumulation in Plants.

The begomovirus C4 protein is required for disease symptom development during virus infection in host plants. It can reprogram the cell cycle process for more efficient virus accumulation. In this study, we showed that the Malvastrum yellow vein virus (MaYVV) C4 protein could cause leaf up-ward curling and flower malformation, and increase virus accumulation in plants using PVX-based transient expression technology. We also demonstrated that, in the presence of its cognate betasatellite DNA (MaYVB), a mutant MaYVV, defective in producing the C4 protein (MaYVVΔC4), caused and alleviated infection in Nicotiana benthamiana. Transgenic plants expressing the MaYVV C4 protein showed upward leaf curling and uneven leaf lamina growth. Microscopic analysis showed that the epidermal cells of the C4 transgenic leaves were much smaller than those in the wild type (WT) leaves, and the mesophyll cells size and arrangement of transgenic plants was significantly altered. Inoculation of C4 transgenic plants with MaYVV or MaYVVΔC4 alone or associated with MaYVB showed that the transgenic C4 protein could promote viral and betasatellite accumulation and rescue the accumulation defect of MaYVVΔC4. Other transient expression assays also confirmed that the MaYVV C4 protein could suppress silencing of a GFP gene. In summary, our results indicate that the MaYVV C4 protein is a determinant of disease symptom and viral DNA accumulation. This protein can also function as a suppressor of RNA silencing and alter cell division and expansion.

Molecular characterization of virus-derived small RNAs in Nicotiana benthamiana plants infected with tobacco curly shoot virus and its ß satellite.

Tobacco curly shoot virus (TbCSV) is a monopartite DNA virus of the genus Begomovirus, which causes leaf curl symptoms in tobacco and tomato. The ß satellite of TbCSV (TbCSB induces more severe symptoms and enhanced virus accumulation when co-infects the host plants with TbCSV. Small interfering RNAs derived from virus(vsiRNAs) induce disease symptoms and promote virus invasion by target and guide the degradation of host transcripts The vsiRNAs derived from TbCSV and TbCSV + TbCSB remained to be explored to elucidate the molecular mechanism of symptoms development in plants. In the present work, two libraries of small RNA from TbCSV-infected and TbCSV + TbCSB-infected N. benthamiana plants were constructed and the vsiRNAs in both samples shared the same characteristics. The size of the vsiRNAs ranged from 18 to 30 nucleotides (nt), with most of them being 21 or 22 nt, which accounted for 29.11% and 23.22% in TbCSV plants and 29.39% and 21.82% in TbCSV + TbCSV plants, respectively. The vsiRNAs with A/U bias at the first site were abundant in both the TbCSV-treated and TbCSV + TbCSB-treated plants. It is discovered that the vsiRNAs continuously, but heterogeneously, distributed through bothe the TbCSV and TbCSB sequences. And the distribution profiles were similar in both the treatments such as mainly in the overlapping region of the AC2/AC3 coding sequences. The host transcripts targeted by vsiRNAs were predicted, and the targeted genes were found to be involved in varied biological processes. It is indicated that the presence of TbCSB does not significantly affect the production of vsiRNAs from TbCSV in plants, the distribution hotsopt of TbCSV vsiRNAs could be useful in designing effective targets for TbCSV resistance exploiting RNA interference.

Molecular identification of tobacco leaf curl disease in Sichuan province of China.

BACKGROUND: Tobacco leaf curl disease (TLCD) is caused by begomoviruses in Geminiviridae, and infected plants exhibit leaf thickening, downward leaf curling, vein swelling as well as stunting symptoms. It is one of the economically important diseases in tropical and subtropical tobacco-growing areas. Seven monopartite begomoviruses have been identified causing TLCD in China. FINDINGS: In this study, two begomoviruses were identified, characterized and polygenetically analyzed to be responsible for TLCD in Sichuan province, China. The complete genomes of two isolates SC230 and SC379 from diseased tobacco samples were cloned and sequenced to be 2738 nucleotides (nts) and 2748 nts in size, respectively. Sequence alignment indicated that SC230 and SC379 were most closely related to Tomato yellow leaf curl China virus (TYLCCNV-CN[CN:Sc226:Mal:12]) and Papaya leaf curl China virus (PaLCuCNV-CN[CN:Gx30:Lyc:03]), with a sequence identity of 99.2 and 99.2 %, respectively. The infection rate of TYLCCNV and PaLCuCNV was 100 and 34.78 %, respectively and the co-infection rate was 34.78 % in fields. Betasatellites of SC230 and SC379 share the highest sequence identity with Tomato yellow leaf curl China betasatellite (TYLCCNB-CN[CN:Sc176:Malva:12]) and TYLCCNB-CN[CN:Yn149:Tom:09], with a sequence identity of 95.2 and 97.2 % respectively. Sequence identity between betasatellites of SC230 and SC379 was 89.6 %. And TYLCCNB was detected in all the samples. CONCLUSION: Co-infection of TYLCCNV and PaLCuCNV was identified in tobacco plants with typical symptoms of TLCD from Sichuan province in China, and this is the first report of PaLCuCNV infecting tobacco in China. TYLCCNV/TYLCCNB disease complex is widespread in tobacco-growing areas in Panzhihua city of Sichuan.