The 3' end of the coding region of senecavirus A contains a highly conserved sequence that potentially forms a stem-loop structure required for virus rescue.

Senecavirus A (SVA) can cause a vesicular disease in swine. It is a positive-strand RNA virus belonging to the genus Senecavirus in the family Picornaviridae. Positive-strand RNA viruses possess positive-sense, single-stranded genomes whose untranslated regions (UTRs) have been reported to contain cis-acting RNA elements. In the present study, a total of 100 SVA isolates were comparatively analyzed at the genome level. A highly conserved fragment (HCF) was found to be located in the 3D sequence and to be close to the 3' UTR. The HCF was computationally predicted to form a stem-loop structure. Eight synonymous mutations can individually disrupt the formation of a single base pair within the stem region. We found that SVA itself was able to tolerate each of these mutations alone, as evidenced by the ability to rescue all eight single-site mutants from their individual cDNA clones, and all of them were genetically stable during serial passaging. However, the replication-competent SVA could not be rescued from another cDNA clone containing all eight mutations. The failure to recover SVA might be attributed to disruption of the predicted stem-loop structure, whereas introduction of a wild-type HCF into the cDNA clone with eight mutations still had no effect on virus recovery. These results suggest that the putative stem-loop structure at the 3' end of the 3D sequence is a cis-acting RNA element that is required for SVA growth.

Identification of cis-acting replication element in VP2-encoding region of Senecavirus A genome.

Picornavirus possesses one positive-sense, single-stranded RNA genome, in which a cis-acting replication element (cre) is located. The cre is a stem-loop structure that harbors a conserved AAACA motif within its loop region. This motif functions as a template for adding two U residues to the viral VPg, therefore generating a VPg-pUpU that is required for viral RNA synthesis. Senecavirus A (SVA) is an emerging picornavirus. Its cre has not been identified as yet. In the present study, one putative cre containing a typical AAACA motif was computationally predicted to exist within the VP2-encoding sequence of SVA. To test the role of this putative cre, 22 SVA cDNA clones with different point mutations in their cre-formed sequences were constructed in an attempt to rescue replication-competent SVAs. A total of 11 viruses were rescued from their individual cDNA clones, implying that some mutated cres exerted lethal impacts on SVA replication. To eliminate these impacts, an intact cre was artificially inserted into those SVA cDNA clones without ability of recovering virus. The artificial cre was proven to be able of compensating for some, but not all, defects caused by mutated cres, leading to successful recovery of SVAs. These results indicated that the putative cre of SVA was functionally similar to those of other picornaviruses, perhaps involved in the uridylylation of VPg.

Only fourteen 3'-end poly(A)s sufficient for rescuing Senecavirus A from its cDNA clone, but inadequate to meet requirement of viral replication.

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. Its genome is a positive-sense, single-strand RNA that has 5' and 3' untranslated regions. There is a poly(A) tail at the 3' end of viral genome. Although the number of poly(A)s is variable, the length of poly(A) tail generally has the minimum nucleotide limit for picornaviral replication. To identify a range limit of poly(A)s for SVA recovery, five SVA cDNA clones, separately containing 25, 20, 15, 10 and 5 poly(A)s, were constructed for rescuing viruses. Replication-competent SVAs could be rescued from the first three cDNA clones, implying the range limit of poly(A)s was (A)15 to (A)10. To recognize the precise limit, four extra cDNA clones, separately containing 14, 13, 12 and 11 poly(A)s, were constructed to rescue SVAs further. The replication-competent SVA was rescued only from the poly(A)14-containing plasmid, indicating that the precise limit was poly(A)14 at the 3' end of cDNA clone for SVA recovery. The rescued SVA was serially passaged in cells. The passage-5 and -10 progenies were independently subjected to the analysis of 3'-rapid amplification of cDNA ends. Both progenies showed their own poly(A) tails far more than 14 (A)s, implying extra (A)s added to the poly(A)14 sequence during viral passaging. It can be concluded that fourteen (A)s are sufficient for rescuing a replication-competent SVA from its cDNA clone, but inadequate for maintaining viral propagation in cells.

Translation of Senecavirus A polyprotein is initiated from the IRES-proximal initiation codon.

To clarify whether Senecavirus A (SVA) has the potential of alternative translation, an extra G residue was inserted into an SVA cDNA clone, resultantly generating an "AUGAUG" motif. The second AUG is the authentic SVA initiation codon, whereas the first AUG is a putative one. Subsequently, eighteen nucleotides were inserted one by one between AUG and AUG for reconstructing cDNA clones. The test of virus recovery showed that three replication-competent SVAs, whose AUG/AUG-flanked sequences were not multiples of three nucleotides, were successfully rescued from their individual cDNA clones. The wild-type SVA possesses a UUUUU motif within the polyprotein-encoding region. Sanger sequencing showed that these three replication-competent SVAs harbored one or two extra U residues in the UUUUU motif, implying that polyprotein translation was initiated from the putative AUG, and the authentic AUG would be inactivated. This is probably attributed to the lack of ribosome scanning along an SVA genome.

The improved effect and its mechanism of phytic acid on DSS-induced UC mice.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) with an unknown etiology that is currently difficult to treat effectively. Phytic acid, a natural compound found in high-fiber foods, such as grains, has been shown to have many pharmacological functions, such as anti-inflammatory and blood-milk barrier maintenance. Our study investigated the effects of phytic acid on UC as well as the underlying mechanisms. We found that phytic acid ameliorated weight loss, shortened colon length, increased clinical scores, and decreased the release of pro-inflammatory factors (Il-1ß, Il-6, and TNF-α) in mice with DSS-induced UC. Phytic acid was also found to protect intestinal barrier integrity in mice with UC by maintaining the expression of tight junction proteins (occludin, claudin-3 and ZO-1) and mucin-2. In LPS-stimulated Caco-2 cells, phytic acid significantly inhibited the upregulation of pro-inflammatory factors and the downregulation of tight junction proteins. Further experimental results confirmed that phytic acid inhibits the activation of the AKT/NF-κB signaling pathway in both mice with UC and Caco-2 cells. To sum up, this study proved that phytic acid has a beneficial ameliorative effect on mice with DSS-induced UC, suggesting that it may be used to develop functional foods for the treatment or prevention of UC.

Pomiferin Exerts Antineuroinflammatory Effects through Activating Akt/Nrf2 Pathway and Inhibiting NF-κB Pathway.

Background: Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, are mainly characterized by progressive motor, sensory, or cognitive dysfunction in patients. Such diseases mostly occur in middle-aged and elderly people, and there is no effective cure. Studies have shown that neurodegenerative diseases are accompanied by neuroinflammation. The proinflammatory mediators produced neuroinflammation further damage neurons and aggravate the process of neurodegenerative diseases. Therefore, inhibiting neuroinflammation might be an effective way to alleviate neurodegenerative diseases. Pomiferin extracted from the fruit of the orange mulberry has a wide range of antioxidation and anti-inflammatory effects in peripheral tissues. However, it is not clear whether it plays a role on neuroinflammation. Methods: In our experiment, we studied the effect of Pomiferin on BV2 cell inflammation and its mechanism with cck-8, LDH, quantitative PCR, and ELISA and methods. We then investigated the effect of Pomiferin on the classical inflammatory pathway by Western blot methods. Results: The results showed that Pomiferin inhibited the production of ROS, NO, and proinflammatory mediators (IL-6, TNF-α, iNOS, and COX2) in BV2 cells. Further mechanism studies showed that Pomiferin activated the Akt/Nrf2 pathway and inhibited the NF-κB pathway. Conclusion: Our study demonstrated that Pomiferin exerts antineuroinflammatory effects through activating Akt/Nrf2 pathway and inhibiting NF-κB pathway.

[Comparative Phosphorus Accumulation and Ca-P Content of Two Submerged Plants in Response to Light Intensity and Phosphorus Levels].

The micro-environment formed by the photosynthesis of submerged plants is conducive to the formation of CaCO3-P from co-precipitation of calcium and phosphorus in water, thereby permanently removing phosphorus from water to the bottom mud and avoiding secondary pollution after plants decay. However, CaCO3-P co-precipitation shows obvious specific-differences and environmental dependencies. In the present study, two different submerged plants, Myriophyllum aquaticum and Potamogeton crispus, were used as the research objects. Two variables, inorganic phosphorus level (0, 0.2, and 2 mg·L-1) and light intensity [66 µmol·(m2·s)-1 and 110 µmol·(m2·s)-1], were set. After cultivating for a week, the plant relative growth rate, plant total phosphorus, plant ash phosphorus, and Ca-P were measured to analyze the actual ability of phosphorus accumulation and clarify the effect of plant corruption on phosphorus increase in the water body. Results revealed that under various culture conditions, the relative growth rates (RGR) of P. crispus were significantly higher than those of M. aquaticum, and RGR reached the maximum at a P level of 2 mg·L-1 and a light intensity of 66 µmol·(m2·s)-1. The addition of inorganic phosphorus significantly affected plant ash phosphorus of the two plants (P. crispus 95.681%, M. aquaticum 85.432%), and the highest value of Ca-P content in the ash phosphorus of the two submerged plants appeared at a high phosphorus level. The total phosphorus in P. crispus was lower than that in M. aquaticum under various treatments, but the total ash phosphorus and Ca-P levels were higher than those in M. aquaticum. Consequently, M. aquaticum and P. crispus can effectively accumulate phosphorus during growth. However, the actual ability of P. crispus of removing phosphorus from water by the formation of CaCO3-P was higher than that of M. aquaticum at a P level of 2 mg·L-1.