Phosphorylation of TG-interacting factor 1 at carboxyl-terminal sites in response to insulin regulates adipocyte differentiation.

TG-interacting factor 1 (TGIF1) contributes to the differentiation of murine white preadipocyte and human adipose tissue-derived stem cells; however, its regulation is not well elucidated. Insulin is a component of the adipogenic cocktail that induces ERK signaling. TGIF1 phosphorylation and sustained stability in response to insulin were reduced through the use of specific MEK inhibitor U0126. Mutagenesis at T235 or T239 residue of TGIF1 in preadipocytes led to dephosphorylation of TGIF1. The reduced TGIF1 stability resulted in an increase in p27kip1 expression, a decrease in phosphorylated Rb expression and cellular proliferation, and a reduced accumulation of lipids compared to the TGIF1-overexpressed cells. These findings highlight that insulin/ERK-driven phosphorylation of the T235 or T239 residue at TGIF1 is crucial for adipocyte differentiation.

An orally available Mpro inhibitor is effective against wild-type SARS-CoV-2 and variants including Omicron.

Emerging SARS-CoV-2 variants continue to cause waves of new infections globally. Developing effective antivirals against SARS-CoV-2 and its variants is an urgent task. The main protease (Mpro) of SARS-CoV-2 is an attractive drug target because of its central role in viral replication and its conservation among variants. We herein report a series of potent α-ketoamide-containing Mpro inhibitors obtained using the Ugi four-component reaction. The prioritized compound, Y180, showed an IC50 of 8.1 nM against SARS-CoV-2 Mpro and had oral bioavailability of 92.9%, 31.9% and 85.7% in mice, rats and dogs, respectively. Y180 protected against wild-type SARS-CoV-2, B.1.1.7 (Alpha), B.1.617.1 (Kappa) and P.3 (Theta), with EC50 of 11.4, 20.3, 34.4 and 23.7 nM, respectively. Oral treatment with Y180 displayed a remarkable antiviral potency and substantially ameliorated the virus-induced tissue damage in both nasal turbinate and lung of B.1.1.7-infected K18-human ACE2 (K18-hACE2) transgenic mice. Therapeutic treatment with Y180 improved the survival of mice from 0 to 44.4% (P = 0.0086) upon B.1.617.1 infection in the lethal infection model. Importantly, Y180 was also highly effective against the B.1.1.529 (Omicron) variant both in vitro and in vivo. Overall, our study provides a promising lead compound for oral drug development against SARS-CoV-2.

Genotyping of Soybean Cultivars With Medium-Density Array Reveals the Population Structure and QTNs Underlying Maturity and Seed Traits.

Soybean was domesticated about 5,000 to 6,000 years ago in China. Although genotyping technologies such as genotyping by sequencing (GBS) and high-density array are available, it is convenient and economical to genotype cultivars or populations using medium-density SNP array in genetic study as well as in molecular breeding. In this study, 235 cultivars, collected from China, Japan, USA, Canada and some other countries, were genotyped using SoySNP8k iSelect BeadChip with 7,189 single nucleotide polymorphisms (SNPs). In total, 4,471 polymorphic SNP markers were used to analyze population structure and perform genome-wide association study (GWAS). The most likely K value was 7, indicating this population can be divided into 7 subpopulations, which is well in accordance with the geographic origins of cultivars or accession studied. The LD decay rate was estimated at 184 kb, where r2 dropped to half of its maximum value (0.205). GWAS using FarmCPU detected a stable quantitative trait nucleotide (QTN) for hilum color and seed color, which is consistent with the known loci or genes. Although no universal QTNs for flowering time and maturity were identified across all environments, a total of 30 consistent QTNs were detected for flowering time (R1) or maturity (R7 and R8) on 16 chromosomes, most of them were corresponding to known E1 to E4 genes or QTL region reported in SoyBase (soybase.org). Of 16 consistent QTNs for protein and oil contents, 11 QTNs were detected having antagonistic effects on protein and oil content, while 4 QTNs soly for oil content, and one QTN soly for protein content. The information gained in this study demonstrated that the usefulness of the medium-density SNP array in genotyping for genetic study and molecular breeding.

A PP2C-1 Allele Underlying a Quantitative Trait Locus Enhances Soybean 100-Seed Weight.

Cultivated soybeans may lose some useful genetic loci during domestication. Introgression of genes from wild soybeans could broaden the genetic background and improve soybean agronomic traits. In this study, through whole-genome sequencing of a recombinant inbred line population derived from a cross between a wild soybean ZYD7 and a cultivated soybean HN44, and mapping of quantitative trait loci for seed weight, we discovered that a phosphatase 2C-1 (PP2C-1) allele from wild soybean ZYD7 contributes to the increase in seed weight/size. PP2C-1 may achieve this function by enhancing cell size of integument and activating a subset of seed trait-related genes. We found that PP2C-1 is associated with GmBZR1, a soybean ortholog of Arabidopsis BZR1, one of key transcription factors in brassinosteroid (BR) signaling, and facilitate accumulation of dephosphorylated GmBZR1. In contrast, the PP2C-2 allele with variations of a few amino acids at the N-terminus did not exhibit this function. Moreover, we showed that GmBZR1 could promote seed weight/size in transgenic plants. Through analysis of cultivated soybean accessions, we found that 40% of the examined accessions do not have the PP2C-1 allele, suggesting that these accessions can be improved by introduction of this allele. Taken together, our study identifies an elite allele PP2C-1, which can enhance seed weight and/or size in soybean, and pinpoints that manipulation of this allele by molecular-assisted breeding may increase production in soybean and other legumes/crops.

The transcriptomic signature of developing soybean seeds reveals the genetic basis of seed trait adaptation during domestication.

Cultivated soybean has undergone many transformations during domestication. In this paper we report a comprehensive assessment of the evolution of gene co-expression networks based on the analysis of 40 transcriptomes from developing soybean seeds in cultivated and wild soybean accessions. We identified 2680 genes that are differentially expressed during seed maturation and established two cultivar-specific gene co-expression networks. Through analysis of the two networks and integration with quantitative trait locus data we identified two potential key drivers for seed trait formation, GA20OX and NFYA. GA20OX encodes an enzyme in a rate-limiting step of gibberellin biosynthesis, and NFYA encodes a transcription factor. Overexpression of GA20OX and NFYA enhanced seed size/weight and oil content, respectively, in seeds of transgenic plants. The two genes showed significantly higher expression in cultivated than in wild soybean, and the increases in expression were associated with genetic variations in the promoter region of each gene. Moreover, the expression of GA20OX and NFYA in seeds of soybean accessions correlated with seed weight and oil content, respectively. Our study reveals transcriptional adaptation during soybean domestication and may identify a mechanism of selection by expression for seed trait formation, providing strategies for future breeding practice.

GmWRKY27 interacts with GmMYB174 to reduce expression of GmNAC29 for stress tolerance in soybean plants.

Soybean (Glycine max) is an important crop for oil and protein resources worldwide. The molecular mechanism of the abiotic stress response in soybean is largely unclear. We previously identified multiple stress-responsive WRKY genes from soybean. Here, we further characterized the roles of one of these genes, GmWRKY27, in abiotic stress tolerance using a transgenic hairy root assay. GmWRKY27 expression was increased by various abiotic stresses. Over-expression and RNAi analysis demonstrated that GmWRKY27 improves salt and drought tolerance in transgenic soybean hairy roots. Measurement of physiological parameters, including reactive oxygen species and proline contents, supported this conclusion. GmWRKY27 inhibits expression of a downstream gene GmNAC29 by binding to the W-boxes in its promoter region. The GmNAC29 is a negative factor of stress tolerance as indicated by the performance of transgenic hairy roots under stress. GmWRKY27 interacts with GmMYB174, which also suppresses GmNAC29 expression and enhances drought stress tolerance. The GmWRKY27 and GmMYB174 may have evolved to bind to neighbouring cis elements in the GmNAC29 promoter to co-reduce promoter activity and gene expression. Our study discloses a valuable mechanism in soybean for regulation of the stress response by two associated transcription factors. Manipulation of these genes should facilitate improvements in stress tolerance in soybean and other crops.

[Identification A novel protein TRIM38 that activate NF-kappaB signaling pathways].

OBJECTIVE: The tripartite motif (TRIM) proteins are a family of more than 70 human members, however only a few of them have been well studied. It has been shown that TRIM proteins are involved in various cellular processes such as cell proliferation, differentiation, apoptosis and antiviral defense. The functions of TRIM38 are largely unknown. In this study we explore the effect of TRIM38 on NF-kappaB signaling pathway. METHODS: 293T cells were transfected with NF-kappaB-Luc and plasmids expressing TRIM38 and its mutants fused to Flag. 24 h after transfection, cells were harvested and luciferase activities were measured. Data are representative of three independent experiments with triplicate samples. The expression of proteins was analyzed by Western Blot. RESULTS: TRIM38 could activate NF-kappaB signaling pathway. The mutants of TRIM38 affected the function of TRIM38. Only the mutant of SPRY domain deletion had no obviously influence of the function of TRIM38. CONCLUSION: Our study reveals that NF-kappaB is activated in response to TRIM38.

[Codon modification of HIV-1 subtype B gag gene and its immunogenicity in mice].

HIV-1 subtype B gag genes were cloned from the infected paid blood donors in Henan, and the consensus sequence based on these prevalent strains was obtained by aligning. The codons of the consensus gag sequence were modified according to mammalian codon usage. Western blot analysis was used to compare the expression level of wild type and codon-modified gag gene. It was found that the expression level of Gag protein was improved largely by codon-modification. Then the mod. gag gene was inserted into the adenovirus vector and the recombinant adenovirus rAdV-mod. gag was constructed. 10(8) PFU or 10(9) PFU rAdV-mod, gag vaccinated mice twicely could elicit high level Gag-specific CTL responses in immunized mice. In conclusion,the codon modification of gag gene is successful. The recombinant adenovirus vaccine harbouring mod. gag can induce robust Gag-specific CTL immune response in mice.

[Immunogenicity of a chimeric adenovirus type 5 vector with type 35 fiber containing HIV-1 gag in mice].

OBJECTIVE: To study the immune effect of a chimeric adenovirus type 5 vector with type 35 fiber (rAd5/F35) vaccine in BALB/c mice. METHODS: The expression of HIV Gag protein was determined using indirect immunofluorescent staining. The rAd5/F35-mod.gag vector was injected intramuscularly to mice. The IgG antibody was detected by ELISA and CTL response was detected by intracellular cytokine stain assay. RESULTS: The rAd5/F35-mod.gag vector could express HIV Gag protein in vitro and generate strong HIV-specific immune responses in vivo. But anti-Ad5 immunity could limit its immunogenicity in vivo. CONCLUSION: The rAd5/F35-mod.gag vector can elicit specific CTL response and IgG antibody in animal model. In mice with high Ad5 vector-specific immunity, Ad5/F35-mod.gag showed lower level of Gag specific CTL and antibody response than in mice without pre-existing adenovirus type 5 immunity. The results indicated that fiber exchange alone does not evade pre-existing Ad5 immunity.

Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virus gag gene.

AIM: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions, and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses. METHODS: A series of chimeric BHIV proviral DNAs differing in the replacement regions in gag gene were constructed, and then were transfected into 293T cells. The expression of chimeric viral genes was detected at the RNA and protein level. The supernatant of 293T cell was ultra centrifuged to detect the probable chimeric virion. Once the chimeric virion was detected, its biological activities were also assayed by infecting HIV-sensitive MT4 cells. RESULTS: Four chimeric BHIV proviral DNAs were constructed. Genes in chimeric viruses expressed correctly in transfected 293T cells. All four constructs assembled chimeric virions with different degrees of efficiency. These virions had complete structures common to retroviruses and packaged genomic RNAs, but the cleavages of the precursor Gag proteins were abnormal to some extent. Three of these virions tested could attach and enter into MT4 cells, and one of them could complete the course of reverse transcription. Yet none of them could replicate in MT4 cells. CONCLUSION: The replacement of partial gag gene of HIV with BIV gag gene is feasible. Genes in chimeric BHIVs are accurately expressed, and virions are assembled. These chimeric BHIVs (proviral DNA together with virus particles) have the potential to become a new kind of HIV/AIDS vaccine.